Untitled record

Antibodies reactive with B7-H3, immunologically active fragments thereof and their uses

Antibodies Reactive with B7-H3, Immunologically Active Fragments Thereof and Uses Thereof

Full description

Background of the invention

The present invention relates to antibodies and fragments thereof that interact immunologically with the mammalian, especially human, B7-H3 receptor and their use, particularly in the treatment of cancer and inflammation. The invention therefore relates in particular to humanized antibodies reacting with B7-H3- and its immunoreactive fragments capable of mediating, and preferably enhancing, activation of the immune system against cancer cells associated with several human cancers.

Referring to the list of sequences

This application contains one or more lists of sequences pursuant to 37 CFR 1.821 et seq., which have been disclosed in both print and computer-readable media, and such disclosures are incorporated throughout the present document by reference.

The growth and spread of tumors depend largely on their ability to evade host immune surveillance and overcome host defenses. Most tumors express antigens that the host's immune system can, to a varying degree, recognize, but in many cases, an inadequate immune response is elicited due to ineffective activation of effector T cells:

(Khawli, LA et al. (2008) Cytokine, Chemokine, and Co-Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors, Exper. Pharmacol. 181:291-328).

CD4+ T-lymphocytes are key regulators of most mammalian immune and autoimmune responses (Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules, Immunolog. Res. 28(1):39-48). It has been found that activation of CD4+ helper T cells can be mediated through co-stimulatory interactions between antigen donor cells and CD4+ T lymphocytes. Two interactions are required:

(Viglietta, V. et al. (2007) Modulating Co-Stimulation, Neurotherapeutics 4:666-675; Korman, AJ et al. (2007) Checkpoint Blockade in Cancer Immunotherapy, Adv. Immunol. 90:297-339)

In the first reaction, antigen donor cells must have the relevant target antigen bound to the cell's MHC so that it can bind to the T-cell receptor (TCR) of a native CD4+ T lymphocyte. In the second reaction, a binding moiety specific to the antigen donor cell must bind to the CD28 receptor of the lymphocyte:

CD4+ T (Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules, Immunolog. Res. 28(1):39-48; Lindley, PS et al. (2009) The Clinical Utility Of Inhibiting CD28-Mediated Costimulation, Immunol Rev. 229:307-321

CD4+ helper T cells that undergo both stimulatory signals are then able to respond to cytokines (such as IL-2 and IL-12) to enter into Th1 cells. These cells produce tumor necrosis factor alpha and interferon-gamma (IFN-γ), which mediate immune responses to target Target cells expressing the target antigen:

(Bernard, A. et al. (2005) T and B Cell Cooperation: A Dance of Life and Death, Transplantation 79:S8-S11)

In the absence of both co-stimulatory signals during TCR interference, T cells enter a functionally unresponsive state, referred to as clonal inactivation:

(Khawli, LA et al. (2008) Cytokine, Chemokine, and Co-Stimulatory Fusion Proteins for the Immunotherapy of Solid Tumors, Exper. Pharmacol. 181:291-328)

In pathological conditions, Th1 cells are key drivers of various autoimmune diseases, such as type 1 diabetes, rheumatoid arthritis, and multiple sclerosis:

(Dong, C. et al. (2003) Immune Regulation by Novel Costimulatory Molecules, Immunolog. Res. 28(1):39-48)

1- The huge family of B7 and B7-H3

Studies of CD28 receptor binding molecules have led to the classification of a group of related molecules known as the large B7 family

(Coyle, AJ et al. (2001) The Expanding B7 Superfamily: Increasing Complexity In Costimulatory Signals Regulating T Cell Function, Nature Immunol. 2(3):203-209; Sharpe, AH et al. (2002) The B7-CD28 Superfamily, Nature Rev. 2:116-126; Greenwald, R. J. et al B7 Family Of Immune-Regulatory Ligands, Genome Biol 6:223.1-223.7; (2004) Emerging Mechanisms Of Immune Regulation: The Extended B7 Family And Regulatory T Cells. Arthritis Res. Ther. 6:208-214; Korman, A. J. et al. (2007) Checkpoint Blockade in Cancer Immunotherapy, Adv. Immunol. 90:297-339; Flies, D. B. et al. (2007) The New B7s: Playing a Pivotal Role in Tumor Immunity, J. Immunother. 30(3):251-260; Agarwal, A. et al. (2008) The Role Of Positive Costimulatory Molecules In Transplantation And Tolerance, Curr. Opin. Organ Transplant. 13:366-372; Lenschow, D.J et al. (1996) CD28/B7 System of T Cell Costimulation, Ann. Rev. Immunol. 14:233-258; Wang, S. et al. (2004) Co-Signaling Molecules Of The B7-CD28 Family In Positive And Negative Regulation Of T Lymphocyte Responses, Microbes Infect. 6:759-766)

There are currently seven known members of this family:

B7.1 (CD80), B7.2 (CD86), inducible co-inducer binding molecule (ICOS-L), programmed death-binding molecule-1 (PD-L1), programmed death-binding molecule-2 (PD-L2), B7-H3, f

B7-H4(Collins, M. et al. (2005) The B7 Family Of Immune-Regulatory Ligands, Genome Biol. 6:223.1-223.7)

The B7 family members are members of a large immunoglobulin superfamily with an IgV-like domain and an IgVIgC-like domain (Sharpe, AH et al. (2002) The B7-CD28 Superfamily, Nature Rev. Immunol. 2:116- 126). Each IgV and IgC domain of B7 family members is encoded by individual exons, with additional exons encoding guide sequences, transmembrane and cytoplasmic domains. The trans-cytoplasmic domains are short, ranging from 19 to 62 amino acid residues in length and can be encoded by multiple exons (Collins, M. et al. (2005) The B7 Family Of Immune-Regulatory Ligands, Genome Biol. 6:223.1-223.7)

As for B7-H3, it is unique in that the main human form contains two tandem IgV-IgC extracellular domains (such as IgVIgCIgVIgC) (Collins, M. et al. (2005) The B7 Family Of Immune-Regulatory Ligands, Genome Biol. 6:223.1- 223.7). Members of the B7 family are predicted to form successive noncovalent homodimers at the cell surface, and such dimers have been found related to B7-1 (CD80) and B7-2 (CD86).

The B7-CD28 Superfamily, Nature Rev. Immunol. 2: 116-126).

Although it was initially believed that it would only include two areas:

Ig (IgVIgC) (Chapoval, A. et al. (2001) B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production, Nature Immunol. 2:269274; Sun, M. et al. (2002) Characterization of Mouse and Human B7-H3 Genes, J. Immunol 168:6294-6297).

However, the extracellular immunoglobulin tetrameric domain (4Ig-B7-H3) has been identified and shown to be a dominant human protein form (Sharpe, A.H. et al. (2002) The B7-CD28 Superfamily, Nature Rev. Immunol. 2: 116-126). No functional differences were observed between these two forms, because the normal mouse form (2Ig) and the human form 4Ig are characterized by similar function:

(Hofmeyer, K. et al. (2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278)

The molecule is characterized by natural killer cell-mediated degradation specific to cancer cells:

Castriconi, R. et al. Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis, Proc. Natl. Acad. Sci. (USA) 101(34): 12640-12645)

Human B7-H3 (Figure 2Ig) has been shown to enhance T cell activation and IFN-γ production by binding to a putative receptor on the activated T cell:

(Chapoval, A. et al. (2001) B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production, Nature Immunol. 2:269274; Expression of Immunoinhibitory Molecule B7-H3: Potential Implications for Immune Based Therapy of Human Solid Tumors, Cancer Res 69(15):5275-6281.

Both B7-H4 and B7-H1 are potent inhibitors of immune function when expressed on tumor cells:

(Flies, DB et al. (2007) The New B7s: Playing a Pivotal Role in Tumor Immunity, J. Immunother. 30(3):251-260).

The mode of action of B7-H3 is complex, with the protein mediating both T cell co-stimulation and T cell co-inhibition (Hofmeyer, K. et al. (2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad. Sci. (USA) 105 (30):10277-10278; Martin-Orozco, N. et al. (2007). ) The Balance of Immune Responses: Costimulation Verse Coinhibition, J. Mol. 83:193-202). B7-H3 binds to TREM-like transcript 2 (TLT-2) and co-stimulates T cell activation, but binds to unidentified receptor(s) causing co-inhibition of T cells. In addition, B7-H3, through interactions With unknown receptor(s), it is an inhibitor of natural killer cells and osteoclasts (Hofmeyer, K. et al. (2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad. Sci. (USA) 105(30) :10277-10278). Inhibition may act through interactions with members of key signaling pathways through which the T-cell receptor (TCR) regulates gene transcription (such as NFTA, NF-κB or AP-1 factors).

B7-H3 co-stimulates CD4+ and CD8+ T cell proliferation. B7-H3 also stimulates IFN-γ production and lytic CD8+ activity:

(Chapoval, A. et al. (2001) B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production, Nature Immunol. 2:269274; Sharpe, A.H. et al. (2002) The B7-CD28 Superfamily, Nature Rev. Immunol 2:116-126)

However, the protein may also act through the factors NFAT (nuclear factor of activated T cells), NF-κB (nuclear factor kappa B) and AP-1 (activating protein-1) to inhibit T cell activation (Yi. KH et al (2009) Fine Tuning The Immune Response Through B7-H3 And B7-H4, Immunol Rev. 229:145-151. B7-H3 is also thought to inhibit Th1, Th2, or Th17 in vivo (Prasad, DV et al. (2004) Murine B7H3 Is A Negative Regulator Of T Cells, J. Immunol. 173:2500-2506; Fukushima , A. et al. (2007) B7H3 Regulates The Development Of Experimental Allergic Conjunctivitis In Mice, Immunol. Lett. 113:52-57; Yi. KH et al. (2009) Fine Tuning The Immune Response Through B7-H3 And B7-H4, Immunol. Rev. 229:145-151). Several independent studies have shown that human malignant tumor cells are characterized by significantly increased expression of the B7-H3 protein and that this overexpression is associated with increased disease severity (Zang, X. et al. (2007) The B7 Family And Cancer Therapy: Costimulation And Coinhibition , Clin. Cancer Res. 13:5271-5279), suggesting that B7-H3 is exploited by tumors as an immune evasion pathway (Hofmeyer, K. et al. (2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad. Sci. (USA). ) 105(30):10277-10278).

Molecules that inhibit the ability of the B7 molecule to bind to the T cell receptor (such as CD28) suppress the immune system and have been proposed as treatments for autoimmune diseases (Linsley, PS et al. (2009) The Clinical Utility Of Inhibiting CD28-Mediated Co-Stimulation, Immunolog. Rev. 229:307-321). 4Ig-B7-H3-expressing neuroblastoma cells treated with anti-4Ig-B7-H3 antibodies were susceptible to NK cells. However, it is unclear whether this activity can be attributed solely to antibodies against the 4Ig-B7-H3 form because all of the aforementioned antibodies were raised against 4Ig-B7-H3 and also bound to the B7H3-like Ig form (Steinberger, P. et al. (2004) Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains, J. Immunol 172(4): 2352-2359 and Castriconi et al. (2004) Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis, Proc. Natl. Acad. Sci. (USA) 101(34):12640-12645).

B7-H3 is not expressed on surviving B or T cells, mononuclear cells, or dendritic cells, but is induced on dendritic cells by IFN-γ, and on mononuclear cells by GM-CSF (Sharpe, A.H. et al (2002) The B7-CD28 Superfamily, Nature Rev. 2:116-126. The receptor(s) that bind to B7-H3 have not been fully characterized. Preliminary studies have indicated that one of these receptors needs to be rapidly and temporarily controlled to be increased on T cells after activation (Loke, P. et al. (2004) Emerging Mechanisms Of Immune Regulation: The Extended B7 Family And Regulatory T Cells. Arthritis Res. Ther. 6:208-214). More recently, the TREM-like transcript 2 receptor (TLT-2 or TREML2) (King, RG et al. (2006) Trem-Like Transcript 2 Is Expressed On Cells Of The Myeloid/Granuloid And B Lymphoid Lineage And Is Up- Regulated In Response to Inflammation, J. Immunol 176:6012-6021; KH et al. (2009) Fine Tuning The Immune Response Through B7-H3 And B7-H4, Immunol. Rev. 229:145-151), which is expressed on myeloid cells has been shown to bind B7-H3, thereby co-stimulating CD8+ T cell activation in particular (Zang, X. et al. (2003) B7x: A Widely Expressed B7 Family Member That Inhibits T Cell Activation, Proc Natl Sci (USA) 100:1038810392; (2008) Triggering Receptor Expressed On Myeloid Cell-Like Transcript 2 (TLT-2) Is A Counter-Receptor For B7H3 And Enhances T Cell Responses, Proc. Natl. Acad. Sci. (USA) 105(30):10495-10500; Hofmeyer, K. et al. (2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad. Sci. (USA) 105(30):10277-10278).

In addition to its expression on neuroblast cells, human B7-H3 is also known to be expressed on many other cancer cells (eg, stomach, ovarian, and non-small cell lung cancer). Expression of B7-H3 protein has also been observed in immune tissues in tumor cell lineages (Chapoval, A. et al. (2001) B7-H3: A Costimulatory Molecule For T Cell Activation and IFN-γ Production, Nature Immunol. 2:269274 Saatian, B. et al. (2004) Expression Of Genes For B7-H3 And Other T Cell Ligands By Nasal Epithelial Cells During Differentiation And Activation, Amer. J. Physiol. Lung Cell. Mol. Physiol. 287:L217L225; Castriconi et al. (2004) Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis, Proc. Natl. Acad. Sci. (USA) 101(34):12640-12645); Sun, M. et al. (2002) Characterization of Mouse and Human B7-H3 Genes, J. Immunol. 168:6294-6297). Expression of mRNA has been shown in heart, kidney, testes, lung, liver, pancreas, prostate, colon, and bone cells (Collins, M. et al. (2005) The B7 Family Of Immune-Regulatory Ligands, Genome Biol. 6: 223.1-223.7). At the protein level, B7-H3 has been found in human liver, lung, bladder, testis, prostate, thorax, placenta, and lymphatic organs (Hofmeyer, K. et al. (2008) The Contrasting Role Of B7-H3, Proc. Natl. Acad Sci (USA) 105(30):10277-10278).

2- Therapeutic antibodies

In addition to their well-known uses in diagnosis, antibodies have been shown to be useful as therapeutic agents. For example, immunotherapy, or the use of antibodies for treatment, has been used in recent years to treat cancer. Passive immunotherapy involves the use of monoclonal antibodies in cancer treatments (see, for example, DeVita, Hellman, and Rosenberg's Cancer: Principles & Practice of Oncology, Eighth Edition (2008), DeVita, V. et al. Eds., Lippincott Williams & Wilkins, Philadelphia, PA, pp. 537-547, 2979-2990)). These antibodies may have inherent biotherapeutic activity either by directly inhibiting the growth or survival of tumor cells or by their ability to utilize the natural cell-killing activity of the body's immune system. These agents may be given alone or in combination with radiotherapy or chemotherapy agents. Rituximab and trastuzumab, approved to treat Hodkin lymphoma and breast cancer, respectively, are examples of such treatments. Alternatively, antibodies can be used to prepare antibody conjugates in which the antibody is attached to a toxic agent and that agent is directed to the tumor by tumor-specific binding. Gemtuzumab ozogamicin is an example of an approved antibody derivative used in the treatment of leukemia.

Monoclonal antibodies have been discovered that bind to cancer cells and can be used in diagnosis and treatment (see, for example, the following patent applications that reveal, among other things, some of the molecular weights of the target proteins: US Patent No. 6,054,561) (200 One kilodalton c-erbB-2 (Her2), and other unknown antigens with a size of 40-200 kilodaltons and US patent No. ((5656444). Examples of antibodies in clinical trials and/or approved for the treatment of solid tumors include: trastuzumab (antigen: 180 kDa, HER2/neu), idercolumab (antigen: 40-50 kDa, Ep-CAM), fat globules Human milk antibodies (HMFGl (antigen: 200 kDa, HMW Mucin), cetuximab (antigen: 150 kDa and 170 kDa, EGF receptor), alemtuzumab (antigen: 21-28 kDa, CD52) and rituximab ( Antigen: 35 kDa, CD20).

The antigenic targets of trastuzumab (Her-2 receptor), used in the treatment of breast cancer, and cetuximab (EGF receptor), used in clinical trials to treat many types of cancer, are present at specific levels that can be detected on a large number of adult tissues from Humans such as skin, colon, lung, ovary, liver, and pancreas. The margin of safety in using these treatments may vary depending on the levels of expression of the antigen or the access or activity of the antibody to those sites.

Another type of immunotherapy is active immunotherapy, or vaccination, with an antigen present on a particular type(s) of cancer or a DNA component that directs the expression of the antigen, which elicits an immune response in the person, that is, to induce the person to actively produce antibodies Against the cancer he suffers from. Active immunization has also often been used as an immunotherapy or immunotoxin.

Numerous models of disease progression (including cancer) have been proposed. Theories have ranged from the causation of an infectious/transforming event to the development of an increasingly “disease-like” or “cancer-like” tissue type ultimately leading to an event with fully pathogenic or malignant potential. Some argue that in the case of cancer, for example, a single mutational event is sufficient to cause malignant disease, while others argue that subsequent modifications are equally necessary. While some have suggested that increased mutational load and tumor grade are important for the initiation and progression of malignancies via persistent mutagenesis-selection events at the cell level. Some cancer targets were found only in tumor tissues, while others were found in normal tissues and were controlledly increased and/or overexpressed in tumor tissues. In these cases, some researchers have suggested that overexpression is associated with the acquisition of malignant disease, while others suggest that overexpression is merely a sign of a trend along the path of increasing disease.

In some cases, cancer targets, such as oncogenic proteins expressed or overexpressed in tumors, have been shown to be present during primitive and embryonic development and act as regulators of growth and differentiation. Some researchers have proven that the expression of these tumor-generating proteins during primitive and embryonic development appears to be limited to certain tissues and to certain stages of development. Conversely, expression of oncogenic proteins in adults has been shown to be associated with overexpression in tumor growth and/or underexpression of tumor suppressor proteins.

The diagnostic and/or therapeutic antibody will be specific for an antigen that is present on many cancers, but not present or present at low levels on any normal tissue. The discovery, classification, and isolation of a new antibody capable of binding to an antigen specifically related to cancer would be beneficial in several ways. First, the antibody will have biological activity against the cells of that cancer and be able to use the immune system's response to treat the disease. The antibody can be given as a treatment either alone, in combination with existing treatments, or used to prepare immunoconjugates linked to toxic agents. An antibody with the same specificity but with low or no biological activity when administered alone can be useful in that the antibody can be used in the preparation of an immunoconjugate with a radioisotope, toxin, chemotherapeutic agent or liposome containing a chemotherapeutic agent, where the The conjugate is biologically active thanks to the directing of the toxin antibody to the cells containing the antigen.

As previously noted, antibodies and other molecules that specifically bind to B7-H3 have been described (see, for example, U.S. Patent Nos. 7,527,969; 7,368,554; 7,358,354; and 7,279,567; and U.S. Patent Application Publications Nos. 20090087416; 20090022747; 20090018315 2008116219; 20080081346; 20030103963; 2002016862; 01381; 2002/32375; 2002/10187 and 2001/094413;

European patent 1,292,619b; Modak, S. et al. (March 1999) Disialoganglioside GD2 And Antigen 8H9: Potential Targets For Antibody-Based Immunotherapy Against Desmoplastic Small Round Cell Tumor (DSRCT) And Rhabdomyosarcoma (RMS), Proceedings Of The American Association For Cancer Research Annual Meeting, Vol. 40:474 (90th Annual Meeting Of The American Association For Cancer Research; Philadelphia, Pennsylvania, US; April 10-14, 1999; Modak, S. et al. (March 2000) Radioimmunotargeting To Human Rhabdomyosarcoma Using Monoclonal Antibody 8H9, Proc. Am. Assoc. Cancer Res.41:724; Modak, S. et al. (2001) Monoclonal Antibody 8H9 Targets A Novel Cell Surface Antigen Expressed By A Wide Spectrum Of Human Solid Tumors, Cancer Res. 61(10):4048-4054; Steinberger, P. et al. (2004) Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains, J. Immunol. 172(4):2352-2359; Xu, H. et al. (2009) MicroRNA miR-29 Modulates Expression of Immunoinhibitory Molecule B7-H3: Potential Implications for Immune Based Therapy of Human Solid Tumors, Cancer.

However, one of the required features of an ideal antibody for diagnosis and/or therapy is the discovery and classification of new antibodies capable of inducing and especially enhancing activation of the immune system against cancer cells (particularly human cancer cells) associated with many cancers. These constructs will be useful for drug discovery (e.g., small molecules) and for classifying cell organization, growth and differentiation.

Therefore, despite all previous advances, there is still a need for improved formulations capable of binding to cancer cells and facilitating or inducing an immune response against cancer cells. These formulations can be used to diagnose and treat these types of cancer. There is also a need, based on the findings of the present document, for new formulations that specifically recognize dual targets on the surface of cells and thus modify, either by reducing or enhancing, the abilities of B7-H3 to cause T cell activation or to recognize killer cancer cells that express B7-H3. The present invention aims to identify those compositions. The invention also aims to provide new compounds for use in the B7-H3 expression experiment.

As will be described in detail, the present invention relates to novel antibodies, in particular dual affinity retargeting agents (DARTS) comprising modulators of T cell activation of B7-H3 and capable of influencing T cell activation as well as novel antibodies that bind to B7-H3 receptors. to cancer cells and facilitating or causing the death of those cells. The present invention relates to these compositions and their use in the diagnosis and treatment of diseases such as cancer.

General description of the invention

The present invention relates to antibodies and fragments thereof that are immunologically cross-reactive with a mammalian, especially human, B7-H3 receptor, and their uses, particularly in the treatment of cancer and inflammation. The invention also relates in particular to humanized antibodies cross-reactive with B7-H3 and its immunoreactive fragments capable of causing, and preferably enhancing, activation of the immune system against cancer cells associated with several types of human cancer.

In particular, the invention relates to an isolated antibody or fragment thereof that is immunologically cross-reactive, wherein the antibody or fragment thereof includes a variable domain that specifically binds to the extracellular domain of B7-H3, whereby the antibody competes for binding to B7-H3 with any of the antibodies. : BRCA69D, BRCA84D, or PRCA157.

The invention also relates to the aforementioned isolated antibody or an immunoreactive fragment thereof, wherein said antibody or its fragment comprises a variable domain comprising:

(A) CDR1 (SEQ: 21), CDR2 (SEQ: 23), and CDR3 (SEQ: 25) of BRCA69D light chain, CDR1 (SEQ: 29), and CDR2 (SEQ: 31), and CDR3 (sequence no. 33), for the heavy chain of BRCA69D;

(B) CDR1 (SEQ: 5), CDR2 (SEQ: 7), and CDR3 (SEQ: 9) of BRCA84D light chain, CDR1 (SEQ: 13), and CDR2 (SEQ: 15), and CDR3 (sequence no. 17), for the heavy chain of BRCA84D; or

(c) CDR1 (SEQ: 37), CDR2 (SEQ: 39), and CDR3 (SEQ: 41) of PRCA157 light chain, CDR1 (SEQ: 45), CDR2 (SEQ: 47), and CDR3 (sequence no. 49), of the heavy chain of PRCA157.

The invention also relates to any of the aforementioned isolated antibodies or their immunoreactive fragments, wherein the antibody binds to B7-H3 endogenously expressed on the surface of a cancer cell.

The invention also relates to any of the aforementioned isolated antibodies or immunoreactive fragments thereof, wherein said antibody binds to B7-H3 which is internalized upon binding to B7-H3 expressed on the surface of a cancer cell.

The invention also relates to any of the aforementioned isolated antibodies or their immunoreactive fragments, being a humanized monoclonal antibody.

The invention also relates to any of the aforementioned isolated antibodies or immunoreactive fragments thereof, wherein the isolated antibody is a modified antibody comprising a different Fc region of human IgG1, wherein the different Fc region of human IgG1 comprises at least one amino acid modification for of the Fc region of the antibody origin, wherein the amino acid modification(s) comprise amino acid modification(s) that alter the affinity or avidity of the different Fc region for binding to the FcγR such that the modified antibody exhibits an effector function Booster relative to the original antibody.

The invention also relates to any of the aforementioned isolated antibodies or their immunoreactive fragments, wherein the modification of the Fc region includes:

(a) At least one substitution chosen from the set consisting of:

(1) F243L;

(5) Y300L;

(2) D270E;

(6) V305I;

(3)R292P;

(7) A330V; And

(4) S298N;

(8) P396L;

(b) At least one substitution of two amino acid residues, where the substitutions are selected from the set consisting of:

(1) F243L and P396L;

(2) F243L and R292P;

(3)R292P and V305I;

(c) At least one substitution of three amino acid residues, where the substitutions are selected from the group consisting of:

(1) F243L, R292P and Y300L;

(2) F243L, R292P, V305I;

(3) F243L and R292P P396L; And

(4) R292P, V305I, P396L;

(d) At least one substitution of four amino acid residues, where the substitutions are selected from the set consisting of:

(1) F243L, R292P, Y300L, P396L;

(2) F243L, R292P, V305I, P396L;

or

(e) At least one substitution of four to five amino acid residues: F243L, R292P, Y300L, V305I, and P396.

The invention also relates to the aforementioned isolated antibody or an immunoreactive fragment thereof, wherein the antibody includes the substitutions:

(A) F243L, R292P, Y300L;

(b) L235V, F243L, R292P Y300L, P396L; or

(c) F243L, R292P, Y300L, V305I, P396L.

The invention also relates to the aforementioned isolated antibody or an immunologically reactive fragment thereof, where the antibody includes:

(A) Variable domain comprising CDR1 (sequence no: 5); and CDR2 (sequence no. 7); and CDR3 (sequence no: 9); for BRCA84D and CDR1 light chain (sequence no: 13); and CDR2 (sequence no. 15); and CDR3 (sequence no. 17); for the heavy chain of BRCA84D; And

(B) Modification of the Fc region includes substitutions: L235V, F243L, R292P, Y300L, and P396L.

The invention also relates to the aforementioned isolated antibody or an immunoreactive fragment thereof, where the antibody is a chimeric or humanized antibody.

The invention also relates to the aforementioned isolated antibodies or their immunoreactive fragments, wherein the antibody includes:

(a) Variable light chain with the amino acid sequence of hBRCA84D-2 VL (sequence no. 89);

(b) Variable heavy chain with amino acid sequence of hBRCA84D-2 VH (sequence no: 99);

(c) Fc region with substitutions: L235V, F243L, R292P, Y300L and P396L.

The invention also relates to a hybridoma that secretes a monoclonal antibody that specifically binds to the extracellular domain of B7-H3, where the antibody competes for binding to said B7-H3 with any of the antibodies: BRCA69D, BRCA84D, or PRCA157.

The invention also relates to a nucleic acid molecule encoding any of the aforementioned antibodies or their immunoreactive fragments.

The invention also relates to a dual affinity retargeting agent (DART), where the agent includes:

(a) A polypeptide chain I comprising a VL immunoglobulin epitope that specifically binds to a B7-H3-binding domain and a VH epitope that specifically binds to a non-B7-H3 molecule-binding domain; And

(b) a polypeptide chain II comprising a VH immunoglobulin epitope that binds specifically to a domain specific for B7-H3 binding and a VL epitope that binds to a domain specific for binding a molecule other than B7-H3;

The polypeptide chains I and II are linked together to form functional epitope-binding domains capable of binding to B7-H3 and non-B7-H3 molecules.

The invention relates to the aforementioned dual affinity retargeting agent (DART), wherein the non-B7-H3 molecule that may bind to DART is a heptane, particularly where the heptane is fluorescein isothiocyanate.

The invention relates to the aforementioned dual affinity retargeting (DART) agent, wherein the non-B7-H3 molecule that may bind to DART is a T cell receptor or NKG2D receptor.

The invention relates to the aforementioned dual affinity retargeting (DART) agent, wherein other than B7-H3 that may bind to DART is a tumor-associated antigen, particularly where said tumor-associated antigen is selected from the set of A33; ADAM-9; ALCAM; BAGE; beta-catenin; CA125; Carboxypeptidase M; CD103; CD19; CD20; CD22; CD23; CD25; CD27; CD28; CD36; CD40/CD154; CD45; CD46; CD5; CD56; CD79a/CD79b; CDK4; CEA; CTLA4; Cytokeratin 8; EGF-R; EphA2; ErbB1; ErbB3; ErbB4; GAGE-1; GAGE-2; GD2/GD3/GM2; gp100; HER-2/neu; HPV E6; HPV E7; Intergrin alpha-V-beta-6; JAM-3; KID3; KID31; KSA(17-1A); LUCA-2; MAGE-1; MAGE-3; MART; MUC-1; MUM-1; N-acetylglucosamine yltransferase; Oncostatin M; p15; PIPA; PSA; PSMA; ROR1; sTn; TNF- receptor; TNF-α receptor; TNF-γ receptor; transferrin receptor; and VEGF.

The invention also relates to a nucleic acid molecule encoding a polypeptide chain specific to any of the aforementioned dual affinity retargeting factors (DARTs).

The invention also relates to a pharmaceutical composition comprising (1) a therapeutically effective amount of the aforementioned antibodies or immunoreactive fragments thereof, or a dual affinity retargeting agent (DART) and (2) a pharmaceutically acceptable carrier.

The invention relates to the previously described pharmaceutical composition, where the antibody is compatible with humans and includes:

(A) Variable domain comprising CDR1 (sequence no: 5); and CDR2 (sequence no. 7); and CDR3 (sequence no: 9); for BRCA84D and CDR1 light chain (sequence no: 13); and CDR2 (sequence no. 15); and CDR3 (sequence no. 17); for the heavy chain of BRCA84D; And

(B) Modification of the Fc region includes substitutions: L235V, F243L, R292P, Y300L, and P396L.

The invention relates to the previously described pharmaceutical composition, where the antibody is compatible with humans and includes:

(a) Variable light chain with the amino acid sequence of hBRCA84D-2 VL (sequence no. 89);

(b) Variable heavy chain amino acid sequence of hBRCA84D-2 VH (sequence no. 99);

And

(c) The F c region has the substitution groups: L235V, F243L, R292P, Y300L, and P396L.

The invention relates to the aforementioned pharmaceutical compositions, which also comprise one or more additional anticancer agents, in particular where the additional anticancer agent is a chemotherapeutic agent, a therapeutic radiation agent, or a hormonal therapeutic agent. agent, toxin, or immunotherapeutic agent.

The invention also relates to the use of any of the previously described antibodies, their immunoreactive fragments, or dual affinity retargeting agents (DARTs) in cancer diagnosis, wherein the isolated antibody, its immunoreactive fragment, or DART are detectably numbered.

The invention relates to the aforementioned use, wherein a cancer is characterized by the presence of a cancer cell selected from the group including an adrenal tumor cell, AIDS-associated cancer, alveolar sarcoma of the soft segment, astrocytic tumor, bladder cancer, carcinoma Bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumors, Cervical cancer, chondrosarcoma, dhordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, cutaneous benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewings tumor, sarcoma Extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder or bile duct cancer, stomach cancer, gestational trophoblastic disease, Germ cell tumor, head and neck cancer, liver cancer, hepatocellular carcinoma, islet cell tumor hepatocellular carcinoma, Kaposis Sarcoma, kidney cancer, leukemia, leukemia, lipoma/benign lipomatous tumor, liposarcoma/malignant lipomatous tumor, liver cancer, cancer lymphoma cancer, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor tumor, pediatric cancer, peripheral nerve sheath tumor, phaeochromocytoma, adenoma pituitary tumor, prostate cancer, posterior melanoma, rare hematologic disorder, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma -tissue sarcoma, squamous cell cancer, stomach cancer, synovial sarcoma, testicular cancer, thymus cancer Thymic carcinoma, thymoma, thyroid metastatic cancer, uterine cancer.

The invention relates to the use of any of the previously described antibodies, their immunoreactive fragments, or dual affinity retargeting agents (DARTs) in the preparation of a drug for the treatment or prevention of cancer in a patient. The invention also relates to those uses in which the cancer is characterized by the presence of a cancer cell selected from the group including an adrenal tumor cell, AIDS-associated cancer, alveolar sarcoma of the soft segment, astrocytic tumor, bladder cancer, Bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, and carotid body tumors tumors, cervical cancer, chondrosarcoma, dhordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign cutaneous fibrous histiocytoma benign fibrous histiocytoma, desmoplastic small round cell tumor, ependymoma, Ewings tumor, Extraskeletal myxoid chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia of the bone, gallbladder or bile duct cancer, stomach cancer, gestational trophoblastic disease , germ cell tumor, head and neck cancer, liver cancer, hepatocellular carcinoma, cell tumor hepatocellular carcinoma, Kaposis Sarcoma, kidney cancer, leukemia, leukemia, lipoma/benign lipomatous tumor, liposarcoma/malignant lipomatous tumor, liver cancer, Lymphoma cancer, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, phaeochromocytoma, tumor pituitary tumor, prostate cancer, posterior melanoma, rare hematologic disorder, renal metastatic cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma soft-tissue sarcoma, squamous cell cancer, stomach cancer, synovial sarcoma, testicular cancer, adenocarcinoma Thymic carcinoma, thymoma, thyroid metastatic cancer, uterine cancer.

The invention also relates to the previously described uses, which are characterized by the fact that the use also includes the administration of one or more additional cancer treatments selected from the group consisting of chemotherapy, immunotherapy, radiotherapy, hormonal therapy, and surgery.

