Immunotherapeutic agent combined with cd37, and its combination with bifunctional chemotherapeutic agent

CROSS-REFERENCE LINKS TO RELATED APPLICATIONS

This application claims priority in accordance with 35 USC. §119 (e) of US Provisional Patent Application No. 61/190067, filed April 11, 2008, this provisional application being incorporated herein in its entirety.

REPORT ON LIST OF SEQUENCES

The sequence listing attached to this application is provided in place of a hard copy in text format and is thus included as a reference in the description. A text file containing a sequence listing is called SEQUENCE_LISTING.txt. A text file of size 295 KB was created on March 26, 2012.

PREREQUISITES

Technical Field

The present description generally relates to compositions and methods for treating B cell disorders, and more particularly to a humanized small molecule modality small molecule (SMIP) molecule for CD37 as well as to drugs with synergistic combinations of molecules specifically bound to CD37, with bifunctional chemotherapeutic agents for use in the treatment or prevention of autoimmune, inflammatory or hyperproliferative diseases associated with B cells.

<u>Description of related field</u>

In general, the human immune system protects the body from penetrating foreign substances and pathogens. One component of the immune system is B-lymphocytes, also referred to as antibody-producing B cells, which protect the body by binding foreign substances or pathogens and, in some cases, mediating their destruction. However, in some cases, the immune system may not function properly and lead to the onset of the disease. For example, there are many types of malignant tumors, autoimmune diseases and inflammatory diseases in which uncontrolled proliferation of B cells is involved.

B cells can be identified by molecules on their cell surface, such as CD37. CD37 is a strongly glycosylated protein of 40-52 kDa, which is a tetraspanine family of transmembrane cell surface antigens that is highly expressed on normal antibody-producing B cells, but not on pre-B cells or plasmocytes. In addition to normal B cells, almost all malignant tumors of B-cell origin, including chronic lymphocytic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and hairy cell leukemia are positive for CD37 expression (Moore et al., J. Pathol. 152: 13 (1987); Merson and Brochier, Immunol. Lett., 19: 269 (1988), and Faure et al., Am., J. Dermatopathol 12: 122 (1990)).

Several CD37-specific immunotherapeutic agents have been developed. Specific CD37 monoclonal antibody IgG1 mouse, MB-1, methylated¹³¹I and tested in clinical trials for the treatment of NHL (see Press et al., J. Clin., Oncol 7: 1027 (1989), Bernstein et al., Cancer Res. (Suppl.) 50: 1017 (1990); et al., Front., Radiat, Onr., 24: 204 (1990), Press et al., Adv. Exp Med Biol 303: 91 (1991) and Brown et al., Nucl. Med. Biol. 24: 657 (1997)). The MB-1 antibody lacks Fc effector functions, such as antibody-dependent cellular cytotoxicity (AZPC), and the "bare" MB-1 antibody does not inhibit tumor growth in the xenotransplantation model<i>in vivo</i> (Buchsbaum et al., Cancer Res. 52: 6476 (1992)). In addition, mice were treated with an immunoconjugate containing adriamycin bound to G28-1 with another mouse monoclonal antibody to CD37 and showed its internalization with adriamycin, which was released intracellularly (see Braslawsky et al., Cancer Immunol. Immunother. 33: 367 (1991)). At present, a constructed fusion protein called a product of an immunopharmaceutical agent based on a small modular protein (SMIP ™) to CD37 is being tested in humans (see, for example, US Patent Application Publication 2003/0133939 and 2007/0059306).

Although a comprehensive study of antibody-based treatments has been conducted, there remains a need in the art for alternative or improved compositions and methods for treating B-cell-associated disorders or diseases.

SUMMARY

In one aspect, the present disclosure relates to humanized molecules specifically binding to CD37 and a method for reducing the number of B cells or treating a disease associated with abnormal B cell activity, comprising administering to a subject in need thereof an effective amount of a humanized molecule specifically binding to CD37 provided under this document.

In certain embodiments, the present disclosure relates to a humanized molecule specifically binding to CD37, which from the N-terminal to the C-terminus comprises: (i) a humanized heavy chain variable region, (ii) a linker as set forth in SEQ ID NO: 229, (iii) a humanized light chain variable region, (iv) the hinge region of IgG1, (v) the C region_H2 human IgG1 and (vi) region C_H3 human IgG1, wherein (a) the humanized heavy chain variable region from the N-terminus to the C-terminus contains: humanized heavy chain FR1, heavy chain CDR1 as indicated in SEQ ID NO: 63, humanized heavy chain FR2, heavy chain CDR2, as indicated in SEQ ID NO: 65, the humanized heavy chain FR3, the heavy chain CDR3 as indicated in SEQ ID NO: 67, 68 or 69, and the humanized heavy chain FR4, and (b) the humanized light chain variable region from the N-terminus up to the C-terminus contains: humanized FR1 light chain, light chain CDR1, as indicated in SEQ ID NO: 61 or 62, humanized FR2 is easy chain, CDR2 of the light chain as set forth in SEQ ID NO: 64, a humanized light chain FR3, and CDR3 of the light chain as set forth in SEQ ID NO: 66 and a humanized light chain FR4.

In certain embodiments of the above humanized molecules specifically binding to CD37, the humanized heavy chain FR1 comprises SEQ ID NO: 144, the humanized heavy chain FR2 contains the heavy chain SEQ ID NO: 151, the heavy chain FR3 contains SEQ ID NO: 158 and the heavy chain FR4 contains SEQ ID NO: 161 or 162.

In certain embodiments, any of the above humanized molecules specifically binding to CD37, the humanized FR1 of the light chain contains light chain SEQ ID NO: 171, FR2 contains light chain SEQ ID NO: 182, FR3 contains light chain SEQ ID NO: 195 and FR4 contains SEQ ID NO: 206.

In a related aspect, the present disclosure relates to a molecule specifically binding to CD37 that contains an amino acid sequence as set forth in SEQ ID NO: 253.

In certain embodiments, the molecule specifically binding to CD37 essentially consists of an amino acid sequence as set forth in SEQ ID NO: 253.

In certain embodiments, the molecule specifically binding to CD37 consists of the amino acid sequence as set forth in SEQ ID NO: 253.

In a related aspect, the present disclosure also relates to an isolated nucleic acid molecule that contains a nucleotide sequence that encodes a humanized molecule specifically binding to CD37 provided herein.

In another related aspect, the present disclosure relates to a vector that contains an isolated nucleic acid molecule that encodes a humanized molecule specifically binding to CD37 provided herein.

In another related aspect, the present disclosure relates to a host cell that contains the above vector.

The present disclosure also relates to a composition that comprises a humanized molecule specifically binding to CD37 provided herein and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure relates to a method for reducing the number of B cells or treating a disease associated with an abnormal B cell activity that comprises administering to an individual in need thereof an effective amount of a humanized molecule specifically binding to CD37 provided herein.

In certain embodiments, the disease associated with abnormal B cell activity is B-cell lymphoma, B-cell leukemia, B-cell myeloma, a disease characterized by the production of autoantibodies or a disease characterized by inadequate T-cell stimulation associated with B- cell pathway.

In certain embodiments, the disease characterized by the production of autoantibodies is idiopathic inflammatory myopathy, rheumatoid arthritis, myasthenia gravis, Graves disease, Type I diabetes mellitus, multiple sclerosis, autoimmune disease, dermatomyositis, polymyositis or Waldenstrom macroglobulinemia.

In certain embodiments, the disease associated with abnormal B-cell activity is chronic lymphocytic leukemia (CLL).

In another aspect, the present disclosure relates to compositions and methods for the combined use of molecules specifically binding to CD37 and bifunctional chemotherapeutic agents for reducing the number of B cells or treating a disease associated with abnormal B cell activity. An unexpected result of the use of this combination was that these compounds acted synergistically, which resulted in a more pronounced decrease in the number of B cells.

For example, the present disclosure relates to a composition that contains a molecule specifically binding to CD37 and bendamustine.

In certain embodiments, the molecule specifically binding to CD37 is a CD37-specific antibody or SMIP, such as a humanized antibody or humanized SMIP.

In certain embodiments, a molecule specifically binding to CD37, by binding specificity to CD37, competes with mAbs G28-1.

In certain embodiments, the molecule specifically binding to CD37 is a humanized molecule specifically binding to CD37 provided herein, such as a humanized molecule specifically binding to CD37 that contains the amino acid sequence as set forth in SEQ ID NO: 253, essentially consists or consists of it.

In a related aspect, the present disclosure relates to a method for reducing the number of B cells or treating a disease associated with an abnormal B cell activity, comprising administering to an individual in need thereof an effective amount of a molecule specifically binding to CD37 and bendamustine.

In certain embodiments, the disease associated with abnormal B-cell activity is B-cell lymphoma, B-cell leukemia, B-cell myeloma, a disease characterized by autoantibody production, or a disease characterized by inadequate T-cell stimulation associated with B cell pathway.

In certain additional embodiments, the disease characterized by the production of autoantibodies is idiopathic inflammatory myopathy, rheumatoid arthritis, myasthenia gravis, Graves disease, type I diabetes, multiple sclerosis, autoimmune disease, dermatomyositis, polymyositis or Waldenstrom macroglobulinemia.

In other specific embodiments, the disease associated with abnormal B-cell activity is chronic lymphocytic leukemia (CLL).

In certain embodiments, a molecule specifically binding to CD37 and bendamustine are administered concomitantly.

In other specific embodiments, a molecule specifically binding to CD37 and bendamustine are administered sequentially.

In certain embodiments, a molecule specifically binding to CD37 and bendamustine are formulated in a single formulation.

In certain embodiments, the molecule specifically binding to CD37 is a CD37-specific antibody or SMIP, such as a humanized antibody or humanized SMIP.

In certain embodiments, a molecule specifically binding to CD37, by binding specificity to CD37, competes with mAbs G28-1.

In certain embodiments, the molecule specifically binding to CD37 is a humanized molecule specifically binding to CD37 provided herein, such as a humanized molecule specifically binding to CD37 that contains the amino acid sequence as set forth in SEQ ID NO: 253, essentially consists or consists of it.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the alignment of the amino acid sequences of the heavy and light chain variable regions of mouse sequences G28.1 (heavy chain variable region: SEQ ID NO: 241, light chain variable region: SEQ ID NO: 236) and mouse CAS-024 (variable region heavy chain: SEQ ID NO: 245, light chain variable region: SEQ ID NO: 238), together with the consensus identical heavy chain variable region sequence and light chain variable region (SEQ ID NO: 270 and 271).

FIGS. 2A-2D show chromatograms CAS-001, CAS-002, CAS-003 and CAS-024 obtained by exclusive chromatography (EC). The interesting peaks (POIs) contain 98-99% purified SMIP molecules. CAS-024 has a very distinct and symmetrical peak (indicates homogeneity), whereas the CAS-001, CAS-002 and CAS-003 peaks have small shoulders (after integration, the shoulder is approximately 35% of POI), indicating a heterogeneous group of molecules .

FIG. 3 is a diagram showing how various CD37-specific SMIP proteins compete with the original molecule CAS-006 (a chimeric SMIP protein to CD37, mV_LmV_H) for binding to CD37 on Ramos line cells, which provides a binding affinity test as compared to the original molecule. CAS-024 (hV_HhV_L) essentially has the same affinity for CD37 as CAS-006, whereas other molecules (CAS-001, CAS-002 and CAS-003, all hV_LhV_H) show a 2-4-fold decrease in affinity.

FIG. 4A and 4B are diagrams of additional competitive binding assays against CAS-006 (marked as SMIP-016 in these diagrams). Here, hybrid mouse-human SMIP molecules (mV_HhV_L CAS-014 and hV_LmV_H CAS-017) have an affinity that is higher than that of CAS-006, whereas CAS-024 exhibits the same binding affinity as CAS-006, and CAS-003 (hV_LhV_H) has a lower binding affinity.

In Fig. 5A-5E shows competitive binding between several different antibodies to CD37 and CAS-006 (a chimeric SMIP molecule to CD37).

In Fig. 6A and 6B, CAS-024 was statistically superior to Rituxan in the treatment<i> in vivo</i> in the model of follicular lymphoma in animals, as shown by (A) the survival rate and (B) lobes without tumors.

In Fig. 7 shows that CAS-024 acts synergistically with chemotherapeutic agents fludarabine and vincristine with destruction of lymphoma cells from cells of the mantle zone (LKMZ), cells Rec-1.

FIG. 8 is a bar graph that shows the level of depletion of peripheral blood lymphocytes in human patients treated with SMIP molecules to CD37 as described herein.

In Fig. 9 shows the depletion of the number of lymphocytes and the course of treatment of the patient BJB. The BJB patient (part of cohort 7) was treated with 3.0 mg / kg on days 1, 3 and 5 of the first week, followed by 3 weekly doses in the first cycle, and the same treatment was performed in the second cycle. The patient BJB demonstrated a sharp drop in the number of lymphocytes (within 48 hours), showed a decrease in palpable lymph nodes on day 4 and continues to respond to treatment.

In Fig. 10 shows the depletion of lymphocytes and the course of treatment of the patient GRP. The GRP patient (part of cohort 4) was treated with 1.0 mg / kg once a week for four weeks in the first cycle, and then treated in the same way in the second cycle two months later. The GRP patient demonstrated a sharp decrease in the number of lymphocytes (within 2 weeks), showed a decrease in the size of the lymph nodes in CT scan by 36%, a decrease in the size of the spleen, an improved level of hemoglobin, and continues to respond to treatment.

In Fig. 11 is a graph of the combination index (CI) for the inhibitory effect of CAS-024 and bendamustine on the growth of Rec-1 cells.

In Fig. 12 shows the inhibitory effect of chlorambucil, alone and in combination with CAS-024, on the growth of SU-DHL-6 cells.

In Fig. 13 is a graph of the combination index for the inhibitory effect of CAS-024 and chlorambucil on SU-DHL-6 cell growth.

In Fig. 14A shows the comparison of tumor volumes in mice bearing tumors resulting from injections of DOHH2 cells and then treated with huIgG (human IgG, R &amp; D Systems), CAS-024, bendamustine and a combination of CAS-024 and bendamustine. In Fig. 14B shows tumor volumes in individual mice at day 13 compared with day 0.

In Fig. 15 shows the average tumor volumes over time in tumor-bearing mice resulting from injections of DOHH2 cells and then treated with huIgG, CAS-024, bendamustine, and a combination of CAS-024 and bendamustine. The values ​​represent the mean ± standard error of the mean for each day of measurement. After one or more mice in the group are sacrificed, the curves for each group end.

In Fig. 16 shows the percentage of survival of mice carrying tumors resulting from injections of DOHH2 cells and then treated with huIgG, CAS-024, bendamustine and a combination of CAS-024 and bendamustine over time.

In Fig. 17 shows the frequency of mice without tumors over time after treatment with huIgG, CAS-024, bendamustine, and a combination of CAS-024 and bendamustine.

DETAILED DESCRIPTION

In one aspect, the present disclosure relates to a molecule of CAS-024 (SEQ ID NO: 253) specifically binding to CD37, which is a humanized version of CAS-006 (a small modular small molecule (SMIP) protein immuno- of the immunoglobulin region from the monoclonal antibody of mouse G28-1 to human CD37). The SMIP protein CAS-024 unexpectedly (1) was expressed approximately to 25 times higher than other humanized versions of CAS-006 (such as CAS-002, CAS-003; see examples 2 and 5), (2) could communicate with CD37, as well as CAS-006, although other humanized versions did not have this capacity (see examples 4 and 5), and (3) was produced as a homogeneous group of molecules compared to the heterogeneous character of other humanized versions (see. example 3). In addition, the present disclosure relates to a molecule of CAS-024 (SEQ ID NO: 253) specifically binding to CD37 for use in methods for reducing the number of B cells or treating a disease associated with abnormal B-cell activity comprising administering to a subject in need thereof , the effective amount of CAS-024 provided herein.

In another aspect, the present disclosure relates to compositions and methods for the combined use of any molecule specifically binding to CD37 and bifunctional chemotherapeutic agents (such as bendamustine) to reduce the number of B cells or treat a disease associated with abnormal B cell activity. An unexpected result of using this combination is that this combination of compounds acts synergistically and essentially leads to a more effective therapeutic regimen.

Before describing this description in more detail, it may be useful to provide a definition of some of the terms used herein in order to understand it. Further definitions are given throughout this specification.

It should be understood that, in this specification, any range of concentrations, percentage range, range of ratios, or numeric range includes the value of any number within the specified range and, where appropriate, its fractional parts (such as one tenth and one-hundredth of an integer), if not specified otherwise. It should also be understood that any numerical range specified herein relating to any physical property, such as a polymer subunit, size or density, includes any number within the specified range unless otherwise indicated. As used herein, "approximately" or "essentially consists of" means ± 20% of the specified range, value or structure, unless otherwise specified. It should be understood that the singular forms, as used herein, indicate "one or more" of the specified components. It should be understood that the use of an alternative (eg, "or") means either one alternative, or both, or any combination thereof. As used herein, the terms "includes" and "contains" are used as synonyms. In addition, it is to be understood that the individual compounds or groups of compounds obtained from the various combinations of structures and substituents described herein are described in the present application to the same extent as if each of these compounds or from a group of compounds are indicated individually. Thus, the choice of specific structures or specific substituents is within the scope of this disclosure. that the use of an alternative (for example, "or") denotes either one alternative, or both, or any combination thereof. As used herein, the terms "includes" and "contains" are used as synonyms. In addition, it is to be understood that the individual compounds or groups of compounds obtained from the various combinations of structures and substituents described herein are described in the present application to the same extent as if each of these compounds or from a group of compounds are indicated individually. Thus, the choice of specific structures or specific substituents is within the scope of this disclosure. that the use of an alternative (for example, "or") denotes either one alternative, or both, or any combination thereof. As used herein, the terms "includes" and "contains" are used as synonyms. In addition, it is to be understood that the individual compounds or groups of compounds obtained from the various combinations of structures and substituents described herein are described in the present application to the same extent as if each of these compounds or from a group of compounds are indicated individually. Thus, the choice of specific structures or specific substituents is within the scope of this disclosure. are used as synonyms. In addition, it is to be understood that the individual compounds or groups of compounds obtained from the various combinations of structures and substituents described herein are described in the present application to the same extent as if each of these compounds or from a group of compounds are indicated individually. Thus, the choice of specific structures or specific substituents is within the scope of this disclosure. are used as synonyms. In addition, it is to be understood that the individual compounds or groups of compounds obtained from the various combinations of structures and substituents described herein are described in the present application to the same extent as if each of these compounds or from a group of compounds are indicated individually. Thus, the choice of specific structures or specific substituents is within the scope of this disclosure.

The "binding domains" or "binding regions" according to the present disclosure can be, for example, any protein, polypeptide, oligopeptide or peptide capable of specifically recognizing and binding a biological molecule (eg, CD37) or a complex of two or more of the same or different molecules or structure or aggregate, stable or temporary. The binding region includes any natural, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule or other target of interest. To identify the binding domains of the present disclosure that specifically bind to a particular target, a variety of assays are known, including Western blotting, ELISA, or Biacore analysis.

The binding domains and their fusion proteins as described herein may have the ability to bind to the desired extent, including "specific or selective binding" of the target, without substantially binding the other components present in the test sample if they bind the target molecule to affinity or K_a (i.e., with an equilibrium association constant of a particular binding interaction with a dimension of 1 / M), for example, greater than or equal to about 10⁵ M^-1, 10^6th M^-1, 10^7th M^-1, 10⁸ M^-1, 10⁹ M^-1, 10¹⁰ M^-1, 10^eleven M^-1, 10¹² M^-1 or 10¹³ M^-1. "High affinity" binding domains refers to binding domains with K_a at least 10^7th M^-1, at least 10⁸ M^-1, at least 10⁹ M^-1, at least 10¹⁰ M^-1, at least 10^eleven M^-1, at least 10¹² M^-1, at least 10¹³ M^-1 or more. "Low affinity" binding domains refer to binding domains with K_a up to 5 × 10^7th M^-1, to 10^7th M^-1, to 10^6th M^-1, to 10⁵ M^-1 or less. Alternatively, the affinity can be defined as the equilibrium dissociation constant (K_d) of the specific binding interaction with the dimension M (for example, from 10^-5 M to 10^-13 M). The affinities of the polypeptides of the binding domains and fusion proteins of the present disclosure can be readily determined using conventional methods (see, for example, Scatchard et al. (1949) Ann.N. Acad. Sci. 51: 660, and US Pat. Nos. 5,283,173, 5468614 or equivalent).

The term "molecules specifically binding to CD37" refers to a protein, polypeptide, oligopeptide or peptide that specifically binds CD37 to K_a at least about 10^6th M^-1 (e.g., at least about 10^7th M^-1, 10⁸ M^-1, 10⁹ M^-1, 10¹⁰ M^-1, 10^eleven M^-1, 10¹² M^-1 or 10¹³ M^-1).

