EP2467165B1

Targeted immunoconjugates

Abstract

This record has no abstract on file.

EP2467165B1, drawing sheet 1 — Sheet 1 of 852

Term

3.9 yearsleft in the term

Expires 13 August 2030.

16 claims: 6 independent, 10 dependent

1
An immunoconjugate comprising:(a) at least a first single-chain effector moiety;and (b) a first and a second antigen binding moiety, wherein said single-chain effector moiety is a cytokine selected from the group consisting of: interleukin-2 (IL-2), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α (INF-α), and interleukin-12 (IL-12);wherein each of said first and second antigen binding moieties is a Fab molecule;wherein said first single-chain effector moiety shares an amino- or carboxy-terminal peptide bond with said first antigen binding moiety;and wherein said second antigen binding moiety shares an amino- or carboxy-terminal peptide bond with said first effector moiety.
4
The immunoconjugate of any one of claims 1-3, wherein the variable regions of said first and second antigen binding moieties are specific for the same antigen.
5
The immunoconjugate of any one of claims 1-3, wherein the variable regions of said first and second antigen binding moieties are specific for different antigens.
11
An isolated polynucleotide encoding a fragment of the immunoconjugate of any one of claims 1 to 3, wherein said polynucleotide encodes (i) the heavy chains of said first and second antigen binding moieties and said first effector moiety;(ii) the light chains of said first and second antigen binding moieties and said first effector moiety;or (iii) one light chain from said first antigen binding moiety, one heavy chain from said second antigen binding moiety and said first effector moiety.
14
A composition comprising the immunoconjugate of any one of claims 1-10 and a pharmaceutical carrier.
15
The immunoconjugate of any one of claims 1-10 for use in the treatment of a disease in a patient in need thereof.

Independent claims6

362 paragraphs in 7 sections, as filed

BACKGROUND OF THE INVENTION

Field of the Invention

0001The present invention relates to an immunoconjugate comprising: (a) at least a first single-chain effector moiety; and (b) a first and a second antigen binding moiety, wherein said single-chain effector moiety is a cytokine selected from the group consisting of: interleukin-2 (IL-2), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α (INF-α), and interleukin-12 (IL-12); wherein each of said first and second antigen binding moieties is a Fab molecule; wherein said first single-chain effector moiety shares an amino- or carboxy-terminal peptide bond with said first antigen binding moiety; and wherein said second antigen binding moiety shares an amino- or carboxy-terminal peptide bond with said first effector moiety. The disclosure generally relates to antigen-specific immunoconjugates for selectively delivering effector moieties that influence cellular activity. In addition, the disclosure relates to nucleic acid molecules encoding such immunoconjugates, and vectors and host cells comprising such nucleic acid molecules. The disclosure further relates to methods for producing the immunoconjugates described herein, and to compositions comprising these immunoconjugates for use in the treatment of disease.

Background Art

0002The selective destruction of an individual cell or a specific cell type is often desirable in a variety of clinical settings. For example, it is a primary goal of cancer therapy to specifically destroy tumor cells, while leaving healthy cells and tissues intact and undamaged. A multitude of signal transduction pathways in the cell are linked to the cell's survival and/or death. Accordingly, the direct delivery of a pathway factor involved in cell survival or death can be used to contribute to the cell's maintenance or destruction.

0003Cytokines are cell signaling molecules that participate in regulation of the immune system. When used in cancer therapy, cytokines can act as immunomodulatory agents that have anti-tumor effects and which can increase the immunogenicity of some types of tumors. However, rapid blood clearance and lack of tumor specificity require systemic administration of high doses of the cytokine in order to achieve a concentration of the cytokine at the tumor site sufficient to activate an immune response or have an anti-tumor effect. These high levels of systemic cytokine can lead to severe toxicity and adverse reactions.

0004One way to deliver a signal transduction pathway factor, such as a cytokine, to a specific site in vivo (e.g., a tumor or tumor microenvironment) is to conjugate the factor to an immunoglobulin specific for the site. Early strategies aimed at delivering signal transduction pathway factors, such as cytokines, to a specific site in vivo included immunoglobulin heavy chains conjugated to various cytokines, including lymphotoxin, tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor (GM-CSF). The immunoglobulin heavy chains were either chemically conjugated to a cytokine or the immunogobulin-cytokine conjugate was expressed as a fusion protein. See<nplcit id="ncit0001" npl-type="s"><text>Nakamura K. and Kubo, A. Cancer Supplement 80:2650-2655 (1997</text></nplcit>); <nplcit id="ncit0002" npl-type="s"><text>Jun, L. et al., Chin. Med. J. 113:151-153 (2000</text></nplcit>); and <nplcit id="ncit0003" npl-type="s"><text>Becker J.C., et al., Proc. Natl. Acad. Sci. USA 93:7826-7831 (1996</text></nplcit>). Researchers observed that, not only were they able to target cytokines to specific sites in vivo, they were also able to take advantage of the fact that monoclonal antibodies have longer serum half-lives than most other proteins. Due to the systemic toxicity associated with high doses of certain unconjugated cytokines, i.e. IL-2, the ability of an immunoglobulin-cytokine fusion protein to maximize immunostimulatory activities at the site of a tumor whilst keeping systemic side effects to a minimum at a lower dose led researchers to believe that immunoglobulin-cytokine immunoconjugates were optimal therapeutic agents. However, one of the major limitations in the clinical utility of immunoglobulins as delivery agents is their inadequate uptake and poor distribution in tumors, partially due to the large size of the immunoglobulin molecule. See<nplcit id="ncit0004" npl-type="s"><text>Xiang, J. et al., Immunol. Cell Biol. 72:275-285 (1994</text></nplcit>). Additionally, it has been suggested that immunoglobulin-cytokine immunoconjugates can activate complement and interact with Fc receptors. This inherent immunoglobulin feature has been viewed unfavorably because therapeutic immunoconjugates may be targeted to cells expressing Fc receptors rather than the preferred antigen-bearing cells. One approach to overcoming these problems is the use of engineered immunoglobulin fragments. Numerous. studies have detailed the characteristics of immunoglobulin fragment-cytokine immunoconjugates. See<nplcit id="ncit0005" npl-type="s"><text>Savage, P. et al., Br. J. Cancer 67:304-310 (1993</text></nplcit>); <nplcit id="ncit0006" npl-type="s"><text>Harvill, E.T. and Morrison S.L., Immunotechnol. 1:95-105 (1995</text></nplcit>); and <nplcit id="ncit0007" npl-type="s"><text>Yang J. et al., Mol. Immunol. 32:873-881 (1995</text></nplcit>). In general, there are two common immunoglobulin fragment-cytokine fusion protein constructs, the F(ab')2-cytokine expressed in mammalian cells and the scFv-cytokine expressed in Escherichia coli. See<nplcit id="ncit0008" npl-type="s"><text>Xiang, J. Hum. Antibodies 9:23-36 (1999</text></nplcit>). Both the tumor-binding reactivity of the immunoglobulin parent molecule and the functional activity of the cytokine are maintained in most of these types of immunoconjugates. Recent preclinical studies have shown that these fusion proteins are able to target cytokines to tumors expressing the tumor-associated antigen in vivo, and to inhibit both primary and metastatic tumors in an immune competent animal model.

0005Examples of immunoglobulin fragment-cytokine immunoconjugates include the scFv-IL-2 immunoconjugate as set forth in <patcit id="pcit0001" dnum="WO2001062298A2A"><text>PCT publication WO 2001/062298 A2</text></patcit>; the immunoglobulin heavy chain fragment-GM-CSF immunoconjugate as set forth in <patcit id="pcit0002" dnum="US5650150A"><text>U.S. Pat. No. 5,650,150</text></patcit>; the immunoconjugate as set forth in <patcit id="pcit0003" dnum="WO2006119897A2A"><text>PCT publication WO 2006/119897 A2</text></patcit>, wherein scFv-IL-12 first subunit shares only disulfide bond(s) with IL-12 second subunit-scFv, and the immunoconjugate as described in <patcit id="pcit0004" dnum="WO9929732A2A2"><text>PCT publication WO 99/29732 A2</text></patcit>, wherein Ig heavy chain fragment-IL-12 first subunit shares only disulfide bond(s) with Ig heavy chain fragment-IL-12 second subunit. Also immunotoxins based on immunoglobulin fragments have been described. <patcit id="pcit0005" dnum="WO9839363A"><text>WO 98/39363</text></patcit> describes immunotoxins wherein two subunits, each consisting of a toxin moiety coupled to an scFv or Fab fragment-based antigen-binding moiety, are linked via a disulfide bond to form divalent molecules. <patcit id="pcit0006" dnum="WO2009017823A"><text>WO 2009/017823</text></patcit> describes diabody-based diphtheria immunotoxins. While these second generation immunoconjugates have some improved properties as compared to the first generation immunoglobulin-cytokine conjugates, development of more and even safer specific therapeutic agents is desirable for greater effectiveness against tumor cells and a decrease in the number and severity of the side effects of these products (e.g., toxicity, destruction of non-tumor cells, etc.). Additionally, it is desirable to identify ways to further stabilize immunoconjugates while maintaining acceptable therapeutic activity levels.

0006The present invention provides immunoconjugates that exhibit improved efficacy, high specificity of action, reduced toxicity, and improved stability in blood relative to known immunoconjugates.

BRIEF SUMMARY OF THE INVENTION

0007Described herein are immunoconjugates that exhibit improved efficacy, high specificity of action, reduced toxicity and improved stability as compared to known immunoconjugates. The immunoconjugates of the present invention can be used to selectively deliver effector moieties to a target site in vivo. In another embodiment, the immunoconjugate delivers a cytokine to a target site, wherein the cytokine can exert a localized biological effect, such as a local inflammatory response, stimulation of T cell growth and activation, and/or activation of B and/or NK cells. The invention relates to the following immunoconjugates, polynucleotides, host cells and methods for producing an immunoconjugate, and compositions comprsising an immunoconjugate: <ol id="ol0001" ol-style=""><li>(1) An immunoconjugate comprising: (a) at least a first single-chain effector moiety; and (b) a first and a second antigen binding moiety, wherein said single-chain effector moiety is a cytokine selected from the group consisting of: interleukin-2 (IL-2), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-α (INF-α), and interleukin-12 (IL-12); wherein each of said first and second antigen binding moieties is a Fab molecule; wherein said first single-chain effector moiety shares an amino- or carboxy-teiminal peptide bond with said first antigen binding moiety; and wherein said second antigen binding moiety shares an amino- or carboxy-terminal peptide bond with said first effector moiety.</li><li>(2) The immunoconjugate of (1), wherein said immunoconjugate consists essentially of a first effector moiety and first and second antigen binding moieties joined by one or more linker sequences.</li><li>(3) The immunoconjugate of (1) or (2), wherein said first Fab molecule is joined at its heavy or light chain carboxy-terminus to the amino-terminal amino acid of said effector moiety, and said effector moiety is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of the heavy or light chain of the second Fab molecule.</li><li>(4) The immunoconjugate of any one of (1) to (3), wherein the variable regions of said first and second antigen binding moieties are specific for the same antigen.</li><li>(5) The immunoconjugate of any one of (1) to (3), wherein the variable regions of said first and second antigen binding moieties are specific for different antigens.</li><li>(6) The immunoconjugate of (4) or (5), wherein said first and/or said second antigen binding moieties are specific for an antigen selected from the group consisting of the Fibroblast Activated Protein (FAP), the Melanoma Chondroitin Sulfate Proteoglycan (MCSP), the A2 domain of Tenascin (TNC-A2), the A1 domain of Tenascin (TNC-A1) and the Extra Domain B of fibronectin (EDB).</li><li>(7) The immunoconjugate of (6), wherein said first and/or second antigen binding moieties are specific for the A2 domain of Tenascin (TNC-A2), and wherein said immunoconjugate comprises a heavy chain variable region sequence selected from the group of SEQ ID NO: 7, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO:195, SEQ ID NO: 199, SEQ ID NO: 203, and SEQ ID NO: 207, and a light chain variable region sequence selected from the group of SEQ ID NO: 3, SEQ ID NO: 5; SEQ ID NO: 177, SEQ ID NO: 181, SEQ ID NO:185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO: 205.</li><li>(8) The immunoconjugate of (6), wherein said first and/or second antigen binding moieties are specific for Fibroblast Activated Protein (FAP), and wherein said immunoconjugate comprises a heavy chain variable region sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171, and SEQ ID NO: 175, and a light chain variable region sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169, and SEQ ID NO: 173.</li><li>(9) The immunoconjugate of (6), wherein said first and/or second antigen binding moieties are specific for Melanoma Chondroitin Sulfate Proteoglycan (MCSP), and wherein said immunoconjugate comprises a heavy chain variable region sequence of SEQ ID NO: 257 or SEQ ID NO: 261, and a light chain variable region sequence of SEQ ID NO: 259 or SEQ ID NO: 271.</li><li>(10) The immunoconjugate of (1), wherein said cytokine is IL-2.</li><li>(11) An isolated polynucleotide encoding a fragment of the immunoconjugate of any one of (1) to (3), wherein said polynucleotide encodes (i) the heavy chains of said first and second antigen binding moieties and said first effector moiety; (ii) the light chains of said first and second antigen binding moieties and said first effector moiety; or (iii) one light chain from said first antigen binding moiety, one heavy chain from said second antigen binding moiety and said first effector moiety.</li><li>(12) A host cell comprising the polynucleotide of (11).</li><li>(13) A method of producing the immunoconjugate of any one of (1) to (10), wherein the method comprises culturing the host cell of claim 16 under conditions suitable for the expression of the immunoconjugate.</li><li>(14) A composition comprising the immunoconjugate of any one of (1) to (10) and a pharmaceutical carrier.</li><li>(15) The immunoconjugate of any one of (1) to (10) for the treatment of a disease in a patient in need thereof.</li><li>(16) The immunoconjugate of (15), wherein said disease is cancer.</li></ol>

0008The disclosure generally relates to an immunoconjugate that comprises at least a first effector moiety and at least a first and a second antigen binding moiety independently selected from the group consisting of an Fv and an Fab, wherein a first effector moiety shares an amino- or carboxy-terminal peptide bond with a first antigen binding moiety and a second antigen binding moiety shares an amino- or carboxy-terminal peptide bond with either i) the first effector moiety or ii) the first antigen binding moiety.

0009Another aspect of the present disclosure is an immunoconjugate that comprises at least a first single-chain effector moiety joined at its amino-terminal amino acid to one or more scFv molecules and wherein the first-single-chain effector moiety is joined at its carboxy-terminal amino acids to one or more scFv molecules.

0010Another aspect of the present disclosure is an immunoconjugate that comprises at least a first single-chain effector moiety and first and second antigen binding moieties, wherein each of the first and second antigen binding moieties comprises an scFv molecule joined at its carboxy-terminal amino acid to a constant region comprising a immunoglobulin constant domain independently selected from the group consisting of: IgG CH1, IgG Ckappa, and IgE CH4, and wherein the first antigen binding moiety is joined at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked through at least one disulfide bond. Another aspect of the present disclosure is an immunoconjugate that comprises at least a first single-chain effector moiety and first and second antigen binding moieties, wherein each of the first and second antigen binding moieties comprises an scFv molecule joined at its carboxy-terminal amino acid to an IgG1 CH3 domain, and wherein the first antigen binding moiety is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked through at least one disulfide bond.

0011Another aspect of the present disclosure is directed to an immunoconjugate that comprises first and second single-chain effector moieties and first and second antigen binding moieties, wherein each of the antigen binding moieties comprises an Fab molecule joined at its heavy or light chain carboxy-terminal amino acid to an IgG1 CH3 domain, and wherein each of the IgG1 CH3 domains is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked through at least one disulfide bond.

0012In one embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 95. In another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 104. In another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 105. In another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 106. In another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 107. In another embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 96. In a further embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 96 and a polypeptide sequence selected from the group consisting of SEQ ID NOs: 95 and 104-107. In another embodiment, the immunoconjugate comprises a polypeptide having a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 95, 96, and 104-107.

0013In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 108. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of SEQ ID NO: 108. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 117. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of SEQ ID NO: 117. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 118. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of SEQ ID NO: 118. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 119. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of SEQ ID NO: 119. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 120. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of SEQ ID NO: 120. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 109. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of SEQ ID NO: 109.

0014In one embodiment, the immunoconjugate comprises a heavy chain variable region that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 13 or SEQ ID NO: 15. In another embodiment, the immunoconjugate comprises a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 9 or SEQ ID NO: 11. In a further embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 13 or SEQ ID NO: 15, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 9 or SEQ ID NO: 11.

0015In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 14 or SEQ ID NO: 16. In another embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is encoded by the polynucleotide sequence of either SEQ ID NO: 14 or SEQ ID NO: 16. In one embodiment, the immunoconjugate comprises a light chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 10 or SEQ ID NO: 12. In another embodiment, the immunoconjugate comprises a light chain variable region sequence that is encoded by the polynucleotide sequence of either SEQ ID NO: 10 or SEQ ID NO: 12.

0016In one embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 99. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 100 or SEQ ID NO: 215. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 101 or SEQ ID NO: 235. In a further embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 100, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 101. In a further embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 215, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 235.

0017In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 112. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of SEQ ID NO: 112. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 113 or SEQ ID NO: 216. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of either SEQ ID NO: 113 or SEQ ID NO: 216. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 114 or SEQ ID NO: 236. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of either SEQ ID NO: 114 or SEQ ID NO: 236.

0018In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 7, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 203 and SEQ ID NO: 207. In another embodiment, the immunoconjugate comprises a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 177, SEQ ID NO: 181, SEQ ID NO: 185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 201 and SEQ ID NO: 205. In a further embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 7, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO:195, SEQ ID NO: 199, SEQ ID NO: 203 and SEQ ID NO: 207 and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 177, SEQ ID NO: 181, SEQ ID NO:185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 201 and SEQ ID NO: 205.

0019In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 8, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 204 and SEQ ID NO: 208. In another embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 8, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 204 and SEQ ID NO: 208. In one embodiment, the immunoconjugate comprises a light chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, SEQ ID NO: 202 and SEQ ID NO: 206. In another embodiment, the immunoconjugate comprises a light chain variable region sequence that is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198; SEQ ID NO: 202 and SEQ ID NO: 206.

0020In one embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 239, SEQ ID NO: 241 and SEQ ID NO: 243. In another embodiment, the conjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 245, SEQ ID NO: 247 and SEQ ID NO:249. In a further embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 239, SEQ ID NO: 241 and SEQ ID NO: 243, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 245, SEQ ID NO: 247 and SEQ ID NO:249.

0021In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 240, SEQ ID NO: 242 and SEQ ID NO: 244. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 240, SEQ ID NO: 242 and SEQ ID NO: 244. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 246, SEQ ID NO: 248 and SEQ ID NO: 250. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence selected from the group of SEQ ID NO: 246, SEQ ID NO: 248 and SEQ ID NO: 250. In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171 and SEQ ID NO: 175. In another embodiment, the immunoconjugate comprises a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169 and SEQ ID NO: 173. In a further embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171 and SEQ ID NO: 175, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169 and SEQ ID NO: 173.

0022In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172 and SEQ ID NO: 176. In another embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172 and SEQ ID NO: 176. In one embodiment, the immunoconjugate comprises a light chain variable region sequence that is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170 and SEQ ID NO: 174. In another embodiment, the immunoconjugate comprises a light chain variable region sequence that is encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170 and SEQ ID NO: 174.

0023In one embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225 and SEQ ID NO: 227. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233 and SEQ ID NO: 237. In a further embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 211, SEQ ID NO: 219, and SEQ ID NO: 221, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 231. In a further embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence selected from the group of SEQ ID NO: 209, SEQ ID NO: 223, SEQ ID NO: 225 and SEQ ID NO: 227, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 229. In a further embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 213, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 233. In a further embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 217, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 237.

0024In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226 and SEQ ID NO: 228. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226 and SEQ ID NO: 228. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234 and SEQ ID NO: 238. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234 and SEQ ID NO: 238.

0025In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 257 or SEQ ID NO: 261. In another embodiment, the immunoconjugate comprises a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 259 or SEQ ID NO: 271. In a further embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to to the sequence of either SEQ ID NO: 257 or SEQ ID NO: 261, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 259 or SEQ ID NO: 271.

0026In one embodiment, the immunoconjugate comprises a heavy chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of either SEQ ID NO: 258 or SEQ ID NO: 262. In another embodiment, the immunoconjugate comprises a heavy chain variable region sequence that is encoded by the polynucleotide sequence of either SEQ ID NO: 258 or SEQ ID NO: 262. In one embodiment, the immunoconjugate comprises a light chain variable region sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of either SEQ ID NO: 260 or SEQ ID NO: 272. In another embodiment, the immunoconjugate comprises a light chain variable region sequence that is encoded by the polynucleotide sequence of either SEQ ID NO: 260 or SEQ ID NO: 272.

0027In one embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 251 or SEQ ID NO: 255. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 253 or SEQ ID NO: 265. In a further embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 251 or SEQ ID NO: 255, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of either SEQ ID NO: 253 or SEQ ID NO: 265.

0028In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of either SEQ ID NO: 252 or SEQ ID NO: 256. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of either SEQ ID NO: 252 or SEQ ID NO: 256. In one embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 254 or SEQ ID NO: 266. In another embodiment, the immunoconjugate comprises a polypeptide sequence that is encoded by the polynucleotide sequence of either SEQ ID NO: 254 or SEQ ID NO: 266.

0029In another embodiment, the immunoconjugate comprises at least one effector moiety, wherein the effector moiety is a cytokine. In a specific embodiment, the effector moiety is a cytokine selected from the group consisting of: interleukin-2 (IL-2), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-a (INF-α), interleukin-12 (IL-12), interleukin-8 (IL-8), macrophage inflammatory protein-1α (MIP-1α), macrophage inflammatory protein-1β (MIP-1β), and transforming growth factor-β (TGF-β). In another embodiment, at least one antigen binding moiety is specific for one of the following antigenic determinants: the Extra Domain B of fibronectin (EDB), the A1 domain of tenascin (TNC-A1), the A2 domain of tenascin (TNC-A2), the Fibroblast Activated Protein (FAP); and the Melanoma Chondroitin Sulfate Proteoglycan (MCSP).

0030In another embodiment, the immunoconjugate described herein binds to an effector moiety receptor with a dissociation constant (KD) that is at least about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 times greater than that for a control effector moiety. In another embodiment, the immunoconjugate inhibits an increase in tumor volume in vivo by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more at the end of an administration period. In another embodiment, the immunoconjugate prolongs the survival of mammals with malignant tumors by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% when administered to a mammal in need thereof, relative to a control effector moiety or an effector moiety in a "diabody" immunoconjugate molecule.

0031Another aspect of the present disclosure is directed to isolated polynucleotides encoding immunoconjugates described herein or fragments thereof. Another aspect of the present disclosure is directed to an expression vector comprising an expression cassette comprising the polynucleotide sequences described herein.

0032Another aspect of the present disclosure is directed to host cells that express the immunoconjugates described herein or fragments thereof.

0033Another aspect of the present disclosure is directed to methods for producing the immunoconjugates described herein or fragments thereof, wherein the method comprises culturing host cells transformed with expression vectors encoding for the immunoconjugates described herein or fragments thereof under conditions suitable for the expression of the same. Another aspect of the present disclosure is directed to the promotion of proliferation and differentiation in an activated T lymphocyte cell, comprising contacting an activated T lymphocyte cell with an effective amount of the immunoconjugates described hererin.

0034Another aspect of the present disclosure is directed to the promotion of proliferation and differentiation in an activated B lymphocyte cell, comprising contacting an activated B lymphocyte cell with an effective amount of the immunoconjugates described herein.

0035Another aspect of the disclosure is directed to the promotion of proliferation and differentiation in a natural killer (NK) cell, comprising contacting a NK cell with an effective amount of the immunoconjugates described herein.

0036Another aspect of the disclosure is directed to the promotion of proliferation and differentiation in a granulocyte, a monocyte or a dendritic cell, comprising contacting a granulocyte, a monocyte or a dendritic cell with an effective amount of the immunoconjugates described herein. Another aspect of the disclosure is directed to the promotion of cytotoxic T lymphocyte (CTL) differentiation, comprising contacting a T lymphocyte cell with an effective amount of the immunoconjugates described herein.

0037Another aspect of the disclosure is directed to the inhibition of viral replication, comprising contacting a virus-infected cell with an effective amount of the immunoconjugates described herein.

0038Another aspect of the disclosure is directed to the upregulation of the expression of major histocompatibility complex I (MHC I), comprising contacting a target cell with an effective amount of the immunoconjugates described herein.

0039Another aspect of the disclosure is directed to the induction of cell death, comprising administering to a target cell an effective amount of the immunoconjugates described herein. Another aspect of the disclosure is directed to the induction of chemotaxis in a target cell, comprising administering to a target cell an effective amount of the immunoconjugate described herein.

0040Another aspect of the disclosure is directed to the treatment of a disease in an individual, comprising the steps of administering to an individual a therapeutically effective amount of a composition comprising the immunoconjugate described herein and a pharmaceutical carrier.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

0041<ul id="ul0001" list-style="none" compact="compact"><li><figref idref="f0001">FIGURE 1</figref>. Schematic overview of the various immunoconjugate fusion formats. All constructs in <figref idref="f0001">FIGURE 1</figref> comprise two antibody scFv fragments (in the antigen binding moiety), and one or two cytokine molecules (as the effector moieties) connected to it. Panels A to E show different connectivities and stoichiometries of the antigen binding moieties and effector moieties. Panel A) depicts a "diabody"-IL-2 fusion. The "diabody" assembles non-covalently from two identical polypeptide chains. Panel B shows an immunoconjugate comprising a heavy chain of an Fab molecule fused at its carboxy-terminus to a cytokine which, in turn, is fused at its carboxy-terminus to a second Fab heavy chain. A light chain is coexpressed with the heavy chain Fab-cytokine-heavy chain Fab polypeptide to form the immunoconjugate. Alternatively, the two light chains can be fused to the cytokine, and the heavy chains are coexpressed. In panel C, the two Fab heavy chains are fused directly to each other. The cytokine shares an amino-terminal peptide bond with the second antigen binding moiety heavy chain. The two molecular formats of panels B and C can be varied such that the Fab chain is replaced by an scFv fragment, as in panels D and E.</li><li><figref idref="f0002">FIGURE 2</figref>. Schematic overview of additional immunoconjugates that comprise two antigen binding moieties and at least one or more effector moieties. Panel A shows a Fab molecule fused through its carboxy-teiminus to an IgG CH3 domain. In order to achieve covalent antigen binding moiety homodimerization, an artificial disulfide bond can be introduced at the carboxy-terminus of the IgG CH3 domain (immunoconjugate on the right within panel A. The IgG1 CH3 domain shown in panel A can be substituted with an IgE CH4 domain. The Fab moieties in panel A are replaced by scFv fragments in panel B. For the immunoconjugate of panel C, the natural IgG hinge was fused C-terminal to the Fab molecules. Since the carboxy-terminus region of the hinge could impose some geometric constraints on the assembly of constant domains that are fused C-terminal to the IgG hinge region, an artificial linker can be introduced between the carboxy-terminal region of the hinge and the amino-terminus of the IgG CH3 domain. The hinge region can also be introduced between a scFv fragment and an immunoglobulin constant domain, as shown in panel D. In panels A to D, an IgG1 CH3 or IgE CH4 domain is used to homodimerize the constructs. Panel E depicts an immunoconjugate in which dimerization is achieved via a CH1/Ckappa heterodimerization interaction. The immunoconjugate of panel D can have one or two cytokines per immunoconjugate.</li><li><figref idref="f0003">FIGURE 3</figref> presents the results of an efficacy experiment with two different interleukin-2 immunoconjugate molecular formats specific for tumor stroma. The F9 teratocarcinoma was subcutaneously injected into 129SvEv mice, and tumor size was measured using a caliper. The "diabody"-IL-2 molecule was compared at two different concentrations to the Fab-interleukin-2-Fab (Fab-IL2-Fab) immunoconjugate, wherein the concentrations reflected similar numbers of immunoconjugate molecules. The Fab-IL2-Fab immunoconjugate is labeled as "Fab-L19", the unconjugated interleukin-2 control is labeled as "Unconj rIL-2," the "diabody"-IL-2 molecule is labeled as "diabody" in <figref idref="f0003">FIGURE 3</figref>. The L19 antibody, directed against Extra Domain B of fibronectin (EDB), was used to generate the antigen binding moieties in both the diabody and the Fab-L19 immunoconjugates. The amount of immunoconjugate injected per mouse (in µg) is indicated in the figure legend.</li><li><figref idref="f0004">FIGURE 4</figref> presents the results of a survival experiment with two different interleukin-2 immunoconjugate molecular formats specific for tumor stroma. Human gastric tumor cell-line LS174T was intrasplenically injected into SCID-beige mice. The Fab-IL2-Fab immunoconjugate is labeled as "Fab-L19", the unconjugated interleukin-2 control is labeled as "Unconj rIL-2", the "diabody"-IL-2 molecule is labeled as "diabody" in <figref idref="f0004">FIGURE 4</figref>. The anti-EDB antibody, L19, was used to generate the antigen binding moieties in both the diabody and the Fab-L19 immunoconjugates. The amount of immunoconjugate injected per mouse (in µg) is indicated in the figure legend, and reflects same numbers of immunoconjugate molecules.</li><li><figref idref="f0005">FIGURE 5</figref> shows immunohistochemical images of human uterus tissue at 100X and 400X magnification. The 2B10 variable region generated by the methods described in Example 3 binds to the A2 domain of human tenascin (TNC-A2). The 2B10 variable region in a Fab fragment was fused to a FLAG fragment (SHD2B10-FLAG). Healthy and cancerous human uterine tissue samples were prepared for immunohistochemical staining. Subsequently, the samples were incubated with the SHD2B10-FLAG Fab fragment. The samples were then washed and incubated with a fluorescent antibody specific for the FLAG epitope. Cancerous tissue samples exhibited higher expression levels of TNC-A2 as compared to the healthy tissue samples.</li><li><figref idref="f0006">FIGURE 6</figref> shows the expression levels of TNC-A2 in various human tissue samples in terms of % of immunofluorescence surface area. Various human tissue samples from healthy individuals and cancer patients were incubated with the SHD2B10-FLAG Fab fragment as described in <figref idref="f0005">FIGURE 5</figref>.</li><li><figref idref="f0007">FIGURE 7</figref> shows the expression levels of Fibroblast Activated Protein (FAP) in various human tissue samples in terms of % of immunofluorescence surface area. Various human tissue samples from healthy individuals and cancer patients were incubated with a commercial antibody against FAP (Abcam). The top portion of each bar on the graph represents tumor expression of FAP, while the bottom portion of each bar on the graph represents normal FAP expression.</li><li><figref idref="f0008">FIGURE 8</figref> presents BIACORE data showing the affinity of a known IgG antibody, L19, for EDB.</li><li><figref idref="f0009">FIGURE 9</figref> presents BIACORE data showing the affinity of an Fab-IL-2-Fab immunoconjugate specific for EDB. The Fab fragments in the immunoconjugate were derived from the L19 antibody.</li><li><figref idref="f0010">FIGURE 10</figref> presents BIACORE data showing the affinity of a "diabody"-IL2 fusion protein specific for EDB. The diabody scFv fragment was derived from the L19 antibody. The "diabody"-IL2 fusion protein includes an 8 amino acid linker located between the scFv fragment and the IL-2 molecule.</li><li><figref idref="f0011">FIGURE 11</figref> presents BIACORE data showing the affinity of a "diabody"-IL2 fusion protein specific for EDB. The diabody scFv fragment was derived from the L19 antibody. The "diabody"-IL2 fusion protein includes a 12 amino acid linker between the scFv fragment and the IL-2 molecule</li><li><figref idref="f0012">FIGURE 12</figref> presents BIACORE data showing the affinity of a known IgG antibody, F16, for immobilized domain A1 of tenascin (TNC-A1). <figref idref="f0012">FIGURE 12</figref> also presents BIACORE data showing the affinity of an Fab fragment of the F16 antibody for TNC-A1. Dissociation constants (KD) calculated for the F16 IgG and Fab molecules are indicated in the figure.</li><li><figref idref="f0013">FIGURE 13</figref> presents BIACORE data showing the affinity of IL-2 for immobilized IL-2 receptor. Heterodimerization of the β and γ chains of IL-2R was achieved by fusing the respective chains to the "knob-into-holes" variants of the Fc portion of a human IgG1 as described in <nplcit id="ncit0009" npl-type="s"><text>Merchant, A.M, et al., Nat. Biotech. 16:677-681 (1998</text></nplcit>). The KD value calculated from the BIACORE data is indicated in the figure.</li><li><figref idref="f0014">FIGURE 14</figref> presents BIACORE data showing the affinity of a "diabody"-IL-2 fusion protein for TNC-A1 and IL-2 receptor. The scFv molecule in the diabody is derived from the F16 antibody. The KD values calculated from the BIACORE data are indicated in the figure.</li><li><figref idref="f0015">FIGURE 15</figref> presents BIACORE data showing the affinity of an Fab-IL-2-Fab immunoconjugate for TNC-A1 and IL-2 receptor. The Fab molecules in the immunoconjugate were derived from the F16 antibody. The KD values calculated from the BIACORE data are indicated in the figure. <figref idref="f0016">FIGURE 16</figref> presents BIACORE data showing the affinity of an scFv-IL-2-scFv immunoconjugate for TNC-A1 and IL-2 receptor. The scFv molecules in the immunoconjugate were derived from the F16 antibody. The KD values calculated from the BIACORE data are indicated in the figure.</li><li><figref idref="f0017">FIGURE 17</figref> is a summary table of the KD values obtained from the BIACORE studies presented in <figref idref="f0012 f0013 f0014 f0015 f0016">Figures 12-16</figref>.</li><li><figref idref="f0018">FIGURE 18</figref> presents the results of an efficacy experiment comparing the "diabody"-IL-2 molecule targeting the EDB domain of fibronectin to the Fab-interleukin-2-Fab immunoconjugate (labeled as "Fab-SH2B10", comprising the heavy and light chain variable regions of SEQ ID NOs: 3 and 7, respectively) targeting the A2 domain of tenascin C. The unconjugated interleukin-2 control is labeled as "Unconj rIL-2," the "diabody"-IL-2 molecule is labeled as "L19 diabody" in <figref idref="f0018">FIGURE 18</figref>. The anti-EDB antibody, L19, was used to generate the antigen binding moiety in the diabody immunoconjugate. The teratocarcinoma cell-line F9 was subcutaneously injected into immunocompetent mice of the 129 strain. The amount of immunoconjugate injected per mouse (in µg) is indicated in the figure legend. Treatment was started at day 6 and 5 injections were performed in total until day 11 of the experiment.</li><li><figref idref="f0019">FIGURE 19</figref> shows the induction of proliferation of NK-92 cells by anti-FAP, or anti-tenascin C, Fab-IL2-Fab immunoconjugates (generated using the VH and VL sequences of the 3F2, 3D9, 4B3 (anti-FAP), 2F11, and 2B10 constructs (anti-tenascin C)) compared to unconjugated human IL-2. Cell proliferation was measured using the CellTiter Glo system after two days of incubation.</li><li><figref idref="f0020 f0021">FIGURE 20</figref> presents the results of an ELISA assay measuring induction of IFN-γ production by various interleukin-12 containing immunoconjugates compared either to unconjugated cytokines, or to immunoconjugates that contain the p35 and p40 domains of IL-12 in separate molecules. Panel A shows the results on fibronectin coated plates. Panel B shows the results in solution.</li><li><figref idref="f0022">FIGURE 21</figref> shows Surface Plasmon Resonance (SPR)-based kinetic analyses of affinity-matured anti-FAP Fab fragments. Processed kinetic data sets are presented for clone 19G1 binding to human (hu) FAP (A) and murine (mu) FAP (B), for clone 20G8 binding to hu FAP (C), mu FAP (D) and for clone 4B9 binding to hu FAP (E) and mu FAP (F). Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0023">FIGURE 22</figref> shows SPR-based kinetic analyses of affinity-matured anti-FAP Fab fragments. Processed kinetic data sets are presented for clone 5B8 binding to hu FAP (A) and mu FAP (B), for clone 5F1 binding to hu FAP (C), mu FAP (D) and for clone 14B3 binding to hu FAP (E) and mu FAP (F). Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0024">FIGURE 23</figref> shows SPR-based kinetic analyses of affinity-matured anti-FAP Fab fragments. Processed kinetic data sets are presented for clone 16F1 binding to hu FAP (A) and mu FAP (B), for clone 16F8 binding to hu FAP (C), mu FAP (D) and for clone O3C9 binding to hu FAP (E) and mu FAP (F). Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0025">FIGURE 24</figref> shows SPR-based kinetic analyses of affinity-matured anti-FAP Fab fragments. Processed kinetic data sets are presented for clone O2D7 binding to hu FAP (A) and mu FAP (B), for clone 28H1 binding to hu FAP (C), mu FAP (D), cyno FAP (E) and for clone 22A3 binding to hu FAP (F), mu FAP (G) and Cynomolgus (cyno) FAP (H). Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0026">FIGURE 25</figref> shows SPR-based kinetic analyses of affinity-matured anti-FAP Fab fragments. Processed kinetic data sets are presented for clone 29B11 binding to hu FAP (A), mu FAP (B), cyno FAP (C) and for clone 23C10 binding to hu FAP (D), mu FAP (E) and cyno FAP (F). Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0027">FIGURE 26</figref> shows SPR-based kinetic analyses of affinity-matured anti-TNC A2 Fab fragments binding to human (hu) TNC A2. Processed kinetic data sets are presented for clone 2B10_C3B6 (A), clone 2B10_6A12 (B), clone 2B10_C3A6 (C), clone 2B10_07D8 (D), clone 2B10_01F7 (E) and clone 2B10_6H10 (F). Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0028">FIGURE 27</figref> gives an overview of the three purification steps performed for the purification of 3F2-based Fab-IL2-Fab.</li><li><figref idref="f0029 f0030">FIGURE 28</figref> shows results from the purification of 3F2-based Fab-IL2-Fab (A and B) and results from the purification of 4G8-based Fab-IL2-Fab (C and D). (A, C) 4-12% Bis-Tris and 3-8% Tris Acetate SDS-PAGE with fractions during the purification procedure and the end product. (B, D) Analytical size exclusion chromatography after the three applied purification steps.</li><li><figref idref="f0031">FIGURE 29</figref> shows results from the purification of the 2B10 Fab-IL2-Fab immunoconjugate. (A) 4-12% Bis-Tris SDS-PAGE with fractions during the purification procedure and the end product. B) Analytical size exclusion chromatography after the three applied purification steps.</li><li><figref idref="f0032">FIGURE 30</figref> shows the stability assessment of anti-fibronectin EDB L19-based Fab-IL2-Fab. L19 Fab-IL2-Fab was formulated in 20 mM histidine HCl, 140 mM NaCl, pH 6.0 at a concentration of 6.3 mg/ml and stored for 4 weeks at room temperature and at 4°C. Samples were analyzed every week for (A) concentration by UV spectroscopy (after centrifugation to pellet potential precipitated material) and (B) aggregate content by analytical size exclusion chromatography.</li><li><figref idref="f0033">FIGURE 31</figref> shows SPR-based kinetic analyses of FAP-targeted 3F2 Fab-IL2-Fab immunoconjugates for human, murine and Cynomolgus (cyno) FAP and human IL-2 receptor-β/γ (IL2R bg) as determined by Surface Plasmon Resonance. Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0034">FIGURE 32</figref> shows SPR-based kinetic analyses of FAP-targeted 4G8 Fab-IL2-Fab immunoconjugates for human, murine and Cynomolgus (cyno) FAP as determined by Surface Plasmon Resonance. Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0035">FIGURE 33</figref> shows SPR-based kinetic analyses of FAP-targeted 4G8 Fab-IL2-Fab constructs for human and murine IL-2 receptor β/γ and α chains, as determined by Surface Plasmon Resonance. Smooth lines represent a global fit of the data to a two-state-reaction model.</li><li><figref idref="f0036">FIGURE 34</figref> shows SPR-based kinetic analyses of FAP-targeted 3D9 Fab-IL2-Fab constructs for human, murine and Cynomolgus (cyno) FAP and human IL-2 receptor- β/γ (IL2R bg) as determined by Surface Plasmon Resonance. Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0037">FIGURE 35</figref> shows SPR-based kinetic analyses of TNC A2-targeted 2B10 Fab-IL2-Fab constructs for human, murine and Cynomolgus (cyno) chimeric TNC A2 fusion proteins and human IL-2 receptor-β/γ (IL2R bg) as determined by Surface Plasmon Resonance. Smooth lines represent a global fit of the data to a 1:1 interaction model.</li><li><figref idref="f0038">FIGURE 36</figref> illustrates the efficacy of targeted IL-2 Fab-IL2-Fab immunoconjugates recognizing TNC A2 (2B10) or FAP (3F2 and 4G8) in inducing proliferation of NK92 cells in comparison to IL-2 (Proleukin) and the L19 diabody recognizing fibronectin-EDB. The x-axis is normalized to the number of IL-2 molecules, as the diabody has two IL-2 effector moieties while the Fab-IL2-Fab constructs contain only one IL-2 effector moiety. Cell proliferation was measured using the Cell Titer Glo system after two days of incubation.</li><li><figref idref="f0039">FIGURE 37</figref> shows the induction of STAT5 phosphorylation as a consequence of IL-2 mediated IL-2 receptor signaling following incubation with a FAP-targeted 4G8-based IL-2 Fab-IL2-Fab immunoconjugate recognizing FAP on different effector cell populations including (A) CD56+ NK cells, (B) CD4+CD25-CD127+ helper T cells, (C) CD3+, CD8+ cytotoxic T cells and (D) CD4+CD25+FOXP3+ regulatory T cells (Tregs) from human PBMCs in solution.</li><li><figref idref="f0040">FIGURE 38</figref> illustrates the efficacy of targeted IL-2 Fab-IL2-Fab immunoconjugates recognizing TNC A1 (2F11), TNC A2 (2B10) or FAP (3F2, 4B3 and 3D9) in inducing IFN-γ release and proliferation of NK92 cells in comparison to IL-2, when the immunoconjugates are either present in solution or immobilized via FAP or TNC A2 coated on microtiter plates.</li><li><figref idref="f0041">FIGURE 39</figref> presents the results of a survival experiment with two different IL-2 immunoconjugate molecular formats specific for tumor stroma. Human colon tumor cell line LS174T was intrasplenically injected into SCID mice. The TNC A2-targeted 2B10 Fab-IL2-Fab immunoconjugate is labeled as "SH2B10", the unconjugated IL-2 control is labeled as "proleukin", the fibronectin EDB-targeted diabody-IL-2 molecule is labeled as "diabody". The amount of immunoconjugate injected per mouse (in µg) is indicated in the figure legend, and reflects same numbers of immunoconjugate molecules.</li><li><figref idref="f0042">FIGURE 40</figref> presents the results of a survival experiment with two different IL-2 immunoconjugate molecular formats specific for tumor stroma. Human renal cell line ACHN was intrarenally injected into SCID mice. The FAP-targeted 3F2 or 4G8 Fab-IL2-Fab immunoconjugates are labeled as "FAP-3F2" and "FAP-4G8", the unconjugated IL-2 control is labeled as "proleukin", the fibronectin EDB-targeted diabody-IL-2 molecule is labeled as "diabody". The amount of immunoconjugates injected per mouse (in µg) is indicated in the figure legend, and reflects same numbers of immunoconjugate molecules.</li><li><figref idref="f0043">FIGURE 41</figref> presents the results of a survival experiment with two different IL-2 immunoconjugate molecular formats specific for tumor stroma. Human NSCLC cell line A549 was injected i.v. into SCID mice. The TNC A2-targeted 2B10 Fab-IL2-Fab immunoconjugate is labeled as "2B10", the fibronectin EDB-targeted diabody-IL-2 molecule is labeled as "diabody". The amount of immunoconjugate injected per mouse (in µg) is indicated in the figure legend, and reflects same numbers of immunoconjugate molecules.</li><li><figref idref="f0044">FIGURE 42</figref> presents (A) an overview of the purification procedure of the Fab-GM-CSF-Fab immunoconjugate with L19 (Fibronectin Ectodomain-B binder) as Fab, and (B) an SDS-PAGE (reduced, non-reduced) of the purified Fab-GM-CSF-Fab immunoconjugate.</li><li><figref idref="f0045">FIGURE 43</figref> presents the results of a GM-CSF-dependent proliferation assay comparing the effect of GM-CSF and the purified Fab-GM-CSF-Fab immunoconjugate with L19 (Fibronectin Ectodomain-B binder) as Fab on TF-1 cells.</li><li><figref idref="f0046">FIGURE 44</figref> presents (A) an overview of the purification procedure of the Fab-IL12-Fab immunoconjugate with 4G8 (FAP binder) as Fab, and (B) an SDS-PAGE (reduced, non-reduced) of the purified Fab-IL12-Fab immunoconjugate.</li><li><figref idref="f0047">FIGURE 45</figref> presents the results of an assay testing IL-12 induced IFN-γ release, comparing the effect of IL-12 and the purified Fab-IL12-Fab immunoconjugate with 4G8 (FAP binder) as Fab, using PBMCs isolated from fresh human blood of a healthy donor.</li><li><figref idref="f0048">FIGURE 46</figref> presents (A) an overview of the purification procedure of the Fab-IFNα2-Fab immunoconjugate with L19 (Fibronectin Ectodomain-B binder) as Fab, and (B) an SDS-PAGE (reduced, non-reduced) of the purified Fab-IFNα2-Fab immunoconjugate.</li><li><figref idref="f0049">FIGURE 47</figref> presents the results of an assay testing IFN-α-induced proliferation inhibition of (A) Jurkat T cells and (B) A549 tumor cells comparing the effect of IFN-α (Roferon A, Roche) and the purified Fab-IFNα2-Fab immunoconjugate with L19 (Fibronectin Ectodomain-B binder) as Fab.</li><li><figref idref="f0050">FIGURE 48</figref> shows (A) the elution profiles from the purification of the MCSP-targeted MHLG based Fab-IL2-Fab and (B) the results from the analytical characterization of the same Fab-IL2-Fab by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer, reduced and non-reduced).</li><li><figref idref="f0051">FIGURE 49</figref> shows (A) the elution profiles from the purification of the MCSP-targeted MHLG1 based Fab-IL2-Fab and (B) the results from the analytical characterization of the same Fab-IL2-Fab by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer, reduced and non-reduced).</li><li><figref idref="f0052">FIGURE 50</figref> presents the results of an assay testing IL-2 induced IFN-γ release comparing the effect of the purified Fab-IL2-Fab immunoconjugate with 4G8 (FAP binder) as Fab, and the purified Fab-IL2-Fab immunoconjugate with MHLG KV9 (MCSP binder) as Fab, using IL-2 starved NK92 cells.</li><li><figref idref="f0053">FIGURE 51</figref> presents the results of an assay testing IL-2 induced IFN-γ release comparing the effect of the purified Fab-IL2-Fab immunoconjugate with 4G8 (FAP binder) as Fab, and the purified Fab-IL12-Fab immunoconjugate with MHLG1 KV9 (MCSP binder) as Fab, using IL-2 starved NK92 cells.</li><li><figref idref="f0054">FIGURE 52</figref> shows the binding of MCSP-targeted MHLG1 KV9 Fab-IL2-Fab immunoconjugate to Colo38 cells, as determined by flow cytometry. Secondary antibody alone or cells only are shown as negative controls.</li><li><figref idref="f0055">FIGURE 53</figref> presents (A) an overview of the purification procedure of the 2B10 Fab-IL2-Fab immunoconjugate with 2B10 (TNC A2 binder) as Fab, and (B) an SDS-PAGE (reduced, non-reduced) of the purified 2B10 Fab-IL2-Fab immunoconjugate.</li></ul>