Brief explanation of the drawings

Figures 1a-b show the results of IHC investigations performed using normal tissue samples from pancreas, liver, lung, and colon with BRCA84D at concentrations of 0.625 μg/ml and 0.078 μg/ml (Figure 1a). and normal tissue from heart, kidney, and adrenal with BRCA84D at a concentration of 625 μg/ml (Figure 1B).

Figure 2 shows the results of IHC studies performed using cancerous pancreas, chest, colon, and lung tissue samples with BRCA84D at concentrations of 0.625 μg/ml and 0.078 μg/ml.

Figure 3a-d illustrates dose-dependent redirected killing caused by the antibodies of the invention. Figures 3A-B show redirected killing of A498 renal adenocarcinoma cells (along with remaining PBMC at 18 h (LDH)) by cross-reactive monoclonal antibodies against B7-H3 (effector:target ratio 20:1). (Figure 3a: BRCA68D, BRCA69D, PRCA157, GB8, TCR-4420; and Figure 3b: OVCA22, BRCA84D, TDH6, TES7, TCR-4420). Figures 3C–3D show dose-dependent redirected killing of A549 lung adenocarcinoma cells (along with remaining PBMC at 18 h (LDH)) by cross-reactive monoclonal antibodies against B7-H3 (effector:target ratio 30:1). (Figure 3c: BRCA84D, OVCA22, PRCA157, TES7; and Figure 3d: TDH6, BRCA68D, BRCA6).

Figures 4A-4B show the abilities of anti-B7-H3 antibodies to bind to soluble B7H3-2Ig (Figure 4A) and soluble B7H3-4Ig (Figure 4B) (antibody concentration 100 nM). Explanatory list: (A) BLA8; (B) BRCA165; (C) BRCA68D; (D) BRCA69D; (E) BRCA84D; (F) GB8; (G) LUCA1; (H)LUCA50; (I) OVCA21; (J) OVCA22; (K)PA20; (L)PRCA123; (M)SG24; (N)SG27; (O)STO9; (P)TDH4(184-192); (Q) TDH4; (R)TDH5; (S)TES7. The vertical position of the list is related to the position of the corresponding curve.

Figures 5a–5s show the binding affinity between antigens in solution and the retained monoclonal antibodies (solid lines: B7-H3(4Ig) 100 nM; dashed lines; B7-H3, 100 nM).

Figures 6a–6i show the results of BIACORE analyzes for B7-H3 antibodies immobilized with B7-H3-2Ig (dashed gray lines) or B7-H3-4Ig (solid black lines). Antibodies were titrated from 0.063 μM to 1 μM. The time was in seconds.

Figure 7 provides a comparison of the BIACORE analysis for PRCA157, BRCA69D, BLA8, PA20, BRCA84D, GB8 and SG27 antibodies.

Figure 8 provides BIACORE analyzes showing that BRCA68D, BRCA69D, and PRCA157 antibodies do not compete with BRCA84D for binding to human B7-H3.

Figures 9a-9b show the results of studies on the ability of anti-B7-H3 antibodies of the present invention to be internalized onto cancer cells (Figure 9a, prostate CSCs; and Figure 9b, pancreatic Hs700t cells).

Figures 9a-9b illustrate the ability of the B7-H3 antibodies of the present invention to cross-block each other and thus detect overlapping or separate binding peaks. A tenfold excess amount of the competitor antibody was used.

Figures 11a-11b show the stacking of amino acid residues of the variable light chains (Figure 11a) or variable heavy chains (Figure 11b) of BRCA84D and its humanized derivative hBRCA84D.

Figure 12 shows the relative binding affinity of the hBRCA84D light chain derivatives BRCA84D-3VL, BRCA84D-4VL, and BRCA84D-5VL for human B7-H3.

Figure 13 shows the relative binding affinity of the hBRCA84D heavy-chain derivatives BRCA84D-2VH, BRCA84D-3VH, and BRCA84D-4VH for human B7-H3.

Figure 14 shows the relative binding affinities of (1) antibodies containing hBRCA84D-2VL and hBRCA84D-2VH (experiments 1 and 2), (2) chimeric BRCA84D, and (3) antibodies containing chimeric hBRCA84D-5VL and BRCA84D-HC. and (4) antibody containing hBRCA84D-5V and hBRCA84D-2VH.

Figure 15 illustrates the ability of humanized Fc-modifying anti-B7-H3 antibody to inhibit tumor growth of HT-1197 tumor cells in vivo in a murine allograft model system. The Fc-modified hBRCA84D-2 antibody (comprising the Fc modifications L235V, F243L, R292P, Y300L, and P396L) was administered to mice (at a dose of 1 μg/kg, 10 μg/kg, or 20 μg/kg) after transplantation. Cancer cells at 7 days, 14 days, 21 days, and 28 days.

Figure 16 shows the ability of humanized Fc-modified anti-B7-H3 antibodies to inhibit tumor growth of renal adenocarcinoma cells in vivo in a murine allograft system. The Fc-modified hBRCA84D antibody (comprising modifications L235V, F243L, R292P, Y300L, and P396L) was administered to mice (at a dose of 1 μg/kg, 10 μg, or 20 μg/kg) 7 days after cancer cell culture. 14 days, 21 days, and 28 days.

Figures 17A-17D show the ability of hBRCA84D-2/TCR antagonist DART to cause redirected killing of lung cancer SK-MES-1 cells, A498 renal carcinoma cells, prostate cancer LNCaP cells, and UACC-melanoma cells. 62.

Figures 18a-18c show the pharmacokinetic decay of the monoclonal antibody B7-H3 Mab1 in the sera of tumor-free male mCD16-/-, hCD16A_FOXN1 mice (Figures 18a-18b). Figure 18c shows the expected pharmacokinetic properties resulting from using a two-compartment model with variants of 5 mg/kg at concentrations of 0.1, 0.5, 1, 5, and 10 mg/kg.

Figure 19 shows the relative expression of HER2 and PRCA135 by bladder cancer strain HT-1197.

Figure 20 shows the photo-binding affinity of hBRCA84D for anti-B7-H3 antibody to HT-1197 cells.

Figures 21a-21c show the results of the murine allograft analysis of HT-1197. A group of 8 female mice received vector, 10 mg/kg IgG comparator, cintuximab at a dose of 1, 5, or 15 mg/kg or the anti-B7-H3 antibody Mab1 at a dose of 0.1, 0.5, 1, 5, or 10 mg/kg (Q7D 5). The tumor was measured every 3-4 days. Figure 21a shows the ability of the anti-B7-H3 antibody Mab1 to prevent or inhibit tumor growth in a mouse allograft model. The IgG versus IgG comparison is shown: Mab1 (1 and 5 mg/kg) versus IgG comparison *** from day 51; and Mab1 (10 mg/kg) vs. IgG comparison ** from day 48. Figure 21B shows the ability of cintuximab to prevent or inhibit tumor growth in a murine allograft model. Centuximab (7 mg/kg) versus IgG comparator** from day 51; and cintuximab (15 mg/kg) versus IgG comparator *** from day 58. Figure 21c compares the results obtained at the maximum doses tested.

Figures 22a-22b show the relative expression of HER2 and PMSA by bladder cancer strain HT-1376.

Figure 23 shows analysis of the murine allograft of HT-1376. Groups of mice received either the vector or 1 mg/kg of anti-B7-H3 antibody Mab1 (Q7D 4).

Figure 24 shows the results of the analysis of murine allografts for AGS. Groups of mice received either the vector or 10 mg/kg of the anti-B7-H3 antibody Mab1 at doses of 0.5, 1, or 5 mg/kg (Q7D 5).

Figure 25 shows the results of an in vitro cytotoxicity experiment for lung cancer A549 cells when incubated with hBRCA84D, chBRCA84D, and hBRCA84 (Fc Var1) anti-B7-H3 antibodies (E:T ratio = 25:1, effector = human PBMC; And read the LDH experiment).

Figure 26 shows analysis of the murine allograft of A549. Groups of mice received either the vector or 1 mg/kg of anti-B7-H3 antibody Mab1 (Q7D 4).

Figure 27 shows the results of the analysis of the mouse allograft for CaLu3. Groups of mice received either the vector or 0.5, 1, or 5 mg/kg of anti-B7-H3 Mab1 antibody (Q7D 5) or IgG conjugate (10 mg/ml).

Figures 28a-28c show the results of analysis of mouse allografts for LOX-IMVI melanoma cells. A group of 8 female mice received the vector, 5 mg/kg/conjugate IgG, ecetaxel at a dose of 5, 10, or 20 mg/kg, or anti-B7-H3 Mab1 antibody at a dose of 0.5, 1, 5, or 10 mg/kg. kg. Figure 28 shows the ability of the anti-B7-H3 antibody Mab1 to prevent or inhibit tumor growth in a mouse allograft model. Figure 28b shows the ability of docetaxel to prevent or inhibit tumor growth in a mouse allograft model. Figure 28c compares the results obtained at the maximum doses tested.

Figure 29 shows the results of analysis of mouse allografts of UACC-62 melanoma cells. Groups of mice received vector, 5 mg/kg IgG comparator, or murine allograft at a dose of 0.5, 1, 5, or 10 mg/kg.

Figures 30a-30c show the results of analysis of mouse allografts of prostate cancer 2rv cells. Groups of 8 female mice received the vector or 10 mg/kg IgG comparator, trastuzumab at a dose of 1, 7, or 15 mg/kg or the anti-B7-H3 antibody Mab1 at a dose of 0.5, 1, 5, or 10 mg/kg. ...Figure 30a shows the ability of the anti-B7-H3 antibody Mab1 to prevent or inhibit tumor growth in a mouse allograft model. Figure 30 shows the ability of trastuzumab to prevent or inhibit tumor growth in a mouse allograft model. Figure 28c compares the results obtained at the maximum doses tested.

Figure 31 shows the results of an in vitro cytotoxicity experiment for kidney cancer A498 cells when incubated with hBRCA84D, chBRCA84D, and hBRCA84 (Fc Var1) anti-B7-H3 antibodies (E:T ratio = 25:1, effector = human PBMC; And read the LDH experiment).

Figure 32 shows the result of analysis of the mouse allograft of kidney cancer A498 cells. Mice groups received the vector, 10 mg/kg of IgG comparator, or anti-B7-H3 antibody at a dose of 0.1, 0-.5, 1, 5, or 10 mg/kg. A control group of mice was given cintuximab (anti-EGRF antibody) at doses of 1, 7, or 15 mg/kg.

Figures 33a-33b Results of murine allograft analysis of 786-0 renal carcinoma cells compared to cintuximab. Mice groups received the vector, 10 mg/kg IgG conjugate, or anti-B7-H3 Mab1 antibody at a dose of 0.1, 0.5, 1, 5, or 10 mg/kg. Cintuximab (anti-EGRF antibody) was administered to the comparison group of mice at doses of 1, 7, or 15 mg/kg.

Figure 34 shows the result of a murine allograft analysis of 786-0 renal cancer cells compared to paclitaxel. Mice groups received the vector, 5 mg/kg IgG comparator, or the anti-B7-H3 antibody Mab1 at a dose of 0.1, 0.5, 1, or 10 mg/kg. A comparison group of eight rats was given paclitaxel at 2.5 mg/kg.

Detailed description

The present invention relates to antibodies and fragments thereof that are immunologically cross-reactive with a mammalian, especially human, B7-H3 receptor, and their uses, particularly in the treatment of cancer or inflammation. The invention relates to humanized B7-H3-reactive antibodies and their immunoreactive fragments capable of causing, and preferably enhancing, activation of the immune system against cancer cells associated with many types of human cancer.

1- General methods

Unless otherwise stated, implementation of the present invention entails the use of conventional methods known in molecular biology (including gene resequencing methods), microbiology, cell biology, biochemistry, and immunology, as are within the skill of the art. These methods are fully explained in monographs, such as Molecular Cloning: A Laboratory Manual, Third Edition (Sambrook et al. Eds., 2001) Cold Spring Harbor Press, Cold Spring Harbor, NY; Oligonucleotide Synthesis: Methods and Applications (Methods in Molecular Biology), Herdewijn, P., Ed., Humana Press, Totowa, NJ; Oligonucleotide Synthesis (Gait, M.J., Ed., 1984); Methods in Molecular Biology, Humana Press, Totowa, NJ; Cell Biology: A Laboratory Notebook (Cellis, J.E., Ed., 1998) Academic Press, New York, NY; Animal Cell Culture (Freshney, RI, Ed., 1987); Introduction to Cell and Tissue Culture (Mather, JP and Roberts (P.E., Eds., 1998) Plenum Press, New York, NY; Cell and Tissue Culture: Laboratory Procedures (Doyle, A. et al., Eds., 1993-8) John Wiley and Sons, Hoboken, NJ; Methods in Enzymology (Academic Press, Inc.) New York, NY; Weirs Handbook of Experimental Immunology (Herzenberg, LA et al. Eds. 1997) Wiley-Blackwell Publishers, New York, NY; Gene Transfer Vectors for Mammalian Cells (Miller, J.M. et al. Eds., 1987) Cold Spring Harbor Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (Ausubel, F.M. et al., Eds., 1987) Greene Pub. Associates, New York, NY; PCR: The Polymerase Chain Reaction, (Mullis, K. et al., Eds., 1994) Birkhuser, Boston MA; Current Protocols in Immunology (Coligan, J.E. et al., eds., 1991) John Wiley and Sons, Hoboken, NJ; Short Protocols in Molecular Biology (John Wiley and Sons, 1999) Hoboken, NJ; Immunobiology 7 (Janeway, CA et al. 2007) Garland Science, London, UK; Antibodies (P. Finch, 1997) Stride Publications, Devoran, UK; Antibodies: A Practical Approach (D. Catty., ed., 1989) Oxford University Press, USA, New York NY); Monoclonal Antibodies: A Practical Approach (Shepherd, P. et al. Eds., 2000) Oxford University Press, USA, New York NY; Using Antibodies: A Laboratory Manual (Harlow, E. et al. Eds., 1998) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; The Antibodies (Zanetti, M. et al. Eds. 1995) Harwood Academic Publishers, London, UK); and DeVita, Hellman, and Rosenberg's Cancer: Principles & Practice of Oncology, Eighth Edition, DeVita, V. et al. Eds. 2008, Lippincott Williams & Wilkins, Philadelphia, PA.

2- Definitions

As used in the present document, the term B7-H3 refers to a member of the human B7 family of proteins, a type 1 member protein with Ig-like domains also known as CD276. The term 2Ig-B7-H3 refers to a B7-H3 form that includes only two Ig-like domains (see, Sun, M. et al. (2002) Characterization of Mouse and Human B7-H3 Genes, J. Immunol. 168:6294 -6297;Steinberger et al. (2004), Molecular Characterization Of Human 4Ig-B7-H3, A Member Of The B7 Family With Four Ig-Like Domains, J. Immunol. 2004, 172(4):2352-2359 and Castriconi et al. (2004) Identification Of 4Ig-B7-H3 As A Neuroblastoma-Associated Molecule That Exerts A Protective Role From An NK Cell-Mediated Lysis, Proc. Natl. Acad. Sci. (USA) 101(34):12640-12645). The TES7 antigen (WO 2008/066691) is an antigen that shares properties of 4Ig-B7-H3. Thus, antibodies that specifically bind to TES7 bind to 4Ig-B7-H3. The TES7 antigen may contain more than one different epitope, and the epitope may be nonlinear. Many anti-B7-H3 antibodies are known to bind to nonlinear epitopes, some of which are found only to the 4Ig-B7-H3 isotype. It is currently accepted that TES7 may be overexpressed in certain cancer cells compared to their counterparts from normal tissues.

Adjuvants, antagonists, and modifiers of B7-H3 function are clearly within the scope of the present invention. These adjuvants, antigens, and modifiers are polypeptides comprising one or more specific B7-H3 antigen sites, or comprising one or more fragments, images, or peptide mimetics of those sites. B7-H37-modifying compounds are provided in linear or cyclic form, optionally comprising at least one amino acid unit not normally found in nature or at least one amide isostere. These compounds can be treated with glycosylation.

More specifically, the term “B7-H3 modifier” as used in the present document is defined as any compound that (i) is capable of inactivating or inhibiting the interaction between human B7-H3 and its native binding molecule or anti-B7-H3 antibody; (2) capable of binding to human B7-H3 and its native binding molecule or anti-B7-H3 antibody; (3) contains an antigenic locus that can be used to elicit antibodies capable of binding to human B7-H3 and its native binding molecules or anti-B7-H3 antibody; and (4) contains an antigenic locus that can be used in screening for antibodies capable of Binding to human B7-H3 and its native binding molecules or anti-B7-H3 antibody; (v) contains an antigenic locus that can be used to elicit antibodies capable of inactivating or blocking the interaction between human B7-H3 and its native binding molecules or anti-B7-H3 antibody; (6) Contains an antigenic locus that can be used to screen for antibodies capable of inactivating or blocking the interaction between human B7-H3 and its native binding molecules or the anti-B7-H3 antibody. B7-H3 modulators can be “B7-H3 adjuvants” or “B7-H3 antagonists” depending on whether their activity enhances T cell activation or inhibits T cell activation, respectively.

B7-H3 adjuvants, antagonists, and modulators include B7-H3 forms, B7-H3 peptide antagonists, peptide mimetics, small molecules, anti-B7-H3 antibodies, immunoglobulin forms, and amino acid forms of human B7-H3 such as amino acid substitutions. , deletions, additions, or any combination thereof, and immunoglobulins. The B7-H3 adjuvants, B7-H3 antagonists and modifiers of the present invention are based on identifying B7-H3 domains present in the binding of human B7-H3 to its native binding molecules or B7-H3 antagonists. Thus, the invention provides B7-H3 adjuvants, B7-H3 antagonists and modifiers with molecular structures that duplicate or mimic one or more of the anti-B7-H3 binding domains of human B7-H3.

As used herein, “B7-H3 motif” means any amino acid motif of human B7-H3 including amino acid substitutions, deletions, insertions, or any combination thereof. The definition includes chimeric molecules such as human/non-human B7-H3 chimeras and other hybrid molecules. The definition also includes any fragment of a B7-H3 image molecule that includes the image or heterohybrid region(s) of the molecule.

As used in the present document, the term “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc. This is through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used in the present document, the term includes not only intact monoclonal or polyclonal antibodies, but also their fragments (such as Fab, Fab', and F(ab')2 Fv), their monoclonal sequences, their mutants, their natural forms, and their fusion proteins. Comprising a portion of an antibody with an antigen recognition locus for the specificity, humanized antibodies, chimeric antibodies, BiTEs, DART molecules and any other modified body of the immunoglobulin molecule comprising the antigen recognition locus for the specificity.

The term BiTEs (bispecific T cell enantiomers) refers to a single polypeptide chain molecule with two antigen-binding domains, one binding to the T cell antigen and the other to an antigen present on the surface of the target (WO 05/061547; Baeuerle, P et al. (2008) BiTE: A New Class Of Antibodies That Recruit T Cells, Drugs of the Future 33: 137-147; Bargou, et al. 2008) Tumor Regression in Cancer Patients by Very Low Doses of a T Cell-Engaging Antibody, Science 321: 974 -977).

The term DART (dual affinity retargeting agent) refers to an immunoglobulin molecule comprising at least two polypeptide chains that combine (particularly by a divalent interaction) to form at least one epitope-binding site, where it may recognize the same or Different adhesive. Each DART polypeptide chain contains an immunoglobulin light-chain variable region and an immunoglobulin heavy-chain variable region, but these two regions do not interact to form a binding site for the epitope. Rather, the immunoglobulin heavy-chain variable region interacts on one (as if the first ) of DART polypeptide chains with the immunoglobulin light-chain variable region of a different DART polypeptide chain (such as the second) to form an antigen-binding position. Also, the immunoglobulin light-chain variable region of one DART polypeptide chain interacts with the heavy-chain variable region of a different DART polypeptide chain (such as the second) to form a binding site for the epitope. DARTs may be mono-specific, bi-specific, tri-specific, etc., and thus able to bind simultaneously to one, two, or three different epitopes (which may be the same or different antigens). DARTs can additionally be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, etc. Then it is able to bind at the same time to one, two, three, four, five, six or more particles. These two characteristics of DARTs (i.e. specificity and equivalence) can be combined, for example to produce antibodies that are bispecific (i.e. able to bind to two epitopes), quadrivalent (i.e. able to bind to four sets of epitopes), etc. DART molecules are disclosed in PCT Publications 113665/2006, 157379/2008, and 080538/2010.

The term "monoclonal antibody" refers to a homogeneous antibody. The monoclonal antibody consists of amino acids (natural and unnatural) that engage in selective binding to the antigen. Monoclonal antibodies are highly specific, as they are directed against a single site of antigen. The term “monoclonal antibody” includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also their fragments (such as Fab, Fab', and F(ab')2 Fv), their single-chain, mutations, and natural forms , fusion proteins thereof comprising an antibody portion, humanized antibodies, chimeric antibodies, and any other modified form of the immunoglobulin molecule comprising an antigen recognition locus for the necessary specificity and ability to bind to an antigen. The aim is not to be limited to the source of the antibody or the method of its preparation (e.g. by hybridomas, phage selection, recombinant expression, transgenic animals, etc.) and the term includes all immunoglobulins and their fragments, as previously described when defining “antibody”. .

The term “humanized antibody” refers to a chimeric molecule, usually prepared using gene recombination techniques, that has an antigen-binding site from a non-human immunoglobulin and the remainder of the immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin. The antigen-binding site may comprise either fully variable domains fused to constant domains or simply complementarity-determining regions (CDRs) grafted onto appropriate frame regions in the variable domains. The antigen binding sites may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immune gene in humans, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A. F. et al. (1989) Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response, Proc. Natl. Acad. Sci. (USA) 86:4220-4224). Other approaches focus not only on providing fixed, human-derived regions, but on modifying variable regions as well as reshaping them as closely as possible to the human image. The variable regions of both heavy and light chains are known to contain three complementary determining regions (CDRs) that range in response to the antigens of interest and determine binding capacity, flanked by four frame regions (FRs) that are relatively conserved in a given species and which are supposed to provide scaffolding. For CDRs. When preparing non-human antibodies for a particular antigen, the variable regions can be “recombined” or “humanized” by grafting CDRs derived from the non-human antibody onto the FRs present in the human antibody to be modified. Applications of this approach to antibodies are presented in Sato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) Reshaping Human Antibodies for Therapy, Nature 332:323-327; Verhoeyen, M. et al. (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239:1534-1536; Kettleborough, CA et al. (1991) Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation, Protein Engineering 4:773-3783; Maeda, H. et al. (1991) Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity, Human Antibodies Hybridoma 2:124-134; Gorman, S. D. et al. (1991) Reshaping A Therapeutic CD4 Antibody, Proc. Natl. Acad. Sci. (USA) 88:4181-4185; Tempest, P. R. et al. (1991) Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in Vivo, Bio/Technology 9:266-271; Co, M.S. et al. (1991) Humanized Antibodies For Antiviral Therapy, Proc. Natl. Acad. Sci. (USA) 88:2869-2873; Carter, P. et al. (1992) Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy, Proc. Natl. Acad. Sci. (USA) 89:4285-4289; and Co, MS et al. (1992) Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen, J. Immunol. 148:1149-1154. In some embodiments, the humanized antibodies preserve all CDR sequences (e.g., a humanized murine antibody containing all six CDRs of the human antibodies). In other embodiments, the humanized antibodies contain one or more CDRs. (one, two, three, four, five, six) are modified relative to the original antibody, where one or more CDRs are “derived from” one or more CDRs of the original antibody.

As used herein, an antibody or polypeptide is thought to “specifically” bind a region of another molecule (i.e., an epitope) if it interacts or binds more frequently, or more rapidly, with greater duration and/or greater affinity with that epitope. Adhesive compared to alternative adhesive tops. For example, an antibody that specifically binds to a B7-H3 epitope is an antibody that binds to that B7-H3 epitope with greater affinity, avidity, ease, and/or longer duration than it binds to other B7-H3 epitope. Or non-sticky B7-H3 peaks. It is also understood from reading this definition that, for example, an antibody (or cleft or epitope) that specifically binds to a first target may or may not bind specifically or preferentially to a second target. Therefore, a “specific link” need not necessarily have (although it may contain) an exclusive link. Generally, but not necessarily, a reference to a link means a “specific” link.

As used throughout the present document, the term “immunoactive” in reference to an epitope that is or “remains immunologically active” refers to the ability of an antibody (eg, anti-B7-H3 antibody) to bind to an epitope under various conditions, for example, after exposure The adhesive top refers to conditions of reducing or removing the natural properties.

Various biological functions are associated with B7-H3 antibodies, including, but not limited to, one or more of the following: The ability to bind specifically to B7-H3 (particularly B7-H3 molecules expressed on the surface of cancer cells, such as, but not limited to, kidney, prostate, or lung cancer cells); the ability to competitively bind to preferentially bind an anti-B7-H3 antibody known as B7-H3, including the ability to preferentially bind to the same epitope of B7-H3 to which the antibody preferentially binds; the ability to bind to a B7-H3 fragment exposed on the surface of a living cell in vitro or in vivo; And the ability to relate to a part B7-H3 expressed on the surface of living cancer cells, such as, but not limited to, prostate, lung, or kidney cancer cells; the ability to deliver a chemotherapy agent to cancer cells (such as kidney, prostate, or lung cancer cells) that express B7-H3 on their surfaces; and/or the ability to deliver a therapeutic agent or landmark that can be detected in cancer cells expressing B7-H3 on their surface. As previously noted, the polypeptides (as well as antibodies) of the invention may have one or more of these properties.

The term “anti-B7-H3 equivalent antibody” or “anti-B7-H3 equivalent polypeptide” refers to an antibody or polypeptide having one or more biological functions associated with an anti-B7-H3 antibody, such as binding recognition.

As used throughout the present document, the term “agent” refers to a biological, pharmaceutical, or chemical compound. Non-restricted examples include a simple organic and inorganic molecule and a compound, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, an antibody fragment, a vitamin derivative, a carbohydrate, and a toxin or therapeutic agent compound. Many compounds can be synthesized, for example, small molecules, oligomers (such as oligopeptides and oligonucleotides), and synthetic organic compounds based on various core structures. In addition, many natural sources can provide compounds for screening, such as plant or animal extracts, etc.

The agents used in the method of the present invention may be randomly selected, logically selected or designed. As used in the present document, an agent is considered to have been selected at random when it is selected without prior consideration or knowledge of the amino acid or other chemical moiety involved in the binding of the molecule to its original binding partner(s) or known antibodies. An example of a randomly selected agent is an agent that is identified through the use and screening of a chemical or peptide combinatorial library.

As used in the present document, an agent is considered to have been rationally selected or designed when it is selected on a non-random basis that takes into account the arrangement and/or configuration of the target site in relation to the effect of the agent. For anti-B7-H3 agents, it is currently believed that there are at least three epitopes on B7-H3 against which antibodies can be raised and thus three sites of action for agents that block the interaction between B7-H3 and anti-B7-H3. The invention also includes factors that interact on the interaction sites between B7-H3 and its original binding partner, although there are other binding molecules and their active sites interacting with B7-H3 that fall within the scope of the present, whether currently known or to be determined later. Agents may be randomly selected or logically designed using peptide sequences that form contact sites for the receptor/binding molecule complex and/or the B7-H3/anti-B7-H3 antibody complex. For example, a randomly selected peptide agent could be a peptide whose amino acid sequence Identical to the epitope appearing on B7-H3 as expressed on the surface of a live cell in the native environment. This agent will reduce or inhibit the binding of the anti-B7-H3 antibody to B7-H3 or the binding of B7-H3 to its native binding molecule, as desired, by binding to the anti-B7-H3 antibody or to its native binding molecule.

As used throughout the present document, the term “labeled,” for an antibody, includes direct labeling of the antibody by conjugating (i.e., physical binding) to a detectable substance, such as a radioactive agent or fluorescence electrophoresis (such as physoerythrin (PE) or fluorescein). Isocyanate (also known as fluoroisocyanate or FITC) of the antibody, as well as indirect labeling of the probe or antibody by reactivity with an detectable substance.

As used throughout the present document, the term “binding” to an antibody includes covalent and noncovalent attachment or attachment of an agent (such as a chemotherapeutic agent) to the antibody. An antibody may bind to an agent (such as a chemotherapy agent) by binding directly or indirectly by contact with a common group, such that the antibody directs the localization of the agent to the cancer cell to which the antibody is bound and where the antibody does not essentially dissociate under physiological conditions so that the agent is not targeted to the same The cancer cell to which the antibody binds so that the strength of the agent is not increased.

The term “biospecimen” includes several types of samples that are obtained from an individual and can be used in a diagnostic or monitoring trial. Identification includes saliva, blood, and other liquid samples of biological origin, solid tissue samples, such as a sample taken from a patient or tissue cultures or cells taken from them, and their products, such as cells obtained from a tissue sample taken from an individual with possible cancer. , and in preferred tissue models of ovary, lung, prostate, pancreas, colon, and chest. The definition also includes samples that have been processed in any way after purchase, such as treatment with reagents, solubilization, enrichment of certain components, such as proteins or polynucleotides, or burying in a semi-solid or solid matrix for partitioning purposes. The term “biological specimen” includes a clinical specimen, as well as cells in culture, cell supernatants, cell lysis product, serum, plasma, biofluid, and tissue specimens.

The term “family cell” also includes an individual cell or cell culture that is or has been a recipient of the vector(s) for insertion of polynucleotide ligands. Host cells include the product of a single host cell, and the results may not necessarily be an exact match (in form or complement of genomic DNA) to the original parent cell due to natural, accidental, or intentional mutation. The host cell includes cells that have been infected within the body of the organism using the polypeptide(s) of the present invention.

As used in the present document, the term “delaying the progression of an outbreak” means postponing, impeding, slowing down, delaying, stabilizing, and/or postponing an outbreak. This delay can be of different lengths of time, depending on the history of the cancer and/or the person being treated. As is clear to someone skilled in the art, a delay that is sufficient or obvious can include prevention, so that the person does not develop the outbreak and spread.

As used in the present document, the term “effective amount” of a pharmaceutical composition, in one embodiment, is the amount sufficient to produce the beneficial effect or desired outcome, including, but not limited to, clinical outcomes such as tumor volume shrinkage (e.g. in the context of cancer , breast cancer, or prostate), impeding the growth of the cancer cell, delaying the growth of the tumor outbreak, reducing the symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, reducing the dose of other medications needed to treat the disease, enhancing the effect of another drug, for example by By targeting and/or internalizing, affecting disease progression, and/or prolonging survival of individuals. The effective amount may be given in one or more administrations. For the purposes of the present invention, the effective amount of the drug, compound, or pharmaceutical composition is an amount sufficient to reduce the proliferation of (or destroy) cancer cells and reduce and/or delay the development or growth of cancer cell outbreaks, either directly or indirectly. In some embodiments, an effective amount of the drug, compound, or pharmaceutical composition can be achieved with another drug, compound, or pharmaceutical composition. Therefore, an “amount may be considered effective” in the context of the administration of one or more therapeutic agents, and a single agent administered in an amount may be considered effective if, in combination with one or more other agents, a desired result can be achieved. Although needs may vary, determining the optimal values ​​of effective quantities for each component falls within the skill of the field. Typical doses include 0.1 to 100 mg/kg/body weight. Preferred doses include 1 to 100 mg/kg/body weight. The best preferred dose includes 10 to 100 mg/kg/body weight.

As used in the present document, a DNA fragment or agent, antibody, construct, cell, etc., is considered "isolated" when it is separated from contaminating DNA molecules, antibodies, agents, constructs, cells, etc. contained in the original source.

The term "individual" refers to a vegetarian animal, preferably a mammal. Mammals include, but are not limited to, humans, farm animals, domestic animals, primates, mice, and rats. In the best examples, the term individual refers to a human being.

The terms "polypeptide", "oligopeptide", and "peptide" are used interchangeably to refer to polymers of amino acids of any length. The polymer may be straight or branched, may include modified amino acids, or may be hindered by non-amino acids. The terms also include an amino acid polymer that has been modified naturally or by intervention; For example, disulfide bond formation, glycosylation, lipolysis, acetylation, phosphorylation, or other intervention or modification, such as conjugation with a numbering component. Also included under this definition are, for example, polypeptides containing one or more amino acid analogues (such as unnatural amino acids, etc.), and other modifications known in the art. It is understood that, because the polypeptides of the present invention are antibody-based, the polypeptides may occur as single chains or as linked chains.

Also within the scope of the present invention are peptidomimetics of B7-H3 peptide adjuvants, antagonists, and modulators (such as anti-B7-H3 antibodies) contained in the present document. These peptidomimetics include peptides in which one or more amino acid residues are replaced by an amino acid residue not normally found in nature, such as the D-isomer of the amino acid or an N-alkyl species of the amino acid. In other embodiments, the peptidomimetics are generated by substituting at least one amide bond (−C(=O)−NH−) in a co-amide, antagonist, or modified B7-H3 amide peptide. Examples of amide enantiomers include CH2−NH−, CH2−S−, CH2−S(O)−, CH2−S(O)2−, CH2−CH2−, CH=CH− (as E or Z), −C( =O)−CH2−, CH(CN)−NH−, C(OH)−CH2−, and −O−C(=O)−NH−. The amide bonds in the B7-H3 adjuvant, antagonist, or modifier are suitable candidates for replacement with amide enantiomers. The bonds are hydrolyzable by endogenous enantiomers or proteases of the subject for processing with the B7-H3 adjuvant, antagonist, or modifier.

As used in the present document, the term “substantially pure” refers to a substance that is at least 50% pure (i.e. free from impurities), preferably at least 90% pure, preferably at least 95% pure, preferably at least 90% At least 98%, and preferably 99% or more.