The term "CD37 specific binding domain" refers to a part or domain of a molecule specifically binding to CD37, which is responsible for the specific binding of the molecule to CD37. The CD37-specific binding domain itself (ie, without all other parts of the molecule specifically binding to CD37) binds to CD37 with K_a at least about 10^6th M^-1 (e.g., at least about 10^7th M^-1, 10⁸ M^-1, 10⁹ M^-1, 10¹⁰ M^-1, 10^eleven M^-1, 10¹² M^-1 or 10¹³ M^-1). The CD37-specific binding domain may itself be sufficient as a molecule specifically binding to CD37. Exemplary binding domains specific for CD37 include scFv and Fab fragments specific for CD37, which can be prepared from antibodies to CD37, such as the monoclonal antibody G28-1.

Unless explicitly defined herein, each of the terms understood by those skilled in the art as referring to antibody technology is given in the meaning used in the art. For example, the terms "V_L"and" V_H"refer to the variable binding regions originating from the light and heavy chain of the antibody, respectively.The variable binding regions are formed from separate, well-defined subregions that are known as" complementarity determining regions "(CDRs) and" framework regions. "The terms" C_L"and" C_H"refer to the" constant region of the immunoglobulin ", i.e., constant regions derived from the light or heavy chain of the antibody, respectively, while it is understood that the latter region is further divided into domains of the constant region C_H1, C_H2, C_H3 and C_H4, depending on the isotype of the antibody (IgA, IgD, IgE, IgG, IgM) from which this region occurs. A portion of the domains in the constant region forms an Fc region (the region of the "crystallizing fragment") that contains the domains responsible for the effector functions of the immunoglobulin, such as AZKZ (antibody-dependent cell cytotoxicity), complement-dependent cytotoxicity and complement fixation, binding to Fc -receptors, an increase in half-life<i>in vivo</i> with respect to a polypeptide lacking the Fc region, binding to protein A, and possibly even transplacental transfer (see Capon et al., Nature, 337: 525 (1989)). In addition, the polypeptide containing the Fc region allows dimerization or multimerization of the polypeptide.

The "hinge region" is an amino acid sequence located between a binding domain specific for CD37 and another region (eg, region C_H2) in the fusion protein and binding them so that the fusion protein still remains capable of specifically binding to CD37 (i.e., with K_a at least about 10^6th M^-1, 10^7th M^-1, 10⁸ M^-1, 10⁹ M^-1, 10¹⁰ M^-1, 10^eleven M^-1, 10¹² M^-1 or 10¹³ M^-1). In certain embodiments, the hinge region is the hinge region of an immunoglobulin.

The "hinge region of an immunoglobulin" refers to the hinge region of a wild-type immunoglobulin or to a modified hinge region of a wild-type immunoglobulin.

According to crystallographic studies, the hinge region of the immunoglobulin can be further functionally divided into three regions: the upper hinge region, the central region and the lower hinge region. The upper hinge region includes amino acids from the carboxyl terminus of C_H1 to the first residue in the hinge region, which limits mobility, usually to the first cysteine ​​residue, which forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental mobility of the antibody. The central hinge region includes the disulfide bonds between the heavy chains, and the lower hinge region connects the N-terminus of the C_H2 and includes residues in C_H2. Also, the central hinge region of human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 264), which upon dimerization by formation of disulfide bonds leads to the formation of a cyclic octapeptide presumably acting as a point of rotation, thus imparting mobility .

As used herein, the "wild type immunoglobulin hinge region" refers to a natural amino acid sequence located between regions C_H1 and C_H2 of one antibody chain and connecting them. It comprises a top hinge region, a central hinge region and a portion of the lower hinge region that is not part of the region C_H2. An exemplary hinge region of a wild-type immunoglobulin is the hinge region of human IgG1, as indicated in SEQ ID NO: 90, wherein from its N-terminus to its C-terminal end, the first ten amino acids (EPKSCDKTHT, SEQ ID NO: 263) the next four amino acids (CPPC, SEQ ID NO: 264) are derived from the centrally hinged region, and the last amino acid (i.e., proline) is the first amino acid in the lower hinge region and is not part of C_H2.

The "modified wild type immunoglobulin hinge region" or "modified immunoglobulin hinge region" refers to (a) a hinge region of a wild-type immunoglobulin containing up to 30% amino acid substitutions (for example, up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), (b) portions of the hinge region of the wild-type immunoglobulin of at least 10 amino acids in length (eg, at least 12, 13, 14 or 15 amino acids) that contains up to 30% amino acid substitutions (e.g., up to 25%, 20%, 15%, 10% or 5% amino acid substitutions or deletions), or (c) parts of the hinge of the wild type immunoglobulin region, which contains a central hinge region (the length of which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or at least 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids)._H2) and connects them, this allows the fusion protein to specifically bind to CD37 (ie, with K_a at least about 10^6th M^-1, 10^7th M^-1, 10⁸ M^-1, 10⁹ M^-1, 10¹⁰ M^-1, 10^eleven M^-1, 10¹² M^-1 or 10¹³ M^-1). In certain embodiments, one or more cysteine ​​residues in the hinge region of the wild-type immunoglobulin can be replaced by one or more other amino acid residues (eg, one or more serine residues). The altered hinge region of the immunoglobulin, alternatively or additionally, may contain a residue where the proline from the hinge region of the wild-type immunoglobulin is substituted by another amino acid residue (eg, a serine residue).

The "linker" refers to the amino acid sequence that connects the heavy chain variable region and the light chain variable region to each other and provides a spacer function corresponding to the interaction of the two binding subdomains so that the resulting polypeptide can specifically bind to CD37.

As used herein, a "derivative" refers to a chemically or biologically modified version of a compound that structurally corresponds to the parent compound and which (in fact or theoretically) can be obtained from the parent compound. As a rule, the "derivative" differs from the "analog" in that the starting compound can be a starting material for the preparation of a "derivative", whereas for the preparation of an "analog" as the starting material, it is not necessary to use the parent compound. The chemical or physical properties of the derivative may differ from the chemical or physical properties of the parent compound. For example, the derivative may be more hydrophobic, or it may be a mutant sequence,

"B-cell related disorder or disease" refers to an abnormal activity of B cells or an activity that deviates from a normal, correct or expected direction. For example, a B cell-related disorder or disease may include inappropriate cell proliferation with damaged or defective DNA or other cellular components. Abnormal B cell activity may include cellular proliferation characterized by inadequately high levels of cell division, inadequately low levels of apoptosis, or both. For such diseases, for example, the presence of a single or multiple foci of abnormal proliferation of cells, groups of cells or tissue (s), malignant or non-malignant, benign or malignant can be inherent. A B cell-related disorder or disease may also include production of abnormal antibodies, such as autoantibody production or overproduction of antibodies more desirable for production at normal levels. It is also believed herein that abnormal B cell activity may occur in certain B cell subpopulations and not occur in other subpopulations, or may include inappropriate stimulation of T cells, for example, by inappropriate antigen presentation to T cells or by another B-cell pathway.

"Treatment" refers to therapeutic treatment or preventive / preventive treatment. Therapeutic treatment may improve at least one symptom of the disease in the individual being treated, or may delay the worsening of the progression of the disease in the individual or prevent the occurrence of additional associated diseases.

The "therapeutically effective amount (or dose)" or "effective amount (or dose)" of a specific binding molecule or compound refers to an amount of a compound that is sufficient to improve one or more of the symptoms of the disease being treated. When applied to a separate active ingredient administered separately, a therapeutically effective dose refers to this ingredient separately. When applied to a combination, a therapeutically effective dose refers to a combined amount of active ingredients that results in a therapeutic effect, with a periodical or simultaneous administration. In the present invention, in particular, it is provided that one or more specific binding molecules can be administered by the methods of the invention, each in an effective dose.

"An individual having or suspected of having a disease associated with abnormal B cell activity" is an individual in whose disease or symptom of impairment may be caused by abnormal B-cell activity or B-cell proliferation, may be aggravated by abnormal B-cell activity or can be attenuated by controlling the activity of B cells. Examples of such diseases are B-cell malignant neoplasm or B-cell malignant tumor (eg, B-cell lymphoma, B-cell leukemia or B-cell myeloma), a disease characterized by autoantibody production, or a disease characterized by inappropriate T-cell stimulation, which is caused by inappropriate antigen presentation by B-cells to T-cells or mediated by other routes involving B cells.

Additional definitions are provided in the following detailed description of the present description.

<u>Humanized molecules specifically binding to CD37,</u>

In one aspect, the present disclosure relates to humanized molecules specifically binding to CD37. These molecules can exist in any form that contains a humanized CD37-specific binding domain, including a humanized anti-CD37 antibody, a Fab fragment of a humanized anti-CD37 antibody, a humanized CD37 single-chain Fv (scFv), a humanized CD37-specific SMIP protein, a humanized specific to CD37, a PIMS protein (a fusion protein containing SMIP components in reverse orientation), a humanized CD32-specific SCORPION protein, and other bi-or polyspecific binding proteins that contain at least Din humanized CD37-specific binding domain to. A detailed description of SMIP proteins and methods for their preparation can be found, for example, in US Patent Publications Nos. 2003/0133939, 2003/0118592, 2005/0136049 and WO 2005017148. The structures and methods for the preparation of PIMS proteins are described in US Application No. 12/168875. Methods of producing SCORPION proteins can be found, for example, in PCT Application Publication No. WO 2007/146968. Other illustrative polyfunctional fusion proteins can be found, for example, in US Patent Application Publication No. 2006/0051844 and US Pat. No. 7166707. Certain bi- or polyspecific binding proteins may contain a CD37-specific scFv and one or more other binding domains that do not originate from immunoglobulin.

<i>Humanized binding domains specific for CD37</i>

An exemplary "humanized CD37 specific binding domain" is an immunoglobulin variable region specific for CD37 that contains at least one human framework region.

The human framework region refers to the framework region of the wild-type immunoglobulin variable (ie, naturally occurring) human framework region, to the altered framework region of the human immunoglobulin variable region where, in this region, deleted or replaced (eg, one or more amino acid residues of a non-immunoglobulin immunoglobulin framework in appropriate positions) of less than about 50% (eg, preferably less than about 45%, 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) of the amino acids, or to the altered framework region of an immunoglobulin variable region other than the human immunoglobulin, where, in the field, deleted or replaced (eg,

In certain embodiments, the human framework region is a framework region of a human wild-type immunoglobulin variable region. In other specific embodiments, the human framework region is an altered human immunoglobulin variable region framework region with deletions or amino acid substitutions at one, two, three, four or five positions. In other specific embodiments, the human framework region is an altered variable region framework region from an immunoglobulin other than a human immunoglobulin, with deletions or substitutions of amino acids at one, two, three, four or five positions.

In certain embodiments, the humanized CD37-specific binding domain comprises at least one, two, three, four, five, six, seven or eight human framework regions (FR) selected from human FR1 light chain, human heavy chain FR1, FR2 human light chain, human heavy chain FR2, human light chain FR3, human heavy chain FR3, human light chain FR4 and human heavy chain FR4.

Representative human FRs are shown in SEQ ID NO: 140-146 (human heavy chain FR1), SEQ ID NO: 147, 150 and 151 (human heavy chain FR2), SEQ ID NO: 154-160 (human heavy chain FR3), SEQ ID: 161-163, 168 and 169 (human heavy chain FR4), SEQ ID NO: 170-172, 175 and 177-181 (human FR1 light chain), SEQ ID NO: 182, 184-188 and 191 (FR2 human light chain), SEQ ID NO: 194-198, 203 and 205 (FR3 human light chain), and SEQ ID NO: 206-210 (FR4 human light chain). Additional illustrative FR regions of a human can be found in the regions FR of the CD37-specific SMIP proteins provided herein, such as CAS-001, CAS-002, CAS-003 or CAS-024.

FR human that may be present in binding domains specific for CD37 also include variants of exemplary FRs provided herein, in which one or two exemplary amino acids have been replaced or deleted.

In certain embodiments, the humanized CD37-specific binding domain comprises (a) a humanized light chain variable region that contains human light chain FR1, human light chain FR2, human light chain FR3 and human light chain FR4, and (b) a humanized variable region of a heavy a chain that contains human heavy chain FR1, human heavy chain FR2, human heavy chain FR3, and human heavy chain FR4.

CD37-specific binding domains provided herein also include one, two, three, four, five or six CDRs. Such CDRs may be CDRs other than human CDRs or altered CDRs other than human CDRs selected from light chain CDR1, CDR2 and CDR3 and heavy chain CDR1, CDR2 and CDR3. In certain embodiments, the CD37-specific binding domain comprises (a) a light chain variable region that contains light chain CDR1, light chain CDR2 and light chain CDR3, and (b) a heavy chain variable region that contains heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3.

Exemplary CDRs include the light chain CDR1 as indicated in SEQ ID NO: 61 (RASENVYSYLA) or in SEQ ID NO: 62 (RTSENVYSYLA), heavy chain CDR1 as indicated in SEQ ID NO: 63 (GYNMN), light chain CDR2 as is indicated in SEQ ID NO: 64 (FAKTLAE), heavy chain CDR2 as indicated in SEQ ID NO: 65 (NIDPYYGGTTYNRKFKG), light chain CDR3 as indicated in SEQ ID NO: 66 (QHHSDNPWT), heavy chain CDR3 as indicated in SEQ ID NO: 67 (SVGPFDY), heavy chain CDR3 as indicated in SEQ ID NO: 68 (SVGPFDS), and heavy chain CDR3 as indicated in SEQ ID NO: 69 (SVGPMDY). The preferred light chain CDR1 is SEQ ID NO: 61 (RASENVYSYLA), and the preferred heavy chain CDR3 comprises SEQ ID NO: 68 (SVGPFDS) or SEQ ID NO: 69 (SVGPMDY).

Additional exemplary CDRs include light chain CDR1 as indicated in SEQ ID NO: 128 (RTSQNVYSYLA), 129 (RTSESVYSYLA), 130 (RASQSVYSYLA), 131 (RASQSVSSYLA) and 132 (RASQSVSYYLA), heavy chain CDR1 as indicated in SEQ ID NO : 133 (SYMNM) and 134 (SYWIG), light chain CDR2 as indicated in SEQ ID NO: 135 (AASSLQS), 136 (GASTRAT) and 137 (DASNRAT), heavy chain CDR2 as indicated in SEQ ID NO: 138 IIYPGDSDTRYSPSFQG) and 139 (RIDPSDSYTNYSPSFQG), light chain CDR3 as indicated in SEQ ID NO: 220 (QHHSDNPWT), and heavy chain CDR3 as indicated in SEQ ID NO: 211 (SVGPMDY), 212 (SVGPFDY), 213 (SVGPMDV) , 214 (SVGPFDS), 215 (SVGPFDP), 216 (SVGPFQH), 217 (SVGPFDV), 218 (SVGPFDI), and 219 (SVGPFDL). Additional exemplary CDRs include CDRs in CD37-specific SMIP proteins provided herein.

In certain embodiments, the binding domains specific for CD37 contain a humanized light chain variable region that, from its N-terminus to the C-terminus, contains: human light chain FR1, light chain CDR1, human light chain FR2, light chain CDR2, human chain, light chain CDR3 and human light chain FR4.

In certain embodiments, binding domains specific for CD37 contain a humanized light chain variable region that from its N-terminus to the C-terminus contains: human light chain FR1, light chain CDR1 as indicated in SEQ ID NO: 61 or 62, FR2 human light chain, light chain CDR2 as indicated in human light chain SEQ ID NO: 64, FR3, light chain CDR3 as indicated in SEQ ID NO: 66 and human light chain FR4. In additional embodiments, the binding domains specific for CD37 contain, essentially consist of or consist of a humanized light chain variable region that from its N-terminus to the C-terminus contains: the human light chain FR1 as indicated in SEQ ID NO: 171, light chain CDR1 as indicated in human light chain SEQ ID NO: 61, FR2 as indicated in SEQ ID NO: 182, light chain CDR2 as indicated in SEQ ID NO: 64, FR3 of the human light chain as indicated in SEQ ID NO: 195, light chain CDR3 as indicated in SEQ ID NO: 66 and FR4 of the human light chain as indicated in SEQ ID NO: 206. Additional illustrative humanized light chains are shown in SEQ ID NO: 237-240 and include light chains in humanized CD37-specific SMIP proteins provided herein.

In certain embodiments, the binding domains specific for CD37 contain a humanized heavy chain variable region that, from its N-terminus to the C-terminus, contains: human heavy chain FR1, heavy chain CDR1, human heavy chain FR2, heavy chain CDR2, human chains, heavy chain CDR3 and human heavy chain FR4.

In certain embodiments, binding domains specific for CD37 contain a humanized heavy chain variable region that from its N-terminus to the C-terminus contains: human heavy chain FR1, heavy chain CDR1 as indicated in SEQ ID NO: 63, FR2 with a heavy human heavy chain CDR2 as indicated in human heavy chain SEQ ID NO: 65, FR3, heavy chain CDR3 as indicated in SEQ ID NO: 67, 68 or 69, and human heavy chain FR4. In additional embodiments, the binding domains specific for CD37 contain, essentially consist of or consist of a humanized heavy chain variable region that from its N-terminal to the C-terminus contains: the human heavy chain FR1 as set forth in SEQ ID NO: 144, heavy chain CDR1 as indicated in human heavy chain SEQ ID NO: 63, FR2 as indicated in SEQ ID NO: 151, heavy chain CDR2, as indicated in human heavy chain SEQ ID NO: 65, FR3, as indicated in SEQ ID NO: 158, heavy chain CDR3 as indicated in SEQ ID NO: 67, 68 or 69 and human heavy chain FR4 as indicated in SEQ ID NO: ID NO: 161. Additional illustrative humanized light chains are shown in SEQ ID NOs: 242-245 and include light chains in humanized CD37-specific SMIP proteins provided herein.

In certain embodiments, binding domains specific for CD37 may be in the form of a Fab or scFv fragment. In a preferred embodiment, the CD37-specific binding domain is a humanized CD37 scFv specificity that comprises a light chain variable region and a heavy chain variable region linked together by a linker. In additional embodiments, the light and heavy chain variable regions are humanized and may comprise a humanized light chain variable region as indicated in SEQ ID NO: 238 and a humanized heavy chain variable region as specified in SEQ ID NO: 245.

In other further embodiments, only the light or heavy chain variable region is humanized. For example, binding domains specific for CD37 may contain a humanized light chain variable region (i.e., a light chain variable region that contains at least one human FR) and a non-human variable region of the heavy chain (eg, mouse or rat). Alternatively, binding domains specific for CD37 may comprise a non-human variable light chain variable region (eg, belonging to a mouse or rat) and a humanized heavy chain variable region (i.e., a heavy chain variable region that contains at least one variable region of the heavy chain, one person FR). Both types of binding domains specific for CD37 can be referred to as "

In certain embodiments, the C-terminus of the light chain variable region in humanized scFv, specific for CD37, is linked to the N-terminus of the variable region of the heavy chain by a linker. Thus, the resulting scFv from its N-terminus to its C-terminus contains: a light chain variable region, a linker and a variable region of the heavy chain. In other specific embodiments, the C-terminus of the heavy chain variable region in the humanized scFv, specific for CD37, is linked to the N-terminus of the light chain variable region by a linker. Thus, the resulting scFv from its N-terminus to its C-terminus contains: a heavy chain variable region, a linker and a variable region of the heavy chain.

In certain embodiments, the linkers contain 5-30 amino acids, for example 15-25 amino acids. In certain embodiments, the linkers comprise (Gly_nSer)_m, where n and m can be an integer independently selected from the range of 1 to 5 (SEQ ID NO: 229, 272-293). For example, in certain embodiments, n is 4 and m is 1, 2, 3, 4 or 5 (SEQ ID NO: 229, 274-275). In certain embodiments, one or two amino acids, other than Gly or Ser, may be present at the N-terminus, the C-terminus or both. In other specific embodiments, to replace Gly or Ser in a linker that contains (Gly_nSer)_m, where m and n are as defined above, one or two amino acids other than Gly or Ser can be used. An exemplary linker comprises a sequence (Gly₄Ser)₅, as indicated in SEQ ID NO: 229. Additional illustrative linker sequences are given in SEQ ID NO: 225-228.

In certain embodiments, humanized binding domains specific for CD37 or molecules specifically binding to CD37 compete for binding to CD37 with mAT G28-1. That is, in such embodiments, binding of mAbs G28-1 to CD37 in the presence of other binding domains specific for CD37 (such as monoclonal antibodies to CD37) or molecules specifically binding to CD37 is reduced compared to binding to mAbs G28 -1 with CD37 in the absence of binding domains specific for CD37, or molecules specifically binding to CD37. Competitive binding assays, such as those described in Examples 4-6, are known in the art and can be used to determine the ability of a CD37 specific binding domain or molecule specifically binding to CD37 to compete with mAT G28-1 for binding to CD37 .

<i>Humanized SMIP polypeptides specific for CD37</i>

In certain embodiments, molecules specifically binding to CD37 are polypeptides of immunopharmaceuticals based on small modular protein (SMIP) specific for CD37. SMIP proteins are the fusion proteins of the binding domain and immunoglobulin, which, usually from their N-termini to the C-terminus, comprise: a binding domain derived from an immunoglobulin (eg, scFv), a hinge region and an effector domain (eg, regions C_H2 and C_H3 IgG). In preferred embodiments, SMIP polypeptides specifically binding to CD37 are humanized.