DETAILED DESCRIPTION OF THE INVENTION

Definitions

0042Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, nucleic acid chemistry and hybridization described below are those well known and commonly employed in the art. Standard techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, <nplcit id="ncit0010" npl-type="b"><text>Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y</text></nplcit>.), which are provided throughout this document.

0043As used herein, the term "immunoconjugate" refers to a polypeptide molecule that includes at least one effector moiety and at least one antigen binding moiety. In one embodiment, the immunoconjugate comprises at least one single-chain effector moiety, and at least two antigen binding moieties. The antigen binding molecule can be joined to the effector moiety by a variety of interactions and in a variety of configurations as described herein.

0044As used herein, the term "effector moiety" refers to a polypeptide, e.g., a protein or glycoprotein, that influences cellular activity, for example, through signal transduction or other cellular pathways. Accordingly, the effector moiety can be associated with receptor-mediated signaling that transmits a signal from outside the cell membrane to modulate a response in a cell bearing one or more receptors for the effector moiety. In one embodiment, an effector moiety can elicit a cytotoxic response in cells bearing one or more receptors for the effector moiety. In another embodiment, an effector moiety can elicit a proliferative response in cells bearing one or more receptors for the effector moiety. In another embodiment, an effector moiety can elicit differentiation in cells bearing receptors for the effector moiety. In another embodiment, an effector moiety can alter expression (i.e., upregulate or downregulate) of an endogenous cellular protein in cells bearing receptors for the effector moiety. Non-limiting examples of effector moieties include cytokines, growth factors, hormones, enzymes, substrates, and cofactors. The effector moiety can be associated with an antigen binding moiety in a variety of configurations to form an immunoconjugate.

0045As used herein, the term "cytokine" refers to a molecule that mediates and/or regulates a biological or cellular function or process (e.g., immunity, inflammation, and hematopoiesis). The term "cytokine" as used herein includes "lymphokines," "chemokines," "monokines," and "interleukins." Examples of useful cytokines include, but are not limited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β.

0046As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In one embodiment, the effector moiety is a single-chain effector moiety. Non-limiting examples of single-chain effector moieties include cytokines, growth factors, hormones, enzymes, substrates, and cofactors. When the effector moiety is a cytokine and the cytokine of interest is normally found as a multimer in nature, each subunit of the multimeric cytokine is sequentially encoded by the single-chain of the effector moiety. Accordingly, non-limiting examples of useful single-chain effector moieties include GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β.

0047As used herein, the term "control effector moiety" refers to an unconjugated effector moiety. For example, when comparing an IL-2 immunoconjugate described herein with a control effector moiety, the control effector moiety is free, unconjugated IL-2. Likewise, e.g., when comparing an IL-12 immunoconjugate described herein with a control effector moiety, the control effector moiety is free, unconjugated IL-12 (e.g., existing as a heterodimeric protein wherein the p40 and p35 subunits share only disulfide bond(s)).

0048As used herein, the term "effector moiety receptor" refers to a polypeptide molecule capable of binding specifically to an effector moiety. For example, where IL-2 is the effector moiety, the effector moiety receptor that binds to a IL-2 (e.g., an immunoconjugate comprising IL-2) is the IL-2 receptor. Similarly, e.g., where IL-12 is the effector moiety of an immunoconjugate, the effector moiety receptor is the IL-12 receptor. Where an effector moiety specifically binds to more than one receptor, all receptors that specifically bind to the effector moiety are "effector moiety receptors" for that effector moiety.

0049As used herein, the term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to an antigenic determinant. In one embodiment, an antigen binding moiety is able to direct the entity to which it is attached (e.g., an effector moiety or a second antigen binding moiety) to a target site, for example to a specific type of tumor cell or tumor stroma bearing the antigenic determinant. Antigen binding moieties include antibodies and fragments thereof as further defined herein. By "specifically binds" is meant that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties with constant regions as further defined herein and known in the art. Useful heavy chain constant regions include any of the five isotypes: α, δ, ε, γ, or µ. Useful light chain constant regions include any of the two isotypes: κ and X.

0050As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope," and refers to a site (e.g., a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding moiety binds, forming an antigen binding moiety-antigen complex.

0051As used herein, the term "control antigen binding moiety" refers to an antigen binding moiety as it would exist free of other antigen binding moieties and effector moieties. For example, when comparing an Fab-IL2-Fab immunoconjugate described herein with a control antigen binding moiety, the control antigen binding moiety is free Fab, wherein the Fab-IL2-Fab immunoconjugate and the free Fab molecule can both specifically bind to the same antigen determinant.

0052As used herein, the terms "first" and "second" with respect to antigen binding moieties, effector moieties, etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the immunoconjugate unless explicitly so stated.

0053In the case where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light polypeptides. This particular region has been described by <nplcit id="ncit0011" npl-type="s"><text>Kabat et al., U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983</text></nplcit>) and by <nplcit id="ncit0012" npl-type="s"><text>Chothia et al., J. Mol. Biol. 196:901-917 (1987</text></nplcit>), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table I as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody. <tables id="tabl0001" num="0001"><table frame="all"><title>TABLE 1. CDR Definitions1</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="16mm" /><colspec colnum="3" colname="col3" colwidth="18mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><thead><row><entry align="center" valign="top">CDR</entry><entry align="center" valign="top">Kabat</entry><entry align="center" valign="top">Chothia</entry><entry align="center" valign="top">AbM2</entry></row></thead><tbody><row><entry align="center">VH CDR1</entry><entry align="center">31-35</entry><entry align="center">26-32</entry><entry align="center">26-35</entry></row><row><entry align="center">VH CDR2</entry><entry align="center">50-65</entry><entry align="center">52-58</entry><entry align="center">50-58</entry></row><row><entry align="center">VH CDR3</entry><entry align="center">95-102</entry><entry align="center">95-102</entry><entry align="center">95-102</entry></row><row><entry align="center">VL CDR1</entry><entry align="center">24-34</entry><entry align="center">26-32</entry><entry align="center">24-34</entry></row><row><entry align="center">VL CDR2</entry><entry align="center">50-56</entry><entry align="center">50-52</entry><entry align="center">50-56</entry></row><row><entry align="center">VL CDR3</entry><entry align="center">89-97</entry><entry align="center">91-96</entry><entry align="center">89-97</entry></row></tbody></tgroup><tgroup cols="4" rowsep="0"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="16mm" /><colspec colnum="3" colname="col3" colwidth="18mm" /><colspec colnum="4" colname="col4" colwidth="16mm" /><tbody><row><entry namest="col1" nameend="col4" align="justify">1Numbering of all CDR definitions in Table 1 is according to the numbering conventions set forth by Kabat et al. (see below). 2 "AbM" with a lowercase "b" as used in Table 1 refers to the CDRs as defined by Oxford Molecular's "AbM" antibody modeling software.</entry></row></tbody></tgroup></table></tables>

0054Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antigen binding moiety described herein are according to the Kabat numbering system. The polypeptide sequences of the sequence listing (i.e., SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 96, 97, etc.) are not numbered according to the Kabat numbering system. However, it is well within the ordinary skill of one in the art to convert the numbering of the sequences of the Sequence Listing to Kabat numbering.

Immunoconjugates

0055Immunoconjugates are polypeptide molecules that comprise at least one effector moiety and at least one antigen binding moiety. In one embodiment, the effector moiety is a single-chain effector moiety. In one embodiment, the immunoconjugate comprises at least two antigen binding moieties. The antigen binding moieties and effector moieties of the immunoconjugate include those that are described in detail herein above and below and in the accompanying figures. The antigen binding moiety of the immunoconjugate can be directed againt a variety of target molecules (e.g., an antigenic determinant on a protein molecule expressed on a tumor cell or tumor stroma). Non-limiting examples of antigen binding moieties are described herein. In one embodiment, the at least one antigen binding moiety is directed to an antigenic determinant of one or more of the polypeptides represented in Table 5, herein below. Immunoconjugates described herein typically exhibit one or more of the following properties: high specificity of action, reduced toxicity and/or improved stability, particularly as compared to known immunoconjugates of different configurations targeting the same antigenic determinants and carrying the same effector moities.

0056In one embodiment, the immunoconjugate comprises at least a first effector moiety and at least a first and a second antigen binding moiety. In a preferred embodiment, the first effector moiety is a single chain effector moiety. In a preferred embodiment, the first and second antigen binding moeity are independently selected from the group consisting of an Fv and an Fab. In a specific embodiment, the first effector moiety shares an amino- or carboxy-terminal peptide bond with a first antigen binding moiety and a second antigen binding moiety shares an amino- or carboxy-terminal peptide bond with either i) the first effector moiety or ii) the first antigen binding moiety. In another embodiment, the immunoconjugate consists essentially of a first single-chain effector moiety and first and second antigen binding moieties.

0057In one embodiment, a first effector moiety, shares a carboxy-terminal peptide bond with a first antigen binding moiety and further shares an amino-terminal peptide bond with a second antigen binding moiety. In another embodiment, a first antigen binding moiety shares a carboxy-terminal peptide bond with a first effector moiety, preferably a single chain effector moiety, and further shares an amino-terminal peptide bond with a second antigen binding moiety. In another embodiment, a first antigen binding moiety shares an amino-terminal peptide bond with a first effector moiety, preferably a single chain effector moiety, and further shares a carboxy-terminal peptide with a second antigen binding moiety.

0058In one embodiment, an effector moiety, preferably a single chain effector moiety, shares a carboxy-terminal peptide bond with a first heavy chain variable region and further shares an amino-terminal peptide bond with a second heavy chain variable region. In another embodiment, an effector moiety, preferably a single chain effector moiety, shares a carboxy-terminal peptide bond with a first light chain variable region and further shares an amino-terminal peptide with a second light chain variable region. In another embodiment, a first heavy or light chain variable region is joined by a carboxy-terminal peptide bond to a first effector moiety, preferably a single chain effector moiety, and is further joined by an amino-terminal peptide bond to a second heavy or light chain variable region. In another embodiment, a first heavy or light chain variable region is joined by an amino-terminal peptide bond to a first effector moiety preferably a single chain effector moiety, and is further joined by a carboxy-terminal peptide bond to a second heavy or light chain variable region.

0059In one embodiment, an effector moiety, preferably a single chain effector moiety, shares a carboxy-terminal peptide bond with a first Fab heavy or light chain and further shares an amino-terminal peptide bond with a second Fab heavy or light chain. In another embodiment, a first Fab heavy or light chain shares a carboxy-terminal peptide bond with a first single-chain effector moiety and further shares an amino-terminal peptide bond with a second Fab heavy or light chain. In other embodiments, a first Fab heavy or light chain shares an amino-terminal peptide bond with a first single-chain effector moiety and further shares a carboxy-terminal peptide bond with a second Fab heavy or light chain.

0060In one embodiment, the immunoconjugate comprises at least a first effector moiety sharing an amino-terminal peptide bond to one or more scFv molecules and wherein the first effector moiety further shares a carboxy-terminal peptide bond with one or more scFv molecules. In a preferred embodiment, the effector moiety is a single chain effector moiety.

0061In another embodiment, the immunoconjugate comprises at least a first effector moiety, preferably a single chain effector moiety, and first and second antigen binding moieties, wherein each of the antigen binding moieties includes an scFv molecule joined at its carboxy-terminal amino acid to a constant region that includes an immunoglobulin constant domain, and wherein the first antigen binding moiety is joined at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of the first effector moiety, and wherein the first and second antigen binding moieties are covalently linked through at least one disulfide bond. In a preferred embodiment, the constant region is independently selected from the group consisting of IgG CH1, IgG CH2, IgG CH3, IgG Ckappa, IgG Clambda and IgE CH4 domains. In one embodiment, the immunoglobulin domain of the first antigen binding moiety is covalently linked to the immunoglobulin domain of the second antigen binding moiety through a disulfide bond. In one embodiment, at least one disulfide bond is located carboxy-terminal of the immunoglobulin domains of the first and second antigen binding moieties. In another embodiment, at least one disulfide bond is located amino-terminal of the immunoglobulin domains of the first and second antigen binding moieties. In another embodiment, at least two disulfide bonds are located amino-terminal of the immunoglobulin domains of the first and second antigen binding moieties. In a specific embodiment, the immunoconjugate comprises first and second antigen binding moieties, each comprising an scFv molecule joined at its carboxy-terminal amino acid to a constant region that comprises an IgG CH1 domain, wherein the first antigen binding moiety is joined at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of the first effector moiety, preferably a single chain effector moiety, and wherein the first and second antigen binding moieties are covalently linked through at least one disulfide bond. The second antigen binding moiety of the immunoconjugate can be further joined at its carboxy-terminal amino acid to the amino-terminal amino acid of a second effector moiety. In one embodiment, the second effector moiety is a single chain effector moiety.

0062In a specific embodiment, the immunoconjugate comprises first and second antigen binding moieties each comprising an scFv molecule joined at its carboxy-terminal amino acid to a constant region that comprises an IgG Ckappa domain, wherein the first antigen binding moiety is joined at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of the first effector moiety, preferably a single chain effector moiety, and wherein the first and second antigen binding moieties are covalently linked through at least one disulfide bond. The second antigen binding moiety of the immunoconjugate can be further joined at its carboxy-terminal amino acid to the amino-terminal amino acid of a second effector moiety. In one embodiment, the second effector moiety is a single chain effector moiety.

0063In another specific embodiment, the immunoconjugate comprises first and second antigen binding moieties, each comprising an scFv molecule joined at its carboxy-terminal amino acid to a constant region that comprises an IgE CH4 domain, wherein the first antigen binding moiety is joined at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of the first effector moiety, preferably a single chain effector moiety, and wherein the first and second antigen binding moieties are covalently linked through at least one disulfide bond. The second antigen binding moiety of the immunoconjugate can be further joined at its carboxy-terminal amino acid to the amino-terminal amino acid of a second effector moiety. In one embodiment, the second effector moiety is a single chain effector moiety.

0064In another specific embodiment, the immunoconjugate comprises first and second antigen binding moieties each, comprising an scFv molecule joined at its carboxy-terminal amino acid to an IgE CH3 domain, wherein the first antigen binding moiety is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of the first effector moiety, preferably a single chain effector moiety, and wherein the first and second antigen binding moieties are covalently linked through at least one disulfide bond. The second antigen binding moiety of the immunoconjugate can be further joined at its carboxy-terminal amino acid to the amino-terminal amino acid of a second effector moiety. In one embodiment, the second effector moiety is a single chain effector moiety.

0065In another embodiment, the immunoconjugate comprises first and second effector moieties, and first and second antigen binding moieties, wherein each of the antigen binding moieties comprises an Fab molecule joined at its heavy or light chain carboxy-terminal amino acid to an IgG1 CH3 domain, and wherein each of the IgG1 CH3 domains is joined at its respective carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked through at least one disulfide bond. In a preferred embodiment, the first and/or second effector moiety is a single chain effector moiety. In a further embodiment, the IgG1 CH3 domains of the antigen binding moieties may be joined by disulfide bond. In another embodiment, at least one disulfide bond is located carboxy-terminal of the IgG1 CH3 domains of the first and second antigen binding moieties. In another embodiment, at least one disulfide bond is located ammo-terminal of the IgG1 CH3 domains of the first and second antigen binding moieties. In another embodiment, at least two disulfide bonds are located amino-terminal of the IgG1 CH3 domains of the first and second antigen binding moieties.

0066In another embodiment, the immunoconjugate comprises one or more proteolytic cleavage sites located between effector moieties and antigen binding moieties.

0067Components of the immunoconjugate (e.g., antigen binding moieties and/or effector moieties) may be linked directly or through various linkers (e.g., peptide linkers comprising one or more amino acids, typically about 2-10 amino acids) that are described herein or are known in the art. In a particular embodiment, the immunoconjugate has improved stability in solution, particularly compared to known immunoconjugate preparations. In one embodiment, the immunoconjugate binds to an antigenic determinant with a dissociation constant (KD) that is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 times lower than that for a control antigen binding moiety. In a more specific embodiment, the immunoconjugate binds to an antigenic determinant with a KD that is about 10 times lower than that for a control antigen binding moiety. In one embodiment the immunoconjugate binds to an antigenic determinant with a KD that is lower than about 10 nM, lower than about 1 nM, or lower than about 0.1 nM.

0068In another embodiment, the immunoconjugate has a superior safety profile compared to known immunoconjugate preparations. The immunoconjugate preferably elicits fewer and less severe side effects, such as toxicity, destruction of non-tumor cells, etc. The decrease in side effects may be attributed to the reduced binding affinity of the immunoconjugates described herein towards effector moiety receptors. In one embodiment, the immunoconjugate binds to an effector moiety receptor with a KD that is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 times greater than that for a control effector moiety. In a more specific embodiment, the immuonconjugate binds to an effector moiety receptor with a KD that is about 2 times greater than that for a control effector moiety. In another embodiment, the immuonconjugate binds to an effector moiety receptor with a KD that is about 10 times greater than that for a control effector moiety. In another embodiment, the immunoconjugate binds to an effector moiety receptor with a KD that is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than that for a corresponding effector moiety in a "diabody" immunoconjugate molecule. In another embodiment, the immunoconjugate binds to an effector moiety receptor with a dissociation constant KD that is about 10 times greater than that for a corresponding effector moiety in a "diabody" immunoconjugate.

0069In another embodiment, the immunoconjugate has superior efficacy, particularly compared to known immunoconjugate preparations. In one embodiment, the immunoconjugate is better able to inhibit increases in tumor volume in vivo and/or better able to prolong survival in mammals with malignant tumors. In one embodiment, the immunoconjugate inhibits an increase in tumor volume in vivo by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% by the end of an administration period of about at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In one embodiment, the immunoconjugate inhibits an increase in tumor volume in vivo by at least 50%, 55%, 60%, 65%, 70%, or 75% by the end of a 13 day administration period. In another embodiment, the immunoconjugate prolongs the survival of mammals with malignant tumors by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% when administered to a mammal in need thereof, relative to a control effector moiety. In another embodiment, the immunoconjugate prolongs the survival of mammals with malignant tumors by at least 30%, 32% or 35% when administered to a mammal in need thereof, relative to a control effector moiety. In another embodiment, the immunoconjugate prolongs the survival of mammals with malignant tumors by about 30% when administered to a mammal in need thereof, relative to a control effector moiety. In another embodiment, the immunoconjugate prolongs the survival of mammals with malignant tumors by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% when administered to a mammal in need thereof, relative to an effector moiety in a "diabody" immunoconjugate molecule. In another embodiment, the immunoconjugate prolongs the survival of mammals with malignant tumors by at least 30%, 32%, or 35% when administered to a mammal in need thereof, relative to an effector moiety in a "diabody" immunoconjugate molecule. In another embodiment, the immunoconjugate prolongs the survival of mammals with malignant tumors by about 30% when administered to a mammal in need thereof, relative to an effector moiety in a "diabody" immunoconjugate molecule. In another embodiment, the immunoconjugate prolongs the survival of mammals with malignant tumors by at least 5%, 10% or 15%, relative to a control effector moiety or an effector moiety in a "diabody" immunoconjugate molecule.

Antigen Binding Moieties

0070The antigen binding moiety of the immunoconjugate described herein is generally a polypeptide molecule that binds to a specific antigenic determinant and is able to direct the entity to which it is attached (e.g., an effector moiety or a second antigen binding moiety) to a target site, for example to a specific type of tumor cell or tumor stroma that bears the antigenic determinant. The immunoconjugate can bind to antigenic determinants found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, free in blood serum, and/or in the extracellular matrix (ECM).

0071Non-limiting examples of tumor antigens include MAGE, MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectal associated antigen (CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2.

0072Non-limiting examples of viral antigens include influenza virus hemagglutinin, Epstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, and HIV gp120.

0073Non-limiting examples of ECM antigens include syndecan, heparanase, integrins, osteopontin, link, cadherins, laminin, laminin type EGF, lectin, fibronectin, notch, tenascin, and matrixin.

0074The immunoconjugates described herein can bind to the following specific non-limiting examples of cell surface antigens: FAP, Her2, EGFR, CD2 (T-cell surface antigen), CD3 (heteromultimer associated with the TCR), CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD25 (IL-2 receptor α chain), CD30 (cytokine receptor), CD33 (myeloid cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R (IL6 receptor), CD20, MCSP, and PDGFβR (β platelet-derived growth factor receptor).

0075In one embodiment, the immunoconjugate comprises two or more antigen binding moieties, wherein each of these antigen binding moieties specifically bind to the same antigenic determinant. In another embodiment, the immunoconjugate comprises two or more antigen binding moieties, wherein each of these antigen binding moieties specifically bind to different antigenic determinants.

0076The antigen binding moiety can be any type of antibody or fragment thereof that retains specific binding to an antigenic determinant. Antibody fragments include, but are not limited to, VH fragments, VL fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies (see, e.g.,<nplcit id="ncit0013" npl-type="s"><text>Hudson and Souriau, Nature Med. 9: 129-134 (2003</text></nplcit>)).

0077In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that are specific for the Extra Domain B of fibronectin (EDB). In another embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody L19 for binding to an epitope of EDB. See, e.g., PCT publication <patcit id="pcit0007" dnum="WO2007128563A1PCT"><text>WO 2007/128563 A1</text></patcit>. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with an IL-2 molecule which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain derived from the L19 monoclonal antibody. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with an IL-12 molecule which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain derived from the L19 monoclonal antibody. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with an IFN α molecule which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain derived from the L19 monoclonal antibody. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with a GM-CSF molecule which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain derived from the L19 monoclonal antibody. In a further embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first scFv derived from the L19 monoclonal antibody shares a carboxy-terminal peptide bond with an IL-2 molecule which in turn shares a carboxy-terminal peptide bond with a second scFv derived from the L19 monoclonal antibody.

0078In a more specific embodiment, the immunoconjugate comprises the polypeptide sequence of SEQ ID NO: 95 or a variant thereof that retains functionality. In another embodiment, the immunoconjugate comprises an Fab light chain derived from the L19 monoclonal antibody. In a more specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 96 or a variant thereof that retains functionality. In yet another embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 95 and SEQ ID NO: 96 or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 104 or a variant thereof that retains functionality. In yet another embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 104 and SEQ ID NO: 96 or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 105 or a variant thereof that retains functionality. In yet another embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 105 and SEQ ID NO: 96 or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 106 or a variant thereof that retains functionality. In yet another embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 106 and SEQ ID NO: 96 or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 107 or a variant thereof that retains functionality. In yet another embodiment, the immunoconjugate comprises a polypeptide sequences that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 107 and SEQ ID NO: 96 or variants thereof that retain functionality. In another specific embodiment, the polypeptides are covalently linked, e.g., by a disulfide bond.

0079In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that are specific for the A1 domain of Tenascin (TNC-A1). In another embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that can compete with monoclonal antibody F16 for binding to an epitope of TNC-A1. See, e.g.,<patcit id="pcit0008" dnum="WO2007128563A1A"><text>PCT Publication WO 2007/128563 A1</text></patcit>. In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that are specific for the A1 and/or the A4 domain of Tenascin (TNC-A1 or TNC-A4 or TNC-A1/A4). In another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain specific for the A1 domain of Tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule, an IL-12 molecule, an IFN α molecule or a GM-CSF molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for the A1 domain of Tenascin. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain specific for the A1 domain of Tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for the A1 domain of Tenascin. In a further embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first scFv specific for the A1 domain of Tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule which in turn shares a carboxy-terminal peptide bond with a second scFv specific for the A1 domain of Tenascin. In a specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 13 or SEQ ID NO: 15, or variants thereof that retain functionality. In another specific embodiment, the antigen binding moieties of the immunoconjugate comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 9 or SEQ ID NO: 11, or variants thereof that retain functionality. In a more specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 13 or SEQ ID NO: 15 or variants thereof that retain functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 9 or SEQ ID NO: 11 or variants thereof that retain functionality. In another specific embodiment, the heavy chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 14 or SEQ ID NO: 16. In yet another specific embodiment, the heavy chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by the polynucleotide sequence of either SEQ ID NO: 14 or SEQ ID NO: 16. In another specific embodiment, the light chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 10 or SEQ ID NO: 12. In yet another specific embodiment, the light chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by the polynucleotide sequence of either SEQ ID NO: 10 or SEQ ID NO: 12. In a specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 99 or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 100 or SEQ ID NO: 215, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 101 or SEQ ID NO: 235 or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 100 and SEQ ID NO: 101 or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 215 and SEQ ID NO: 235 or variants thereof that retain functionality. In a specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 112. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of SEQ ID NO: 112. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 113 or SEQ ID NO: 216. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of either SEQ ID NO: 113 or SEQ ID NO: 216. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either SEQ ID NO: 114 or SEQ ID NO: 236. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of either SEQ ID NO: 114 or SEQ ID NO: 236.

0080In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that are specific for the A2 domain of Tenascin (TNC-A2). In another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain specific for the A2 domain of Tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule, an IL-12 molecule, an IFN α molecule or a GM-CSF molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for the A2 domain of Tenascin. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain specific for the A2 domain of Tenascin shares a carboxy-terminal peptide bond with an IL-2 molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for the A2 domain of Tenascin. In a specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 7, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO:195, SEQ ID NO: 199, SEQ ID NO: 203 and SEQ ID NO: 207, or variants thereof that retain functionality. In another specific embodiment, the antigen binding moieties of the immunoconjugate comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 3, SEQ ID NO: 5; SEQ ID NO: 177, SEQ ID NO: 181, SEQ ID NO:185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 201 and SEQ ID NO: 205, or variants thereof that retain functionality. In a more specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 7, SEQ ID NO: 179, SEQ ID NO: 183, SEQ ID NO: 187, SEQ ID NO: 191, SEQ ID NO:195, SEQ ID NO: 199, SEQ ID NO: 203 and SEQ ID NO: 207, or variants thereof that retain functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 3, SEQ ID NO: 5; SEQ ID NO: 177, SEQ ID NO: 181, SEQ ID NO:185, SEQ ID NO: 189, SEQ ID NO: 193, SEQ ID NO: 197, SEQ ID NO: 201 and SEQ ID NO: 205, or variants thereof that retain functionality. In another specific embodiment, the heavy chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 8, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 204 and SEQ ID NO: 208. In yet another specific embodiment, the heavy chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 8, SEQ ID NO: 180, SEQ ID NO: 184, SEQ ID NO: 188, SEQ ID NO: 192, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 204 and SEQ ID NO: 208. In another specific embodiment, the light chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, SEQ ID NO: 202 and SEQ ID NO: 206. In yet another specific embodiment, the light chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence selected from the group of of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 178, SEQ ID NO: 182, SEQ ID NO: 186, SEQ ID NO: 190, SEQ ID NO: 194, SEQ ID NO: 198, SEQ ID NO: 202 and SEQ ID NO: 206. In a specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 239, SEQ ID NO: 241 and SEQ ID NO: 243, or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 245, SEQ ID NO: 247 and SEQ ID NO:249, or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 239, SEQ ID NO: 241, and SEQ ID NO: 243 or variants thereof that retain functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 245, SEQ ID NO: 247 and SEQ ID NO:249 or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 239 and either SEQ ID NO: 247 or SEQ ID NO: 249, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 241 and either SEQ ID NO: 245 or SEQ ID NO: 247, or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 243 and SEQ ID NO: 245, or variants thereof that retain functionality. In a specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 240, SEQ ID NO: 242 and SEQ ID NO: 244. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of of SEQ ID NO: 240, SEQ ID NO: 242 and SEQ ID NO: 244. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 246, SEQ ID NO: 248 and SEQ ID NO: 250. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of of SEQ ID NO: 246, SEQ ID NO: 248 and SEQ ID NO: 250.

0081In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that are specific for the Fibroblast Activated Protein (FAP). In another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain specific for FAP shares a carboxy-terminal peptide bond with an IL-2 molecule, an IL-12 molecule, an IFN α molecule or a GM-CSF molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for FAP. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain specific for FAP shares a carboxy-terminal peptide bond with an IL-2 molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for FAP. In another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain specific for FAP shares a carboxy-terminal peptide bond with an IL-12 molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for FAP.In a specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171 and SEQ ID NO: 175, or variants thereof that retain functionality. In another specific embodiment, the antigen binding moieties of the immunoconjugate comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169 and SEQ ID NO: 173, or variants thereof that retain functionality. In a more specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85, SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 123, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 135, SEQ ID NO: 139, SEQ ID NO: 143, SEQ ID NO: 147, SEQ ID NO: 151, SEQ ID NO: 155, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 167, SEQ ID NO: 171, and SEQ ID NO: 175, or variants thereof that retain functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO: 121, SEQ ID NO: 125, SEQ ID NO: 129, SEQ ID NO: 133, SEQ ID NO: 137, SEQ ID NO: 141, SEQ ID NO: 145, SEQ ID NO: 149, SEQ ID NO: 153, SEQ ID NO: 157, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 169, and SEQ ID NO: 173, or variants thereof that retain functionality. In another specific embodiment, the heavy chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, and SEQ ID NO: 176. In yet another specific embodiment, the heavy chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 22, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 70, SEQ ID NO: 74, SEQ ID NO: 78, SEQ ID NO: 82, SEQ ID NO: 86, SEQ ID NO: 90, SEQ ID NO: 94, SEQ ID NO: 124, SEQ ID NO: 128, SEQ ID NO: 132, SEQ ID NO: 136, SEQ ID NO: 140, SEQ ID NO: 144, SEQ ID NO: 148, SEQ ID NO: 152, SEQ ID NO: 156, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 172, and SEQ ID NO: 176. In another specific embodiment, the light chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170, and SEQ ID NO: 174. In yet another specific embodiment, the light chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 38, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 76, SEQ ID NO: 80, SEQ ID NO: 84, SEQ ID NO: 88, SEQ ID NO: 92, SEQ ID NO: 122, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 134, SEQ ID NO: 138, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 150, SEQ ID NO: 154, SEQ ID NO: 158, SEQ ID NO: 162, SEQ ID NO: 166, SEQ ID NO: 170, and SEQ ID NO: 174. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225 and SEQ ID NO: 227, or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233 and SEQ ID NO: 237 or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 211, SEQ ID NO: 219 and SEQ ID NO: 221 or variants thereof that retain functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 231 or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group of SEQ ID NO: 209, SEQ ID NO: 223, SEQ ID NO: 225 and SEQ ID NO: 227 or variants thereof that retain functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 229 or variants thereof that retain functionality. In a further specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 213 and SEQ ID NO: 233 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 217 and SEQ ID NO: 237 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 221 and SEQ ID NO: 231 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 223 and SEQ ID NO: 229 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 225 and SEQ ID NO: 229 or variants thereof that retain functionality. In yet another specific embodiment, the immunoconjugate comprises two polypeptide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 227 and SEQ ID NO: 229 or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, and SEQ ID NO: 228. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, and SEQ ID NO: 228. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a sequence selected from the group of SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, and SEQ ID NO: 238. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence selected from the group of SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, and SEQ ID NO: 238.