As used in the present document, “toxin” refers to any substance that induces an adverse response within a cell. For example, a toxin directed at a cancer cell will have an adverse, and sometimes dangerous, effect on the cancer cell. Examples of toxins include, but are not limited to, taxanes, metansinoids, and auristatin (e.g., monomethyl auristatin (MMAE), monomethyl auristatin F (MMAF), auristatin E (AE), etc.) (such as these Which are disclosed in US Patent Nos. (5208020; 5416064; 6333410; 6340701; 6372738; 6436931; 6441163; 6596757; 7267497; 75857; or 7851432), and an anthracycline (e.g., doxo-rubi Syn), CC-1065 analog, and Docetakel ;mathepsin B or E; ricin, and gelonin gelonin, Pseudomonas exotoxin, diphtheria toxin, and RNase; and radiolabeled antibodies (e.g., conjugated to peptioacetane or labeled with a radioactive isotope (e.g. 90Y; 131I, 177Lu, 186Re, 188Re, 211At, 212Bi, 213Bi, 225Ac, etc.).

As used throughout the present document, the term “treatment” or “treatment” refers to a method of obtaining a beneficial or desired result including and preferably a beneficial or desired clinical result. These beneficial or desired results include, but are not limited to, one or more of the following: reducing the proliferation (or destruction) of cancer cells, or other diseased cells, reducing the spread of cancer cells present in any type of cancer, reducing the size of the tumor, reducing the resulting symptoms. treatment of the disease, increasing the quality of life for those with the disease, reducing the dose of other medications needed to treat the disease, delaying the progression of the disease, and/or prolonging the survival of individuals.

As used in the present document, the term carcinoma includes cancers characterized by the presence of a cancer cell selected from the group composed of an adrenal tumor cell, AIDS-associated carcinoma, soft alveolar carcinoma, astrocytoma, and bladder cancer. Squamous and transitional cell carcinoma), brain and spinal cord cancer, metastatic brain tumor, breast cancer, arterial body tumors, cervical cancer, chondroma, and dhordoma, chromatophobic renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign dermatofibrous histiocytoma, small round cell tumor, ependymoma, Ewing's tumor, skeletal mucinous chondroma, fibrogenesis Osteochondrosis, fibrous bone dysplasia, gallbladder or bile duct cancer, stomach cancer, trophoblastic disease of pregnancy, germ cell tumor, and head and neck cancer, liver cancer, hepatocellular carcinoma, Kaposi's sarcoma, kidney cancer (nephroblastoma, papillary renal cell carcinoma), leukemia, lipoma, lipoma/malignant lipoma, liver cancer ( Hepatoblastoma, liver cancer, lymphoma, lung cancer, medulloblastoma, skin cancer, meningioma, multiple endocrine neoplasia, multiple myeloma and malignant syndrome. Myeloid growth, neurogenic tumors, neurohormonal tumors, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, phaeochromocytoma, pituitary tumor, prostate cancer, cancer Posterior non-acute skin cancer, rare blood disorder, renal metastatic cancer, rhomboid tumor, rhabdomyosarcoma, skin cancer, and sarcoma Soft tissue, squamous cell cancer, stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thymoma, thyroid cancer, uterine cancer (cervical cancer, endometrial cancer, tumor smooth muscle).

3- Methods of preparing antibodies and polypeptides

Methods for preparing monoclonal antibodies are known in the art. One of the methods used is the method of Koehler G. et al. (1975) Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity, Nature 256:495-497 or a modification thereof. Typically, monoclonal antibodies are developed in non-human species, such as mice. Generally, a mouse or rat is used for vaccination, but other animals may be used. Antibodies are produced by immunizing mice with an immunogenic amount of cells, cell extracts, or protein preparations containing B7-H3. The immunogen can be, but is not limited to, primate cells, cultured cell lines, cancer cells, nucleic acids, or tissues. In one embodiment, human lung cancer tumor cells are used. The cells used for immunization, for example, testicular cells, pancreatic adenocarcinoma or stomach cells, can be cultured for a period of time (eg at least 24 hours) before being used as an immunogen. Cells (such as testicular cells, pancreatic adenocarcinoma or stomach cells) can be used as immunogens on their own in combination with a non-denaturing adjuvant, such as Ribi. In general, cell integrity and preferably viability must be maintained when used as immunogens. Intact cells may allow detection of antigens better than cells disrupted by the immunized animal. The use of natural or dry deburring aids, such as Freud's Aid, may lead to cell disruption and is therefore not recommended. The immunogen can be given several times at periodic intervals, for example twice a week, or weekly, or it can be given in a way that maintains the animal's viability (for example, as a result of tissue reattachment).

In one embodiment, a monoclonal antibody that binds to B7-H3 is obtained by using host cells that overexpress B7-H3 as an immunogen. These cells include, but are not limited to, human lung cancer cells and human colon cancer cells.

To monitor an antibody response, a small biological sample (such as blood) may be obtained from the animal and tested for the antibody titer against the immunogen. Lymph nodes of the spleen and/or ovary may be removed and separated into individual cells. If desired, spleen cells can be examined (after non-specific adherent cells have been removed) by applying the cell suspension to an antigen-coated plate or speck. B cells, expressing an antigen-specific membrane-bound immunoglobulin, will attach to the dish, not sliding with the rest of the suspension. The remaining B cells, or detached spleen cells, can then be combined with myeloma cells (eg, X63-Ag8.653 and those from SaIk Institute, Cell Distribution Center, San Diego, CA). Polyethylene glycol (PEG) can be used to fuse spleen or lymphocytes with myeloma cells to form a hybridoma. The hybridoma is then cultured in a selective medium (such as hypoxanthin, aminopeptin, thymidine, or other media known in the art as HAT). The resulting hybridomas are then plated at a specific dilution, and tested for production of antibodies that specifically bind to the immunogen, using, for example, FACS (fluorescently activated cell storage) or immunohistochemistry (IHC) screening. Hybridomas secreting the selected monoclonal antibody are then grown either in vitro (eg in tissue culture flasks or hollow fiber reactors) or in vivo (eg ascites in mice).

As another alternative to the cell fusion method, Epstein-Eber-induced B cells can be used to produce the monoclonal antibodies of the present invention. If desired, hybridomas are expanded and subcloned, and the supernatants are tested for antigenic activity using conventional testing procedures (eg, FACS, IHC, radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, etc.).

Alternatively, the B7-H3 monoclonal antibody and other equivalent antibodies can be sequenced and produced by reconjugation by any method known in the art (eg, humanization, use of transgenic mice to produce full-length antibodies, phage display technology, etc.). ). In one embodiment, sequences for the B7-H3 monoclonal antibody are generated and the polypeptide sequence is then transcribed into a vector for expression or replication. The sequence encoding the relevant antibody can be preserved in a vector in a host cell and the host cell can then be expanded and frozen for future use.

The polynucleotide sequence of the B7-H3 monoclonal antibody and any equivalent antibodies can be used in gene therapy to generate a “humanized” antibody, for improved affinity, or other properties of the antibody. The general principle in adapting an antibody to humans includes retaining the basic sequence of the part of the antibody that binds the antigen, while swapping the non-human part of the antibody for the human antibody sequence. There are four main steps to making a monoclonal antibody compatible with humans. They are: (1) Determination of the expected nucleotide and amino acid sequences of the light and heavy variable domains of the initiating antibody (2) Design of a humanized antibody, i.e. determining which framework region of the antibody is used during the human approval process (3) Methods/Methods Compatibility with actual humans and (iv) transfection and expression of humanized antibody. See, for example, US Patent Nos. (4,816,567); (5807175); (5866692); And (6331415).

A number of “humanized” antibody molecules containing an antigen-binding motif derived from non-human immunoglobulins have been described, including chimeric antibodies containing rodent V regions or modified variants thereof and associated complementarity-determining regions (CDRs) fused with fixed human regions (see, e.g., Winter et al. (1991) Man-made Anditbodies, Nature 349:293-299; Lobuglio et al. (1989) Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response ,Proc. Natl. Sci. (USA) 86:4220-4224 (1989), Shaw et al. (1987) Characterization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor-Associated Antigen, J. Immunol. 138:4534-4538, and Brown et al. (1987) Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody, Cancer Res. 47:3577-3583). Other references have described rat CDRs grafted into a supporting human FR region before fusion with the constant domain of an appropriate human antibody (see, e.g., Riechmann, L. et al. (1988) Reshaping Human Antibodies for Therapy, Nature 332:323-327; Verhoeyen, M. et al. (1988) Reshaping Human Antibodies: Grafting An Antilysozyme Activity, Science 239:1534-1536; and Jones et al. (1986) Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse, Nature 321:522-525). Other references have described rodent CDRs supported by rodent framework regions spliced ​​in a back-linked manner. These “humanized” molecules are designed to reduce the unwanted immune response to anti-human rodent antibody molecules, which determines the effectiveness of therapeutic applications of these molecules in human recipients. Other methods could also be used to cross-match antibodies to humans, such as those revealed by Daugherty et al. (1991) Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins, Nucl. Acids Res. 19:2471-2476 and also in US Patent Nos. (6180377); (6054297); (5997867); And (5866692).

The invention also includes fragments of single-chain variable regions (scFv) of antibodies of the invention, such as murine anti-B7-H3. Single-chain variable region fragments are prepared by linking light and/or heavy chain variable regions using a short linker peptide. Bird et al. describe (1988) Single-Chain Antigen-Binding Proteins, Science 242:423-426 is an example of binding peptides that bridge approximately 3.5 nM between the carboxy terminus of a variable region and the amino terminus of another variable region. Linkers to other sequences have been designed and used (Bird et al. (1988) Single-Chain Antigen-Binding Proteins, Science 242:423-426). The ligands can be modified for other additional functions, such as drug delivery or contact with solid carrier materials. It has been possible to produce different single-chain images, whether through genetic reconnection or synthetically. For synthetic production of scFv, automated synthesis can be used. For recombinant production of scFv, a plasmid containing a polynucleotide encoding scFv can be introduced into a suitable host cell, whether it is a eucalyptus, such as yeast, plants, insects, or mammalian cells, or a prokaryotic, such as Escherichia coli. Polynucleotides encoding scFv can be prepared for routine processing such as ligation of polypeptides. scFv can be isolated using standard protein purification methods known in the art.

The invention includes modifications of antibodies and polypeptides that bind B7-H3 to its chaperones, antagonists, and modifiers, including functionally equivalent antibodies and polypeptides that do not obviously affect their properties and forms that have enhanced or decreased activity. Modification of polypeptides is a routine procedure in the field and does not need a detailed description here. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly or detrimentally alter the functional activity, or the use of chemical analogues. Examples of amino acid residues that can be conservatively replaced with each other include, but are not limited to: glycine/alanine; Valine/Isoleucine/Leucine; asparagine/glutamine; aspartic acid/glutamic acid; Serine/Threonine; Lysine/Arginine; and phenyl/triosine. These polypeptides also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation with various sugars, acetylation, and phosphorylation. Preferably, amino acid substitutions should be conservative, that is, the replaced amino acid should have chemical properties similar to the original amino acid. These conservative substitutions are known in the field, and the above are examples. Amino acid modifications may range from changing or modifying one or more amino acids to completely redesigning a region, such as a variable region. Changes in the variable region may alter binding affinity and/or specificity. Other methods of modification include the use of conjugation techniques known in the art, such as, but not limited to, enzymatic methods, oxidative substitution, and chelation. The modifications can be used, for example, to deliver markers for an immunoassay, such as delivering radioactive moieties to a radioimmunoassay. The modified polypeptides are prepared using well-established procedures in the art and can be screened using standard experiments known in the art.

The invention also includes fusion proteins containing one or more fragments or regions of polypeptides or antibodies of the present invention. In one embodiment, a fusion polypeptide is provided comprising at least 10 contiguous amino acids of a light-chain variable region and at least 10 amino acids of a heavy-chain variable region. In one embodiment, the fusion polypeptide contains a heterogeneous immunoglobulin constant region. In another embodiment, the fusion polypeptide contains a light-chain constant region and a heavy-chain variable region for the antibody generated by a generic deposit hybridoma. In the present invention, the antibody fusion protein contains one or more polypeptide domains that specifically bind to B7-H3 and another amino acid sequence to which it does not bind in the original molecule, e.g., a heterologous sequence or a homologous sequence from another region.

Anti-B7-H3 polypeptide and other B7-H3 adjuvants, antagonists, and modulators can be generated using methods known in the art, such as synthesis or gene recombination. One method for producing B7-H3 adjuvants, antagonists, and modifiers involves chemical synthesis of the polypeptide, then treatment under appropriate oxidation conditions to obtain the original form, which is the correct disulfide bond bonds. This can be accomplished using methods known to those skilled in the art (see, for example, Kelley, R. F. et al. (1990) In: Genetic Engineering Principles and Methods, Setlow, J. K. Ed., Plenum Press, NY, vol. 12, pp. 1-19; Stewart, J.M. et al. (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, see also US Patent Nos. 4,105,603; 3972859; 3842067; And 3862925).

The polypeptides of the invention can be conveniently prepared using solid phase peptide synthesis:

(Merrifield, B. (1986) Solid Phase Synthesis, Science 232(4748):341-347; Houghten, RA (1985) General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen-Antibody Interaction At The Level Of Individual Amino Acids, Proc Natl Acad Sci (USA) 82(15):5131-5135; Chem.6(1):3-10)

In another alternative example, fully human antibodies can be obtained through the use of commercially available mice that have been genetically modified to express specific human immunoglobulin proteins. Transgenic animals are engineered to produce (eg fully human) antibodies or a better or more robust immune response can be used to produce humanized or humanized antibodies. Examples of these technologies include Xenomouse (Abgenix, Inc., Fremont, CA), HuMAb-Mouse, and TC Mouse (both Abgenix, Inc., Princeton, NJ).

In an alternative embodiment, antibodies may be prepared by gene religation and expressed by any method known in the art. Antibodies can be prepared by genetic reassignment by first isolating antibodies taken from host animals (such as CHO cells). Another method can be used to express the antibody sequence in plants (eg tobacco) or transgenic milk. A convenient method for expressing antibodies is gene recombination in plants or milk, which have been described (e.g., Peeters et al. (2001) Production Of Antibodies And Antibody Fragments In Plants, Vaccine 19:2756; Lonberg, N. et al. (1995) Human Antibodies From Transgenic Mice, Int. Rev. Immunol 13:65-93; and Pollock et al.(1999) Transgenic Milk As A Method For The Production Of Recombinant Antibodies, J. Immunol Methods 231:147-157). Suitable methods for preparing antibody derivatives, such as humanized, single-chain, etc. are known in the art. In an alternative embodiment, antibodies may be obtained from a gene-replicase phage display technique (see, for example, US Patent Nos. 5,565,323; 5,580,717; 5,733,743; 6,265,150 and

Winter, G. et al. (1994) Making Antibodies By Phage Display Technology, Annu. Rev. Immunol. 12.433-455

The antibody or protein of interest can be subjected to the formation of Edman hemolytic sequences, known to those skilled in the art. Polypeptide information generated by mass spectrometry or Edman degradation can be used to design probes or primers to be used to transcribe the port of interest.

An alternative method for transcribing the protein of interest is by “spin-washing” with purified B7-H3 or fragments thereof to cells expressing the antibody or protein of interest. B7-H3 exists as 2Ig and 4Ig. The amino acid sequence of the 4Ig form of human B7-H3 is (sequence number: 1):

SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL

AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE

PNKDLRPGDT VTITCSSYRG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG

LFDVHSVLRV VLGANGTYSC LVRNPVLQQD AHGSVTITGQ PMTFPPEALW

VTVGLSVCLI ALLVALAFVC WRKIKQSCEE ENAGAEDQDG EGEGSKTALQ

PLKHSDSKED DGQEIA

The cDNA sequence encoding the 2Ig motif of human B7-H3 (Sequence No.: 2):

The amino acid sequence of image 2Ig of human B7-H3 (sequence no. 1) (shown in dark and underlined) fits entirely within image 4Ig of B7-H3 (sequence no. 76):

MLRRRGSPGM GVHVGAALGA LWFCLTGALE VQVPEDPVVA LVGTDATLCC

SFSPEPGFSL AQLNLIWQLT DTKQLVHSFA EGQDQGSAYA NRTALFPDLL

AQGNASLRLQ RVRVADEGSF TCFVSIRDFG SAAVSLQVAA PYSKPSMTLE

PNKDLRPGDT VTITCSSYQG YPEAEVFWQD GQGVPLTGNV TTSQMANEQG

LFDVHSILRV VLGANGTYSC LVRNPVLQQD AHSSVTITPQ RSPTGAVEVQ

VPEDPVVALV GTDATLRCSF SPEPGFSLAQ LNLIWQLTDT KQLVHSFTEG

RDQGSAYANR TALFPDLLAQ GNASLRLQRV RVADEGSFTC FVSIRDFGSA

AVSLQVAAPY SKPSMTLEPN KDLRPGDTVT ITCSSYRGYP EAEVFWQDGQ

GVPLTGNVTT SQMANEQGLF DVHSVLRVVL GANGTYSCLV RNPVLQQDAH

GSVTITGQPM TFPPEALWVT VGLSVCLIAL LVALAFVCWR KIKQSCEEEN

AGAEDQDGEG EGSKTALQPL KHSDSKEDDG QEIA

The cDNA sequence encoding the 4Ig motif of human B7-H3 is (sequence no: 77); The residues that encode B7-H3 in 2Ig are shown below in dark color and underlined:

“Spin-washing” can be performed by obtaining a pool of cDNA from B7-H3-expressing tissues or cells, overexpressing the cDNA in a second cell type, and screening transfected cells from the second cell type for specific binding to B7-H3. Methods used to clone mammalian genes encoding cell surface proteins can be found in the prior art by “spin washing” (see, for example, Aruffo, A. et al. (1987) Molecular Cloning Of A CD28 cDNA By A High-Efficiency COS Cell Expression System, Proc. Natl. Sci. (USA) 84:8573-8577 and Stephan, J. et al. (1999) Selective Cloning Of Cell Surface Proteins Involved In Organ Development: Epithelial Glycoprotein Is Involved In Normal Epithelial Differentiation, Endocrinol. 140:5841-5854).

cDNAs encoding anti-B7-H3 antibodies, other B7-H3 peptide chaperones, antagonists, and mRNA reverse transcriptase modifiers from a given cell type can be found according to industry standard methods. Specifically, mRNA can be isolated using various lytic enzymes or chemical solutions according to the procedures described in Sambrook et al. indicated above or extracted with chemically available DNA binding resins following the accompanying instructions provided by the manufacturer (e.g., Qiagen, Invitrogen). The cDNAs are then synthesized and inserted into an expression vector to produce the antibody or protein of interest in type II cells. It is understood that the expression vector must be able to be transcribed into host cells as liposomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adenoviruses, retroviruses, and cosmids.

Vectors containing the polynucleotides of interest may be introduced into a host cell by any suitable means, such as electrophoresis, transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other materials; Bombardment with precision projectiles; sebaceous infections, and infections (eg where the carrier is an infectious agent such as variola virus). The choice of introducing vectors or polynucleotides often depends on the characteristics of the host cell.

[00142] Any host cells capable of overexpressing heterologous DNAs may be used for the purpose of isolating genes encoding the antibody, polynucleotide or protein of interest. Nonspecific examples of human host cells include COS, HeLa, and CHO cells. Preferably, host cells express cDNAs at a level approximately 5-fold, ideally 10-fold, and preferably 20-fold greater than the level of the corresponding endogenous antibody or protein of interest, if present, in the host cells. Screening of host cells for specific binding to B7-H3 is influenced by autoimmune testing or FACS. A cell overexpressing the antibody or protein in question can be identified.

There are also various methods that can currently be used to produce mutagenic B7-H3 peptide adjuvants, antagonists, or modifiers that encode additions, deletions, or changes in the amino acid sequence of the resulting protein relative to the original adjuvant, antagonist, or modifying molecule. For peptide B7-H3.

The invention includes a polypeptide comprising an amino acid sequence of antibodies of the invention. The polypeptides of the present invention can be prepared using procedures known in the art. Polypeptides can be produced by resolving methods of protein or other antibodies, by reconjugation methods (i.e. single or fusion polypeptides) as previously described, or by chemical synthesis. Antibody polypeptides, especially the best polypeptides of about 50 amino acids, are prepared by chemical synthesis. Chemical synthesis methods are known in the art and commercially available. For example, the anti-B7-H3 polypeptide can be produced with an automatic polypeptide synthesis device that uses the solid-phase method.

4- Methods for screening polypeptides and monoclonal antibodies

Several methods can be used to screen polypeptides and monoclonal antibodies that bind to B7-H3. It is understood that “binding” refers to biological or autoimmune specific binding, and does not refer to non-specific binding that may occur, for example, when high concentration immunoglobulin is used against a non-specific target.

In one embodiment, the monoclonal antibody is screened for its binding to B7-H3 using standard screening methods. In this way, a monoclonal antibody to B7-H3 was obtained. The hybridomas of the present invention are those produced by BRCA69D, BRCA84D, and PRCA157 antibodies.

Additional antibodies that bind to B7-H3 can be identified. Therefore, monoclonal antibodies are screened for their discriminatory ability to bind to cancerous tissue but not to non-cancerous cells. In one embodiment, a monoclonal antibody is selected that binds to B7-H3 and cross-reacts with cancerous human cells or tissues, but not with more or less normal cells or tissues. One method that can be used for this examination is immunohistochemistry (IHC). Immunohistochemistry methods are known to those with intermediate skill in the field. See, for example, Animal Cell Culture Methods (J.P. Mather and D. Barnes, eds., Academic Press, NY, Vol. 57, Ch. 18 and 19, pp. 314-350, 1998). Biological samples (such as tissue) can be obtained from biopsies, anatomical incisions, or anatomical specimens. To ensure that B7-H3 is only present on cancer cells, anti-B7-H3 antibodies can be used to detect the presence of B7-H3 on tissue from individuals with cancer even though other non-cancerous tissue from the individual with cancer or tissue from non-cancer individuals is used as a comparison sample. . Tissues can be immersed in a solid or semi-solid material that prevents damage during freezing (eg, agarose gel or OCT) and then sectioned for staining. Cancers from different organs and at different grades can be used to screen for monoclonal antibodies. Examples of tissues that can be used for examination purposes include, but are not limited to, ovary, breast, lung, prostate, colon, kidney, skin, thyroid, blood vessels, bone, upper gastrointestinal tract, and pancreas. Examples of the different types of cancer that can be used for screening include, but are not limited to, carcinomas, adenocarcinomas, sarcomas, adenosarcoma, lymphomas, and leukemias.

In another alternative, cancer cell lines such as HMEC (BioWhittaker CC- 2251), HUVEC (primary endothelial cells), BT-474 (ATCC# HTB-20), MCF7 (ATCC# HTB22), MDA-MB-175-VII can be used. (ATCC# HB-25), MDA-MB-361 (ATCC# HB-27), SKBR3 (ATCC# HTB-30), A549 (ATCC# CCL- 185), Calu-3 (ATCC# HTB-55), SKMES-I (ATCC# HTB-58), ES-2 (ATCC# CRL-1978), SKOV3 (ATCC# HTB- 77), Panc-1 (ATCC# CRL-1469), AsPC-I (ATCC# CRL- 1682), HPAF-II (ATCC# CRL-1997), Hs700T (ATCC# HTB-174), Colo205 (ATCC# CCL-222), HT-29 (ATCC# HTB-38), SW480 (ATCC# CCL-228), SW948 (ATCC# CCL- 237), 293 (ATCC# CRL-1573), 786-O (ATCC# CRL-1932), A498 (ATCC# HTB-44), Caki-2 ( ATCC# HTB-47), COS-7 (ATCC# CRL-1651), RL-65 (ATCC# CRL-10345), SV-T2 (ATCC# CCL-163.1), 22RV1 (ATCC# CRL-2505), DU145 (ATCC# HTB-81), LNCaP (ATCC# CRL-1740), PC-3 (ATCC# CRL-1435), HT29 (ATCC# HTB-38), Hs746T (ATCC# HTB-135), NCI-N87 ( ATCC# CRL-5822) and normal cells from their corresponding tissues in a cancer tissue-specific monoclonal antibody assay. It is possible to use primary, or low-passage, cell cultures derived from normal tissue from various organs, including, but not limited to, kidney, ovary, breast, lung, prostate, colon, kidney, skin, thyroid, aortic soft muscle, and endothelial cells. These are negative comparison samples. Cancerous or noncancerous cells may be cultured on glass slides, cover slides, or on plastic surfaces, or prepared in a CellArray device, as described in the international application (01/43869), and examined for antibody binding using IHC as previously described for tissue. Alternatively, cells can be removed from the growth surface using proteolytic media, spun into pellets, then embedded and processed as tissue for IHC analysis as previously described. The cells can be inoculated into immunodeficient animals, allowed for tumor growth, and then that tumor is collected, encapsulated, and used as a tissue source for IHC analysis. Alternatively, individual cells can be examined by incubation with the primary antibody, a secondary “guide” antibody linked to a fluorescent molecule and then analyzed using a fluorescence-activated cell storage (FACS) machine.

Any of various monitoring systems can be used to monitor the binding of antibodies to a tissue fragment. Typically, immunohistochemistry involves binding a primary antibody to the tissue and then producing a secondary antibody cross-reactive against a variant of the primary antibody and conjugating it to a detectable marker (such as hot watercress peroxidase, HRP, or diaminobenzidine, DAB). Alternative methods that can be used are polyclonal mirror image complement antibodies or PoIyMICA (PoIyMICA; The Binding Site Limited, Birmingham, UK; Mangham, DC et al. (1999) A Novel Immunohistochemical Detection System Using Mirror Image Complementary Antibodies (MICA), Histopathology 35(2):129-33). The PoIyMICA method can be used to test the binding of key antibodies (such as anti-B7-H3 antibodies) to normal and cancerous tissues. Several types of polyMICA monitoring kits are commercially available: Product No. HK004.D is a polyMICA monitoring kit that uses DAB chromagen; Product No. HK004.A is a polyMICA monitoring kit using AEC Chromagen. Alternatively, the primary antibody can be directly labeled with a detectable marker.

The first step in B7-H3 screening to select the appropriate antibody is to bind key antibodies arising in mice (eg, anti-B7-H3 antibodies) to one or more immunogens (eg, cell or tissue samples). In another embodiment, the tissue sample is frozen tissue sections from various organs. Cell or tissue samples can be either cancerous or noncancerous.

Frozen tissue may be prepared, dissociated, with or without fixation, and performed + by any method known to a person familiar with the art (see, e.g., Stephan et al. (1999) Distribution And Function Of The Adhesion Molecule BEN During Rat Development, Dev. Biol. 212:264-277 and Stephan et al. (1999) Selective Cloning Of Cell Surface Proteins Involved In Organ Development: Epithelial Glycoprotein Is Involved In Normal Epithelial Differentiation, Endocrinology 140:5841-5854).

5- Methods of classifying antibodies to B7-H3

Any method can be used to classify anti-B7-H3 antibodies. One of these methods is to determine the sticky peak to which it is attached. Sticky peak mapping is commercially available from many sources, such as Pepscan Systems (Lelystad, The Netherlands). The mapping of binding peaks can be used to identify the sequence to which the anti-B7-H3 antibody binds. The sticky epitope can be straight, i.e., located in a single stretch of amino acid, or conformable, formed by a three-dimensional interaction of amino acids that may not necessarily be located in a single stretch.

Peptides of different lengths (preferably at least 4-6 amino acids long) can be isolated or synthesized (eg by gene recombination) and used in binding experiments with an anti-B7-H3 antibody. The epitope to which the anti-B7-H3 antibody binds can be identified in a regular assay using overlapping peptides derived from the extracellular sequence and the binding to the anti-B7-H3 antibody determined.

Another method for classifying anti-B7-H3 antibodies is to use competition experiments with other antibodies known to bind to the same antigen, i.e. B7-H3, to determine whether anti-B7-H3 antibodies bind to the same epitope as other antibodies. Some models of anti-B7-H3 antibodies may be commercially available and can be identified using the binding experiments described herein. Competition experiences are known to those skilled in the field, and these procedures and explanatory data are detailed in examples. B7-H3 antibodies can be classified according to the tissue, type of cancer, or type of tumor to which they are associated.

Another way to classify B7-H3 antibodies is by the antigen to which they bind. Anti-B7-H3 antibodies have been used in Western blots with cell lysis products from various types of human cancer. As is known to a person of skill in the art, Western blots can involve running cell lysis products and/or cell fragments on a gel to alter their normal or other properties, transferring the proteins to nitrocellulose paper, and then probing the blot with an antibody (such as anti-B7-H3 antibody ) to find out which proteins the antibody binds to. B7-H3 is associated with various human cancers of various tissues, including but not limited to colon cancer, breast, ovary, pancreas, and lung.

6- Methods for diagnosing cancer using anti-B7-H3 antibodies and B7-H3 modifiers

The anti-B7-H3 monoclonal antibodies disclosed herein may be used to determine the presence or absence of cancer cells in many tissues, including, but not limited to, ovarian, breast, lung, prostate, colon, kidney, pancreas, Skin, thyroid, brain, heart, liver, stomach, nerve, blood vessels, bones, and upper gastrointestinal tract, for diagnostic purposes. Anti-B7-H3 antibodies prepared by the methods disclosed herein can also be used to determine the presence or absence and level of cancer cells circulating in the blood after their release from a solid tumor. This circulating antigen may be an intact B7-H3 antigen, or a fragment thereof that has the potential to be detected according to the methods described in the present document. This monitoring can be influenced by FACS analysis using standard methods commonly used in the field.

These uses may include formation of a complex between B7-H3 and an antibody that binds specifically to B7-H3. Examples of such antibodies include, but are not limited to, anti-B7-H3 monoclonal antibodies produced by BRCA84D, BRCA69D, and PRCA157 hybridomas. This complex can be formed in a laboratory or inside the body of a living organism. Without being theoretically restricted, a monoclonal antibody can bind to B7-H3 through the ectodomain of B7-H3 and then internalize it.

In a preferred embodiment of the diagnostic methods of the invention, the antibody carries a detectable marker. An example of a marker that can be used is a radioactive agent or a fluorophore, such as fisyrethrin or fluorescein isothiocyanate (also known as fluoroisocyanate or FITC).

As with other known antibodies used commercially in diagnosis and therapy, the target antigen of the present invention is generally expressed in normal tissue. It also has a controlled increase in some tumors. Therefore, the doses and methods of administration for the antibodies of the present invention as they are used as diagnostic or therapeutic agents will be modified depending on the tumors and disease state, as well as the individual being treated.

One of the methods that uses antibodies in diagnosis is to image the tumor inside the body, attaching the antibody to a radioactive or radioactive agent, giving the antibody to a person, and using X-rays or other imaging machines to see the location of the labeled antibody on the surface of cancer cells expressing the antigen. The antibody is given at a concentration that enhances binding under physiological conditions.

Laboratory methods for monitoring B7-H3 are routine in the field and include enzyme-linked immunosorbent tests (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blot analysis.

In other aspects of the present invention, methods for radiographic imaging of tumors or recent growth of tumors, or measuring the efficiency of a radiolabeled antibody treatment method, comprise the step of administering a tumor-specific radiolabeled antibody to an individual following an implementation of the present invention. The radiolabeled antibody may be a monoclonal or polyclonal antibody containing a radioactive broth, preferably chosen from the group consisting of technetium-99m, indium-111, iodine-131, ruthenium-186, samarium-153, luteium-177, and copper. -64, scandium-47, yttrium-90. In particular, monoclonal antibodies labeled with therapeutic nucleotides such as day-131, rhenium-188, holmium-166, samarium-153, and scandium-47 are preferred, as they do not involve the immunoreactivity of the antibodies and do not inactivate in vivo. A person of skill in the art will recognize that other radioactive isotypes are known, which may be suitable for particular applications. Radiography can be performed using single photon emission computed tomography (SPECT), position emission tomography (PET), computed tomography (CT), or magnetic resonance imaging (MRI). Correlative imaging can also be used, which allows for increased anatomical localization of metastatic tumors determined by radioimmunoimaging.

In other methods, tumor cells are removed and the tissue is prepared for immunohistochemistry using methods known in the art (eg, immersion in a freezing compound, freezing and sectioning, with or without fixation; fixation and paraffin immersion with or without various antigen retrieval and reverse staining methods). Monoclonal antibodies can also be used to identify cancer cells at different stages of development. Antibodies can also be used to identify which person's tumors express an antigen on their surface at a predetermined level and are therefore candidates for immunotherapy using antibodies directed against that antigen. The antibodies may recognize both primary and metastatic cancers that express B7-H3. As used in the present document, monitoring may include qualitative and/or quantitative monitoring and may include comparing the measured level to a normal cell to determine the increased expression level of B7-H3 in cancer cells.

The invention also provides methods to aid in the diagnosis of cancer, as it is characterized by the fact that cancer cells that express B7-H3 in an individual use any antibody that binds to B7-H3 and any other methods that can be used to determine the level of expression of B7-H3. As used in the present document, “aid-diagnostic” methods mean those methods that assist in arriving at a clinical decision regarding the classification or nature of the cancer, which may or may not be definitive as to the final diagnosis. Therefore, a route to aid in the diagnosis of cancer may include the step of monitoring the level of B7-H3 in a biological sample from an individual and/or determining the level of expression of B7-H3 in the sample. Antibodies that recognize the antigen or part of it can also be used to create diagnostic immunological tests to monitor the antigen that is released or secreted from living or dead cancer cells in body fluids, such as, but not limited to, blood, saliva, urine, and blood fluids. Lung, or ascitic fluid.