The hinge region of the humanized SMIP polypeptide specifically binding to CD37 can be the hinge region of an immunoglobulin. In certain embodiments, the hinge region is a hinge region of a wild-type immunoglobulin such as an IgG hinge region, an IgA hinge region, an IgD hinge region, an IgE hinge region, or a fragment thereof (for example, a length of 4 to 20 or 5 to 15 amino acids) , which contains the central hinge region. In certain preferred embodiments, the hinge region may be a hinge region of an antibody selected from the hinge regions of human IgG1, human IgG2, human IgG3, human IgG4, or fragments or variants thereof. In certain embodiments, the hinge region is a hinge region of a wild-type immunoglobulin or part thereof, such as a hinge region of a human immunoglobulin. Illustrative hinge regions for such embodiments are the human wild-type IgG1 hinge region as set forth in SEQ ID NO: 90, the human wild-type IgA hinge region as set forth in SEQ ID NO: 115, the hinge region of human wild-type IgA2 as indicated in SEQ ID NO: 116, the human wild-type IgG3 hinge region as set forth in SEQ ID NO: 118, part of the human IgG3 articulation region as set forth in SEQ ID NO: 258, and the human IgD articulation as specified in SEQ ID NO : 127. In certain embodiments, one or more amino acid residues can be added at the N- or C-terminus of the hinge region of the wild-type immunoglobulin as part of the structure of the fusion protein structure. Such amino acid residues are referred to as "connecting amino acids" (see Table 4).

In certain embodiments, the hinge region is an altered (mutated) hinge region of the wild-type immunoglobulin, such as a modified hinge region of the wild-type IgG or a modified portion of the hinge region of the wild-type immunoglobulin. For example, the hinge region of human wild-type IgG1 contains three cysteine ​​residues - the cysteine ​​closest to the N-terminus is designated as the first cysteine ​​residue, while the most cysteine ​​close to the C-terminus is the third cysteine ​​residue. In certain embodiments, the human IgG1 mutant hinge region contains only two cysteine ​​residues, for example, the hinge region of human IgG1, in which the first cysteine ​​residue is replaced with serine. In other specific embodiments, the human IgG1 mutant hinge region contains only one cysteine ​​residue. In certain embodiments, proline, located closer to the C-terminus relative to the third cysteine ​​in the hinge region of human IgG1, is replaced, for example, with serine. Exemplary mutant hinge regions of human IgG1 are shown in SEQ ID NO: 92, 94, 102, 104, 255, 256, 106, 108, 257, 96, 110, 112, 98 and 100. Exemplary mutant parts of human IgG3 hinge regions are shown in SEQ ID NO: 120, 126, 259-261, 122 and 124. In certain embodiments, one or more amino acid residues may be added at the N- or C-terminus of the mutant hinge region of the immunoglobulin as part of the structure of the fusion protein structure. Examples of such modified hinge regions are italicized in SEQ ID NO: 231-235.

In certain embodiments, the hinge region comprises or has a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least by at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % identical to the ball of the wild type of immunoglobulin, such as the hinge regions of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD and IgE.

In additional embodiments, the altered hinge regions may be based on the hinge region of a wild-type immunoglobulin (for example, on the hinge region of IgG1) and, in comparison to the hinge region of the wild-type immunoglobulin, comprise one or more inserts, for example 1, 2, 3 or 4, one or more (eg 1, 2, 3 or 4) deletions, one or more (eg, 1, 2, 3 or 4) amino acid changes (eg conservative amino acid substitutions or non-conservative amino acid substitutions) or a combination of these mutations provided that h a modified hinge region retains the flexibility or rigidity suitable for the correct orientation of the binding domain of a fusion binding protein to interact with its target. Insert (s),

As described herein, SMIP-specific polypeptides specific for CD37 may contain region C_H2 immunoglobulin. In certain embodiments, the region C_H2 immunoglobulin is the region of C_H2 wild-type immunoglobulin, such as region C_H2 wild type human immunoglobulin, including the C region_H2 IgA1, IgA2, IgD, IgE, IgG1, IgG2, IgG3, IgG4 and IgM of human wild type. In certain embodiments, the region C_H2 immunoglobulin is the region of C_H2 human IgG1.

In other specific embodiments, the region C_H2 immunoglobulin is a modified region of C_H2 wild-type immunoglobulin. For example, the modified region C_H2, the wild-type immunoglobulin can be a C region_H2 human IgG1, but with one, two, three, four or five mutations at positions 234 to 238, 253, 279, 310, 318, 320, 322 and 331 (EU numbering, Ward et al., 1995 Therap. Immunol. 2: 77-94). Mutations in such positions reduce or eliminate the activity of antibody-dependent cellular cytotoxicity (AZPC), the ability to bind to the Fc receptor and / or the ability to bind complement.

As described herein, humanized SMIP-specific polypeptides specific for CD37 may contain region C_H3 immunoglobulin. In certain embodiments, the polypeptide of region C_H3 immunoglobulin is a polypeptide of region C_H3 wild-type immunoglobulin, including region C_H3 wild type from the immunoglobulin of any isotype (eg IgA, IgD, IgG1, IgG2, IgG3, IgG4, IgE or IgM) from different species (ie, human, mouse, rat or other mammals). In other embodiments, the polypeptide of region C_H3 immunoglobulin is a mutant polypeptide of region C_H3 immunoglobulin. Mutations in the C region_H3, the immunoglobulin can be in one or more positions that participate in the complement fixation reaction, for example at positions H433 or N434.

In certain embodiments, humanized SMIP polypeptides specific for CD37 may contain one or more additional regions. Such additional regions may be the leader sequence at the N-terminus for secretion of the expressed SMIP polypeptide, an additional subregion of Fc (eg, region C_H4 wild type or mutant region C_H4 IgM or IgE), the terminal sequence at its C-terminus for identification or purification (eg, epitope tags for detection or purification, including the tag of 6 histidine residues or the FLAG epitope) or additional amino acid residues that result from the use of specific expression systems . Exemplary leader peptides of the present disclosure include naturally occurring leader sequences or other sequences, such as the sequences set forth in SEQ ID NOs: 223 and 224.

The present disclosure includes SMIP polypeptides specific for CD37, whose identity to the polypeptide as specified in SEQ ID NO: 2 is at least 80 percent (eg 82%, 84%, 85%, 86%, 88% 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99%) where the CD37-specific SMIP polypeptide binds to CD37. In additional embodiments, such polypeptides whose identity with SEQ ID NO: 2 is at least 80% can be further humanized. Exemplary humanized SMIP polypeptides specific for CD37 contain, essentially consist of or consist of any amino acid sequence selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 52, 80, 82, 84, 86, 88 and 222,

As used herein, "sequence identity" refers to the proportion of amino acid residues in one sequence that are identical to the amino acid residues in another reference polypeptide sequence after aligning the sequences and, if necessary, inserting omissions to achieve the maximum percent sequence identity, and not considering any conservative replacements as a proportion of sequence identity. Sequence identity percentages were obtained by NCBI software BLAST 2.0, as determined by Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402, with the parameters set to the default values.

In a preferred embodiment, the present disclosure relates to a humanized SMIP polypeptide specific for CD37, which from the N-terminus to the C-terminus comprises: a humanized heavy chain variable region (V_H), the linker (G₄S)₅ (SEQ ID NO: 229), a humanized light chain variable region (V_L), the modified hinge region IgG1, the region C_H2 human IgG1 and region C_H3 IgG1 human. The humanized heavy chain variable region from its N-terminus to its C-terminus contains: human heavy chain FR1, heavy chain CDR1 as indicated in human heavy chain SEQ ID NO: 63, FR2, CDR2 as indicated in SEQ ID NO: 65 , FR3 of the human heavy chain, CDR3 as indicated in SEQ ID NO: 67, 68 or 69, and FR4 of the human heavy chain. The humanized light chain variable region from its N-terminus to its C-terminus contains: human light chain FR1, light chain CDR1 as indicated in SEQ ID NO: 61 or 62, human light chain FR2, light chain CDR2 as indicated in SEQ ID NO: 64, FR3 of the human light chain and light chain CDR3 as indicated in SEQ ID NO: 66 and FR4 of the human light chain.

In certain of the above preferred embodiments, the human heavy chain FR1, FR2 and FR3 comprise SEQ ID NO: 144, 151 and 158, respectively, and the heavy chain FR4 comprises SEQ ID NO: 161 or 162. In further preferred embodiments, FR1, FR2, FR3 and FR4 of the human light chain contain SEQ ID NOs: 171, 182, 195 and 206, respectively. Alternatively, these sequences contain both heavy and light chains.

The SMIP protein CAS-024 unexpectedly (1) was expressed approximately to 25 times higher than other humanized versions of CAS-006 (such as CAS-002, CAS-003; see examples 2 and 5), (2) could communicate with CD37, as well as CAS-006, although other humanized versions did not have this capacity (see examples 4 and 5), and (3) was produced as a homogeneous group of molecules compared to the heterogeneous character of other humanized versions (see example 3). In a preferred embodiment, a protein specifically binding to CD37 that contains or consists of CAS-024 (SEQ ID NO: 253) is provided herein. In particular, this humanized molecule specifically binding to CD37 has substantially the same binding affinity to CD37 as the original chimeric molecule (CAS-006, SMIP protein, containing immunoglobulin variable regions from a monoclonal antibody of mouse G28-1 to human CD37), unlike other humanized molecules, is expressed at high levels compared to other humanized molecules and / or exhibits a high degree of homogeneity after purification, for example, by means of exclusive chromatography (EC) , in contrast to other humanized molecules. In addition, it was shown that this molecule of CAS-024, specifically binding to CD37, effectively inhibits tumor growth and causes a prolonged remission of the tumor. for example, by means of exclusion chromatography (EC), unlike other humanized molecules. In addition, it was shown that this molecule of CAS-024, specifically binding to CD37, effectively inhibits tumor growth and causes a prolonged remission of the tumor. for example, by means of exclusion chromatography (EC), unlike other humanized molecules. In addition, it was shown that this molecule of CAS-024, specifically binding to CD37, effectively inhibits tumor growth and causes a prolonged remission of the tumor.

The present disclosure also relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding humanized molecules specifically binding to CD37 and components thereof including human or humanized FR, CDR, humanized light chain variable regions, humanized heavy chain variable regions, humanized scFv and humanized SMIP polypeptides. Illustrative isolated nucleic acid molecules encoding humanized SMIP-specific polypeptides specific for CD37 include nucleic acid molecules comprising SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 , 31, 33, 35, 37, 39, 41, 43, 45, 47, 51, 79, 81, 83, 85, 87 and 221. In one embodiment, the present disclosure relates to vectors comprising these nucleic acid molecules,

The present disclosure also relates to a method for producing polypeptides described herein that includes culturing the host cells under conditions suitable for the expression of polypeptides and optionally isolating the polypeptides from the culture.

<u>Compounds suitable for combination treatment</u>

The present disclosure also relates to combined treatment using any molecule specifically binding to CD37, known in the art or described herein, and bifunctional chemotherapeutic agents (e.g., bendamustine).

Molecules that specifically bind to CD37 that can be used for combination treatment may exist in any form that contains a CD37 specific binding domain, including an anti-CD37 antibody, a CD37 antibody fragment to CD37, a CD37 single-chain Fv (scFv) specific, CD37-specific SMIP, CD37-specific PIMS, CD37-specific SCORPION, and other bi- or polyspecific binding proteins that contain at least one protein specifically binding to CD37.

In certain embodiments, molecules specifically binding to CD37, suitable for combination treatment with a bifunctional chemotherapeutic agent, are CD37-specific antibodies. Such antibodies include antibodies used to characterize the CD37 antigen on Thrid HLDA Workshop, i.e., HD28, G28-1, HH1, BI14, WR17 and F93G6 (see Ling and MacLennan, pp. 302-335 in "Leucocyte Typing III White Cell Differentiation Antigens ", Oxford University Press (1987)). Other CD37 specific antibodies suitable for combination treatment include RFB-7, Y29 / 55, MB-1, M-B371, M-B372 and IPO-24 (see Moldenhaurer, J. Biol., Regul. Homeost. Agents , 14: 281-283 (2000), which indicates that all these antibodies recognize only one CD37 epitope, and Schwartz-Albiez et al., 14: 905-914 (1988), where it is indicated that this epitope is in the carbohydrate residue of CD37). Another specific CD37 antibody suitable for combination treatment is S-B3 (Biosys).

In certain embodiments, molecules specifically binding to CD37 suitable for combined treatment with a bifunctional chemotherapeutic agent are SMIP polypeptides specific for CD37. An exemplary SMIP polypeptide comprises SEQ ID NO: 2. Additional exemplary SMIP polypeptides include the polypeptides described in WO2005017148, such as (1) scFv G28-1 (SSS-S) H WC_H2 WC_H3 containing scFv G28-1, a modified hinge region of human IgG1 in which all three cysteine ​​residues and proline located closer to the C-terminus than the third cysteine ​​in the hinge region of human IgG1 are replaced by serine residues and the C_H2 and C_H3 IgG1 wild-type human; (2) scFv G28-1 IgAH WC_H2 WC_H3 containing scFv G28-1, part of the hinge region of human IgA and domains C_H2 and C_H3 IgG1 human; (3) scFv G28-1 VHL11S (SSS-S) H WC_H2 C_H3 containing scFv G28-1, a modified hinge region of human IgG1 in which all three cysteine ​​residues and proline located closer to the C-terminus than the third cysteine ​​in the hinge region are replaced with serine residues and the C_H2 and C_H3 human IgG1, where leucine at position 11 of the heavy chain variable region is replaced by serine; (4) scFv G28-1 VHL11S (CSS-S) H WC_H2 C_H3 containing scFv G28-1, a modified hinge region of human IgG1 in which cysteine ​​residues in the second and third position and proline located closer to the C-terminus than the third cysteine ​​are replaced by serine residues and the C_H2 and C_H3 human IgG1, where leucine at position 11 of the heavy chain variable region is replaced by serine; (5) scFv G28-1 VHL11S (CSC-S) H WC_H2 C_H3 containing scFv G28-1, a modified hinge region of human IgG1 in which the cysteine ​​residue at the second position and proline located closer to the C-terminus than the cysteine ​​at the third position are replaced with serine residues and the C_H2 and C_H3 human IgG1, where leucine at position 11 of the heavy chain variable region is replaced with serine; (6) scFv G28-1 VH11S (SSC-P) H WC_H2 WC_H3 containing scFv G28-1, a modified hinge region of human IgG1 in which the first and second cysteine ​​residues in the hinge region are replaced with serine residues and the C_H2 and C_H3 human IgG1, where leucine at position 11 of the heavy chain variable region is replaced with serine; (7) scFv G28-1 VH11S (SCS-S) H WC_H2 WC_H3 containing scFv G28-1, a modified hinge region of human IgG1 in which the first and third cysteine ​​residues and proline located closer to the C-terminus than the third cysteine ​​in the hinge regions are replaced with serine residues and the C_H2 and C_H3 human IgG1, where leucine at position 11 of the heavy chain variable region is replaced with serine; (8) scFv G28-1 VHL11S (CCS-P) H WC_H2 WC_H3 containing scFv G28-1, a modified hinge region of human IgG1 in which the third cysteine ​​residue in the hinge region is replaced by serine, and the C_H2 and C_H3 human IgG1, where leucine at position 11 of the heavy chain variable region is replaced by serine (9) scFv G28-1 VHL11S (SCC-P) H WC_H2 WC_H3 containing scFv G28-1, a modified human IgG1 hinge region in which the first cysteine ​​is replaced by serine, and the C_H2 and C_H3 people, where leucine at position 11 of the heavy chain variable region is replaced with serine; (10) scFv G28-1 VHL11S mIgE C_H2 C_H3 C_H4, containing scFv G28-1 and region C_H2, C_H3 and C_H4 IgE mouse, where leucine at position 11 of the heavy chain variable region is replaced with serine; (11) scFv G28-1 VHL11S mIgA WIgAC_H2 T4C_H3, containing scFv G28-1, the hinge region of mouse IgA and C_H2 wild-type IgA and a truncated domain C_H3 IgA with no 4 C-terminal amino acids GTCY (SEQ ID NO: 265); (12) scFv G28-1 VHL11S hIgE C_H2 C_H3 C_H4, containing scFv G28-1 and region C_H2, C_H3 and C_H4 of human IgE, where leucine at position 11 of the heavy chain variable region is replaced by serine; and (13) scFv G28-1 VHL11S hIgAH WIgAC_H2 TC_H3, containing scFv G28-1, part of the hinge region of human IgA, C_H2 wild-type IgA and a truncated domain C_H3 IgA with the absence of 4 C-terminal amino acids GTCY (SEQ ID NO: 265), where leucine at position 11 of the heavy chain variable region is replaced with serine.

In certain embodiments, molecules specifically binding to CD37 suitable for combination treatment with a bifunctional chemotherapeutic agent are humanized molecules specifically binding to CD37 as described herein including humanized anti-CD37 antibodies, Fab fragments of humanized anti-CD37 antibodies, humanized a CD37-specific PIMS protein, a humanized CD37-specific SCORPION protein and other bi- or polyspecifically binding proteins that contain, shey least one humanized protein that specifically binds to CD37, in particular, a humanized single chain Fv (scFv), specific for CD37, polypeptides and humanized SMIP, specific for CD37.

Certain molecules specifically binding to CD37, as discussed herein, have affinities for CD37 of about 0.5 to about 10 nM. Another indication of certain CD37 binding molecules contemplated herein is that they have a half-life in the bloodstream of about 5 to about 30 days.

In certain embodiments, molecules specifically binding to CD37 have the ability to compete for binding specificity to CD37 with mAT G28-1.

Bendamustine (4- [5- [bis (2-chloroethyl) amino] -1-methylbenzimidazol-2-yl] butanoic acid) is a nitrogen mustard with alkylating and antimetabolic activity. Bendamustine contains an alkylating group and a benzimidazole ring. The alkylating group allows the metabolites of bendamustine to alkylate and crosslink macromolecules, which leads to inhibition of the synthesis of DNA, RNA and protein, and subsequently to apoptosis. The benzimidazole ring can allow bendamustine to act as an analogue of purines. Bendamustine hydrochloride has trade names THREANDA® and RIBOMUSTIN®.

Although bendamustine or its salts are preferred therapeutic agents that can be used in combination with molecules specifically binding to CD37, also in combination with molecules specifically binding to CD37, as described herein, other therapeutic agents that contain one or more alkylating groups and are able to function as an analogue of purines.

As used herein, the term "alkylating group" refers to a group that allows a compound containing this group to attach an alkyl group to the DNA. A compound that contains an alkylating group can be referred to as an "alkylating agent". In certain embodiments, the alkylating agents are nitrogen mustards.

The term "purine analog" refers to an antimetabolite that mimics the structure of metabolic purines (eg, adenine and guanine) and contains one, two, three or four substituents in the purine ring that are different from metabolic purines. Exemplary purine analogs include azathioprine, mercaptopurine, thioguanine, fludarabine, pentostatin and cladribine.

The therapeutic agent "is capable of functioning as a purine analog" if it has at least one function of a purine analog. Exemplary functions of purine analogs include impairment or inhibition of purine nucleotide synthesis, purine nucleotide metabolism, nucleic acid synthesis, nucleic acid processing or nucleic acid function, for example, inhibition of ribonucleotide reductase, DNA polymerase, adenosine deaminase, and incorporation into DNA or RNA.

<u>Compositions and Methods</u>

In one aspect, the present disclosure relates to a method for reducing the number of B cells or treating a disease associated with an abnormal B cell activity, comprising administering to a subject in need thereof (i.e., an individual suffering or suspected of having a disease associated with abnormal activity B cells) of an effective amount of a humanized molecule specifically binding to CD37 provided herein (eg, CAS-024).

In another aspect, the present disclosure relates to a method for reducing the number of B cells or treating a disease associated with an abnormal B cell activity, comprising administering to an individual in need thereof an effective amount of a molecule specifically binding to CD37 (eg, CAS-024) and a bifunctional chemotherapeutic agents (e.g., bendamustine). As described above, molecules specifically binding to CD37 suitable for combination treatment with a bifunctional chemotherapeutic agent are not limited to humanized molecules specifically binding to CD37, but also include other molecules specifically binding to CD37 that are not humanized.

In one embodiment, the composition comprises a therapeutic agent binding to CD37 and a bifunctional chemotherapeutic agent that act synergistically and reduce the number of B cells or treat a disease associated with abnormal B cell activity. Two or more compounds that act synergistically interact so that the combined effect of the compounds exceeds the sum of the individual effects of each compound when administered separately (see, for example, Berenbaum, Pharmacol. Rev. 41:93, 1989). For example, the interaction between an immunopharmaceutical agent based on a small modular protein directed to CD37 and another agent or compound can be analyzed using a variety of theoretical and empirical models (see, for example, Ouzounov et al., Antivir Res 55: 425 , 2002)._a, d_b) is indicated by a point on the graph whose axes are the dose axes of the individual agents (see, for example, Ouzounov et al., supra; also see Tallarida, J. Pharmacol. Exp. Therap. 298: 865, 2001).