0082In one embodiment, the immunoconjugate comprises at least one, typically two or more antigen binding moieties that are specific for the Melanoma Chondroitin Sulfate Proteoglycan (MCSP). In another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain specific for MCSP shares a carboxy-terminal peptide bond with an IL-2 molecule, an IL-12 molecule, an IFN α molecule or a GM-CSF molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for MCSP. In yet another embodiment, the immunoconjugate comprises a polypeptide sequence wherein a first Fab heavy chain specific for MCSP shares a carboxy-terminal peptide bond with an IL-2 molecule, which in turn shares a carboxy-terminal peptide bond with a second Fab heavy chain specific for MCSP. In a specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of either SEQ ID NO: 257 or SEQ ID NO: 261 or variants thereof that retain functionality. In another specific embodiment, the antigen binding moieties of the immunoconjugate comprise a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of either SEQ ID NO: 259 or SEQ ID NO: 271 or variants thereof that retain functionality. In a more specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of either SEQ ID NO: 257 or SEQ ID NO: 261, or variants thereof that retain functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of either SEQ ID NO: 259 or SEQ ID NO: 271, or variants thereof that retain functionality. In a more specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 257, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 259. In another specific embodiment, the antigen binding moieties of the immunoconjugate comprise a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 261, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 259. In another specific embodiment, the heavy chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of either SEQ ID NO: 258 or SEQ ID NO: 262. In yet another specific embodiment, the heavy chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by the polynucleotide sequence of either SEQ ID NO: 258 or SEQ ID NO: 262. In another specific embodiment, the light chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of either SEQ ID NO: 260 or SEQ ID NO: 272. In yet another specific embodiment, the light chain variable region sequence of the antigen binding moieties of the immunoconjugate is encoded by the polynucleotide sequence of either SEQ ID NO: 260 or SEQ ID NO: 272. In a specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 251 or SEQ ID NO: 255, or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 253 or SEQ ID NO: 265, or variants thereof that retain functionality. In a more specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 251 or SEQ ID NO: 255 or variants thereof that retain functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to either SEQ ID NO: 253 or SEQ ID NO: 265, or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 251 or variants thereof that retain functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 253 or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 255 or variants thereof that retain functionality, and a polypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 253 or variants thereof that retain functionality. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of either SEQ ID NO: 252 or SEQ ID NO: 256. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of either SEQ ID NO: 252 or SEQ ID NO: 256. In another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by a polynucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of either SEQ ID NO: 254 or SEQ ID NO: 266. In yet another specific embodiment, the immunoconjugate comprises a polypeptide sequence encoded by the polynucleotide sequence of either SEQ ID NO: 254 or SEQ ID NO: 266.

0083In one embodiment the antigen binding moieties comprise at least a variable region capable of binding an antigenic determinant. Non-limiting variable regions useful in the immunoconjugates described herein can be of murine, primate, or human origin. Human variable regions can be derived from human monoclonal antibodies made by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor et al., J Immunol. 133:3001-3005 (1984) and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987). Human variable regions may also be produced by transgenic animals (e.g. mice) that are capable, upon immunization, of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigenic challenge. See, e.g.,<nplcit id="ncit0014" npl-type="s"><text>Jakobovits et al., Nature 362:255-258 (1993</text></nplcit>).

0084Alternatively, phage display can be used to produce human antibodies and human variable regions in vitro from immunoglobulin variable (V) domain gene repertoires e.g., from unimmunized donors. (<nplcit id="ncit0015" npl-type="s"><text>McCafferty et al., Nature 348:552-554 (1990</text></nplcit>).) In one example of this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle.

0085Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody/antibody fragments also result in selection of the gene encoding the antibody/antibody fragments exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats. For a review of phage display formats, see<nplcit id="ncit0016" npl-type="s"><text>Hoogenboom et al., Nucleic Acids Res. 19:4133-4137 (1991</text></nplcit>). Several sources of V-gene segments can be used for phage display. Clackson et al., isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. See<nplcit id="ncit0017" npl-type="s"><text>Clackson et al., Nature 352:624-628 (1991</text></nplcit>). A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by <nplcit id="ncit0018" npl-type="s"><text>Marks et al., J. Mol. Bio. 222:581-597 (1991</text></nplcit>). In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes introduced will confer higher affinity, and B cells displaying high-affinity surface immunoglobulin are preferentially replicated and differentiated during subsequent antigen challenge. This natural process can be mimicked by employing the technique known as "chain shuffling." See <nplcit id="ncit0019" npl-type="s"><text>Marks et al., Biotech. 10:779-783 (1992</text></nplcit>). In this method, the affinity of "primary" human antibodies or variable regions obtained by phage display can be improved by sequentially replacing the heavy and light chain V region genes with repertoires of naturally occurring variants (repertoires) of V domain genes obtained from unimmunized donors. This technique allows the production of antibodies and variable regions with affinities in the nM range. A strategy for making very large phage antibody repertoires has been described by <nplcit id="ncit0020" npl-type="s"><text>Waterhouse et al., Nucl. Acids Res. 21:2265-2266 (1993</text></nplcit>), and the isolation of a high affinity human antibody directly from such large phage library is reported by <nplcit id="ncit0021" npl-type="s"><text>Griffith et al., J. Cell. Bio. 120:885-896 (1993</text></nplcit>). Gene shuffling can also be used to derive human antibodies and variable regions from rodent antibodies, where the human antibody or variable region has similar affinities and specificities to the starting rodent antibody or variable region. According to this method, which is also referred to as "epitope imprinting," the heavy or light chain V domain gene of rodent antibodies obtained by phage display techniques is replaced with a repertoire of human V domain genes, creating rodent-human chimeras. Selection with antigen results in the isolation of human variable regions capable of restoring a functional antigen binding site, i.e., the epitope governs (imprints) the choice of partner. When the process is repeated to replace the remaining rodent V domain, a human antibody is obtained (see <patcit id="pcit0009" dnum="WO9306213A"><text>PCT publication WO 93/06213</text></patcit>). Unlike traditional humanization of rodent antibodies, the epitope imprinting technique provides completely human antibodies or variable regions, which have no framework or CDR residues of rodent origin.

0086Variable regions that can be used also include murine variable region sequences that have either been primatized or humanized or primate variable region sequences that have been humanized. As used herein, the term "humanized" refers to an antigen-binding moiety variable region sequence derived from a non-human antibody, for example, a murine antibody, that retains or substantially retains the antigen-binding properties of the parent molecule but which is less immunogenic in humans. This may be achieved by various methods including (a) grafting only the non-human CDRs onto human framework regions with or without retention of critical framework residues (e.g., those that are important for retaining good antigen binding affinity or antibody functions) and (b) "cloaking" the non-human variable regions with a human-like section by replacement of surface residues. Such methods are disclosed by Jones et al.,<nplcit id="ncit0022" npl-type="s"><text>Morrison et al,. Proc. Natl. Acad. Sci., 81:6851-6855 (1984</text></nplcit>); <nplcit id="ncit0023" npl-type="s"><text>Morrison and Oi, Adv. Immunol., 44:65-92 (1988</text></nplcit>); <nplcit id="ncit0024" npl-type="s"><text>Verhoeyen et al., Science, 239:1534-1536 (1988</text></nplcit>); <nplcit id="ncit0025" npl-type="s"><text>Padlan, Molec. Immun., 28:489-498 (1991</text></nplcit>); <nplcit id="ncit0026" npl-type="s"><text>Padlan, Molec. Immun., 31(3):169-217 (1994</text></nplcit>). There are generally 3 complementarity determining regions, or CDRs, (CDR1, CDR2, and CDR3) in each of the heavy and light chain variable regions of an antibody, which are flanked by four framework subregions (i.e., FR1, FR2, FR3, and FR4) in each of the heavy and light chain variable domains of an antibody: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A discussion of antibodies with humanized variable regions can be found, inter alia, in <patcit id="pcit0010" dnum="US6632927B"><text>U.S. Patent No. 6,632,927</text></patcit>, and in <patcit id="pcit0011" dnum="US20030175269A" dnum-type="L"><text>U.S. Patent Application Publication No. 2003/0175269</text></patcit>.

0087Similarly, as used herein, the term "primatized" is used to refer to an antigen-binding moiety variable region derived from a non-primate antibody, for example, a murine antibody, that retains or substantially retains the antigen-binding properties of the parent molecule but which is less immunogenic in primates.

0088The choice of human variable domains, both heavy and light, in making humanized antigen binding moieties is very important to reduce antigenicity. According to the so-called "best fit" method, the sequence of the variable region of a rodent antigen binding moiety is screened against the entire library of known human variable-region sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework region (FR) for the humanized antigen binding moiety (<nplcit id="ncit0027" npl-type="s"><text>Sims et al, J. Immunol., 151:2296 (1993</text></nplcit>); <nplcit id="ncit0028" npl-type="s"><text>Chothia et al., J. Mol. Biol., 196:901 (1987</text></nplcit>)). Another method of selecting the human framework sequence is to compare the sequence of each individual subregion of the full rodent framework (i.e., FR1, FR2, FR3, and FR4) or some combination of the individual subregions (e.g., FR1 and FR2) against a library of known human variable region sequences that correspond to that framework subregion (e.g., as determined by Kabat numbering), and choose the human sequence for each subregion or combination that is the closest to that of the rodent (<patcit id="pcit0012" dnum="US20030040606A1" dnum-type="L"><text>U.S. Patent Application Publication No. 2003/0040606A1</text></patcit>). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antigen binding moieties (<nplcit id="ncit0029" npl-type="s"><text>Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992</text></nplcit>); <nplcit id="ncit0030" npl-type="s"><text>Presta et al., J. Immunol., 151:2623 (1993</text></nplcit>)).

0089Generally, the antigen binding moieties of the immunoconjugate described herein retain high affinity for specific antigenic determinants and other favorable biological properties. Accordingly, humanized variable regions are prepared by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformation structures of selected candidate immunoglobulin variable region sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin variable region sequence, i.e., the analysis of residues that influence the ability of the candidate variable region sequence to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antigen binding moiety characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

0090In another embodiment, the antigen binding molecules described herein are engineered to have enhanced binding affinity according to, for example, the methods disclosed in <patcit id="pcit0013" dnum="US20040132066A" dnum-type="L"><text>U.S. Pat. Appl. Publ. No. 2004/0132066</text></patcit>. The ability of the immunoconjugate described herein to bind to either an effector moiety receptor or to a specific antigenic determinant can be measured either through an enzyme linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., surface plasmon resonance technique (analyzed on a BIACORE T100 system) (<nplcit id="ncit0031" npl-type="s"><text>Liljeblad, et al., Glyco. J. 17:323-329 (2000</text></nplcit>)), and traditional binding assays (<nplcit id="ncit0032" npl-type="s"><text>Heeley, R. P., Endocr. Res. 28:217-229 (2002</text></nplcit>)).

Effector Moieties

0091The effector moieties for use in the immunoconjugates described herein are generally polypeptides that influence cellular activity, for example, through signal transduction pathways. Accordingly, the effector moiety of the immunoconjugate described herein can be associated with receptor-mediated signaling that transmits a signal from outside the cell membrane to modulate a response within the cell. For example, an effector moiety of the immunoconjugate can be a cytokine. In a particular embodiment, the effector moiety is a single-chain effector moiety as defined herein. In one embodiment, one or more effector moieties, typically single-chain effector moieties, of the immunoconjugates described herein are cytokines selected from the group consisting of: IL-2, GM-CSF, IFN-α, and IL-12. In another embodiment, one or more single-chain effector moieties of the immunoconjugates are cytokines selected from the group consisting of: IL-8, MIP-1α, MIP-1β, and TGF-β.

0092In one embodiment, the effector moiety, preferably a single-chain effector moiety, of the immunoconjugate is IL-2. In a specific embodiment, the IL-2 effector moiety can elicit one or more of the cellular responses selected from the group consisting of: proliferation in an activated T lymphocyte cell, differentiation in an activated T lymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in an activated B cell, differentiation in an activated B cell, proliferation in a natural killer (NK) cell, differentiation in a NK cell, and NK/lymphocyte activated killer (LAK) antitumor cytotoxicity. In one embodiment, the effector moiety, preferably a single-chain effector moiety, of the immunoconjugate is GM-CSF. In a specific embodiment, the GM-CSF effector moiety can elicit proliferation and/or differentiation in a granulocyte, a monocyte or a dendritic cell. In one embodiment, the effector moiety, preferably a single-chain effector moiety, of the immunoconjugate is IFN-α. In a specific embodiment, the IFN-α effector moiety can elicit one or more of the cellular responses selected from the group consisting of: inhibiting viral replication in a virus-infected cell, and upregulating the expression of major histocompatibility complex I (MHC I). In another specific embodiment, the IFN α effector moiety can inhibit proliferation in a tumor cell. In one embodiment, the effector moiety, preferably a single-chain effector moiety, of the immunoconjugate is IL-12. In a specific embodiment, the IL-12 effector moiety can elicit one or more of the cellular responses selected from the group consisting of: proliferation in a NK cell, differentiation in a NK cell, proliferation in a T cell, and differentiation in a T cell. In one embodiment, the effector moiety, preferably a single-chain effector moiety, of the immunoconjugate is IL-8. In a specific embodiment, the IL-8 effector moiety can elicit chemotaxis in neutrophils. In one embodiment, the effector moiety, preferably a single-chain effector moiety, of the immunoconjugate, is MIP-1α. In a specific embodiment, the MIP-1α effector moiety can elicit chemotaxis in monocytes and T lymphocyte cells. In one embodiment, the effector moiety, preferably a single-chain effector moiety, of the immunoconjugate is MIP-1β. In a specific embodiment, the MIP-1β effector moiety can elicit chemotaxis in monocytes and T lymphocyte cells. In one embodiment, the effector moiety, preferably a single-chain effector moiety, of the immunoconjugate is TGF-β. In a specific embodiment, the TGF-β effector moiety can elicit one or more of the cellular responses selected from the group consisting of: chemotaxis in monocytes, chemotaxis in macrophages, upregulating the expression of IL-1 in activated macrophages, and upregulating the expression of IgA in activated B cells.

Immunoconjugate Polypeptides and Polynucleotides

0093The immunoconjugates described herein comprise polypeptides and fragments thereof. As used herein, term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

0094A polypeptide as described herein may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three-dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.

0095By an "isolated" polypeptide or a variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the present disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

0096Also included as polypeptides according to the present disclosure are derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "variant," "derivative" and "analog" when referring to polypeptides described herein include any polypeptides that retain at least some of the biological, antigenic, or immunogenic properties of the corresponding native polypeptide. Variants of polypeptides described herein include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions or additions. Derivatives of polypeptides described herein, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins. Variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein a "derivative" of a polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine. Alternatively, recombinant variants encoding these same or similar polypeptides can be synthesized or selected by making use of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system. Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, interchain affinities, or degradation/turnover rate.

0097Preferably, amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements. "Conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. "Insertions" or "deletions" are preferably in the range of about 1 to about 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

0098By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence described herein, it is intended that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a query amino acid sequence, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted, or substituted with another amino acid. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the references sequence.

0099As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96,%, 97%, 98%, or 99% identical to a reference polypeptide can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence described herein) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of <nplcit id="ncit0033" npl-type="s"><text>Brutlag et al., Comp. Appl. Biosci. 6:237-245 (1990</text></nplcit>). In a sequence alignment the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty-0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter.

0100If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present disclosure. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence.

0101For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present disclosure.

0102Polypeptides described herein include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set forth in Tables 3 and 4, below, including functional fragments or variants thereof. The disclosure also encompasses polypeptides comprising sequences of Tables 3 or 4 with conservative amino acid substitutions.

0103The polypeptides described herein may be encoded by a single polynucleotide. Alternatively, the may be encoded by multiple (e.g., two or more) polynucleotides, so that the polypeptides are co-expressed. Polypeptides that are co-expressed from multiple polynucleotides may associate through, e.g., disulfide bonds or other means to form a functional immunoconjugate. For example, the heavy chain portion of an antigen binding moiety may be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the light chain portion of the antigen binding moiety and the effector moiety. When co-expressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the antigen binding moiety. Alternatively, in another example, the light chain portion of the antigen binding moiety could be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the heavy chain portion of the antigen binding moiety and the effector moiety.

0104Immunoconjugates described herein and fragments thereof are generally encoded by polynucleotides. The term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a therapeutic polypeptide contained in a vector is considered isolated for the purposes of the present disclosure. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts, as well as positive and negative strand forms, and double-stranded forms, of pestivirus vectors disclosed herein.

0105Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

0106As used herein, a "coding region" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns or 5' and 3' non-translated regions, are not part of a coding region. Two or more coding regions described herein can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. Furthermore, any vector may contain a single coding region, or may comprise two or more coding regions, e.g., a vector described herein may encode one or more polyproteins, which are post- or co-translationally separated into the final proteins via proteolytic cleavage. In addition, a vector, polynucleotide, or nucleic acid described herein may encode heterologous coding regions, either fused or unfused to a first or second nucleic acid encoding the immunoconjugate described herein, or variant or derivative thereof. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

0107In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of DNA, a polynucleotide comprising a nucleic acid, which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" if induction of promoter function results in the transcription of mRNA encoding the desired gene product and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. The promoter may be a cell-specific promoter that directs substantial transcription of the DNA only in predetermined cells. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the polynucleotide to direct cell-specific transcription. Suitable promoters and other transcription control regions are disclosed herein.

0108A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions, which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (e.g., the immediate early promoter, in conjunction with intron-A), simian virus 40 (e.g., the early promoter), and retroviruses (such as, e.g., Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

0109Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

0110In other embodiments, a polynucleotide described herein is RNA, for example, in the form of messenger RNA (mRNA). RNA described herein may be single stranded or double stranded.

0111Polynucleotide and nucleic acid coding regions described herein may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide described herein. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or "full length" polypeptide to produce a secreted or "mature" form of the polypeptide. In certain embodiments, the native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, or a functional derivative thereof, may be used. For example, the wild-type leader sequence may be substituted with the leader sequence of human tissue plasminogen activator (TPA) or mouse β-glucuronidase.

0112The term "expression cassette" refers to a polynucleotide generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In one embodiment, the expression cassette described herein comprises polynucleotide sequences that encode immunoconjugates described herein or fragments thereof.

0113The term "expression vector" is synonymous with "expression contsruct" and refers to a DNA molecule that is used to introduce and direct the expression of a specific gene to which it is operably associated into a target cell. The expression vector described herein comprises an expression cassette. Expression vectors allow transcription of large amounts of stable mRNA. Once the expression vector is inside the target cell, the ribonucleic acid molecule or protein that is encoded by the gene is produced by the cellular transcription and/or translation machinery. In one embodiment, the expression vector described herein comprises an expression cassette comprises polynucleotide sequences that encode immunoconjugates described herein or fragments thereof.

0114The term "artificial" refers to a synthetic, or non-host cell derived composition, e.g., a chemically-synthesized oligonucleotide.

0115By a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence described herein, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.

0116As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptide sequence described herein can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence described herein) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of <nplcit id="ncit0034" npl-type="s"><text>Brutlag et al., Comp. Appl. Biosci. 6:237-245 (1990</text></nplcit>). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty-30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject nucleotide sequences, whichever is shorter.

0117If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present disclosure. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

0118For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matached/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present disclosure.

0119Polynucleotides described herein include those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences set forth in Tables 6 and 8, below, including functional fragments or variants thereof. The polynucleotides may be expressed as a single polynucleotide that encodes the entire immunoconjugate or as multiple (e.g., two or more) polynucleotides that are coexpressed. Polypeptides encoded by polynucleotides that are co-expressed may associate through, e.g., disulfide bonds or other means to form a functional immunoconjugate. For example, the heavy chain portion of an antigen binding moiety may be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the light chain portion of the antigen binding moiety and the effector moiety. When coexpressed, the heavy chain polypeptides will associate with the light chain polypeptides to form the antigen binding moiety. Alternatively, in another example, the light chain portion of the antigen binding moiety could be encoded by a separate polynucleotide from the portion of the immunoconjugate comprising the heavy chain portion of the antigen binding moiety and the effector moiety.

0120In a specific embodiment, an isolated polynucleotide described herein encodes a fragment of an immunoconjugate comprising at least one effector moiety, preferably a single-chain effector moiety, and at least one, preferably two or more antigen binding moieties, wherein a first effector moiety shares an amino- or carboxy-terminal peptide bond with a first antigen binding moiety and a second antigen binding moiety shares an amino- or carboxy-terminal peptide bond with either the first effector moiety or the first antigen binding moiety. In a preferred embodiment, the antigen binding moieties are independently selected from the group consisting of Fv and Fab. In another specific embodiment, the polynucleotide encodes the heavy chains of two of the antigen binding moieties and one of the effector moieties. In another specific embodiment, the polynucleotide encodes the light chains of two of the antigen binding moieties and one of the effector moieties. In another specific embodiment, the polynucleotide encodes one light chain from one of the antigen binding moieties, one heavy chain from a second antigen binding moiety and one of the effector moieties.

0121In another specific embodiment, an isolated polynucleotide described herein encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the heavy chains of two Fab molecules and an effector moiety, preferably a single-chain effector moiety. In another specific embodiment, an isolated polynucleotide described herein encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the light chains of two Fab molecules and an effector moiety, preferably a single-chain effector moiety. In another specific embodiment an isolated polynucleotide described herein encodes a fragment of an immunoconjugate, wherein the polynucleotide encodes the heavy chain of one Fab molecule, the light chain of second Fab molecule and an effector moiety, preferably a single-chain effector moiety.

0122In one embodiment, an isolated polynucleotide described herein encodes an immunoconjugate comprising at least one effector moiety, preferably a single-chain effector moiety, joined at its amino- and carboxy-terminal amino acids to one or more scFv molecules.

0123In one embodiment, an isolated polynucleotide described herein encodes an immunoconjugate fragment comprising at least one effector moiety, preferably a single-chain effector moiety and at least first and second antigen binding moieties, wherein each of the antigen binding moieties comprises an scFv molecule joined at its carboxy-terminal amino acid to a constant region comprising an immunoglobulin constant domain independently selected from the group consisting of IgG1 CH1, IgG Ckappa, and IgE CH4, and wherein one of the antigen binding moieties is joined at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked through a disulfide bond. In a further embodiment, the polynucleotide described herein encodes one of the antigen binding moieties and an effector moiety, preferably a single-chain effector moiety.

0124In one embodiment, an isolated polynucleotide described herein encodes an immunoconjugate fragment comprising first and second effector moieties and two antigen binding moieties, wherein each of the antigen binding moieties comprises an scFv molecule joined at its carboxy-terminal amino acid to a constant region comprising an immunoglobulin constant domain, and wherein one of the antigen binding moieties is joined at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the second antigen binding moiety is joined at its constant region carboxy-terminal amino acid to the amino-terminal amino acid of the second effector moiety, and wherein the first and second antigen binding moieties are covalently linked through a disulfide bond. In a preferred embodiment, the first and/or second effector moieties are single chain effector moieties. In a preferred embodiment, the constant domain is independently selected from the group consisting of IgG1 CH1, IgG Ckappa, and IgE CH4. In a further embodiment, the polynucleotide described herein encodes one of the antigen binding moieties and one of the effector moieties.

0125In one embodiment, an isolated polynucleotide described herein encodes an immunoconjugate fragment comprising at least one effector moiety, preferably a single-chain effector moiety, and at least first and second antigen binding moieties, wherein each of the antigen binding moieties comprises an scFv molecule joined at its carboxy-terminal amino acid to an IgG CH3 domain, and wherein one of the antigen binding moieties is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked through a disulfide bond. In a further embodiment, the polynucleotide described herein encodes one of the antigen binding moieties and an effector moiety, preferably a single chain effector moiety.

0126In one embodiment, an isolated polynucleotide described herein encodes an immunoconjugate fragment comprising two effector moieties and two antigen binding moieties, wherein each of the antigen binding moieties comprises an scFv molecule joined at its carboxy-terminal amino acid to an IgG CH3 domain, and wherein one of the antigen binding moieties is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the second antigen binding moiety is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of the second effector moiety, and wherein the first and second antigen binding moieties are covalently linked through a disulfide bond. In a preferred embodiment, the first and/or second effector moieties are single chain effector moieties. In a further embodiment, the polynucleotide described herein encodes one of the antigen binding moieties and one of the effector moieties, preferably a single chain effector moiety.

0127In one embodiment, an isolated polynucleotide described herein encodes an immunoconugate fragment comprising two effector moieties and two antigen binding moieties, wherein each of the antigen binding moieties comprises an Fab molecule joined at its heavy or light chain carboxy-terminal amino acid to an IgG1 CH3 domain, and wherein each of the IgG1 CH3 domains is joined at its carboxy-terminal amino acid to the amino-terminal amino acid of one of the effector moieties, and wherein the first and second antigen binding moieties are covalently linked through a disulfide bond. In a preferred embodiment, the first and/or second effector moieties are single chain effector moieties. In a further embodiment, the polynucleotide described herein comprises a sequence encoding the heavy chain variable region of one of the antigen binding moieties and one of said effector moieties, preferably a single chain moiety. In yet another embodiment, the polynucleotide described herein comprises a sequence encoding the light chain variable region of one of the antigen binding moieties and one of the effector moieties, preferably a single chain effector moiety.