Not all cells in a given tumor express B7-H3, and cancer cells in other tissues may express B7-H3, so the individual must be screened for the presence of B7-H3 on the cancer cells to neutralize the benefit of immunotherapy in that individual. Anti-B7-H3 antibodies prepared by the methods disclosed herein may be used to adjudicate a person as having cancer or as a candidate for immunotherapy using antibodies directed against B7-H3. In one embodiment, a cancerous tumor or biopsy specimen can be tested for expression of B7-H3, using antibodies directed against B7-H3. Individuals with cancer cells that express B7-H3 are suitable candidates for immunotherapy using antibodies directed against B7-H3. Staining with anti-B7-H3 antibodies can also be used to distinguish cancerous tissue from normal tissue.

Methods that use antibodies to B7-H3 for diagnostic purposes are useful before or after any form of cancer treatment, such as chemotherapy or radiotherapy, and to identify tumors that respond most to a particular treatment, and to predict a person's likelihood of developing cancer, the subtype of the tumor or the origin of the disease. Rampant, disease progression or response to treatment.

The compositions of the present invention are also suitable for diagnosing conditions other than cancer, using the methods described above in application to other diseased cells (with a disease other than cancer). Disease conditions for which the methods of the present invention are appropriate include, but are not limited to, diseases or disorders accompanied by inflammation or autoimmune responses in individuals. The methods described above can be used to modify inflammatory or autoimmune responses in individuals. Diseases and conditions resulting from inflammation and autoimmune disorders that can be diagnosed and/or treated using the compositions and methods of the invention include, but are not limited to, multiple sclerosis, meningitis, encephalitis, stroke, other brain injuries, and inflammatory bowel disease such as ulcerative colitis, Crohn's disease, myasthenia gravis, lupus, arthritis, asthma, acute juvenile onset diabetes, dementia, AIDS, atherosclerosis, nephritis, retinitis, eczema, psoriasis, and hypoxia. Myocardial perfusion and leukocyte-mediated acute lung injury.

In other indications for diagnostic and/or therapeutic purposes antibodies and other therapeutic agents of the invention are administered to persons at risk of organ or transplant rejection. During recent years there has been a noticeable improvement in the efficiency of surgical methods for transferring tissues and organs such as skin, kidneys, liver, heart, lung, pancreas, and bone marrow. Perhaps the most prominent problem is the lack of pathogenic factors to create immune tolerance in the person receiving the transferred or transplanted organ. When homologous cells or organs are transferred into a host (i.e. the donor and donor are two different individuals of the same species), it is expected that the host's immune system will develop an immune response to foreign antigens in the transplanted organ (host rejection disease of the transplanted organ), which leads to the destruction of the transferred tissue. .

Uses listed in any part of the application for B7-H3 antibodies may also include the use of B7-H3 cofactors, antagonists, and modifiers as previously described. In such embodiments, the B7-H3 adjuvant, antigen, or other non-antibody modifier is substituted for the antibody in the described steps, and modifications are made within the scope of the skilled practitioner to modify the method for the modified substituted B7-H3 formulation.

Anti-B7-H3 monoclonal antibodies prepared as described herein can be used to determine the presence or absence of human cancer stem cells in various tissues. It has been hypothesized that human CSCs play a role in tumor growth and metastasis (Ghotra, VP et al. (2009) The Cancer Stem Cell Microenvironment And Anti-Cancer Therapy, Int. J. Radiat. Biol. 85(11): 955-962; Gupta, PB et al. (2009) Cancer Stem Cells: Mirage or Reality? (2009) Cancer Stem Cells In Breast Cancer And Metastasis, Breast Cancer Res. Treat. 118(2):241-254; Hermann, P. C. et al. (2009) Pancreatic Cancer Stem Cells--Insights And Perspectives, Expert Opinion. Biol. Ther. 9(10):1271-1278; Schatton, T. et al. (2009) Identification And Targeting Of Cancer Stem Cells, Bioessays 31(10):1038-1049; Mittal, S. et al. (2009) Cancer Stem Cells: The Other Face Of Janus, Amer. J.Med. Sci. 338(2):107-112; Alison, M.R. et al. (2009) Stem Cells and Lung Cancer: Future Therapeutic Targets? Expert Opin. Biol. Ther. 9(9):1127-1141; Charafe-Jauffret, E. et al. (2009) Breast Cancer Stem Cells: Tools And Models To Rely On, BMC Cancer 9:202; Scopelliti, A. et al. (2009) Therapeutic Implications Of Cancer Initiating Cells, Expert Opin. Biol. Ther. 9(8):1005-1016;

and International Application for PCT Circular No. 2008/091908). Under this hypothesis, CSCs provide a small, distinct subset of cells within each tumor capable of nonspecific self-renewal and development into more mature tumor cell(s) that are relatively specific in transcriptional capacity. It was hypothesized that cancer stem cells may be more resistant to chemotherapy agents, radiation, or other toxic conditions, and thus persist after clinical treatment and subsequently develop into secondary or metastatic tumors, or may be responsible for collapse. It has been proposed that CSCs may arise either from stem cells of “normal” tissues or from progenitor cells of a more differentiated tissue.

Cancer stem cells have several characteristics. These characteristics are described in PCT Circular Application No. 2008/091908 and are included in the current document by reference. Monoclonal antibodies to cell surface targets on cancer stem cells can be used to determine the presence of cancer stem cells in many tissues. Anti-B7-H3 monoclonal antibodies prepared using the methods disclosed herein can also be used to determine the presence or level of cancer stem cells in a specimen or tissue or in the circulation after their release from a solid tumor. This circulating antigen may be an intact B7-H3 antigen, or a fragment thereof that retains the ability to be detected according to the methods of the invention herein. This monitoring can be performed by FACS analysis using standard methods commonly used in immunohistochemical analysis and tissue samples using standard methods commonly used in the field.

These uses include a complex between B7-H3 and an antibody that specifically binds to B7-H3 on cancer stem cells. Examples of such antibodies include, but are not limited to, anti-B7-H3 monoclonal antibodies produced by BRCA84D, BRCA69D and PRCA157 hybridomas. The formation of this complex can occur in the laboratory or in the body of a living organism.

The uses described in the present application that mention their use in anti-B7-H3 antibodies also include the use of other B7-H3 adjuvants, antagonists, and modulators as described in the present document for use in the identification and treatment of cancer stem cells. In these embodiments, adjuvants, antagonists, and B7-H3 modulators are used to diagnose or treat cancer stem cells using similar described methods, and modifications are within the scope of a practitioner of ordinary skill to modify the method to modify/diagnose or treat cancer stem cells.

7- Preferred compositions of the present invention

The present invention comprises compositions, such as pharmaceutical compositions, comprising anti-B7-H3 antibodies, polypeptides derived from anti-B7-H3 antibodies, polynucleotides comprising a sequence encoding anti-B7-H3 antibodies, and other agents described herein. As used, the compositions further comprise one or more antibodies, polypeptides and/or proteins that bind B7-H3, and/or B7-H3 chaperones, antagonists, modulators, and/or one or more Polynucleotides comprising sequences encoding one or more antibodies, polypeptides, and proteins, which bind to B7-H3.

The invention also provides conjugates for any peptide adjuvant, antagonist, or B7-H3 modifier, and additional chemical structural formulas that support the desired function or functions of the B7-H3 adjuvant, antagonist, or modifier.

These conjugates include an adjuvant, antagonist, or B7-H3 modifier covalently attached to a macromolecule such as any solid insoluble support matrix used in the diagnostic, screening, or purification procedures described herein. Suitable matrix materials include any chemically inert, highly porous material with a large number of functional groups capable of forming covalent bonds with peptide binding molecules. Examples of matrix materials and procedures for preparing matrix-binding molecule conjugates are given in Dean et al. (Eds) Affinity Chromatography: A Practical Approach, IRL Press (1985); Lowe, An Introduction to Affinity Chromatography, in Work et al. (eds) Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 7, Part II, North-Holland (1979); Porath et al., Biospecific Affinity Chromatography, in Neurath, H. et al. (eds), The Proteins, 3rd ed., Vol. 1, pp. 95-178 (1975); and Schott, H. Affinity Chromatography, Macel Dekker, Inc. NY (1984).

Conjugates of the adjuvant, antagonist, or modifier of the B7-H3 peptide and any guide moiety used in the diagnostic procedures are also provided herein. The cofactors, antagonists or modulators of the B7-H3 peptide, polypeptides and proteins of the invention are further identified and classified by (one or more of) the following criteria:

(a) the ability to bind specifically to B7-H3 (particularly B7-H3 molecules expressed on the surface of cancer cells, such as, but not limited to, kidney, prostate, or lung cancer cells);

(b) the ability to competitively bind preferentially to a known anti-B7-H3 antibody known as B7-H3, including the ability to preferentially bind to the same epitope of B7-H3 to which the antibody preferentially binds;

(c) the ability to bind to the B7-H3 fragment exposed on the surface of a living cell in vitro or in vivo;

(d) the ability to bind to the B7-H3 fragment expressed on the surface of living cancer cells, such as, but not limited to, prostate, lung, or kidney cancer cells;

(e) the ability to deliver a chemotherapy agent to cancer cells (such as kidney, prostate, or lung cancer cells) expressing B7-H3 on their surfaces; wow

(f) The ability to deliver a therapeutic agent or landmark that can be detected in cancer cells (including but not limited to prostate cancer cells) expressing B7-H3 on their surface.

The preferred antibody of the invention will feature differential IHC staining of tumor tissue relative to normal, noncancerous tissue, and will be able to be tested in primate (particularly baboons) models of antibody efficacy. The antibodies of the empty invention will also have desired levels of immunoregulatory activity and cellular internalization.

In some embodiments, the antibody of the invention is an antibody produced by the hybridoma-specific BRCA84D, BRCA69D, or PRCA157 or its product. The present invention also includes various antibody formulations produced by these deposited hybridomas, equivalent antibodies or polypeptide fragments (such as Fab, Fab', F(ab')2 Fv, F(ab')2 Fv, Fc, etc.), and antibodies Chimeric antibodies, single chain (scFv), mutations thereof, fusion proteins including a portion of the antibody, or humanized antibodies, and any other modified design of any of these antibodies or their equivalents that include antigen (B7-H3), website Know the necessary definition. The invention also provides human antibodies characterized by one or more biological properties of a member of the anti-B7-H3 family. Antibodies equivalent to the B7-H3 family (including humanized and humanized antibodies), polypeptide fragments, and polypeptides including polypeptides comprising those fragments are identified and classified by one (or more) of those criteria described above. Representative murine and humanized variable domain sequences from the anti-B7-H3 antibody are provided in Request Publication No. 06691/2008. These sequences are provided for illustration and are not limited to, and fragments and images of the given sequences also fall within the scope of the invention.

BRCA84D, BRCA69D, and PRCA157 are the anti-B7-H3 antibodies of choice due to their pure normal tissue IHC properties, strong tumor/normal IHC differentiation, moderate to strong binding (BIACORE/IHC), and strong baboons and baboons B7-H3 cross-reactivity. To DART molecules (UDARTs) relative to other antibodies. In one particularly preferred embodiment, the invention comprises chimeric and humanized forms of those preferred antibodies, as well as native, chimeric and humanized forms of those preferred antibodies having modified regions as described below. The invention also includes DART molecules having binding regions for the epitope binding regions of such antibodies, specifically in coordination with the epitope binding region(s) that bind to a T cell receptor, an NKG2D receptor, a tumor-associated antigen, or a heptane such as fluorescein (such as fluorescein isocyanate (known as Also fluoroisocyanate (FITC).

In some embodiments, the antibodies, polypeptides and proteins of the invention that bind to B7-H3 are the antibodies, polypeptides and proteins that competitively inhibit the differential binding of the anti-B7-H3 antibody identified herein to B7-H3. In some embodiments, antibodies, polypeptides and proteins preferentially bind to a specific epitope on B7-H3 and the murine anti-B7-H3 antibody preferentially binds.

Therefore, the invention provides any of the following (or compositions, such as pharmaceutical compositions, comprising any of the following): (a) an antibody produced by the host cell with the application number specified above or its product; (b) a humanized form of that antibody; (c) an antibody comprising one or more light- and/or heavy-chain variable regions of that antibody; (d) A chimeric antibody comprising variable regions that are homologous or derived from the heavy or light chain variable regions of that antibody, and constant regions that are homologous or derived from constant regions that are homologous to or derived from the heavy or light chain variable regions of that antibody. Heavy and light chain of a human antibody; (e) an antibody comprising one or more light and/or heavy chain CDRs (at least two, three, four, five or six) of that antibody; (f) an antibody comprising a heavy and/or light chain of that antibody; (g) An antibody equivalent to that antibody. The humanized form of the antibody may or may not contain CDRs identical to that of the original antibody, or an antibody produced by a family cell with a deposit number specified above. The identification of CDR regions is well known in the field. In some embodiments, the invention provides an antibody comprising at least a CDR that is substantially identical to at least one, at least two, at least three, at least four, or at least five CDRs of the antibody produced by one or More than one of the above-mentioned precipitated hybridomas (or, in some embodiments substantially identical to all six CDRs of one such antibody, or derived from one or more such antibodies), or an antibody produced by the host cell with a previously mentioned deposit number. Other embodiments include antibodies having at least two, three, four, five, or six CDRs of the antibody resulting from, or derived from, the deposited hybridoma as previously described. It is understood that for the purposes of the present invention, identification of the general binding and/or activity (which may be in terms of delivering a chemotherapeutic agent to or in cancer cells to reduce the growth and/or proliferation of cancer cells, to induce cell death by disappearing in the cancer cell, is generally reserved) To retard the growth of cancer outbreaks, and/or to treat disease, although the extent of activity may vary compared to the antibody produced by a deposited hybridoma (and perhaps more or less). The invention also provides methods for preparing any of these antibodies. Methods for preparing antibodies are known in the art and are described in the present document.

The invention also provides polypeptides comprising the amino acid sequence of antibodies of the invention. In some embodiments, the polypeptides comprise one or more light- and/or heavy-chain variable regions of the antibody. In some embodiments, the polypeptide comprises one or more light- and/or heavy-chain CDRs of the antibody. In some embodiments, the polypeptide comprises three light- and/or heavy-chain CDRs of the antibody. In some embodiments, the polypeptide comprises an antibody amino acid sequence characterized by any of the following: at least 5 contiguous amino acids to the parent antibody sequence, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, about 15 contiguous amino acids on The least, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids, where at least 3 amino acids are from a variable region of the antibody. In one embodiment, the variable region is a light chain of the antibody. In another embodiment, the variable region is a heavy chain of the antibody. In another embodiment, the five contiguous amino acids are from the complementary determining region (CDR) of the antibody.

In some embodiments of the present invention, cells of the present invention expressing B7-H3, a B7-H3 fragment, anti-B7-H3 antibodies, or other B7-H3-binding polypeptides of the present invention are administered directly to an individual to modulate B7- activity. H3 biosynthesis within an organism.

The preferred anti-B7-H3 antibodies of the present invention are BRCA84D, BRCA69D, and PRCA157, all of which are murine and react with the human B7-H3 molecule. The following is the amino acid encoding the polynucleotide sequence of the light and heavy variable chains of BRCA84D, BRCA69D, and PRCA157 with the CDR1, CDR2, and CDR3 domains for each chain. Therefore, those skilled in the art will be able to create antibodies with CDRs, and their derivatives, that are able to bind to epitopes recognized by BRCA84D, BRCA69D, and PRCA157.

a. BRCA84D sequences

(1) BRCA84D light chain sequences

The amino acid sequence of the light chain variant of BRCA84D (Sequence No.: 3)

DIAMTQSQKF MSTSVGDRVS VTCKASQNVD TNVAWYQQKP GQSPKALIYS

ASYRYSGVPD RFTGSGSGTD FTLTINNVQS EDLAEYFCQQ YNNYPFTFGS

GTKLEIK

Polynucleotide sequence encoding the light chain variable of BRCA84D (Sequence No.: 4)

gacattgcga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagc

gtcacctgca aggccagtca gaatgtggat actaatgtag cctggtatca acagaaacca

gggcaatctc ctaaagcact gatttactcg gcatcctacc ggtacagtgg agtccctgat

cgcttcacag gcagtggatc tgggacagat ttcactctca ccatcaacaa tgtgcagtct

gaagacttgg cagagtattt ctgtcagcaa tataacaact atccattcac gttcggctcg

gggacaaagt tggaaataaa a

CDR1 light chain variant of BRCA84D (Sequence No.: 5): KASQNVDTNVA

The polynucleotide sequence encoding the CDR1 light-chain variant of BRCA84D (SEQ No. 6): aaggccagtc agaatgtgga tactaatgta gcc

CDR2 light chain variant of BRCA84D (sequence no: 7): SASYRYS

The polynucleotide sequence encoding the CDR2 light-chain variant of BRCA84D (Sequence No. 8): tcggcatcct accggtacag t

CDR3 light chain variant of BRCA84D (sequence no.: 9): QQYNNYPFT

The polynucleotide sequence encoding the CDR3 light-chain variant of BRCA84D (SEQ No. 10): cagcaatata acaactatcc attcacg

(2) BRCA84D heavy chain sequences

The amino acid sequence of the heavy variable chain of BRCA84D (Sequence No. 11):

DVQLVESGGG LVQPGGSRKL SCAASGFTFS SFGMHWVRQA PEKGLEWVAY

ISSSSAIYY ADTVKGRFTI SRDNPKNTLF LQMTSLRSED TAMYYCGRGR

ENIYYGSRLD YWGQGTTLTV SS

Polynucleotide sequence encoding the heavy variable chain of BRCA84D (Sequence No. 12):

gatgtgcagc tggtggagtc tgggggaggc ttagtgcagc ctggagggtc ccggaaactc

tcctgtgcag cctctggatt cactttcagt agctttggaa tgcactgggt tcgtcaggct

ccagagaagg ggctggagtg ggtcgcatac attagtagtg acagtagtgc catctactat

gcagacacag tgaagggccg attcaccatc tccagagaca atcccaagaa caccctgttc

ctgcaaatga ccagtctaag gtctgaggac acggccatgt attactgtgg aagagggagg

gaaaacattt actacggtag taggcttgac tactggggcc aaggcaccac tctcacagtc

tcctca

CDR1 heavy chain variant of BRCA84D (Sequence No. 13): FGMH

Polynucleotide sequence encoding CDR1 for the BRCA84D heavy variable chain (SEQ#: 14): tttggaatgcac

CDR2 heavy chain variant of BRCA84D (sequence no: 15): YISSDSSAIYYADTVK

The polynucleotide sequence CDR2 encoding the heavy variable chain of BRCA84D (SEQ NO. 16): tacattagta gtgacagtag tgccatctac tatgcagaca cagtgaag

CDR3 heavy chain variant of BRCA84D (sequence no: 17): GRENIYYGSRLDY

The polynucleotide sequence encoding the CDR3 heavy variable chain of BRCA84D (SEQ NO: 18): gggagggaaa acatttacta cggtagtagg cttgactac

B. BRCA69D sequences

(1) BRCA69D light chain sequences

Amino acid sequences of the BRCA69D light chain variant (SEQ NO: 19):

DIQMTQTTSS LSASLGDRVT ISCRASQDIS NYLNWYQQKP DGTVKLLIYY

TSRLHSGVPS RFSGSGSGTD YSLTIDNLEQ EDIATYFCQQ GNTLPPTFGG

GTKLEIK

The polynucleotide sequence encoding the light chain variant of BRCA69D (Sequence No.: 20):

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc

atcagttgca gggcaagtca ggacattagt aattatttaa actggtatca gcagaaacca

gatggaactg ttaaactcct gatctactac acatcacgat tacactcagg agtcccatca

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattgacaa cctggagcaa

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttcctccgac gttcggtgga

ggcaccaaac tggaaatcaa a

CDR1 light chain variant of BRCA69D (Sequence No.: 21): RASQDISNYLN

The polynucleotide sequence encoding the CDR1 light-chain variant of BRCA69D (SEQ No. 22): agggcaagtc aggacattag taattattta aac

CDR2 light chain variant of BRCA69D (sequence no: 23): YTSRLHS

The polynucleotide sequence encoding the CDR2 light-chain variant of BRCA69D (Sequence No. 24): tacacatcac gattacactc a

CDR3 light chain variant of BRCA69D (sequence no: 25): QQGNLTLPT

The polynucleotide sequence encoding the CDR3 light-chain variant of BRCA69D (SEQ #: 26): caacagggta atacgcttcc tccgacg

(2) BRCA69D heavy chain sequences

Amino acid sequences of the heavy variable chain of BRCA69D (Sequence No.: 27):

QVQLQQSGAE LARPGASVKL SCKASGYTFT SYWMQWVKQR PGQGLEWIGT

IYPGDGDTRY TQKFKGKATL TADKSSSTAY MQLSSLASED SAVYYCARRG

IPRLWYFDVW GAGTTVTVSS

Polynucleotide sequence encoding the heavy variable chain of BRCA69D (Sequence No.: 28):

caggttcagc tccagcagtc tggggctgag ctggcaagac ctggggcttc agtgaagttg

tcctgcaagg cttctggcta cacctttact agctactgga tgcagtgggt aaaacagagg

cctggacagg gtctggaatg gattggggact atttatcctg gagatggtga tactaggtac

actcagaagt tcaagggcaa ggccacattg actgcagata aatcctccag cacagcctac

atgcaactca gcagcttggc atctgaggac tctgcggtct attactgtgc aagaagaggg

attccacggc tttggtactt cgatgtctgg ggcgcaggga ccacggtcac cgtctcctca

CDR1 heavy chain variant of BRCA69D (Sequence No.: 29): SYWMQ

The polynucleotide sequence encoding the CDR1 heavy-chain variant of BRCA69D (SEQ NO: 30): agctactgga tgcag

CDR2 heavy chain variant of BRCA69D (sequence no: 31): TIYPGDGDTR YTQKFKG

The polynucleotide sequence encoding the CDR2 heavy-chain variant of BRCA69D (Sequence No. 32): actatttatc ctggagatgg tgatactagg tacactcag aagttcaagg gc

CDR3 heavy chain variant of BRCA69D (Sequence No.: 33): RGIPRLWYFD V

The polynucleotide sequence encoding the CDR3 heavy-chain variant of BRCA69D (Sequence No. 34): agagggattc cacggctttg gtacttcgat gtc

C- PRCA157 sequences

(1) PRCA157 light chain sequences

The amino acid sequence of the variable light chain of PRCA157 (Sequence No.: 35):

DIQMTQSPAS LSVSVGETVT ITCRASESIY SYLAWYQQKQ GKSPQLLVYN

TKTLPEGVPS RFSGSGSGTQ FSLKINSLQP EDFGRYYCQH HYGTPPWTFG

GGTNLEIK

Variable light chain polynucleotide sequence of PRCA157 (SEQ NO: 36):

gacatccaga tgactcagtc tccagcctcc ctatctgtat ctgtgggaga

aactgtcacc attacatgtc gagcaagtga gagtatttac agttatttag

catggtatca gcagaaacag ggaaaatctc ctcagctcct ggtctataat

acaaaaacct taccagaggg tgtgccatca aggttcagtg gcagtggatc

aggcacacag ttttctctga agatcaacag cctgcagcct gaagattttg

ggagatatta ctgtcaacat cattatggta ctcctccgtg gacgttcggt

ggaggcacca acctggaaat caaa

CDR1 light chain variant of PRCA157 (SEQ NO: 37): RASESIYSYLA

The polynucleotide sequence encoding the CDR1 light-chain variant of PRCA157 (SEQ NO: 38): cgagcaagtg agagtatttta cagttattta gca

CDR2 light chain variant of PRCA157 (Sequence No.: 39): NTKTLPE

The polynucleotide sequence encoding the CDR2 light-chain variant of PRCA157 (SEQ NO: 40): aatacaaaaa ccttaccaga g

CDR3 light chain variant of PRCA157 (sequence no. 41): QHHYGTPPW

The polynucleotide sequence encoding the CDR3 light-chain variant of PRCA157 (SEQ NO: 42): caacatcatt atggtactcc tccgtgg.

(2) PRCA157 heavy chain sequences

The amino acid sequence of the variable heavy chain of PRCA157 (Sequence No.: 43):

EVQQVESGGD LVKPGGSLKL SCAASGFTFS SYGMSWVRQT PDKRLEWVAT

INSGGSNTYY PDSLKGRFTI SRDNAKNTLY LQMRSLKSED TAMYYCARHD

GGAMDYWGQG TSVTVSS

Polynucleotide sequence encoding the variable heavy chain of PRCA157 (Sequence No. 44):

gaggtgcagc aggtggagtc ggggggagac ttagtgaagc ctggagggtc

cctgaaactc tcctgtgcag cctctggatt cactttcagt tcctatggca

tgtcttgggt tcgccagact ccagacaaga ggctggagtg ggtcgcaacc

attaatagtg gtggaagtaa cacctactat ccagacagtt tgaaggggcg

attcaccatc tccagagaca atgccaagaa caccctttac ctgcaaatgc

gcagtctgaa gtctgaggac acagccatgt attactgtgc aagacatgac

gggggagcta tggactactg gggtcaagga acctcagtca ccgtctcctc a

CDR1 of PRCA157 heavy chain variant (SEQ NO: 45): SYGMS

The CDR1-encoding polynucleotide sequence of the PRCA157 heavy chain variant (SEQ NO: 46): tcctatggca tgtct

PRCA157 heavy chain variant CDR2 (sequence no: 47): TYYPDSLKG

The CDR2-encoding polynucleotide sequence of the PRCA157 heavy chain variant (SEQ No. 48): gtcgcaacca ttaatagtgg tggaagtaac acctactatc cagacagttt gaagggg

CDR3 heavy chain variant of PRCA157 (sequence no. 49): HDGGAMDY

The CDR3-encoding polynucleotide sequence of the PRCA157 heavy chain variant (SEQ NO: 50): catgacgggg gagctatgga ctac

Dr.. Anti-B7-H3 antibodies transgenic with Fc

In classical immune function, the interaction of antibody-antigen complexes with cells of the immune system leads to numerous responses, ranging from effector functions such as antibody-dependent cytotoxicity, mast cell degranulation, and mitophagy to immunomodulatory signals such as regulation of lymphocyte proliferation and secretion. antibody. All of these interactions are initiated by the binding of the Fc domain of antibodies or immune complexes to specialized cell surface receptors on hematopoietic cells. The diversity of cellular responses stimulated by antibodies and immune complexes results from the heterogeneity of origin of these three Fc receptors: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) are activating receptors (i.e., enhance the immune system); and FcγRIIB (CD32B) are inhibitory receptors (i.e., suppress the immune system). The following is the amino acid sequence of the Fc region of IgG1 (as sequence no. 51, numbering according to Kabat et al., Sequence of Proteins of Immunological Interest, 5th Ed. Public Health Service, NIH, MD (1991), expressly incorporated herein by reference, and hereafter referred to as Kabat EU):

Sequence No.: 51

PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV

230 240 250 260 270

DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP

280 290 300 310 320

APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV

330 340 350 360 370

EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH

380 390 400 410 420

EALHNHYTQK SLSLSPGK

430 440

Residues 230-241 are the FcCH2 region. Residues 243-447 are the FcCH3 region.

The present invention includes an antibody that binds specifically to B7-H3 comprising a form of an Fc region having one or more amino acid modifications (such as substitutions, deletions, and insertions) in one or more moieties, such modifications increasing the affinity and affinity of the region. Different types of FcγR (including activating and inhibitory FcγR types). In some embodiments, one or more modifications increase the affinity of the different Fc region for FcγRIIIA and/or FcγRIIA. In another embodiment, the different Fc region specifically binds FcγRIIB with lower affinity than the Fc region of the like native antibody (i.e., an antibody that has the amino acid sequence of the antibody of the invention except for one or more amino acid modifications in the Fc region). In some embodiments, these modifications increase the affinity of the different Fc region of FcγRIIIA and/or FcγRIIA and further enhance the affinity of the different Fc region of FcγRIIB relative to the original antibody. In other embodiments, said amino acid modifications increase the affinity of the different Fc regions of FcγRIIIA and/or FcγRIIA but do not change the affinity of the Fc regions of FcγRIIB relative to the Fc region of the original antibody. In another embodiment, the one or more modifications to said amino acid enhance the affinity of the Fc region of FcγRIIIA and FcγRIIA but reduce the affinity of FcγRIIB relative to the original antibody. Increased affinity and/or avidity results in detectable binding to FcγR or FcγR-binding activity in cells expressing low levels of FcγR when binding activity of the native molecule (body of the modified Fc region) cannot be detected in cells. In other embodiments, the modified molecule has binding that can be detected in cells expressing non-FcγR receptor-targeting antigens at an intensity of 30,000 to 20,000 molecules/cell, an intensity of 20,000 to 10,000 molecules/cell, or an intensity of 10,000 to 5,000 molecules. /cell, or at a strength of 5000 to 1000 molecules/cell, or at a strength of 1000 to 200 molecules/cell, or at a strength of 200 molecules/cell or less (but at least 10, 50, 100, or 150 molecules/cell).

In another embodiment, one or more modifications to the amino acids of the Fc region can reduce the affinity and avidity of the antibody for one or more FcγR receptors. In a particular embodiment, the invention comprises antibodies comprising a different Fc region, wherein that region includes at least one amino acid modification relative to the wild-type Fc region, wherein the different Fc region binds only one FcγR, wherein said FcγR is an FcγRIIIA . In another certain embodiment, the invention comprises antibodies comprising a different Fc region, wherein said different Fc region includes a modification of at least one amino acid relative to the wild-type Fc region, wherein said different Fc region binds only one FcγR, and wherein said FcγR It is FcγRIIA.

Preferably, the binding properties of the molecules of the invention may be characterized by in vitro functional experiments to determine one or more effector cell-mediated functions of the FcγR (see Section 5.2.7). The affinity and binding properties of molecules, such as antibodies, of the invention for the FcγR can be determined using in vitro experiments (biochemical or immunological experiments) known in the art to determine antibody-antigen or Fc-FcγR interactions, i.e. specific binding of antigen to antibody or specific binding to the Fc region of FcγR, respectively, including, but not limited to, ELISA, surface plasmon resonance, and immunoprecipitation tests. In best embodiments, the molecules of the invention have similar binding properties in in vivo embodiments (such as those described and disclosed herein) as reported in in vitro experiments. However, the present invention does not exclude molecules of the invention that do not have the desired phenotype in vitro but have the desired phenotype in vivo.

In some embodiments, molecules of the invention comprising a different Fc region have at least one amino acid modification (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications). In the CH3 domain of the Fc region, which is identified as spanning amino acids 212-447. In other embodiments, molecules of the invention comprising a different Fc region have at least one amino acid modification (e.g., containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications). In the CH2 domain of the Fc region, which is identified as extending from amino acids 231-341. In some embodiments, molecules of the invention having at least two amino acid modifications (e.g. having 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications), where one of these modifications is At least one in the CH3 region and at least one in the CH2 region. The invention also includes modification of the amino acid in the joint area. In a particular embodiment, the invention includes an amino acid modification in the CH1 domain of the Fc region, identified as extending from amino acids 216-230.

In particularly preferred embodiments, the invention comprises molecules containing a different Fc region that confer said different profile or have increased ADCC activity and/or increased binding to FRIIA (CD32A), as measured by methods known to a person skilled in the art and represented In the current document. The ADCC assays used according to the methods of the invention may be NK-based or macrophages-based.

In particularly preferred embodiments, the invention comprises molecules comprising a different Fc region that confer said different profile and have increased ADCC activity and/or increased binding to FcγRIIIA (CD16A), as measured by methods known to a person of skill in the art and represented herein. The ADCC assays used according to the methods of the invention may be NK-based or macrophages-based.

Various Fc images of the present invention may be combined with other Fc modifications, such as those disclosed in U.S. Patent Nos. 7,632,494; 7,521,542; 7,425,619; 7,416,727; 7,371,826; 7,355,008; 7,335,742; 7,332,581; 7.183.387; 7,122,637; and 6,737,056; In PCT Publications International Patent Numbers 105886/2008; 002933/2008; 021841/2007; 2007/106707; 06/088494; 05/115452; 05/110474; 04/1032269; and 04/063351; Also in Presta, LG et al. (2002) Engineering therapeutic antibodies for improved function, Biochem. Soc. Trans. 30(4):487-490; Shields, R.L. et al. (2002) Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity, J. Biol. Chem. 26;277(30):26733-26740 and Shields, R.L. et al. (2001) High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R, J. Biol. Chem. 276(9):6591-6604). The invention includes combining various Fc forms of the invention with other Fc modifications to provide additional, synergistic, or novel properties to the modified antibody. Preferably, the Fc images of the invention enhance the modification phenotype with which they are associated. For example, if the Fc motifs of the invention are fused to a mutant known to bind FcγRIIIA with higher affinity than the wild-type Fc region; The combination with the mutant of the invention results in a greater enhancement in the affinity of FcγRIIIA.

The invention includes antibodies that bind specifically to B7-H3 comprising a different Fc region, wherein the different Fc region comprises at least one amino acid modification (e.g. having 1, 2, 3, 4, 5, 6, 7, 8, 9, or more than two amino acid modifications) relative to the wild-type Fc region, such that the molecule has enhanced effector function relative to the molecule comprising the wild-type Fc region, provided that the different Fc region contains or is merely a substitution at one or more of the positions 243, 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331, 333, 334, 335, 33 7, 338, 339, 340, 359, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438, 439. In a particular embodiment, the invention comprises antibodies comprising a different Fc region, wherein the different Fc region comprises at least one amino acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to the wild-type Fc region, such that the molecule has enhanced effector function relative to a molecule containing the wild-type Fc region, provided that the heterologous Fc region does not contain or is merely a substitution at any of the positions 243, 255, 258 , 267, 269, 270, 276, 278, 280, 283, 285, 289, 292, 293, 294, 295, 296, 300, 303, 305, 307, 309, 320, 322, 329, 332, 331, 337, 338, 340, 373, 376, 416, 419, 434, 435, 437, 438, 439 do not contain an alanine at any of the positions 256 , 290, 298, 312, 326, 333, 334, 359, 360, or 430; asparagine at position 268; and glutamine at position 272; glutamine, serine or aspartic acid at position 286; serine at position 290; methionine at position 301; methionine, glutamine, glutamic acid, or arginine at position 320; glutamic acid at position 322; Asparagine, serine, glutamic acid, or acid Aspartic at position 326; lysine at position 330; and glutamine at position 334; glutamic acid at position 334; methionine at position 334; histidine at position 334; Valine at position 334; leucine at position 334; and glutamine at position 335; lysine at position 335; Or threonine at position 339.