Another method known in the art for the analysis of drug interactions (antagonism, additivity, synergism) includes the definition of combinatorial indices (CI), corresponding to the principle of the mean effect, providing estimates of IC values₅₀ compounds administered alone and in combination (see, for example, Chou. In Synergism and Antagonism Chemotherapy, Eds., Chou and Rideout, Academic Press, San Diego Calif., pages 61-102, 1991; CalcuSyn ™ software). The value of CI less than one means a synergistic action, equal to one means additive action, and exceeding one means antagonism.

Another exemplary method is a method of independent action (Pritchard and Shipman, Antiviral Res 14: 181, 1990, Pritchard and Shipman, Antiviral Therapy 1: 9, 1996; MACSYNERGY ™ II software, University of Michigan, Ann Arbor, Mich.) . The MACSYNERGY ™ II software allows a 3D (3D) analysis of the interactions of compounds by comparing the calculated additive surface with those observed to produce differential plots that reveal areas (in the form of a volume region) statistically greater than expected (synergistic) or less than expected (antagonistic) interactions of the compounds. For example, a composition comprising a molecule specifically binding to CD37 and a bifunctional chemotherapeutic agent that modifies viral replication,

In additional embodiments, a molecule specifically binding to CD37 and a bifunctional chemotherapeutic agent may be administered to provide synergistic effects in the treatment of B-cell malignancies or B-cell malignancies. B-cell malignancies or B-cell malignancies include B-cell lymphomas [such as various forms of Hodgkin's disease, non-Hodgkin's lymphoma (NHL) or central nervous system lymphoma], leukemias [such as acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia CLL), hairy cell leukemia and chronic myoblastic leukemia] and myeloma (such as multiple myeloma). Additional types of B-cell malignancies include small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmocytic lymphoma, lymphoma of the marginal zone of the spleen, plasmacytic myeloma, solitary plasmacytoid bone, extraossal plasmacytoma, extranodal B-cell lymphoma of the marginal zone of the lymphoid tissue of the mucous membranes, nodal B-cell lymphoma of the marginal zone, follicular lymphoma, mantle cell lymphoma, diffuse B-cell large-cell lymphoma , mediastinal (thymic) B-cell large-cell lymphoma, intravascular large-cell B-cell lymphoma, primary exudative lymphoma, lymphoma / leukemia Ber kitta, B-cell proliferation of an indeterminate oncogenic potential, lymphomatoid granulomatosis and a post-transplant lymphoproliferative disorder. extranodal B-cell lymphoma of the marginal zone of lymphoid tissue of the mucous membranes, nodal B-cell lymphoma of the marginal zone, follicular lymphoma, mantle cell lymphoma, diffuse B-cell large cell lymphoma, mediastinal (thymic) B cell large cell lymphoma, intravascular B-cell large-celled lymphoma, primary exudative lymphoma, Burkitt's lymphoma / leukemia, B-cell proliferation of an indeterminate oncogenic potential, lymphomatoid granulomatosis and post-transplantation lymphoproliferative disorder. extranodal B-cell lymphoma of the marginal zone of the lymphoid tissue of the mucous membranes, nodal B-cell lymphoma of the marginal zone, follicular lymphoma, lymphoma from the cells of the mantle zone, diffuse B-cell large cell lymphoma, mediastinal (thymic) B cell large cell lymphoma, intravascular B-cell large-celled lymphoma, primary exudative lymphoma, Burkitt's lymphoma / leukemia, B-cell proliferation of an indeterminate oncogenic potential, lymphomatoid granulomatosis and post-transplantation lymphoproliferative disorder.

Burkitt's lymphoma (or "B-cell malignant neoplasm of Burkitt", or "Burkitt's tumor," or "Burkitt's malignant lymphoma") is a malignant tumor of the lymphatic system (in particular, B-lymphocytes). It can be divided into three main clinical options: epidemic, sporadic and immunodeficient variants.

Neberquitt's B-cell malignancies include, but are not limited to, B-cell chronic lymphocytic leukemia (CLL) / small cell lymphoma, B-cell prolymphocytic leukemia, acute lymphoblastic leukemia (ALL), lymphoplasmocytic lymphoma (including, but not limited to, Waldenstrom's macroglobulinemia), lymphomas (including, but not limited to, B-cell lymphoma of the marginal zone of the spleen, nodal lymphoma of the marginal zone and extranodal B-cell lymphomas mu marginal zone mucosal lymphoid tissue), hairy cell leukemia, plazmotsitarnuyu myeloma / plasmacytoma, follicular lymphoma, mantle cell lymphoma zone (LKMZ), B-cell diffuse large cell lymphoma, transforming B-cell large cell lymphoma,

Disorders characterized by the production of autoantibodies are often considered autoimmune diseases. Autoimmune diseases include, but are not limited to, arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, myositis with included corpuscles, inflammatory myositis, toxic epidermal necrolysis, systemic sclerosis and sclerosis, syndrome Tibierja-Weissenbach, reactions associated with inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, respiratory distress syndrome, oslyh (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions that involve infiltration of T-cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, impaired leukocyte adhesion, systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus, myelitis with lupus, lupus encephalitis, juvenile diabetes, multiple sclerosis, allergic encephalomyelitis, optic neuritis, rheumatic attack, Sydenham's chorea, immune reactions associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis, including Wegener's granulomatosis and Cherdja-Strauss disease, ag (including vasculitis / angiitis in hypersensitivity, ANCA and rheumatoid vasculitis), aplastic anemia, Diamond-Blackfen anemia, immune hemolytic anemia, including autoimmune hemolytic anemia (AIGA), pernicious anemia, true erythrocyte aplasia (IEA) insufficiency of the VIII factor, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, inflammatory diseases of the central nervous system (CNS), polyorganic insufficiency syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, antiglomerular basement membrane disease , the syndrome of antiphospholipid antibodies, allergic neuritis, Behcet's disease, Castleman syndrome, Goodpasture's syndrome, myasthenic Lambert-Eaton syndrome, illness eyno, Sjorgen's syndrome, Stevens-Johnson syndrome, organ transplant rejection solidngo reaction "graft versus host" disease (GVHD), bullous pemphigoid, pemphigus vulgaris, autoimmune poliendokrinopatii, seronegative spondyloarthropathies, Reiter's syndrome, chained man syndrome, giant cell arteritis, immune nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), Shenlen-Henoch disease, autoimmune thrombocytopenia, autoimmune diseases of the testicles or ovaries, including autoimmune orchitis and oophoritis , primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathic syndromes), type I diabetes, also referred to as insulin-dependent diabetes mellitus (IDDM) and Shihan syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), obliterans bronchitis (non-transplant) versus NSIP, Guillain-Barre syndrome, vasculitis of large vessels (including rheumatic polymyalgia and giant cell arteritis (Takayasu)), vascular vasculitis (including Kawasaki disease and nodular polyarteritis), nodular polyarteritis (UPA), ankylosing spondylitis, disease Berger (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, gluten disease, cryoglobulinemia, cryoglobulinemia associated with hepatitis, amyotrophic lateral sclerosis (ALS), disease to ronarnyh arteries, familial Mediterranean fever, microscopic polyangiitis, Cogan's syndrome, Wiskott-Aldrich syndrome and thromboangiitis obliterans, autoimmune thyroid disease (such as Graves' disease and Hashimoto's thyroiditis), Sjogren's syndrome, and idiopathic inflammatory myopathy (INM) including dermatomyositis (DM) and polymyositis (PM). The above autoimmune diseases can also be treated with humanized molecules specifically binding to CD37, or a combination of molecules specifically binding to CD37 and a bifunctional chemotherapeutic agent.

In one aspect of the present disclosure, a humanized molecule specifically binding to CD37 or a combination of a molecule specifically binding to CD37 with a bifunctional chemotherapeutic agent is administered in a pharmaceutical composition. To introduce a humanized molecule specifically binding to CD37 or a combination of a molecule specifically binding to CD37 and a bifunctional chemotherapeutic agent to a human or test animal, it is preferable to formulate a binding molecule or combination into a composition containing one or more pharmaceutically acceptable carriers. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecules and compositions that do not cause allergic or other adverse reactions when administered using routes, well known in the art, as described below. "Pharmaceutically acceptable carriers" include any or all of the clinics used, solvents, dispersants, coatings, antimicrobial and antifungal agents, isotonic and suction retarding agents, and the like. In addition, the compounds can form solvates with water or conventional organic solvents. Such solvates are also provided.

Pharmaceutical compositions of the present disclosure comprising a humanized molecule specifically binding to CD37 or a combination of a molecule specifically binding to CD37 and a bifunctional chemotherapeutic agent and used in the method of the present disclosure may contain pharmaceutically acceptable carriers or additives depending on the route of administration. Examples of such carriers or additives include water, a pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, casein, gelatin, agar, diglycerin, glycerol, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HAC), mannitol, sorbitol, lactose, a pharmaceutically acceptable surfactant, and the like. The additives used are selected, but are not limited to, the above substances or combinations thereof, as necessary, depending on the dosage form of the present disclosure.

The composition of the pharmaceutical composition varies according to the method of administration chosen (eg, solution, emulsion). A suitable composition containing an antibody for administration may be prepared in a physiologically acceptable excipient or carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or hydroalcoholic solutions, emulsions or suspensions, including saline and buffer media. Parenteral carriers may include a solution of sodium chloride, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate or non-volatile oils. Intravenous carriers can include various adjuvants, preservatives or liquids, nutrients or compensators for electrolytes.

A variety of aqueous vehicles, for example water, buffered water, 0.4% sodium chloride solution, 0.3% glycine or aqueous suspensions, may contain the active compound with the addition of excipients suitable for the production of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and gum arabic; dispersing agents or humectants may be a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with aliphatic long chain alcohols, for example heptadecaethyleneoxycetanol or condensation products of ethylene oxide with partial esters, such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate.

A molecule specifically binding to CD37, a combination of a molecule specifically binding to CD37, with a bifunctional chemotherapeutic agent, or a composition containing a binding molecule or combination, can be lyophilized for storage and reconstitution in a suitable carrier before use. It was shown that this method is effective against standard immunoglobulins. Any suitable method of lyophilization and reduction may be used. Those skilled in the art will understand that lyophilization and reduction can lead to loss of activity to varying degrees and that, possibly, the dosage used should be adjusted for compensation.

Dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide the active compound in admixture with a dispersing agent or humectant, a suspending agent and one or more preservatives. Examples of suitable dispersing agents or humectants and suspending agents are those already mentioned above.

The concentration of molecules specifically binding to CD37 or the bifunctional chemotherapeutic agent in these formulations can vary over a very wide range, for example, from less than about 0.5%, typically from or at least about 1% to 15 or 20% by weight, and is generally selected based on the volume of fluid, viscosity, etc., according to the particular mode of administration chosen. Thus, a typical pharmaceutical composition for parenteral administration can be formulated to contain 1 ml of sterile buffered water and 50 mg of antibody. A typical composition for intravenous administration may be formulated so that it contains up to 250 ml of sterile Ringer's solution and 150 mg of antibody. Particular methods for preparing compositions for parenteral administration are known or apparent to those skilled in the art and are described in greater detail, for example, in Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980). The effective dose of molecules specifically binding to CD37 (including humanized molecules specifically binding to CD37) is in the range of 0.01 mg to 1000 mg per kg of body weight per administration.

The pharmaceutical compositions may be in the form of a sterile aqueous solution for injection, oily suspension, dispersions or sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The slurry can be formulated in accordance with the prior art using suitable dispersing agents or humectants and suspending agents as described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in non-toxic parenterally acceptable diluents and solvents, for example as a solution in 1,3-butanediol. The carrier may be a solvent or dispersing medium containing, for example, water, ethanol, a polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol, etc.), their suitable mixtures, vegetable oils, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are traditionally used as the solvent or suspending medium. For this purpose, any light non-volatile oil can be used, including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectable agents.

In all cases, the form should be sterile and must be liquid to such an extent that it can easily be injected through a syringe. Proper fluidity can be maintained, for example, by using a coating, such as lecithin, by maintaining the desired particle size in the case of dispersion and by using surfactants. It must be stable under production and storage conditions and should be protected from the polluting effects of microorganisms, such as bacteria and fungi. The prevention of the activity of microorganisms can be effected by various antimicrobial or antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is desirable to include isotonic agents, for example, sugar or sodium chloride.

Compositions suitable for administration may be formulated using capture or suction enhancers to increase their effectiveness. Such enhancers include, for example, salicylate, glycocholate / linoleate, glycocholate, aprotinin, bacitracin, SDS, caprate and the like. See, for example, Fix (J. Pharm Sci., 85: 1282-1285, 1996) and Oliyai and Stella (Ann. Rev. Pharmacol. Toxicol., 32: 521-544, 1993).

In addition, the hydrophilic and hydrophobic properties of the compositions proposed for use herein are well balanced, thereby increasing their applicability <i>in vitro</i> and especially <i>in vivo</i>, while other compositions that do not have such a balance are substantially less applicable. In particular, the compositions provided for use herein have an appropriate degree of solubility in aqueous media that provides absorption and bioavailability in the body, while also having a certain degree of lipid solubility that allows these compounds to pass through the cell membrane to the intended site of action . Thus, the present antibody compositions are most effective when they can be delivered to the site of activity of the target antigen.

In one aspect, the methods of the present disclosure include the step of administering the composition to a molecule specifically binding to CD37. In certain embodiments, combinations of the compounds can be administered simultaneously, together in one pharmaceutically acceptable carrier or separately (but simultaneously). In other embodiments, the immunotherapeutic agent for CD37 (i.e., a molecule specifically binding to CD37) and the bifunctional chemotherapeutic agent may be administered sequentially in any order and in any combination.

The binding molecule, bifunctional chemotherapeutic agent or combined compositions can be administered orally, topically, transdermally, parenterally, by inhalable spray, vaginally, rectally or by intracranial injection, or by any combination of these routes. In one embodiment, the molecule specifically binding to CD37 and the bifunctional chemotherapeutic agent are administered parenterally, simultaneously or sequentially. As used herein, the term parenteral includes subcutaneous injections, intravenous, intramuscular, intracisternal injection or infusion techniques. Also, administration is envisaged by intravenous, intradermal, intramuscular, intrasternal, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and / or surgical implantation at a particular site. Generally, the compositions do not substantially contain pyrogens, as well as other impurities that may be dangerous to the recipient. Preferred is injection, in particular, intravenous.

In one embodiment, the administration is performed in a portion of a malignant tumor or diseased tissue that needs to be treated by direct administration to this site, or by a sustained delivery or sustained release mechanism that allows the composition to be delivered to the body. For example, the compositions described herein implanted adjacent to the malignant tumor site may include biodegradable microspheres or capsules or biodegradable polymer configurations with a sustained delivery of the composition (eg, soluble polypeptide, antibody, or low molecular weight compound).

Therapeutic compositions can also be delivered to a patient in several areas. You can conduct several injections simultaneously or after a certain period of time. In certain cases, it is useful to provide a continuous flow of the therapeutic composition. Periodically, you can enter an additional drug, for example, hourly, daily, weekly or monthly.

The binding molecule, bifunctional chemotherapeutic agent, or combination compositions of the present disclosure may contain one or more binding molecules, bifunctional chemotherapeutic agents, or any combination thereof. Also, the present disclosure provides for the administration of a binding molecule, a bifunctional chemotherapeutic agent, or combination compositions in combination with an additional therapeutic agent. The additional therapeutic agents provided for in this description are listed in the sections below.

The additional therapeutic agent may be a molecule bound to B-cells. Other molecules bound to B-cells contemplated herein include binding molecules that bind to molecules on the surface of B cells that are not CD37. B cell-related molecules include CD19 (CD19 B-cell antigen, also referred to as B4 B-lymphocyte surface antigen or Leu-12), CD20, CD21, CD22 (B cell receptor CD22, also referred to as Leu-14 , the cell adhesion molecule of B-lymphocytes or BL-CAM), CD23, CD40 (surface antigen of B cells CD40, also referred to as representative of the 5 tumor necrosis factor receptor superfamily, CD40L or Bp50 receptor), CD80 (T80 antigen activation antigen CD80, also referred to as activation antigen B7-1, B7, B7-1 or BB1), CD86 (antigen activation of T-lymphocytes CD86, also referred to as activation antigen B7-2, B70, FUN-1 or BU63), CD137 (also referred to as representative of the 9 tumor necrosis factor receptor superfamily), CD152 (also referred to as the cytotoxic T- lymphocytes 4 or CTLA-4), L6 (tumor-associated L6 antigen, also referred to as representative of the 1 superfamily of transmembrane proteins 4, surface marker of membrane component 1, or M3S1), CD30 (antigen of CD30 lymphocyte activation, also referred to as representative of the 8 receptor superfamily factor tumor necrosis, CD30L or Ki-1 receptor), CD50 (also referred to as intercellular adhesion factor 3 (ICAM3) or ICAM-R), CD54 (also referred to as intercellular adhesion factor 1 (ICAM1) or the main retrovirus group receptor), B7 -H1 (an immunoinhibitory receptor ligand, expressed by activated T cells, B cells and myeloid cells, also referred to as PD-L1; see Dong, et al., "B7-H1, and the third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion", Nat. Med., 5: 1365-1369 (1999), CD134 (also referred to as representative of the 4 tumor necrosis factor receptor superfamily, OX40, OX40L receptor, ACT35 antigen or transcriptional activated TAX glycoprotein 1 receptor), 41BB (ligand receptor 4-1BB, T cell antigen 4-1 BB or T-cell antigen ILA), CD153 (also referred to as the representative of the 8 tumor necrosis factor-ligand superfamily, ligand CD30 or CD30-L), CD154 (also referred to as representative of the 5 superfamily of tumor necrosis factor TNF activation protein, TRAP, or T-cell antigen Gp39),

Examples of chemotherapeutic agents provided as additional therapeutic agents include alkylating agents such as nitrogen mustards (eg, mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil); nitrosoureas (e.g., carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU)); ethylenimines and methylmelamines (for example, triethylene melamine (TEM), triethylenethiophosphoramide (thiotepa), and hexamethylmelamine (HMM, altretamine)); alkylsulfonates (e.g., busulfan); and triazines (e.g., dacarbazine (DTIC)); antimetabolites, such as folic acid analogues (eg, methotrexate, trimetrexate and pemetrexed (multitarget antifolate)); pyrimidine analogs (such as 5-fluorouracil (5-FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, and 2,2'-difluorodeoxycytidine); and purine analogues (for example, 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonadenine (EHNA), fludarabine phosphate, 2-chlorodeoxyadenosine (cladribine, 2-CdA)); Type I topoisomerase inhibitors, such as camptothecin (CPT), topotecan and irinotecan; natural products, such as epipodophyllotoxins (eg, etoposide and teniposide); and vinca alkaloids (eg, vinblastine, vincristine and vinorelbine); antineoplastic antibiotics such as actinomycin D, doxorubicin, and bleomycin; radiosensitizing agents such as 5-bromodeoxyuridine, 5-iododeoxyuridine and bromodeoxycytidine; coordination complexes of platinum, such as cisplatin, carboplatin and oxaliplatin; substituted urea derivatives, such as hydroxyurea; and methylhydrazine derivatives such as N-methylhydrazine (MIH) and procarbazine.

The additional therapeutic agents provided herein for the treatment of autoimmune diseases are referred to as immunosuppressants that suppress or mask the immune system of the individual being treated. Immunosuppressants include, for example, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, glucocorticoids, antirheumatic disease modifying drugs (DMARD) for the treatment of arthritis or biological response modifiers. The compositions in the DMARD Description are also suitable for the treatment of many other autoimmune diseases other than PA.

Exemplary NSAIDs are selected from the group consisting of ibuprofen, naproxen, naproxen sodium, COX-2 inhibitors such as viox and tselebrex and sialylates. Exemplary analgesics are selected from the group consisting of acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Illustrative glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone or prednisone. Illustrative biological response modifiers include molecules directed against surface cell markers (eg, CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (eg, etanercept (enbrel, Enbrel), adalimumab (Humira, Humira), and infliximab (Remicade, Remicade)), chemokine inhibitors and inhibitors of adhesion molecules. The biological response modifiers include monoclonal antibodies, as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral form (auranofin) and intramuscular form) and minocycline.

It is believed that the composition of the binding molecule and the additional therapeutic agent can be administered simultaneously in the same formulation. Alternatively, these agents are administered in separate formulations, but simultaneously, while "simultaneously" refers to agents administered, for example, within minutes, hours or days relative to each other.

In another aspect, an additional therapeutic agent is administered prior to administration of a binding molecule, a bifunctional chemotherapeutic agent, or a combination composition. "Before administration" refers to the administration of an additional therapeutic agent in the range of several minutes, hours or one week prior to treatment using a binding molecule, a bifunctional chemotherapeutic agent, or a combination composition. It is further provided that the additional therapeutic agent is administered after administration of the binding molecule composition. "Sequential administration" is intended to describe administration over an interval of more than a few minutes, hours or weeks after treatment or administration of a binding molecule, a bifunctional chemotherapeutic agent, or a combination composition.