0128In another embodiment, the present disclosure is directed to an isolated polynucleotide encoding an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a variable region sequence as shown in Table 3 below. In another embodiment, the present disclosure is directed to an isolated polynucleotide encoding an immunoconjugate or fragment thereof, wherein the polynucleotide comprises a sequence that encodes a polypeptide sequence as shown in Table 4. In another embodiment, the disclosure is further directed to an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence shown in Tables 6 and 8 below. In another embodiment, the disclosure is directed to an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a nucleic acid sequence shown in Tables 6 and 8. In another embodiment, the disclosure is directed to an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a sequence that encodes a variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in Table 3. In another embodiment, the disclosure is directed to an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a sequence that encodes a polypeptide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence in Table 4. The disclosure encompasses an isolated nucleic acid encoding an immunoconjugate or fragment thereof, wherein the nucleic acid comprises a sequence that encodes the variable region sequences of Table 3 with conservative amino acid substitutions. The disclosure also encompasses an isolated nucleic acid encoding an immunoconjugate of the invention or fragment thereof, wherein the nucleic acid comprises a sequence that encodes the polypeptide sequences of Table 4 with conservative amino acid substitutions. <tables id="tabl0002" num="0002"><table frame="all"><title>TABLE 2.</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="37mm" /><colspec colnum="2" colname="col2" colwidth="117mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><thead><row><entry align="center" valign="top">Construct</entry><entry align="center" valign="top">NUCLEOTIDE SEQUENCE</entry><entry align="center" valign="top">SEQ ID NO</entry></row></thead><tbody><row><entry align="center">Library Template</entry><entry align="center"><img file="EP2467165B1_D0001.tif" /></entry><entry align="center">1</entry></row><row><entry align="center">for DP47-3 library; complete Fab coding region comprising PelB leader sequence + Vk3_20 kappa V-domain + CL constant domain for light chain and PelB + VH3_23 V-domain + CH1 constant domain for heavy chain; pMS25opt</entry><entry><img file="EP2467165B1_D0002.tif" /></entry><entry align="center" /></row><row><entry align="center">Library Template for DP88-3 library; complete Fab coding region comprising PelB leader sequence + Vk1_17 kappa V-domain + CL constant domain for light chain and PelB + VH1_69 V-domain + CH1 constant domain</entry><entry><img file="EP2467165B1_D0003.tif" /></entry><entry align="center">2</entry></row><row><entry align="center">for heavy chain; pRJH32</entry><entry><img file="EP2467165B1_D0004.tif" /></entry><entry /></row></tbody></tgroup></table></tables><tables id="tabl0003" num="0003"><table frame="all"><title>TABLE 3.</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="33mm" /><colspec colnum="2" colname="col2" colwidth="120mm" /><colspec colnum="3" colname="col3" colwidth="13mm" /><thead><row><entry align="center" valign="top">Construct</entry><entry align="center" valign="top">POLYPEPTIDE SEQUENCE</entry><entry align="center" valign="top">SEQ ID NO</entry></row></thead><tbody><row><entry align="center">2B10; VL</entry><entry><img file="EP2467165B1_D0005.tif" /></entry><entry align="center">3</entry></row><row><entry align="center">2B10(GS); VL</entry><entry><img file="EP2467165B1_D0006.tif" /></entry><entry align="center">5</entry></row><row><entry align="center">2B10; VH</entry><entry><img file="EP2467165B1_D0007.tif" /></entry><entry align="center">7</entry></row><row><entry align="center">2F11; VL</entry><entry><img file="EP2467165B1_D0008.tif" /></entry><entry align="center">9</entry></row><row><entry align="center">2F11(VI); VL</entry><entry><img file="EP2467165B1_D0009.tif" /></entry><entry align="center">11</entry></row><row><entry align="center">2F11; VH</entry><entry><img file="EP2467165B1_D0010.tif" /></entry><entry align="center">13</entry></row><row rowsep="0"><entry align="center">2F11(MT); VH</entry><entry><img file="EP2467165B1_D0011.tif" /></entry><entry align="center">15</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0012.tif" /></entry><entry align="center" /></row><row><entry align="center">3F2; VL</entry><entry><img file="EP2467165B1_D0013.tif" /></entry><entry align="center">17</entry></row><row><entry align="center">3F2(YS); VL</entry><entry><img file="EP2467165B1_D0014.tif" /></entry><entry align="center">19</entry></row><row><entry align="center">3F2; VH</entry><entry><img file="EP2467165B1_D0015.tif" /></entry><entry align="center">21</entry></row><row><entry align="center">3D9, VL</entry><entry><img file="EP2467165B1_D0016.tif" /></entry><entry align="center">23</entry></row><row><entry align="center">3D9, VH</entry><entry><img file="EP2467165B1_D0017.tif" /></entry><entry align="center">25</entry></row><row><entry align="center">3D9(TA); VH</entry><entry><img file="EP2467165B1_D0018.tif" /></entry><entry align="center">27</entry></row><row><entry align="center">4G8; VL</entry><entry><img file="EP2467165B1_D0019.tif" /></entry><entry align="center">29</entry></row><row><entry align="center">4G8; VH</entry><entry><img file="EP2467165B1_D0020.tif" /></entry><entry align="center">31</entry></row><row><entry align="center">4B3; VL</entry><entry><img file="EP2467165B1_D0021.tif" /></entry><entry align="center">33</entry></row><row><entry align="center">4B3; VH</entry><entry><img file="EP2467165B1_D0022.tif" /></entry><entry align="center">35</entry></row><row><entry align="center">4D6; VL</entry><entry><img file="EP2467165B1_D0023.tif" /></entry><entry align="center">37</entry></row><row><entry align="center">4D6; VH</entry><entry><img file="EP2467165B1_D0024.tif" /></entry><entry align="center">39</entry></row><row><entry align="center">2C6; VL</entry><entry><img file="EP2467165B1_D0025.tif" /></entry><entry align="center">41</entry></row><row><entry align="center">2C6; VH</entry><entry><img file="EP2467165B1_D0026.tif" /></entry><entry align="center">43</entry></row><row><entry align="center">5H5; VL</entry><entry><img file="EP2467165B1_D0027.tif" /></entry><entry align="center">45</entry></row><row><entry align="center">5H5; VH</entry><entry><img file="EP2467165B1_D0028.tif" /></entry><entry align="center">47</entry></row><row><entry align="center">2C4; VL</entry><entry><img file="EP2467165B1_D0029.tif" /></entry><entry align="center">49</entry></row><row rowsep="0"><entry align="center">2C4; VH</entry><entry><img file="EP2467165B1_D0030.tif" /></entry><entry align="center">51</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0031.tif" /></entry><entry align="center" /></row><row><entry align="center">2D9; VL</entry><entry><img file="EP2467165B1_D0032.tif" /></entry><entry align="center">67</entry></row><row><entry align="center">2D9; VH</entry><entry><img file="EP2467165B1_D0033.tif" /></entry><entry align="center">69</entry></row><row><entry align="center">4B8; VL</entry><entry><img file="EP2467165B1_D0034.tif" /></entry><entry align="center">71</entry></row><row><entry align="center">4B8; VH</entry><entry><img file="EP2467165B1_D0035.tif" /></entry><entry align="center">73</entry></row><row><entry align="center">7A1; VL</entry><entry><img file="EP2467165B1_D0036.tif" /></entry><entry align="center">75</entry></row><row><entry align="center">7A1; VH</entry><entry><img file="EP2467165B1_D0037.tif" /></entry><entry align="center">77</entry></row><row><entry align="center">13C2; VL</entry><entry><img file="EP2467165B1_D0038.tif" /></entry><entry align="center">79</entry></row><row><entry align="center">13C2; VH</entry><entry><img file="EP2467165B1_D0039.tif" /></entry><entry align="center">81</entry></row><row><entry align="center">13E8; VL</entry><entry><img file="EP2467165B1_D0040.tif" /></entry><entry align="center">83</entry></row><row><entry align="center">13E8; VH</entry><entry><img file="EP2467165B1_D0041.tif" /></entry><entry align="center">85</entry></row><row><entry align="center">14C10; VL</entry><entry><img file="EP2467165B1_D0042.tif" /></entry><entry align="center">87</entry></row><row><entry align="center">14C10; VH</entry><entry><img file="EP2467165B1_D0043.tif" /></entry><entry align="center">89</entry></row><row><entry align="center">17All; VL</entry><entry><img file="EP2467165B1_D0044.tif" /></entry><entry align="center">91</entry></row><row><entry align="center">17A11; VH</entry><entry><img file="EP2467165B1_D0045.tif" /></entry><entry align="center">93</entry></row><row><entry align="center">19G1; VL</entry><entry><img file="EP2467165B1_D0046.tif" /></entry><entry align="center">121</entry></row><row><entry align="center">19G1; VH</entry><entry><img file="EP2467165B1_D0047.tif" /></entry><entry align="center">123</entry></row><row><entry align="center">20G8; VL</entry><entry><img file="EP2467165B1_D0048.tif" /></entry><entry align="center">125</entry></row><row><entry align="center">20G8; VH</entry><entry><img file="EP2467165B1_D0049.tif" /></entry><entry align="center">127</entry></row><row><entry align="center">4B9; VL</entry><entry><img file="EP2467165B1_D0050.tif" /></entry><entry align="center">129</entry></row><row><entry align="center">4B9; VH</entry><entry><img file="EP2467165B1_D0051.tif" /></entry><entry align="center">131</entry></row><row><entry align="center">5B8; VL</entry><entry><img file="EP2467165B1_D0052.tif" /></entry><entry align="center">133</entry></row><row><entry align="center">5B8; VH</entry><entry><img file="EP2467165B1_D0053.tif" /></entry><entry align="center">135</entry></row><row><entry align="center">5F1; VL</entry><entry><img file="EP2467165B1_D0054.tif" /></entry><entry align="center">137</entry></row><row><entry align="center">5F1; VH</entry><entry><img file="EP2467165B1_D0055.tif" /></entry><entry align="center">139</entry></row><row><entry align="center">14B3; VL</entry><entry><img file="EP2467165B1_D0056.tif" /></entry><entry align="center">141</entry></row><row><entry align="center">14B3; VH</entry><entry><img file="EP2467165B1_D0057.tif" /></entry><entry align="center">143</entry></row><row><entry align="center">16F1; VL</entry><entry><img file="EP2467165B1_D0058.tif" /></entry><entry align="center">145</entry></row><row><entry align="center">16F1; VH</entry><entry><img file="EP2467165B1_D0059.tif" /></entry><entry align="center">147</entry></row><row><entry align="center">16F8; VL</entry><entry><img file="EP2467165B1_D0060.tif" /></entry><entry align="center">149</entry></row><row><entry align="center">16F8; VH</entry><entry><img file="EP2467165B1_D0061.tif" /></entry><entry align="center">151</entry></row><row><entry align="center">O3C9; VL</entry><entry><img file="EP2467165B1_D0062.tif" /></entry><entry align="center">153</entry></row><row><entry align="center">O3C9; VH</entry><entry><img file="EP2467165B1_D0063.tif" /></entry><entry align="center">155</entry></row><row><entry align="center">O2D7; VL</entry><entry><img file="EP2467165B1_D0064.tif" /></entry><entry align="center">157</entry></row><row><entry align="center">O2D7; VH</entry><entry><img file="EP2467165B1_D0065.tif" /></entry><entry align="center">159</entry></row><row rowsep="0"><entry align="center">28H1; VL</entry><entry><img file="EP2467165B1_D0066.tif" /></entry><entry align="center">161</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0067.tif" /></entry><entry align="center" /></row><row><entry align="center">28H1; VH</entry><entry><img file="EP2467165B1_D0068.tif" /></entry><entry align="center">163</entry></row><row><entry align="center">22A3; VL</entry><entry><img file="EP2467165B1_D0069.tif" /></entry><entry align="center">165</entry></row><row><entry align="center">22A3; VH</entry><entry><img file="EP2467165B1_D0070.tif" /></entry><entry align="center">167</entry></row><row><entry align="center">29B11; VL</entry><entry><img file="EP2467165B1_D0071.tif" /></entry><entry align="center">169</entry></row><row><entry align="center">29B11; VH</entry><entry><img file="EP2467165B1_D0072.tif" /></entry><entry align="center">171</entry></row><row><entry align="center">23C10; VL</entry><entry><img file="EP2467165B1_D0073.tif" /></entry><entry align="center">173</entry></row><row><entry align="center">23C10; VH</entry><entry><img file="EP2467165B1_D0074.tif" /></entry><entry align="center">175</entry></row><row><entry align="center">2B10_C3B6; VL</entry><entry><img file="EP2467165B1_D0075.tif" /></entry><entry align="center">177</entry></row><row><entry align="center">2B10_C3B6; VH</entry><entry><img file="EP2467165B1_D0076.tif" /></entry><entry align="center">179</entry></row><row><entry align="center">2B10_6A12; VL</entry><entry><img file="EP2467165B1_D0077.tif" /></entry><entry align="center">181</entry></row><row><entry align="center">2B10_6A12; VH</entry><entry><img file="EP2467165B1_D0078.tif" /></entry><entry align="center">183</entry></row><row><entry align="center">2B10_C3A6; VL</entry><entry><img file="EP2467165B1_D0079.tif" /></entry><entry align="center">185</entry></row><row><entry align="center">2B10_C3A6; VH</entry><entry><img file="EP2467165B1_D0080.tif" /></entry><entry align="center">187</entry></row><row><entry align="center">2B10_D1A2_wt; VL</entry><entry><img file="EP2467165B1_D0081.tif" /></entry><entry align="center">189</entry></row><row><entry align="center">2B10_D1A2_wt; VH</entry><entry><img file="EP2467165B1_D0082.tif" /></entry><entry align="center">191</entry></row><row><entry align="center">2B10 D1A2 VD; VL</entry><entry><img file="EP2467165B1_D0083.tif" /></entry><entry align="center">193</entry></row><row><entry align="center">2B10_D1A2_VD; VH</entry><entry><img file="EP2467165B1_D0084.tif" /></entry><entry align="center">195</entry></row><row rowsep="0"><entry align="center">2B10_O7D8; VL</entry><entry><img file="EP2467165B1_D0085.tif" /></entry><entry align="center">197</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0086.tif" /></entry><entry align="center" /></row><row><entry align="center">2B10_O7D8; VH</entry><entry><img file="EP2467165B1_D0087.tif" /></entry><entry align="center">199</entry></row><row><entry align="center">2B10_O1F7; VL</entry><entry><img file="EP2467165B1_D0088.tif" /></entry><entry align="center">201</entry></row><row><entry align="center">2B10_O1F7; VH</entry><entry><img file="EP2467165B1_D0089.tif" /></entry><entry align="center">203</entry></row><row><entry align="center">2B10_6H10; VL</entry><entry><img file="EP2467165B1_D0090.tif" /></entry><entry align="center">205</entry></row><row><entry align="center">2B10_6H10; VH</entry><entry><img file="EP2467165B1_D0091.tif" /></entry><entry align="center">207</entry></row><row><entry align="center">MHLG1; VH</entry><entry><img file="EP2467165B1_D0092.tif" /></entry><entry align="center">257</entry></row><row><entry align="center">KV9; VL</entry><entry><img file="EP2467165B1_D0093.tif" /></entry><entry align="center">259</entry></row><row><entry align="center">MHLG;VH</entry><entry><img file="EP2467165B1_D0094.tif" /></entry><entry align="center">261</entry></row><row><entry align="center">KV1; VL</entry><entry><img file="EP2467165B1_D0095.tif" /></entry><entry align="center">269</entry></row><row><entry align="center">KV7; VL</entry><entry><img file="EP2467165B1_D0096.tif" /></entry><entry align="center">271</entry></row></tbody></tgroup></table></tables><tables id="tabl0004" num="0004"><table frame="all"><title>TABLE 4.</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="37mm" /><colspec colnum="2" colname="col2" colwidth="117mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><thead><row><entry align="center" valign="top">Construct</entry><entry align="center" valign="top">POLYPEPTIDE SEQUENCE</entry><entry align="center" valign="top">SEQ ID NO</entry></row></thead><tbody><row rowsep="0"><entry align="center">Fab heavy chain derived from L19 monoclonal antibody-C125A variant of IL2-Fab heavy chain derived from L19 monoclonal antibody</entry><entry><img file="EP2467165B1_D0097.tif" /></entry><entry align="center">95</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0098.tif" /></entry><entry align="center" /></row><row><entry align="center">Fab light chain derived from L19 monoclonal antibody</entry><entry><img file="EP2467165B1_D0099.tif" /></entry><entry align="center">96</entry></row><row><entry align="center">scFv derived from L19 monoclonal antibody-8 amino acid linker-C125A variant of IL2</entry><entry><img file="EP2467165B1_D0100.tif" /></entry><entry align="center">97</entry></row><row><entry align="center">F16-diabody-IL2 protein</entry><entry><img file="EP2467165B1_D0101.tif" /></entry><entry align="center">98</entry></row><row><entry align="center">scFv-IL2-scFv (F16, protein)</entry><entry><img file="EP2467165B1_D0102.tif" /></entry><entry align="center">99</entry></row><row rowsep="0"><entry align="center">Fab-IL2-Fab (F16, heavy chain cytokine fusion construct, protein)</entry><entry><img file="EP2467165B1_D0103.tif" /></entry><entry align="center">100</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0104.tif" /></entry><entry align="center" /></row><row><entry align="center">F16, light chain, protein</entry><entry><img file="EP2467165B1_D0105.tif" /></entry><entry align="center">101</entry></row><row><entry align="center">IL2R-beta-Fc(hole) fusion protein, protein</entry><entry><img file="EP2467165B1_D0106.tif" /></entry><entry align="center">102</entry></row><row><entry align="center">IL2R-gamma-Fc(knob), protein</entry><entry><img file="EP2467165B1_D0107.tif" /></entry><entry align="center">103</entry></row><row rowsep="0"><entry align="center">Fab-IL12-Fab L19 antibody, murine scIL12, protein</entry><entry><img file="EP2467165B1_D0108.tif" /></entry><entry align="center">104</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0109.tif" /></entry><entry align="center" /></row><row><entry align="center">Fab-IL12-Fab L19 antibody, human scIL12, protein</entry><entry><img file="EP2467165B1_D0110.tif" /></entry><entry align="center">105</entry></row><row><entry align="center">Fab-GMCSF-Fab L19 antibody, human GM-CSF, protein</entry><entry><img file="EP2467165B1_D0111.tif" /></entry><entry align="center">106</entry></row><row rowsep="0"><entry align="center">Fab-IFNα2-Fab, L19 antibody, protein</entry><entry><img file="EP2467165B1_D0112.tif" /></entry><entry align="center">107</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0113.tif" /></entry><entry align="center" /></row><row><entry align="center">3F2 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0114.tif" /></entry><entry align="center">209</entry></row><row><entry align="center">4G8 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0115.tif" /></entry><entry align="center">211</entry></row><row rowsep="0"><entry align="center">3D9 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0116.tif" /></entry><entry align="center">213</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0117.tif" /></entry><entry align="center" /></row><row><entry align="center">2F11 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0118.tif" /></entry><entry align="center">215</entry></row><row><entry align="center">4B3 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0119.tif" /></entry><entry align="center">217</entry></row><row rowsep="0"><entry align="center">4G8 Fab-IL12-Fab (murine IL-12; heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0120.tif" /></entry><entry align="center">219</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0121.tif" /></entry><entry align="center" /></row><row><entry align="center">28H1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0122.tif" /></entry><entry align="center">221</entry></row><row><entry align="center">29B11 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0123.tif" /></entry><entry align="center">223</entry></row><row><entry align="center">19G1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0124.tif" /></entry><entry align="center">225</entry></row><row rowsep="0"><entry align="center">20G8 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0125.tif" /></entry><entry align="center">227</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0126.tif" /></entry><entry align="center" /></row><row><entry align="center">3F2 light chain</entry><entry><img file="EP2467165B1_D0127.tif" /></entry><entry align="center">229</entry></row><row><entry align="center">4G8 light chain</entry><entry><img file="EP2467165B1_D0128.tif" /></entry><entry align="center">231</entry></row><row><entry align="center">3D9 light chain</entry><entry><img file="EP2467165B1_D0129.tif" /></entry><entry align="center">233</entry></row><row><entry align="center">2F11 light chain</entry><entry><img file="EP2467165B1_D0130.tif" /></entry><entry align="center">235</entry></row><row><entry align="center">4B3 light chain</entry><entry><img file="EP2467165B1_D0131.tif" /></entry><entry align="center">237</entry></row><row rowsep="0"><entry align="center">2B10 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0132.tif" /></entry><entry align="center">239</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0133.tif" /></entry><entry align="center" /></row><row><entry align="center">C3B6 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0134.tif" /></entry><entry align="center">241</entry></row><row><entry align="center">6A12 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0135.tif" /></entry><entry align="center">243</entry></row><row><entry align="center">2B10 light chain</entry><entry><img file="EP2467165B1_D0136.tif" /></entry><entry align="center">245</entry></row><row><entry align="center">D1A2 light chain</entry><entry><img file="EP2467165B1_D0137.tif" /></entry><entry align="center">247</entry></row><row><entry align="center">O7D8 light chain</entry><entry><img file="EP2467165B1_D0138.tif" /></entry><entry align="center">249</entry></row><row rowsep="0"><entry align="center">MHLG1 Fab-IL2-Fab (heavy chain</entry><entry><img file="EP2467165B1_D0139.tif" /></entry><entry align="center">251</entry></row><row><entry align="center">cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0140.tif" /></entry><entry align="center" /></row><row><entry align="center">KV9 light chain</entry><entry><img file="EP2467165B1_D0141.tif" /></entry><entry align="center">253</entry></row><row><entry align="center">MHLG Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0142.tif" /></entry><entry align="center">255</entry></row><row><entry align="center">KV1 light chain</entry><entry><img file="EP2467165B1_D0143.tif" /></entry><entry align="center">263</entry></row><row><entry align="center">KV7 light chain</entry><entry><img file="EP2467165B1_D0144.tif" /></entry><entry align="center">265</entry></row></tbody></tgroup></table></tables><tables id="tabl0005" num="0005"><table frame="all"><title>TABLE 5.</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="37mm" /><colspec colnum="2" colname="col2" colwidth="117mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><thead><row><entry align="center" valign="top">Construct</entry><entry align="center" valign="top">POLYPEPTIDE SEQUENCE</entry><entry align="center" valign="top">SEQ ID NO</entry></row></thead><tbody><row rowsep="0"><entry align="center">Human FAP ectodomain+poly-lys-tag+his6-tag</entry><entry><img file="EP2467165B1_D0145.tif" /></entry><entry align="center">53</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0146.tif" /></entry><entry align="center" /></row><row><entry align="center">Murine FAP ectodomain+poly-lys-tag+his6-tag</entry><entry><img file="EP2467165B1_D0147.tif" /></entry><entry align="center">55</entry></row><row><entry align="center">Human TNC-A2+avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0148.tif" /></entry><entry align="center">57</entry></row><row><entry align="center">Human TNC-A1+avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0149.tif" /></entry><entry align="center">59</entry></row><row><entry align="center">Murine TNC-A1+avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0150.tif" /></entry><entry align="center">61</entry></row><row><entry align="center">Human TNC-A4+ avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0151.tif" /></entry><entry align="center">63</entry></row><row><entry align="center">Murine TNC-A4 +avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0152.tif" /></entry><entry align="center">65</entry></row></tbody></tgroup></table></tables><tables id="tabl0006" num="0006"><table frame="all"><title>TABLE 6.</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="32mm" /><colspec colnum="2" colname="col2" colwidth="115mm" /><colspec colnum="3" colname="col3" colwidth="19mm" /><thead><row><entry align="center" valign="top">Construct</entry><entry align="center" valign="top">POLYNUCLEOTIDE SEQUENCE</entry><entry align="center" valign="top">SEQ ID NO</entry></row></thead><tbody><row><entry align="center">2B10; VL</entry><entry><img file="EP2467165B1_D0153.tif" /></entry><entry align="center">4</entry></row><row><entry align="center">2B10(GS); VL</entry><entry><img file="EP2467165B1_D0154.tif" /></entry><entry align="center">6</entry></row><row><entry align="center">2B10; VH</entry><entry><img file="EP2467165B1_D0155.tif" /></entry><entry align="center">8</entry></row><row><entry align="center">2F11; VL</entry><entry><img file="EP2467165B1_D0156.tif" /></entry><entry align="center">10</entry></row><row><entry align="center">2F11(VI); VL</entry><entry><img file="EP2467165B1_D0157.tif" /></entry><entry align="center">12</entry></row><row rowsep="0"><entry align="center">2F11; VH</entry><entry><img file="EP2467165B1_D0158.tif" /></entry><entry align="center">14</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0159.tif" /></entry><entry align="center" /></row><row><entry align="center">2F11(MT); VH</entry><entry><img file="EP2467165B1_D0160.tif" /></entry><entry align="center">16</entry></row><row><entry align="center">3F2; VL</entry><entry><img file="EP2467165B1_D0161.tif" /></entry><entry align="center">18</entry></row><row><entry align="center">3F2(YS); VL</entry><entry><img file="EP2467165B1_D0162.tif" /></entry><entry align="center">20</entry></row><row><entry align="center">3F2; VH</entry><entry><img file="EP2467165B1_D0163.tif" /></entry><entry align="center">22</entry></row><row><entry align="center">3D9, VL</entry><entry><img file="EP2467165B1_D0164.tif" /></entry><entry align="center">24</entry></row><row rowsep="0"><entry align="center">3D9, VH</entry><entry><img file="EP2467165B1_D0165.tif" /></entry><entry align="center">26</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0166.tif" /></entry><entry align="center" /></row><row><entry align="center">3D9(TA); VH</entry><entry><img file="EP2467165B1_D0167.tif" /></entry><entry align="center">28</entry></row><row><entry align="center">4G8; VL</entry><entry><img file="EP2467165B1_D0168.tif" /></entry><entry align="center">30</entry></row><row><entry align="center">4G8; VH</entry><entry><img file="EP2467165B1_D0169.tif" /></entry><entry align="center">32</entry></row><row><entry align="center">4B3; VL</entry><entry><img file="EP2467165B1_D0170.tif" /></entry><entry align="center">34</entry></row><row><entry align="center">4B3; VH</entry><entry><img file="EP2467165B1_D0171.tif" /></entry><entry align="center">36</entry></row><row rowsep="0"><entry align="center">4D6; VL</entry><entry><img file="EP2467165B1_D0172.tif" /></entry><entry align="center">38</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0173.tif" /></entry><entry align="center" /></row><row><entry align="center">4D6; VH</entry><entry><img file="EP2467165B1_D0174.tif" /></entry><entry align="center">40</entry></row><row><entry align="center">2C6; VL</entry><entry><img file="EP2467165B1_D0175.tif" /></entry><entry align="center">42</entry></row><row><entry align="center">2C6; VH</entry><entry><img file="EP2467165B1_D0176.tif" /></entry><entry align="center">44</entry></row><row><entry align="center">5H5; VL</entry><entry><img file="EP2467165B1_D0177.tif" /></entry><entry align="center">46</entry></row><row><entry align="center">5H5; VH</entry><entry><img file="EP2467165B1_D0178.tif" /></entry><entry align="center">48</entry></row><row rowsep="0"><entry align="center">2C4; VL</entry><entry><img file="EP2467165B1_D0179.tif" /></entry><entry align="center">50</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0180.tif" /></entry><entry align="center" /></row><row><entry align="center">2C4; VH</entry><entry><img file="EP2467165B1_D0181.tif" /></entry><entry align="center">52</entry></row><row><entry align="center">2D9; VL</entry><entry><img file="EP2467165B1_D0182.tif" /></entry><entry align="center">68</entry></row><row><entry align="center">2D9; VH</entry><entry><img file="EP2467165B1_D0183.tif" /></entry><entry align="center">70</entry></row><row><entry align="center">4B8; VL</entry><entry><img file="EP2467165B1_D0184.tif" /></entry><entry align="center">72</entry></row><row><entry align="center">4B8; VH</entry><entry><img file="EP2467165B1_D0185.tif" /></entry><entry align="center">74</entry></row><row rowsep="0"><entry align="center">7A1; VL</entry><entry><img file="EP2467165B1_D0186.tif" /></entry><entry align="center">76</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0187.tif" /></entry><entry align="center" /></row><row><entry align="center">7A1; VH</entry><entry><img file="EP2467165B1_D0188.tif" /></entry><entry align="center">78</entry></row><row><entry align="center">13C2; VL</entry><entry><img file="EP2467165B1_D0189.tif" /></entry><entry align="center">80</entry></row><row><entry align="center">13C2; VH</entry><entry><img file="EP2467165B1_D0190.tif" /></entry><entry align="center">82</entry></row><row><entry align="center">13E8; VL</entry><entry><img file="EP2467165B1_D0191.tif" /></entry><entry align="center">84</entry></row><row><entry align="center">13E8; VH</entry><entry><img file="EP2467165B1_D0192.tif" /></entry><entry align="center">86</entry></row><row rowsep="0"><entry align="center">14C10; VL</entry><entry><img file="EP2467165B1_D0193.tif" /></entry><entry align="center">88</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0194.tif" /></entry><entry align="center" /></row><row><entry align="center">14C10; VH</entry><entry><img file="EP2467165B1_D0195.tif" /></entry><entry align="center">90</entry></row><row><entry align="center">17A11; VL</entry><entry><img file="EP2467165B1_D0196.tif" /></entry><entry align="center">92</entry></row><row><entry align="center">17A11; VH</entry><entry><img file="EP2467165B1_D0197.tif" /></entry><entry align="center">94</entry></row><row><entry align="center">19G1; VL</entry><entry><img file="EP2467165B1_D0198.tif" /></entry><entry align="center">122</entry></row><row><entry align="center">19G1; VH</entry><entry><img file="EP2467165B1_D0199.tif" /></entry><entry align="center">124</entry></row><row rowsep="0"><entry align="center">20G8; VL</entry><entry><img file="EP2467165B1_D0200.tif" /></entry><entry align="center">126</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0201.tif" /></entry><entry align="center" /></row><row><entry align="center">20G8; VH</entry><entry><img file="EP2467165B1_D0202.tif" /></entry><entry align="center">128</entry></row><row><entry align="center">4B9; VL</entry><entry><img file="EP2467165B1_D0203.tif" /></entry><entry align="center">130</entry></row><row><entry align="center">4B9; VH</entry><entry><img file="EP2467165B1_D0204.tif" /></entry><entry align="center">132</entry></row><row><entry align="center">5B8; VL</entry><entry><img file="EP2467165B1_D0205.tif" /></entry><entry align="center">134</entry></row><row><entry align="center">5B8; VH</entry><entry><img file="EP2467165B1_D0206.tif" /></entry><entry align="center">136</entry></row><row><entry align="center">5F1; VL</entry><entry><img file="EP2467165B1_D0207.tif" /></entry><entry align="center">138</entry></row><row><entry align="center">5F1; VH</entry><entry><img file="EP2467165B1_D0208.tif" /></entry><entry align="center">140</entry></row><row><entry align="center">14B3; VL</entry><entry><img file="EP2467165B1_D0209.tif" /></entry><entry align="center">142</entry></row><row><entry align="center">14B3; VH</entry><entry><img file="EP2467165B1_D0210.tif" /></entry><entry align="center">144</entry></row><row><entry align="center">16F1; VL</entry><entry><img file="EP2467165B1_D0211.tif" /></entry><entry align="center">146</entry></row><row rowsep="0"><entry align="center">16F1; VH</entry><entry><img file="EP2467165B1_D0212.tif" /></entry><entry align="center">148</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0213.tif" /></entry><entry align="center" /></row><row><entry align="center">16F8; VL</entry><entry><img file="EP2467165B1_D0214.tif" /></entry><entry align="center">150</entry></row><row><entry align="center">16F8; VH</entry><entry><img file="EP2467165B1_D0215.tif" /></entry><entry align="center">152</entry></row><row><entry align="center">O3C9; VL</entry><entry><img file="EP2467165B1_D0216.tif" /></entry><entry align="center">154</entry></row><row><entry align="center">03C9; VH</entry><entry><img file="EP2467165B1_D0217.tif" /></entry><entry align="center">156</entry></row><row><entry align="center">02D7; VL</entry><entry><img file="EP2467165B1_D0218.tif" /></entry><entry align="center">158</entry></row><row rowsep="0"><entry align="center">O2D7; VH</entry><entry><img file="EP2467165B1_D0219.tif" /></entry><entry align="center">160</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0220.tif" /></entry><entry align="center" /></row><row><entry align="center">28H1; VL</entry><entry><img file="EP2467165B1_D0221.tif" /></entry><entry align="center">162</entry></row><row><entry align="center">28H1; VH</entry><entry><img file="EP2467165B1_D0222.tif" /></entry><entry align="center">164</entry></row><row><entry align="center">22A3; VL</entry><entry><img file="EP2467165B1_D0223.tif" /></entry><entry align="center">166</entry></row><row><entry align="center">22A3; VH</entry><entry><img file="EP2467165B1_D0224.tif" /></entry><entry align="center">168</entry></row><row><entry align="center">29B11; VL</entry><entry><img file="EP2467165B1_D0225.tif" /></entry><entry align="center">170</entry></row><row rowsep="0"><entry align="center">29B11; VH</entry><entry><img file="EP2467165B1_D0226.tif" /></entry><entry align="center">172</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0227.tif" /></entry><entry align="center" /></row><row><entry align="center">23C10; VL</entry><entry><img file="EP2467165B1_D0228.tif" /></entry><entry align="center">174</entry></row><row><entry align="center">23C10; VH</entry><entry><img file="EP2467165B1_D0229.tif" /></entry><entry align="center">176</entry></row><row><entry align="center">2B10_C3B6; VL</entry><entry><img file="EP2467165B1_D0230.tif" /></entry><entry align="center">178</entry></row><row><entry align="center">2B10_C3B6; VH</entry><entry><img file="EP2467165B1_D0231.tif" /></entry><entry align="center">180</entry></row><row><entry align="center">2B10_6A12; VL</entry><entry><img file="EP2467165B1_D0232.tif" /></entry><entry align="center">182</entry></row><row rowsep="0"><entry align="center">2B10_6A12; VH</entry><entry><img file="EP2467165B1_D0233.tif" /></entry><entry align="center">184</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0234.tif" /></entry><entry align="center" /></row><row><entry align="center">2B10_C3A6; VL</entry><entry><img file="EP2467165B1_D0235.tif" /></entry><entry align="center">186</entry></row><row><entry align="center">2B10_C3A6; VH</entry><entry><img file="EP2467165B1_D0236.tif" /></entry><entry align="center">188</entry></row><row><entry align="center">2B10_D1A2_wt; VL</entry><entry><img file="EP2467165B1_D0237.tif" /></entry><entry align="center">190</entry></row><row><entry align="center">2B10_D1A2_wt; VH</entry><entry><img file="EP2467165B1_D0238.tif" /></entry><entry align="center">192</entry></row><row><entry align="center">2B10_D1A2_VD; VL</entry><entry><img file="EP2467165B1_D0239.tif" /></entry><entry align="center">194</entry></row><row><entry align="center">2B10_D1A2_VD; VH</entry><entry><img file="EP2467165B1_D0240.tif" /></entry><entry align="center">196</entry></row><row><entry align="center">2B10_O7D8; VL</entry><entry><img file="EP2467165B1_D0241.tif" /></entry><entry align="center">198</entry></row><row><entry align="center">2B10_O7D8; VH</entry><entry><img file="EP2467165B1_D0242.tif" /></entry><entry align="center">200</entry></row><row><entry align="center">2B10_O1F7; VL</entry><entry><img file="EP2467165B1_D0243.tif" /></entry><entry align="center">202</entry></row><row><entry align="center">2B10_O1F7; VH</entry><entry><img file="EP2467165B1_D0244.tif" /></entry><entry align="center">204</entry></row><row rowsep="0"><entry align="center">2B10_6H10; VL</entry><entry><img file="EP2467165B1_D0245.tif" /></entry><entry align="center">206</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0246.tif" /></entry><entry align="center" /></row><row><entry align="center">2B10_6H10; VH</entry><entry><img file="EP2467165B1_D0247.tif" /></entry><entry align="center">208</entry></row><row><entry align="center">MHLG1; VH</entry><entry><img file="EP2467165B1_D0248.tif" /></entry><entry align="center">258</entry></row><row><entry align="center">KV9; VL</entry><entry><img file="EP2467165B1_D0249.tif" /></entry><entry align="center">260</entry></row><row><entry align="center">MHLG; VH</entry><entry><img file="EP2467165B1_D0250.tif" /></entry><entry align="center">262</entry></row><row><entry align="center">KV1; VL</entry><entry><img file="EP2467165B1_D0251.tif" /></entry><entry align="center">270</entry></row><row rowsep="0"><entry align="center">KV7; VL</entry><entry><img file="EP2467165B1_D0252.tif" /></entry><entry align="center">272</entry></row><row><entry /><entry><img file="EP2467165B1_D0253.tif" /></entry><entry /></row></tbody></tgroup></table></tables><tables id="tabl0007" num="0007"><table frame="all"><title>TABLE 7.</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="37mm" /><colspec colnum="2" colname="col2" colwidth="117mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><thead><row><entry align="center" valign="top">Construct</entry><entry align="center" valign="top">POLYNUCLEOTIDE SEQUENCE</entry><entry align="center" valign="top">SEQ ID NO</entry></row></thead><tbody><row rowsep="0"><entry align="center">Human FAP ectodomain+poly-lys-tag+his6-tag</entry><entry><img file="EP2467165B1_D0254.tif" /></entry><entry align="center">54</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0255.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">Murine FAP ectodomain+poly-lys-tag+his6-tag</entry><entry><img file="EP2467165B1_D0256.tif" /></entry><entry align="center">56</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0257.tif" /></entry><entry align="center" /></row><row><entry align="center">Human TNC-A2+avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0258.tif" /></entry><entry align="center">58</entry></row><row><entry align="center">Human TNC-A1+avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0259.tif" /></entry><entry align="center">60</entry></row><row><entry align="center">Murine TNC-A1+avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0260.tif" /></entry><entry align="center">62</entry></row><row rowsep="0"><entry align="center">Human TNC-A4+ avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0261.tif" /></entry><entry align="center">64</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0262.tif" /></entry><entry align="center" /></row><row><entry align="center">Murine TNC-A4 +avi-tag +his6-tag</entry><entry><img file="EP2467165B1_D0263.tif" /></entry><entry align="center">66</entry></row></tbody></tgroup></table></tables><tables id="tabl0008" num="0008"><table frame="all"><title>TABLE 8.</title><tgroup cols="3"><colspec colnum="1" colname="col1" colwidth="38mm" /><colspec colnum="2" colname="col2" colwidth="117mm" /><colspec colnum="3" colname="col3" colwidth="12mm" /><thead><row><entry align="center" valign="top">Construct</entry><entry align="center" valign="top">POLYNUCLEOTIDE SEQUENCE</entry><entry align="center" valign="top">SEQ ID NO</entry></row></thead><tbody><row rowsep="0"><entry align="center">Fab heavy chain derived from L19 monoclonal antibody-C125A variant of IL2-Fab heavy chain derived from L19 monoclonal antibody</entry><entry><img file="EP2467165B1_D0264.tif" /></entry><entry align="center">108</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0265.tif" /></entry><entry align="center" /></row><row><entry align="center">Fab light chain derived from L19 monoclonal antibody</entry><entry><img file="EP2467165B1_D0266.tif" /></entry><entry align="center">109</entry></row><row rowsep="0"><entry align="center">scFv derived from L19 monoclonal antibody-8 amino acid linker-C125A variant of IL2</entry><entry><img file="EP2467165B1_D0267.tif" /></entry><entry align="center">110</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0268.tif" /></entry><entry align="center" /></row><row><entry align="center">F16-diabody-IL2 DNA</entry><entry><img file="EP2467165B1_D0269.tif" /></entry><entry align="center">111</entry></row><row rowsep="0"><entry align="center">scFv-IL2-scFv (F16, DNA)</entry><entry><img file="EP2467165B1_D0270.tif" /></entry><entry align="center">112</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0271.tif" /></entry><entry /></row><row rowsep="0"><entry align="center">Fab-IL2-Fab (F16, heavy chain cytokine fusion construct, DNA)</entry><entry><img file="EP2467165B1_D0272.tif" /></entry><entry align="center">113</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0273.tif" /></entry><entry align="center" /></row><row><entry align="center">F16, light chain, DNA</entry><entry><img file="EP2467165B1_D0274.tif" /></entry><entry align="center">114</entry></row><row rowsep="0"><entry align="center">IL2R-beta-Fc(hole) fusion protein, DNA</entry><entry><img file="EP2467165B1_D0275.tif" /></entry><entry align="center">115</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0276.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">IL2R-gamma-Fc(knob), DNA</entry><entry><img file="EP2467165B1_D0277.tif" /></entry><entry align="center">116</entry></row><row><entry /><entry><img file="EP2467165B1_D0278.tif" /></entry><entry /></row><row rowsep="0"><entry align="center">Fab-IL12-Fab L19 antibody, murine scIL12, DNA</entry><entry><img file="EP2467165B1_D0279.tif" /></entry><entry align="center">117</entry></row><row><entry /><entry><img file="EP2467165B1_D0280.tif" /></entry><entry /></row><row rowsep="0"><entry align="center">Fab-IL12-FabL19 antibody, human scIL12, DNA</entry><entry><img file="EP2467165B1_D0281.tif" /></entry><entry align="center">118</entry></row><row rowsep="0"><entry /><entry><img file="EP2467165B1_D0282.tif" /></entry><entry /></row><row><entry align="center" /><entry><img file="EP2467165B1_D0283.tif" /></entry><entry align="center" /></row><row><entry align="center">Fab-GMCSF-Fab L19 antibody, human GM-CSF, DNA</entry><entry><img file="EP2467165B1_D0284.tif" /></entry><entry align="center">119</entry></row><row rowsep="0"><entry align="center">Fab-IFNα2-Fab, L19 antibody, DNA</entry><entry><img file="EP2467165B1_D0285.tif" /></entry><entry align="center">120</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0286.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">3F2 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0287.tif" /></entry><entry align="center">210</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0288.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">4G8 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0289.tif" /></entry><entry align="center">212</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0290.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">3D9 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0291.tif" /></entry><entry align="center">214</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0292.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">2F11 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0293.tif" /></entry><entry align="center">216</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0294.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">4B3 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0295.tif" /></entry><entry align="center">218</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0296.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">4G8 Fab-IL12-Fab (murine IL-12; heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0297.tif" /></entry><entry align="center">220</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0298.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">28H1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0299.tif" /></entry><entry align="center">222</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0300.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">29B11 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0301.tif" /></entry><entry align="center">224</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0302.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">19G1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0303.tif" /></entry><entry align="center">226</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0304.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">20G8 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0305.tif" /></entry><entry align="center">228</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0306.tif" /></entry><entry align="center" /></row><row><entry align="center">3F2 light chain</entry><entry><img file="EP2467165B1_D0307.tif" /></entry><entry align="center">230</entry></row><row><entry align="center">4G8 light chain</entry><entry><img file="EP2467165B1_D0308.tif" /></entry><entry align="center">232</entry></row><row rowsep="0"><entry align="center">3D9 light chain</entry><entry><img file="EP2467165B1_D0309.tif" /></entry><entry align="center">234</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0310.tif" /></entry><entry align="center" /></row><row><entry align="center">2F11 light chain</entry><entry><img file="EP2467165B1_D0311.tif" /></entry><entry align="center">236</entry></row><row><entry align="center">4B3 light chain</entry><entry><img file="EP2467165B1_D0312.tif" /></entry><entry align="center">238</entry></row><row rowsep="0"><entry align="center">2B10 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0313.tif" /></entry><entry align="center">240</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0314.tif" /></entry><entry align="center" /></row><row rowsep="0"><entry align="center">C3B6 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0315.tif" /></entry><entry align="center">242</entry></row><row><entry /><entry><img file="EP2467165B1_D0316.tif" /></entry><entry /></row><row rowsep="0"><entry align="center">6A12 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0317.tif" /></entry><entry align="center">244</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0318.tif" /></entry><entry align="center" /></row><row><entry align="center">2B10 light chain</entry><entry><img file="EP2467165B1_D0319.tif" /></entry><entry align="center">246</entry></row><row rowsep="0"><entry align="center">D1A2 light chain</entry><entry><img file="EP2467165B1_D0320.tif" /></entry><entry align="center">248</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0321.tif" /></entry><entry align="center" /></row><row><entry align="center">O7D8 light chain</entry><entry><img file="EP2467165B1_D0322.tif" /></entry><entry align="center">250</entry></row><row rowsep="0"><entry align="center">MHLG1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0323.tif" /></entry><entry align="center">252</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0324.tif" /></entry><entry align="center" /></row><row><entry align="center">KV9 light chain</entry><entry><img file="EP2467165B1_D0325.tif" /></entry><entry align="center">254</entry></row><row rowsep="0"><entry align="center">MHLG Fab-IL2-Fab (heavy chain cytokine fusion construct)</entry><entry><img file="EP2467165B1_D0326.tif" /></entry><entry align="center">256</entry></row><row><entry align="center" /><entry><img file="EP2467165B1_D0327.tif" /></entry><entry align="center" /></row><row><entry align="center">KV1 light chain</entry><entry><img file="EP2467165B1_D0328.tif" /></entry><entry align="center">264</entry></row><row rowsep="0"><entry align="center">KV7 light chain</entry><entry><img file="EP2467165B1_D0329.tif" /></entry><entry align="center">266</entry></row><row><entry /><entry><img file="EP2467165B1_D0330.tif" /></entry><entry /></row></tbody></tgroup></table></tables>

Host Cells

0129As used herein, the term "host cell" refers to any kind of cellular system which can be engineered to generate the immunoconjugates described herein or fragments thereof. In one embodiment, the host cell is engineered to allow the production of an immunoconjugate fragment. Host cells include cultured cells, e.g., mammalian cultured cells, such as CHO cells, HEK, BHK cells, NS0 cells, Sp2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, bacterial cells and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one embodiment, the host cell described herein comprises an expression vector comprising polynucleotide sequences that encode immunoconjugates described herein or fragments thereof. Host cells described herein may be eukaryotic or prokaryotic.

Purification of Immunoconjugate Polypeptides and Fragments Thereof

0130The immunoconjugates described herein or fragments thereof can be purified by art-known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography or size exclusion chromatography. The actual conditions used to purify a particular protein will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those having skill in the art.

0131For affinity chromatography purification, a matrix with protein A or protein G may be used. Alternatively, for affinity chromatography purification, any antibody which specifically binds the single-chain effector moiety of the immunoconjugate may be used. For the production of antibodies, various host animals, including, but not limited to rabbits, mice, rats, etc., may be immunized by injection with a immunoconjugate described herein or a fragment thereof. The immunoconjugate may be attached to a suitable carrier, such as bovine serum albumin (BSA), by means of a side chain functional group or linkers attached to a side chain functional group. Various adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhold limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Cornyebacterium parvum. Accordingly, one embodiment includes a method for producing the immunoconjugates described herein by culturing a host cell comprising an expression vector comprising polynucleotide sequences that encode immunoconjugates described herein or fragments thereof under conditions suitable for the expression of the same.

Methods of Using Immunoconjugates

0132The immunoconjugates described herein are useful for targeting specific antigenic determinants and eliciting various cellular responses in target and recruited cells. The immunoconjugate described herein is also useful as a diagnostic reagent. The binding of an immunoconjugate to an antigenic determinant can be readily detected by using a secondary antibody specific for the effector moiety. In one embodiment, the secondary antibody and the immunoconjugate facilitate the detection of binding of the immunoconjugate to an antigenic determinant located on a cell or tissue surface.

0133In some embodiments, an effective amount of the immunoconjugates described herein are administered to a cell. In other embodiments, a therapeutically effective amount of the immunoconjugate described herein is administered to an individual for the treatment of disease. The term "effective amount" as used herein is defined as the amount of the immunoconjugate described herein that is necessary to result in a physiological change in the cell or tissue to which it is administered. The term "therapeutically effective amount" as used herein is defined as the amount of the immunoconjugate described herein that eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

0134The immunoconjugates described herein may be administered to a subject per se or in the form of a pharmaceutical composition. In one embodiment, the disease is a proliferative disorder, such as cancer. Non-limiting examples of proliferative disorders such as cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other cell proliferation disorders that can be treated using an immunoconjugate described herein include, but are not limited to neoplasms located in the: abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic region, and urogenital system. Also included are pre-cancerous conditions or lesions and cancer metastases. Similarly, other cell proliferation disorders can also be treated by the immunoconjugates described herein. Examples of such cell proliferation disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other cell proliferation disease, besides neoplasia, located in an organ system listed above. In another embodiment, the disease is related to autoimmunity, transplantation rejection, post-traumatic immune responses and infectious diseases (e.g. HIV). More specifically, the immunoconjugates may be used in eliminating cells involved in immune cell-mediated disorders, including lymphoma; autoimmunity, transplantation rejection, graft-versus-host disease, ischemia and stroke. A skilled artisan readily recognizes that in many cases the immunoconjugates may not provide a cure but may only provide partial benefit. In some embodiments, a physiological change having some benefit is also considered therapeutically beneficial. Thus, in some embodiments, an amount of immunoconjugate that provides a physiological change is considered an "effective amount" or a "therapeutically effective amount."

0135The subject, patient, or individual in need of treatment is typically a mammal, more specifically a human.

Compositions, Formulations, Dosages, and Routes of Administration

0136Pharmaceutical compositions described herein comprise an effective amount of one or more immunoconjugates dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one immunoconjugate and optionally an additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by <nplcit id="ncit0035" npl-type="b"><text>Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990</text></nplcit>. Moreover, for animal (e.g., human) administration, it will be understood that preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards or corresponding authorities in other countires.

0137As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, buffers, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, <nplcit id="ncit0036" npl-type="b"><text>Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329</text></nplcit>). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

0138The immunoconjugates may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection. The immunoconjugates described herein can be administered intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrasplenically, intrarenally, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intraperitoneally, subcutaneously, subconjunctivally, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, topically, locally, inhalation (e.g. aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example,<nplcit id="ncit0037" npl-type="b"><text>Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990</text></nplcit>). Parenteral administration, in particular intravenous injection, is most commonly used for administering polypeptide molecules such as the immunoconjugates described herein.

0139The actual dosage amount of a composition described herein administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject.

0140In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1 % of the immunoconjugate described herein. In other embodiments, the immunoconjugates may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein. In other non-limiting examples, a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 nucrogram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of a derivable range from the numbers listed herein, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc., can be administered, based on the numbers described above.

0141In any case, the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

0142The immunoconjugates may be formulated into a composition in a free base, neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.