The present invention also includes antibodies that bind specifically to B7-H3 comprising a different Fc region, wherein the different Fc region includes those antibodies comprising a different Fc region, wherein the different Fc region does not contain or is merely a substitution at any of the positions 268 , 269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305, 307, 309, 320, 331, 333, 334, 335, 337, 338, 340, 360 , 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 and do not contain histidine, glutamine at position or tyrosine 280; serine, glycine, threonine or tyrosine at position 290, asparagine at position 294, or lysine at position 295; proline at position 296; or proline, asparagine, aspartic acid, or valine at position 298; Or leucine or iso-leucine at position 300. In another embodiment, the invention includes antibodies comprising a different Fc region, wherein the different Fc region includes at least one amino acid modification relative to the wild-type Fc region, such that the FR molecule binds with reduced affinity relative to the molecule comprising the wild-type Fc region Wild type, provided that it does not contain the wild-type Fc region, provided that the different Fc region does not contain or is merely a substitution at one or more of the positions 243, 252, 254, 265, 268, 269, 270, 278, 289, 292, 293, 294, 295. , 296, 298, 300, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, ​​388, 389, 414, 416, 419, 434, 435, 437, 438, or 439. In another embodiment, the invention comprises antibodies comprising a different Fc region, wherein the different Fc region includes at least one amino acid modification relative to the wild-type Fc region, such that the molecule binds FR with enhanced affinity relative to a molecule comprising the Fc region of Wild type provided that the different Fc region does not contain or is merely a substitution at one or more of the positions 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315, 331 , 333, 334, 337, 340, 360, 378, 398, or 430.

The invention also includes antibodies that bind specifically to B7-H3 comprising a different Fc region, wherein the different Fc region does not include or is merely a substitution at one or more positions 330, 243, 247, 298, 241, 240, 244, 263, 262 , 235, 269, or 328 and does not contain leucine at position 243, asparagine at position 298, leucine at position 241, isoleucine or alanine at position 240, histidine at position 244, valine at position 330, or isoleucine at position 328. .

In particular, the invention includes antibodies specifically binding to B7-H3 comprising a different Fc region having enhanced effector function and/or heterogeneous affinity for activating and/or inhibitory receptors, wherein the different Fc region includes: (a) either 1, or 2, or 3, 4, 5, or 6 of the following substitutions:; S239D, S298A, A330L, I332E, E333A or K334A; or (b) any combination of substitutions: (i) S298A, E333A, and K334A; (2) S239D and I332E; Or (3) S239D, A330L, and I332E.

The invention also specifically includes antibodies that bind specifically to B7-H3, comprising a different Fc region with enhanced effector function and/or heterogeneous affinity for activating and/or inhibitory receptors, wherein the different Fc region includes a substitution:

(1) at position 288 busparagine, at position 330 serine and at position 396 pliocene;

(2) at position 334 with glutamic acid, at position 359 with asparagine, and at position 336 with serine;

(3) at position 316 with aspartic acid, at position 378 with valine, and at position 399 with glutamic acid;

(4) at position 247 with pliocene, substitution at position 421 with plycine;

(5) at position 392 in threonine, and at position 396 in pliocene;

(6) at position 221 with glutamic acid, at position 270 with glutamic acid, at position 308 with alanine, at position 311 with histidine, at position 396 with leucine, and at position 402 with aspartic acid;

(7) at position 419 with a histidine, and a replacement at position 396 with a leucine;

(8) at position 240 in alanine, and at position 396 in leucine;

(9) at position 410 with histidine, and at position 396 with leucine;

(10) at position 243 with leucine, at position 305 with iso-leucine, at position 378 with aspartic acid, at position 404 with serine, and at position 396 with leucine;

(11) At position 255 iso-leucine, and at position 396 pleucine;

(12) at position 370 with glutamic acid and at position 396 with leucine;

(13) at position 270 with glutamic acid; or

(14) Any combination of the previous substitutions (1)-(12).

In a particular embodiment, the invention includes an antibody that specifically binds to B7-H3 comprising a heterologous Fc region comprising the substitution: F243L, R292P, and Y300L. In another embodiment, the invention includes an antibody that specifically binds to B7-H3 comprising a different Fc region comprising the substitution: L235V, F243L, R292P, Y300L and P396L. In another embodiment, the invention comprises an antibody that specifically binds to B7-H3 and which includes a different Fc region including the substitution F243L, R292P, Y300L, V305I, and P396L.

In a particular embodiment, the invention comprises an antibody that specifically binds to B7-H3 comprising a different Fc region comprising a replacement at position 396 with a leucine, at position 270 with a glutamic acid, and at position 243 with a leucine. In another specified embodiment, the molecule further includes one or more amino acid modifications such as those disclosed herein.

In particular, the invention includes antibodies specifically binding to B7-H3, comprising a different Fc region with enhanced effector function and/or modified affinity for activating and/or inhibitory receptors, having an amino acid modification at one or more of the following positions, 119, 125 , 132, 133, 141, 142, 147, 149, 162, 166, 185, 192, 202, 205, 210, 214, 215, 216, 217, 218, 219, 221, 222, 223, 224, 225, 227 , 229, 231, 232, 233, 235, 240, 241, 242, 243, 244, 246, 247, 248, 250, 251, 252, 253, 254, 255, 256, 258, 261, 262, 263, 268 , 269, 270, 272, 274, 275, 276, 279, 280, 281, 282, 284, 287, 288, 289, 290, 291, 292, 293, 295, 298, 301, 303, 304, 305, 306, 307, 30 8, 309, 310, 311, 312, 313, 315, 316, 317, 318, 319, 320, 323, 326, 327, 328, 330, 333, 334, 335, 337, 339, 340, 343, 344, 34 5, 347, 348, 352, 353, 354, 355, 358, 359, 360, 361, 362, 365, 366, 367, 369, 370, 371, 372, 375, 377, 378, 379, 380, 381, 38 2, 383, 384, 385, 386, 387, 388, 389, 390, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 404, 406, 407, 408, 409, 41 0, 411, 412, 414, 415, 416, 417, 419, 420, 421, 422, 423, 424, 427, 428, 431, 433, 435, 436, 438, 440, 441, 442, 443, 446or 44 7. And who Preferably, such mutations would produce molecules that would have conferred a function caused by an effector cell and, optionally, with modified affinity for FcR as determined by the methods disclosed and represented in the present document and known to a person of skill in the art.

The invention also particularly includes antibodies that bind specifically to B7-H3, comprising a different Fc region with enhanced effector function and/or modified affinity for activating and/or inhibitory receptors, which are characterized by:

(1) An amino acid modification at one or more of the following positions: 235, 240, 241, 243, 244, 247, 262, 263, 269, 298, 328, or 330, and preferably one or more of the following: V240A, V240I. , F241L, F243L, P244H, S298N, L328I, A330V; These antibodies have modified effector function relative to antibodies containing the wild-type Fc region that lack these modifications;

(2) Amino acid modification at one or more of the following positions: 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, 303, 305, 307, 309, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439 and preferably one or more of the following amendments: D280H, D280Q, D280Y, K290G, K290S, K290T, K290Y, E294N, Q295K, Y296P, S298D, S298N, S298P, S298V, Y300I, Y300L; These antibodies have modified effector function relative to antibodies with a wild-type Fc region that lack these modifications;

(3) Amino acid modification at one or more of the following positions: 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331, 333, 334, 335, 337, 338, 339, 34 0, 359, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438, 439, and preferably one or more of the following amendments: T256A, H268N, E272Q, N286D, N286Q, N286S, K290A, K290S, S298A, R301M, D312A, K320E, K320M, K320Q, K320R, K322E, K326A, K326D, K326E, K326N, K32 6S, A330K, A339T, E333A, K334A, K334E, K334H, K334L, K334M, K334Q, K334V, T335K, T335Q, T359A, K360A, E430A; These antibodies have modified effector function relative to antibodies containing the wild-type Fc region that lack these modifications;

(4) Amino acid modification at one or more of the following positions: 252, 254, 265, 268, 269, 270, 278, 289, 292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, ​​388, 389, 414, 416, 419, 434, 435, 437, 438, or 439; These antibodies have reduced effector function relative to antibodies containing the wild-type Fc region lacking these modifications;

(5) Amino acid modification at one or more of the following positions: 280, 283, 285, 286, 290, 294, 295, 298, 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398, or 430; These antibodies have enhanced effector function relative to antibodies that have the wild-type Fc region and lack that modification;

or

(6) Amino acid modification at one or more of the following positions: R255A, T256A, E258A, S267A, H268A, H268N, E272A, E272Q, N276A, D280A, E283A, H285A, N286A, N286D, N286Q, N286S, K290A, K290S , R301M, K320E, K320M, K320Q, K320R, K322E, K326A, K326D, K326E, K326S, A330K, P331A, T335Q, S337A, E430A; These antibodies have enhanced effector function relative to antibodies that have the wild-type Fc region and lack that modification;

In other embodiments, the invention comprises the use of any Fc motif known in the art such as that disclosed in Jefferis, B.J. et al. (2002) Interaction Sites On Human IgG-Fc For FcgammaR: Current Models, Immunol. Lett. 82:57-65; Presta, L.G. et al. (2002) Engineering Therapeutic Antibodies For Improved Function, Biochem. Soc. Trans. 30:487-90; Idusogie, E. E. et al. (2001) Engineered Antibodies With Increased Activity To Recruit Complement, J. Immunol. 166:2571-75; Shields, R.L. et al. (2001) High Resolution Mapping Of The Binding Site On Human IgG1 For Fc Gamma RI, Fc Gamma RII, Fc Gamma RIII, And FcRn And Design Of IgG1 Variants With Improved Binding To The Fc Gamma R, J. Biol. Chem. 276:6591-6604; Idusogie, E. E. et al. (2000) Mapping Of The C1q Binding Site On Rituxan, A Chimeric Antibody With A Human IgG Fc, J. Immunol. 164:4178-84; Reddy, M. P. et al. (2000) Elimination Of Fc Receptor-Dependent Effector Functions Of A Modified IgG4 Monoclonal Antibody To Human CD4, J. Immunol. 164:1925-1933; Xu, D. et al. (2000) In Vitro Characterization of Five Humanized OKT3 Effector Function Variant Antibodies, Cell. Immunol. 200:16-26; Armour, K. L. et al. (1999) Recombinant human IgG Molecules Lacking Fcgamma Receptor I Binding And Monocyte Triggering Activities, Eur. J. Immunol. 29:2613-24; Jefferis, R. et al. (1996) Modulation Of Fc(Gamma)R And Human Complement Activation By IgG3-Core Oligosaccharide Interactions, Immunol. Lett. 54:101-04; Lund, J. et al. (1996) Multiple Interactions Of IgG With Its Core Oligosaccharide Can Modulate Recognition By Complement And Human Fc Gamma Receptor I And Influence The Synthesis Of Its Oligosaccharide Chains, J. Immunol. 157:4963-4969; Hutchins et al. (1995) Improved Biodistribution, Tumor Targeting, and Reduced Immunogenicity In Mice With A Gamma 4 Variant Of Campath-1H, Proc. Natl. Acad. Sci. (USA) 92:11980-84; Jefferis, R. et al. (1995) Recognition Sites On Human IgG For Fc Gamma Receptors: The Role Of Glycosylation, Immunol. Lett. 44:111-17; Lund, J. et al. (1995) Oligosaccharide-Protein Interactions In IgG Can Modulate Recognition By Fc Gamma Receptors, FASEB J. 9:115-19; Alegre, M. L. et al. (1994) A Non-Activating “Humanized” Anti-CD3 Monoclonal Antibody Retains Immunosuppressive Properties In Vivo, Transplantation 57:1537-1543; Lund et al. (1992) Multiple Binding Sites On The CH2 Domain Of IgG For Mouse Fc Gamma R11, Mol. Immunol. 29:53-59; Lund et al. (1991) Human Fc Gamma RI And Fc Gamma RII Interact With Distinct But Overlapping Sites On Human IgG, J. Immunol. 147:2657-2662; Duncan, A. R. et al. (1988) Localization Of The Binding Site For The Human High-Affinity Fc Receptor On IgG, Nature 332:563-564; US Patent Numbers: 5,624,821; 5885573; 6,194,5517276586 and 7,317,091 and PCT publications 00/42072 and 99/58572.

The invention comprises molecules comprising various Fc regions consisting of or comprising any of the mutations described in Table 1 below.

Table 1

Representative Fc modifications

Replacements for a single site

S132I

F241W

D265N

D280Q

Y296T

D312A

L328I

K334E

A162V

F241Y

D265Q

D280Y

N297D

W313F

L328K

K334H

S219Y

F241Y

D265T

G281D

N297E

N315I

L328M

K334I

K222N

F243D

D265V

G281K

N297I

E318K

L328N

K334L

H224L

F243H

D265Y

G281P

N297S

K320E

L328P

K334M

T225S

F243L

V266A

G281Y

S298A

K320M

L328Q

K334N

P228E

F243L

V266I

V282M

S298D

K320Q

L328R

K334Q

P228G

F243Q

V266M

E283A

S298N

K320R

L328S

K334V

P228K

F243R

V266T

V284E

S298N

K322E

L328T

T335K

P228Y

F243W

S267A

V284L

S298N

V323I

L328V

T335Q

P230A

F243Y

H268A

V284N

S298P

N325A

L328W

I336E

P230E

P244H

H268N

V284T

S298V

N325D

L328Y

I336K

P230G

P245A

D270E

V284Y

T299A

N325E

A330i

I336Y

P230Y

P247G

P271A

H285A

T299D

N325F

A330K

S337A

A231E

P247L

P271D

N286A

T299E

N325G

A330L

A339T

A231G

P247V

P271E

N286D

T299F

N325H

A330S

M352L

A231K

K248M

P271F

N286S

T299G

N325I

A330V

T359A

A231P

R255A

P271G

K288N

T299H

N325K

A330Y

T359N

A231Y

T256A

P271H

K290A

T299I

N325L

P331A

K360A

P232E

E258A

P271I

K290G

T299K

N325M

I332A

T366N

P232G

V262A

P271K

K290S

T299L

N325P

I332D

T366S

P232K

V262E

P271L

K290T

T299M

N325R

I332E

F372Y

P232Y

V262F

P271M

K290Y

T299N

N325S

I332F

F372Y

E233D

V262I

P271N

P291D

T299P

N325T

I332G

I377F

E233G

V262T

P271Q

P291E

T299Q

N325V

I332H

I377N

L234I

V263A

P271R

P291G

T299R

N325W

I332K

V379L

L235D

V263I

P271S

P291H

T299S

N325Y

I332L

V379M

S239D

V263M

P271T

P291I

T299V

K326A

I332M

K392R

S239E

V263T

P271V

P291Q

T299W

K326D

I332N

P396H

S239N

V264A

P271W

P291T

T299Y

K326E

I332P

P396L

S239Q

V264E

P271Y

R292G

Y300I

K326E

I332Q

L398V

V240A

V264F

E272A

R292L

Y300L

K326N

I332R

S400P

V240I

V264I

E272Q

E294N

R301M

K326S

I332S

D401V

V240M

V264R

V273I

Q295K

R301M

K326T

I332T

S407I

V240T

V264T

F275L

Y296D

V302I

L328A

I332V

K414N

F241E

V264W

F275W

Y296E

S304D

L328D

I332W

E430A

V241I

D265F

F275Y

Y296H

S304H

L328E

I332Y

F241L

D265H

N276A

Y296N

S304L

L328F

E333A

F241R

D265I

D280A

Y296P

S304N

L328G

K334A

F241S

D265L

D280H

Y296Q

S304T

L328H

K334E

Replacements for two sites

I332E, A330L

S239N/I332Q

V279L, P395S

P396L, P217S

I332E, L328D

S239Q/I332D

V284A, F372L

P396L, P227S

I332E, L328E

S239Q/I332E

K288N, K326N

P396L, V323I

I332E, L328H

S239Q/I332N

K288N, A330S

P396L, V240A

I332E, L328I

S239Q / I332Q

K290E, L142P

P396L, L242F

I332E, L328M

V240I, V281M

K290E, P227S

P396L, P244H

I332E, L328N

F241L, E258G

K290T, G371D

P396L, T250A

I332E, L328Q

F241L / V262I

P291S, P353Q

P396L, R255L

I332E, L328T

F243L, E318K

R292P, V305I

P396L, E258D

I332E, L328V

F243I, V379L

S298A/I332E

P396L, H268D

I332E, N297D

P243L / V264I

S298N, W381R

P396L, H268N

I332E, N297E

K246T, Y319F

S298N, S407R

P396L, V303I

I332E, N297S

K246T, P396H

K317N, F423-DEL

P396L, K326I

S166N, K409R

P247H, G285E

K326E, K320E

P396L, V305L

P232S, S304G

P247L, I377F

K326E, A330T

P396L, L358P

S239D / I332D

P247L, E389G

K326E, G385E

P396L, K370E

S239D / I332E

P247S, P396L

A330V, Q419H

P396L, S375C

S239D/I332N

P247L, L398Q

K334E, E233D

P396L, V379M

S239D / I332Q

P247L, L406F

K334N, K246I

P396L, N384K

S239E/D265N

P247L, N421K

K334E, K288M

P396L, K392T

S239E/D265Q

L251F, F372L

K334E, R292L

P396L, S400F

S239E/I332D

L251F, S415I

K334E, E308D

P396L, L410H

S239E / I332E

R255L, E318K

K334E, E380D

P396L, Q419H

S239E/I332N

R255Q, K326E

K334N, P396L

P396L, Q419L

S239E / I332Q

E258D, N384K

A339V, Q347H

P396L, V427A

S239N/I332D

V263Q, E272D

K370N, S440N

D399E, G402D

S239N/I332E

V264I/I332E

T394M, V397M

D399E, M428L

S239N/I332N

H268D, E318D

P396L, K210M

Replacements for three sites

V185M, R292L, D399E

P217S, A378V, S408R

K218R, G281D, G385R

S192T, M252L, R301C

P247L, I253N, K334N

P247L, A330T, S440G

V125L, V215I, S408I

D312E, K327N, I378S

T355N, P387S, H435Q

R292L, T359N, P396L

E216D, E345K, S375I

P247L, A431V, S442F

F275I, K334N, V348M

K288N, A330S, P396L

A378V, N390I, V422I

F243L, R255L, E318K

G316D, A378V, D399E

V282E, V369I, L406F

K334E, T359N, T366S

N315I, V379M, T394M

V397M, T411A, S415N

K288N, A330S, P396L

P247L,W313R,E388G

T223I, T256S, L406F

F243I, V379L, G420V

R301H, K340E, D399E

K246N, P396L, Q419R

A231V, Q386H, V412M

K326I, P396L, S408N

P217A, T359A, P396L

E216D,K334R,S375I

K210M, K261N, P396L

V215I, K290V, P396L

T335N, P387S, H435Q

A330V, G427M, K438R

V263Q, E272D, Q419H

K246I, Q362H, K370E

K222E, V263Q, S298N

N276Y, T393N, W417R

K334E, E380D, G446V

E233G, P247S, L306P

D270E, G316D, R416G

V303I, V369F, M428L

S219T, T225K, D270E

D270E, K392T, P396L

K246E, V284M, V308A

R292P, F243L, V305I

R255L, D270E, P396L

E293V, Q295E, A327T

V284M, R292L, K370N

V240A, D270E, P396L

Y319F, P352L, P396L

D270E, K370E, P396L

270E, P396L, Q419HD

K290T, N390I, P396L

P247L, D270E, N421K

S239D, A330L, I332E

N297D, A330Y, I332E

Y296D, N297D, I332E

S239D, A330Y, I332E

N297D, T299L, I332E

Y296E, N297D, I332E

S239D, I332E, A330I

N297D, T299I, I332E

Y296H, N297D, I332E

S239D, N297D, I332E

N297D, T299L, I332E

Y296N, N297D, I332E

S239D, S298A, I332E

N297D, T299V, I332E

Y296Q, N297I, I332E

S239D, V2641I, I332E

F243L, V262I, V264W

Y296T, N297D, I332E

S239E, N297D, I332E

D265F, N297E, I332E

P230A, E233D, I332E

S239E, V264I, I332E

D265Y, N297D, I332E

P244H, P245A, P247V

S239N, A330L, I332E

V264E, N297D, I332E

V264I, A330Y, I332E

S239N, A330Y, I332E

V264I, A330L, I332E

V264I, S298A, I332E

S239Q, V264I, I332E

Replacements of four sites

A141V, H268L, K288E, P291S

T256S, V305I, K334E, N390S

E258D, T289A, H310Y, Y407V

D280E, S354F, A431D, L441I

K334E, T359N, T366S, Q386R

P343S, P353L, S375I, S383N

K326Q, K334E, T359N, T366S

E269K, K290N, Q311R, H433Y

K288R, T307A, K344E, P396L

K290E, V369A, T393A, P396L

V273I, K326E, L328I, P396L

K210N, K222I, K320M, P396L

F275L, Q362H, N384K, P396L

S219T, T225K, D270E, K360R

V282L, A330V, H433Y, T436R

P243L, S254T, A330V, N361D

R255L, D270E, Y300L, P396L,

F243L, D270E, K392N, P396L

R255L, D270E, R292G, P396L

F243L, R255L, D270E, P396L

V284M, S298N, K334E, R355W

S239D, D265F, N297D, I332E

D265Y, N297D, T299L, I332E

S239D, D265H, N297D, I332E

F241E, F2430, V262T, V264F

S239D, D265I, N297D, I332E

F241E, F243R, V262E, V264R

S239D, 0265L, N297D, I332E

F241E, F243Y, V262T, V264R

S239D, D265T, N297D, I332E

F241L, F243L, V262I, V264I

S239D, D265V, N297D, I332E

F241R, F2430, V262T, V264R

S239D, D265Y, N297D, I332E

F241W, F243W, V262A, V264A

S239D, N297D, I332E, A330Y

F241Y, F243Y, V262T, V264T

S239D, N297D, I332E, K326E

N297D, I332E, S239D, A330L

S239D, N297D, I332E, L235D

N297D, S298A, A330Y, I 332E

S239D, V264I, A330L, I332E

S239D, A330Y, I332E, K326E

S239D, V264I, S298A, I332E

S239D, A330Y, I332E, K326T

S239E, V264I, A330Y, I332E

S239D, A330Y, I332E, L234I

S239D, A330Y, I332E, V264T

S239D, A330Y, I332E, L235D

S239D, A330Y, I332E, V266I

S239D, A330Y, I332E, V240I

Five site replacements

V284M, S298N, K334E, R355W, R416T

K147T, Y202M, F275I, K334N, V348M

P217S, V305I, I309L, N390H, P396L

T335N, K370E, A378V, T394M, S424L

F243L, V305I, A378D, P396L, F404S

P244H, L358M, V379M, N384K, V397M

K222N, T335N, K370E, A378V, T394M

P244A, K326I, C367R, S375I, K447T

L235P, S304G, V305I, V323I, V382M

C229Y, A287T, V379M, P396L, L443V

F241E, F2430, V262T, V264E, I332E

F241R, F243Q, V262T, V264R, I332E

F241E, F243R, V262E, V264R, I332E

S239E, V264I, S298A, A330Y, I332E

F241E, F243Y, V262T, V264R, I332E

Replacements of more than five locations

D221E, D270E, V308A, Q311H, P396L, G402D

T215P, K274N, A287G, K334N, L365V, P396L

F241Y, F243Y, V262T, V264T, N297D, I332E

N297D, T299F, I332E, N297D, T299H, I332E

D221Y, M252I, A330G, A339T, T359N, V422I, H433L

S239D, N297D, I332E, A330Y, F241S, F243H, V262T, V264T

K133M, F149Y, K205E, R214I, K218E, S383N, N384K, T256N, V262L

In certain embodiments, the different Fc region of such anti-B7-H3 antibodies is characterized by:

Leucine at position 247, and lysine at position 247

(1) leucine at position 247, lysine at position 421 and glutamic acid at position 270;

(2) Threonine at position 392, leucine at position 396, glutamic acid at position 270, and leucine at position 243

(3) histidine at position 419, leucine at position 396, and glutamic acid at position 270;

(4) histidine at position 419, leucine at position 396, glutamic acid at position 270, and leucine at position 243;

(5) alanine at position 240, leucine at position 396, and glutamic acid at position 270;

(6) lysine at position 255 and leucine at position 396;

(7) lysine at position 255, leucine at position 396, and glutamic acid at position 270;

(8) lysine at position 255, leucine at position 396, glutamic acid at position 270, and lysine at position 300;

(9) lysine at position 255, leucine at position 396, glutamic acid at position 270, and glycine at position 292;

(10) lysine at position 255, leucine at position 396, glutamic acid at position 270, and leucine at position 243;

(11) glutamic acid at position 370, leucine at position 396, and glutamic acid at position 270;

(12) glutamic acid at position 270, aspartic acid at position 316, and glycine at position 416;

(13) leucine at position 243, proline at position 292, iso-lysine at position 305, and leucine at position 396;

(14) leucine at position 243, glutamic acid at position 270, asparagine at position 392 and leucine at position 396;

(15) leucine at position 243, leucine at position 255, glutamic acid at position 270 and leucine at position 396;

(16) Glutamine at position 297;

(17) Any combination of the previous substitutions (1)-(16).

In some embodiments, the molecules of the invention further comprise one or more glycosylation sites, such that one or more carbohydrate moieties are covalently attached to the molecule. Preferably, the molecules of the invention may be endowed with one or more glycosylation sites and/or one or more modifications in the Fc region or have an enhanced effector function induced by the antibody, such as enhanced ADCC activity, compared to the original antibody. In some embodiments, the invention further includes molecules comprising one or more amino acid modifications known to directly or indirectly interact with the carbohydrate moiety of the antibody, including but not limited to amino acids at positions 241, 243, 244, 245. , 245, 249, 256, 258, 260, 262, 264, 265, 296, 299, and 301.

In another embodiment, the invention comprises molecules that have been modified by introducing one or more glycosylation sites into one or more molecule sites, preferably without changing the function of the molecules, such as binding activity to the target antigen or FcR. The glycosylation sites may be inserted into a variable and/or constant region of the molecules of the invention. As used, the term “glycosylation sites” includes any relevant amino acid sequence in the antibody to which an oligosaccharide (i.e., a carbohydrate containing two or more simple sugars linked together) will be specifically or covalently attached. The saccharide side chains are typically attached to the antibody structure via either N- or O-linkages. N-linked glycosylation indicates contact of the oligosaccharide moiety with the side chain of the asparagine residue. O-linked glycosylation indicates the connection of the oligosaccharide moiety to a hydroxyamino acid, such as serine, and threonine. The molecules of the invention may include one or more glycosylation sites, such as N- or O-linked glycosylation sites. Any N- or O-linked glycosylation site known in the art may be used in accordance with the present invention. An example of an N-linked glycosylation site useful according to the methods of the present invention is the amino acid sequence: Asn-X-Thr/Ser, where X can be any amino acid and Thr/Ser refers to threonine or serine. Such site or sites may be introduced into the molecule of the invention using methods known in the art of this invention (see, for example, In Vitro Mutagenesis, Recombinant DNA: A Short Course, J. D. Watson, et al. W. H. Freeman and Company, New York, 1983 , chapter 8, pp. 106-116, which is incorporated throughout the present invention by reference. Examples of the glycosylation site in a molecule of the present invention may include: modifying or mutating the amino acid sequence of the molecule such that an Asn-X-Thr/Ser sequence is obtained.

In some embodiments, the invention includes methods for modifying the proportion of carbohydrates in the molecule of the invention by adding or deleting a glycosylation site. Methods for modifying the proportion of carbohydrates are known in the art and are within the scope of invention. See, for example, US Patent 6,218,149; and European 096 359 0; American 0028486/2002, international order 035835/03; American Publication No. 0115414/2003; US Patent No. 6,218,149; and 6,472,511; They are all included in their entirety in the present document as references. In other embodiments, the invention includes methods for modifying the proportion of carbohydrates in the molecule of the invention by deleting one or more carbohydrate moieties in the molecule. In a specific embodiment, the invention includes modifying the glycosylation site of the Fc region of the antibody, by modifying positions close to 297. In a specific embodiment, the invention includes modifying position 296 such that position 296 but not position 297 is glycosylated.

The function of the effector can also be modified using methods such as introducing one or more cysteine ​​residues into the Fc region, thus allowing the formation of a disulfide bond between chains in that region, generating a homodimeric antibody that can improve internalization capacity and/or increase Complement-Mediated Cell Killing and ADCC (Caron, PC et al. (1992) Engineered Humanized Dimeric Forms Of IgG Are More Effective Antibodies, J. Exp. Med. 176:1191-1195; Shopes, B. (1992) A Genetically Engineered Human IgG Mutant With Enhanced Cytolytic Activity, J. Immunol. 148(9):2918-2922. Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using bifunctional heterogeneous crosslinkers as described in EA et al. (1993) Monoclonal Antibody Homodimers: Enhanced Antitumor Activity In Nude Mice, Cancer Research 53:2560-2565. Alternatively, an antibody could be modified with dual Fc regions which could have enhanced load degradation and ADCC capabilities. Stevenson, G. T. et al. (1989) A Chimeric Antibody With Dual Fc Regions (bisFabFc) Prepared By Manipulations At The IgG Hinge, Anti-Cancer Drug Design 3:219-230).

E- (double affinity retargeting reactants)

As discussed above, the present invention further comprises “DART” (double affinity retargeting reactants) molecules comprising at least two polypeptide chains that form at least two epitope binding sites, one of which specifically binds to B7- H3.

In preferred embodiments, the polypeptide chain of DART comprises:

(i) domain (A) comprising the binding region of the variable domain of immunoglobulin light chain I (VL1) of the epitope (1);

(ii) domain (b) containing the binding region of the variable domain of immunoglobulin heavy chain II (VH2) for binding to the epitope (2); And

(iii) Scope (c).

The second polypeptide chain of DART includes:

(i) domain (d) comprising the binding region of the variable domain of immunoglobulin light chain II (VL2) of the epitope (2);

(ii) domain (e) containing the binding region of the immunoglobulin heavy chain I (VH1) variable domain for binding to the epitope (1); And

(iii) Scope (f).

Domains A and B do not bind to each other to form the epitope binding site. Similarly, domains D and E do not bind to each other to form the epitope binding site. In addition, the DART domains (a) and (b) link to form a binding site that binds the epitope (1); The DART domains (b) and (d) bind to form a binding site specific to the epitope (2). Domain (c) and (f) are covalently bonded to each other.

Each polypeptide chain of the DART molecule includes a VL domain and a VH domain, which are covalently linked to each other so that the domains are prevented from self-assembling. The interaction of two polypeptide chains will produce two VL-VH pairs, which form two epitope binding positions, i.e., a divalent molecule. Neither the VH nor VL domains are restricted to any position within the polypeptide chain, i.e., restricted to any amine (N) or carboxy (C) terminus, or any restriction positions are in positions relative to each other, i.e., the VL domain can serve as the N terminus. For the VH domain and vice versa. The only limitation is the complementary polypeptide chain that can be available for functional DARTs to be formed. Since the VL and VH domain are derived from the same antibody, the two complementary polypeptide chains can be identical. For example, when the binding domains are derived from an antibody specific for epitope A (i.e., the binding domain formed by the VLA-VHA interaction), each polypeptide will include VHA and VLA. Homodimerization of two polypeptide chains of the antibody will result in the formation of two VLA-VHA binding sites resulting in a monospecific antibody. Whereas the VL and VH domains are derived from antibodies specific for different antigens, the functional DART dimer is formed, requiring the interaction of two different polypeptides, i.e., the formation of different dimers. For example, for bispecific DART, one of the polypeptide chains includes VLA and VLB; The homodimer will result in the formation of two VLA-VHB binding sites with either no association or an unpredictable association. Conversely, two different polypeptide chains are free to interact, i.e., in the recombinant expression system, one comprising VLA and VHB and the other comprising VLB and VHA and thus two binding sites are formed: VLA-VHA and VLB-VHB. For all pairs of DART polypeptide chains, the possibility of misalignment or lack of association of two chains, i.e. interaction of VL-VL or VH-VH domains, can exist; However, purification of dimers based on immunolabeling of the appropriate dimeric binding site can easily be performed using any affinity-based method known in the art and described herein, e.g., affinity chromatography.