It is further contemplated that, upon administration of a binding molecule in combination with an additional therapeutic agent, wherein the additional therapeutic agent is a cytokine or a growth factor or a chemotherapeutic agent, administration may also include the use of a radiotherapeutic agent or radiation therapy. Radiation therapy performed in conjunction with the antibody composition is carried out in accordance with the instructions of the attending physician and in the doses usually prescribed to patients undergoing treatment for a malignant tumor.

These compositions can be administered in a single dose or in multiple doses. In standard dose-effect studies, first in animal models and then in clinical testing, optimal doses were found for specific pathological conditions and patient groups.

The introduction of a binding molecule, a bifunctional chemotherapeutic agent, or a combination composition reduces the B cell population after treatment with a single dose of at least 20%. In one embodiment, the B cell population is decreased by at least about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or about 100 %. Reduction in the number of B cells is defined as a decrease in the absolute number of B cells below the lower limit of the normal range. B cell recovery is defined as the return of the absolute number of B cells, for example, up to 70%, 80%, 90% of the original value of the individual or normal range. In addition, the introduction of a binding molecule,

In certain embodiments, patients suffering from a disease associated with an abnormal B cell activity treated as described herein may exhibit a general favorable response to treatment based on clinical criteria well known and widely used in the art as described below.

For example, in patients suffering from rheumatoid arthritis, administration can improve a patient's condition through a clinically significant amount [eg, "American College of Rheumatology Preliminary Detection of Improvement" (ACR20)] and / or 20% improvement in painful and swollen joints and 20% improvement in 3/5 of the remaining ACR criteria (Felson et al., Arthritis Rheum. 1995, 38: 727-35). Biological criteria for improvement in patients with RA after administration of molecules specifically binding to CD37 and specifically binding to CD20 include measuring changes in cytokine levels measured by protein or RNA levels. Cytokines of interest include, but are not limited to, TNF-α, IL-1, interferons, Blys and APRIL. Changes in cytokine levels may occur due to a decrease in the number of B cells or a decrease in activated T cells. In patients with RA, markers related to bone metabolism (bone resorption or erosion) are measured before and after the administration of CD20-specifically binding molecules. Suitable markers include, but are not limited to, alkaline phosphatase, osteocalcin, fragments of collagen degraded, hydroxyproline, tartrate-resistant acid phosphatase, and RANK ligand (RANKL). Other criteria related to the improvement of RA include measurement of C-reactive protein (CRP) levels, erythrocyte sedimentation rate (ESR), rheumatoid factor, anti-CCP (cyclic citrulinated peptide) and systemic B cell and lymphocyte count by flow cytometry .

In an appropriate aspect, it is possible to measure the effect of combined administration on other diseases in accordance with standards known in the art. For example, it is believed that patients with Crohn's disease treated with the invention achieved an improvement in the activity index for Crohn's disease (CDAI) in the range of about 50 to about 70 units, where remission is 150 units (Simonis et al, Scand. J Gastroent. 1998, 33: 283-8). A score of 150 or 200 is considered normal, while an estimate of 450 is considered an estimate corresponding to a serious illness. In addition, it is desirable that in individuals affected by an inflammatory bowel disease, the administration of molecules specifically binding to CD37 and specifically binding to CD20,<i>Saccharomyces cervisiae</i> (ASCA).

In addition, it is believed that in patients with juvenile myositis and adult myositis treated as described herein, it is possible to achieve an improvement in estimates from the baseline, for example 3 of 6 baseline assessments improved by approximately 20% with no more than 2 baseline estimates of approximately 25% (see Rider et al., Arthritis Rheum 2004, 50: 2281-90).

In addition, it is believed that in patients with SLE treated as described herein, an improvement in the evaluation of the SLAM or SLEDA activity index by at least 1 unit can be achieved (Gladman et al , J Rheumatol 1994, 21: 1468-71) (Tan et al., Arthritis Rheum. 1982, 25: 1271-7). SLAM score below 5 or SLEDAI score below 2 is considered appropriate for a clinically active disease. Response to treatment can be defined as an improvement or stabilization according to 2 criteria of disease activity (SLEDAI activity index and activity criterion for systemic lupus) and 2 quality of life criteria (general examination of the patient and Krupp fatigue severity scale) (Petri et al. , Arthritis Rheum 2004, 50: 2858-68.) In addition, it is believed, that the introduction of a binding molecule to patients with SLE leads to a decrease in the number of antibodies to double-stranded DNA. Alternatively, the improvement can be measured using the group of lupus assessment criteria of the British Isles (BILAG).

In addition, it is believed that in patients with multiple sclerosis treated as described herein, an improvement in the clinical evaluation of the extended incidence of Kurtzke (EDSS) (Kurtzke, F., Neurology 1983, 33: 1444-52) at least 0.5, or delayed deterioration of the clinical state by at least 1.0 on the Kurtzke scale (Rudick et al., Neurology 1997, 49: 358-63).

In addition, it is expected that in patients suffering from IPM who have been treated as described herein, it is possible to achieve a reduction in at least one of the five criteria established in the evaluation of the criteria for idiopathic inflammatory myopathy (IIMC) (Miller, F., supra) . In addition, it is believed that administration to patients with an ICM can lead to a reduction in the factors associated with ICM that are selected from the group consisting of creatine kinase (CC), lactate dehydrogenase, aldolase, C-reactive protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and antinuclear autoantibodies (ANA), myositis specific antibodies (MCA), and antibodies to extractable nuclear antigens. Alternatively, patients meet three of the six criteria given in Rider et al., Arthritis Rheum., 50 (7): 2281-2290 (2004),

In certain embodiments, patients suffering from B-cell malignancy undergoing treatment as described herein may exhibit a general favorable response to treatment based on well-known and frequently used clinical criteria in the field, for example, reducing the size of the tumor, reducing the number of tumors and / or improvement in the symptoms of the disease, as described below.

Exemplary clinical criteria are provided by the National Cancer Institute (NCI) USA, in which some classes of malignant tumors are divided into clinical categories of "slow growing" and "aggressive" lymphomas. Slowly growing lymphomas include lymphomas from follicular cells, subdivided by cytological "degrees," diffuse small cell lymphoma / chronic lymphocytic leukemia (CLL), Waldenstrom lymphoplasmacytoid / macroglobulinemia, marginal zone lymphoma and hairy cell leukemia. Aggressive lymphomas include diffuse mixed and large cell lymphomas, Burkitt's lymphoma / diffuse small cell lymphoma with uncleaved nucleus, lymphoblastic lymphoma, mantle cell lymphoma, and AIDS-associated lymphoma. In some cases, in cases of aggressive and follicular lymphoma, use the international prognostic index (IPI). The factors considered in the IPI include age (age &lt;60 years in comparison with the age&gt; 60 years), serum lactate dehydrogenase (normal levels versus elevated levels), general condition (0 or 1 vs 2-4) (definition of cm below), the stage of the disease (I or II versus III or IV), and the involvement of extranodal localization (0 or 1 vs 2-4). Patients with 2 or more risk factors have a chance of no relapse and an overall survival rate of less than 50% within 5 years. general condition (0 or 1 vs. 2-4) (see definition below), stage of the disease (I or II versus III or IV), and involvement of extranodal localization (0 or 1 vs 2-4). Patients with 2 or more risk factors have a chance of no relapse and an overall survival rate of less than 50% within 5 years. general condition (0 or 1 vs. 2-4) (see definition below), stage of the disease (I or II versus III or IV), and involvement of extranodal localization (0 or 1 vs 2-4). Patients with 2 or more risk factors have a chance of no relapse and an overall survival rate of less than 50% within 5 years.

The general condition for aggressive IPI is defined as follows: degree description: 0 - full activity, ability without restrictions to perform all the same actions as before the disease; 1 - restriction of heavy physical activity, but outpatient and able to perform light or sedentary work, for example, easy work at home, work in the office; 2 - outpatient and able to care for themselves, but the lack of ability to perform any work up to and approximately more than 50% of the waking time; 3 - the ability of limited self-care, lying in a lying or sitting position for more than 50% of the waking time; 4 - total loss of ability to work, inability to perform any care for themselves, complete chained to a bed or chair; and 5 - death. (see The International Non-Hodgkin's Lymphoma Prognostic Factors Project. A predictive model for aggressive non-Hodgkin's lymphoma. N. Engl. J. Med. 329: 987-94, 1993).

Typically, the degree of lymphoma is clinically evaluated using the criterion that high-grade lymphoma is usually present as a nodal disease and often grows slowly or slightly. Disease with medium and low degree of differentiation is usually present in the form of a much more aggressive disease with large volume extranodal tumors.

To measure the progression of tumors, in particular, non-Hodgkin's lymphomas, the Ann Arbor classification system is also used. In this system, stages I, II, III and IV of mature NHL can be divided into categories A and B, depending on whether the patient has clearly expressed generalized symptoms (B) or not (A). The designation B is given to patients with the following symptoms: unexplained loss of more than 10% of body weight 6 months before diagnosis, unexplained fever with a temperature above 38 ° C and heavy night sweating. The stages have the following definitions: Stage I - involvement of one lymph node region or localized involvement of one extralymphatic organ or site. Stage II - involving areas of two or more lymph nodes on one side of the diaphragm or localized involvement of one bound extralimphatic organ or region and its regional lymph nodes with other areas of lymph nodes on the same side of the diaphragm or without them. Stage III - involvement of the lymph nodes on both sides of the diaphragm, possibly accompanied by a localized involvement of the extralymphatic organ or site, involvement of the spleen or both. Stage IV - dissesuric (multifocal) involvement of one or more extralymphatic sites involving or without the bound lymph node, or involvement of a separate extralymphatic organ with involvement of remote (non-regional) nodes. For more details see The International Non-Hodgkin ' s Lymphoma Prognostic Factors Project: A predictive model for aggressive non-Hodgkin's lymphoma, New England J. Med. (1993) 329: 987-994.

In one aspect, the therapeutic effect of the method of the present disclosure is determined by the level of response, for example, a partial response is defined as a tumor reduction less than half its original size. A complete response is defined as the complete elimination of the disease, confirmed by clinical or radiological studies. In one embodiment, the individual undergoing treatment according to the invention exhibits at least a partial response to treatment.

According to Chaison's criteria for the evaluation of NHL, which were developed in conjunction with the National Cancer Institute (Cheson et al., J Clin Oncol., 1999: 17: 1244; Grillo-Lopez et al., Ann Oncol. 2000, 11: 399-408) the answer is achieved with the complete disappearance of the detected clinical and radiographic signs of the disease and disease-related symptoms, the return of all lymph nodes to a normal size, a decrease in the size of the spleen, and cleansing of the bone marrow from lymphoma.

An unconfirmed complete response is achieved when the patient demonstrates the complete disappearance of the disease and the spleen decreases in size, and the lymph nodes decrease by more than 75%, and the bone marrow is in an uncertain state. An unconfirmed complete response meets the criteria for a partial response and surpasses them. The overall response is defined as a reduction in the total tumor burden by at least 50 percent.

For various other forms of malignant tumors or hyperproliferative diseases, similar criteria have been developed that are readily available to a professional in the field. See, for example, Cheson et al., Clin Adv Hematol Oncol. 2006, 4: 4-5, which describes the evaluation criteria for CLL; Cheson et al., J Clin Oncol. 2003, 21: 4642-9, which describes the criteria for AML; Cheson et al., Blood 2000, 96: 3671-4, which describes the criteria for myelodysplastic syndromes.

In another aspect, the therapeutic response of a patient with a B-cell malignant tumor is manifested as a delay in the development of the disease as compared to patients who do not undergo treatment. Measuring the delayed development of the disease or any of the above factors can be accomplished using methods that are well known in the art, including bone scans, CT scans, gallium scans, lymphangiograms, NMR tomography, PET scans, ultrasound, P.

As an additional aspect, the present disclosure includes kits that comprise one or more compounds or compositions useful in the methods of the present disclosure packaged in a manner that facilitates their use for practicing the methods of the present disclosure. In a simplest embodiment, such a kit comprises a compound or composition described herein as a practical method of this disclosure that is packaged in a container, such as a sealed bottle or bottle, with a label attached to the container or enclosed in a package in which describes the use of a compound or composition for practicing the method of the present disclosure. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further comprise a device that can be used to administer the composition in accordance with a preferred route of administration or for practical screening. The kit may contain a label that describes the use of the binding molecule (s) composition in the method of the present disclosure.

EXAMPLES

EXAMPLE 1

MOLECULES SPECIFICALLY CONNECTING WITH CD37

Various proteins specifically binding to CD37 can be prepared using the illustrative components provided in Tables 2-4. For example, it is possible to obtain antibodies or SMIP molecules, and these molecules can be chimeric, humanized or belonging to a human. More specifically, preferred light chain variable region CDRs are shown in SEQ ID NO: 236-240 and 247-254, and preferred heavy chain variable region CDRs include SEQ ID NOs: 241-245 and 247-254. In addition, preferred light and heavy chain variable regions are indicated in SEQ ID NO: 236-240 and SEQ ID NO: 241-245, respectively. Preferred light and heavy chain variable regions are also provided in SEQ ID NOs: 247-254. Preferred variable domain linkers include SEQ ID NO: 225-229,

A particularly preferred embodiment is CAS-024 [G28-1 V_H (M99F, Y102S) -V_L (T25A) scFv (SSC-P) H WC_H2 WC_H3], which is a recombinant single-chain fusion protein of 483 amino acids that binds to human CD37. The binding domain contains humanized scFv, based on the CDR of the variable region of antibody G28-1, including mutations in the heavy chain CDR3 and in the light chain CDR1. Variable domains are connected to a sequence (G₄S)₅ (25 amino acids) (SEQ ID NO: 229), which is linked through a three-amino acid (GDQ) compound site to the N-terminus of the modified upper and central hinge region of IgG1 (where the first two cysteines of the three found in these hinge regions are replaced by serine). The C-terminus of the hinge region is fused to the effector domain containing the C_H2 and C_H3 from IgG1. The amino acid sequence of CAS-024 is shown in SEQ ID NO: 253. In Fig. 1 shows the alignment of the amino acid sequences of the heavy and light chain variable regions from murine sequences G28.1 and CAS-024, together with the consensus sequence.

<tables num="1"><table frame="all"><tgroup cols="5" rowsep="1" colsep="1"><colspec colname="c0" colwidth="22mm" /><colspec colname="c1" colwidth="76mm" /><colspec colname="c2" colwidth="25mm" /><colspec colname="c3" colwidth="19mm" /><colspec colname="c4" colwidth="35mm" /><tbody><row><entry align="right" namest="c0" nameend="c4" rowsep="1" colsep="0">Table 1 SMIP-specific illustrative constructions, CD37-specific</entry></row><row><entry align="justify" rowsep="1" colsep="1">Design</entry><entry align="center" rowsep="1" colsep="1">Description†</entry><entry align="center" rowsep="1" colsep="1">Linker</entry><entry align="justify" rowsep="1" colsep="1">Hinge area *</entry><entry align="center" rowsep="1" colsep="0">SEQ ID NO: amino acid sequence</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-001</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5-51</entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">6th</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-002</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 JH4 <i>CDRL1 (T25A);</i><i>CDRH3 (M99F)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">48</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-003</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 JH5a <i>CDRL1 (T25A); CDRH3 (M99F; Y102S)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">52</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-007</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5-51 (<i>Linker TG → SS)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₃S</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">8</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-008</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5-51 <i>VH V11S</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">10</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-009</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5-51 <i>CDRL1 (E27Q)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">12</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-010</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5-51 <i>CDRL1 (N28S)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">14</entry></row><row><entry align="justify" rowsep="0" colsep="1">CAS-011</entry><entry align="center" rowsep="0" colsep="1">Vk3: VH5-51 <i>CDRL1 (T25A)</i></entry><entry align="center" rowsep="0" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="0" colsep="1">SSC-P</entry><entry align="center" rowsep="0" colsep="0">16</entry></row></tbody></tgroup></table></tables>

<tables num="2"><table frame="all"><tgroup cols="5" rowsep="1" colsep="1"><colspec colname="c0" colwidth="22mm" /><colspec colname="c1" colwidth="76mm" /><colspec colname="c2" colwidth="25mm" /><colspec colname="c3" colwidth="19mm" /><colspec colname="c4" colwidth="35mm" /><tbody><row><entry align="justify" rowsep="1" colsep="1">CAS-012</entry><entry align="center" rowsep="1" colsep="1">mVk: VH5-5a</entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">18</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-013</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 <i>VH3 FW1</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">22</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-014</entry><entry align="center" rowsep="1" colsep="1">mVH: Vk3</entry><entry align="center" rowsep="1" colsep="1">22 a.k. (G₄S)₄AS</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">24</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-015</entry><entry align="center" rowsep="1" colsep="1">Vk3: mVH <i>(Leader sequence 2H7)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">26th</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-016</entry><entry align="center" rowsep="1" colsep="1">mVH: Vk3</entry><entry align="center" rowsep="1" colsep="1">22aa (G₄S)₄AS</entry><entry align="center" rowsep="1" colsep="1">SCC-P</entry><entry align="center" rowsep="1" colsep="0">28</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-017</entry><entry align="center" rowsep="1" colsep="1">Vk3: mVH</entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">thirty</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-018</entry><entry align="center" rowsep="1" colsep="1">Vk3: mVH</entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SCC-P</entry><entry align="center" rowsep="1" colsep="0">32</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-019</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 <i>VH3 FW1</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SCC-P</entry><entry align="center" rowsep="1" colsep="0">34</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-020</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 <i>VH3-13 FW1</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">38</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-021</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 <i>VH3-13 FW1</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SCC-P</entry><entry align="center" rowsep="1" colsep="0">40</entry></row><row><entry align="justify" rowsep="0" colsep="1">CAS-022</entry><entry align="center" rowsep="0" colsep="1">Vk3: VH5 <i>VH3-13 V11S FW1</i></entry><entry align="center" rowsep="0" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="0" colsep="1">SSC-P</entry><entry align="center" rowsep="0" colsep="0">42</entry></row></tbody></tgroup></table></tables>

<tables num="3"><table frame="all"><tgroup cols="5" rowsep="1" colsep="1"><colspec colname="c0" colwidth="22mm" /><colspec colname="c1" colwidth="76mm" /><colspec colname="c2" colwidth="25mm" /><colspec colname="c3" colwidth="19mm" /><colspec colname="c4" colwidth="35mm" /><tbody><row><entry align="justify" rowsep="1" colsep="1">CAS-023</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 <i>VH3-13 V11S FW1</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SCC-P</entry><entry align="center" rowsep="1" colsep="0">44</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-024</entry><entry align="center" rowsep="1" colsep="1">VHVL</entry><entry align="center" rowsep="1" colsep="1">25 a.k. (G₄S)₅</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">253</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-060</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 <i>VH3 FW1</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">36</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-061</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 <i>CDRL1 (T25A, E27Q)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">46</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-062</entry><entry align="center" rowsep="1" colsep="1">Vk3: CDR-H3 JH6 <i>CDRL1 (T25A); CDRH3 (Y102V)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">254</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-063</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 JH5b <i>CDRL1 (T25A); CDRH3 (M99F; Y102P)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">266</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-064</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 JH1 <i>CDRL1 (T25A) CDRH3 (D101E; Y102H)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">267</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-065</entry><entry align="center" rowsep="1" colsep="1">Vk3: CDR-H3 JH3a <i>CDRL1 (T25A) CDRH3 (M99F; Y102V)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">268</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-066</entry><entry align="center" rowsep="1" colsep="1">Vk3: CDR-H3 JH3b <i>CDRL1 (T25A) CDRH3 (M99F; Y102I)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">269</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-067</entry><entry align="center" rowsep="1" colsep="1">Vk3: CDR-H3 JH2 <i>CDRL1 (T25A) CDRH3 (M99F; Y102L)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄T) G</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">80</entry></row><row><entry align="justify" rowsep="0" colsep="1">CAS-068</entry><entry align="center" rowsep="0" colsep="1">Vk3: VH5 JH2 <i>CDRL1 (T25A) CDRH2 (T59N; N61A; R62Q; K65Q)</i></entry><entry align="center" rowsep="0" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="0" colsep="1">SSC-P</entry><entry align="center" rowsep="0" colsep="0">82</entry></row></tbody></tgroup></table></tables>

<tables num="4"><table frame="all"><tgroup cols="5" rowsep="1" colsep="1"><colspec colname="c0" colwidth="22mm" /><colspec colname="c1" colwidth="76mm" /><colspec colname="c2" colwidth="25mm" /><colspec colname="c3" colwidth="19mm" /><colspec colname="c4" colwidth="35mm" /><tbody><row><entry align="justify" rowsep="1" colsep="1">CAS-069</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 JH2 <i>CDRL1 (T25A) CDRH2 (T59G; N61A; R62Q; K65Q)</i></entry><entry align="center" rowsep="1" colsep="1">16 a.k. (G₄S)₂(G₄A) S</entry><entry align="center" rowsep="1" colsep="1">SSC-P</entry><entry align="center" rowsep="1" colsep="0">262</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-070</entry><entry align="center" rowsep="1" colsep="1">Vk3: VH5 JH5a <i>CDRL1 (T25A); CDRH3 (M99F; Y102S)</i></entry><entry align="center" rowsep="1" colsep="1">20 a.k. (G₄S)₃(G₃A) S</entry><entry align="center" rowsep="1" colsep="1">CPPCP</entry><entry align="center" rowsep="1" colsep="0">84</entry></row><row><entry align="left" namest="c0" nameend="c4" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" namest="c0" nameend="c4" rowsep="0" colsep="0">^* Records denote abbreviations related to the hinge regions of IgG1, containing mutations only in the first or first and second cysteines located in the upper and central region. The only exception is SEQ ID NO: 84, which represents the full size amino acid sequence used for the hinge region (CPPCP, SEQ ID NO: 230) (essentially just the central IgG1 sequence with proline at the end). † CDR mutation numbering is based on the Kabat numbering scheme .</entry></row></tbody></tgroup></table></tables>

The table below provides additional hinge regions that can be used in molecules specifically binding to CD37, such as SMIP molecules or antibodies.