0143In embodiments where the composition is in a liquid form, a carrier can be a solvent or dispersion medium comprising but not limited to, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes) and combinations thereof. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin; by the maintenance of the required particle size by dispersion in carriers such as, for example liquid polyol or lipids; by the use of surfactants such as, for example hydroxypropylcellulose; or combinations thereof such methods. In many cases, it will be preferable to include isotonic agents, such as, for example, sugars, sodium chloride or combinations thereof, and/or buffering agents to maintain physiologically acceptable pH values. In other embodiments, one may use eye drops, nasal solutions or sprays, aerosols or inhalants. Such compositions are generally designed to be compatible with the target tissue type. In a non-limiting example, nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions are prepared so that they are similar in many respects to nasal secretions, so that normal ciliary action is maintained. Thus, in some embodiments the aqueous nasal solutions usually are isotonic or slightly buffered to maintain a pH of about 5.5 to about 6.5. In addition, antimicrobial preservatives, similar to those used in ophthalmic preparations, drugs, or appropriate drug stabilizers, if required, may be included in the formulation. For example, various commercial nasal preparations are known and include drugs such as antibiotics or antihistamines.

0144In certain embodiments, the immunoconjugate is prepared for administration by such routes as oral ingestion. In these embodiments, the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof. Oral compositions may be incorporated directly with the food of the diet. Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof. In other aspects of the disclosure, the oral composition may be prepared as a syrup or elixir. A syrup or elixir, and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.

0145In certain embodiments, an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof. In certain embodiments, a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations thereof the foregoing. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.

0146Additional formulations which are suitable for other modes of administration include suppositories. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%. Sterile injectable solutions are prepared by incorporating the immunoconjugates described herein in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and/or the other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, suspensions or emulsion, the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof. The liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose. The preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.

0147The composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.

0148In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.

0149Pharmaceutical compositions comprising the immunoconjugates described herein may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the proteins into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

0150For topical administration the immunoconjugates described herein may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.

0151Systemic formulations include those designed for administration by injection, e.g. subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, inhalation, oral or pulmonary administration.

0152For injection, the immunoconjugates described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. The solution may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

0153Alternatively, the immunoconjugates may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

0154For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

0155For oral administration, the immunoconjugates can be readily formulated by combining the immunoconjugates with pharmaceutically acceptable carriers well known in the art. Such carriers enable the immunoconjugates described herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries or suspensions, for oral ingestion by a patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, e.g. lactose, sucrose, mannitol and sorbitol; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

0156If desired, solid dosage forms may be sugar-coated or enteric-coated using standard techniques. For oral liquid preparations such as, for example, suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives or coloring agents may be added.

0157For buccal administration, the immunoconjugates may take the form of tablets, lozenges, etc. formulated in conventional manner.

0158For administration by inhalation, the immunoconjugates for use according to the disclosure are conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the immunoconjugate and a suitable powder base such as lactose or starch.

0159The immunoconjugates may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

0160In addition to the formulations described previously, the immunoconjugates may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the immunoconjugates may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

0161Alternatively, other pharmaceutical delivery systems may be employed. Liposomes and emulsions are well known examples of delivery vehicles that may be used to deliver immunoconjugates described herein. Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity. Additionally, the immunoconjugates may be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. Various of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the immunoconjugates for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the immunoconjugates, additional strategies for immunoconjugates stabilization may be employed. As the immunoconjugates described herein may contain charged side chains or termini, they may be included in any of the above-described formulations as the free acids or bases or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts which substantially retain the biologic activity of the free bases and which are prepared by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous and other protic solvents than are the corresponding free base forms.

0162The immunoconjugates described herein will generally be used in an amount effective to achieve the intended purpose. For use to treat or prevent a disease condition, the immunoconjugates described herein, or pharmaceutical compositions thereof, are administered or applied in a therapeutically effective amount. A therapeutically effective amount is an amount effective to ameliorate or prevent the symptoms, or prolong the survival of, the patient being treated. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

0163For systemic administration, a therapeutically effective dose can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

0164Initial dosages can also be estimated from in vivo data, e.g., animal models, using techniques that are well known in the art. One having ordinary skill in the art could readily optimize administration to humans based on animal data.

0165Dosage amount and interval may be adjusted individually to provide plasma levels of the immunoconjugates which are sufficient to maintain therapeutic effect. Usual patient.dosages for administration by injection range from about 0.1 to 50 mg/kg/day, typically from about 0.5 to 1 mg/kg/day. Therapeutically effective serum levels may be achieved by administering multiple doses each day.

0166In cases of local administration or selective uptake, the effective local concentration of the immunoconjugates may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.

0167The amount of immunoconjugate administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.

0168The therapy may be repeated intermittently while symptoms detectable or even when they are not detectable. The therapy may be provided alone or in combination with other drugs. In the case of autoimmune disorders, the drugs that may be used in combination with immunoconjugates described herein include, but are not limited to, steroid and non-steroid antiinflammatory agents.

Toxicity

0169A therapeutically effective dose of the immunoconjugates described herein will generally provide therapeutic benefit without causing substantial toxicity. Toxicity of the immunoconjugates can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. In one embodiment, the immunoconjugate exhibits a high therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic, for example, for use in human. The dosage of the immunoconjugates described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g.,<nplcit id="ncit0038" npl-type="b"><text>Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1</text></nplcit>).

Other Agents and Treatments

0170It is contemplated that other agents may be used in combination with the immunoconjugates described herein to improve the therapeutic efficacy of treatment. These additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers. Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1β, MCP-1, RANTES, and other chemokines. It is further contemplated that the upregulation of cell surface receptors or their ligands such as Fas/Fas ligand, DR4 or DR5 /TRAIL would potentiate the apoptotic inducing abililties of the immunoconjugates described herein by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases in intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population. In other embodiments, cytostatic or differentiation agents can be used in combination with the immunoconjugates described herein to improve the anti-hyperproliferative efficacy of the treatments. Inhibitors of cell adhesion are contemplated, to improve the efficacy of the immunoconjugates described herein. Examples of cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody C225, could be used in combination with the immunoconjugates described herein to improve the treatment efficacy.

0171Hormonal therapy may also be used in conjunction with the immunoconjugates described herein or in combination with any other cancer therapy previously described. The use of hormones may be employed in the treatment of certain cancers such as breast, prostate, ovarian, or cervical cancer to lower the level or block the effects of certain hormones such as testosterone or estrogen. This treatment is often used in combination with at least one other cancer therapy as a treatment option or to reduce the risk of metastases.

0172The immunonconjugates described herein may also be administred in conjunction with chemotherapy, radiation therapy or other immunotherapies. Anti-cancer agents for such combination therapy may, e.g., be selected from the groups of microtubule disruptors (e.g. vinca alkaloids such as vinblastine or vincristine, taxanes such as docetaxel or paclitaxel, epothilones such as ixabepilone), antimetabolites (e.g. anti-folates such as methotrexate or aminopterin, anti-purines such as fludarabine, 6-mercaptopurine or 6-thioguanine, anti-pyrimidines such as 5-fluorouracil, capecitabine or gemcitabine, hydroxyurea), topoisomerase inhibitors (e.g. camptothecin, irinotecan, topotecan, or podophyllotoxins such as etoposide), DNA intercalators (e.g. doxorubicin, daunorubicin, actinomycin, bleomycin), alkylating agents (e.g. cyclophosphamide, chlorambucil, nitrosureas such as carmustine or nimustine, streptozocin, busulfan, cisplatin, oxaliplatin, triethylenemelamine, dacarbazine), hormonal therapies (e.g. glucocorticoids, aromatase inhibitors such as tamoxifene, antiandrogens such as flutamide, gonadotropin-releasing hormone (GnRH) analogs such as leuprolide), antibiotics, kinase inhibitors (e.g. erlotinib, gefitinib, imatinib), receptor antagonists (e.g. antibodies targeting cell surface receptors known to promote carcinogenesis and tumor growth), enzyme inhibitors (e.g. cyclin-dependent kinase (CDK) inhibitors), amino acid-depleting enzymes (e.g. asparaginase), leucovorin, retinoids, activators of tumor cell apoptosis, and antiangiogenic agents.

EXAMPLES

Example 1

0173When antibody-mediated delivery of cytokines was first performed by Harvill E.T., and Morrison S.L. Immunotech. 1(2):95-105 (1995), the constructs had two antigen binding moieties for tumor antigen targeting and two cytokine moieties for inducing lymphocyte activation (an IL-2 molecule is fused to each of the heavy chain C-termini of IgG3). The affinity of the cytokine is generally high towards its receptor. A molecule carrying two cytokine units could have an even higher affinity towards cytokine receptors because of avidity (or multivalency) effects. Such a molecule could therefore easily activate lymphocytes in the blood stream, even before the targeting to the tumor takes place. This effect would not be desired for the patient. In contrast, an immunoconjugate molecule carrying only one cytokine moiety and two or more targeting domains would be less likely to activate lymphocytes in the circulation and could direct the entire immunoconjugate molecule to the tumor, where lymphocyte activation can take place at a lower speed. Therefore, molecules as depicted in <figref idref="f0001">Figures 1</figref> and <figref idref="f0002">2</figref>, were constructed with interleukin-2 (IL-2) as a model cytokine. All of the generated molecules are bivalent for the tumor antigen, and are either bivalent or monovalent for the IL-2 cytokine as indicated in the drawings. Affinities towards the antigen were compared for two different immunoconjugate formats using the L19 antibody as an example. As a reference, this antibody was cloned into the human IgG1 format, the diabody format (<figref idref="f0001">Figure 1A</figref>), and the Fab-IL2-Fab fusion (<figref idref="f0001">Figure 1B</figref>). One variable was tested within the diabody format such that the linker peptide between the VH and the VL was either eight or twelve amino acids in length. The purified antigen Extra Domain B of fibronectin (EDB) was immobilized on a BIACORE chip, and the antibody fusion construct was used as the soluble analyte for affinity determination. <figref idref="f0008 f0009 f0010 f0011">Figures 8 to 11</figref> show the results of this experiment. The IgG was considered to be the ideal case of a bivalent binding event. Here, an affinity constant of 260 pM was observed. The Fab-IL2-Fab fusion construct gave an affinity of 310 pM, which is essentially identical to the IgG. The two variants of the diabody (shown in <figref idref="f0010">Figures 10</figref> and <figref idref="f0011">11</figref>) had measured affinities of 270 pM and 360 pM, respectively. Therefore, all of these constructs have similar affinities towards the antigen. The affinity towards the antigen and the IL-2 receptor was addressed using similar constructs based on the F16 antibody sequence (<nplcit id="ncit0039" npl-type="s"><text>Brack, S.S. et al., Clin. Canc. Res. 12(10):3200-3208 (2006</text></nplcit>)). <figref idref="f0012">Figure 12</figref> shows the BIACORE sensograms of the F16 IgG and its corresponding monovalent Fab fragment, when the TNC-A1 domain was immobilized on the BIACORE chip. Under these particular experimental conditions, the IgG molecule showed an affinity constant of 2.6 nM, and the Fab molecules showed an affinity constant of 50 nM. Here, the increase of affinity attributed to bivalency is a factor of 20. The diabody, the Fab-IL2-Fab and the scFv-IL2-scFv immunoconjugates showed affinities toward the antigen of 5 nM, 4.8 nM, and 12 nM, respectively. All of these constructs therefore have affinities toward the antigen that more closely resemble the bivalent character of an IgG molecule than the monovalent behavior of the Fab fragment.

0174The differences were more pronounced when looking at affinities toward the IL-2 receptor. To study IL-2 receptor binding affinity, a tool was generated that allowed for the expression of a heterodimeric IL-2 receptor; the β chain of the IL-2 receptor was fused to an Fc molecule that was engineered to heterodimerize (Fc(knob)) using the "knobs-into-holes" technology (<nplcit id="ncit0040" npl-type="s"><text>Merchant, A.M. et al., Nat. Biotech. 16:677-681 (1998</text></nplcit>)). The gamma chain of the IL-2 receptor was then fused to the Fc(hole) variant, which heterodimerized with Fc(knob). This heterodimeric Fc-fusion protein was then used as a substrate for analyzing the IL-2/IL-2 receptor interaction. <figref idref="f0013">Figure 13</figref> shows the BIACORE sensogram of commercially available IL-2 (Proleukin), as the analyte, with the immobilized IL-2 receptor. The measured affinity of ~0.5 nM is in accordance with previously published values. The affinities of the various constructs towards the IL-2 receptor are summarized in <figref idref="f0017">Figure 17</figref>. An important result that was observed was the difference between the diabody (F16 dia IL2), which is bivalent with respect to the cytokine, and the Fab-IL2-Fab molecule which carries only one IL-2 moiety. The IL-2 receptor binding affinity of the diabody (0.8 nM) was similar to that of unconjugated IL-2 (Proleukin) (0.5 nM), despite the diabody being bivalent and the IL-2 being monovalent. The Fab-IL2-Fab fusion had an IL-2 receptor binding affinity almost reduced by a factor of 10 compared to the diabody, which was reflected in a reduced capacity to induce proliferation of NK92 cells as shown in <figref idref="f0019">Figure 19</figref>.

Example 2

Construction of Generic Fab-Libraries

0175Generic antibody libraries in the Fab-format were constructed on the basis of human germline genes using the following V-domain pairings: Vk3_20 kappa light chain with VH3_23 heavy chain for the DP47-3 library and Vk1_17 kappa light chain with VH1_69 heavy chain for the DP88-3 library. See Table 2. Both libraries were randomized in CDR3 of the light chain (L3) and CDR3 of the heavy chain (H3) and were assembled from 3 fragments per library by splicing by overlapping extension (SOE) PCR. Fragment 1 comprises the 5' end of the antibody gene including randomized L3, fragment 2 is a central constant fragment spanning from L3 to H3, whereas fragment 3 comprises randomized H3 and the 3' portion of the antibody gene.

0176The following primer combinations were used to generate library fragments for DP47-3 library: fragment 1 (LMB3 - LibL1b_new), fragment 2 (MS63 - MS64), fragment 3 (Lib2H - fdseqlong). See Table 9. The following primer combinations were used to generate library fragments for the DP88-3 library: fragment 1 (LMB3 - RJH_LIB3), fragment 2 (RJH31 - RJH32) and fragment 3 (LIB88_2 - fdseqlong). See Table 10. <tables id="tabl0009" num="0009"><table frame="all"><title>TABLE 9.</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="23mm" /><colspec colnum="2" colname="col2" colwidth="149mm" /><thead><row><entry namest="col1" nameend="col2" align="center" valign="top">Primers Used In the DP47-3 Library</entry></row></thead><tbody><row><entry>LMB3</entry><entry>CAGGAAACAGCTATGACCATGATTAC</entry></row><row><entry>LibL1b_new</entry><entry><img file="EP2467165B1_D0331.tif" /></entry></row><row><entry>MS63</entry><entry>TTTCGCACAGTAATATACGGCCGTGTCC</entry></row><row><entry>MS64</entry><entry>ACGTTCGGCCAGGGGACCAAAGTGG</entry></row><row><entry>Lib2H</entry><entry><img file="EP2467165B1_D0332.tif" /></entry></row><row><entry>fdseqlong</entry><entry>GACGTTAGTAAATGAATTTTCTGTATGAGG</entry></row></tbody></tgroup></table></tables><tables id="tabl0010" num="0010"><table frame="all"><title>TABLE 10.</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="18mm" /><colspec colnum="2" colname="col2" colwidth="148mm" /><thead><row><entry namest="col1" nameend="col2" align="center" valign="top">Primers Used in DP88-3 Library</entry></row></thead><tbody><row><entry>LMB3</entry><entry>CAGGAAACAGCTATGACCATGATTAC</entry></row><row><entry>RJH_LIB3</entry><entry><img file="EP2467165B1_D0333.tif" /></entry></row><row><entry>RJH31</entry><entry>ACGTTTGGCCAGGGCACCAAAGTCGAG</entry></row><row><entry>RJH32</entry><entry>TCTCGCACAGTAATACACGGCGGTGTCC</entry></row><row><entry>LIB88_2</entry><entry><img file="EP2467165B1_D0334.tif" /></entry></row><row><entry>fdseqlong</entry><entry>GACGTTAGTAAATGAATTTTCTGTATGAGG</entry></row></tbody></tgroup></table></tables>

0177The PCR protocol for the production of library fragments included: 5 minutes of initial denaturation at 94°C; 25 cycles of 1 minute at 94°C, 1 minute at 58°C, and 1 minute at 72°C; and terminal elongation for 10 minutes at 72 °C. For assembly PCR, equimolar ratios of the 3 fragments were used as template. The assembly PCR protocol included: 3 minutes of initial denaturation at 94°C; and 5 cycles of 30 seconds at 94°C, 1 minute at 58°C, and 2 minutes at 72°C. At this stage, primers complementary to sequence outside fragments 1-3 were added and an additional 20 cycles were performed prior to a terminal elongation for 10 min at 72 °C.

0178After assembly of sufficient amounts of full length randomized Fab constructs, the Fab constructs were digested with NcoI / NotI for the DP47-3 library and with NcoI / NheI for the DP88-3 library alongside with similarly treated acceptor phagemid vector. For the DP47-3 library, 22.8 µg of Fab library was ligated with 16.2 µg of phagemid vector. For the DP88-3 library, 30.6 µg of Fab library was ligated with 30.6 µg of phagemid vector.

0179Purified ligations were used for 68 transformations for the DP47-3 library and 64 transformations for the DP88-3 library, respectively, to obtain final library sizes of 4.2 × 1010 for DP47-3 and 3.3 × 109 for DP88-3.

0180Phagemid particles displaying the Fab libraries were rescued and purified by PEG/NaCl purification to be used for selections.

Example 3

Selection of Anti-TNC A2 Clone 2B10

0181Selections were carried out against E. coli-expressed human TNC-A2 which was subcloned 5' of an avi-tag and 6xhis-tag. See SEQ ID NO: 57 in Table 5. The antigen was biotinylated in vivo upon expression. Selections have been carried out in solution according to the following protocol: (i) binding of ~ 1012 phagemid particles of library DP88-3 and 100nM biotinylated human TNC A2 for 0.5 hours in a total volume of 1ml; (ii) capture of biotinylated human TNC-A2 and attached phage by the addition of 5.4 × 107 streptavidin-coated magnetic beads for 10 minutes; (iii) washing of beads using 5x 1ml PBS/Tween20 and 5x 1ml PBS; (iv) elution of phage particles by the addition of 1 mL 100mM TEA (triethylamine) for 10 minutes and neutralization by the addition of 500 µL 1M Tris/HCl pH 7.4; and (v) re-infection of log-phase E. coli TG1 cells, infection with helperphage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles to be used in subsequent selection rounds.

0182Selections were carried out over 3 rounds using constant antigen concentrations at 100 nM. In round 2, capture of antigen:phage complexes was performed on neutravidin plates instead of streptavidin beads. Specific binders were identified by ELISA as follows using: 100 µl of 100 nM biotinylated human TNC-A2 was coated in each well of neutravidin plates.

0183Fab-containing bacterial supernatants were added and binding Fabs were detected via their Flag-tags by using an anti-Flag/HRP secondary antibody. Once identified, clone 2B10 was bacterially expressed in a 0.5 litre culture volume, affinity purified and further characterized by SPR-analysis using BIACORE T100. See SEQ ID NOs: 3 and 7 of Table 3.

Example 4

Selection of Anti-TNC A1/A4 Clone 2F11

0184Selections were carried out against E. coli expressed human TNC A1 which was subcloned 5' of an avi-tag and 6xhis-tag. See SEQ ID NO: 59 of Table 5. The antigen was biotinylated in vivo upon expression. Selections were carried out in solution according to the following protocol: (i) binding of ~ 1012 phagemid particles of library DP47-3 and 100 nM biotinylated human TNC-A1 for 0.5 hours in a total volume of 1 mL; (ii) capture of biotinylated human TNC-A1 and attached phage by the addition of 5.4 × 107 streptavidin-coated magnetic beads for 10 minutes; (iii) washing of beads using 5x 1 mL PBS/Tween20 and 5x 1ml PBS; (iv) elution of phage particles by the addition of 1 mL 100 mM TEA (triethylamine) for 10 minutes and neutralization by the addition of 500 µl 1M Tris/HCl pH 7.4; and (v) re-infection of log-phase E. coli TG1 cells, infection with helperphage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles to be used in subsequent selection round.

0185Selections were carried out over 3 rounds using constant antigen concentrations at 100 nM. In round 2, capture of antigen:phage complexes was performed on neutravidin plates instead of streptavidin beads.

0186All binding reactions were supplemented with 100 nM non-biotinylated human IgG CH3 constant domain comprising a carboxy-terminal avi-tag and 6xhis-tag in order to compete for unwanted clones recognizing the tags of the antigen.

0187In a first screening step, specific binders were identified by ELISA as follows: 100 µl of 100 nM biotinylated human TNC-A1 was coated in each of neutravidin plates. Fab-containing bacterial supernatants were added and Fabs that specifically bound to human TNC-A1 were detected via their Flag-tags by using an anti-Flag/HRP secondary antibody.

0188In a second screening step, the above ELISA was repeated using also murine TNC-A1, human TNC A4 and mu TNC A4 as antigens in order to determine cross-reactivity. All antigens comprised the same avi-tag and 6xhis-tag at their C-terminus and were in vivo biotinylated. See SEQ ID NOs: 50 and 61 of Table 5.

0189Once identified, clone 2F11 was bacterially expressed in a 0.5 litre culture volume, affinity purified and further characterized by SPR-analysis using BIACORE T100. See SEQ ID NOs: 9 and 13 of Table 3.

Example 5

Selection of anti-FAP clones (primary selections)

0190Selections were carried out against the ectodomain of human or murine fibroblast activating protein (FAP) which were cloned upstream a poly-lysine and a 6xhis-tag. See SEQ ID NOs: 53 and 55 of Table 5. Prior to selections, the antigens were coated into immunotubes at a concentration of either 10 µg/ml or 5 µg/ml, depending on round of selection. Selections were carried out according to the following protocol: (i) binding of ~ 1012 phagemid particles of library DP47-3 to immobilized human or murine FAP for 2 hours; (ii) washing of immunotubes using 5 × 5mL PBS/Tween20 and 5 × 5ml PBS; (iii) elution of phage particles by addition of 1mL 100mM TEA (triethylamine) for 10 minutes and neutralization by the addition of 500 µL 1M Tris/HCl pH 7.4; and (iv) re-infection of log-phase E. coli TG1 cells, infection with helperphage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles to be used in subsequent selection rounds.

0191Selections have been carried out over three or four rounds using decreasing antigen concentrations of human FAP and in some cases using murine FAP at 5ug/ml in the final selection round. Specific binders were defined as signals 5 × higher than background and were identified by ELISA. NUNC maxisorp plates were coated with 10ug/ml of human or murine FAP followed by addition of Fab-containing bacterial supernatants and detection of specifically binding Fabs via their Flag-tags by using an anti-Flag/HRP secondary antibody.

0192ELISA-positive clones were bacterially expressed as 1 mL cultures in 96-well format and supernatants were subjected to a kinetic screening experiment using BIACORE T100.

Example 6

Construction of Anti-FAP Affinity Maturation Libraries

0193Three affinity maturation libraries were constructed on basis of pre-selected antibodies from the primary anti-FAP selections. More precisely, they were based on (i) anti-human FAP clone 2D9 (library a.m.FAP2D9) (see SEQ ID NOs: 67 and 69 of Table 3), (ii) anti-murine FAP clone 4B8 (library a.m.FAP4B8) (see SEQ ID NOs: 71 and 73 of Table 3) and (iii) cross-reactive clones 7A1, 13B2, 13C2, 13E8, 14C10 and 17A11 (library a.m.FAPpool) (see SEQ ID NOs: 75 and 77 of Table 3 corresponding to the variable region sequences of 7A1; SEQ ID NOs: 79 and 81 of Table 3 corresponding to the variable region sequences of 13C2; SEQ ID NOs: 83 and 85 corresponding to the variable region sequences of 13E8; SEQ ID NOs: 87 and 89 corresponding to the variable region sequences of 14C10; and SEQ ID NOs: 91 and 93 corresponding to the variable region sequences of 17A11).

0194Each of these libraries consists of two sublibraries, randomized in either CDR1 and CDR2 of the light chain (L1/L2) or CDR1 and CDR2 of the heavy chain (H1/H2), respectively. These sublibraries were pooled upon transformation. Each of these sublibraries was constructed by four subsequent steps of amplification and assembly.

0195For L1/L2 libraries, the amplification and assembly protocol included: (i) amplification of fragment 1 (LMB3 - DPK22_CDR1_rand_ba_opt) and fragment 2 (DPK22_CDR1_fo - DPK22_Ck_BsiWI_ba); (ii) assembly of fragments 1 and 2 using outer primers LMB3 and DPK22_Ck_BsiWI_ba to create the template for fragment 3; (iii) amplification of fragment 3 (LMB3 - DPK22_CDR2_rand_ba) and fragment 4 (DPK22_CDR2_fo - DPK22_Ck_BaiWI_ba); and (iv) final assembly of fragments 3 and 4 using the same outer primers as above. See Table 11 for primer sequences. <tables id="tabl0011" num="0011"><table frame="all"><title>TABLE 11.</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="46mm" /><colspec colnum="2" colname="col2" colwidth="121mm" /><thead><row><entry namest="col1" nameend="col2" align="center" valign="top">Primers Used in L1/L2 Affinity Maturation Libraries for anti-FAP Affinity Maturation</entry></row></thead><tbody><row><entry>LMB3</entry><entry>CAGGAAACAGCTATGACCATGATTAC</entry></row><row><entry>DPK22_CDR1_rand_ba_opt</entry><entry><img file="EP2467165B1_D0335.tif" /></entry></row><row><entry>DPK22_CDR1_fo</entry><entry>TTAGCCTGGTACCAGCAGAAACCTG</entry></row><row><entry>DPK22_Ck_BsiWI_ba</entry><entry>GGTGCAGCCACCGTACGTTTGATTTCC</entry></row><row><entry>DPK22_CDR2_rand_ba</entry><entry><img file="EP2467165B1_D0336.tif" /></entry></row><row><entry>DPK22_CDR2_fo</entry><entry>AGGGCCACTGGCATCCCAGACAG</entry></row></tbody></tgroup><tgroup cols="2" rowsep="0"><colspec colnum="1" colname="col1" colwidth="46mm" /><colspec colnum="2" colname="col2" colwidth="121mm" /><tbody><row><entry namest="col1" nameend="col2" align="justify">Bold: 60% original base and 40% randomization as M Underline: 60% original base and 40% randomization as N</entry></row></tbody></tgroup></table></tables>

0196For H1/H2 libraries, the amplification and assembly protocol included: (i) amplification of fragment 1 (RJH53 - DP47_CDR1_rand_ba_opt) and fragment 2 (DP47_CDR1_fo - MS52); (ii) assembly of fragments 1 and 2 using outer primers RJH53 and MS52 to create the template for fragment 3; (iii) amplification of fragment 3 (RJH53 - DP47_CDR2_rand_ba) and fragment 4 (DP47_CDR2_fo - MS52); and (iv) final assembly of fragments 3 and 4 using the same outer primers as above. See Table 12 for primer sequences. <tables id="tabl0012" num="0012"><table frame="all"><title>TABLE 12.</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="44mm" /><colspec colnum="2" colname="col2" colwidth="121mm" /><thead><row><entry namest="col1" nameend="col2" align="center" valign="top">Primers Used in H1/H2 Affinity Maturation Libraries for anti-FAP Affinity Maturation</entry></row></thead><tbody><row><entry>RJH53</entry><entry>CATCAGGGCCTGAGCTCGCCCGTCAC</entry></row><row><entry>DP47_CDR1_rand_ba_opt</entry><entry><img file="EP2467165B1_D0337.tif" /></entry></row><row><entry>DP47_CDR1_fo</entry><entry>ATGAGCTGGGTCCGCCAGGCTC</entry></row><row><entry>MS52</entry><entry>GAAGACCGATGGGCCTTTGGTGCTAG</entry></row><row><entry>DP47_CDR2_rand_ba</entry><entry><img file="EP2467165B1_D0338.tif" /></entry></row><row><entry>DP47_CDR2_fo</entry><entry>ACATACTACGCAGACTCCGTGAAGG</entry></row></tbody></tgroup><tgroup cols="2" rowsep="0"><colspec colnum="1" colname="col1" colwidth="44mm" /><colspec colnum="2" colname="col2" colwidth="121mm" /><tbody><row><entry namest="col1" nameend="col2" align="justify">Bold: 60% original base and 40% randomization as M Underline: 60% original base and 40% randomization as N</entry></row></tbody></tgroup></table></tables>

0197Final assembly products have been digested with NcoI / BsiWI for L1/L2 sublibraries of a.m.FAP2D9 and a.m.FAP4B8, with MunI and NheI for H1/H2 sublibraries of a.m.FAP2D9 and a.m.FAP4B8 as well as with NcoI / BamHI for L1/L2 library of a.m.FAPpool and with BspEI/PstI for H1/H2 libraries of a.m.FAPpool, respectively, alongside with similarly treated acceptor vectors based on plasmid preparations of clones 2D9, 4B8 or an equimolar mixture of clones 7A1, 13B2, 13C2, 13E8, 14C10 and 17A11, respectively. The following amounts of digested randomized (partial) V-domains and digested acceptor vector(s) were ligated for the respective libraries (µg V-domain/µg vector): a.m.FAP2D9 L1/L2 sublibrary (5.7/21.5), a.m.FAP2D9 H1/H2 sublibrary (4.1/15.5), a.m.FAP4B8 L1/L2 sublibrary (6.5/24.5), a.m.FAP4B8 H1/H2 sublibrary (5.7/21.5), a.m.FAPpool L1/L2 sublibrary (4.4/20), a.m.FAPpool H1/H2 sublibrary (3.4/15.5).

0198Purified ligations of L1/L2 and H1/H2 sublibraries were pooled and used for 60 transformations for each of the 3 affinity maturation libraries, to obtain final library sizes of 6.2 × 109 for a.m.FAP2D9, 9.9 × 109 for a.m.FAP4B8 and 2.2 × 109 for a.m.FAPpool.

0199Phagemid particles displaying these Fab libraries were rescued and purified by PEG/NaCl purification to be used for secondary selections.

Example 7

Selection of Affinity-Matured Anti-FAP Clones

0200Selections were carried out against the ectodomain of human or murine fibroblast activating protein (FAP) which were cloned 5' of a poly-lysine and a 6xhis-tag. See SEQ ID NOs: 53 and 55 of Table 5. Prior to selections, the antigens were coated into immunotubes at a concentration of either 10 µg/mL, 5 µg/mL or 0.2 µg/mL, depending on the library and round of selection. Selections were carried out according to the following protocol: (i) binding of ~ 1012 phagemid particles of library a.m.FAP2D9, a.m.FAP4B8 or a.m.FAPpool to immobilized human or murine FAP for 2 hours; (ii) washing of immuno tubes using 10 - 20 × 5 mL PBS/Tween20 and 10 - 20 × 5 mL PBS (depending on library and selection round); (iii) elution of phage particles by addition of 1 mL 100m M TEA (triethylamine) for 10 minutes and neutralization by addition of 500 µL 1M Tris/HCl pH 7.4; and (iv) re-infection of log-phase E. coli TG1 cells, infection with helperphage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles to be used in subsequent selection rounds.

0201Selections were carried out over 2 rounds and conditions were adjusted for each of the 3 libraries individually. In detail, selection parameters were: a.m.FAP2D9 (5 µg /mL human FAP and 20 washes in total for round 1, 1 µg/mL human FAP and 30 washes in total for round 2), a.m.FAP4B8 (1 µg/mL murine FAP and 30 washes in total for round 1, 0.2 µg/mL human FAP and 40 washes in total for round 2) and a.m.FAPpool (5 µg/mL human FAP and 30 washes in total for round 1, 5 µg/mL murine FAP and 30 washes in total for round 2). Specific binders were defined as signals 5 × higher than background and were identified by ELISA. NUNC maxisorp plates were coated with 1 µg/mL or 0.2 µg/mL of human or murine FAP followed by addition of Fab-containing bacterial supernatants and detection of specifically binding Fabs via their Flag-tags by using an anti-Flag/HRP secondary antibody.

0202ELISA-positive clones were bacterially expressed as 1ml cultures in 96-well format and supernatants were subjected to a kinetic screening experiment using BIACORE T100.

Example 8

Efficacy Studies of Different Formats of Targeted IL-2

0203An efficacy experiment was performed using two different Interleukin-2 immunoconjugate molecular formats specific for tumor stroma. The F9 teratocarcinoma was subcutaneously injected into 129SvEv mice, and tumor size was measured using a caliper. The "diabody"-IL-2 molecule was compared at two different concentrations to the Fab-interleukin-2-Fab (Fab-IL2-Fab) immunoconjugate, wherein the concentrations reflected similar numbers of immunoconjugate molecules. Results are shown in <figref idref="f0003">Figure 3</figref>. The Fab-IL2-Fab immunoconjugate shows a significant tumor growth inhibition and is better than the diabody format at two different concentrations and better than controls.

0204Survival of mice treated with two different Interleukin-2 immunoconjugate molecular formats specific for tumor stroma was also examined. Human gastric tumor cell-line LS174T was intrasplenically injected into SCID-beige mice. The "diabody"-IL-2 molecule was compared at two different concentrations to the Fab-IL-2-Fab immunoconjugate, wherein the concentrations reflected similar numbers of immunoconjugate molecules. Results are shown in <figref idref="f0004">Figure 4</figref>. The Fab-IL-2-ab format resulted in a higher percent survival compared to the diabody format and controls.

Example 9

Recombinant DNA Techniques

0205Standard methods were used to manipulate DNA as described in <nplcit id="ncit0041" npl-type="b"><text>Sambrook, J. etal., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989</text></nplcit>. The molecular biological reagents were used according to the manufacturer's instructions.

0206General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: <nplcit id="ncit0042" npl-type="b"><text>Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIH Publication No 91-3242</text></nplcit>.

DNA Sequencing

0207DNA sequences were determined by double strand sequencing.

Gene Synthesis

0208Desired gene segments were prepared by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. The gene segments which are flanked by singular restriction endonuclease cleavage sites were cloned into pGA18 (ampR) plasmids. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. Gene Segments were designed with suitable restriction sites to allow sub-cloning into the respective expression vectors. All constructs were designed with a 5'-end DNA sequence coding for a leader peptide which targets proteins for secretion in eukaryotic cells. Table 13 and 14 give exemplary leader peptides and polynucleotide sequences encoding them, respectively. <tables id="tabl0013" num="0013"><table frame="all"><title>TABLE 13.</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="63mm" /><colspec colnum="2" colname="col2" colwidth="23mm" /><thead><row><entry namest="col1" nameend="col2" align="center" valign="top">Leader Sequences for Secretion: Polypeptide Sequences.</entry></row><row><entry valign="top">Polypeptide Sequence</entry><entry align="center" valign="top">SEQ ID NO</entry></row></thead><tbody><row><entry>MDWTWRILFLVAAATGAHS</entry><entry align="center">273</entry></row><row><entry>MDMRVPAQLLGLLLLWFPGARC</entry><entry align="center">276</entry></row><row><entry>MGWSCIILFLVATATGVHS</entry><entry align="center">278</entry></row></tbody></tgroup></table></tables><tables id="tabl0014" num="0014"><table frame="all"><title>TABLE 14. Leader Sequences for Secretion: Polynucleotide Sequences.</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="124mm" /><colspec colnum="2" colname="col2" colwidth="21mm" /><thead><row><entry valign="top">Polynucleotide Sequence</entry><entry align="center" valign="top">SEQ ID NO</entry></row></thead><tbody><row><entry><img file="EP2467165B1_D0339.tif" /></entry><entry align="center">274</entry></row><row><entry><img file="EP2467165B1_D0340.tif" /></entry><entry align="center">275</entry></row><row><entry><img file="EP2467165B1_D0341.tif" /></entry><entry align="center">277</entry></row><row><entry><img file="EP2467165B1_D0342.tif" /></entry><entry align="center">279</entry></row><row><entry><img file="EP2467165B1_D0343.tif" /></entry><entry align="center">280</entry></row><row><entry><img file="EP2467165B1_D0344.tif" /></entry><entry align="center">281</entry></row></tbody></tgroup></table></tables>

Preparation of Immunoconjugates

0209The resulting DNA sequences were subcloned into mammalian expression vectors (one for the light chain and one for the heavy chain/fusion protein) under the control of the MPSV promoter and upstream of a synthetic polyA site, each vector carrying an EBV OriP sequence. Immunoconjugates as applied in the examples below were produced by co-transfecting exponentially growing HEK293-EBNA cells with the mammalian expression vectors using a calcium phosphate-transfection. Alternatively, HEK293 cells growing in suspension were transfected by polyethylenimine (PEI) with the expression vectors, or stably transfected CHO cell pools were used. While 3F2 and 4G8 based FAP-targeted Fab-IL2-Fab constructs can be purified by affinity chromatography using a protein A matrix, 2B10 based TNC A2-targeted Fab-IL2-Fab constructs have to be purified by affinity chromatography on a protein G matrix. Briefly, TNC A2-targeted 2B10 Fab-IL2-Fab was purified from supernatants by one affinity step (protein G) followed by size exclusion chromatography (Superdex 200, GE Healthcare). The protein G column was equilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5, supernatant was loaded, and the column was washed with 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. Fab-IL2-Fab was eluted with 8.8 mM formic acid pH 3. The eluted fractions were pooled and polished by size exclusion chromatography in the final formulation buffer: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7. <figref idref="f0055">Figure 53</figref> shows the exemplary results from purification and analytics.

0210FAP-targeted 3F2 Fab-IL2-Fab or 4G8 Fab-IL2-Fab were purified by a similar method composed of one affinity step (protein A) followed by size exclusion chromatography (Superdex 200, GE Healthcare). The protein A column was equilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5, supernatant was loaded, and the column was washed with 20 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 7.5, followed by a wash with 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45. A third wash with 10 mM MES, 50 mM sodium chloride pH 5 was optionally performed. Fab-IL2-Fab was eluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3. The eluted fractions were pooled and polished by size exclusion chromatography in the final formulation buffer: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7.