One or more polypeptide chains of DART can optionally comprise an Fc domain or a portion thereof (e.g., CH2 domain or CH3 domain). The Fc domain, or part thereof, may be derived from any corresponding immunoglobulin fragment or homolog including, but not limited to, IgA, IgD, IgG, IgE, and IgM. In preferred embodiments, the Fc domain (or portion thereof) is derivatized in order to form an IgG. In certain embodiments, the IgG isotype is IgG1, IgG2, IgG3, IgG4, or any allele thereof. In one embodiment, the dimeric antibody molecule comprises an Fc domain, the Fc domain comprising a CH2 domain and a CH3 domain independently selected from the corresponding type of immunoglobulin (i.e., the Fc domain comprising a CH2 domain that is derived from an IgG and a CH3 domain derived from IgE or CH2 domain which is derived from IgG1 and CH3 domain or derived from IgG2, etc.). The Fc domain of the polypeptide chain comprising the dimeric body molecule of the present invention may be modified at any position relative to other domains or parts of said polypeptide chain (i.e., the Fc domain or part thereof which may have the c-terminus of both the VL and VH domains of The polypeptide of the chain; or it can have the n-terminus of both the VL and VH domains; or it can be the N-terminus of one domain and the c-terminus of the other (i.e., between two domains of the polypeptide chain).

The Fc domains in the polypeptide chains of DART molecules are preferably dimerized, resulting in the formation of a DART molecule that displays immunoglobulin-like properties, for example, Fc-FcγR interactions. Fc may comprise dimers which may be dimers, for example, comprising two polypeptide chains each comprising a VH domain, a VL domain and an Fc domain. Dimerization of the aforementioned polypeptide results in a bivalent DART containing an Fc domain, albeit with a structure different from that of the bivalent antibody. Such that DART molecules exhibit an altered immunoglobulin-specific phenotype that has not been processed, e.g., altered serum half-life, binding properties, etc. In other embodiments, the DART molecules include Fc domains which may be tetramers. Said quaternary units comprise two “heavier” polypeptide chains, i.e. the polypeptide chains comprising the VL, VH and Fc domain and two “lighter” polypeptide chains, i.e. the polypeptide chains comprising the VL and VH domain. The lighter and heavier chains interact to form a monomer and the said monomer interacts through the unpaired Fc domains to form an Ig-like molecule. Ig-like DARTs are tetrameric molecules, they can be monomeric, they can be multimeric, they can be tetrameric and specific.

The formation of a specific dimeric tetrameric antibody molecule requires the interaction of four different polypeptide chains. This reaction is difficult to achieve with the desired efficiency in a single gene recombination cell production system due to the many variants of potential chain pairs that are not good. One solution involves increasing the possibility of missing pairs in modifying “blocks in holes” in the desired polypeptide chain pairs. These mutations require the presence of the heterodimer as opposed to the homodimer. For example, for Fc-Fc interactions, an amino acid substitution (preferably a substitution using an amino acid with a bulky side group that forms a “cluster” e.g., a crystalline amino acid) can be introduced into the CH2 or CH3 domain such as a stereointerference that prevents interaction with Similarly mutagenic domains which will pair the mutagenic domain with a domain in complementary mutagenesis or positional mutagenesis that is modified, i.e., “punched” (e.g., replaced with glycine). Mutagenic groups can be modified in any pair of polypeptides comprising the body dimer and processing can also take place in any part of the polypeptide chains of the said pair. Methods for protein modification by heterodimerization as opposed to homodimerization are known in the art, specifically for the modification of immunoglobulin-like molecules that are included herein (see, for example, Ridgway et al. (1996) “‘Knobs-Into-Holes’ Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization, Protein Engr. 9:617-621, Atwell et al. (1997) Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library, J. Mol Biol 270: 26-35, and Xie et al. (2005) A New Format of Bispecific Antibody: Highly Efficient Heterodimerization, Expression and Tumor Cell Lysis, J. Methods 296:95-101” Each of these references is incorporated throughout this document by reference.

The present invention also includes binary antibody molecules comprising the variable form of Fc or variable hinge domains of Fc (or part thereof) and the variable domains of Fc comprising at least one amino acid modification (e.g., substitution, insertion, or deletion) of the cognate unprocessed form of the Fc domain or of the hinge Fc domain (or part thereof). Molecules with an altered form of Fc domains or Fc hinge domains (or parts thereof) (for example, antibodies) naturally have variable phenotypes relative to molecules containing the unprocessed form of Fc domains or Fc hinge domains or parts thereof . An altered phenotype may be expressed as altered serum half-life, altered stability, altered exposure to cellular enzymes, or altered neurotransmitter function as tested in a non-NK-dependent assay or a macrophage-dependent assay. The Fc domain is specified in the form of a neurotransmitter function as disclosed above.

The present invention also includes molecules comprising the hinge domain. The hinge domain can be derived from any immunoglobulin isotype or allele, which includes IgA, IgD, IgG, IgE, and IgM. In preferred embodiments, the hinge domains are derived from IgG, wherein the IgG isotype is IgG1, IgG2, IgG3, IgG4, or an allele thereof. Said hinge domain may be modified into any polypeptide chain comprising a dimer with an Fc domain such as a dimer with an Fc domain. In certain embodiments, the Fc domain is independently selected from any similar type of immunoglobulin mentioned in the domain or described herein. In other embodiments, the hinge domain and the Fc domain are separated by at least one domain of the polypeptide chain, e.g., a VL domain. The hinge domain or optionally the Fc hinge domain can be modified and processed in the polypeptide of the invention at any position relative to other domains or portions of the polypeptide chain. In certain embodiments, the polypeptide chain of the present invention comprises the hinge domain, wherein the hinge domain is at the C terminus of the polypeptide chain, and wherein the polypeptide chain does not include the Fc domain. In yet another embodiment, the polypeptide chain of the present invention comprises an Fc hinge domain, wherein the Fc hinge domain is the C terminus of the polypeptide chain. In other embodiments, the polypeptide chain of the invention comprises an Fc hinge domain, wherein the Fc hinge domain is at the N terminus of the polypeptide chain.

Each domain of the polypeptide chain of DART, i.e., the VL, VH, and Fc domains, can be separated by a peptide linker. The peptide linker can be 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in length. In certain embodiments, the amino acid linker sequence is GGGSGGGG (sequence 52) which is encoded by the amino acid sequence ggaggcggat ccggaggcgg aggc (sequence 53). The polypeptide chains of the DART molecule can be modified to include at least one cysteine ​​residue that interacts with a corresponding cysteine ​​residue on the second polypeptide chain of DART to form a disulfide bond within the chain. Intra-chain disulfide bonds are used to stabilize the DART molecule, thus improving expression and extraction in recombinant systems, which results in a stable and compatible formulation and improved stability of the isolated and/or purified product in vivo. The cysteine ​​residue may be inserted as a single amino acid or part of a larger amino acid sequence, e.g., a hinge domain, into any part of the polypeptide chain. In a particular embodiment, the cysteine ​​residue can be modified to access the C terminus of the polypeptide chain. In some embodiments, the cysteine ​​residue in the polypeptide chain is inserted within the amino acid sequence LGGC. In a particular embodiment, the C terminus of the polypeptide chains comprises the DART molecule of the invention comprising the amino acid sequence LGGC (of sequence No. 54). In one embodiment, the cysteine ​​residue is inserted into an amino acid sequence comprising the hinge domain, e.g., EPKSCDKTHTCPP (sequence 55), or ESKYGPPCPS (sequence 56). In a specific embodiment, the C-terminus of the polypeptide chain of the DART molecule of the present invention comprises an amino acid sequence of the IgG hinge domain, e.g., sequence No. 55 or sequence No. 56. In another embodiment, the C-terminus of the polypeptide comprises A DART molecule of the invention comprising the amino acid sequence VEPKSC (sequence 57), which can be encoded by the nucleotide sequence gttgagccca aatcttgt (sequence 58). In another embodiment, the residue inserted into the polypeptide chain of the amino acid sequence LGGCFNRGEC (sequence No. 59) may be encoded by the nucleotide sequence ctgggaggct gcttcaacag gggagagtgt (sequence No. 60). In a preferred embodiment, the C terminus of the DART polypeptide chain of the present invention comprises the amino acid sequence LGGCFNFRGEC (sequence No. 59). In still other embodiments the cysteine ​​residue is inserted into the polypeptide chain through the amino acid sequence FNRGEC (sequence No. 61), which may be encoded by the nucleotide sequence ttcaacaggg gagagtgt (sequence No. 2). In one embodiment, the C terminus of the DART polypeptide of the invention comprises the amino acid sequence FNRGEC (sequence No. 61).

In certain embodiments, the dimeric body molecule comprises at least two polypeptide chains, each comprising an amino acid sequence LGGC (sequence No. 54) and covalently linked through a disulfide bond between the cysteine ​​residues of the LGGC sequence ( Sequence No. 54). In another certain embodiment, the dimeric body molecule comprises at least two polypeptide chains, one of which comprises an FNRGEC sequence (sequence No. 61) and the other comprises a hinge domain (comprising at least one residue), wherein two of the FNRGEC sequences are linked Said polypeptide is at least covalently linked to a disulfide bond between the cysteine ​​residue of FNRGEC (sequence No. 61) and the cysteine ​​residue of the hinge domain. In a particular feature, the cysteine ​​residue is responsible for the disulfide bond located in the hinge domain which is Cys-128 (as numbered according to Kabat EU; located in the hinge domain of the unmodified heavy chain, IgG heavy chain) and the The corresponding cysteine ​​in sequence 23 is Cys -114 (as numbered according to Kabat EU; located at the C-terminus of the unmodified light chain, IgG light chain) (Elkabetz et al. (2005), “Cysteines In CH1 Underlie Retention Of Unassembled Ig Heavy Chains, J. Biol. Chem. 280:14402-14412). In yet another embodiment, a cysteine ​​residue at the N terminus of the amino acid chain is modified. In still other embodiments, a cysteine ​​residue is modified so that it is located in the linker portion of the polypeptide chain of the dimeric body molecule. In a further embodiment, the VH or VL domain is modified to include at least one amino acid modification specific to the VH or VL core domain, such as said amino acid modification comprising the replacement of an amino acid with a cysteine.

In another aspect of the present invention, domain (c) of the first polypeptide chain includes the amino acid sequence VEPKSC (sequence No. 57), which is derived from the hinge domain of human IgG and which can be encoded by the nucleotide sequence gttgagccca aatcttgt (Sequence No. 58). In another aspect of the embodiment, domain (f) of the second polypeptide chain includes the amino acid sequence VEPKSC (sequence 57). In certain features of said embodiment, domain (c) of the first polypeptide chain comprises the C terminus, which has 6 amino acids of human light chain kappa, FNRGEC (sequence No. 61); Domain (F) of the second polypeptide chain includes the amino acid sequence VEPKSC (sequence No. 57) or the hinge domain. In other features of this motif, domain (f) of the second polypeptide chain comprises the C terminus of 6 amino acids of the kappa light chain, FNRGEC (sequence no. 61); Domain (c) of the first polypeptide chain contains the amino acid sequence VEPKSC (sequence No. 57) or the hinge domain.

As can be seen from the previous description, the non-individual polypeptides of bispecific DART can form two types of homodimers and one type of heterodimers. In one embodiment of the present invention, a charged polypeptide can be added to the C terminus of one and preferably each of the DART polypeptides. By selecting charged polypeptides of variable charges with respect to individual polypeptides from the specific DART, inclusion of relevant polypeptides will give good formation of heterodimers and reduce formation of homodimers. The in situ charged polypeptide preferably has a substantially higher content of arginine, glutamine, histidine and/or lysine (or mixtures of said amino acids) and the negatively charged polypeptide has a substantially higher content of aspartate or glutamate (or mixtures of the mentioned amino acids). Specifically, positively charged polypeptides with a high lysine content and negatively charged polypeptides with a high glutamate content are specifically preferred. In order to increase the electrostatic attraction between oppositely charged polypeptides, it is preferable to use helical polypeptides.

Thus in a preferred embodiment, the positively charged portions of the Escherichia coli would be attached to one of the polypeptides used in the formation of the specific DART and negatively charged a K-helix, which would be attached to the second DART polypeptide.

It is usefully preferable for Escherichia coli to have the sequence: (EVAALEK)4 [i.e., (sequence No. 63) EVAALEKEVAALEKEVAALEKEVAALEK].

A particularly favored K-helix will have the sequence: (KVAALKE)4 [i.e., (sequence #64) KVAALKEKVAALKEKVAALKEKVAALKE].

The preferred DART polypeptide comprises Escherichia coli and has the general sequence: [VL domain] [ GGGSGGGG]-[VH domain] [ (EVAALEK)4] - GGGNS, the VL being the variable light Ig domain of the DARTs, GGGSGGGG, sequence #52 , VH is the variable Ig heavy domain of DARTs, (EVAALEK)4 is sequence 63 and GGGNS is sequence 65. The DART polypeptide preferably includes the aforementioned K helix and will have the following general sequence: [VL domain] [GGGSGGGG] [VH domain]-[(KVAALKE)4] - GGGNS, where VL is the Ig variable domain of DARTs, and GGGSGGGG is sequence 52, VH is the variable heavy chain Ig domain of DARTs, (KVAALKE)4 is sequence 64 and GGGNS is sequence 65.

In an additional embodiment, the Fc regions may be linked to helices E and/or K of each helix E or K of the DARTs. Additionally, separation of the Fc regions from the DART VH domain is desirable in cases where a less separated arrangement of the domains results in little interaction between These domains and compounds bind or overlap with the DART assembly. Although terminators can be used for any amino acid sequence, it is preferable to use terminators that form turns of the α-helix, so that the increase is maximal and is expressed in the Fc domain distant from the variable domains. Because helical polypeptides with opposite charges interact to improve the heterodimer shape, these molecules are preferred as specific separation media. Fc-DART molecules containing a profile provide benefits similar to those of Fc-DARTS, including improved half-life and improved neurotransmitter function. Polypeptides from helix E or helix K are specifically preferred for this purpose. Thus, in a preferred embodiment, a DART comprising an Fc having helix E would have the general sequence: [VL domain]-[GGGSGGGG]-[VH domain]-[(EVAALEK)4]-GGG Fc starting at D234 (numbering Kabat), where VL is the variable light Ig domain of DARTs, GGGSGGGG is sequence 52, VH is the variable heavy Ig domain of DARTs and (EVAALEK)4 is sequence 63. Similarly, in a preferred embodiment, a DART having Fc and including coil K will have the general sequence: [VL domain]-[GGGSGGGG]-[VH domain]-[(KVAALKE)4]-GGG and an Fc domain starting at D234 (Kabat numbering), where VL is the variable Ig domain of DARTs, GGGSGGGG is sequence 51, VH is the variable heavy Ig domain of DARTs and (KVAALKE)4 is sequence 64.

As described above, a DART molecule with a helix or a DART-Fc molecule with a helix may comprise one single helix or a helix separation means, or may comprise more than one of said separation means (e.g., two separation means The reverse charge is favored by which one molecule is linked to another through the VH domain of DARTs polypeptides. By binding the Fc region to said separating molecule(s), the ability of divalent or tetravalent molecule or transcriptional enhancement of Fc-DART molecules through chain exchange is enhanced. Thus, Fc-DART molecules can be produced to form monomers or dimers depending on whether the Fc domain is linked to one or more DART VH domains.

1-Diversity in B7-H3 DART molecules

Specific DARTs can simultaneously be linked to two separate or distinct epitopes. In certain embodiments, the epitopes are from the same antigen. In another embodiment, the epitope is from different antigens. In preferred embodiments, the binding site of at least one epitope is specific for a determinant expressed on the immune neurotransmitter cell (e.g., CD3, CD16, CD32, CD64, T-cell receptor, etc.), which is expressed on lymphocytes. T and natural killing (NK) cells or other mononuclear cells. In one embodiment the DART molecule binds to a specific neurotransmitter cell and further activates said neurotransmitter cell. In this regard, the DART molecules of the invention can independently display functions similar to those of Ig if they include an Fc domain (for example, as has been tested in connection with testing a neurotransmitter function known in the art and an example of which is illustrated in this Document (for example, ADCC test). In certain embodiments the bispecific DART of the invention binds to both a cancer-specific antigen on a tumor cell and a means of identifying a neurotransmitter cell during said cell activation. In alternative embodiments, the DART or DART molecule of the invention can offer to activate a target, e.g., a neurotransmitter or cell through simultaneous binding and thus binding to an activating and inhibitory receptor on the same cell (e.g., binding CD32A, CD32B, BCR, CD32B or IgERI and CD32B) as described above (see Technical Background section). In an additional aspect of the invention, bi-specific DART can exhibit antiviral properties by simultaneously binding to two neutralizing epitopes on a virus (e.g., RSV peaks, WNV peaks such as E16 and E53).

2- Comprehensive B7-H3 DART molecules

In one embodiment, an antibody can be constructed comprising an epitope-binding domain that specifically binds to B7-H3 and an epitope-binding domain that specifically includes a deficient antigen, e.g., fluorescein or isothiocinate (also known as iso thiocinate or FITC). Such as DART is used as a universal adapter (“UDART”) which is able to bind to B7-H3 using molecules that interact with fluorescein-conjugated binding posts. For example, the FITC-reactive arm of DART can be used to bind to a FITC-labeled antibody that binds to a target other than B7-H3 including intracellular localization, intracellular localization, cell-free localization, multi-targeting, etc. The mouse chimeric version of Fv/human Fc version of the anti-fluorescein MAb, 4420, can be used as a source for the CDR domains of FITC (Gruber, M. et al. (1994) Efficient Tumor Cell Lysis Mediated By A Bispecific Single Chain Antibody Expressed in Escherichia coli, J. Immunol. 152(11): 5368-5374).

3- B7-H3 DART molecules specific to the target cell

The bispecific DART molecules of the invention display unique properties that target all cell types. For example, a dual-specific DART molecule, or DART, can be modified to include epitope binding sites that recognize a set of unique antigens targeting the cell or tissue type. Additionally, when any or all of the individual antigens are co-segregated in a tissue and/or cell type, low affinity binding domains can be used for a DART or DART molecule to be generated. Binding domains with low affinity to bind to a single epitope or antigen will not have sufficient activity for therapeutic purposes. However, since all epitopes or antigens are present in the target cell or tissue, the activity of the cell or tissue-specific DART or DART molecule is appropriate for the cell or tissue that only expresses the antigens and will increase as said cell or tissue is Target it effectively by invention. The desired bispecific molecule may display enhanced binding to one or all of the target antigens on cells expressing each of the said antigens related to the monospecific DART or an antibody that has specificity for one or more of the antigens missing.

For example, B7-H3-specific DARTS can be constructed according to the present invention to include a domain that is a natural killer group 2D (NKG2D) receptor binding molecule. The NKG2D receptor is expressed on all natural killer cells (Bauer, S. et al. (1999) Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA, Science 285(5428):727-729; Jamieson , AM et al. (2002) The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing, Immunity 17(1):19-29) as well as on all CD8+ T cells (Groh, V. et al. (2001) Costimulation Of CD8αβ T cells Cells By NKG2D Via MIC Induced On Virus-Infected Cells, Nat. 2(3):255-260; Immunity 17(1):19-29. These binding complexes, especially those that are not expressed on normal cells, include histocompatibility complex molecule 60 (H60), the retinoic acid-induced early gene product (RAE-1), and transcription protein UL16 binding 1 (MULT1)) (Raulet DH (2003 ) Roles Of The NKG2D Immunoreceptor And Its Ligands, Nature Rev Immunol 3:781-790; 106:1711-1717). Additional ligands reactive with human NKG2D include the polymorphic MHC class I chain-related molecules MICA and MICB (Diefenbach, A. et al. (1999) Natural Killer Cells: Stress Out, Turn On, Tune In, Curr. Biol. 9(22) :R851-R8533; MICA And MICB Genes: Can The Enigma Of Their Polymorphism Be Resolved? The MICA sequence is sequence number: 66:

MGLGPVFLLL AGIFPFAPPG AAAEPHSLRY NLTVLSWDGS VQSGFLTEVH

LDGQPFLRCD RQKCRAKPQG QWAEDVLGNK TWDRETRDLT GNGKDLRMTL

AHIKDQKEGL HSLQEIRVCE IHEDNSTRSS QHFYYDGELF LSQNLETKEW

TMPQSSRAQT LAMNVRNFLK EDAMKTKTHY HAMHADCLQE LRRYLKSGVV

LRRTVPPMVN VTRSEASEGN ITVTCRASGF YPWNITLSWR QDGVSLSHDT

QQWGDVLPDG NGTYQTWVAT RICQGEEQRF TCYMEHSGNH STHPVPSGKV

LVLQSHWQTF HVSAVAAAAI FVIIIFYVRC CKKKTSAAEG PELVSLQVLD

QHPVGTSDHR DATQLGFQPL MSDLGSTGST EGA

The MICB sequence is sequence number: 67:

PHSLRYNLMV LSQDGSVQSG FLAEGHLDGQ PFLRYDRQKR RAKPQGQWAE

DVLGAKTWDT ETEDLTENGQ DLRRTLTHIK DQKGGLHSLQ EIRVCEIHED

SSTRGSRHFY YDGELFLSQN LETQESTVPQ SSRAQTLAMN VTNFWKEDAM

KTKTHYRAMQ ADCLQKLQLP PMVNVICSEV SEGNITVTCR ASSFYPRNIT

LTWRQDGVSL SHNTQQWGDV LPDGNGTYQT WVATRIRQGE EQRFTCYMEH

SGNHGTHPVP SGKALVLQSQ RTDFPYVSAA MPCFVIIIIL CVPCCKKKTS

AAEGP

Alternatively, the DART molecules of the invention can be constructed to include a domain that is a T cell receptor (TCR) or CD3 (T cell co-receptor) binding molecule. The native expression of the TCR is by CD4+ or CD8+ T cells, and allows these cells to recognize bound antigenic peptides represented by MHC class 1 or 2 antigen-representing cell MHC proteins. Recognition of the pMHC (peptide-MHC) complex by the TCR initiates the generation of an immune response leading to the production of cytokines and cell lysis product representing the antigen (see, for example, Armstrong, K.M. et al. (2008) Conformational Changes And Flexibility In T-Cell Receptor Recognition Of PeptideMHC Complexes, Biochem. J. 415(Pt 2):183196; Willemsen, R. (2008) Selection Of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes For Adoptive T-Cell Therapy, Cytometry A. 73(11):1093-1099; Beier, K. C. et al. (2007) Master Switches Of T-Cell Activation And Differentiation, Eur. Respir. J. 29:804-812; Mallone, R. et al. (2005) Targeting T Lymphocytes For Immune Monitoring And Intervention In Autoimmune Diabetes, Am. J.Ther. 12(6):534550). CD3 is the receptor that binds to the TCR (Thomas, S. et al. (2010) Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer, Immunology 129(2):170-177; Guy, CS et al. (2009) ) Organization Of Proximal Signal Initiation At The TCR:CD3 Complex, Immunol Rev 232(1):7-21; St Clair, EW (Epub 2009 Oct 12) Novel Targeted Therapies For Autoimmunity, Curr. Opin. Immunol. 21(6):648-657; Baeuerle, P. A. et al. (Epub 2009 Jun 9) Bispecific T-Cell Engaging Antibodies For Cancer Therapy, Cancer Res. 69(12):4941-4944; Smith-Garvin, J.E. et al. (2009) T Cell Activation, Annu. Rev. Immunol. 27:591-619; Renders, L. et al. (2003) Engineered CD3 Antibodies For Immunosuppression, Clin. Exp. Immunol. 133(3):307-309).

By establishing that DART molecules also include at least one epitope-binding domain capable of binding, for example, to a receptor located on the surface of a target cell, such DART molecules will then be able to bind to target cells and thus render the target cells viewable. The binding molecule of the natural killer group 2D (NKG2D) receptor or the TCR (whichever is present on a DART bound to the target cell) (see, e.g., Germain, C. et al. (2008) Redirecting NK Cells Mediated Tumor Cell Lysis By A New Recombinant Bifunctional Protein, Prot. Engineer. Design Selection 21(11):665-672. These DARTs can be used to redirect any desired target cell into a target cell for the product of NK cell-mediated cell lysis or T cell-mediated cytotoxicity. In one embodiment, the epitope-binding domain of DART capable of binding to a receptor located on the surface of a target cell is an epitope that binds to a tumor-associated antigen in order to redirect those tumor cells to substrates for the product of NK cell-mediated cell lysis or T cell-mediated cytotoxicity. It is noteworthy that the tumor-associated antigen is an antigen against breast cancer, an antigen against ovarian cancer, an antigen against prostate cancer, an antigen against cervical cancer, an antigen against pancreatic cancer, an antigen against lung cancer. Lung cancer, bladder cancer antigen, rectal cancer antigen, testicular cancer antigen, primary glioma cancer antigen, antigen associated with B-cell malignancy, lymphoma, or antigen associated with Chronic lymphocytic leukemia.

Tumor-associated antigens suitable for this use include A33 (a colorectal carcinoma antigen; Almqvist, Y. 2006, Nucl Med Biol. Nov; 33(8):991-998); B1 (Egloff, AM et al. 2006, Cancer Res. 66(1):6-9); BAGE (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84); beta-catenin (Prange W. et al. 2003 J Pathol. 201(2):250-9); CA125 (Bast, RC Jr. et al. 2005 Int J Gynecol Cancer 15 Suppl 3:274-81); CD5 (Calin, GA et al. 2006 Semin Oncol. 33(2):167-73); CD19 (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48; CD20 (Thomas, DA et al. 2006 Hematol Oncol Clin North Am. 20(5):1125-36); CD22 (Kreitman, RJ 2006 AAPS J. 18;8(3):E532-51); CD23 (Rosati, S. et al. 2005 Curr Top Microbiol Immunol. 5;294:91-107); CD25 (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48); CD27 (Bataille, R. 2006 Haematologica 91(9):1234-40); CD28 (Bataille, R. 2006 Haematologica 91(9):1234-40); CD36 (Ge, Y. 2005 Lab Hematol. 11(1):31-7); CD40/CD154 (Messmer, D. et al. 2005 Ann NY Acad Sci. 1062:51-60); CD45 (Jurcic, JG 2005 Curr Oncol Rep. 7(5):339-46); CD56 (Bataille, R. 2006 Haematologica 91(9):1234-40); CD79a/CD79b (Troussard, CD103 (Troussard, X. et al. 1998 Hematol Cell Ther. 40(4):139-48); CDK4 (Lee, YM et al. 2006 Cell Cycle 5(18):2110-4); CEA (carcinoembryonic antigen; Mathelin, C. 2006 Gynecol Obstet Fertil. 34(7-8):638-46; Tellez-Avila, F.I. et al. 2005 Rev Invest Clin. 57(6):814-9; CTLA4 (Peggs, KS et al. 2006 Curr Opin Immunol. 18(2):206-13); EGF-R (epidermal growth factor receptor; Adenis, A. et al. 2003 Bull Cancer. 90 Spec No:S228-32); Erb (ErbB1; ErbB3; ErbB4; Zhou, H. et al. 2002 Oncogene 21(57):8732-40; Rimon, E. et al. 2004 Int J Oncol. 24(5):1325-38); GAGE (GAGE-1; GAGE-2; Akcakanat, A. et al. 2006 Int J Cancer. 118(1):123-8); GD2/GD3/GM2 (Livingston, P. O. et al. 2005 Cancer Immunol Immunother. 54(10):1018-25; gp100 (Lotem, M. et al. 2006 J Immunother. 29(6):616-27); HER-2/neu (Kumar, Pal S et al. 2006 Semin Oncol. 33(4):386-91); human papillomavirus-E6/human papillomavirus-E7 (DiMaio, D. et al. 2006 Adv Virus Res. 66:125-59; KSA (17-1A) (Ragupathi, G. 2005 Cancer Treat Res. 123:157-80) MAGE (MAGE-1; MAGE-3; (Bodey, B. 2002 Expert Opin Biol Ther. 2(6):577-84); MART (Kounalakis, N. et al. 2005 Curr Oncol Rep. 7(5) :377-82 (Mathelin, C. 2006 Gynecol Obstet Fertil. 34(7-8):638-46; MUM-1 (Castelli, C. et al. 2000 J Cell Physiol. 182(3):323-31); N-acetylglucosaminyltransferase (Dennis, JW 1999 Biochim Biophys Acta. 6;1473(1):21-34); p15 (Gil, J. et al. 2006 Nat Rev Mol Cell Biol. 7(9):667-77); PSA (prostate specific antigen; Cracco, CM et al. 2005 Minerva Urol Nefrol. 57(4):301-11); PSMA (Ragupathi, G. 2005 Cancer Treat Res. 123:157-80); sTn (Holmberg, LA 2001 Expert Opin Biol Ther. 1(5):881-91); TNF-receptor (TNF-α receptor, TNF- receptor; or TNF-γ receptor; van Horssen, R. et al. 2006 Oncologist. 11(4):397-408; Gardnerova, M. et al. 2000 Curr Drug Targets 1(4):327-64; or VEGF receptor (ODwyer. PJ 2006 Oncologist. 11(9):992-8).

Other tumor-associated antigens suitable for this use (and publications specifically disclosing antibodies cross-reactive to those antigens) include ADAM-9 (US Patent Publication No. 2006/0172350; International Application Publication No. 084075/06); ALCAM (International Application Circular No. 093443/03); Carboxypeptidase M (US Patent Publication No. 2006/0166291); CD46 (US Patent No. 7,148,038; International Application Publication No. 032814/03); Cytokeratin 8 (International Application Circular No. 024191/03); Ephrin receptors (esp EphA2 (US Patent No. 7,569,672; International Application Publication No. 084226/06); Integrin Alpha-V-Beta-6 (International Order Circular No. 087340/05); JAM-3 (International Application Circular No. 084078/06); KID3 (International Application Circular No. 028498/05); KID31 (International Application Circular No. 076584/06); LUCA-2 (US Patent Publication No. 2006/0172349; International Application Publication No. 083852/06); Oncostatin M (Oncostatin Receptor Beta) (US Patent No. 7,572,896; International Application Publication No. 084092/06); PIPA (Patent American No. 7,405,061; International Application Circular No. 043239/04); ROR1 (US Patent No. 5,843,749); and the Transferrin Receptor (US Patent No. 7,572,895; International Application Publication No. 121179/05.

It is also worth noting the antigens specific to certain infectious agents, such as viral agents, including, but not limited to, human immunodeficiency virus (HIV), hepatitis B virus (HBV), influenza, human papillomavirus (HPV), and foot infections. Oral infections (coxsackieviruses), rabies virus, herpes simplex virus (HSV), and causative agents of gastroenteritis, such as retroviruses, adenoviruses, caliciviruses, astroviruses, and Norwalk virus; Bacterial agents, including but not limited to, Escherichia cholerae, Salmonella Typhimurium, Pseudomonas aeruginosa, Vibrio cholerae, Neisseria gonorrhoeae, Helicobacter pylori, Haemophilus influenzae, Shigella dysentery, Staphylococcus aureus, Mycobacterium tuberculosis and Streptococcus pneumoniae, and fungal agents and parasites such as Giardi.

In some embodiments, the molecules of the invention are genetically modified to include a modified glycosylation profile or glycosylation profile different from the comparator portion of the typical molecule. Transgenic sugars may be useful for many purposes, including, but not limited to, enhancing effector function. Transgenic glycoforms may also be produced by any method known to the person skilled in the art, for example by using different or different genetically modified expression strains, or by co-expression with one or more enzymes, such as DI N-acetylglucosamine transferase 3 (GnTI11), or by expression The invention applies to DART in various organisms or cell lines of various organisms, or to modification of carbohydrates after expression and purification of DART. Methods for producing genetically modified glycosylated images are well known in the field, and include, but are not limited to, those described in Umana et al. (1999) Engineered Glycoforms Of An Antineuroblastoma IgG1 With Optimized Antibody-Dependent Cellular Cytotoxic Activity, Nat. Biotechnol 17:176-180; Davies et al. (2001) Expression Of GnTIII In A Recombinant Anti-CD20 CHO Production Cell Line: Expression Of Antibodies With Altered Glycoforms Leads To An Increase In Adcc ​​Through Higher Affinity For Fc Gamma RIII, Biotechnol Bioeng 74:288-294; Shields et al. (2002) Lack Of Fucose On Human IgG1 N-Linked Oligosaccharide Improves Binding To Human Fcgamma RIII And Antibody-Dependent Cellular Toxicity, J Biol Chem 277:26733-26740; Shinkawa et al. (2003) The Absence Of Fucose But Not The Presence Of Galactose Or Bisecting N-Acetylglucosamine Of Human IgG1 Complex-Type Oligosaccharides Shows The Critical Role Of Enhancing Antibody-Dependent Cellular Cytotoxicity, J Biol Chem 278:3466-3473) US 6,602,684; USSN 10/277,370; USSN 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent technology (Biowa, Inc.) Princeton, NJ); GlycoMAb glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland); each of which is incorporated herein by reference in its entirety. See,eg,WO 00061739; EA01229125; US 20030115614; Okazaki et al. (2004) Fucose Depletion From Human IgG1 Oligosaccharide Enhances Binding Enthalpy And Association Rate Between IgG1 And FcGammaRIIIA, JMB, 336: 1239-49, which are incorporated in their entirety into the present document by reference.