<tables num="5"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="38mm" /><colspec colname="c1" colwidth="93mm" /><colspec colname="c2" colwidth="31mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 2 Illustrative hinge regions for proteins specifically binding to CD37</entry></row><row><entry align="center" rowsep="1" colsep="1">Description of the hinge region</entry><entry align="center" rowsep="1" colsep="1">The amino acid sequence</entry><entry align="justify" rowsep="1" colsep="0">SEQ ID NO:</entry></row><row><entry align="justify" rowsep="1" colsep="1">ccc (p) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSCDKTHTCPPCP</entry><entry align="center" rowsep="1" colsep="0">90</entry></row><row><entry align="justify" rowsep="1" colsep="1">scc (p) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSSDKTHTCPPCP</entry><entry align="center" rowsep="1" colsep="0">92</entry></row><row><entry align="justify" rowsep="1" colsep="1">scc (s) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSSDKTHTCPPCS</entry><entry align="center" rowsep="1" colsep="0">94</entry></row><row><entry align="justify" rowsep="1" colsep="1">csc (p) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSCDKTHTSPPCP</entry><entry align="center" rowsep="1" colsep="0">102</entry></row><row><entry align="justify" rowsep="1" colsep="1">csc (s) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSCDKTHTSPPCS</entry><entry align="center" rowsep="1" colsep="0">104</entry></row><row><entry align="justify" rowsep="1" colsep="1">ccs (p) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSCDKTHTCPPSP</entry><entry align="center" rowsep="1" colsep="0">255</entry></row><row><entry align="justify" rowsep="1" colsep="1">ccs (s) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSCDKTHTCPPSS</entry><entry align="center" rowsep="1" colsep="0">256</entry></row><row><entry align="justify" rowsep="1" colsep="1">ssc (p) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSSDKTHTSPPCP</entry><entry align="center" rowsep="1" colsep="0">106</entry></row><row><entry align="justify" rowsep="1" colsep="1">ssc (s) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSSDKTHTSPPCS</entry><entry align="center" rowsep="1" colsep="0">108</entry></row><row><entry align="justify" rowsep="1" colsep="1">scs (p) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSSDKTHTCPPSP</entry><entry align="center" rowsep="1" colsep="0">257</entry></row><row><entry align="justify" rowsep="1" colsep="1">scs (s) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSSDKTHTCPPSS</entry><entry align="center" rowsep="1" colsep="0">96</entry></row><row><entry align="justify" rowsep="1" colsep="1">css (p) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSCDKTHTSPPSP</entry><entry align="center" rowsep="1" colsep="0">110</entry></row><row><entry align="justify" rowsep="1" colsep="1">css (s) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSCDKTHTSPPSS</entry><entry align="center" rowsep="1" colsep="0">112</entry></row><row><entry align="justify" rowsep="1" colsep="1">sss (p) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKSSDKTHTSPPSP</entry><entry align="center" rowsep="1" colsep="0">98</entry></row><row><entry align="justify" rowsep="1" colsep="1">sss (s) -hIgG1</entry><entry align="left" rowsep="1" colsep="1">EPKS SDKTHTSPPSS</entry><entry align="center" rowsep="1" colsep="0">100</entry></row><row><entry align="justify" rowsep="0" colsep="1">hIgA1</entry><entry align="left" rowsep="0" colsep="1">VPSTPPTPSPSTPPTPSPS</entry><entry align="center" rowsep="0" colsep="0">115</entry></row></tbody></tgroup></table></tables>

<tables num="6"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="38mm" /><colspec colname="c1" colwidth="93mm" /><colspec colname="c2" colwidth="31mm" /><tbody><row><entry align="justify" rowsep="1" colsep="1">hIgA2</entry><entry align="left" rowsep="1" colsep="1">VPPPPP</entry><entry align="center" rowsep="1" colsep="0">116</entry></row><row><entry align="justify" rowsep="1" colsep="1">hIgG3</entry><entry align="left" rowsep="1" colsep="1">ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP</entry><entry align="center" rowsep="1" colsep="0">118</entry></row><row><entry align="justify" rowsep="1" colsep="1">hIgG3 (ccc)</entry><entry align="left" rowsep="1" colsep="1">EPKSCDTPPPCPRCP</entry><entry align="center" rowsep="1" colsep="0">258</entry></row><row><entry align="justify" rowsep="1" colsep="1">hIgG3 (scc)</entry><entry align="left" rowsep="1" colsep="1">EPKSSDTPPPCPRCP</entry><entry align="center" rowsep="1" colsep="0">120</entry></row><row><entry align="justify" rowsep="1" colsep="1">hIgG3 (csc)</entry><entry align="left" rowsep="1" colsep="1">EPKSCDTPPPSPRCP</entry><entry align="center" rowsep="1" colsep="0">126</entry></row><row><entry align="justify" rowsep="1" colsep="1">hIgG3 (ccs)</entry><entry align="left" rowsep="1" colsep="1">EPKSCDTPPPCPRSP</entry><entry align="center" rowsep="1" colsep="0">259</entry></row><row><entry align="justify" rowsep="1" colsep="1">hIgG3 (ssc)</entry><entry align="left" rowsep="1" colsep="1">EPKSCDTPPPSPRCP</entry><entry align="center" rowsep="1" colsep="0">260</entry></row><row><entry align="justify" rowsep="1" colsep="1">hIgG3 (scs)</entry><entry align="left" rowsep="1" colsep="1">EPKSCDTPPPCPRSP</entry><entry align="center" rowsep="1" colsep="0">261</entry></row><row><entry align="justify" rowsep="1" colsep="1">hIgG3 (css)</entry><entry align="left" rowsep="1" colsep="1">EPKSCDTPPPSPRSP</entry><entry align="center" rowsep="1" colsep="0">122</entry></row><row><entry align="justify" rowsep="1" colsep="1">hIgG3 (sss)</entry><entry align="left" rowsep="1" colsep="1">EPKSSDTPPPSPRSP</entry><entry align="center" rowsep="1" colsep="0">124</entry></row><row><entry align="justify" rowsep="0" colsep="1">hIgD</entry><entry align="left" rowsep="0" colsep="1">ESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTP</entry><entry align="center" rowsep="0" colsep="0">127</entry></row></tbody></tgroup></table></tables>

The following tables show additional framework regions that can be used in molecules specifically binding to CD37, such as SMIP molecules or antibodies.

<tables num="7"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="35mm" /><colspec colname="c1" colwidth="95mm" /><colspec colname="c2" colwidth="31mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 3A Frame regions of human heavy chains for proteins specifically binding to CD37</entry></row><row><entry align="justify" rowsep="1" colsep="1">V-area</entry><entry align="justify" rowsep="1" colsep="1">Frame regions of V_H rights</entry><entry align="justify" rowsep="1" colsep="0">SEQ ID NO:</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1"><b>FR1</b></entry><entry align="left" rowsep="1" colsep="0"> </entry></row><row><entry align="center" rowsep="1" colsep="1">VH1</entry><entry align="center" rowsep="1" colsep="1">QVQLVQSGAEVKKPGASVKVSCKASGYTFT</entry><entry align="center" rowsep="1" colsep="0">140</entry></row><row><entry align="center" rowsep="1" colsep="1">VH1</entry><entry align="center" rowsep="1" colsep="1">QVQLVQSGAEVKKPGSSVKVSCKASGGTFS</entry><entry align="center" rowsep="1" colsep="0">141</entry></row><row><entry align="center" rowsep="1" colsep="1">VH1</entry><entry align="center" rowsep="1" colsep="1">EVQLVQSGAEVKKPGATVKISCKVSGYTFT</entry><entry align="center" rowsep="1" colsep="0">143</entry></row><row><entry align="center" rowsep="0" colsep="1">VH5</entry><entry align="center" rowsep="0" colsep="1">EVQLVQSGAEVKKPGESLKISCKGSGYSFT</entry><entry align="center" rowsep="0" colsep="0">144</entry></row></tbody></tgroup></table></tables>

<tables num="8"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="35mm" /><colspec colname="c1" colwidth="95mm" /><colspec colname="c2" colwidth="31mm" /><tbody><row><entry align="center" rowsep="1" colsep="1">VH5</entry><entry align="center" rowsep="1" colsep="1">EVQLVQSGAEVKKPGESLRISCKGSGYSFT</entry><entry align="center" rowsep="1" colsep="0">145</entry></row><row><entry align="center" rowsep="1" colsep="1">VH7</entry><entry align="center" rowsep="1" colsep="1">QVQLVQSGSELKKPGASVKVSCKASGYTFT</entry><entry align="center" rowsep="1" colsep="0">146</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1"><b>FR2</b></entry><entry align="left" rowsep="1" colsep="0"> </entry></row><row><entry align="center" rowsep="1" colsep="1">VH1</entry><entry align="center" rowsep="1" colsep="1">WVRQAPGQGLEWMG</entry><entry align="center" rowsep="1" colsep="0">147</entry></row><row><entry align="center" rowsep="1" colsep="1">VH1</entry><entry align="center" rowsep="1" colsep="1">WVQQAPGKGLEWMG</entry><entry align="center" rowsep="1" colsep="0">150</entry></row><row><entry align="center" rowsep="1" colsep="1">VH5</entry><entry align="center" rowsep="1" colsep="1">WVRQMPGKGLEWMG</entry><entry align="center" rowsep="1" colsep="0">151</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1"><b>FR3</b></entry><entry align="left" rowsep="1" colsep="0"> </entry></row><row><entry align="center" rowsep="1" colsep="1">VH1</entry><entry align="center" rowsep="1" colsep="1">RVTMTTDTSTSTAYMELRSLRSDDTAVYYCAR</entry><entry align="center" rowsep="1" colsep="0">154</entry></row><row><entry align="center" rowsep="1" colsep="1">VH1</entry><entry align="center" rowsep="1" colsep="1">RVTITADESTSTAYMELSSLRSEDTAVYYCAR</entry><entry align="center" rowsep="1" colsep="0">155</entry></row><row><entry align="center" rowsep="1" colsep="1">VH1</entry><entry align="center" rowsep="1" colsep="1">RVTITADKSTSTAYMELSSLRSEDTAVYYCAR</entry><entry align="center" rowsep="1" colsep="0">156</entry></row><row><entry align="center" rowsep="1" colsep="1">VH1</entry><entry align="center" rowsep="1" colsep="1">RVTITADTSTDTAYMELSSLRSEDTAVYYCAT</entry><entry align="center" rowsep="1" colsep="0">157</entry></row><row><entry align="center" rowsep="1" colsep="1">VH5</entry><entry align="center" rowsep="1" colsep="1">QVTISADKSISTAYLQWSSLKASDTAMYYCAR</entry><entry align="center" rowsep="1" colsep="0">158</entry></row><row><entry align="center" rowsep="1" colsep="1">VH5</entry><entry align="center" rowsep="1" colsep="1">HVTISADKSISTAYLQWSSLKASDTAMYYCAR</entry><entry align="center" rowsep="1" colsep="0">159</entry></row><row><entry align="center" rowsep="1" colsep="1">VH7</entry><entry align="center" rowsep="1" colsep="1">RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR</entry><entry align="center" rowsep="1" colsep="0">160</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1"><b>FR4</b></entry><entry align="left" rowsep="1" colsep="0"> </entry></row><row><entry align="center" rowsep="1" colsep="1">JH1, JH4, JH5a, JH5b</entry><entry align="center" rowsep="1" colsep="1">WGQGTLVTVSS</entry><entry align="center" rowsep="1" colsep="0">161</entry></row><row><entry align="center" rowsep="1" colsep="1">JH2</entry><entry align="center" rowsep="1" colsep="1">WGRGTLVTVSS</entry><entry align="center" rowsep="1" colsep="0">162</entry></row><row><entry align="center" rowsep="1" colsep="1">JH3a, JH3b</entry><entry align="center" rowsep="1" colsep="1">WGQGTMVTVSS</entry><entry align="center" rowsep="1" colsep="0">163</entry></row><row><entry align="center" rowsep="1" colsep="1">JH6</entry><entry align="center" rowsep="1" colsep="1">WGQGTTVTVSS</entry><entry align="center" rowsep="1" colsep="0">168</entry></row><row><entry align="left" rowsep="0" colsep="1"> </entry><entry align="center" rowsep="0" colsep="1">WGKGTTVTVSS</entry><entry align="center" rowsep="0" colsep="0">169</entry></row></tbody></tgroup></table></tables>

<tables num="9"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="38mm" /><colspec colname="c1" colwidth="95mm" /><colspec colname="c2" colwidth="29mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">TABLE 3B Frame regions of human light chains for proteins specifically binding to CD37</entry></row><row><entry align="center" rowsep="1" colsep="1">V-area</entry><entry align="justify" rowsep="1" colsep="1">Human VK frame areas</entry><entry align="center" rowsep="1" colsep="0">SEQ ID NO:</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1"><b>FR1</b></entry><entry align="left" rowsep="1" colsep="0"> </entry></row><row><entry align="center" rowsep="1" colsep="1">VK3</entry><entry align="center" rowsep="1" colsep="1">EIVMTQSPATLSVSPGERATLSC</entry><entry align="center" rowsep="1" colsep="0">170</entry></row><row><entry align="center" rowsep="1" colsep="1">VK3</entry><entry align="center" rowsep="1" colsep="1">EIVLTQSPATLSLSPGERATLSC</entry><entry align="center" rowsep="1" colsep="0">171</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">DIQMTQSPSSLSASVGDRVTITC</entry><entry align="center" rowsep="1" colsep="0">172</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">NIQMTQSPSAMSASVGDRVTITC</entry><entry align="center" rowsep="1" colsep="0">175</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">AIQLTQSPSSLSASVGDRVTITC</entry><entry align="center" rowsep="1" colsep="0">177</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">DIQLTQSPSFLSASVGDRVTITC</entry><entry align="center" rowsep="1" colsep="0">178</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">AIRMTQSPFSLSASVGDRVTITC</entry><entry align="center" rowsep="1" colsep="0">179</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">AIQMTQSPSSLSASVGDRVTITC</entry><entry align="center" rowsep="1" colsep="0">180</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">DIQMTQSPSTLSASVGDRVTITC</entry><entry align="center" rowsep="1" colsep="0">181</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1"><b>FR2</b></entry><entry align="left" rowsep="1" colsep="0"> </entry></row><row><entry align="center" rowsep="1" colsep="1">VK3</entry><entry align="center" rowsep="1" colsep="1">WYQQKPGQAPRLLIY</entry><entry align="center" rowsep="1" colsep="0">182</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">WYQQKPGKAPKLLIY</entry><entry align="center" rowsep="1" colsep="0">184</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">WYQQKPGKVPKLLIY</entry><entry align="center" rowsep="1" colsep="0">185</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">WYQQKPGKAPKRLIY</entry><entry align="center" rowsep="1" colsep="0">186</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">WFQQKPGKVPKHLIY</entry><entry align="center" rowsep="1" colsep="0">187</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">WFQQKPGKAPKSLIY</entry><entry align="center" rowsep="1" colsep="0">188</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">WYQQKPAKAPKLFIY</entry><entry align="center" rowsep="1" colsep="0">191</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1"><b>FR3</b></entry><entry align="left" rowsep="1" colsep="0"> </entry></row><row><entry align="center" rowsep="1" colsep="1">VK3</entry><entry align="center" rowsep="1" colsep="1">GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC</entry><entry align="center" rowsep="1" colsep="0">194</entry></row><row><entry align="center" rowsep="0" colsep="1">VK3</entry><entry align="center" rowsep="0" colsep="1">GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC</entry><entry align="center" rowsep="0" colsep="0">195</entry></row></tbody></tgroup></table></tables>

<tables num="10"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="38mm" /><colspec colname="c1" colwidth="95mm" /><colspec colname="c2" colwidth="29mm" /><tbody><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC</entry><entry align="center" rowsep="1" colsep="0">196</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC</entry><entry align="center" rowsep="1" colsep="0">197</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC</entry><entry align="center" rowsep="1" colsep="0">198</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC</entry><entry align="center" rowsep="1" colsep="0">203</entry></row><row><entry align="center" rowsep="1" colsep="1">VK1</entry><entry align="center" rowsep="1" colsep="1">GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC</entry><entry align="center" rowsep="1" colsep="0">205</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1"><b>FR4</b></entry><entry align="left" rowsep="1" colsep="0"> </entry></row><row><entry align="center" rowsep="1" colsep="1">JK1</entry><entry align="center" rowsep="1" colsep="1">FGQGTKVEIK</entry><entry align="center" rowsep="1" colsep="0">206</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1">FGQGTKLEIK</entry><entry align="center" rowsep="1" colsep="0">207</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1">FGPGTKVDIK</entry><entry align="center" rowsep="1" colsep="0">208</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1">FGGGTKVEIK</entry><entry align="center" rowsep="1" colsep="0">209</entry></row><row><entry align="left" rowsep="0" colsep="1"> </entry><entry align="center" rowsep="0" colsep="1">FGQGTRLEIK</entry><entry align="center" rowsep="0" colsep="0">210</entry></row></tbody></tgroup></table></tables>

Preferred illustrative constituents of CD37-specific SMIP molecules (including leader sequences used for expression and export but that are removed from the mature fusion protein upon export from the cell, linker sequences used to join the light and heavy chain variable domains to obtain scFv binding domains , hinge regions used to connect the binding domains of scFv to effector domains, and effector domains), as well as certain SMIP molecules specific for CD37, including the preferred CAS-024 fusion protein, are provided in Table 4.