Example 10

Construction of Additional Anti-FAP Affinity Maturation Libraries (based on clones 3F2,3D9,4G8,4B3 and 2C6)

0211Four additional affinity maturation libraries were constructed on the basis of pre-selected cross-reactive antibodies from the first affinity-maturation campaign of anti-FAP antibodies, namely clones 3F2, 3D9, 4G8, 4B3 and 2C6 (see SEQ ID NOs: 17 and 21 of Table 3 corresponding to the variable region sequences of 3F2; SEQ ID NOs: 23 and 25 of Table 3 corresponding to the variable region sequences of 3D9; SEQ ID NOs: 33 and 35 of Table 3 corresponding to the variable region sequences of 4B3; SEQ ID NOs: 41 and 43 of Table 3 corresponding to the variable region sequences of 2C6). More precisely, the four libraries were based on 1) anti-FAP clones 3F2, 4G8 and 4B3 (VH library, randomized in CDRs 1 and 2 of variable heavy chain, i.e. H1/H2 library), 2) anti-FAP clones 3D9 and 2C6 (VL library, randomized in CDRs 1 and 2 of variable light chain, i.e. L1/L2 library), 3) anti-FAP clone 3F2 (L3 library with soft randomization in CDR3 of light chain, i.e. L3 library) and 4) anti-FAP clone 3F2 (H3 library with soft randomization in CDR3 of heavy chain, i.e. H3 library). The first two libraries were constructed exactly the same way as outlined for the first affinity-maturation campaign of anti-FAP antibodies, for the L1/L2 and H1/H2 libraries, respectively. In contrast, for the L3 and H3 affinity-maturation libraries based on clone 3F2, two new primers were used to introduce soft randomization in L3 (AM_3F2_DPK22_L3_ba: CACTTTGGTCCCCTGGCCGAACGT CGGGGGAAGCA TAATACCCTGCTGACAGTAATACACTGC with underlined bases being 60% given base and 40% mixture N (mixture of the four nucleotides A, C, G, and T)) and H3 (AM_3F2_DP47_H3_fo: GGCCGTATATTACTGTGCG AAA GGG TGG TTTGGT GGT TTT AAC TACTGGGGCCAAGGAAC with underlined bases being 60% given base and 40% mixture N, bases in italics being 60% given base and 40% G, as well as underlined bases in italics being 60% given base and 40% mixture K (mixture of the two nucleotides G and T)) of the parental clone. Library sizes were as follows: H1/H2 library (1.13 × 1010), L1/L2 library (5.6 × 109), L3 library (2.3 × 1010) and H3 library (2.64 × 1010).

Example 11

Selection of Affinity-Matured Anti-FAP Clones

0212Selections were carried out against the ectodomain of human and murine fibroblast activating protein (FAP) which were cloned upstream a 6x-lysine and a 6x-his tag (see SEQ ID NOs: 53 and 55 of Table 5). Prior to selections, the antigens were coated into immunotubes at a concentration of either 1 µg/ml, 0.2 µg/ml or 0.02 µg/ml, depending on the library and round of selection. Selections and ELISA-based screenings were carried out as described for the first affinity-maturation campaign of anti-FAP antibodies. Secondary screenings were carried out using a ProteOn XPR36 biosensor (Biorad), and kinetic rate constants and affinities were determined analyzing affinity-purified Fab preparations on the same instrument. The following affinity-matured clones were identified: 19G1 (see SEQ ID NOs: 121 and 123 of Table 3), 20G8 (see SEQ ID NOs: 125 and 127 of Table 3), 4B9 (see SEQ ID NOs: 129 and 131 of Table 3), 5B8 (see SEQ ID NOs: 133 and 135 of Table 3), 5F1 (see SEQ ID NOs: 137 and 139 of Table 3), 14B3 (see SEQ ID NOs: 141 and 143 of Table 3), 16F1 (see SEQ ID NOs: 145 and 147 of Table 3), 16F8 (see SEQ ID NOs: 149 and 151 of Table 3), O3C9 (see SEQ ID NOs: 153 and 155 of Table 3), 22A3 (see SEQ ID NOs: 165 and 167 of Table 3) and 29B11 (see SEQ ID NOs: 169 and 171 of Table 3) (all these clones were selected from the H1/H2 library and are derived from parental clone 3F2), O2D7 (see SEQ ID NOs: 157 and 159 of Table 3) (selected from the L3 library based on parental clone 3F2), and 28H1 (see SEQ ID NOs: 161 and 163 of Table 3) and 23C10 (see SEQ ID NOs: 173 and 175 of Table 3) (these two clones were selected from the H1/H2 library and are derived from parental clone 4G8).

0213<figref idref="f0022 f0023 f0024 f0025 f0026">Figures 21-25</figref> show the Surface Plasmon Resonance sensorgrams of the selected affinity matured Fabs against FAP and Table 15 gives the respective affinities. The selected Fabs span a high affinity range in the pM to nM range and are cross-reactive for human (hu) and murine (mu) FAP, as well as Cynomolgus (cyno) FAP as determined for selected clones. The affinity matured anti-FAP Fabs were converted into the Fab-IL2-Fab format. Specificity of binding was shown by lack of binding to DPPIV as close homologue of FAP, expressed on HEK293 or CHO cells. <tables id="tabl0015" num="0015"><table frame="all"><title>TABLE 15.</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="43mm" /><colspec colnum="2" colname="col2" colwidth="40mm" /><colspec colnum="3" colname="col3" colwidth="41mm" /><colspec colnum="4" colname="col4" colwidth="43mm" /><thead><row><entry namest="col1" nameend="col4" align="center" valign="top">Summary of kinetic equilibrium constants (KD) of affinity-matured anti-FAP antibodies as Fab fragments (monovalent binding).</entry></row><row><entry align="center" valign="top">antibody</entry><entry align="center" valign="top">affinity (KD) to hu FAP [pM]</entry><entry align="center" valign="top">affinity (KD) to mu FAP [pM]</entry><entry align="center" valign="top">affinity (KD) to cyno FAP [pM]</entry></row></thead><tbody><row><entry align="center">19G1</entry><entry align="center">76</entry><entry align="center">2600</entry><entry align="center">n.d.</entry></row><row><entry align="center">20G8</entry><entry align="center">69</entry><entry align="center">2800</entry><entry align="center">n.d.</entry></row><row><entry align="center">4B9</entry><entry align="center">157</entry><entry align="center">3300</entry><entry align="center">n.d.</entry></row><row><entry align="center">5B8</entry><entry align="center">690</entry><entry align="center">3200</entry><entry align="center">n.d.</entry></row><row><entry align="center">5F1</entry><entry align="center">243</entry><entry align="center">4100</entry><entry align="center">n.d.</entry></row><row><entry align="center">14B3</entry><entry align="center">377</entry><entry align="center">3800</entry><entry align="center">n.d.</entry></row><row><entry align="center">16F1</entry><entry align="center">193</entry><entry align="center">3400</entry><entry align="center">n.d.</entry></row><row><entry align="center">16F8</entry><entry align="center">301</entry><entry align="center">3800</entry><entry align="center">n.d.</entry></row><row><entry align="center">O3C9</entry><entry align="center">160</entry><entry align="center">3700</entry><entry align="center">n.d.</entry></row><row><entry align="center">O2D7</entry><entry align="center">619</entry><entry align="center">8300</entry><entry align="center">n.d.</entry></row><row><entry align="center">28H1</entry><entry align="center">200</entry><entry align="center">9</entry><entry align="center">3600</entry></row><row><entry align="center">22A3</entry><entry align="center">34</entry><entry align="center">655</entry><entry align="center">522</entry></row><row><entry align="center">29B11</entry><entry align="center">35</entry><entry align="center">436</entry><entry align="center">23</entry></row><row><entry align="center">23C10</entry><entry align="center">1600</entry><entry align="center">125</entry><entry align="center">990</entry></row></tbody></tgroup></table></tables>

Example 12

Construction of Anti-TNC A2 Affinity Maturation Libraries (based on clone 2B10)

0214An affinity maturation library was constructed on the basis of a pre-selected antibody from the primary TNC A2 selections. More precisely, it was based on parental clone 2B10 and consisted of two sub-libraries: 1) VL sub-library, randomized in CDR1 and CDR2 of the light chain (L1/L2) and 2) VH sub-library, randomized in CDR1 and CDR2 of the heavy chain (H1/H2). These sub-libraries were pooled upon transformation. Each of these sub-libraries was constructed by four subsequent steps of amplification and assembly. For L1/L2 libraries: 1) amplification of fragment 1 (LMB3 - AM_Vk1A30_L1_ba) and fragment 2 (RJH50 (Vk1A30_L1/L2_fo) - RJH51 (Vk1A30_BsiWI_ba)), 2) assembly of fragments 1 and 2 using outer primers LMB3 and RJH51 (Vk1A30_BsiWI_ba) to create the template for 3) amplification of fragment 3 (LMB3 - AM_Vk1A30_L2_ba) and fragment 4 (RJH52 (Vk1A30_L2/L3) - RJH51 (Vk1A30_BsiWI_ba)) and 4) final assembly of fragments 3 and 4 using the same outer primers as above. For H1/H2 libraries: 1) amplification of fragment 1 (RJH53 - AM_DP88_H1_ba_opt) and fragment 2 (RJH54(DP88_H1/H2_fo) - MS52), 2) assembly of fragments 1 and 2 using outer primers RJH53 and MS52 to create the template for 3) amplification of fragment 3 (RJH53 - AM_DP88_H2_ba) and fragment 4 (RJH55 (DP88_H2H3_fo) - MS52) and 4) final assembly of fragments 3 and 4 using the same outer primers as above. Final assembly products have been digested NcoI / BsiWI for VL sub-libraries and MunI and NheI for VH sub-libraries and were cloned in similarly digested acceptor vectors. Library size resulted in 1.16 × 1010 independent clones. <tables id="tabl0016" num="0016"><table frame="all"><title>TABLE 16.</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="44mm" /><colspec colnum="2" colname="col2" colwidth="122mm" /><thead><row><entry namest="col1" nameend="col2" align="center" valign="top">Primers used in L1/L2 Affinity Maturation Libraries for Anti-TNC A2 binder 2B10</entry></row></thead><tbody><row><entry>LMB3</entry><entry>CAGGAAACAGCTATGACCATGATTAC</entry></row><row><entry>AM_Vk1A30_L1_ba</entry><entry><img file="EP2467165B1_D0345.tif" /></entry></row><row><entry>RJH50(Vk1A30_L1/L2_fo)</entry><entry>GCTGGTACCAGCAGAAGCCAGGGAAAG</entry></row><row><entry>RJH51(Vk1A30_BsiWI_ba)</entry><entry>GGTGCAGCCACCGTACGCTTGATCTC</entry></row><row><entry>AM_Vk1A30_L2_ba</entry><entry><img file="EP2467165B1_D0346.tif" /></entry></row><row><entry>RJH52(Vk1A30_L21L3)</entry><entry>TTGCAGAGTGGCGTCCCATCAAGGTTC</entry></row></tbody></tgroup><tgroup cols="2" rowsep="0"><colspec colnum="1" colname="col1" colwidth="44mm" /><colspec colnum="2" colname="col2" colwidth="122mm" /><tbody><row><entry namest="col1" nameend="col2" align="justify">Underline: 60% original base and 40% randomization as V Bold: 60% original base and 40% randomization as N</entry></row></tbody></tgroup></table></tables><tables id="tabl0017" num="0017"><table frame="all"><title>TABLE 17.</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="41mm" /><colspec colnum="2" colname="col2" colwidth="120mm" /><thead><row><entry namest="col1" nameend="col2" align="left" valign="top">Primers used in H1/H2 Affinity Maturation Libraries for Anti-TNC A2 binder 2B10</entry></row></thead><tbody><row><entry>RJH53</entry><entry>CATCAGGGCCTGAGCTCGCCCGTCAC</entry></row><row><entry>AM_DP88_H1_ba_opt</entry><entry><img file="EP2467165B1_D0347.tif" /></entry></row><row><entry>RJH54(DP88_H1/H2_fo)</entry><entry>ATAAGCTGGGTGCGACAGGCCCCTGGAC</entry></row><row><entry>MS52</entry><entry>GAAGACCGATGGGCCTTTGGTGCTAG</entry></row><row><entry>AM_DP88_H2_ba</entry><entry><img file="EP2467165B1_D0348.tif" /></entry></row><row><entry>RJH55 (DP88_H2H3_fo)</entry><entry>TACGCACAGAAGTTCCAGGGCAGGGTCAC</entry></row></tbody></tgroup><tgroup cols="2" rowsep="0"><colspec colnum="1" colname="col1" colwidth="41mm" /><colspec colnum="2" colname="col2" colwidth="120mm" /><tbody><row><entry namest="col1" nameend="col2" align="justify">Underligned: 60% original base and 40% randomization as V Bold: 60% original base and 40% randomization as N</entry></row></tbody></tgroup></table></tables>

Example 13

Selection of Affinity-Matured Anti-TNC A2 Clones

0215Selections were carried out against E. coli expressed human TNC A2 which was cloned upstream an avi-tag and 6xhis-tag (see SEQ ID NO: 57 of Table 5). The antigen was biotinylated in vivo upon expression. Selections have been carried out in solution as described for the primary TNC A2 selections using decreasing concentrations of human TNC A2 ranging from 100 to 2 nM. After identification of affinity-matured clones by ELISA, secondary screenings were carried out using a ProteOn XPR36 biosensor (Biorad) and kinetic rate constants and affinities were determined analyzing affinity-purified Fab preparations on the same instrument. The following affinity-matured clones were identified: 2B10_O1F7 (see SEQ ID NOs: 201 and 203 of Table 3), 2B10_6H10 (see SEQ ID NOs: 205 and 207 of Table 3), 2B10_C3A6 (see SEQ ID NOs: 185 and 187 of Table 3), 2B10_D1A2 (see SEQ ID NOs: 189 and 191 of Table 3), and 2B10_O7D8 (see SEQ ID NOs: 197 and 199 of Table 3) (all of these are derived from the VL sub-library), as well as 2B10_C3B6 (see SEQ ID NOs: 177 and 179 of Table 3) and 2B10_6A12 (see SEQ ID NOs: 181 and 183 of Table 3) (these two clones are derived from the VH sub-library). Moreover, for clone 2B10_D1A2, a V32D mutant was generated (see SEQ ID NOs: 193 and 195 of Table 3) (numbering according to Kabat).

0216<figref idref="f0027">Figure 26</figref> shows the Surface Plasmon Resonance sensorgrams of the selected affinity matured Fabs against TNC A2 and Table 18 gives the respective affinities. The selected Fabs span a high affinity range in the pM range. <tables id="tabl0018" num="0018"><table frame="all"><title>TABLE 18.</title><tgroup cols="2"><colspec colnum="1" colname="col1" colwidth="86mm" /><colspec colnum="2" colname="col2" colwidth="80mm" /><thead><row><entry namest="col1" nameend="col2" align="center" valign="top">Summary of kinetic equilibrium constants (KD) of affinity-matured anti-TNC A2 antibodies as Fab fragments (monovalent binding).</entry></row><row><entry align="center" valign="top">antibody</entry><entry align="center" valign="top">affinity (KD) to hu TNC A2 [pM]</entry></row></thead><tbody><row><entry align="center">2B10_C3B6</entry><entry align="center">191</entry></row><row><entry align="center">2B10_6A12</entry><entry align="center">290</entry></row><row><entry align="center">2B10_C3A6</entry><entry align="center">497</entry></row><row><entry align="center">2B10_O7D8</entry><entry align="center">147</entry></row><row><entry align="center">2B10_O1F7</entry><entry align="center">56</entry></row><row><entry align="center">2B10_6H10</entry><entry align="center">810</entry></row></tbody></tgroup></table></tables>

Example 14

Purification of 2B10, 3F2 and 4G8-based Fab-IL2-Fab Constructs

0217Another purification method (in addition to the one described in Example 9) was developed for 2B10, 3F2 and 4G8 based Fab-IL2-Fab constructs. While 3F2 and 4G8 based Fab-IL2-Fab constructs can be purified by affinity chromatography using a protein A matrix (e.g. MabSelect Sure), 2B10 based Fab-IL2-Fab constructs have to be purified by affinity chromatography on a protein G matrix. The purification procedure is based on the following four steps: <ol id="ol0002" compact="compact" ol-style=""><li>1. Affinity chromatography with MabSelect Sure or protein G</li><li>2. Low pH hold for retroviral inactivation</li><li>3. Anion exchange chromatography - CaptoQ chromatography, to remove DNA</li><li>4. Cation exchange chromatography - SP Sepharose FF chromatography, to remove aggregates</li></ol>

0218For removal of aggregates in small scale, size exclusion chromatography on a Superdex 200 column (GE Healthcare) can be alternatively used.

0219An example of the purification procedure is given subsequently for 3F2-based Fab-IL2-Fab. In a first step supernatant from transiently PEI-transfected HEK293 cells in Freestyle medium (Invitrogen) was adjusted to pH 7 and applied to a MabSelect protein A column (GE Healthcare), washed with 100 mM NaPO4, 250 mM NaCl pH 7 and eluted with 8.8 mM sodium formiate pH 3. Selected fractions were exchanged in wash buffer and applied to a CaptoQ column (GE Healthcare), washed with 10 mM NaPO4, 40 mM NaCl pH 6.5 and eluted with 2 M NaCl. The flowthrough was adjusted to pH 5 and applied to a SP Sepharose FF column (GE Healthcare), washed with 25 mM sodium acetate, 25 mM NaCl, pH 5 and eluted with 25 mM sodium acetate, 300 mM NaCl pH 5. Fractions were exchanged into final formulation buffer. <figref idref="f0028">Figure 27</figref> shows an overview of the purification procedure.

0220The purified 3F2-based Fab-IL2-Fab was pure after purification (<figref idref="f0029">Figure 28A</figref>) and contained no aggregates (<figref idref="f0029">Figure 28B</figref>). The described purification procedure was applied to 4G8-based Fab-IL2-Fab. 4G8-based Fab-IL2-Fab behaved similarly to the 3F2-based Fab-IL2-Fab. The purified material was pure after purification and contained no aggregates (<figref idref="f0029 f0030">Figure 28</figref>, C-D).

0221Purification was performed for the Fab-IL2-Fab format with 2B10 (TNC A2 binder) as Fab fragment. In a first step supernatant from transiently PEI-transfected HEK293 cells in Freestyle medium (Invitrogen) was adjusted to pH 7 and applied to a protein G column (GE Healthcare), washed with 100 mM NaPO4, 250 mM NaCl pH 7, and eluted with 8.8 mM Na formiate pH 3. Selected fractions were exchanged in wash buffer and applied to a CaptoQ column (GE Healthcare), washed with 10 mM NaPO4, 40 mM NaCl pH 6.5 and eluted with 2 M NaCl. The flowthrough was adjusted to pH 5 and applied to a SP Sepharose FF column (GE Healthcare), washed with 25 mM sodium acetate, 25 mM NaCl, pH 5 and eluted with 25 mM sodium acetate, 300 mM NaCl pH 5. Fractions were exchanged into final formulation buffer.

0222<figref idref="f0031">Figure 29</figref> shows the results from (A) the analytical characterization of the product by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer, reduced and non-reduced) and (B) analytical size exclusion chromatography of the product after each of the three purification steps. 2.3% aggregates were detected in the final product.

Example 15

Stability Testing

0223Stability testing was performed for the Fab-IL2-Fab format with the fibronectin Ectodomain-B binder L19 as Fab fragment. For stability tests the Fab-IL2-Fab construct was purified by protein A affinity chromatography with an elution step at pH 3, followed by size exclusion chromatography on a Superdex 200 column (GE Healthcare) as described. Three different buffers were tested and 20 mM histidine HCl, 140 mM NaCl, pH 6.0 was identified as a suitable buffer. Subsequently, L19 Fab-IL2-Fab was formulated in 20 mM HistidineHCl, 140 mM NaCl, pH 6.0 at a concentration of 6.3 mg/ml and stored for four weeks at room temperature and at 4°C. <figref idref="f0032">Figure 30</figref> shows exemplary stability data: Probes were analyzed every week for concentration by UV spectroscopy (<figref idref="f0032">Figure 30A</figref>) (after centrifugation to pellet potential precipitated material) and for aggregate content by analytical size exclusion chromatography on a Superdex 200 column (<figref idref="f0032">Figure 30B</figref>). The results show that no aggregation and no degradation occured, when the construct was stored at 4°C or at room temperature for 28 days as well as after a freeze/thaw cycle at 6 mg/ml concentration. These data show that the Fab-IL2-Fab format is highly stable and behaves comparably to IgG antibodies.

Example 16

FAP-targeted Fab-IL2-Fab Binding Affinities by Surface Plasmon Resonance (Biacore)

0224Binding affinities of the three FAP-targeted Fab-IL2-Fab constructs, 3F2 Fab-IL2-Fab, 4G8 Fab-IL2-Fab and 3D9 Fab-IL2-Fab, were determined by Surface Plasmon Resonance.

0225For determination of FAP binding, FAP was captured by an immobilized anti-His antibody (Penta His, Qiagen #34660) and the constructs were used as analytes. Temperature of analysis was 25°C and Fab-IL2-Fab constructs were diluted 1:5 from 10 nM to 3.2 pM. The following measurement parameters were applied: Association time 180 s, dissociation 900 s, flow 90 µl/min. The chip was regenerated with 10 mM glycine pH 2 for 60 s. The curves were fitted with the 1:1 model to get the KD values (Rmax local, RI=0).

0226To determine the affinity for IL-2 receptor (IL-2R) chains, the β and γ chains (b/g; knob-into-hole construct) or the α chain (a) of IL-2R were immoblized on the chip and the Fab-IL2-Fab constructs were used as analytes. Temperature of analysis was 25°C. Fab-IL2-Fab constructs 3F2 and 3D9 were diluted 1:2 from 25 nM to 0.78 nM and the following measurement parameters were applied: Association time 100 s, dissociation 180 s, flow 90 µl/min. Regeneration was done with 10 mM glycine pH 1.5 for 20 s. Fab-IL2-Fab constructs 4G8 were diluted from 100 nM to 3.125 nM and the following measurement parameters were applied: Association time 180 s, dissociation 180 s, flow 40 µl/min. Regeneration with 3 M MgCl2 for 30 s.

0227Table 19 gives a summary of binding affinities of the 3F2 Fab-IL2-Fab, 4G8 Fab-IL2-Fab and 3D9 Fab-IL2-Fab immunoconjugates. Picomolar values of affinity reach the limit of detection of the Biacore. <figref idref="f0033 f0034 f0035 f0036">Figures 31-34</figref> show the respective Biacore sensorgrams and affinities. <tables id="tabl0019" num="0019"><table frame="all"><title>TABLE 19.</title><tgroup cols="6"><colspec colnum="1" colname="col1" colwidth="26mm" /><colspec colnum="2" colname="col2" colwidth="20mm" /><colspec colnum="3" colname="col3" colwidth="20mm" /><colspec colnum="4" colname="col4" colwidth="27mm" /><colspec colnum="5" colname="col5" colwidth="41mm" /><colspec colnum="6" colname="col6" colwidth="35mm" /><thead><row><entry namest="col1" nameend="col6" align="center" valign="middle">Summary of kinetic equilibrium constants (KD) of FAP-targeted Fab-IL2-Fab constructs for FAP from different species and IL-2 receptor as determined by Surface Plasmon Resonance</entry></row><row><entry align="center" valign="middle">Construct</entry><entry align="center" valign="middle">Human FAP</entry><entry align="center" valign="middle">Murine FAP</entry><entry align="center" valign="middle">Cynomolgus FAP</entry><entry align="center" valign="middle">Affinity to IL-2 receptor (b/g) Receptor immobilized</entry><entry align="center" valign="middle">Affinity to IL-2 receptor (a) Receptor immobilized</entry></row></thead><tbody><row><entry align="center" valign="middle">Fab-IL2-Fab 3F2</entry><entry align="center" valign="middle">Avidity: 25 pM</entry><entry align="center" valign="middle">Avidity: 49 pM</entry><entry align="center" valign="middle">Avidity: 24 pM</entry><entry align="center" valign="middle">2.7 nM hu IL-2R bg</entry><entry align="center" valign="middle">ND</entry></row><row><entry align="center" valign="middle">Fab-IL2-Fab 4G8</entry><entry align="center" valign="middle">Avidity: 83 pM</entry><entry align="center" valign="middle">Avidity: 2.3 pM</entry><entry align="center" valign="middle">Avidity: 74 pM</entry><entry align="center" valign="middle">3.8 nM hu IL-2R bg 45.6 nM mu IL-2R bg</entry><entry align="center" valign="middle">4.5 nM hu IL-2R a 29 nM mu IL-2R a</entry></row><row><entry align="center" valign="middle">Fab-IL2-Fab 3D9</entry><entry align="center" valign="middle">Avidity: 96 pM</entry><entry align="center" valign="middle">Avidity: 63 pM</entry><entry align="center" valign="middle">Avidity: 105 pM</entry><entry align="center" valign="middle">2.7 nM hu IL-2R bg</entry><entry align="center" valign="middle">ND</entry></row></tbody></tgroup></table></tables>

Example 17

TNC A2-targeted Fab-IL2-Fab Binding Affinities by Surface Plasmon Resonance (Biacore)

0228For determination of TNC A2 binding the biotinylated antigens (TNC fn5-A1-A2-A3 domains, fused together, expressed in E.coli. While the fn5 and the A3 domains are always of human origin, A1 and A2 domains are human, murine or cynomolgus) were immobilized on a streptavidin chip and the immunoconjugate constructs were used as analytes. Temperature of analysis was 25°C. Fab-IL2-Fab were diluted 1:2 from 25 nM to 0.39 nM and the following measurement parameters were applied: Association time 180 s, dissociation 180 s, flow 50 µl/min. Regeneration with 10 mM glycine pH 1.5 for 60 s. The curves were fitted with the 1:1 model to get the KD values. As a negative control, TNC domains 1 to 8 produced in HEK cells were applied (TNC 1-8 HEK).

0229To determine the affinity for IL-2 receptor (IL-2R) β and γ chains (b/g; knob-into-hole construct) the IL-2R construct was immoblized on the chip and the Fab-IL2-Fab immunoconjugates were used as analytes. Temperature of analysis was 25°C. Fab-IL2-Fab immunoconjugates were diluted 1:2 from 25 nM to 0.78 nM and the following measurement parameters were applied: Association time 100 s, dissociation 180 s, flow 90 µl/min. Regeneration was done with 10 mM glycine pH 1.5 for 20 s. Fab-IL2-Fab constructs were diluted from 100 nM to 3.125 nM and the following measurement parameters were applied: Association time 180 s, dissociation 180 s, flow 40 µl/min. Regeneration with 3 M MgCl2 for 30 s. Table 20 gives a summary of binding affinities for the 2B10 Fab-IL2-Fab immunoconjugate, <figref idref="f0037">Figure 35</figref> shows the respective Biacore sensorgrams and affinities. <tables id="tabl0020" num="0020"><table frame="all"><title>TABLE 20.</title><tgroup cols="5"><colspec colnum="1" colname="col1" colwidth="34mm" /><colspec colnum="2" colname="col2" colwidth="27mm" /><colspec colnum="3" colname="col3" colwidth="27mm" /><colspec colnum="4" colname="col4" colwidth="35mm" /><colspec colnum="5" colname="col5" colwidth="46mm" /><thead><row><entry namest="col1" nameend="col5" align="center" valign="middle">Summary of kinetic equilibrium constants (KD) of TNCA2-targeted Fab-IL2-Fab constructs for TNC A2 from different species and IL-2 receptor-β/γ as determined by Surface Plasmon Resonance</entry></row><row><entry align="center" valign="middle">Construct</entry><entry align="center" valign="middle">Human TNC</entry><entry align="center" valign="middle">Murine TNC</entry><entry align="center" valign="middle">Cynomolgus TNC</entry><entry align="center" valign="middle">Affinity to hu IL-2 receptor (β/γ) Receptor immobilized</entry></row></thead><tbody><row rowsep="0"><entry align="center" valign="middle">Fab-IL2-Fab 2B10 Immob. E. coli Antigen</entry><entry align="center" valign="middle">Avidity: 0.12 nM</entry><entry align="center" valign="middle">Avidity: 0.77 nM</entry><entry align="center" valign="middle">Avidity: 0.12 nM</entry><entry align="center" valign="middle">2.7 nM</entry></row><row><entry align="center" valign="middle">TNC 1-8 HEK (neg. cont)</entry><entry align="center" valign="middle">no binding</entry><entry align="center" valign="middle" /><entry align="center" valign="middle" /><entry align="center" valign="middle" /></row></tbody></tgroup></table></tables>

Example 18

Biological Activity Assays with Targeted IL-2 Fab-IL2-Fab Immunoconjugates

0230The biological activity of targeted IL-2 Fab-IL2-Fab immunoconjugates was investigated in several cellular assays in comparison to IL-2 (Proleukin).

Induction of Proliferation of NK92 Cells

0231Targeted IL-2 Fab-IL2-Fab molecules recognizing TNC A2 (2B10) or FAP (3F2 and 4G8) were investigated for their potential to induce proliferation of NK92 cells in comparison to IL-2 (Proleukin) and the IL-2 L19 diabody recognizing Fibronectin-EDB.

02322 µg/ml of human Tenascin, FAP or Fibronectin were coated over night. at 4°C in a 96-well flat bottom ELISA plate. After blocking the plate, the Fab-IL2-Fab constructs or the diabody were titrated into the plate and incubated for 90 min at room temperature (RT) for binding. After intensive washing to remove the unbound constructs, IL-2 starved NK92 cells (10000 cells/well) were added. As positive control, Proleukin was added in solution to some of the wells. The cells were incubated for 2 days at 37°C in a humidified incubator (with 5 % CO2) before lysing the cells to determine proliferation by ATP measurement using the CellTiter Glo Kit (Promega). The results in <figref idref="f0038">Figure 36</figref> show that all Fab-IL2-Fab constructs were able to activate IL-2R signaling on NK92 cells and stimulate their proliferation. Due to the reduced binding affinity for the IL-2R β/γ heterodimer required for signaling, the potency of induction of cell growth was reduced by a factor of approximately 10 or more compared to IL-2 (Proleukin). However, the overall efficacy at higher doses was retained and comparable to IL-2 (Proleukin).

Induction of STAT5 Phosphorylation

0233In another experiment we tested the induction of STAT5 phosphorylation as a consequence of IL-2 mediated IL-2R signaling following incubation with an IL-2 Fab-IL2-Fab molecule recognizing FAP (based on 4G8) on different effector cell populations, including A) CD56+ NK cells, B) CD4+CD25-CD127+ helper T cells, C) CD3+, CD8+ cytotoxic T cells and D) CD4+CD25+FOXP3+ regulatory T cells (Tregs) from human PBMCs in solution.

0234PBMCs isolated from blood of healthy donors were treated for 20 min with different concentrations of Proleukin or Fab-IL2-Fab before fixing/permeabilizing them and staining them with anti-PhosphoSTAT5 antibody (Becton Dickinson) according to the instructions of the supplier. After intracellular staining of phosphorylated STAT5 as well as FOXP3, surface markers (CD3, CD4, CD8, CD56 and CD127) were stained for determination of the different subpopulations by flow cytometry (FACS Canto II).

0235The results in <figref idref="f0039">Figure 37</figref> confirm the findings from <figref idref="f0038">Figure 36</figref> and show that the 4G8 Fab-IL2-Fab construct was able to activate IL-2R signaling on various IL-2R positive effector cells and induce IL-2R downstream signaling and STAT5 phosphorylation. Due to the reduced binding affinity for the IL-2R β/γ heterodimer required for signaling, the potency of induction of STAT5 phosphorylation was reduced by a factor of approximately 10 or more compared to IL-2 (Proleukin). However, the overall efficacy at higher doses was retained and comparable to IL-2 (Proleukin).

IFN-

Release and Proliferation Induction in Solution and upon Immobilization

0236In another experiment we aimed to mimic the situation as it will occur in a tumor where the targeted IL-2 immunoconjugate is bound and immobilized on the tumor cells or tumor stroma and can activate effector cells. In order to do this, we performed the IFN-γ release assay with NK92 cells as well as a proliferation assay with the immunoconjugates in solution or we coated microtiter plates with TNC or FAP antigen so that the targeted IL-2 immunoconjugates are immobilized upon binding to TNC or FAP.

02372 µg/ml of human Tenascin or FAP were coated over night at 4°C in a 96-well flat bottom ELISA plate. After blocking the plate, the Fab-IL2-Fab constructs were titrated into the plate and incubated for 90 min at RT for binding. After intensive washing to remove the unbound constructs, IL-2 starved NK92 cells (10000 cells/well) were added. As positive control, Proleukin was added in solution to some of the wells. For determination of proliferation the cells were incubated for 2 days at 37°C in a humidified incubator (with 5 % CO2) before lysing the cells to determine proliferation by ATP measurement using the CellTiter Glo Kit (Promega). The release of IFN-γ was measured in a separate approach after 24 h of incubation with Fab-IL-2-Fab in the supernatant of the cells with the human IFN-γ ELISA Kit from Becton Dickinson.

0238The results confirmed that all Fab-IL2-Fab constructs targeting FAP, TNC A1 or TNC A2 were able to activate IL-2R signaling on NK92 cells and induce proliferation (<figref idref="f0040">Figure 38A</figref>) of the cells as well as IFN-γ secretion (<figref idref="f0040">Figure 38C</figref>) when present in solution. Due to the reduced binding affinity for the IL-2R β/γ heterodimer required for signaling the potency of induction of IFN-γ release was reduced by a factor of approximately 10 or more compared to IL-2 (Proleukin). However, the overall efficacy at higher doses was retained and comparable to IL-2 (Proleukin). If the microtiter plates were coated with FAP or TNC and the constructs immobilized on the plate, all Fab-IL2-Fab constructs targeting FAP, TNC A1 or TNC A2 were still able to activate IL-2R signaling on NK92 cells and induce cell growth (<figref idref="f0040">Figure 38B</figref>) and IFN-γ release (<figref idref="f0040">Figure 38D</figref>). In comparison to the assay performed in solution the difference of efficacy between uncoated IL-2 (Proleukin) and the immobilized Fab-IL2-Fab constructs was an order of magnitude higher, however, the overall efficacy at higher doses was retained and comparable to IL-2 (Proleukin).

0239These data strongly support the concept of generating targeted immunoconjugates with low systemic exposure, but accumulation at the site of disease where they mediate their function.

0240In the following example we investigated whether these in vitro properties translate into superior efficacy in vivo in xenograft models.

Example 19

In vivo Efficacy of Targeted Fab-IL2-Fab Immunoconjugates Against FAP and TNC A2 in Xenografts of Human Tumor Cell Lines

0241Targeted Fab-IL2-Fab immunoconjugates against FAP and TNC A2 were tested for their antitumoral efficacy in several xenograft models.

LS174T Xenograft Model

0242The TNC A2-targeted 2B10 Fab-IL2-Fab immunoconjugate was tested in the human colorectal LS174T cell line, intrasplenically injected into SCID mice.

0243LS174T cells (human colon carcinoma cells) were originally obtained from ECACC (European Collection of Cell Culture) and after expansion deposited in the Glycart internal cell bank. LS174T were cultured in MEM Eagle's medium containing 10% FCS (PAA Laboratories, Austria), 1% Glutamax and 1% MEM Non-Essential Amino Acids (Sigma). The cells were cultured at 37 °C in a water-saturated atmosphere at 5 % CO2. In vitro passage 15 was used for intrasplenic injection, at a viability of 92.8%. A small incision was made at the left abdominal site of anesthetized SCID/beige mice. Fifty microliters (3x106 LS174T cells in AimV medium) cell suspension was injected through the abdominal wall just under the capsule of the spleen. Skin wounds were closed using clamps.

0244Female SCID mice; aged 8-9 weeks at the start of the experiment (purchased from Taconics, Denmark) were maintained under specific-pathogen-free conditions with daily cycles of 12 h light / 12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 2008016). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.

0245Mice were injected intrasplenically on study day 0 with 3x106 LS174T cells, randomized and weighed. One week after the tumor cell injection mice were injected i.v. TNC A2-targeted 2B10 Fab-IL2-Fab (Fab-IL2-Fab-SH2B10), Fibronectin-EDB targeted L19 IL-2-diabody, or Proleukin twice weekly for three weeks.

0246All mice were injected i.v. with 200 µl of the appropriate solution. The mice in the vehicle group were injected with PBS and the treatment groups with the Fab-IL2-Fab construct, the diabody, or Proleukin. To obtain the proper amount of immunoconjugate per 200 µl, the stock solutions were diluted with PBS when necessary.

0247<figref idref="f0041">Figure 39</figref> shows that the TNC A2 targeted 2B10 Fab-IL2-Fab immunoconjugate mediated superior efficacy in terms of enhanced median survival compared to naked IL-2 (Proleukin) and the IL-2 diabody molecule targeting fibronectin-EDB. <tables id="tabl0021" num="0021"><table frame="all"><title>TABLE 21.</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="53mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="65mm" /><colspec colnum="4" colname="col4" colwidth="35mm" /><thead><row><entry valign="middle">Compound</entry><entry valign="middle">Dose</entry><entry valign="middle">Formulation buffer</entry><entry valign="middle">Concentration (mg/mL)</entry></row></thead><tbody><row><entry>L19 IL-2 diabody</entry><entry>12 µg</entry><entry>20 mM sodium citrate, 190 mM sucrose, 20 mM arginine, pH 6.5</entry><entry>2.00 (= stock solution)</entry></row><row><entry>Proleukin (IL-2)</entry><entry>4 µg</entry><entry>mannitol sodium laurylsulphate sodium phosphate</entry><entry>1.0 (= stock solution)</entry></row><row><entry>huTNC A2 2B10 (G65S) Fab-IL2-Fab = SH2B10</entry><entry>16 µg</entry><entry>25 mM potassium phosphate, 125 mM NaCl, 100 mM glycine, pH 6.7</entry><entry>1.86 (= stock solution)</entry></row></tbody></tgroup></table></tables>

ACHN Xenograft Model

0248The FAP-targeted 3F2 or 4G8 Fab-IL2-Fab immunoconjugates were tested in the human renal cell line ACHN, intrarenally injected into SCID mice.

0249ACHN cells (human renal adenocarcinoma cells) were originally obtained from ATCC (American Type Culture Collection) and after expansion deposited in the Glycart internal cell bank. ACHN were cultured in DMEM containing 10% FCS. The cells were cultured at 37°C in a water-saturated atmosphere at 5 % CO2. In vitro passage 9 was used for intrarenal injection, at a viability of 97.7%. A small incision (2 cm) was made at the right flank and peritoneal wall of anesthetized SCID mice. Fifty µl (1x106 ACHN cells in AimV medium) cell suspension was injected 2 mm subcapsularly in the kidney. Skin wounds and peritoneal wall were closed using clamps.

0250Female SCID mice; aged 8-9 weeks at the start of the experiment (purchased from Charles River, Sulzfeld, Germany) were maintained under specific-pathogen-free conditions with daily cycles of 12 h light / 12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 2008016). After arrival, animals were maintained for one week to get accustomed to new environment and for observation. Continuous health monitoring was carried out on a regular basis.

0251Mice were injected intrarenally on study day 0 with 1x06 ACHN cells, randomized and weighed. One week after the tumor cell injection, mice were injected i.v. with Proleukin, L19 IL-2 diabody or FAP-targeted 4G8 or 3F2 Fab-IL2-Fab immunoconjugates twice weekly for three weeks.

0252All mice were injected i.v. with 200 µl of the appropriate solution. The mice in the vehicle group were injected with PBS and the treatment groups with Proleukin, L19 IL-2 diabody or FAP-targeted 4G8 or 3F2 Fab-IL2-Fab immunoconjugates. To obtain the proper amount of immunoconjugate per 200 µl, the stock solutions were diluted with PBS when necessary.