The invention also includes the introduction of unnatural amino acids to produce the DARTs of the invention. This method, known to those skilled in the art, involves the use of natural biosynthetic machinery to allow the introduction of amino acids into proteins, see, for example, Wang et al. (2002) Expanding The Genetic Code, Chem. Comm. 1: 1-11; Wang et al. (2001) Expanding The Genetic Code Of Escherichia coli, Science, 292: 498-500; van Heest et al. (2001) Protein-Based Materials, Toward A New Level Of Structural Control," Chem. Comm. 19: 1897-1904, each of which is incorporated herein by reference in its entirety. Alternative strategies focus on the enzymes responsible for the biosynthesis of amino acyl-tRNA, see, eg, Tang et al. (2001) Biosynthesis Of A Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine In An Engineered Bacterial Host, J. Am. Chem. Soc. 123(44): 11089-11090; Kiick et al. (2001) Identification Of An Expanded Set Of Translationally Active Methionine Analogues In Escherichia coli, FEBS Lett. 502(1-2):25-30, which are incorporated in their entirety into the present document by reference. Alternative strategies focus on enzymes responsible for aminoacyl-tRNA biosynthesis, see, for example, Tang et al. (2001) Biosynthesis Of A Highly Stable Coiled-Coil Protein Containing Hexafluoroleucine In An Engineered Bacterial Host, J. Am. Chem. Soc. 123(44): 11089-11090; Kiick et al. (2001) Identification Of An Expanded Set Of Translationally Active Methionine Analogues In Escherichia coli, FEBS Lett. 502(1-2):25-30; Each of them is included in the present document in its entirety by reference. In some embodiments, the invention includes methods for modifying the VL, VH or Fc domain of the molecule of the invention by adding or deleting a glycosylation site. Methods for modifying protein carbohydrates are known in the art and within the scope of the invention, see, for example, US Patent No. (6,218,149); And the European patent 096 359 0 (0359149); US Application No. 0028486/2002 2002/0028486; International Application No. 035835/03, and US Publication No. 0115614/2003; US Patent No. 6,218,149 and US Patent No. 4,672,511; They are all included in the entire current document as references.

8- Ways to use B7-H3 modifiers and B7-H3 antibodies for therapeutic purposes

Anti-B7-H3 monoclonal antibodies may be used for therapeutic purposes in individuals with cancer or other diseases. Treatment with anti-B7-H3 antibodies may involve the formation of complexes either in vitro or in vivo, as previously described. In one embodiment, monoclonal antibodies to B7-H3 can bind to or reduce the proliferation of cancer cells. It is understood that the antibody is administered at a concentration that enhances physiological binding (eg in vitro). In another embodiment, the monoclonal anti-B7-H3 antibody can be used in targeted immunotherapy of cancer cells from various tissues such as colon, lung, breast, prostate, ovarian, pancreas, kidney, and other cancers and sarcomas. In another embodiment, the monoclonal B7-H3 antibody can bind only to and reduce cell division in the cancer cell. In another embodiment, the anti-B7-H3 monoclonal antibody can bind to cancer cells and delay the progression of tumor outbreaks. In another embodiment, an individual with cancer is given palliative treatment with the anti-B7-H antibody. Palliative treatment for an individual with cancer includes treating or reducing adverse symptoms of the disease, or symptoms of pharmacological origin3 resulting from treatments for the disease without directly affecting the progression of the cancer. This includes pain relief, nutritional support, sexual problems, psychological pain, depression, fatigue, psychological disorders, nausea and vomiting, etc.

In these situations, the anti-B7-H3 antibody may be given with agents that enhance or direct the individual's immune response, such as an agent to potentiate ADCC.

In another embodiment, the anti-B7-H3 antibody may be conjugated to or linked to a radioactive molecule, toxin (such as calicheamicin), chemotherapeutic molecule, liposome, or other vector containing chemotherapeutic compounds and administered to an individual in need of such treatment To target these compounds to the cancer cell that contains an antigen that the antibody recognizes and thus eliminates the cancerous or disease-infected cells. Without being limited to theoretical aspects, the anti-B7-H3 antibody is internalized by B7-H3-bearing cells on their surface, thus delivering the conjugated moiety to the cell to induce the therapeutic effect. In another embodiment, the antibody may be used as an adjuvant therapy during surgical removal of a cancer expressing the antigen in order to effect progression of disease. The antibody can also be administered before surgery (neoadjuvant therapy) in a monkey with a tumor that expresses the antigen in order to reduce the size of the tumor and thus facilitate or simplify surgery, save tissue during surgery, and/or reduce the resulting disfigurement.

Dosing for the cell cycle may be considered when implementing the invention. In this model, a chemotherapeutic agent is used to synchronize the cell cycle of tumor or other target infected cells at a predetermined stage. Thus, the anti-B7-H3 antibody of the present invention is administered (either alone or with an additional therapeutic moiety). In alternative embodiments, an anti-B7-H3 antibody is used to synchronize the cell cycle and reduce cell division before a second course of treatment is administered; A second course of anti-B7-H3 antibody and/or an additional treatment regimen may be given.

Chemotherapy agents include radioactive particles, toxins, also referred to as cytotoxins or cytotoxic agents, which include any agent necessary for the survival of cancer cells, agents, liposomes or other carriers containing chemotherapy compounds. Examples of suitable chemotherapy agents include, but are not limited to:

1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin, alkylating agents, allopurinol sodium, altretamine, amifostine, anastrozole, anthramycin (AMC), anti-mitotic agents, cis-dichlorodiamine platinum (II) (DDP) cisplatin), diaminodichloroplatinum, anthracyclines, antibiotics, antimetabolites, asparaginase, BCG live (intravesical), betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bleomycin sulfate, Busulfan, calcium leucouorin, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), Chlorambucil, Cisplatin, Cladribine, Colchicin, conjugated estrogens, Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine, cytochalasin B, Cytoxan, Dacarbazine, Dactinomycin, cin (formerly actinomycin), daunirubicin HCL, daunorucbicin citrate, denileukin diftitox, Dexrazoxane, Dibromomannitol, dihydroxyanthracindione, Docetaxel, dolasetronmesylate, doxorubicin HCL, dronabinol, E. coli, flutamide, folinic acid, gemcitabine HCL, glucocorticoids, goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea, idarubicin HCL, ifosfamide, interferon alpha-2b, irinotecan HCL, letrozole, leucovorin calcium, leuprolide acetate, levamisole HCL, lidocaine, lomustine, maytansinoid, mechlorethamine HCL, medroxyprogesterone acetate, megestrol acetate, melphalan HCL, mercaptipurine, mesna, methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate , ondansetron HCL, paclitaxel, pamidronate disodium, pentostatin, pilocarpine HCL, plimycin, polifeprosan 20 (with carmustine implant), porfimer sodium, procaine, procarbazine HCL, propranolol, rituximab, sargramostim, streptozotocin, tamoxifen, taxol, teniposide, tenoposide, testolactone, tetracaine, thioepa chlorambucil, thioguanine, thiotepa, topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.

In a preferred embodiment, the toxin is particularly effective in dividing or rapidly dividing cells, so that relatively non-dividing cells are spared the toxic effect.

The antibodies of the invention can be internalized into diseased or cancerous cells to which they are bound and thus be useful in therapeutic applications, for example, delivering toxins to be internalized due to their reversible activity into cells. Examples of these toxins include, but are not limited to, saporin, calicheamicin, auristatin, and maytansinoid.

The antibodies or polypeptides of the invention may be attached (including conjugated or conjugated) to a radioactive molecule, toxoid, or other therapeutic agent, or to liposomes or other carriers containing therapeutic agents either covalently or non-covalently, or directly Or indirectly. The antibody can bind to a radioactive molecule, toxin, or chemotherapy molecule at any site with the antibody as long as the antibody is able to bind the target B7-H3.

A toxin or chemotherapeutic agent may be administered concomitantly (before, after, or during administration), or conjugated (e.g. covalently linked) to a suitable monoclonal antibody either directly or indirectly (e.g. via a linker group, or Alternatively, by means of a linker molecule with a suitable contact site, such as a platform molecule as described in US Patent No. 5,552,391). The toxin and chemotherapeutic agent of the invention can be conjugated directly to target proteins using methods known in the art. For example, a nucleophilic group, such as an amino or sulfhydryl, may be able to react on the one hand with a carbonyl-containing group, such as an anhydride or acid halide, or with an alkyl group containing a trace group (such as a halide) on the other hand.

Antibodies or polypeptides can also bind to a chemotherapeutic agent via a microcarrier. The term “microcarrier” refers to a biodegradable, insoluble, water-soluble body with a volume of less than about 150 microliters, 120 microliters, or 100 microliters, most commonly less than about 50-60 microliters, preferably less. From about 10, 2.5, 2 or 1.5 micrometers. Microcarriers include “nano-sized carriers”, which are fine carriers with a size of less than about 1 micrometer, preferably less than about 500 nanometers. These particles are known in the field. Solid-phase microcarriers may be particles composed of biocompatible natural polymers, synthetic polymers or synthetic copolymers, and may or may not include microcarriers composed of agarose or cross-linked agarose, as well as other biodegradable materials known in the field. Solid-phase biodegradable microcarriers can consist of biodegradable polymers (e.g., poly(lactic acid), poly(glycolic acid), and copolymers thereof), or erodible poly(ortho esters) (e.g., 3,9-di Ethylidene-2,4,8,10-tetraoxaspyro[5.5]unidecane (DETOSU) or poly(anhydride), such as sebacic acid poly(anhydrides) under physiological conditions of a mammalian organism. Microcarriers can be liquid phase (e.g. oil, lipid base), such as liposomes, ESCOM species (which are stable complexes of cholesterol, phospholipids, and co-active saponins) without antigen or droplets found in emulsions. Oil-in-water or water-in-oil, provided that the liquid phase carriers are biodegradable. Biodegradable liquid phase carriers typically include biodegradable oil, some of which are known in the art, such as sequalene and vegetable oils. Microcarriers are typically spherical in shape, but microcarriers that deviate from spherical shape are also acceptable (e.g., oval, rod, etc.). Due to its insoluble (with respect to water) nature, fine carriers are leached from water and water-based (aqueous) solutions.

The antibody or urine peptide conjugates of the present invention may comprise a bifunctional linker containing a group capable of conjugation to a toxic agent or chemotherapeutic agent and a group capable of conjugation to the antibody. The linker may act as a spacer to separate the antibody from an agent to avoid interfering with binding capabilities. The link can be fissionable or non-fissionable. The linker may also increase the chemical reactivity of the substance to an agent or antibody, thus increasing the efficiency of conjugation. Increased chemical reactivity may also facilitate the use of agents, or functional groups on agents, that would not be possible without them. The bifunctional linker may also be conjugated to the antibody by methods known in the art. For example, a linker containing an active ester moiety, such as an N-hydroxysuccinimide ester, can be used to conjugate two lysine structural units in an antibody via an amide bond. In another example, a linker containing a nucleophilic amine or a hydrazine residue may be conjugated to aldehyde groups resulting from glycolytic oxidation of the carbohydrate residues of the antibody. In addition to these direct methods, the linker can be indirectly conjugated to the antibody via a carrier intermediate such as aminodextran. In these models, the modified bond is either through a lysine, a carbohydrate, or an intermediate carrier. In one embodiment, the ligand is site-selectively coupled to free thiol units in proteins. Suitable moieties for selective conjugation to thiol groups on proteins are known in the art. Examples of disulfide compounds are α-halocarbonyl and α-halocarboxyl. and maleimides. When the amine function is attached to the nucleus in the same molecule, such as α Halocarbonyl group, or carboxyl group, there is a possibility that cyclization occurs via intramolecular alkylation of the amine. There are known ways to prevent this problem known to a person of ordinary skill in the art, for example by preparing molecules in which the amine and “halo” functions are separated by inflexible groups, such as aryl or transalkene groups, which makes unwanted cyclization undesirable from a chemical standpoint. Stereotypic. See, for example, US Patent No. + (6,441,163) for separation of maytansinoid and antibody conjugates by a disulfide radical.

One of the cleavable bonds that can be used in the preparation of antibody-drug conjugates is an acid-independent linker based on cis-acenic acid that takes advantage of the acidic environment of various intracellular compartments such as endosomes found during receptor-mediated endocytosis and lysosomes. See, for example, Shen, W. C. et al. (1981) (cis-Aconityl Spacer Between Daunomycin And Macromolecular Carriers: A Model Of pH-Sensitive Linkage Releasing Drug From A Lysosomotropic Conjugate, Biochem. Biophys. Res. Comtnun. 102:1048-1054 (1981), for the preparation of daunorbucin conjugates with macromolecular carriers; And Yang et al. (1988) To prepare conjugates of daunorubicin with a melanoma antibody; and Dillman et al. (1988) (Superiority Of An Acid-Labile Daunorubicin-Monoclonal Antibody Immunoconjugate Compared To Free Drug, Cancer Res. 48:6097-6102) to use an acid-stabilized linker in a manner similar to preparing conjugates of daunorubicin with a T-cell antibody; and Trouet et al. (1982) A Covalent Linkage Between Daunorubicin And Proteins That Is Stable In Serum And Reversible By Lysosomal Hydrolases, As Required For A Lysosomotropic Drug-Carrier Conjugate: In Vitro And In Vivo Studies, Proc. Natl. Acad. Sci. (USA) 79:626-629) to bind daunorubicin to an antibody via a peptide spacer arm.

The antibody (or polypeptide) of this invention may be conjugated to a radioactive molecule or toxin using any method known in the art. For a discussion of the antibody radiolabeling method (see Cancer Therapy with Monoclonal Antibodies, D. M. Goldenberg (Ed.) CRC Press, Boca Raton, 1995). Suitable toxins include taxanes, maytansinoids, auristatins (eg, monomethyl auristatin (MMAE), monomethyl auristatin F (MMAF), auristatin E (AE), etc.). (Such as those disclosed in U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333,410; 6,340,701; 6,372,738; 6,436,931; 6,441,163; 6,596,757; 7,276,497; 7,585.85 7; calicheamicin, anthracyclines (such as doxorubicin), CC-1065, and docetaxel; cathepsin B or E; ricin, gelonin, Pseudomonas exotoxin, diphtheria toxin, diphtheria toxin, and RNase; tiuxetan, or toxic radioisotope (such as 90Y; 131I, 177Lu, 186Re, 188Re, 211At, 212Bi, 213Bi, 225Ac, etc.)

Alternatively, the antibody may be coupled to a second antibody to form a heterologous antibody conjugate as described in US Patent 467680. The formation of cross-linked antibodies can target the immune system of certain cell types, such as cancer or infected cells that express About B7-H3.

The present invention also provides methods for delaying the progression of a cancer outbreak in a person with the disease (such as, but not limited to, prostate, lung, or kidney cancer) using an anti-B7-H3 antibody or other models that bind to B7-H3. In combination with a chemotherapy agent or in combination with a chemotherapy agent. In some embodiments, the antibody is humanized or chimeric, or non-human or humanized.

In another embodiment, the antibody may be used as an adjuvant therapy during surgical removal of a cancer expressing the antigen in order to delay the progression of cancer metastasis. The antibody or antibody combined with a chemotherapeutic agent may also be administered preoperatively (neoadjuvant therapy) in an individual with a tumor expressing the antigen in order to reduce the size of the tumor and thus facilitate or simplify surgery, save tissue during surgery and/or resultant disfigurement.

In another embodiment, any of the B7-H3 binding compositions described herein can bind to B7-H3-expressing cancer cells and induce an effective immune response against the B7-H3-expressing cancer cells. In some cases, an effective immune response can cause cancer cell death (such as an antibody binding to the cancer cells that induces cell death by fading), or growth inhibition (such as impeding cell cycle progression) of the cancer cells. In other cases, the new antibodies described in the present document can bind to cancer cells and antibody-dependent cellular cytotoxicity (ADCC) can remove cancer cells to which anti-B7-H3 has bound. Thus, the invention provides methods for inducing an immune response comprising administration of any of the compositions described herein.

In some cases, an antibody can activate both cellular and immune responses and utilize more natural killer cells or increase the production of cytokines (such as IL-2, IFN-gamma, IL-12, TNF-alpha, and TNF-α). - Beta, etc.) which activates the individual’s immune system and destroys cancer cells. In another embodiment, the anti-B7-H3 antibody can bind to cancer cells, macrophages or other cytophages that can expose the cancer cells to the action of phagocytes.

Various formulations of anti-B7-H3 antibody or fragments thereof may be used for administration. In some cases, only antibodies to B7-H3 or its fragments may be given. In addition to the pharmaceutically active agent, the compositions of the present invention may contain pharmaceutically acceptable carriers comprising excipients and excipients known in the art and relatively inert that facilitate the administration of the pharmaceutically active material or assist the processing of the active compounds into pharmaceutically usable preparations to the site of action. For example, the excipient can give a shape or texture, or act as a diluent. Suitable excipients include, but are not limited to, stabilizing, wetting, and emulsifying agents, osmosis-modifying salts, encapsulation agents, buffer solutions, and skin penetration enhancers.

Suitable formulations for non-gastrointestinal administration are aqueous solutions of the active compounds in a water-soluble form, such as water-soluble salts. In addition, suspensions of active compounds are suitable for administration of oil-based injections. Suitable lipophilic solvents or carriers include vegetable fats, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension and include, for example, sodium carboxymethyl cellulose, sorbiol, and/or dextran. Optionally, the suspension may contain stabilizers. Liposomes can also be used to encapsulate the delivery agent into the cell.

The pharmaceutical formulation for administration according to the invention may be formulated for enteral, non-gastrointestinal, or topical administration. All three formulation types can be used simultaneously to achieve systemic administration of the active ingredient. Excipients and formulations for non- and intragastrointestinal drug delivery are described in Remington: The Science And Practice Of Pharmacy, 21st Edition, Lippincott Williams & Wilkins Publishing (2005). Forms suitable for intranasal administration include hard or soft gelatin capsules, tablets, and pills, such as coated tablets, elixirs, suspensions, syrups, inhalers and their forms for metered release. In general, these agents are formulated for parenteral administration (eg, intraperitoneal, intravenous, subcutaneous, etc.), although other forms of administration may be used (eg intraoral , intramuscular, etc.). Thus, anti-B7-H3 antibodies are preferably combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, dextrose solution, and the like.

The dosing regimen, i.e. dose, timing, and frequency, will depend on the individual and his or her medical history. In general, a dose of at least about 100 microg/kg of body weight is given, preferably at least about 250 microg/kg of body weight, preferably at least about 750 microg/kg of body weight, and even better about At least 3 mg/kg of body weight, preferably at least 5 mg/kg of body weight, and even better at least at least 10 mg/kg of body weight.

Experimental considerations, such as half-life, will generally contribute to determining dosage. Antibodies that are compatible with the human immune system, such as humanized antibodies or fully anti-human antibodies, can be used to prolong the half-life of the antibody and prevent the antibody from attacking the host's immune system. The number of times of administration can be determined and adjusted during the treatment period, and it depends on reducing the number of cancer cells, maintaining the decline of cancer cells, reducing their proliferation, and delaying the development of the disease outbreak. Alternatively, administration of sustained-release forms of anti-B7-H3 antibodies may be appropriate. In the field there are many different formats and interfaces to achieve continuous release.

In one embodiment, doses of anti-B7-H3 antibodies can be determined experimentally in individuals given one or more times. Individuals are given excess doses of the B7-H3 antibody. To evaluate the efficacy of anti-B7-H3 antibodies, a guide to the cancer pathology can be followed. They include direct measurements of tumor size by touch or visual observation, or indirect measurement of tumor size using X-rays or other imaging methods; Improvement is shown by taking a direct sample from the tumor and microscopic examination of a sample of the tumor. or measurement of an indirect tumor marker (such as PSA for prostate cancer), a reduction in pain or paralysis; Improving speech, vision, breathing and other types of disability associated with the tumor. Increase appetite; and an increase in quality of life as measured by accepted tests or a prolongation of survival. It will become clear to the person skilled in the field because the incidence will depend on the individual, the type of cancer, the stage of the cancer, whether the cancer has begun to spread to another location in the same individual, and the past and concurrent treatments used.

Other formulations include suitable forms of administration known in the art, including, but not limited to, carriers such as liposomes. See, for example, Mahato et al. (1997) Cationic Lipid-Based Gene Delivery Systems: Pharmaceutical Perspectives, Pharm. Res. 14:853-859. Liposomal preparations include, but are not limited to, cytofectin, multilamellar and unilamellar vesicles.

In some embodiments, a single antibody may be present. Antibodies may be monoclonal or polyclonal, and such combinations may contain at least one, at least two, at least three, at least four, or at least five different antibodies that react against cancerous tumors or adenomas. , or sarcoma, or adenocarcinoma. The B7-H3 antibody can be mixed with one or more antibodies cross-reactive against cancers, adenomas, sarcomas, or adenomas in organs such as the ovary, breast, lung, prostate, colon, kidney, skin, and thyroid. , bones, upper digestive system, and pancreas. In one embodiment, a mixture of various anti-B7-H3 antibodies is used. It may be particularly useful to use a mixture of antibodies, as is often observed in the field in the treatment of multiple individuals

Now that the invention has been described in general, it will be easier to understand it by referring to the following examples, which are but not limited to, and do not limit the scope of the invention, unless otherwise specified.

Example 1

Immunohistocompatibility studies

A panel of 49 mAbs were generated from tumor cell/embryonic progenitor cell immunizations. The ability of antibodies to show differential IHC staining of tumor tissue relative to normal, noncancerous tissue, and potential for use in primate models (particularly baboons) were evaluated for antibody efficacy, levels of affinity and antigenicity, levels of immunomodulatory activity, and cellular internalization. 21 mAbs were initially identified by MS analysis and/or binding to B7-H3-CHO cells. The remaining 28 mAbs were identified by re-screening the collection with ELISA with B7-H3 protein. Table 2 shows the characteristics of 46 and 49 members of the Commission.

Table 2

the name

Isotype

IHC

ATCC matrix

Comprehension

U-DART

BIACORE analysis

Engagement

Cyno B7-H3

BRCA84D

IgG1/k

2a

2

+

+

+

++

TDH6

IgG1/k

2a

+

+

+/−

+

TES7

IgG1/k

2a

+

+

+

−

BRCA68D

IgG1/k

2b

3

+

+

++

++

BRCA69D

IgG1/k

2b

3

+

+

++

++

GB8

IgG1/k

2b

3

+

+

+

++

SG27

IgG2b/k

2b

+

+

+

OVCA22

IgG1/k

2c

3

+

+

+/−

+

PRCA157

IgG1/k

2c

2

+

+

++

BLA8

IgG1/k

2c

+/−

+

++

++

KID35

IgG1/k

2c

2

++

LUCA50

IgG2a/k

2c

+

++

OVCA21

IgG1/k

2c

+

+

+

PRCA135

IgG1/k

2c

3

+

++

SG24

IgG2a/k

2c

3

++

++

TDH5

IgG1/k

2c

3

+

++

++

BCCA66

IgG1/k

2c

2

+

−

RECA13

IgG1/k

2c

3

+

−

RECA9

IgG1/k

2c

3

+

−

PRCA123

IgG1/k

2c/3

3

+

++

BRCA126

IgG1/k

3/F

BRCA192

IgG1/k

3/F

BRCA34

IgG1/k

3/F

KID1

IgG1/k

3/F

N.D

+

+

+

KID13

IgG2a/k

3/F

3

++

LU14

IgG2b/k

3/F

−

LUCA1

IgG1/k

3/F

+

+

++

++

MCLY42

IgG2a/k

3/F

++

MCLY46

IgG1/k

3/F

++

OVCA40

IgG1/k

3/F

++

PA20

IgG1/k

3/F

+

++

−

PA40

IgG2b/k

3/F

3

−

PA41

IgG1/k

3/F

3

PRO6

IgG1/k

3/F

2

−

RECA22

IgG1/k

3/F

3

−

+

SAL3

IgG2a/k

3/F

+++

++

SG20

IgG1/k

3/F

+

SG29

IgG1/k

3/F

++

SKIN2

IgG1/k

3/F

3

+++

++

STO5

IgG2b/k

3/F

3

++

+

TDH36

IgG1/k

3/F

2

++

TDH37

IgG1/k

3/F

3

+

TDH4

IgG1/k

3/F

+++

++

++

TDH40

IgG2b/k

3/F

3

++

TDH44

IgG2b/k

3/F

++

OVCA25

IgG1/k

3/F

3

+

IHC staining demonstrated that the panel included antibodies that caused a strong tumor to normal tissue that bound differentially in several specific antibodies, showed a range of binding properties by BIACORE analysis, showed reactivity with a number of overlapping and non-overlapping adhesive peaks, and showed specificity values ​​for 4Ig versus 2Ig B7- H3. Figures 3 and 4 show the properties of nine of the best candidates.

Table 3

the name

Natural fabric

Colon cancer

Lung cancer

Prostate cancer

breast cancer

BRCA84D

Colon 1+

Lung 1+

Liver 1+

1231

1130

112

1111

TDH6

Colon 1+

Panc 1+

Kidney 1+

Lung 1+

Liver 1+

*

TES7

1110

1010

111

1011

BRCA68D

Pancreas 1+

Kidney 1+

Lung 1+

Liver 2+

*

BRCA69D

Colon 1+

Pancreas 1+

Kidney 1+

Liver 1+

1.5

1.75

3

3

GB8

2321

3332

333

3333

SG27

Colon 1+

Pancreas 1+

Kidney 1+

Liver 1+

*

OVCA22

Colon 2+

Pancreas 2+

Liver 2+

2231

3231

333

3333

PRCA157

Colon 2+

Liver 2+

Skin 2+

*

* +str also; ** str 3+

Table 4

the name

Determine 2Ig/4Ig

Sticky top set

BRCA84D

4Ig/2Ig

TDH6

4Ig/2Ig

2

TES7

4Ig

3

BRCA68D

4Ig/2Ig

4

BRCA69D

4Ig/2Ig

4

GB8

4Ig/2Ig

5

SG27

4Ig/2Ig

6

OVCA22

4Ig

7

PRCA157

4Ig/2Ig

8

Table 5 provides a summary of the activity properties of these antibodies

Table 5

the name

Natural fabric staining

Tumor/normal differential

Positive tumor tissue

Profitable exchange interaction

IHC

*

Link

BIACORE

UDART activity

BRCA84D

Connective tissue tumor bv

positive

(not 1:1)

78

++

++

TES7

Tumor connective tissue

negative

1250

+

++

BRCA68D

3

3

tumor

positive

(1:1)

20

+++

++

BRCA69D

3

3

Tumor connective tissue

positive

(1:1)

20

+++

++

GB8

2/3

Adrenal ND)

3/4

Tumor connective tissue

ND, + link return product

625

+

+

SG27

2/3

N.D

N.D

ND, + link return product

20000

+

+

OVCA22

Tumor connective tissue

negative

+ Return link output

2500

+

++

PRCA157

2

3

Tumor connective tissue

positive

(1:1)

20

N.D

++

* Potential concentration in nanog/ml; ND, not specified

Analysis of the results for the antibodies shown in Table 6 reveals that their properties differed and that each antibody was associated with advantages and disadvantages (Table 6).

Table 6

antibody

Advantages

Defects

BRCA84D

#1 Natural texture stain

#1 The tumor/normal difference

Macular tumor, connective tissue, BV

mid familiarity medium; Unique link location (calibratable link)

The cross-reaction is not 1:1

BRCA68D

#3 Stain natural texture

#3 The tumor/normal difference

Profitable cross-reaction: 1:1

High familiarity

UDART activity is strong

Macular tumor only

BRCA69D

#3 Stain natural texture

#3 The tumor/normal difference

Profitable cross-reaction: 1:1

macular tumor, connective tissue, BV

High familiarity

UDART activity is strong

PRCA157

#2 Stain natural texture

#3 The tumor/normal difference

Profitable cross-reaction: 1:1

macular tumor, connective tissue, BV

UDART activity is strong

BIACORE

TES7

#1/2 natural texture stain

#1/2Difference between tumor and normal

macular tumor, connective tissue, BV

Special for 4Ig

UDART activity is strong

There is no mutual profit interaction

Low familiarity

OVCA22

#1/2 natural texture stain

#1/2 Tumor/normal difference for low affinity

macular tumor, connective tissue

Special for 4Ig

UDART activity is strong

There is no mutual profit interaction

Low familiarity

GB8

#2/3 normal tissue stain (adrenal unspecified)

#3/4 Tumor/normal difference for low familiarity

macular tumor, connective tissue

UDART activity is moderate

Non-specific mutual gain interaction

Low familiarity

SG27

#2/3 Texture stain

The tumor/normal difference for low affinity is not specified

UDART activity is moderate

Non-specific mutual gain interaction

Low familiarity

Because BRCA84D, BRCA68D, BRCA68D, and PRCA157 showed clearer IHC specificities for normal tissue, stronger tumor/normal IHC difference, moderate to strong correlation (BIACORE/IHC), cross-reactivity with B7-H3 of baboons, and strong UDART activity, UDART was chosen. Those types of antibodies are for further development.

These antibodies differed from TES7 and OVCA2, which showed low affinity (in the BIACORE trial) and no cross-reactivity with B7-H3 of baboons. These antibodies differed from SG27, which was characterized by lower affinity (in the BIACORE trial), poor IHC performance (poor binding) and lack of UDART activity. These antibodies also differed from GB8, which was characterized by lower affinity (in the BIACORE trial), poor tumor/normal IHC difference, and lack of UDART activity. Using Caki-2 and Hs700T positive control cells, IHC studies revealed that each antibody had a different optimal concentration. The differential concentration is different for the other (Table 7).

Table 7

antibody

Ideal IHC concentration

Differential IHC concentration

BRCA84D

0.625 µg/ml

0.078 µg/ml

BRCA68D

0.156 µg/ml

0.0195 µg/ml

BRCA69D

0.156 µg/ml

0.0195 µg/ml

PRCA157

0.078 µg/ml

0.0195 µg/ml

TES7

5 µg/ml

1.25 µg/ml

OVCA22

10 µg/ml

2.5 mcg/ml*

GB8

1.25 µg/ml

0.625 µg/ml

SG27

20 µg/ml

Not specified **

TDH6

20 µg/ml

Unspecified ***

* OVCA22 only showed binding to caki2 cells, and did not show binding to Hs700T cells.

The optimization decision was based on the correlation with Caki2 cells.

**Because SG27 did not show consistent results from calibration analysis between two operators, low affinity and concentration difference were not identified.

***TDH6 was not studied due to low affinity for positive control cells.

Using the optimal and differential concentrations shown in Table 7, IHC responses to anti-B7-H3 antibodies in human tissues were determined. The results of these analyzes for the adrenal, liver, pancreas, kidney, lung, and colon are shown in Tables 8a-8b and Tables 9a and 9b (all antibodies showed negative IHC responses for heart tissue).

Table 8a: B7H3 mAb IHC at optimal concentration in human tissue

mAb

Adrenals

Liver

Pancreas

BRCA84D

0.625 microg/ml

negative

Sinusoidal endothelial cells++

Hepatocytes +, 5-10%

Endothelial tissue +5%

Fiber++

BRCA68D

0.156 microg/ml

Veneer +++

Sinusoidal endothelial cells++

Hepatocytes++ (g)

Blanket fabric +

Fiber++

BRCA69D

0.156 µg/ml

Veneer +++

Hepatocytes++ (g)

Blanket fabric +

Fiber++

TES7

5 µg/ml

Veneer +

Sinusoidal endothelial cells +

Endothelial tissue +5%

Fiber++

OVCA22

10 µg/ml

Veneer +

Sinusoidal endothelial cells++

Hepatocytes + (g)

Endothelial tissue +5%

Fiber++

PRCA157

0.078 microg/ml

Veneer++

Sinusoidal endothelial cells++

Hepatocytes + (g)

Endothelial tissue +5%

Fiber++

GB8

1.25 µg/ml

undefined

Sinusoidal endothelial cells++

Hepatocytes + (g)

Blanket fabric +

Fiber++

Table 8b: B7H3 mAb IHC at optimal concentration in human tissue

mAb

Kidney

Lung

Colon

BRCA84D

0.625 µg/ml

negative

Endothelial tissue + (5-10%)

Blanket fabric +

BRCA68D

0.156 µg/ml

Protofibroblast+, rare

Blanket fabric +

Mucous membrane++

BRCA69D

0.156 µg/ml

Protofibroblast+, rare

Blanket fabric +

Mucous membrane +

TES7

5 µg/ml

negative

negative

Blanket fabric +

OVCA22

10 µg/ml

Primary fibroblast +

negative

Blanket fabric +

PRCA157

0.078 µg/ml

negative

negative

Mucous membrane +

GB8

1.25 µg/ml

negative

Blanket fabric +

Mucous membrane +

Table 9a: B7H3 mAb IHC at differential concentration in human tissue

mAb

Adrenals

Liver

Pancreas

BRCA84D

0.078 microg/ml

negative

Sinusoidal endothelial cells +

Fiber + (rare)

BRCA68D

0.0195 µg/ml

Veneer++

Hepatocytes + (g)

Fiber +

BRCA69D

0.0195 microg/ml

Veneer++

Hepatocytes + (g)

Fiber +

TES7

1.25 µg/ml

Primary fibroblast +

Sinusoidal endothelial cells +

Endothelial tissue+, 5%

Fiber++

OVCA22

2.5 microg/ml

Primary fibroblast +

Sinusoidal endothelial cells +

Fiber +

PRCA157

00.0195µg/ml

undefined

Sinusoidal endothelial cells +

Hepatocytes + (g)

Fiber +

GB8

0.625 microg/ml

undefined

Sinusoidal endothelial cells++

Hepatocytes + (g)

Fiber +

Table 9b: B7H3 mAb IHC at differential concentration in human tissue

mAb

Kidney

Lung

Colon

BRCA84D

0.078 microg/ml

negative

negative

Blanket fabric +

BRCA68D

0.0195 microg/ml

negative

Levin + (rare)

Mucous membrane +

BRCA69D

0.0195 microg/ml

negative

negative

Mucous membrane +

TES7

1.25 microg/ml

negative

negative

Blanket fabric +

OVCA22

2.54 microg/ml

negative

negative

Blanket fabric +

PRCA157

0.0195 microg/ml

negative

negative

Mucous membrane +

GB8

0.625 microg/ml

negative

negative

Mucous membrane +

IHC studies performed using cancer specimens have shown that the B7-H3 antibodies of the present invention can be used to identify and diagnose cancer in several tissue sources (Table 10). In Table 10, the numbers indicate the number of positive signs (1=+, 2=++, and 3+=+++); Where each number refers to a different test sample.