<tables num="11"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="44mm" /><colspec colname="c1" colwidth="16mm" /><colspec colname="c2" colwidth="103mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 4 Components of SMIP and selected SMIP specific polypeptides specific for CD37</entry></row><row><entry align="justify" rowsep="1" colsep="1">Structural Number</entry><entry align="center" rowsep="1" colsep="1">SEQ ID NO:</entry><entry align="justify" rowsep="1" colsep="0">The amino acid sequence</entry></row><row><entry align="center" rowsep="1" colsep="1">Leader sequence</entry><entry align="center" rowsep="1" colsep="1">223</entry><entry align="justify" rowsep="1" colsep="0">MDFQVQIFSFLLISASVIIARGV</entry></row><row><entry align="center" rowsep="1" colsep="1">Leader sequence</entry><entry align="center" rowsep="1" colsep="1">224</entry><entry align="justify" rowsep="1" colsep="0">MEAPAQLLFLLLLWLPDTTG</entry></row><row><entry align="center" rowsep="1" colsep="1">Variable domain linker</entry><entry align="center" rowsep="1" colsep="1">225</entry><entry align="justify" rowsep="1" colsep="0">GGGGSGGGGSGGGGSS</entry></row><row><entry align="center" rowsep="1" colsep="1">Variable domain linker</entry><entry align="center" rowsep="1" colsep="1">226</entry><entry align="justify" rowsep="1" colsep="0">GGGGSGGGGSGGGGAS</entry></row><row><entry align="center" rowsep="1" colsep="1">Variable domain linker</entry><entry align="center" rowsep="1" colsep="1">227</entry><entry align="justify" rowsep="1" colsep="0">GGGGSGGSGSGGGGAS</entry></row><row><entry align="center" rowsep="1" colsep="1">Variable domain linker</entry><entry align="center" rowsep="1" colsep="1">228</entry><entry align="justify" rowsep="1" colsep="0">GGGGSGGGGSGGGGTG</entry></row><row><entry align="center" rowsep="1" colsep="1">Variable domain linker</entry><entry align="center" rowsep="1" colsep="1">229</entry><entry align="justify" rowsep="1" colsep="0">GGGGSGGGGSGGGGSGGGGSGGGGS</entry></row><row><entry align="center" rowsep="1" colsep="1">Hinge area</entry><entry align="center" rowsep="1" colsep="1">230</entry><entry align="justify" rowsep="1" colsep="0">CPPCP</entry></row><row><entry align="center" rowsep="0" colsep="1">Hinge region (connecting amino acids in italics)</entry><entry align="center" rowsep="0" colsep="1">231</entry><entry align="justify" rowsep="0" colsep="0"><i>S</i>EPKSSDKTHTSPPCP</entry></row></tbody></tgroup></table></tables>

<tables num="12"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="44mm" /><colspec colname="c1" colwidth="16mm" /><colspec colname="c2" colwidth="103mm" /><tbody><row><entry align="center" rowsep="1" colsep="1">Hinge region (connecting amino acids in italics)</entry><entry align="center" rowsep="1" colsep="1">232</entry><entry align="justify" rowsep="1" colsep="0"><i>DL</i>EPKSSDKTHTSPPCP</entry></row><row><entry align="center" rowsep="1" colsep="1">Hinge region (connecting amino acids in italics)</entry><entry align="center" rowsep="1" colsep="1">233</entry><entry align="justify" rowsep="1" colsep="0"><i>DQ</i>EPKSSDKTHTSPPCP</entry></row><row><entry align="center" rowsep="1" colsep="1">Hinge region (connecting amino acids in italics)</entry><entry align="center" rowsep="1" colsep="1">234</entry><entry align="justify" rowsep="1" colsep="0"><i>GDQ</i>EPKSSDKTHTSPPCP</entry></row><row><entry align="center" rowsep="1" colsep="1">Hinge region (connecting amino acids in italics)</entry><entry align="center" rowsep="1" colsep="1">235</entry><entry align="justify" rowsep="1" colsep="0"><i>GSS</i>EPKSSDKTHTSPPCP</entry></row><row><entry align="center" rowsep="1" colsep="1">V_L mice to CD37 (CDR isolated)</entry><entry align="center" rowsep="1" colsep="1">236</entry><entry align="justify" rowsep="1" colsep="0">DIQMTQSPASLSASVGETVTITC<b><i>RTSENVYSYLA</i></b>WYQQKQGKSPQLLVS<b><i>FAKTLAE</i></b>GVPSRFSGSGSGTQFSLKISSLQPEDSGSYFC<b><i>QHHSDNPWT</i></b>FGGGTTELEIK</entry></row><row><entry align="center" rowsep="1" colsep="1">Humanized V_L to CD37 (CDR highlighted) a</entry><entry align="center" rowsep="1" colsep="1">237</entry><entry align="justify" rowsep="1" colsep="0">EIVLTQSPATLSLSPGERATLSC<b><i>RTSENVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK</entry></row><row><entry align="center" rowsep="0" colsep="1">Humanized V_L to CD37 (CDR highlighted) b</entry><entry align="center" rowsep="0" colsep="1">238</entry><entry align="justify" rowsep="0" colsep="0">EIVLTQSPATLSLSPGERATLSC<b><i>RASENVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEP</entry></row></tbody></tgroup></table></tables>

<tables num="13"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="44mm" /><colspec colname="c1" colwidth="16mm" /><colspec colname="c2" colwidth="103mm" /><tbody><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="justify" rowsep="1" colsep="0">EDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK</entry></row><row><entry align="center" rowsep="1" colsep="1">Humanized V_L to CD37 (CDR highlighted) c</entry><entry align="center" rowsep="1" colsep="1">239</entry><entry align="justify" rowsep="1" colsep="0">EIVLTQSPATLSLSPGERATLSC<b><i>RTSQNVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK</entry></row><row><entry align="center" rowsep="1" colsep="1">Humanized V_L to CD37 (CDR isolated) d</entry><entry align="center" rowsep="1" colsep="1">240</entry><entry align="justify" rowsep="1" colsep="0">EIVLTQSPATLSLSPGERATLSC<b><i>RTSESVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK</entry></row><row><entry align="center" rowsep="1" colsep="1">V_H mice to CD37 (CDR isolated)</entry><entry align="center" rowsep="1" colsep="1">241</entry><entry align="justify" rowsep="1" colsep="0">AVQLQQSGPESEKPGASVKISCKASGYSFT<b><i>GYNMN</i></b>WVKQNNGKSLEWIG<b><i>NIDPYYGGTTYNRKFKG</i></b>KATLTVDKSSSTAYMQLKSLTSEDSAVYYCAR<b><i>SVGPMDY</i></b>WGQGTSVTVSS</entry></row><row><entry align="center" rowsep="1" colsep="1">Humanized V_H to CD37 (CDR highlighted) a</entry><entry align="center" rowsep="1" colsep="1">242</entry><entry align="justify" rowsep="1" colsep="0">EVQLVQSGAEVKKPGESLKISCKGSGYSFT<b><i>GYNMN</i></b>WVRQMPGKGLEWMG<b><i>NIDPYYGGTTYNRKFKG</i></b>QVTISADKSISTAYLQWSSLKASDTAMYYCAR<b><i>SVGPMDY</i></b>WGQGTLVTVSS</entry></row><row><entry align="center" rowsep="1" colsep="1">Humanized V_H to CD37 (CDR highlighted) a</entry><entry align="center" rowsep="1" colsep="1">243</entry><entry align="justify" rowsep="1" colsep="0">EVQLVQSGAEVKKPGESLKISCKGSGYSFT<b><i>GYNMN</i></b>WVRQMPGKGLEWMG<b><i>NIDPYYGGTTYNRKFKG</i></b>QVTISADKSISTAYLQWSSLKASDTAMYYCAR<b><i>SVGPMDV</i></b>WGQGTLVTVSS</entry></row><row><entry align="center" rowsep="1" colsep="1">Humanized V_H to CD37 (CDR highlighted) b</entry><entry align="center" rowsep="1" colsep="1">244</entry><entry align="justify" rowsep="1" colsep="0">EVQLVQSGAEVKKPGESLKISCKGSGYSFT<b><i>GYNMN</i></b>WVRQMPGKGLEWMG<b><i>NIDPYYGGTTYNRKFKG</i></b>QVTISADKSISTAYLQWSSLKASDTAMYYCAR<b><i>SVGPFDY</i></b>WGQGTLVTVSS</entry></row><row><entry align="center" rowsep="1" colsep="1">Humanized V_H to CD37 (CDR highlighted) c</entry><entry align="center" rowsep="1" colsep="1">245</entry><entry align="justify" rowsep="1" colsep="0">EVQLVQSGAEVKKPGESLKISCKGSGYSFT<b><i>GYNMN</i></b>WVRQMPGKGLEWMG<b><i>NIDPYYGGTTYNRKFKG</i></b>QVTISADKSISTAYLQWSSLKASDTAMYYCAR<b><i>SVGPFDS</i></b>WGQGTLVTVSS</entry></row><row><entry align="justify" rowsep="0" colsep="1"> CH2CH3 IgG1</entry><entry align="center" rowsep="0" colsep="1">246</entry><entry align="justify" rowsep="0" colsep="0">APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN</entry></row></tbody></tgroup></table></tables>

<tables num="14"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="44mm" /><colspec colname="c1" colwidth="16mm" /><colspec colname="c2" colwidth="103mm" /><tbody><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="left" rowsep="1" colsep="1"> </entry><entry align="justify" rowsep="1" colsep="0">YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</entry></row><row><entry align="left" rowsep="1" colsep="1">CAS-006 (chimeric SMIP to CD37)</entry><entry align="center" rowsep="1" colsep="1">247</entry><entry align="justify" rowsep="1" colsep="0">DIQMTQSPASLSASVGETVTITC<b><i>RTSENVYSYLA</i></b>WYQQKQGKSPQLLVS<b><i>FAKTLAE</i></b>GVPSRFSGSGSGTQFSLKISSLQPEDSGSYFC<b><i>QHHSDNPWT</i></b>FGGGTTELEIK<i>GGGGSGGGGSGGGGSS</i>AVQLQQSGPESEKPGASVKISCKASGYSFT<b><i>GYNMN</i></b>WVKQNNGKSLEWIG<b><i>NIDPYYGGTTYNRKFKG</i></b>KATLTVDKSSSTAYMQLKSLTSEDSAVYYCAR<b><i>SVGPMDY</i></b>WGQGTSVTVSS<i><u>DL</u>EPKSSDKTHTSPPCP</i>APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</entry></row><row><entry align="justify" rowsep="0" colsep="1">CAS-001</entry><entry align="center" rowsep="0" colsep="1">248</entry><entry align="justify" rowsep="0" colsep="0">EIVLTQSPATLSLSPGERATLSC<b><i>RTSENVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK<i>GGGGSGGGGSGGGGTG</i>EVQLVQSGAEVKKPGESLKISCKGSGYSFT<b><i>GYNMN</i></b>WVRQMPGKGLEWMG<b><i>NIDPYYGGTTYNRKFKG</i></b>QVTISADKSISTAYLQWSSLKASDTAMYYCAR<b><i>SVGPMDY</i></b>WGRGTLVTVSS<i><u>DQ</u>EPKSSDKTHTSPPCP</i>APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</entry></row></tbody></tgroup></table></tables>

<tables num="15"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="44mm" /><colspec colname="c1" colwidth="16mm" /><colspec colname="c2" colwidth="103mm" /><tbody><row><entry align="justify" rowsep="1" colsep="1">CAS-002</entry><entry align="center" rowsep="1" colsep="1">249</entry><entry align="justify" rowsep="1" colsep="0">EIVLTQSPATLSLSPGERATLSC<b><i>RASENVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK<i>GGGGSGGGGSGGGGTG</i>EVQLVQSGAEVKKPGESLKISCKGSGYSFT<b><i>GYNMN</i></b>WVRQMPGKGLEWMG<b><i>NIDPYYGGTTYNRKFKG</i></b>QVTISADKSISTAYLQWSSLKASDTAMYYCAR<b><i>SVGPFDY</i></b>WGQGTLVTVSS<i><u>DQ</u>EPKSSDKTHTSPPCP</i>APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</entry></row><row><entry align="justify" rowsep="0" colsep="1">CAS-003</entry><entry align="center" rowsep="0" colsep="1">250</entry><entry align="justify" rowsep="0" colsep="0">EIVLTQSPATLSLSPGERATLSC<b><i>RASENVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK<i>GGGGSGGGGSGGGGTG</i>EVQLVQSGAEVKKPGESLKISCKGSGYSFT<b><i>GYNMN</i></b>WVRQMPGKGLEWMG<b><i>NIDPYYGGTTYNRKFKG</i></b>QVTISADKSISTAYLQWSSLKASDTAMYYCAR<b><i>SVGPFDS</i></b>WGQGTLVTVSS<i><u>DQ</u>EPKSSDKTHTSPPCP</i>APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</entry></row></tbody></tgroup></table></tables>

<tables num="16"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="44mm" /><colspec colname="c1" colwidth="16mm" /><colspec colname="c2" colwidth="103mm" /><tbody><row><entry align="justify" rowsep="1" colsep="1">CAS-014 (human-mouse hybrid)</entry><entry align="center" rowsep="1" colsep="1">251</entry><entry align="justify" rowsep="1" colsep="0">AVQLQQSGPESEKPGASVKISCKASGYSFT<b><i>GYNMN</i></b>WVKQNNGKSLEWIG<b><i>NIDPYYGGTTYNRKFKG</i></b>KATLTVDKSSSTAYMQLKSLTSEDSAVYYCAR<b><i>SVGPMDY</i></b>WGQGTSVTVSS<i>GGGGSGGGGSGGGGSGGGGSAS</i>EIVLTQSPATLSLSPGERATLSC<b><i>RTSENVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK<i><u>GSS</u>EPKSSDKTHTSPPCP</i>APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</entry></row><row><entry align="justify" rowsep="0" colsep="1">CAS-017 (human-mouse hybrid)</entry><entry align="center" rowsep="0" colsep="1">252</entry><entry align="justify" rowsep="0" colsep="0">EIVLTQSPATLSLSPGERATLSC<b><i>RTSENVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK<i>GGGGSGGGGSGGGGAS</i>AVQLQQSGPESEKPGASVKISCKASGYSFT<b><i>GYNMN</i></b>WVKQNNGKSLEWIG<b><i>NIDPYYGGTTYNRKFKG</i></b>KATLTVDKSSSTAYMQLKSLTSEDSAVYYCAR<b><i>SVGPMDY</i></b>WGQGTSVTVSS<i><u>S</u>EPKSSDKTHTSPPCP</i>APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</entry></row></tbody></tgroup></table></tables>

<tables num="17"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="44mm" /><colspec colname="c1" colwidth="16mm" /><colspec colname="c2" colwidth="103mm" /><tbody><row><entry align="justify" rowsep="0" colsep="1">CAS-024</entry><entry align="center" rowsep="0" colsep="1">253</entry><entry align="justify" rowsep="0" colsep="0">EVQLVQSGAEVKKPGESLKISCKGSGYSFT<b><i>GYNMN</i></b>WVRQMPGKGLEWMG<b><i>NIDPYYGGTTYNRKFKG</i></b>QVTISADKSISTAYLQWSSLKASDTAMYYCAR<b><i>SVGPFDS</i></b>WGQGTLVTVSS<i>GGGGSGGGGSGGGGSGGGGSGGGGS</i>EIVLTQSPATLSLSPGERATLSC<b><i>RASENVYSYLA</i></b>WYQQKPGQAPRLLIY<b><i>FAKTLAE</i></b>GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC<b><i>QHHSDNPWT</i></b>FGQGTKVEIK<i><u>GDQ</u>EPKSSDKTHTSPPCP</i>APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK</entry></row></tbody></tgroup></table></tables>

EXAMPLE 2

EXPRESSION OF CAS-024 AND OTHER PROTEINS SPECIFICALLY BINDING TO CD37

CAS-024 and other SMIP molecules specifically binding to CD37 were cloned into the mammalian cell expression system in Chinese hamster ovary (CHO) cells. Transfected CHO cells producing SMIP molecules were cultured in shake flasks and the harvested cell culture supernatants were titrated using an Octec Q Protein A sensor.

Table 5 shows that the design of CAS-024 (in V format_HV_L with a 25 amino acid variable domain linker) had an unexpectedly very high level of expression, up to about 10 times that of other humanized SMIP molecules to CD37 (mainly in V format_LV_H with a variable domain 15 amino acid linker). Indeed, all the fully humanized V_LV_H were poorly expressed (data not shown), as well as hybrid mouse-human molecules in any orientation (see Example 5).

<tables num="18"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="20mm" /><colspec colname="c1" colwidth="73mm" /><colspec colname="c2" colwidth="86mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 5Expansion of SMIP</entry></row><row><entry align="center" rowsep="1" colsep="1">Protein SMIP</entry><entry align="center" rowsep="1" colsep="1">The clones were screened</entry><entry align="center" rowsep="1" colsep="0">Protein titre range (μg / ml)</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-001</entry><entry align="center" rowsep="1" colsep="1">492</entry><entry align="center" rowsep="1" colsep="0">65-80</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-002</entry><entry align="center" rowsep="1" colsep="1">425</entry><entry align="center" rowsep="1" colsep="0">200-280</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-003</entry><entry align="center" rowsep="1" colsep="1">611</entry><entry align="center" rowsep="1" colsep="0">300-360</entry></row><row><entry align="center" rowsep="0" colsep="1">CAS-024</entry><entry align="center" rowsep="0" colsep="1">203</entry><entry align="center" rowsep="0" colsep="0">500-650</entry></row></tbody></tgroup></table></tables>

EXAMPLE 3

CLEANING AND EXCLUSIVE CHROMATOGRAPHY CAS-024 AND OTHER PROTEINS SPECIFICALLY BINDING WITH CD37

To obtain more protein, the nucleic acid encoding CAS-024 and some other SMIP molecules specifically binding to CD37 were cloned into the mammalian cell expression system in Chinese hamster ovary (CHO) cells. Transfected CHO cells producing SMIP molecules were cultured in shake flasks.

All SMIP molecules specifically binding to CD37 were purified from the supernatants of the CHO culture by affinity chromatography with protein A. A 50 ml column of rProtein A Sepharose FF (GE Healthcare) was equilibrated at a flow rate of 5.0 ml / min (150 cm / hr) in 1 , 5 column volumes (OK) with buffer dPBS. The culture supernatant was loaded onto a column of rProtein A Sepharose FF at a flow rate of 1.7 ml / min using AKTA Explorer 100 Air (GE healthcare), capturing recombinant SMIP molecules. The column was washed with 5 column volumes (OK) with buffer dPBS followed by 1.0 M NaCl, 20 mM sodium phosphate, pH 6.0 and then 25 mM NaCl, 25 mM NaOAc, pH 5.0. Recombinant molecules specifically binding to CD37 were eluted from the column with 100 mM glycine, pH 3.5. Fractions (10 ml) of the eluted product were collected and then the pH was adjusted to 5.0 °, 5 M 2- (N-morpholino) ethanesulfonic acid (MES) in a volume of 20% of the eluted volume, pH 6.0. This eluted product was concentrated to about 25 mg / ml protein and sterilized by filtration.

This concentrated and sterilized protein was further purified by GPC exclusion chromatography (ECH) to produce a SMIP molecule (dimer) separated from the high molecular weight aggregates. Column XK 50/100 (GE healthcare) containing 1 L of Superdex 200 Sepharose FF was equilibrated at a rate of 12.6 ml / min (38 cm / h) in 1.5 column volumes (OK) with buffer dPBS. The column was filled with a maximum volume of 54 ml (3% OK). The flow rate in the column was continued at 12.6 ml / min and the eluted protein was divided into 40 ml fractions. In each fraction, the quality of the product was analyzed using analytical HPLC, and the eluted fractions were combined to an amount of approximately more than 95% of the protein of interest (non-aggregated). The resulting pooled sample was sterilized by filtration through a 0.22 μm filter, concentrated,

In Fig. 2A-2D are EC curves showing peaks containing the protein of interest for the CAS-001 (SEQ ID NO: 6), CAS-002 (SEQ ID NO: 48), CAS-003 (SEQ ID NO: 52) and CAS-024 (SEQ ID NO: 253), respectively. The CAS-024 peak is the narrowest and more symmetrical than in the samples CAS-001, CAS-002 and CAS-003 (wider and unbalanced). Molecule CAS-006 (chimeric) gives an acute peak similar to CAS-024. Samples CAS-001, CAS-002 and CAS-003 have a small terminal arm, which during integration accounts for approximately 35% of the POI area. This "shoulder" is difficult to separate from the POI and, perhaps, it represents misconfigured conformers or a heterogeneous group of molecules (for example, having different levels of glycosylation). This indicates that CAS-024 is not only better expressed,

EXAMPLE 4

Binding of CAS-024 to cells is unexpectedly high relative to other proteins specific to CD37

To compare the binding affinity of various molecules of immunopharmaceuticals based on small modular protein (SMIP) to CD37, found on Ramos line cells (B-lymphoblastoid cell line derived from Burkitt's lymphoma), a competitive binding assay was used. The purified ECX chimeric SMIP molecule to CD37 (CAS-006, SEQ ID NO: 247) was labeled with the FMAT Blue® fluorescent dye (Applied Biosystems) and used as a standard for competition with the purified unlabeled SMIP chimeric molecule to CD37 (positive control) and purified by unlabeled humanized SMIP test molecules to CD37. The higher affinity was manifested as a weaker fluorescence signal, and the FL1 fluorescence value was used to obtain a competitive binding curve. In summary, labeled with the FMAT Blue® reagent, the chimeric SMIP molecule to CD37 was diluted to a concentration of 2 μg / ml with FACS blocking buffer and purified protein samples (CAS-001 (SEQ ID NO: 6), CAS-002 (SEQ ID NO: 48), CAS-003 (SEQ ID NO: 52) and CAS-024 (SEQ ID NO: 253)) were serially diluted 1: 2 to a concentration in the range of 50 μg / ml to 0.02 μg / ml. Ramos cells were harvested at a rate of 1000 rpm for 5 minutes and resuspended in blocking buffer FACS at a concentration of 4 × 10^6th cells / 10 ml buffer. To each well of a black 96-well plate, the following was added: 50 μl of sample, 50 μl of a chimeric SMIP molecule to CD37 labeled with FMAT Blue® and 50 μl of Ramos cells (4 × 10⁴/ well). The plates were incubated at room temperature for 30 minutes and read into the 8200 Cellular Detection System (Applied Biosystems), tuned for the average cell size and weak signal.

This competitive binding assay with FMAT has shown that CAS-024 (a humanized SMIP molecule to CD37 containing V_HV_L scFv with a 25 amino acid variable domain linker) has the same affinity for CD37 as the original chimeric SMIP molecule to CD37 and, unlike it, has an unexpectedly higher up to 4 fold affinity for CD37 as compared to humanized SMIP molecules to CD37, containing the inverse structure of V_LV_H and a shorter linker of a variable domain of 16 amino acids in length (see Figure 3). The best binding, although still significantly less than that of CAS-006 or CAS-024, was revealed in the design of V_LV_H, not containing any mutations in the CDR (CAS-001). However, CAS-001 was the most poorly expressed construct and gave a non-homogeneous group of purified molecules - even in comparison with this design, CAS-024 bound 1.5-2 times better than CAS-001.