0253<figref idref="f0042">Figure 40</figref> shows that the FAP targeted 3F2 and 4G8 Fab-IL2-Fab immunoconjugates mediated superior efficacy in terms of enhanced median survival compared to naked IL-2 (Proleukin) and the IL-2 diabody molecule targeting fibronectin-EDB. The 4G8-based Fab-IL2-Fab, which has a higher affinity for murine FAP, mediated superior efficacy than 3F2-based Fab-IL2-Fab. <tables id="tabl0022" num="0022"><table frame="all"><title>TABLE 22.</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="39mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="75mm" /><colspec colnum="4" colname="col4" colwidth="40mm" /><thead><row><entry valign="middle">Compound</entry><entry valign="middle">Dose</entry><entry valign="middle">Formulation buffer</entry><entry valign="middle">Concentration (mg/mL)</entry></row></thead><tbody><row><entry>L19 IL-2 diabody</entry><entry>12 µg</entry><entry>20 mM sodium citrate, 190 mM sucrose, 20 mM arginine, pH 6.5</entry><entry>2.00 (= stock solution)</entry></row><row><entry>Proleukin (IL2)</entry><entry>4 g</entry><entry>mannitol sodium laurylsulphate sodium phosphate</entry><entry>1.0 (= stock solution)</entry></row><row><entry>FAP 3F2 Fab-IL2-Fab = FAP 3F2</entry><entry>16 µg</entry><entry>25 mM potassium phosphate, 125 mM NaCl, 100 mM glycine, pH 6.7</entry><entry>2.46 (= stock solution)</entry></row><row><entry>FAP 4G8 Fab-IL2-Fab = FAP 4G8</entry><entry>16 µg</entry><entry>25 mM potassium phosphate, 125 mM NaCl, 100 mM glycine, pH 6.7</entry><entry>11.8 (= stock solution)</entry></row></tbody></tgroup></table></tables>

A549 Xenograft Model

0254The TNC A2-targeted 2B10 Fab-IL2-Fab immunoconjugate was tested in the human NSCLC cell line A549, injected i.v. into SCID-human FcγRIII transgenic mice.

0255The A549 non-small cell lung carcinoma cells were originally obtained from ATCC (CCL-185) and after expansion deposited in the Glycart internal cell bank. The tumor cell line was routinely cultured in DMEM containing 10 % FCS (Gibco) at 37 °C in a water-saturated atmosphere at 5% CO2. Passage 2 was used for transplantation, at a viability of 98%. 2x106 cells per animal were injected i.v. into the tail vein in 200 µl of Aim V cell culture medium (Gibco).

0256Female SCID-FcγRIIII mice (GLYCART-RCC), aged 8-9 weeks at the start of the experiment (bred at RCC, Switzerland) were maintained under specific-pathogen-free condition with daily cycles of 12 h light / 12 h darkness according to committed guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by local government (P 2008016). After arrival, animals were maintained for one week to get accustomed to the new environment and for observation. Continuous health monitoring was carried out on a regular basis.

0257Mice were injected i.v. on study day 0 with 5x106 of A549 cells, randomized and weighed. One week after the tumor cell injection, mice were injected i.v. with 2B10 Fab-IL2-Fab or L19 IL-2 diabody twice weekly for three weeks.

0258All mice were injected i.v. with 200 µl of the appropriate solution. The mice in the vehicle group were injected with PBS and the treatment group with the 2B10 Fab-IL2-Fab construct or the L19 IL-2 diabody. To obtain the proper amount of immunoconjugate per 200 µl, the stock solutions were diluted with PBS when necessary.

0259<figref idref="f0043">Figure 41</figref> shows that the TNC A2 targeted 2B10 Fab-IL2-Fab immunoconjugate mediated superior efficacy in terms of enhanced median survival compared to the IL-2 diabody molecule targeting fibronectin-EDB. <tables id="tabl0023" num="0023"><table frame="all"><title>TABLE 23.</title><tgroup cols="4"><colspec colnum="1" colname="col1" colwidth="51mm" /><colspec colnum="2" colname="col2" colwidth="14mm" /><colspec colnum="3" colname="col3" colwidth="67mm" /><colspec colnum="4" colname="col4" colwidth="36mm" /><thead><row><entry valign="top">Compound</entry><entry valign="top">Dose</entry><entry valign="top">Formulation buffer</entry><entry valign="top">Concentration (mg/mL)</entry></row></thead><tbody><row><entry>L19 IL-2 diabody</entry><entry>12 µg</entry><entry>20 mM sodium citrate, 190 mM sucrose, 20 mM arginine, pH 6.5</entry><entry>2.00 (= stock solution)</entry></row><row><entry>huTNC A2 2B10 (G65S) Fab-IL2-Fab = 2B10</entry><entry>16 µg</entry><entry>25 mM potassium phosphate, 125 mM NaCl, 100 mM glycine, pH 6.7</entry><entry>1.86 (= stock solution)</entry></row></tbody></tgroup></table></tables>

Example 20

Purification of Targeted GM-CSF Fab-GM-CSF-Fab Immunoconjugate

0260Initial purification of the Fab-GM-CSF-Fab immunoconjugate with L19 (Fibronectin Ectodomain-B binder) as Fab fragment was performed from supernatants of transiently transfected HEK 293 EBNA cells. Briefly, Fab-GM-CSF-Fab was purified by protein A followed by size exclusion chromatography. The Protein A column was equilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. The supernatant was loaded and the column washed first with 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5, followed by 20 mM sodium phosphate, 20 mM sodium citrate, 100 mM sodium chloride, pH 7.5. Targeted GM-CSF was eluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine pH 3 and subsequently neutralized. For formulation the following buffer was applied: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7.

0261<figref idref="f0044">Figure 42</figref> shows the elution profiles from the purification and the results from the analytical characterization of the product by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer, reduced and non-reduced). The yield was 4.8 mg/L.

Example 21

Biological Activity Assay with Targeted GM-CSF Fab-GM-CSF-Fab Immunoconjugate

0262The purified Fab-GM-CSF-Fab immunoconjugate with L19 (Fibronectin Ectodomain-B binder) as Fab was subsequently analyzed in a GM-CSF-dependent proliferation assay. Briefly, TF-1 cells, which grow GM-CSF-dependent, were seeded after over night starvation at 10000 cells/well into a 96-well flat bottom plate. Human recombinant GM-CSF (Miltenyi #130-093-862) or Fab-GM-CSF-Fab immunoconjugate were titrated onto the cells in solution. After 2 days of proliferation at 37°C in a humidified incubator with 5 % CO2, the cells were lysed and ATP content was measured with the CellTiter Glo assay from Promega. GM-CSF-untreated cells were set as 0% growth for calculation. Results in <figref idref="f0045">Figure 43</figref> show that the Fab-GM-CSF-Fab immunoconjugate induced strong proliferation of TF-1 cells.

Example 22

Purification of Targeted-IL-12 Fab-IL12-Fab Immunoconjugate

0263Initial purification of the Fab-IL12-Fab immunoconjugate with 4G8 (FAP binder) as Fab fragment was performed from supernatants of transiently transfected HEK 293 EBNA cells. Briefly, Fab-IL12-Fab was purified by protein A followed by size exclusion chromatography. The protein A column was equilibrated with 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. Supernatant was loaded and the column washed with 20 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride pH 7.5. A second wash was performed with 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45. After a third wash with 10 mM MES, 50 mM sodium chloride pH 5, targeted IL-12 was eluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3 and subsequently neutralized. For formulation the following buffer was applied: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7.

0264<figref idref="f0046">Figure 44</figref> shows the elution profiles from the purification and the results from the analytical characterization of the product by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer, reduced and non-reduced). The yield was 43 mg/L.

Example 23

Biological Activity Assay with Targeted-IL-12 Fab-IL12-Fab Immunoconjugate

0265The purified Fab-IL12-Fab immunoconjugate with 4G8 (FAP binder) as Fab was subsequently analyzed for IL-12 induced IFN-γ release, comparing the effect of IL-12 and the purified 4G8 Fab-IL12-Fab immunoconjugate, using PBMCs isolated from fresh human blood of a healthy donor.

0266Briefly, PBMCs were isolated from fresh human blood of a healthy donor and seeded in a 96-well U-bottom plate (1.5x105 cells/well) in AIM V medium. A constant concentration of 10 ng/ml hu IL-2 (Peprotech) was added to all wells. The Fab-IL12-Fab construct was diluted in medium and titrated onto the PBMCs. Supernatants were collected after approximately 20 hours to determine the IFN-γ concentrations using the hu IFN-γ ELISA Kit II from Becton Dickinson (#550612).

0267Results in <figref idref="f0047">Figure 45</figref> show that A) the chosen amount of human (hu) IL-2 alone as well as IL-12 alone were not able to induce significant IFN-γ release by human PBMCs whereas the combination of both cytokines led to significant IFN-γ release by PBMCs. B) The Fab-IL-12-Fab construct induced IFN-γ release by human PBMCs in a concentration-dependent manner in the presence of 10 ng/ml human IL-2.

Example 24

Purification of Targeted IFN-α Fab-IFNα2-Fab Immunoconjugate

0268Initial purification of the Fab-IFNα2-Fab immunoconjugate with L19 (Fibronectin Ectodomain-B binder) as Fab fragment was performed from supernatants of transiently transfected HEK 293 EBNA cells. Briefly, Fab-IFNα2-Fab was purified by protein A followed by size exclusion chromatography. The protein A column was equilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5. The supernatant was loaded and the column washed first with 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5, followed by 20 mM sodium phosphate, 20 rum sodium citrate, 100 mM sodium chloride, pH 7.5. Fab-IFNα2-Fab was eluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine pH 3 and subsequently neutralized. For formulation the following buffer was applied: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7.

0269<figref idref="f0048">Figure 46</figref> shows the elution profiles from the purification and the results from the analytical characterization of the product by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer, reduced and non-reduced). The yield was 8.4 mg/L.

Example 25

Biological Activity Assay with IFN-α Fab-IFNα2-Fab Immunoconjugate

0270The purified Fab-IFNa2-Fab immunoconjugate with L19 (Fibronectin Ectodomain-B binder) as Fab was subsequently analyzed for IFN-α-induced proliferation inhibition of Jurkat T cells and A549 tumor cells, comparing the effect of IFN-α (Roferon A, Roche) and the purified L19 Fab-IFNα2-Fab immunoconjugate. Briefly, A549 and Jurkat T cells which are susceptible for IFN-α-induced proliferation inhibition were seeded at 5000 cells/well (A549) or 10000 cells/well (Jurkat) into 96-well flat bottom plates. Dilutions of Roferon A (Roche) or Fab-IFNα2-Fab in the appropriate cell culture medium were titrated onto the cells in solution. After two days of proliferation at 37°C in a humidified incubator with 5 % CO2, the cells were lysed and ATP content was measured with the CellTiter Glo assay from Promega. IFN-α-untreated cells were set as 0% growth for calculation.

0271Results in <figref idref="f0049">Figure 47</figref> show that Fab-IFNα2-Fab constructs inhibited proliferation of A) Jurkat T cells and B) A549 cells in a concentration-dependent manner comparable to IFN-α (Roferon A).

Example 26

Preparation of MCSP targeted Fab-IL2-Fab Immunoconjugates

0272The humanized anti-MCSP MHLG antibody was generated as described in <patcit id="pcit0014" dnum="WO2006100582A"><text>WO 2006/100582</text></patcit> (see in particular Example 1 therein), and converted into the Fab-IL2-Fab format (see SEQ ID NOs: 255, 256, 261, 262).

0273The humanized anti-MCSP MHLG1 antibody was generated as follows: The murine amino acid sequence of anti-MCSP antibody 225.28 (light chain, and heavy chain, see below) was aligned to a collection of human germ-line antibody V-genes, and sorted according to sequence identity and homology.

0274225.28 light chain; GenBank Acc. No. CAA65007 (SEQ ID 267): <img file="EP2467165B1_D0349.tif" />

0275225.28 heavy chain; no GenBank Acc. No. available (SEQ ID 268): <img file="EP2467165B1_D0350.tif" />

0276The potential acceptor sequence was selected based on high overall homology, and the presence of the right canonical residues already in the acceptor sequence. The human germ-line sequence IGHV3-15 (IMGT Acc. No. X92216) was chosen as the acceptor for the heavy chain and sequence IGKV1-9 (IMGT Acc. No. Z00013) was chosen for the light chain. The humanized constructs were denoted M-KV1 (see SEQ ID NOs: 263, 264, 269, 270), 7 (SEQ ID NOs: 265, 266, 271, 272), and 9 (SEQ ID NOs: 253, 254, 259, 260) for the light chain, and MHLG1 (see SEQ ID NOs: 251, 252, 257, 258), for the heavy chain.

0277The genes for those designed antibody sequences were generated by conventional PCR techniques and fused to human IgG1 and kappa constant domains for the construction of the expression plasmids.

0278Antibodies were expressed either as IgG or as Fab-IL2-Fab fusion proteins in mammalian cell culture systems like HEK or CHO, and purified via protein A and size exclusion chromatography. Comparison of the binding data of light chain variants M-KV1, and M-KV7 revealed that a proline residue at Kabat position 46 is essential for functional binding to the antigen. Two different approaches were taken to ensure the presence of this amino acid: A) A so-called back-mutation was introduced into the human framework of IGKV1-9. And B) To avoid the presence of back-mutations, the entire framework 2 region (Kabat positions 35 to 49) was exchanged by the corresponding region of the human antibody with GenBank entry AAA17574. This antibody has naturally a proline residue at position 46.

0279MCSP-targeted MHLG or MHLG1 KV9 Fab-IL2-Fab was purified by the method described above (Example 9) composed of one affinity step (protein A) followed by size exclusion chromatography (Superdex 200, GE Healthcare). The protein A column was equilibrated in 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5, supernatant was loaded and the column was washed with 20 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 7.5, followed by a wash with 13.3 mM sodium phosphate, 20 mM sodium citrate, 500 mM sodium chloride, pH 5.45. A third wash with 10 mM MES, 50 mM sodium chloride, pH 5 was optionally included. Fab-IL2-Fab was eluted with 20 mM sodium citrate, 100 mM sodium chloride, 100 mM glycine, pH 3. The eluted fractions were pooled and polished by size exclusion chromatography in the final formulation buffer: 25 mM potassium phosphate, 125 mM sodium chloride, 100 mM glycine pH 6.7.

0280<figref idref="f0050">Figure 48</figref> (for MHLG Fab-IL2-Fab) and <figref idref="f0051">Figure 49</figref> (for MHLG1 Fab-IL2-Fab) show (A) the elution profiles from the purification and (B) the results from the analytical characterization of the MCSP-targeted MHLG or MHLG1 Fab-IL2-Fab by SDS-PAGE (NuPAGE Novex Bis-Tris Mini Gel, Invitrogen, MOPS running buffer, reduced and non-reduced).

Example 27

Biological Activity Assay with MCSP targeted Fab-IL2-Fab Immunoconjugates

0281The purified Fab-IL2-Fab immunoconjugates with MHLG KV9 or MHLG1 KV9 (MCSP binders) as Fab were subsequently analyzed for IL-2 induced IFN-γ release, comparing the effect of purified 4G8 Fab-IL2-Fab (FAP binder) and MHLG or MHLG1 Fab-IL2-Fab on NK92 cells. IL-2 starved NK92 cells (pre-incubated for 2 hours without IL-2) were seeded in 96-well U bottom plates (105 cells/well) in NK cell medium (MEMa + 10 % FCS + 10 % horse serum + 0.1 mM 2-mercaptoethanol + 0.2 mM inositol + 0.02 mM folic acid). The MCSP-targeted Fab-IL-2-Fab immunoconjugates were diluted in NK cell medium and titrated onto the NK92 cells in direct comparison to the FAP-targeted 4G8-based Fab-IL2-Fab immunoconjugate. Supernatants were collected after 22 to 24 hours to determine the IFN-γ concentrations using the human IFN-γ ELISA Kit II from Becton Dickinson (#550612).

0282The results in <figref idref="f0052">Figure 50</figref> (for MHLG KV9 Fab-IL2-Fab) and <figref idref="f0053">Figure 51</figref> (for MHLG1 KV9 Fab-IL2-Fab) show that all Fab-IL2-Fab immunoconjugates, targeted against MCSP or FAP, induced comparable IFN-γ secretion in NK92 cells in a concentration dependent manner, independent of the antigen binding moiety used.

Example 28

Cellular Binding Assay with the MCSP-targeted MHLG1 KV9 Fab-IL2-Fab Immunoconjugate

0283The purified MCSP-targeted MHLG1-KV9 Fab-IL2-Fab immunoconjugate was tested by flow cytometry for binding to human MCSP-expressing Colo38 melanoma cells. Briefly, cells were harvested, counted and checked for viability. Cells were adjusted to 1.112 x 106 (viable) cells/ml in PBS/0.1 % BSA and aliquoted 180 µl/well (200'000 cells/well) in a round-bottom 96-well plate. 20 µl MHLG1 KV9 Fab-IL2-Fab immunocytokine (in different dilutions) was added to the cell containing wells and incubated for 30 min at 4°C. Cells were subsequently collected by centrifugation (4 min, 400 x g), washed with 150 µl/well PBS/0.1 % BSA, resuspended and incubated for 30 min at 4°C with 12 µl/well secondary antibody (FITC-conjugated AffiniPure F(ab')2 Fragment goat anti-human F(ab')2 (Jackson Immuno Research Lab #109-096-097), dissolved in 1.5 ml of a 1:1 mixture of water and glycerol = stock solution), diluted 1:20 in PBS/0.1% BSA. Cells were subsequently washed in 150 µl/well PBS/0.1% BSA, followed by a washing step in PBS, collected by centrifugation (4 min, 400 x g), and resuspended with 200 µl/well PBS/0.1% BSA containing propidium iodide (PI). Measurements were performed using a FACSCantoII machine (Software FACS Diva). Results are presented in <figref idref="f0054">Figure 52</figref>, which shows that the MCSP-targeted MHLG1 KV9 Fab-IL2-Fab immunoconjugate bound very well, in a dose-dependent manner, to Colo38 cells.