Table 10

mAbs

µg/ml

Prostate cancer

breast cancer

Lung Cancer

Colon Cancer

BRCA84D

0.625 µg/ml

2، 2، 1

3، 3، 3، 3

2, 3, 2(stromal), 1(blood volume)

2(stromal), 3, 3, 3(stromal)

0.078 µg/ml

0، 2، 3، 2

1(stromal), 1(stromal), 1(stromal), 2, 3

1، 1، 0، 1

2, 2(stromal), 1(stromal), 2(stromal)

BRCA68D

0.156 µg/ml

2، 3، 3، 3

2، 3، 3، 3، 3

3، 3، 2، 2

3، 3، 3، 3

0.0195 µg/ml

0، 1، 1، 1

0، 0، 2، 2، 1

0، 1، 1، 0

1، 1، 1، 1

BRCA69D

0.156 µg/ml

3، 3، 3

3، 3، 3، 3، 3

3, 3, 2, 2(stromal)

3، 3، 3، 3

0، 1، 2، 1

1، 2، 1، 1

1، 1، 0، 1

1(blood volume), 2(stromal), 1, 1

GB8

1.25 µg/ml

2، 3، 1

2، 2، 1، 2

3، 3، 0، 0

2(stromal), 2, 2, 2

0.625 µg/ml

0، 1، 1

0، 0، 0، 0، 1

1، 0، 0، 0

1(stromal), 1(stromal), 0, 0

TES7

5 µg/ml

2، 3، 2، 3

1(stromal), 3, 3, 3, 2

3, 2, 1(stromal), 1(blood volume)

3، 3، 2، 2

1.25 µg/ml

1، 2، 2، 3

1(stromal), 2, 3, 3, 2

3, 1, 1(stromal), 1(blood volume)

3, 2(stromal), 2(stromal), 2

OVCA22

10 µg/ml

3، 2، 2، 1

1(stromal), 2, 2, 3, 3

3, 2, 1(stromal), 0

1(stromal), 1, 1, 2(stromal)

2.5 microg/ml

1، 1، 3، 1

1(stromal), 1(stromal), 1(stromal), 3, 2, 2

2، 1، 0، 0

1, 0, 2(stromal), 0

PRCA157

0.078 µg/ml

2، 2، 2، 3

1، 2، 2، 3، 3

2, 2, 1(stromal), 1(blood volume)

3, 3, 2(stromal), 2(stromal)

0.0195 µg/ml

0، 1، 2، 1

1(stromal), 0, 2,

1(stromal)

0، 1، 0، 0

1, 1, 1(stromal), 1(stromal)

For prostate, breast, colon, and lung cancer cells treated with the B7-H3 antibody BRCA84D, mottling of the tumor sample was present in tumor cells and stromal cells, including the vascular system. In some tumor samples, the staining of stromal was stronger than that of tumor cells. When BRCA84D mAb was titrated to the lower concentration, some cases showed decreased staining in tumor cells, but it remained strong in stromal cells. When the concentration of BRCA84D was 0.625 μg/ml, prostate cancer cells showed an IHC of 3/3+; Breast cancer cells showed an IHC of 4/4+; Colon cancer cells showed an IHC of 4/4+; +; Lung cancer cells showed an IHC of 4/4+. When the concentration of BRCA84D was 0.078 μg/ml, prostate cancer cells showed an IHC of 3/4+; Breast cancer cells showed an IHC of 5/5+; Colon cancer cells showed an IHC of 4/4+; +; Lung cancer cells showed an IHC of 3/4+.

Normal livers were treated with the anti-B7-H3 antibody BRCA68D, and speckling was seen in hepatocytes and cells lining the sinusoids. Normal pancreas stained with the anti-B7-H3 antibody BRCA68D showed multifocal staining in collagen fibers and endothelial tissue. Normal adrenal cells stained with the anti-B7-H3 antibody BRCA68D showed mottling in the cortex. When stained with BRCA68D at a concentration of 0.156, both kidney and ovarian cancer cells showed an IHC of 5/5+.

Additional IHC staining analyzes were performed on gastric, renal, and ovarian cancer tissue samples. The results of these analyzes are shown in Table 12. In Table 11, the numbers indicate the number of positive signs (1=+, 2=++, 3=+++); Where each number indicates the number of samples tested.

Table 11

mAbs

µg/ml

stomach cancer stomach cancer

kidney cancer

Ovarian cancer

BRCA84D

0.625 µg/ml

2، 1، 2، 2، 2

1، 2، 1، 1، 1

0، 3، 1، 2، 2

0.078 µg/ml

1، 0، 0، 1، 0

0، 1، 0، 1، 0، 1

0، 2، 0، 1، 1

BRCA68D

0.156 µg/ml

3، 2، 3، 3، 3

3، 3، 2، 3، 3، 3

2، 3، 3، 2، 2

0.0195 µg/ml

2، 1، 2، 1، 1

2، 2، 2، 2، 2، 2

1، 2، 2، 1، 1

OVCA22

10 µg/ml

3، 1، 3، 1، 1

3، 1، 2، 3، 0، 2

2، 3، 2، 1، 1

2.5 microg/ml

2، 0، 2، 1، 0

2، 1، 1، 2، 0، 1

1، 2، 1، 0، 1

TES7

5 µg/ml

2، 1، 3، 2، 1

2، 3، 1، 2، 2، 1

1، 3، 1، 2، 2

1.25 µg/ml

2، 0، 2، 1، 1

2، 2، 1، 1، 1، 1

1، 3، 1، 2، 2

In summary, all B7-H3 tested showed varying degrees of staining severity in normal liver, pancreas, colon and lung tissue. Figure 1A shows the results of IHC studies performed using samples in normal liver, pancreas, colon and lung tissue with BRCA84D at concentrations of 0.625 μg/ml and 0.078 μg/ml. Liver staining was relatively restricted to cells lining the sinusoids (protofibroblast and Kupffer cells) with BRCA84D and TES7. OVCA22 showed hepatocyte staining as well as staining of cells lining the sinusoids at an ideal concentration. However, the spotting in hepatocytes disappeared at differential concentration. All other mAbs showed staining in hepatocytes including membrane or cytoplasmic staining at both optimal and differential concentrations. Pancreatic mottling was observed mainly in collagen fibers and a small percentage of endothelial tissue (acinar cells and/or intercalated duct cells). The mottling in the endothelial tissue decreased or disappeared at differential concentration. Colon staining is relatively restricted to the apical membrane of the interstitial endothelial tissue and the protofibroblast in the mucosa. No association was observed in colon lymph nodes. The lung showed very weak and patchy staining in the endothelial tissue when BRCA84D, BRCA68D, BRCA69D and GB8 were used. However, the mottling disappeared at differential concentration. No staining was observed in the lung with TES7, OVCA22, and PRCA157. At both concentrations. Adrenal cortical spotting was observed with almost all mAbs at optimal concentrations, except for BRCA84D. Adrenal spotting disappeared clearly with TES7 and OVCA22 at differential concentration. Neither the heart nor the kidney showed obvious staining with all mAbs (Figure 1B). In light of these properties, BRCA84D was considered the best mAb, followed by (2), (3), (4) the group of BRCA68D, BRCA69D, and PRCA157, and finally (5) GB8.

All mAbs generated in the study showed positive staining in 4 types of cancer at the optimal concentration. At differential concentration, BRCA84D maintained good staining in prostate cancer, breast cancer, and colon. TES7 maintained good staining in 4 types of cancer in the experiment. The remaining mAbs showed different degrees of severity in different tumor types. Spotting of tumor samples has been observed in tumor cells and stromal cells, such as the vascular system. Some tumor samples showed spotting only in the vascular system, i.e., BRCA84D, BRCA69D, TES7, and PRCA157. Some tumors showed stronger stromal staining than tumor cell staining. When mAbs were titrated to a lower concentration on these samples, some cases showed reduced or no staining in tumor cells, but remained strong in stromal staining. In general, in terms of expression in normal human tissues and differential expression in normal versus tumor tissues, the ranking of mAbs in terms of best IHC performance to least performance is as follows: (1)BRCA84D, (2)TES7, (3)OVCA22, ( 4) BRCA68D, BRCA69D and PRCA157 cluster, and finally (5). Table 12 and Figure 2 show the results for the BRCA84D antibody.

Table 12

Type of cancerous tissue

BRCA84D

0.625 microg/ml

BRCA84D

0.078 microg/ml

Prostate

3/3+

3/4+

the breast

4/4+

5/5+

Colon

4/4+

4/4+

Lung

4/4+

3/4+

Example 2

Cross-reactivity of B7-H3 to baboons

The B7-H3 sequence of baboons shares approximately 90% homology with its human counterpart, indicating that baboons are an excellent model for human B7-H3 interactions. Studies were performed to evaluate the cross-reactivity of the B7-H3 candidates BRCA84D, BRCA68D, BRCA69D, TES7, OVCA22 and PRCA157 with adrenal, liver, kidney, pancreas and lung, as well as whole placenta from one case of a baboon, in order to compare any cross-reactivity with the intensity and patterns of spotting observed in human tissue. .

The spotting concentration for each MAb tested is the ideal concentration determined in Caki-2 and Hs700T positive control cells (see Table 8). A commercial goat anti-human B7-H3 (cross-reacting with baboons) was chosen as a positive comparison antibody for spotting of baboons placental tissue. Two comparison eyes were applied to the corresponding isotype in each experimental session. The results of these experiments are shown in Table 13.

Table 13

mAb

Adrenal(2)

Liver(2)

Pancreas(2)

Kidney(2)

Lung(2)

Sakhdi(1)

BRCA84D

0.625 µg/ml

negative

negative

negative

negative

1/2

Blanket fabric 1+

Decidual cells 2+

Mesenchymal cells negative

BRCA68D

0.156 µg/ml

Veneer 3+

2/2

Hepatocytes 1+ (g)

Sinusoidal endothelial cells 1+

1/2

Forgot 2+

Blanket fabric 1+

Primary fibroblast 1+

negative

Decidual cells, villi, mesenchymal cells3+

BRCA69D

0.156 µg/ml

Cortex 2+

1/2

Hepatocytes 1+ (g)

1/2

Blanket fabric 1+

Primary fibroblast 1+

rare

negative

Decidual cells 2+, villi, mesenchymal cells 2+

TES7

5 µg/ml

negative

negative

negative

negative

negative

negative

OVCA22

10 µg/ml

negative

negative

negative

negative

negative

negative

PRCA157

0.078 µg/ml

Veneer 1+

1/2

Hepatocytes 1+ (g)

negative

negative

negative

Decidual cells 2+, villi, mesenchymal cells 1+

Note: BRCA84D showed negative spots in the liver and pancreas reaching 5 μg/ml. Although OVCA22 did not bind to trophozoites in IHC, weak binding was observed in B7-H3 trophozoites resulting from rebinding on CHO cells. The IHC score in normal tissue was a negative grading system of 1+, 2+, 3+, and 4; W = membrane; And 2/2 = 2 out of 2 cases, and 1/2 = 1 out of 2 cases.

IHC spotting studies for BR/CA84D (0.625 μg/ml) in baboon placentas showed spotting in flagellar cells but not in villi. Mottling was not observed in the liver and pancreas, however, mottling of cells lining the sinusoids was observed in the human liver and local fibers, and epithelial mottling was observed in human pancreatic tissue.

IHC spotting studies for BRCA68D (0.156 μg/ml) in baboon placentas showed spotting in flagellar cells, mesenchymal cells (endothelial and progenitor fibroblasts), and villi. Mottling was present in the membrane of hepatocytes and the cytoplasm of hepatic fibroblasts, as well as in the pancreatic fibers and the cytoplasm of the pancreatic endothelial tissue. Therefore, human liver, pancreas, and basal tissues are characterized by similar staining patterns with BRCA68D.

In summary, BRCA84D, BRCA68D, BRCA69D, and PRCA157 all showed cross-reactivity in baboons tissues. BRCA84D did not show spots in the simian liver and pancreas; This mottling has been observed in human liver and pancreas tissue. BRCA68D and BRCA69D showed similar mottling intensity and patterns in monkey tissues. Although BRCA68D, BRCA69D, and PRCA157 showed a spotting pattern similar to human tissue, the intensity of spotting was not identical to human tissue under the best conditions. TES7 and OVCA22 did not show any spotting in monkey tissues under optimal conditions.

Table 14 shows a summary of the comparative results of IHC staining in baboons and human tissues.

Table 14

mAb

Adrenals

Liver

Pancreas

Kidney

Lung

Placenta

BRCA84D

0.625 µg/ml

My profit

negative

negative

negative

negative

1/2

Blanket fabric 1+

Decidual cells 2+

Negative mesenchymal cells

BRCA84D

0.625 microg/ml

Human

negative

2/2

Sinusoidal endothelial cells 2+, hepatocytes 1+

5-5-10٪

lining fabric 1+,

5٪،

Fiber2+

negative

lining fabric 1+,

5-10٪

Decidual cells 1+, villi, mesenchymal cells 1+

BRCA68D

0.156 microg/ml

My profit

Veneer 3+

2/2

Hepatocytes 1+ (g)

Sinusoidal endothelial cells 1+

1/1

Lev 2+

Blanket fabric 1+

Primary fibroblast 1+

negative

Decidual cells, villi, mesenchymal cells3+

BRCA68D

0.156 microg/ml

Human

Veneer 3+

sinusoidal endothelial cells 2+, hepatocytes 2+ (g)

Blanket fabric 1+

Fiber+2

Primary fibroblast 1+

rare

Blanket fabric 1+

Decidual cells 3+, villi, mesenchymal cells 3+

BRCA69D

0.156 µg/ml

My profit

Cortex 2+

1/2

Hepatocytes 1+ (g)

1/2

Blanket fabric 1+

Primary fibroblast 1+

rare

negative

Decidual cells2+, villi, mesenchymal cells2+

BRCA69D

0.156 µg/ml

Human

Veneer 3+

Hepatocytes 2+ (G)

Blanket fabric 1+

Fiber2+

Primary fibroblast 1+

rare

Blanket fabric 1+

Decidual cells 3+, villi, mesenchymal cells 3+

PRCA157

0.078 microg/ml

My profit

Veneer 1+

1/2

Hepatocytes 1+ (g)

negative

negative

negative

Decidual cells 2+, villi, mesenchymal cells 1+

PRCA157

0.078 µg/ml

Human

Cortex 2+

Sinusoidal endothelial cells 2+, hepatocytes 1+ (g)

Endothelial tissue 1+5%

Fiber+2

negative

negative

undefined

Example 3

The B7-H3 monoclonal antibody binds to multiple ATCC cancer cell lineages

The antibodies of the present invention are found to be able to bind to multiple ATCC cancer cell lineages found in populations of an “American-type culture collection.” Tables 15 and 16 summarize the correlation results.

Table 15

antibody

Cell lineages

BLA08

BRCA68D

BRCA69D

BRCA84D

PRCA157

Natural human strains

HMEC

++/+++

+++

+++

++

HUVEC

N.D

++

+/++

+/−

+/++

Human breast cancer strains

BT474

+++

++

++/+++

+/++

++/+++

MCF7

+++

++

++/+++

+

++

MDA175

N.D

MDA361

N.D

++

+/+/−

++

SKBR3

+++

++

Human lung cancer strains

A549

+++

+/+/−

+/−

+/−

Calu3

+++

+/++

+

+/++

SKMES1

+++

++

++/+++

+/++

++

Human ovarian cancer strains

ES-2

+++

+/−

SKOV3

+++

++

+/++

+/+/−

++

Human pancreatic cancer strains

Pancreas-1

++/+++

+/++

+/++

+/+/−

+/++

AsPC-1

+++

HPAFII

+++

Hs700T

+++

++/+++

+++

+++

Human colon cancer strains

Colo205

N.D

HT-29

+++

+

+

+

SW480

+++

+/−

+/−

SW948

N.D

+

+

Human kidney cancer strains

293

+++

++

++

+

++/+++

786-0

+++

++

++

+

++/+++

A498

+++

++

++

++

++/+++

Caki2

+++

+++

+++

++

++/+++

Non-human cell lines

Cos7

+++

+

+/++

+/−

+/++

RL65

−

SVT2

N.D

Human prostate cancer strains

22Rv1

+++

DU145

+++

+

+

+

+/+/−

LNCaP

+++

++

++

+/++

++/+++

PC3

+++

+/+/−

+/−

+/−

+/−

TDH

N.D

+/+/−

+/+/−

+

Human stomach cancer strains

HS746T

N.D

+/++

+/++

+

++

N87

N.D

+/++

+/++

+/−

+/++

Table 16

antibody

Cell lineages

TDH06

OVCA22

GB8

SG27

TES7

Natural human strains

HMEC

HUVEC

+/+/−

+/−

+/−

Human breast cancer breast cancer strains

BT474

+/++

+

++

+/++

+/++

MCF7

+

+

++

+/+/−

+

MDA175

++

MDA361

+/+/−

+

SKBR3

++

Human lung cancer strains

A549

Calu3

+

SKMES1

+/++

+/−

+/++

+

+

Human ovarian cancer strains

ES-2

SKOV3

+

+/+/−

Human pancreatic cancer strains

Pancreas-1

+/+/−

+

+/−

+/+/−

AsPC-1

HPAFII

+

Hs700T

+

+++

+++

+

+++

Human colon cancer strains

Colo205

+

HT-29

+

+/+/−

+/+/−

SW480

+/−

+++

SW948

+/−

+

Human kidney cancer strains

293

+/+/−

+

+/+/−

+

786-0

+

+*

+/−

+

A498

+

+/++

+/++

Caki2

++

+

+++

+/++

++

Non-human cell lines

Cos7

+

+/+/− *

+/−

RL65

SVT2

Human prostate cancer strains

22Rv1

+

DU145

+/−

+

LNCaP

+/+/−

+

+*

+/+/−

+

PC3

TDH

+++

+/−

+/−

Human stomach cancer strains

HS746T

+

+/+/−

+/−

+/−

N87

+/+/−

+/−

Example 4

Monoclonal antibody B7-H3 redirects killing

The antibodies of the present invention bind to B7-H3 present on the surface of cancer cells. Using conventional methods, these antibodies can be labeled with fluorescein, as previously described. When these numbered molecules are incubated in the presence of UDART molecules with a T-cell receptor-binding domain and a fluorescein-binding domain (TCR-UDART), they may bind to DART molecules, thus localizing to the surface of B7-H3-expressing cells and inducing cross-killing. His guidance.

a. Redirecting the killing of A498 renal adenocarcinoma cells

To elucidate this redirected killing, fluorescein-labeled B7-H3 antibodies were incubated with TCR-UDART molecules and the ability of the molecules to induce cytotoxicity to A498 renal adenocarcinoma cells was assessed (Table 17). Based on the results obtained, the top candidates were reached: RECA13, BRCA68D, BRCA69D, and TDH6.

Table 17: Redirected killing of A498 renal carcinoma cells

mAb

UDART not found

With TCR-UDART

FACS

middle

middle

MFI

BCCA66

-1.04

46.39

43.3

BLA8

1.35

49.19

50

BRCA165

0

5.11

5.46

BRCA52

0

55.53

41.7

BRCA68D

0

36.89

83.7

BRCA69D

0

54.71

84.1

BRCA84D

0

72.4

30.6

GB8

4

42

17.9

KID1

0.38

52.08

18.5

KID13

26.39

58.2

KID35

-1.68

7.62

LUCA1

9.85

52.73

52.9

OVCA21

-0.85

47.59

6.04

OVCA22

0.36

38.66

53.9

OVCA25

-2.86

16.7

PA40

-0.46

40.54

PRCA123

0

56

130

PRCA135

0

55

127

PRCA157

0

39.14

58.8

RECA13

0

38.62

39.8

RECA22

-0.24

51.74

99.9

RECA9

0

62

50.1

SAL3

4.94

52.23

60.5

SG24

-2.25

42

SG27

-3.98

0.21

SKIN2

3.11

56.44

45.8

STO5

2.91

37.84

36.7

TDH36

-1.03

53.52

155

TDH37

0.05

65.21

47.5

TDH4

5.09

50.63

45.9

TDH40

-0.65

44.55

TDH5

2.92

49.6

28.8

TDH6

0

70.1

19.5

TES7

6.23

52.89

17.5

A498 renal carcinoma cells were incubated with various concentrations of monoclonal antibodies cross-reactive against B7-H3 to determine dose-dependent redirected killing due to the antibodies. The results (Figures 3a and 3b) show that redirected killing was dose dependent.

B. Redirecting the killing of lung cancer A549 cells

To further elucidate this redirected killing, fluorescein-labeled anti-B7-H3 antibodies were incubated with previously described TCR-UDART molecules or with UDART molecules that have an epitope-binding domain that binds to CD16 and an epitope-binding domain that binds to fluorescein (CD16-UDART), and quantitation Ability of molecules to induce cytotoxicity of lung cancer A549 cells (Table 18). The results of the experiments (Figures 3C and 3D) show that the redirected killing depended on the dose. Based on the results obtained, the top candidates are BLA8, BRCA68D, BRCA69D, and BRCA84D.

Table 18: Redirected killing of lung cancer A549 cells

mAb

Without DART

With TCR-UDART

With CD16-UDART

FACS

middle

middle

middle

MFI

BCCA66

1.89

25.17

8.22

36.1

BLA8

-7.7

10.97

3.68

34.7

BRCA52

0

27.63

37

BRCA68D

-4.42

13.45

15.95

58.3

BRCA69D

0

24.25

60.5

BRCA84D

0

15.33

25

GB8

-8.68

2.44

-4.65

17

KID1

0

22.93

41

LUCA1

0

14.65

53

OVCA21

-2.43

18.9

7.22

31.5

OVCA22

0

32.9

61

PRCA123

7.68

29.88

17.31

79.4

PRCA135

-6.58

22.72

8.14

75.6

PRCA157

0.02

18.63

18.24

44.3

PSMA

-0.7

5.58

9.94

RECA13

0.86

17.39

11.9

34.4

RECA22

3.71

20.49

19.35

74.3

RECA9

7.01

26.89

31.8

44.3

SAL3

0

31.8

67.4

SKIN2

-0.08

8.65

9.33

41.9

STO5

-10.36

9.28

1.71

54.7

TDH36

6.79

24.12

24.08

107

TDH37

6.93

22.57

23.37

42.3

TDH4

-6.26

10.07

2.21

32.4

TDH40

4.87

22.01

24.9

53.3

TDH5

-5.08

9.35

-2.85

27.1

TDH6

0

19.09

21.3

TES7

0

19.35

15.7

C. Redirecting the killing of prostate cancer cells (LNcap).

To further elucidate this redirected killing, fluorescein-labeled anti-B7-H3 antibodies were incubated with previously described TCR-UDART molecules or with UDART molecules that have an epitope-binding domain that binds to CD16 and an epitope-binding domain that binds to fluorescein (CD16-UDART), and quantitation Ability of molecules to induce cytotoxicity of prostate cancer LNcap cells (Table 19). Based on the results obtained, the top candidates are BRCA68D, BRCA69D, BRCA84D, and PRCA157.

Table 19: Redirected killing of prostate cancer cells

mAb

Without DART

With TCR-UDART

With CD16-UDART

FACS

middle

middle

middle

MFI

BCCA4

-2.96

13.29

2.47

5.1

BCCA66

-2.13

13.42

16.4

41

BLA8

4.32

14.97

24

48.4

BRCA165

3.59

57.26

12.02

7.6

BRCA183D

-4.65

43.09

35.3

7.6

BRCA52

32.34

71.23

48.28

42.5

BRCA68D

-1.4

23

21.91

86.9

BRCA69D

40.08

78.02

60.55

92.4

BRCA84D

20.11

78.7

41.27

16.4

GB8

-6.25

14.04

10.76

22

KID1

54.65

91.87

67.86

44.8

KID13

15.86

69.21

47.85

KID133

27.51

45.65

47.12

120

KID24

-4.26

34.13

41.17

14.5

KID35

14.17

64.01

33.05

KID47

11.34

39.49

15.02

10.8

KID8

16.98

58.8

34.77

5.5

LUCA1

47.4

89.31

67.15

73

LUCA17

23.18

26.9

35.87

11.1

LUCAT1

8.25

22.36

21.49

6.9

LUCAT7

26.5

38.29

44.77

8.7

MCL12

26.62

35.59

46.38

17.6

MEL2

6.57

29.9

31.4

19

OVCA21

12.07

26.81

31.3

41

OVCA22

45.09

96.5

77.3

113

OVCA25

16.14

63.26

32.39

PA22

1.73

57.7

9.89

8.9

PA33

8.99

34.49

48.14

9.4

PA40

38.42

73.07

63.65

PRCA123

9.96

14.39

18.38

125

PRCA135

-3.75

8.89

13.64

123

PRCA157

1.05

17.07

15.43

16.4

PSMA

11.52

31.38

34.79

PSMA

52.82

71.19

66.04

RECA13

5.86

22.55

15.4

37

RECA22

7.33

24.65

23.54

22.5

RECA9

27.67

52.54

45.14

5.3

SAL1

2.76

17.87

44.52

6.5

SAL2

8.71

30.68

29.17

14.5

SAL3

43.79

92.6

76.46

105

SG24

12.64

66.82

44.99

SG27

1.37

55.3

16.96

SKIN2

-2.04

14.81

24.23

73.8

SPL16

9.97

29.9

23.74

5.2

STO5

-1.48

21.11

24.97

61.3

TDH28

-4.23

18.55

15.04

13.3

TDH36

3.58

19.61

19.79

199

TDH37

7.9

18.78

25.22

57.3

TDH4

14.48

37.96

54.64

45.2

TDH40

8.51

44.55

43.87

79.3

TDH5

7.35

48.71

38.15

29.1

TDH6

4.5

54.59

19.73

41.7

TES7

50.15

94.47

73.4

22.4

Example 5

Ability of B7-H3 monoclonal antibodies to bind to soluble B7H3-2Ig and soluble B7H3-4Ig

As previously explained, B7-H3 exists in both a 4-Ig domain-containing form (B7H3-4Ig) and a 2-Ig domain-containing form (B7H3-2Ig). The anti-B7-H3 antibodies of the present invention have been tested for their ability to bind to soluble B7H3-2Ig (Figure 4a) and soluble B7H3-4Ig (Figure 4b). It has been found that antibodies have a wide range of binding properties. PRCA123, TDH5, BLA8, BRCA68D and SG24 antibodies were found to have the strongest binding to soluble B7H3-2Ig. TES7, LUCA50, BRCA165, OVCA22, STO9 and PA20 antibodies were found to have the strongest binding to B7H3-2Ig, and TES7 antibodies , OVCA21, and BRCA165, which have the weakest binding to soluble STO9.

Example 6

Affinity binding of antigens in solution to captured monoclonal antibodies

To demonstrate the binding affinity between antigens in solution and trapped monoclonal antibodies, antibodies were trapped on specific Fab2 IgG Fc fragments at the level of 100–200 RU. B7-H3 and B7-H3(4Ig) antigens were injected onto the retained antibodies at a concentration of 100 nM (flow rate 20 μL/min for 120 s, and binding was measured. Responses were normalized (flow rate 20 μL/min for 120 s titration to the same level of retained mAb and antibody binding response (mIgG1) and the comparator was subtracted as a blank sample. The results of this analysis (Figures 5A-5R; solid strains; B7-H3(4Ig), 100 nM; cut strains; B7-H3, 100 nM) indicate that the antibodies of the present invention have strong binding to B7-H3(4Ig). .

Example 7

BIACORE Analysis: Calibration of B7-H3 mAbs to immobilized B7-H3

To demonstrate the relative binding affinity of B7-H3-2Ig and B7-H3-4Ig to the antibodies of the present invention, a BIACORE analysis was performed. The B7-H3 antibodies of the present invention were allowed to bind to B7-H3-2Ig or B7-H3-4Ig and the titration of binding over time was estimated (Figures 6a-i). DH5, PRCA123, BLA8, and BRCA69 were found to have high affinity for both B7-H3-2Ig and B7-H3-4Ig. However, its binding peak(s) are the most abundant in the B7-H3-4Ig molecule, as only a few are present. It was found that OVCA22 has very low affinity for both B7-H3-2Ig and B7-H3-4Ig as its binding peak is equally available on both molecules. However, only the B7-H3-4Ig profile is expected to give sufficient proximity to bind bivalently to the antibody (low exclusion rate), as B7-H3-2Ig can only bind monovalently. It was found that TDH6 has almost no affinity for this format, as the binding to Ig2 is likely non-specific. TES7 and PA20 were found to be low-affinity B7-H4-4Ig specific antibodies. TES7 probably had a lower inclusion rate and a higher exclusion rate than PA20. BRCA84D was found to be an intermediate-affinity antibody with potentially multiple binding sites on both B7-H3-2Ig and B7-H3-4Ig. Based on BIACORE analysis, BRCA84D, due to its unusual binding site, is considered a preferred antibody. TES7 and PA20 have been considered candidates for specific binding to high-affinity antigen surfaces and low-specific, high-affinity antibody (eg BRCA69D or other).

Figure 7 shows a comparative BIACORE analysis of the PRCA157, BRCA69D, BLA8, PA20, BRCA84D, GB8 and SG27 antibodies, showing that the anti-B7-H3 antibody of the present invention can exhibit a range of binding properties.

Figure 8 shows the non-competitive identification of several anti-B7-H3 antibodies of the present invention. In the experiment, the anti-B7-H3 antibodies of the invention were incubated. In the experiment, human B7-H3 molecules were incubated in the presence of the BRCA84D antibody and subjected to BIACORE analysis. After about 3 minutes, a second anti-B7-H3 antibody was added to the reaction. If the second antibody competes with BRCA84D, it will find the B7-H3 sites blocked and will not be able to bind. The results indicate that BRCA68D, BRCA69D, and PRCA157 antibodies do not compete with BRCA84D for binding to human B7-H3.

Example 8

Internalization of anti-B7-H3 mAbs on CSC and ATCC cell lineages

The ability of the anti-B7-H3 antibodies of the present invention to be internalized upon binding to cancer cells has been studied. Prostate CSCs and pancreatic Hs700t cells were incubated with anti-B7-H3 antibody. The viability of the cells was measured after incubation in the presence of a secondary cytotoxic saporin-conjugated mouse antibody when it bound to the primary antibody and was internalized. The results of this study of prostate CSCs (Figure 9a) and pancreatic Hs700t cells (Figure 9b) demonstrate the ability of antibodies of the present invention to be internalized into cells.

Example 9

Binding of B7-H3 mAb and cross-inactivation analysis by ELISA

To study the cross-reactivity of the antibodies of the present invention and the agglutination peaks recognized by those antibodies, the extent of binding occurring in the presence of a competing B7-H3 antibody was measured. The results of this analysis are shown in Tables 10a-10f, and indicate that BRCA68D competes with BRCA69D. It was also found that TES7 and OVCA22 compete with each other, but TES7, but not OVCA22, was found to compete with both BRCA68D and BRCA69D. GB8 was found to compete with SG27 for binding to B7-H3-2Ig but not to B7-H3-4Ig. The data are summarized in Table 20 and show at least four adherent peaks for B7-H3-4Ig (i.e., the epitope recognized by SG27, the epitope recognized by GB8, the epitope recognized by OVCA22 and TES7, and the epitope recognized by BRCA68D and BRCA69D and TES7) and at least two epitopes of B7-H3-2Ig (i.e., the epitope recognized by SG27 and GB8, and the epitope recognized by BRCA68D and BRCA69D).

Table 20

Summary ELISA analysis of cross-inactivation of B7-H3 mAb

Competitive antibody

antibody

(Correlation Ratio vs. MIgG)

B7-H3 4Ig

B7-H3-2Ig

GB8

BRCA

69D

BRCA

68D

TES7

OVCA

GB8

BRCA

69D

BRCA

68D

GB8

50.211

119.105

108.948

87.48

98.142

26.618

84.408

94.71

TES7

111.234

109.39

108.425

1.605

16.268

100.645

90.734

99.515

OVCA22

121.783

112.322

100.813

3.371

2.048

100.423

87.991

102.766

TDH6

105.591

105.065

100.494

99.839

96.701

100.089

66.086

100.728

SG27

101.266

103.021

97.763

78.331

87.789

64.421

89.927

94.225

BRCA68D

105.934

40.284

43.144

4.815

102.655

98.888

7.635

7.425

BRCA69D

102.558

66.291

71.441

4.334

96.928

94.952

17.346

17.059

MIgG

100000

100000

100000

100000

100000

100000

100000

100000

The main antibody properties of B7-H3 of the present invention are shown in Table 21. Based on the apparent differential staining of normal and cancerous tissues, it was judged to be the best antibody based on its ability to bind B7-H3-4Ig and B7-H3-2Ig, and its binding affinity It was measured by previous BIACORE analysis and its ability to bind to B7-H3 ligands, and BRCA68D, BRCA69D, BRCA84D, and PRCA157 antibodies.

Table 21

MAb

BRCA

84D

TDH

TES7

BRCA

68D

BRCA

69D

GB8

SG

OVCA

PRCA

157

Isotype

G1/k

G1/k

G1/k

G1/k

G1/k

G1/k

2b/k

G1/k

G1/k

IHC

2a

2a

2a

2b

2b

2b

2b

2c

2c

ATCC matrix

2

3

3

3

3

2

Natural fabric

Colon

1+

1+

1+

1+

2+

2+

Lung

1+

1+

1+

Liver

1+

1+

2+

2+

1+

2+

2+

Kidney

1+

1+

1+

1+

Pancreas

1+

1+

1+

2+

Skin

2+

Cancerous tissue

Colon

1231*

1110*

1.5

2321*

2231*

1221*

1122

2231*

Lung

1130

1010

1.75

3332

3231

1120

3131**

3231

Prostate

112

111

3

333

333

222

222

333

the chest

1111