SEQUENCE LISTING

0284<ul id="ul0002" list-style="none"><li><110> Roche Glycart AG</li><li><120> Targeted Immunoconjugates</li><li><130> 26306</li><li><150> <patcit id="pcit0015" dnum="US61234584B"><text>US 61/234,584</text></patcit> <151> 2009-08-17</li><li><150> <patcit id="pcit0016" dnum="EP10162410A"><text>EP 10162410.4</text></patcit> <151> 2010-05-10</li><li><160> 281</li><li><170> PatentIn version 3.5</li><li><210> 1 <211> 1611 <212> DNA <213> Artificial Sequence</li><li><220> <223> Library Template for DP47-3 library</li><li><400> 1 <img file="EP2467165B1_D0351.tif" /><img file="EP2467165B1_D0352.tif" /></li><li><210> 2 <211> 1617 <212> DNA <213> Artificial Sequence</li><li><220> <223> Library Template for DP88-3 library</li><li><400> 2 <img file="EP2467165B1_D0353.tif" /><img file="EP2467165B1_D0354.tif" /></li><li><210> 3 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10; VL</li><li><400> 3 <img file="EP2467165B1_D0355.tif" /></li><li><210> 4 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10; VL</li><li><400> 4 <img file="EP2467165B1_D0356.tif" /><img file="EP2467165B1_D0357.tif" /></li><li><210> 5 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10 (GS); VL</li><li><400> 5 <img file="EP2467165B1_D0358.tif" /></li><li><210> 6 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10 (GS) ; VL</li><li><400> 6 <img file="EP2467165B1_D0359.tif" /><img file="EP2467165B1_D0360.tif" /></li><li><210> 7 <211> 121 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2BIO; VH</li><li><400> 7 <img file="EP2467165B1_D0361.tif" /></li><li><210> 8 <211> 363 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10; VH</li><li><400> 8 <img file="EP2467165B1_D0362.tif" /><img file="EP2467165B1_D0363.tif" /></li><li><210> 9 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2F11; VL</li><li><400> 9 <img file="EP2467165B1_D0364.tif" /></li><li><210> 10 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2F11; VL</li><li><400> 10 <img file="EP2467165B1_D0365.tif" /></li><li><210> 11 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2F11(VI); VL</li><li><400> 11 <img file="EP2467165B1_D0366.tif" /></li><li><210> 12 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2F11(VI); VL</li><li><400> 12 <img file="EP2467165B1_D0367.tif" /></li><li><210> 13 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2F11; VH</li><li><400> 13 <img file="EP2467165B1_D0368.tif" /></li><li><210> 14 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2F11; VH</li><li><400> 14 <img file="EP2467165B1_D0369.tif" /></li><li><210> 15 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2F11(MT); VH</li><li><400> 15 <img file="EP2467165B1_D0370.tif" /></li><li><210> 16 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2F11(MT); VH</li><li><400> 16 <img file="EP2467165B1_D0371.tif" /></li><li><210> 17 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 3F2; VL</li><li><400> 17 <img file="EP2467165B1_D0372.tif" /></li><li><210> 18 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 3F2; VL</li><li><400> 18 <img file="EP2467165B1_D0373.tif" /></li><li><210> 19 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 3F2(YS); VL</li><li><400> 19 <img file="EP2467165B1_D0374.tif" /></li><li><210> 20 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 3F2 (YS) ; VL</li><li><400> 20 <img file="EP2467165B1_D0375.tif" /></li><li><210> 21 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 3F2; VH</li><li><400> 21 <img file="EP2467165B1_D0376.tif" /><img file="EP2467165B1_D0377.tif" /></li><li><210> 22 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 3F2; VH</li><li><400> 22 <img file="EP2467165B1_D0378.tif" /></li><li><210> 23 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 3D9, VL</li><li><400> 23 <img file="EP2467165B1_D0379.tif" /><img file="EP2467165B1_D0380.tif" /></li><li><210> 24 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 3D9, VL</li><li><400> 24 <img file="EP2467165B1_D0381.tif" /></li><li><210> 25 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 3D9, VH</li><li><400> 25 <img file="EP2467165B1_D0382.tif" /><img file="EP2467165B1_D0383.tif" /></li><li><210> 26 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 3D9, VH</li><li><400> 26 <img file="EP2467165B1_D0384.tif" /></li><li><210> 27 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2D9(TA); VH</li><li><400> 27 <img file="EP2467165B1_D0385.tif" /><img file="EP2467165B1_D0386.tif" /></li><li><210> 28 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2D9(TA); VH</li><li><400> 28 <img file="EP2467165B1_D0387.tif" /></li><li><210> 29 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4G8; VL</li><li><400> 29 <img file="EP2467165B1_D0388.tif" /><img file="EP2467165B1_D0389.tif" /></li><li><210> 30 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4G8; VL</li><li><400> 30 <img file="EP2467165B1_D0390.tif" /></li><li><210> 31 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4G8; VH</li><li><400> 31 <img file="EP2467165B1_D0391.tif" /><img file="EP2467165B1_D0392.tif" /></li><li><210> 32 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4G8; VH</li><li><400> 32 <img file="EP2467165B1_D0393.tif" /></li><li><210> 33 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4B3; VL</li><li><400> 33 <img file="EP2467165B1_D0394.tif" /><img file="EP2467165B1_D0395.tif" /></li><li><210> 34 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4B3; VL</li><li><400> 34 <img file="EP2467165B1_D0396.tif" /></li><li><210> 35 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4B3; VH</li><li><400> 35 <img file="EP2467165B1_D0397.tif" /><img file="EP2467165B1_D0398.tif" /></li><li><210> 36 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4B3; VH</li><li><400> 36 <img file="EP2467165B1_D0399.tif" /></li><li><210> 37 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4D6; VL</li><li><400> 37 <img file="EP2467165B1_D0400.tif" /><img file="EP2467165B1_D0401.tif" /></li><li><210> 38 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4D6; VL</li><li><400> 38 <img file="EP2467165B1_D0402.tif" /></li><li><210> 39 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4D6; VH</li><li><400> 39 <img file="EP2467165B1_D0403.tif" /></li><li><210> 40 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4D6; VH</li><li><400> 40 <img file="EP2467165B1_D0404.tif" /></li><li><210> 41 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2C6; VL</li><li><400> 41 <img file="EP2467165B1_D0405.tif" /></li><li><210> 42 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2C6; VL</li><li><400> 42 <img file="EP2467165B1_D0406.tif" /></li><li><210> 43 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2C6; VH</li><li><400> 43 <img file="EP2467165B1_D0407.tif" /></li><li><210> 44 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2C6; VH</li><li><400> 44 <img file="EP2467165B1_D0408.tif" /></li><li><210> 45 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 5H5; VL</li><li><400> 45 <img file="EP2467165B1_D0409.tif" /></li><li><210> 46 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 5H5; VL</li><li><400> 46 <img file="EP2467165B1_D0410.tif" /></li><li><210> 47 <211> 116 <212> PRT <213> Artificial Sequence</li><li><220> <223> 5H5; VH</li><li><400> 47 <img file="EP2467165B1_D0411.tif" /></li><li><210> 48 <211> 348 <212> DNA <213> Artificial Sequence</li><li><220> <223> 5H5; VH</li><li><400> 48 <img file="EP2467165B1_D0412.tif" /><img file="EP2467165B1_D0413.tif" /></li><li><210> 49 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2C4; VL</li><li><400> 49 <img file="EP2467165B1_D0414.tif" /></li><li><210> 50 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2C4; VL</li><li><400> 50 <img file="EP2467165B1_D0415.tif" /><img file="EP2467165B1_D0416.tif" /></li><li><210> 51 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2C4; VH</li><li><400> 51 <img file="EP2467165B1_D0417.tif" /></li><li><210> 52 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2C4; VH</li><li><400> 52 <img file="EP2467165B1_D0418.tif" /><img file="EP2467165B1_D0419.tif" /></li><li><210> 53 <211> 748 <212> PRT <213> Artificial Sequence</li><li><220> <223> Human FAP ectodomain+poly-lys-tag+his6-tag</li><li><400> 53 <img file="EP2467165B1_D0420.tif" /><img file="EP2467165B1_D0421.tif" /><img file="EP2467165B1_D0422.tif" /><img file="EP2467165B1_D0423.tif" /></li><li><210> 54 <211> 2244 <212> DNA <213> Artificial Sequence</li><li><220> <223> Human FAP ectodomain+poly-lys-tag+his6-tag</li><li><400> 54 <img file="EP2467165B1_D0424.tif" /><img file="EP2467165B1_D0425.tif" /></li><li><210> 55 <211> 749 <212> PRT <213> Artificial Sequence</li><li><220> <223> Murine FAP ectodomain+poly-lys-tag+his6-tag</li><li><400> 55 <img file="EP2467165B1_D0426.tif" /><img file="EP2467165B1_D0427.tif" /><img file="EP2467165B1_D0428.tif" /><img file="EP2467165B1_D0429.tif" /></li><li><210> 56 <211> 2247 <212> DNA <213> Artificial Sequence</li><li><220> <223> Murine FAP ectodomain+poly-lys-tag+his6-tag</li><li><400> 56 <img file="EP2467165B1_D0430.tif" /><img file="EP2467165B1_D0431.tif" /></li><li><210> 57 <211> 119 <212> PRT <213> Artificial Sequence</li><li><220> <223> Human TNC-A2+avi-tag +his6-tag</li><li><400> 57 <img file="EP2467165B1_D0432.tif" /><img file="EP2467165B1_D0433.tif" /></li><li><210> 58 <211> 357 <212> DNA <213> Artificial Sequence</li><li><220> <223> Human TNC-A2+avi-tag +his6-tag</li><li><400> 58 <img file="EP2467165B1_D0434.tif" /></li><li><210> 59 <211> 116 <212> PRT <213> Artificial Sequence</li><li><220> <223> Human TNC-A1+avi-tag +his6-tag</li><li><400> 59 <img file="EP2467165B1_D0435.tif" /><img file="EP2467165B1_D0436.tif" /></li><li><210> 60 <211> 348 <212> DNA <213> Artificial Sequence</li><li><220> <223> Human TNC-A1+avi-tag +his6-tag</li><li><400> 60 <img file="EP2467165B1_D0437.tif" /></li><li><210> 61 <211> 124 <212> PRT <213> Artificial Sequence</li><li><220> <223> Murine TNC-A1+avi-tag +his6-tag</li><li><400> 61 <img file="EP2467165B1_D0438.tif" /><img file="EP2467165B1_D0439.tif" /></li><li><210> 62 <211> 372 <212> DNA <213> Artificial Sequence</li><li><220> <223> Murine TNC-A1+avi-tag +his6-tag</li><li><400> 62 <img file="EP2467165B1_D0440.tif" /></li><li><210> 63 <211> 116 <212> PRT <213> Artificial Sequence</li><li><220> <223> Human TNC-A4+ avi-tag +his6-tag</li><li><400> 63 <img file="EP2467165B1_D0441.tif" /><img file="EP2467165B1_D0442.tif" /></li><li><210> 64 <211> 348 <212> DNA <213> Artificial Sequence</li><li><220> <223> Human TNC-A4+ avi-tag +his6-tag</li><li><400> 64 <img file="EP2467165B1_D0443.tif" /></li><li><210> 65 <211> 124 <212> PRT <213> Artificial Sequence</li><li><220> <223> Murine TNC-A4 +avi-tag +his6-tag</li><li><400> 65 <img file="EP2467165B1_D0444.tif" /><img file="EP2467165B1_D0445.tif" /></li><li><210> 66 <211> 372 <212> DNA <213> Artificial Sequence</li><li><220> <223> Murine TNC-A4 +avi-tag +his6-tag</li><li><400> 66 <img file="EP2467165B1_D0446.tif" /></li><li><210> 67 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2D9; VL</li><li><400> 67 <img file="EP2467165B1_D0447.tif" /><img file="EP2467165B1_D0448.tif" /></li><li><210> 68 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2D9; VL</li><li><400> 68 <img file="EP2467165B1_D0449.tif" /></li><li><210> 69 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2D9; VH</li><li><400> 69 <img file="EP2467165B1_D0450.tif" /><img file="EP2467165B1_D0451.tif" /></li><li><210> 70 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2D9; VH</li><li><400> 70 <img file="EP2467165B1_D0452.tif" /></li><li><210> 71 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4B8; VL</li><li><400> 71 <img file="EP2467165B1_D0453.tif" /><img file="EP2467165B1_D0454.tif" /></li><li><210> 72 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4B8; VL</li><li><400> 72 <img file="EP2467165B1_D0455.tif" /></li><li><210> 73 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4B8; VH</li><li><400> 73 <img file="EP2467165B1_D0456.tif" /><img file="EP2467165B1_D0457.tif" /></li><li><210> 74 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4B8; VH</li><li><400> 74 <img file="EP2467165B1_D0458.tif" /></li><li><210> 75 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 7A1; VL</li><li><400> 75 <img file="EP2467165B1_D0459.tif" /><img file="EP2467165B1_D0460.tif" /></li><li><210> 76 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 7A1; VL</li><li><400> 76 <img file="EP2467165B1_D0461.tif" /></li><li><210> 77 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 7A1; VH</li><li><400> 77 <img file="EP2467165B1_D0462.tif" /><img file="EP2467165B1_D0463.tif" /></li><li><210> 78 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 7A1; VH</li><li><400> 78 <img file="EP2467165B1_D0464.tif" /></li><li><210> 79 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 13C2; VL</li><li><400> 79 <img file="EP2467165B1_D0465.tif" /><img file="EP2467165B1_D0466.tif" /></li><li><210> 80 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 13C2; VL</li><li><400> 80 <img file="EP2467165B1_D0467.tif" /></li><li><210> 81 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 13C2; VH</li><li><400> 81 <img file="EP2467165B1_D0468.tif" /><img file="EP2467165B1_D0469.tif" /></li><li><210> 82 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 13C2; VH</li><li><400> 82 <img file="EP2467165B1_D0470.tif" /></li><li><210> 83 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 13E8; VL</li><li><400> 83 <img file="EP2467165B1_D0471.tif" /></li><li><210> 84 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 13E8; VL</li><li><400> 84 <img file="EP2467165B1_D0472.tif" /></li><li><210> 85 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 13E8; VH</li><li><400> 85 <img file="EP2467165B1_D0473.tif" /></li><li><210> 86 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 13E8; VH</li><li><400> 86 <img file="EP2467165B1_D0474.tif" /></li><li><210> 87 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 14C10; VL</li><li><400> 87 <img file="EP2467165B1_D0475.tif" /></li><li><210> 88 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 14C10; VL</li><li><400> 88 <img file="EP2467165B1_D0476.tif" /></li><li><210> 89 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 14C10; VH</li><li><400> 89 <img file="EP2467165B1_D0477.tif" /></li><li><210> 90 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 14C10; VH</li><li><400> 90 <img file="EP2467165B1_D0478.tif" /></li><li><210> 91 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 17A11; VL</li><li><400> 91 <img file="EP2467165B1_D0479.tif" /></li><li><210> 92 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 17A11; VL</li><li><400> 92 <img file="EP2467165B1_D0480.tif" /><img file="EP2467165B1_D0481.tif" /></li><li><210> 93 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 17A11; VH</li><li><400> 93 <img file="EP2467165B1_D0482.tif" /></li><li><210> 94 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 17A11; VH</li><li><400> 94 <img file="EP2467165B1_D0483.tif" /><img file="EP2467165B1_D0484.tif" /></li><li><210> 95 <211> 603 <212> PRT <213> Artificial Sequence</li><li><220> <223> Fab heavy chain derived from L19 monoclonal antibody-C125A variant of IL2-Fab heavy chain derived from L19 monoclonal antibody</li><li><400> 95 <img file="EP2467165B1_D0485.tif" /><img file="EP2467165B1_D0486.tif" /><img file="EP2467165B1_D0487.tif" /></li><li><210> 96 <211> 215 <212> PRT <213> Artificial Sequence</li><li><220> <223> Fab light chain derived from L19 monoclonal antibody</li><li><400> 96 <img file="EP2467165B1_D0488.tif" /><img file="EP2467165B1_D0489.tif" /></li><li><210> 97 <211> 382 <212> PRT <213> Artificial Sequence</li><li><220> <223> scFv derived from L19 monoclonal antibody-8 amino acid linker-C125A variant of IL2</li><li><400> 97 <img file="EP2467165B1_D0490.tif" /><img file="EP2467165B1_D0491.tif" /><img file="EP2467165B1_D0492.tif" /></li><li><210> 98 <211> 377 <212> PRT <213> Artificial Sequence</li><li><220> <223> F16-diabody-IL2 protein</li><li><400> 98 <img file="EP2467165B1_D0493.tif" /><img file="EP2467165B1_D0494.tif" /></li><li><210> 99 <211> 641 <212> PRT <213> Artificial Sequence</li><li><220> <223> scFv-IL2-scFv (F16, protein)</li><li><400> 99 <img file="EP2467165B1_D0495.tif" /><img file="EP2467165B1_D0496.tif" /><img file="EP2467165B1_D0497.tif" /></li><li><210> 100 <211> 603 <212> PRT <213> Artificial Sequence</li><li><220> <223> Fab-IL2-Fab (F16, heavy chain cytokine fusion construct, protein)</li><li><400> 100 <img file="EP2467165B1_D0498.tif" /><img file="EP2467165B1_D0499.tif" /><img file="EP2467165B1_D0500.tif" /><img file="EP2467165B1_D0501.tif" /></li><li><210> 101 <211> 214 <212> PRT <213> Artificial Sequence</li><li><220> <223> F16, light chain, protein</li><li><400> 101 <img file="EP2467165B1_D0502.tif" /><img file="EP2467165B1_D0503.tif" /></li><li><210> 102 <211> 466 <212> PRT <213> Artificial Sequence</li><li><220> <223> IL2R-beta-Fc(hole) fusion protein, protein</li><li><400> 102 <img file="EP2467165B1_D0504.tif" /><img file="EP2467165B1_D0505.tif" /><img file="EP2467165B1_D0506.tif" /></li><li><210> 103 <211> 492 <212> PRT <213> Artificial Sequence</li><li><220> <223> IL2R-gamma-Fc(knob), protein</li><li><400> 103 <img file="EP2467165B1_D0507.tif" /><img file="EP2467165B1_D0508.tif" /><img file="EP2467165B1_D0509.tif" /></li><li><210> 104 <211> 991 <212> PRT <213> Artificial Sequence</li><li><220> <223> Fab-IL12-Fab, L19 antibody, murine scIL12, protein</li><li><400> 104 <img file="EP2467165B1_D0510.tif" /><img file="EP2467165B1_D0511.tif" /><img file="EP2467165B1_D0512.tif" /><img file="EP2467165B1_D0513.tif" /><img file="EP2467165B1_D0514.tif" /></li><li><210> 105 <211> 987 <212> PRT <213> Artificial Sequence</li><li><220> <223> Fab-IL12-Fab L19 antibody, human scIL12, protein</li><li><400> 105 <img file="EP2467165B1_D0515.tif" /><img file="EP2467165B1_D0516.tif" /><img file="EP2467165B1_D0517.tif" /><img file="EP2467165B1_D0518.tif" /><img file="EP2467165B1_D0519.tif" /></li><li><210> 106 <211> 597 <212> PRT <213> Artificial Sequence</li><li><220> <223> Fab-GMCSF-Fab, L19 antibody, human GM-CSF, protein</li><li><400> 106 <img file="EP2467165B1_D0520.tif" /><img file="EP2467165B1_D0521.tif" /><img file="EP2467165B1_D0522.tif" /></li><li><210> 107 <211> 636 <212> PRT <213> Artificial Sequence</li><li><220> <223> Fab-IFNa2-Fab, L19 antibody, protein</li><li><400> 107 <img file="EP2467165B1_D0523.tif" /><img file="EP2467165B1_D0524.tif" /><img file="EP2467165B1_D0525.tif" /><img file="EP2467165B1_D0526.tif" /></li><li><210> 108 <211> 1812 <212> DNA <213> Artificial Sequence</li><li><220> <223> Fab heavy chain derived from L19 monoclonal antibody-C125A variant of IL2-Fab heavy chain derived from L19 monoclonal antibody</li><li><400> 108 <img file="EP2467165B1_D0527.tif" /><img file="EP2467165B1_D0528.tif" /></li><li><210> 109 <211> 648 <212> DNA <213> Artificial Sequence</li><li><220> <223> Fab light chain derived from L19 monoclonal antibody</li><li><400> 109 <img file="EP2467165B1_D0529.tif" /></li><li><210> 110 <211> 1149 <212> DNA <213> Artificial Sequence</li><li><220> <223> scFv derived from L19 monoclonal antibody-8 amino acid linker-C125A variant of IL2</li><li><400> 110 <img file="EP2467165B1_D0530.tif" /></li><li><210> 111 <211> 1134 <212> DNA <213> Artificial Sequence</li><li><220> <223> F16-diabody-IL2</li><li><400> 111 <img file="EP2467165B1_D0531.tif" /><img file="EP2467165B1_D0532.tif" /></li><li><210> 112 <211> 1926 <212> DNA <213> Artificial Sequence</li><li><220> <223> scFv-IL2-scFv (F16, DNA)</li><li><400> 112 <img file="EP2467165B1_D0533.tif" /><img file="EP2467165B1_D0534.tif" /></li><li><210> 113 <211> 1812 <212> DNA <213> Artificial Sequence</li><li><220> <223> Fab-IL2-Fab (F16, heavy chain cytokine fusion construct, DNA)</li><li><400> 113 <img file="EP2467165B1_D0535.tif" /><img file="EP2467165B1_D0536.tif" /></li><li><210> 114 <211> 645 <212> DNA <213> Artificial Sequence</li><li><220> <223> F16, light chain, DNA</li><li><400> 114 <img file="EP2467165B1_D0537.tif" /><img file="EP2467165B1_D0538.tif" /></li><li><210> 115 <211> 1401 <212> DNA <213> Artificial Sequence</li><li><220> <223> IL2R-beta-Fc(hole) fusion protein, DNA</li><li><400> 115 <img file="EP2467165B1_D0539.tif" /><img file="EP2467165B1_D0540.tif" /></li><li><210> 116 <211> 1479 <212> DNA <213> Artificial Sequence</li><li><220> <223> IL2R-gamma-Fc(knob), DNA</li><li><400> 116 <img file="EP2467165B1_D0541.tif" /></li><li><210> 117 <211> 2976 <212> DNA <213> Artificial Sequence</li><li><220> <223> Fab-IL12-Fab, L19 antibody, murine scIL12, DNA</li><li><400> 117 <img file="EP2467165B1_D0542.tif" /><img file="EP2467165B1_D0543.tif" /></li><li><210> 118 <211> 2964 <212> DNA <213> Artificial Sequence</li><li><220> <223> Fab-IL12-Fab, L19 antibody, human scIL12, DNA</li><li><400> 118 <img file="EP2467165B1_D0544.tif" /><img file="EP2467165B1_D0545.tif" /><img file="EP2467165B1_D0546.tif" /></li><li><210> 119 <211> 1794 <212> DNA <213> Artificial Sequence</li><li><220> <223> Fab-GMCSF-Fab, L19 antibody, human GM-CSF, DNA</li><li><400> 119 <img file="EP2467165B1_D0547.tif" /><img file="EP2467165B1_D0548.tif" /></li><li><210> 120 <211> 1911 <212> DNA <213> Artificial Sequence</li><li><220> <223> Fab-IFNa2-Fab, L19 antibody, DNA</li><li><400> 120 <img file="EP2467165B1_D0549.tif" /><img file="EP2467165B1_D0550.tif" /></li><li><210> 121 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 19G1; VL</li><li><400> 121 <img file="EP2467165B1_D0551.tif" /><img file="EP2467165B1_D0552.tif" /></li><li><210> 122 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 19G1; VL</li><li><400> 122 <img file="EP2467165B1_D0553.tif" /></li><li><210> 123 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 19G1; VH</li><li><400> 123 <img file="EP2467165B1_D0554.tif" /><img file="EP2467165B1_D0555.tif" /></li><li><210> 124 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 19G1; VH</li><li><400> 124 <img file="EP2467165B1_D0556.tif" /></li><li><210> 125 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 20G8; VL</li><li><400> 125 <img file="EP2467165B1_D0557.tif" /><img file="EP2467165B1_D0558.tif" /></li><li><210> 126 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 20G8; VL</li><li><400> 126 <img file="EP2467165B1_D0559.tif" /></li><li><210> 127 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 20G8; VH</li><li><400> 127 <img file="EP2467165B1_D0560.tif" /><img file="EP2467165B1_D0561.tif" /></li><li><210> 128 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 20G8; VH</li><li><400> 128 <img file="EP2467165B1_D0562.tif" /></li><li><210> 129 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4B9; VL</li><li><400> 129 <img file="EP2467165B1_D0563.tif" /></li><li><210> 130 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4B9; VL</li><li><400> 130 <img file="EP2467165B1_D0564.tif" /></li><li><210> 131 <211> 117 <212> PRT <213> Artificial Sequence ;</li><li><220> <223> 4B9; VH</li><li><400> 131 <img file="EP2467165B1_D0565.tif" /></li><li><210> 132 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4B9; VH</li><li><400> 132 <img file="EP2467165B1_D0566.tif" /></li><li><210> 133 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 5B8; VL</li><li><400> 133 <img file="EP2467165B1_D0567.tif" /></li><li><210> 134 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 5B8; VL</li><li><400> 134 <img file="EP2467165B1_D0568.tif" /></li><li><210> 135 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 5B8; VH</li><li><400> 135 <img file="EP2467165B1_D0569.tif" /></li><li><210> 136 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 5B8; VH</li><li><400> 136 <img file="EP2467165B1_D0570.tif" /></li><li><210> 137 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 5F1; VL</li><li><400> 137 <img file="EP2467165B1_D0571.tif" /></li><li><210> 138 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 5F1; VL</li><li><400> 138 <img file="EP2467165B1_D0572.tif" /><img file="EP2467165B1_D0573.tif" /></li><li><210> 139 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 5F1; VH</li><li><400> 139 <img file="EP2467165B1_D0574.tif" /></li><li><210> 140 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 5F1; VH</li><li><400> 140 <img file="EP2467165B1_D0575.tif" /><img file="EP2467165B1_D0576.tif" /></li><li><210> 141 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 14B3; VL</li><li><400> 141 <img file="EP2467165B1_D0577.tif" /></li><li><210> 142 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 14B3; VL</li><li><400> 142 <img file="EP2467165B1_D0578.tif" /><img file="EP2467165B1_D0579.tif" /></li><li><210> 143 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 14B3; VH</li><li><400> 143 <img file="EP2467165B1_D0580.tif" /></li><li><210> 144 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 14B3; VH</li><li><400> 144 <img file="EP2467165B1_D0581.tif" /><img file="EP2467165B1_D0582.tif" /></li><li><210> 145 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 16F1; VL</li><li><400> 145 <img file="EP2467165B1_D0583.tif" /></li><li><210> 146 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 16F1; VL</li><li><400> 146 <img file="EP2467165B1_D0584.tif" /></li><li><210> 147 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 16F1; VH</li><li><400> 147 <img file="EP2467165B1_D0585.tif" /></li><li><210> 148 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 16F1; VH</li><li><400> 148 <img file="EP2467165B1_D0586.tif" /></li><li><210> 149 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 16F8; VL</li><li><400> 149 <img file="EP2467165B1_D0587.tif" /></li><li><210> 150 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 16F8; VL</li><li><400> 150 <img file="EP2467165B1_D0588.tif" /></li><li><210> 151 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 16F8; VH</li><li><400> 151 <img file="EP2467165B1_D0589.tif" /></li><li><210> 152 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 16F8; VH</li><li><400> 152 <img file="EP2467165B1_D0590.tif" /></li><li><210> 153 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> O3C9; VL</li><li><400> 153 <img file="EP2467165B1_D0591.tif" /></li><li><210> 154 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> O3C9; VL</li><li><400> 154 <img file="EP2467165B1_D0592.tif" /></li><li><210> 155 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> O3C9; VH</li><li><400> 155 <img file="EP2467165B1_D0593.tif" /><img file="EP2467165B1_D0594.tif" /></li><li><210> 156 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> O3C9; VH</li><li><400> 156 <img file="EP2467165B1_D0595.tif" /></li><li><210> 157 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> O2D7; VL</li><li><400> 157 <img file="EP2467165B1_D0596.tif" /><img file="EP2467165B1_D0597.tif" /></li><li><210> 158 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> O2D7; VL</li><li><400> 158 <img file="EP2467165B1_D0598.tif" /></li><li><210> 159 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> O2D7; VH</li><li><400> 159 <img file="EP2467165B1_D0599.tif" /><img file="EP2467165B1_D0600.tif" /></li><li><210> 160 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> O2D7; VH</li><li><400> 160 <img file="EP2467165B1_D0601.tif" /></li><li><210> 161 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 28H1; VL</li><li><400> 161 <img file="EP2467165B1_D0602.tif" /><img file="EP2467165B1_D0603.tif" /></li><li><210> 162 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 28H1; VL</li><li><400> 162 <img file="EP2467165B1_D0604.tif" /></li><li><210> 163 <211> 116 <212> PRT <213> Artificial Sequence</li><li><220> <223> 28H1; VH</li><li><400> 163 <img file="EP2467165B1_D0605.tif" /><img file="EP2467165B1_D0606.tif" /></li><li><210> 164 <211> 348 <212> DNA <213> Artificial Sequence</li><li><220> <223> 28H1; VH</li><li><400> 164 <img file="EP2467165B1_D0607.tif" /></li><li><210> 165 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 22A3; VL</li><li><400> 165 <img file="EP2467165B1_D0608.tif" /><img file="EP2467165B1_D0609.tif" /></li><li><210> 166 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 22A3; VL</li><li><400> 166 <img file="EP2467165B1_D0610.tif" /></li><li><210> 167 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 22A3; VH</li><li><400> 167 <img file="EP2467165B1_D0611.tif" /><img file="EP2467165B1_D0612.tif" /></li><li><210> 168 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 22A3; VH</li><li><400> 168 <img file="EP2467165B1_D0613.tif" /></li><li><210> 169 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 29B11; VL</li><li><400> 169 <img file="EP2467165B1_D0614.tif" /><img file="EP2467165B1_D0615.tif" /></li><li><210> 170 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 29B11; VL</li><li><400> 170 <img file="EP2467165B1_D0616.tif" /></li><li><210> 171 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 29B11; VH</li><li><400> 171 <img file="EP2467165B1_D0617.tif" /><img file="EP2467165B1_D0618.tif" /></li><li><210> 172 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 29B11; VH</li><li><400> 172 <img file="EP2467165B1_D0619.tif" /></li><li><210> 173 <211> 108 <212> PRT <213> Artificial Sequence</li><li><220> <223> 23C10; VL</li><li><400> 173 <img file="EP2467165B1_D0620.tif" /></li><li><210> 174 <211> 324 <212> DNA <213> Artificial Sequence</li><li><220> <223> 23C10; VL</li><li><400> 174 <img file="EP2467165B1_D0621.tif" /></li><li><210> 175 <211> 117 <212> PRT <213> Artificial Sequence</li><li><220> <223> 23C10; VH</li><li><400> 175 <img file="EP2467165B1_D0622.tif" /></li><li><210> 176 <211> 351 <212> DNA <213> Artificial Sequence</li><li><220> <223> 23C10; VH</li><li><400> 176 <img file="EP2467165B1_D0623.tif" /></li><li><210> 177 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_C3B6; VL</li><li><400> 177 <img file="EP2467165B1_D0624.tif" /></li><li><210> 178 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_C3B6; VL</li><li><400> 178 <img file="EP2467165B1_D0625.tif" /></li><li><210> 179 <211> 121 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_C3B6; VH</li><li><400> 179 <img file="EP2467165B1_D0626.tif" /></li><li><210> 180 <211> 363 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10-C3B6; VH</li><li><400> 180 <img file="EP2467165B1_D0627.tif" /></li><li><210> 181 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_6A12; VL</li><li><400> 181 <img file="EP2467165B1_D0628.tif" /></li><li><210> 182 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_6A12; VL</li><li><400> 182 <img file="EP2467165B1_D0629.tif" /></li><li><210> 183 <211> 121 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_6A12; VH</li><li><400> 183 <img file="EP2467165B1_D0630.tif" /></li><li><210> 184 <211> 363 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_6A12; VH</li><li><400> 184 <img file="EP2467165B1_D0631.tif" /><img file="EP2467165B1_D0632.tif" /></li><li><210> 185 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_C3A6; VL</li><li><400> 185 <img file="EP2467165B1_D0633.tif" /></li><li><210> 186 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_C3A6; VL</li><li><400> 186 <img file="EP2467165B1_D0634.tif" /><img file="EP2467165B1_D0635.tif" /></li><li><210> 187 <211> 121 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_C3A6; VH</li><li><400> 187 <img file="EP2467165B1_D0636.tif" /></li><li><210> 188 <211> 363 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_C3A6; VH</li><li><400> 188 <img file="EP2467165B1_D0637.tif" /><img file="EP2467165B1_D0638.tif" /></li><li><210> 189 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_D1A2_wt; VL</li><li><400> 189 <img file="EP2467165B1_D0639.tif" /></li><li><210> 190 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_D1A2_wt; VL</li><li><400> 190 <img file="EP2467165B1_D0640.tif" /><img file="EP2467165B1_D0641.tif" /></li><li><210> 191 <211> 121 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_D1A2_wt; VH</li><li><400> 191 <img file="EP2467165B1_D0642.tif" /></li><li><210> 192 <211> 363 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_D1A2_wt; VH</li><li><400> 192 <img file="EP2467165B1_D0643.tif" /><img file="EP2467165B1_D0644.tif" /></li><li><210> 193 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_D1A2_VD; VL</li><li><400> 193 <img file="EP2467165B1_D0645.tif" /></li><li><210> 194 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_D102_VD; VL</li><li><400> 194 <img file="EP2467165B1_D0646.tif" /></li><li><210> 195 <211> 121 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_D1A2_VD; VH</li><li><400> 195 <img file="EP2467165B1_D0647.tif" /></li><li><210> 196 <211> 363 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_D1A2_VD; VH</li><li><400> 196 <img file="EP2467165B1_D0648.tif" /></li><li><210> 197 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_O7D8; VL</li><li><400> 197 <img file="EP2467165B1_D0649.tif" /></li><li><210> 198 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_07D8; VL</li><li><400> 198 <img file="EP2467165B1_D0650.tif" /></li><li><210> 199 <211> 121 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_O7D8; VH</li><li><400> 199 <img file="EP2467165B1_D0651.tif" /></li><li><210> 200 <211> 363 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_O7D8; VH</li><li><400> 200 <img file="EP2467165B1_D0652.tif" /></li><li><210> 201 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_O1F7; VL</li><li><400> 201 <img file="EP2467165B1_D0653.tif" /></li><li><210> 202 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_O1F7; VL</li><li><400> 202 <img file="EP2467165B1_D0654.tif" /></li><li><210> 203 <211> 121 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_O1F7; VH</li><li><400> 203 <img file="EP2467165B1_D0655.tif" /></li><li><210> 204 <211> 363 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_O1F7; VH</li><li><400> 204 <img file="EP2467165B1_D0656.tif" /></li><li><210> 205 <211> 107 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_6H10; VL</li><li><400> 205 <img file="EP2467165B1_D0657.tif" /></li><li><210> 206 <211> 321 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_6H10; VL</li><li><400> 206 <img file="EP2467165B1_D0658.tif" /></li><li><210> 207 <211> 121 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10_6H10; VH</li><li><400> 207 <img file="EP2467165B1_D0659.tif" /><img file="EP2467165B1_D0660.tif" /></li><li><210> 208 <211> 363 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10_6H10; VH</li><li><400> 208 <img file="EP2467165B1_D0661.tif" /></li><li><210> 209 <211> 605 <212> PRT <213> Artificial Sequence</li><li><220> <223> 3F2 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 209 <img file="EP2467165B1_D0662.tif" /><img file="EP2467165B1_D0663.tif" /><img file="EP2467165B1_D0664.tif" /></li><li><210> 210 <211> 1818 <212> DNA <213> Artificial Sequence</li><li><220> <223> 3F2 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 210 <img file="EP2467165B1_D0665.tif" /><img file="EP2467165B1_D0666.tif" /></li><li><210> 211 <211> 605 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4G8 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 211 <img file="EP2467165B1_D0667.tif" /><img file="EP2467165B1_D0668.tif" /><img file="EP2467165B1_D0669.tif" /></li><li><210> 212 <211> 1818 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4G8 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 212 <img file="EP2467165B1_D0670.tif" /><img file="EP2467165B1_D0671.tif" /></li><li><210> 213 <211> 605 <212> PRT <213> Artificial Sequence</li><li><220> <223> 3D9 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 213 <img file="EP2467165B1_D0672.tif" /><img file="EP2467165B1_D0673.tif" /><img file="EP2467165B1_D0674.tif" /></li><li><210> 214 <211> 1818 <212> DNA <213> Artificial Sequence</li><li><220> <223> 3D9 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 214 <img file="EP2467165B1_D0675.tif" /><img file="EP2467165B1_D0676.tif" /></li><li><210> 215 <211> 605 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2F11 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 215 <img file="EP2467165B1_D0677.tif" /><img file="EP2467165B1_D0678.tif" /><img file="EP2467165B1_D0679.tif" /><img file="EP2467165B1_D0680.tif" /></li><li><210> 216 <211> 1818 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2F11 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 216 <img file="EP2467165B1_D0681.tif" /><img file="EP2467165B1_D0682.tif" /></li><li><210> 217 <211> 605 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4B3 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 217 <img file="EP2467165B1_D0683.tif" /><img file="EP2467165B1_D0684.tif" /><img file="EP2467165B1_D0685.tif" /></li><li><210> 218 <211> 1818 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4B3 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 218 <img file="EP2467165B1_D0686.tif" /><img file="EP2467165B1_D0687.tif" /></li><li><210> 219 <211> 993 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4G8 Fab-IL12-Fab (murine IL-12; heavy chain cytokine fusion construct)</li><li><400> 219 <img file="EP2467165B1_D0688.tif" /><img file="EP2467165B1_D0689.tif" /><img file="EP2467165B1_D0690.tif" /><img file="EP2467165B1_D0691.tif" /></li><li><210> 220 <211> 2982 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4G8 Fab-IL12-Fab (murine IL-12; heavy chain cytokine fusion construct)</li><li><400> 220 <img file="EP2467165B1_D0692.tif" /><img file="EP2467165B1_D0693.tif" /></li><li><210> 221 <211> 603 <212> PRT <213> Artificial Sequence</li><li><220> <223> 28H1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 221 <img file="EP2467165B1_D0694.tif" /><img file="EP2467165B1_D0695.tif" /><img file="EP2467165B1_D0696.tif" /><img file="EP2467165B1_D0697.tif" /></li><li><210> 222 <211> 1812 <212> DNA <213> Artificial Sequence</li><li><220> <223> 28H1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 222 <img file="EP2467165B1_D0698.tif" /><img file="EP2467165B1_D0699.tif" /></li><li><210> 223 <211> 605 <212> PRT <213> Artificial Sequence</li><li><220> <223> 29B11 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 223 <img file="EP2467165B1_D0700.tif" /><img file="EP2467165B1_D0701.tif" /><img file="EP2467165B1_D0702.tif" /></li><li><210> 224 <211> 1818 <212> DNA <213> Artificial Sequence</li><li><220> <223> 29B11 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 224 <img file="EP2467165B1_D0703.tif" /><img file="EP2467165B1_D0704.tif" /></li><li><210> 225 <211> 605 <212> PRT <213> Artificial Sequence</li><li><220> <223> 19G1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 225 <img file="EP2467165B1_D0705.tif" /><img file="EP2467165B1_D0706.tif" /><img file="EP2467165B1_D0707.tif" /><img file="EP2467165B1_D0708.tif" /></li><li><210> 226 <211> 1818 <212> DNA <213> Artificial Sequence</li><li><220> <223> 19G1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 226 <img file="EP2467165B1_D0709.tif" /><img file="EP2467165B1_D0710.tif" /></li><li><210> 227 <211> 605 <212> PRT <213> Artificial Sequence</li><li><220> <223> 20G8 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 227 <img file="EP2467165B1_D0711.tif" /><img file="EP2467165B1_D0712.tif" /><img file="EP2467165B1_D0713.tif" /></li><li><210> 228 <211> 1818 <212> DNA <213> Artificial Sequence</li><li><220> <223> 20G8 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 228 <img file="EP2467165B1_D0714.tif" /></li><li><210> 229 <211> 215 <212> PRT <213> Artificial Sequence</li><li><220> <223> 3F2 light chain</li><li><400> 229 <img file="EP2467165B1_D0715.tif" /></li><li><210> 230 <211> 648 <212> DNA <213> Artificial Sequence</li><li><220> <223> 3F2 light chain</li><li><400> 230 <img file="EP2467165B1_D0716.tif" /></li><li><210> 231 <211> 215 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4G8 light chain</li><li><400> 231 <img file="EP2467165B1_D0717.tif" /><img file="EP2467165B1_D0718.tif" /></li><li><210> 232 <211> 648 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4G8 light chain</li><li><400> 232 <img file="EP2467165B1_D0719.tif" /></li><li><210> 233 <211> 215 <212> PRT <213> Artificial Sequence</li><li><220> <223> 3D9 light chain</li><li><400> 233 <img file="EP2467165B1_D0720.tif" /></li><li><210> 234 <211> 648 <212> DNA <213> Artificial Sequence</li><li><220> <223> 3D9 light chain</li><li><400> 234 <img file="EP2467165B1_D0721.tif" /></li><li><210> 235 <211> 215 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2F11 light chain</li><li><400> 235 <img file="EP2467165B1_D0722.tif" /><img file="EP2467165B1_D0723.tif" /></li><li><210> 236 <211> 648 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2F11 light chain</li><li><400> 236 <img file="EP2467165B1_D0724.tif" /></li><li><210> 237 <211> 215 <212> PRT <213> Artificial Sequence</li><li><220> <223> 4B3 light chain</li><li><400> 237 <img file="EP2467165B1_D0725.tif" /><img file="EP2467165B1_D0726.tif" /></li><li><210> 238 <211> 648 <212> DNA <213> Artificial Sequence</li><li><220> <223> 4B3 light chain</li><li><400> 238 <img file="EP2467165B1_D0727.tif" /></li><li><210> 239 <211> 613 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 239 <img file="EP2467165B1_D0728.tif" /><img file="EP2467165B1_D0729.tif" /><img file="EP2467165B1_D0730.tif" /> Pro Lys Ser Cys Asp 610</li><li><210> 240 <211> 1842 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 240 <img file="EP2467165B1_D0731.tif" /><img file="EP2467165B1_D0732.tif" /></li><li><210> 241 <211> 613 <212> PRT <213> Artificial Sequence</li><li><220> <223> C3B6 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 241 <img file="EP2467165B1_D0733.tif" /><img file="EP2467165B1_D0734.tif" /><img file="EP2467165B1_D0735.tif" /></li><li><210> 242 <211> 1842 <212> DNA <213> Artificial Sequence</li><li><220> <223> C3B6 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 242 <img file="EP2467165B1_D0736.tif" /><img file="EP2467165B1_D0737.tif" /></li><li><210> 243 <211> 613 <212> PRT <213> Artificial Sequence</li><li><220> <223> 6A12 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 243 <img file="EP2467165B1_D0738.tif" /><img file="EP2467165B1_D0739.tif" /><img file="EP2467165B1_D0740.tif" /></li><li><210> 244 <211> 1842 <212> DNA <213> Artificial Sequence</li><li><220> <223> 6A12 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 244 <img file="EP2467165B1_D0741.tif" /><img file="EP2467165B1_D0742.tif" /></li><li><210> 245 <211> 214 <212> PRT <213> Artificial Sequence</li><li><220> <223> 2B10 light chain</li><li><400> 245 <img file="EP2467165B1_D0743.tif" /><img file="EP2467165B1_D0744.tif" /></li><li><210> 246 <211> 645 <212> DNA <213> Artificial Sequence</li><li><220> <223> 2B10 light chain</li><li><400> 246 <img file="EP2467165B1_D0745.tif" /></li><li><210> 247 <211> 214 <212> PRT <213> Artificial Sequence</li><li><220> <223> D1A2 light chain</li><li><400> 247 <img file="EP2467165B1_D0746.tif" /><img file="EP2467165B1_D0747.tif" /></li><li><210> 248 <211> 645 <212> DNA <213> Artificial Sequence</li><li><220> <223> D1A2 light chain</li><li><400> 248 <img file="EP2467165B1_D0748.tif" /></li><li><210> 249 <211> 214 <212> PRT <213> Artificial Sequence</li><li><220> <223> O7D8 light chain</li><li><400> 249 <img file="EP2467165B1_D0749.tif" /><img file="EP2467165B1_D0750.tif" /></li><li><210> 250 <211> 645 <212> DNA <213> Artificial Sequence</li><li><220> <223> O7D8 light chain</li><li><400> 250 <img file="EP2467165B1_D0751.tif" /><img file="EP2467165B1_D0752.tif" /></li><li><210> 251 <211> 615 <212> PRT <213> Artificial Sequence</li><li><220> <223> MHLG1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 251 <img file="EP2467165B1_D0753.tif" /><img file="EP2467165B1_D0754.tif" /><img file="EP2467165B1_D0755.tif" /></li><li><210> 252 <211> 1848 <212> DNA <213> Artificial Sequence</li><li><220> <223> MHLG1 Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 252 <img file="EP2467165B1_D0756.tif" /><img file="EP2467165B1_D0757.tif" /></li><li><210> 253 <211> 214 <212> PRT <213> Artificial Sequence</li><li><220> <223> KV9 light chain</li><li><400> 253 <img file="EP2467165B1_D0758.tif" /><img file="EP2467165B1_D0759.tif" /></li><li><210> 254 <211> 645 <212> DNA <213> Artificial Sequence</li><li><220> <223> KV9 light chain</li><li><400> 254 <img file="EP2467165B1_D0760.tif" /><img file="EP2467165B1_D0761.tif" /></li><li><210> 255 <211> 615 <212> PRT <213> Artificial Sequence</li><li><220> <223> MHLG Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 255 <img file="EP2467165B1_D0762.tif" /><img file="EP2467165B1_D0763.tif" /><img file="EP2467165B1_D0764.tif" /></li><li><210> 256 <211> 1848 <212> DNA <213> Artificial Sequence</li><li><220> <223> MHLG Fab-IL2-Fab (heavy chain cytokine fusion construct)</li><li><400> 256 <img file="EP2467165B1_D0765.tif" /><img file="EP2467165B1_D0766.tif" /></li><li><210> 257 <211> 122 <212> PRT <213> Artificial Sequence</li><li><220> <223> MHLG1; VH <img file="EP2467165B1_D0767.tif" /></li><li><210> 258 <211> 366 <212> DNA <213> Artificial Sequence</li><li><220> <223> MHLG1; VH</li><li><400> 258 <img file="EP2467165B1_D0768.tif" /></li><li><210> 259 <211> 109 <212> PRT <213> Artificial Sequence</li><li><220> <223> MHLG KV9; VL <img file="EP2467165B1_D0769.tif" /></li><li><210> 260 <211> 327 <212> DNA <213> Artificial Sequence</li><li><220> <223> MHLG KV9; VL</li><li><400> 260 <img file="EP2467165B1_D0770.tif" /></li><li><210> 261 <211> 122 <212> PRT <213> Artificial Sequence</li><li><220> <223> MHLG; VH</li><li><400> 261 <img file="EP2467165B1_D0771.tif" /><img file="EP2467165B1_D0772.tif" /></li><li><210> 262 <211> 366 <212> DNA <213> Artificial Sequence</li><li><220> <223> MHLG; VH</li><li><400> 262 <img file="EP2467165B1_D0773.tif" /></li><li><210> 263 <211> 214 <212> PRT <213> Artificial Sequence</li><li><220> <223> KV1 light chain</li><li><400> 263 <img file="EP2467165B1_D0774.tif" /><img file="EP2467165B1_D0775.tif" /></li><li><210> 264 <211> 645 <212> DNA <213> Artificial Sequence</li><li><220> <223> KV1 light chain</li><li><400> 264 <img file="EP2467165B1_D0776.tif" /><img file="EP2467165B1_D0777.tif" /></li><li><210> 265 <211> 214 <212> PRT <213> Artificial Sequence</li><li><220> <223> KV7 light chain</li><li><400> 265 <img file="EP2467165B1_D0778.tif" /><img file="EP2467165B1_D0779.tif" /></li><li><210> 266 <211> 645 <212> DNA <213> Artificial Sequence</li><li><220> <223> KV7 light chain</li><li><400> 266 <img file="EP2467165B1_D0780.tif" /></li><li><210> 267 <211> 107 <212> PRT <213> Mus musculus</li><li><400> 267 <img file="EP2467165B1_D0781.tif" /><img file="EP2467165B1_D0782.tif" /></li><li><210> 268 <211> 122 <212> PRT <213> Mus musculus</li><li><400> 268 <img file="EP2467165B1_D0783.tif" /></li><li><210> 269 <211> 109 <212> PRT <213> Artificial Sequence</li><li><220> <223> KV1; VL</li><li><400> 269 <img file="EP2467165B1_D0784.tif" /></li><li><210> 270 <211> 327 <212> DNA <213> Artificial Sequence</li><li><220> <223> KV1; VL</li><li><400> 270 <img file="EP2467165B1_D0785.tif" /></li><li><210> 271 <211> 109 <212> PRT <213> Artificial Sequence</li><li><220> <223> KV7; VL</li><li><400> 271 <img file="EP2467165B1_D0786.tif" /></li><li><210> 272 <211> 327 <212> DNA <213> Artificial Sequence</li><li><220> <223> KV7; VL</li><li><400> 272 <img file="EP2467165B1_D0787.tif" /></li><li><210> 273 <211> 19 <212> PRT <213> Artificial Sequence</li><li><220> <223> leader sequence</li><li><400> 273 <img file="EP2467165B1_D0788.tif" /><img file="EP2467165B1_D0789.tif" /></li><li><210> 274 <211> 57 <212> DNA <213> Artificial Sequence</li><li><220> <223> leader sequence</li><li><400> 274 <img file="EP2467165B1_D0790.tif" /></li><li><210> 275 <211> 57 <212> DNA <213> Artificial Sequence</li><li><220> <223> leader sequence</li><li><400> 275 <img file="EP2467165B1_D0791.tif" /></li><li><210> 276 <211> 22 <212> PRT <213> Artificial Sequence</li><li><220> <223> leader sequence</li><li><400> 276 <img file="EP2467165B1_D0792.tif" /></li><li><210> 277 <211> 66 <212> DNA <213> Artificial Sequence</li><li><220> <223> leader sequence</li><li><400> 277 <img file="EP2467165B1_D0793.tif" /></li><li><210> 278 <211> 19 <212> PRT <213> Artificial Sequence</li><li><220> <223> leader sequence</li><li><400> 278 <img file="EP2467165B1_D0794.tif" /></li><li><210> 279 <211> 57 <212> DNA <213> Artificial Sequence</li><li><220> <223> leader sequence</li><li><400> 279 <img file="EP2467165B1_D0795.tif" /></li><li><210> 280 <211> 57 <212> DNA <213> Artificial Sequence</li><li><220> <223> leader sequence</li><li><400> 280 <img file="EP2467165B1_D0796.tif" /></li><li><210> 281 <211> 57 <212> DNA <213> Artificial Sequence</li><li><220> <223> leader sequence</li><li><400> 281 <img file="EP2467165B1_D0797.tif" /></li></ul>

Contents7

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Every citation, both ways

Citations
Document	Relation	Office	Cited during
US12152073B2	Cited by	United States of America	Applicant
US12351632B2	Cited by	United States of America	Applicant
US12286477B2	Cited by	United States of America	Applicant
US12486326B2	Cited by	United States of America	Applicant
US12247060B2	Cited by	United States of America	Applicant
US12384842B2	Cited by	United States of America	Applicant
US12358982B2	Cited by	United States of America	Applicant
US11965025B2	Cited by	United States of America	Applicant
WO9929732A2	Cites	World Intellectual Property Organization (WIPO)	Examiner
WO9839363A2	Cites	World Intellectual Property Organization (WIPO)	–
WO9929732A2	Cites	World Intellectual Property Organization (WIPO)	–
WO2006119897A2	Cites	World Intellectual Property Organization (WIPO)	–
WO2009017823A2	Cites	World Intellectual Property Organization (WIPO)	–

36 members in 24 offices

This recordMember, by publication datePriority claim14 more offices are in the list below

Priority claims5

Priority claims
Document	Office	Kind	Date
234584P	United States of America	–	2009-08-17
23458409	United States of America	P	2009-08-17
10162410	European Patent Office (EPO)	–	2010-05-10
10162410	European Patent Office (EPO)	A	2010-05-10
2010061810	European Patent Office (EPO)	W	2010-08-13

Members36

Family members, oldest first
Document	Office	Kind
CA2769619A1	Canada	A1	2011-02-24
WO2011020783A2	World Intellectual Property Organization (WIPO)	A2	2011-02-24
US2011064751A1	United States of America	A1	2011-03-17
WO2011020783A3	World Intellectual Property Organization (WIPO)	A3	2011-04-28
TW201119672A	Taiwan Province of China	A	2011-06-16
AR077879A1	Argentina	A1	2011-09-28
AU2010285071A1	Australia	A1	2012-02-02
CR20120042A	Costa Rica	A	2012-03-22
CR20120042A	Costa Rica	A	2012-03-22
IL217844A0	Israel	A0	2012-03-29
ECSP12011685A	Ecuador	A	2012-03-30
SG178443A1	Singapore	A1	2012-04-27
CO6430436A2	Colombia	A2	2012-04-30
KR20120053042A	Republic of Korea	A	2012-05-24
PE20120630A1	Peru	A1	2012-05-26
MX2012002014A	Mexico	A	2012-06-13
MX2012002014A	Mexico	A	2012-06-13
EP2467165A2	European Patent Office (EPO)	A2	2012-06-27
CN102573922A	China	A	2012-07-11
CL2012000430A1	Chile	A1	2012-08-31
MA33717B1	Morocco	B1	2012-11-01
JP2013502209A	Japan	A	2013-01-24
HK1169318A	Hong Kong, China	A	2013-01-25
HK1169318A1	Hong Kong, China	A1	2013-01-25
RU2012110127A	Russian Federation	A	2013-09-27
ZA201200767B	South Africa	B	2014-07-30
EP2467165B1This record	European Patent Office (EPO)	B1	2015-01-07
US8945571B2	United States of America	B2	2015-02-03
ES2534085T3	Spain	T3	2015-04-17
KR20150088328A	Republic of Korea	A	2015-07-31
CN102573922B	China	B	2015-08-05
JP5764127B2	Japan	B2	2015-08-12
KR101588598B1	Republic of Korea	B1	2016-01-29
RU2583876C2	Russian Federation	C2	2016-05-10
CA2769619C	Canada	C	2019-04-30
BR112012003739A2	Brazil	A2	2020-08-11

81 legal events, as 10 offices reported them to INPADOC

Over the term

Point at a mark for the event

Events

Legal events, newest first
	Event	Code	Office
2025-10-21	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2025-10-20	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2025-10-16	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2025-10-15	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2025-10-15	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2025-10-10	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2025-10-09	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2024-10-21	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2024-10-16	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2024-10-14	Annual fee paid to national office [announced via postgrant information from national office to epo]GrantedPGFP	PGFP	EP
2018-10-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2018-07-18	Fee paymentPLFP	PLFP	FR
2018-06-29	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2018-06-29	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2017-07-20	Fee paymentPLFP	PLFP	FR
2017-06-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2017-05-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2017-05-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2017-03-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2016-11-29	Amendment of ipc main classPREVIOUS MAIN CLASS: A61K0047480000R079	R079	DE
2016-07-29	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2016-07-19	Fee paymentPLFP	PLFP	FR
2016-06-01	Patent lapsedLapsedMM4A	MM4A	IE
2016-05-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2016-03-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2016-03-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2016-02-29	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-12-16	No opposition filedOpposition26N	26N	EP
2015-11-13	No opposition filed within time limitOppositionORIGINAL CODE: 0009261PLBE	PLBE	EP
2015-11-13	Information on the status of an ep patent application or granted ep patentGrantedSTATUS: NO OPPOSITION FILED WITHIN TIME LIMITSTAA	STAA	EP
2015-10-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-10-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-10-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-10-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-10-30	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-10-08	No opposition filed against granted patent, or epo opposition proceedings concluded without decisionGrantedR097	R097	DE
2015-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-08-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-07-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-07-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-07-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-07-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-07-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-07-31	Lapsed in a contracting state [announced via postgrant information from national office to epo]LapsedPG25	PG25	EP
2015-06-25	Invalidated european patentMG4D	MG4D	LT
2015-06-24	Fee paymentPLFP	PLFP	FR
2015-06-15	Deletion acc. to par. 5 (withdrawal of the translation of the ep patent)MK05	MK05	AT
2015-05-20	Discontinued in the netherlands as no translation has been filedVDEP	VDEP	NL
2015-04-17	Definitive protectionFG2A	FG2A	ES
2015-02-26	Dpma publication of mentioned ep patent grantGrantedR096	R096	DE
2015-02-15	Reference to at number (ep patent validated in austria)REF	REF	AT
2015-01-28	European patents granted designating irelandGrantedFG4D	FG4D	IE
2015-01-15	European patent takes effect as a national patent in ch/liEP	EP	CH
2015-01-07	Designated contracting statesAK	AK	EP
2015-01-07	Request for extension of the european patentAX	AX	EP
2015-01-07	European patent grantedGrantedFG4D	FG4D	GB
2014-12-17	Intention to grant announcedINTG	INTG	EP
2014-12-12	(expected) grantORIGINAL CODE: 0009210GRAA	GRAA	EP
2014-11-29	Grant fee paidORIGINAL CODE: EPIDOSNIGR3GRAS	GRAS	EP
2014-11-18	Despatch of communication of intention to grant a patentORIGINAL CODE: EPIDOSNIGR1GRAP	GRAP	EP
2014-09-03	Intention to grant announcedINTG	INTG	EP
2014-09-03	Information on inventor provided before grant (corrected)RIN1	RIN1	EP
2014-09-03	Information on inventor provided before grant (corrected)RIN1	RIN1	EP
2014-09-03	Information on inventor provided before grant (corrected)RIN1	RIN1	EP
2014-09-03	Information on inventor provided before grant (corrected)RIN1	RIN1	EP
2014-08-05	Despatch of communication of intention to grant a patentORIGINAL CODE: EPIDOSNIGR1GRAP	GRAP	EP
2014-07-16	Information provided on ipc code assigned before grantRIC1	RIC1	EP
2014-07-16	Information provided on ipc code assigned before grantRIC1	RIC1	EP
2014-07-16	Information provided on ipc code assigned before grantRIC1	RIC1	EP
2014-07-16	Information provided on ipc code assigned before grantRIC1	RIC1	EP
2014-07-16	Information provided on ipc code assigned before grantRIC1	RIC1	EP
2014-07-16	Information provided on ipc code assigned before grantRIC1	RIC1	EP
2013-04-24	First examination report despatched17Q	17Q	EP
2012-06-27	Request for examination filed17P	17P	EP
2012-06-27	Designated contracting statesAK	AK	EP
2012-06-27	Request for extension of the european patentAX	AX	EP
2012-05-25	Public reference made under article 153(3) epc to a published international application that has entered the european phaseORIGINAL CODE: 0009012PUAI	PUAI	EP