This result was unexpected also because M99 and Y102 in the CDR3 of the heavy chain CAS-024 underwent a mutation - the position of Y102 is usually conservative, and one would expect that a change only in this position will weaken or even stop binding (for example, CAS-062, mutant at position Y102, has a detectable but significantly less binding than CAS-001 or CAS-024, while each from CAS-063 to CAS-067 in this assay has a poorly detectable up to absent binding activity when introducing a mutation into the put M99 or D101, the data are not shown). Thus, the structure of CAS-024 provides a molecule that is unexpectedly bound in the same way as the chimeric molecule CAS-006.

EXAMPLE 5

CAS-024 EXPRESSION AND BINDING WITH CELLS COMPARED WITH HYBRID PROTEINS OF THE MOUSE-HUMAN MUSIC SPECIFICALLY BINDING WITH CD37

CAS-024 and other SMIP molecules specifically binding to CD37 were obtained by recombinant DNA technology and transfected into HEK293 cells for 7 days. Supernatants of cell cultures were harvested on day 7 and titrated using an Octec Q Protein A sensor.

Similar to the results obtained in Example 2, here in Table 6 it is shown that CAS-024 (in V format_HV_L with a 25 amino acid variable domain linker) was expressed approximately 5-27 times better than other humanized or hybrid SMIP mouse-human molecules to CD37. Hybrid mouse human molecules were not expressed well enough regardless of the orientation of V_HV_L or V_LV_H.

<tables num="19"><table frame="all"><tgroup cols="2" rowsep="1" colsep="1"><colspec colname="c0" colwidth="51mm" /><colspec colname="c1" colwidth="128mm" /><tbody><row><entry align="right" namest="c0" nameend="c1" rowsep="1" colsep="0">Table 6Expansion of SMIP</entry></row><row><entry align="center" rowsep="1" colsep="1">Protein SMIP</entry><entry align="center" rowsep="1" colsep="0">Titer of protein (μg / ml)</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-002 (hVLhVH)</entry><entry align="center" rowsep="1" colsep="0">0.47</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-003 (hVLhVH)</entry><entry align="center" rowsep="1" colsep="0">2.39</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-014 (mVHhVL)</entry><entry align="center" rowsep="1" colsep="0">2.16</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-017 (hVLmVH)</entry><entry align="center" rowsep="1" colsep="0">0.70</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-006 (mVLmVH)</entry><entry align="center" rowsep="1" colsep="0">9.3</entry></row><row><entry align="center" rowsep="0" colsep="1">CAS-024 (hVHhVL)</entry><entry align="center" rowsep="0" colsep="0">12.7</entry></row></tbody></tgroup></table></tables>

To compare the binding affinities of various human SMIP hybrid molecules to CD37 as compared to the binding of CAS-024 to Ramos cells, the competitive binding assay described in Example 4 was used. The ECX purified chimeric SMIP molecule to CD37 (CAS-006, SEQ ID NO: NO: 247) was labeled with the FMAT Blue® fluorescent dye (Applied Biosystems) and used as a standard for competition with the purified unlabeled SMIP chimeric molecule to CD37 (CAS-006, positive control) and purified unlabeled human SMIP test molecules for CD37-CAS-002 (SEQ ID NO: 48), CAS-003 (SEQ ID NO: 52), CAS-014 (SEQ ID NO: 251), CAS-017 (SEQ ID NO: 252) and CAS-024 (SEQ ID NO: 253).

This competitive binding assay with FMAT again showed that CAS-024 (a humanized V molecule_HV_L with a linker length of 25 amino acids) had the same affinity for CD37 as the original chimeric SMIP molecule to CD37 (CAS-006), whereas CAS-002 and CAS-003 (humanized V molecules_LV_H with a linker length of 16 amino acids) did not bind well either (demonstrating a 2-3-fold decrease) (see Figure 4A). Hybrid molecules of human mouse, regardless of the orientation of the variable domain (V_H mouse-humanized V_L with a linker of length 22 amino acids or humanized V_L-V_H mice with a linker length of 16 amino acids) were also bound or even better (1.5-2 times) than CAS-006 and CAS-024 (see Figure 4B). These data show that a hybrid mouse-human molecule without mutations, regardless of orientation, also binds or even better than CAS-006, and that a fully humanized V_LV_H the design without mutations in the CDR binds better than the humanized molecules, but still have reduced binding as compared to CAS-006 or CAS-024. Together, these data indicate that those molecules that do not contain mutations in the CDR have a stronger binding. Also, apparently, a specific order (V_LV_H or V_HV_L) does not solve the problem of expression even when a longer variable domain linker is used (see CAS-014). Thus, the choice of a molecule with the structure of CAS-024 and properties similar to the original molecule of CAS-006 did not yield to the forecast.

EXAMPLE 6

CAS-006 AND VARIOUS ANTIBODIES SPECIFICED TO CD37 CONNECT WITH ONE OR WITH OVERCURRENT EPITHOPOM ON CD37

To identify the epitope CD37, to which CAS-006 binds and other previously described antibodies specific for CD37, experiments were performed. Unconjugated MB371 (# 555457) and conjugated to FITC MB371 (# 555456) were obtained from BD Pharmingen (San Jose, CA) conjugated with FITC BL14 (# 0457) from Immunotech / Beckman Coulter (Fullerton, CA) conjugated to FITC NMN46 # RDI-CBL 136FT) and unconjugated NMN46 (# RDI-CBL 136) from RDI (Flanders, NJ) conjugated to FITC IPO24 (# 186-040) and unconjugated IPO-24 (# 186-020) from Ancell Corporation (Bayport , M_N) conjugated to FITC HHI (# 3081) and unconjugated HH1 (# 3080) from DiaTec.Com (Oslo, Norway) and conjugated to FITC WR17 (YSRTMCA483F) and unconjugated WR17 (YSRTMCA483S) from Accurate Chemical &amp; Scientific (Westbury, NY) . The SMIP protein CAS-006 was prepared as described in Example 2.

CAS-006 was conjugated to FITC using the Molecular Probes Flouroreporter FITC Labeling kit (F6434) according to the manufacturer's instructions as follows: the peak of the CAS-006 protein of interest (13.5 mg / ml) was adjusted to 5 mg / ml by PBS. One mg (200 μl) was added to the test tubes from the kit containing the stirrer and 1 M NaHCO₃ (adjusted to pH 8.5 with 6N NaOH) to a final concentration of 0.1 M. To 370 μg of FITC, 50 μl of DMSO was added and added to the tubes at a molar ratio of FITC: protein 15, 20, 30 and 40 s following formula to determine the amount of μL FITC to be added: [the number of μl FITC solution for addition = 5 mg / ml protein × 0.2 ml × 389 × 100 × the desired molar ratio / molecular weight CAS-006 (110,000)].

The reaction mixtures were protected from light and continuously stirred for 75 minutes at room temperature. The reaction mixtures were added to centrifugal columns prepared as described in the kit and centrifuged at 1100 g for 5 minutes, replacing the buffer on PBS with azide and removing the unconjugated FITC. OD at 280 nM and 494 nM was determined using 2 μl of drops on Nanodrop; the extinction coefficient for CAS-016 for this device was determined experimentally by reading the dilutions of the original unconjugated SMIP molecule, the concentration of each conjugate was 4.25 mg / ml, and the following FITC: protein: 2.7 FITC / CAS-016 ratios at a ratio of 15; 3.7 FITC / CAS-016 at a ratio of 20; 4.4 FITC / CAS-016 at a ratio of 30 and 5.1 FITC / CAS-016 at a ratio of 40.

BSA was added to 3 mg / ml to help stabilize the protein. The binding of each fraction was evaluated at dilutions in the range of 100-24300 × Ramos and 3200-25600 × per human PBMC. All fractions were bound, but for further use, the MR30 ratio was chosen because it gave a high MFI that was well maintained throughout the used titration range, indicating that in this reaction, the binding avidity was least affected.

To determine the optimal amount for use in blocking studies in the initial binding assay, conjugates of labeled FITC antibodies were titrated from 10 ng / ml to 10 μg / ml. The selected level was slightly below the saturating amount and remained constant in subsequent tests, while the levels of the blocking antibody increased in the 10-fold range. Data graphs were constructed as a percentage of maximum binding, depending on the concentration of the blocking antibody, so higher levels indicate less effective blocking, while lower levels indicate more effective blocking activity. All tested antibodies exhibited blocking activity of maximum binding, which was observed without unlabelled reagents (Fig. 5).

Then, using the panel of various mAb clones to CD37, including MB371, BL14, NMN46, IPO24, HH1, WR17 and chimeric SMIP CAS-006 stained BJAB cells, lymphoblastoid B cell lines.

For the competitive binding assay, 2.5 × 10⁵ BJAB cells in 96-well V-bottomed plates in a staining medium (PBS with 2% mouse serum) conjugated with FITC mAb to CD37 at a concentration of 1.25 μg / ml in the presence of unconjugated mAb to CD37 at the indicated concentrations (2 , 5, 1.25, 0.6 or 0.3 μg / ml) or in a medium for staining for 45 minutes on ice in the dark. Prior to the addition of the cells, blocking antibodies and conjugates of labeled FITC antibodies were added to the reaction mixtures. The cells were then washed in 2.5-fold PBS and fixed in 1% paraformaldehyde (USB, Cleveland, Ohio). The treated cells were analyzed by flow cytometry using the FACsCalibur device and the CellQuest software (BD Biosciences, San Jose, CA).

For the analysis of cross-blocking, FACs were incubated 2.5 × 10⁵ of BJAB cells in 96-well V-bottomed plates in a staining medium (PBS with 2% mouse serum) in the presence of unconjugated mAb to CD37 at a concentration of 5 μg / ml in a staining medium for 45 minutes at room temperature in the dark. FITC-conjugated mAb to CD37 was added to a final concentration of 2 μg / ml, which resulted in dilution of unlabelled reagents to 3.3 μg / ml. The reaction mixtures were further incubated in the dark for 45 minutes at room temperature, then washed 2.5 times in PBS and finally fixed in 1% paraformaldehyde in PBS (USB, Cleveland, Ohio). Cells were analyzed by flow cytometry on a FACsCalibur device using the Cell Quest software (BD Biosciences, San Jose, CA).

To assay for binding to cells, the cells were suspended in PBS (Gibco / Invitrogen, Grand Island NY) containing 2% FBS (Gibco / Invitrogen) (staining medium) at a concentration of approximately 4 × 10^6th cells / ml. The cells were then transferred to plates, and then 1: 1 test samples diluted with the coloring medium to a certain final concentration were added. The reaction mixtures were incubated for 45 minutes on ice. Samples were centrifuged and washed 2 times in PBS. FITC-labeled goat anti-human IgG antibody (CalTag, Burlingame CA) was added at a final dilution of 1:50 and incubated for 45 minutes on ice. Samples were centrifuged, washed in PBS, then fixed in 200 μl of 1% paraformaldehyde in PBS (USB, Cleveland, Ohio). Cells were analyzed by flow cytometry on a FACsCalibur device using the Cell Quest software (BD Biosciences, San Jose, CA).

Each antibody demonstrated a dose-dependent inhibition of binding, indicating that all tested molecules were bound to the same or strongly similar epitopes. Each antibody was characterized by a different inhibition of binding activity. SMIP CAS-006 had the highest level of blocking activity among all tested molecules, while HH1 provided an intermediate level of blocking activity, and WR17, IPO24 blocked better than MB371, but showed less effective blocking than the other two unlabeled molecules (Figure 5) .

In addition to the analysis of blocking activity, similar series of experiments were conducted in which various CD37 directed antibodies were tested for their ability to compete with other antibodies for binding to the CD37 receptor. The results of these experiments, similar to the results obtained in blocking studies for all tested molecules, indicate that different CD37-targeted antibodies and CAS-006 have the same or closely overlapping epitopes.

EXAMPLE 7

EFFECT OF CAS-024 DOSE IN THE MODEL OF INTRODUCED SUBCUTANEUM XENOTRANSPLANTATE OF HUMAN TUMOR (DOHH2) ON SCID MICE

The purpose of this experiment was to check the effect of dose in the treatment using CAS-024 in the model of injected human subcutaneous tumor xenograft (DOHH2) on SCID mice. DOHH2 is a CD20⁺CD37⁺ Human B-lymphoblastoid cell line obtained from a patient with follicular lymphoma (Kluin-Nelemans et al., Leukemia 5: 221, 1991). Thus, DOHH2 was obtained from a patient with a non-Kirkitt NHL.

Five million DOHH2 cells were subcutaneously injected into the female CB-17SCID mice (Harlan, Somerville, NJ) subcutaneously at the age of 6.5 weeks and at an average weight of 18.0 ± 0.1 g (ranging from 14.6 to 22.6 grams ). At day 8 after tumor inoculation, palpable tumors were distinguishable in most mice. The tumor carriers of the mice were sorted into four groups with equal average tumor volumes (n = 14 in the group, 2 cells in 5 mice and 1 cell with 4 mice for each group). The day, when sorting was performed, was determined as day 0. Diameters of tumors were determined with a pair of circulations, and tumor volumes were calculated using the formula: V = 1/2 [length × (width)²]. The initial mean tumor volume was 228 mm³, the median baseline tumor size was 224 mm³, and the range was from 179 to 284 mm³.

<tables num="20"><table frame="all"><tgroup cols="4" rowsep="1" colsep="1"><colspec colname="c0" colwidth="14mm" /><colspec colname="c1" colwidth="15mm" /><colspec colname="c2" colwidth="62mm" /><colspec colname="c3" colwidth="87mm" /><tbody><row><entry align="right" namest="c0" nameend="c3" rowsep="1" colsep="0">Table 7 Reagents for use <i>in vivo</i></entry></row><row><entry align="center" rowsep="1" colsep="1">Reagent</entry><entry align="center" rowsep="1" colsep="1">% POI</entry><entry align="center" rowsep="1" colsep="1">Concentration and endotoxin</entry><entry align="center" rowsep="1" colsep="0">The drug for injection</entry></row><row><entry align="center" rowsep="0" colsep="1">PBS</entry><entry align="center" rowsep="0" colsep="1">n.p.</entry><entry align="left" rowsep="0" colsep="1">1 × Endotoxin &lt;0.03 EE / mg</entry><entry align="center" rowsep="0" colsep="0">n.p.</entry></row></tbody></tgroup></table></tables>

<tables num="21"><table frame="all"><tgroup cols="4" rowsep="1" colsep="1"><colspec colname="c0" colwidth="14mm" /><colspec colname="c1" colwidth="37mm" /><colspec colname="c2" colwidth="40mm" /><colspec colname="c3" colwidth="87mm" /><tbody><row><entry align="center" rowsep="1" colsep="1">Human IgG (huIgG)</entry><entry align="center" rowsep="1" colsep="1">Not tested</entry><entry align="left" rowsep="1" colsep="1">10 mg / ml Endotoxin = 10 OE / mg</entry><entry align="left" rowsep="1" colsep="0">Diluted to 1.0 mg / ml in PBS</entry></row><row><entry align="center" rowsep="0" colsep="1">CAS-024</entry><entry align="center" rowsep="0" colsep="1">100</entry><entry align="left" rowsep="0" colsep="1">9.6 mg / ml Endotoxin = 0.01 EE / mg</entry><entry align="left" rowsep="0" colsep="0">Diluted to 1.0 mg / ml in PBS to obtain a dose of 200 μg; then diluted 1: 2 to obtain a dose of 100 μg, then serially diluted 1: 3 to obtain solutions of other doses.</entry></row></tbody></tgroup></table></tables>

The tumor-bearing groups of SCID mice were treated on day 0, 4 and 8 by intraperitoneal injection of 0.2 ml of PBS containing 200 μg of huIgG (negative control) or 200, 100, 30, 10 or 3 μg of CAS-024. On the day of administration, two solutions with the lowest dose of CAS-024 were obtained to avoid the need to add a carrier protein to the most dilute solutions. Drug solutions had color coding, as described below (see Table 8 below).

<tables num="22"><table frame="all"><tgroup cols="6" rowsep="1" colsep="1"><colspec colname="c0" colwidth="21mm" /><colspec colname="c1" colwidth="48mm" /><colspec colname="c2" colwidth="26mm" /><colspec colname="c3" colwidth="27mm" /><colspec colname="c4" colwidth="28mm" /><colspec colname="c5" colwidth="27mm" /><tbody><row><entry align="right" namest="c0" nameend="c5" rowsep="1" colsep="0">Table 8 Experimentation</entry></row><row><entry align="center" rowsep="1" colsep="1">Group ID</entry><entry align="center" rowsep="1" colsep="1">Number of mice, route of administration and day of administration</entry><entry align="center" rowsep="1" colsep="1">Dose for injection (mcg)</entry><entry align="center" rowsep="1" colsep="1">mg / kg per injection^a</entry><entry align="center" rowsep="1" colsep="1">Total dose (μg)</entry><entry align="center" rowsep="1" colsep="0">The total dose (~ mg / kg)^a</entry></row><row><entry align="center" rowsep="1" colsep="1">huIgG</entry><entry align="justify" morerows="5" rowsep="1" colsep="1">14 per group and / n injectioninjection at day 0, 4 and 8</entry><entry align="center" rowsep="1" colsep="1">200</entry><entry align="center" rowsep="1" colsep="1">11.1</entry><entry align="center" rowsep="1" colsep="1">600</entry><entry align="center" rowsep="1" colsep="0">33</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-024200</entry><entry align="center" rowsep="1" colsep="1">200</entry><entry align="center" rowsep="1" colsep="1">11.1</entry><entry align="center" rowsep="1" colsep="1">600</entry><entry align="center" rowsep="1" colsep="0">33</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-024100</entry><entry align="center" rowsep="1" colsep="1">100</entry><entry align="center" rowsep="1" colsep="1">5.6</entry><entry align="center" rowsep="1" colsep="1">300</entry><entry align="center" rowsep="1" colsep="0">16.7</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-02430</entry><entry align="center" rowsep="1" colsep="1">thirty</entry><entry align="center" rowsep="1" colsep="1">1.7</entry><entry align="center" rowsep="1" colsep="1">90</entry><entry align="center" rowsep="1" colsep="0">5.0</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-02410</entry><entry align="center" rowsep="1" colsep="1">10</entry><entry align="center" rowsep="1" colsep="1">0.6</entry><entry align="center" rowsep="1" colsep="1">thirty</entry><entry align="center" rowsep="1" colsep="0">1.7</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-0243</entry><entry align="center" rowsep="1" colsep="1">3</entry><entry align="center" rowsep="1" colsep="1">0.2</entry><entry align="center" rowsep="1" colsep="1">9</entry><entry align="center" rowsep="1" colsep="0">0.5</entry></row><row><entry align="justify" namest="c0" nameend="c5" rowsep="0" colsep="0">^a It should be noted that huIgG and CAS-024 were delivered in μg per mouse, not in mg / kg. Approximate mg / kg are given for convenience and are based on the average weight (18.0 ± 0.1 g) of mice per day 0. The mass range in this experiment was 14.6 to 22.6 g.</entry></row></tbody></tgroup></table></tables>

Dosage solutions were obtained in similar volumes and the contents of the tubes indicated on the removed labels. A researcher who did not participate in the treatment or evaluation of mice placed a color code on each tube and indicated the code and the corresponding contents of the tube in the laboratory log. The mice were daily monitored by visual inspection. The mass was determined weekly, and the diameter of the tumors was determined at least 3 times a week (Mon, Wed, Fri) by an observer who did not know the information about the treatment groups (see above). The tumor volume was calculated as described above. The mouse was euthanized if the tumor volume was more than 1500 mm³ (or 1200 mm³ On Friday). Death was not the endpoint in the protocols of tumor progression and, unless indicated otherwise, the "survival" of the mouse was determined by the time it was euthanized due to a tumor exceeding the volume of predetermined limits. (According to the protocol, euthanasia mice were required to be exposed if (1) tumor volumes exceeded the above parameters, (2) tumor ulceration occurred, (3) the tumor interfered with the mobility of the mouse, and (4) weight loss exceeded 20 percent of body weight.

One of the mice in the CAS-024 treatment group 100 μg was euthanized on day 35 because of a weight loss&gt; 20%. At this point in time, the tumor volume in this mouse was 266 mm³, and it was treated as censored data for survival analysis (the mouse was not euthanized as of day 35 due to tumor growth). When calculating the frequency of the absence of tumors at the end of the study, this mouse was classified as a mouse that was euthanized during the study due to the growth of its tumor (its tumor grew again at the time of its death). No other mouse was found dead and no one was euthanized due to weight loss, tumor ulceration, or decreased mobility. No clear signs of toxicity or weight loss were observed in any treatment group (data not shown).

All statistical studies were conducted using the GraphPad Prism software. Significant differences in mean tumor volumes and mean relative tumor volumes were determined using a single-factor variance analysis for nonparametric data (the Kruskal-Wallis criterion) with the a posteriori Dunn's multiple comparison test. To check the differences between all treatment groups CAS-024 and the huIgG group, all groups were compared. For comparison, only the CAS-024 groups were excluded from the huIgG group. In addition, groups with high and medium doses (200, 100 and 30 μg) were analyzed as one set of data, and groups with medium and low doses (30, 10 and 3 μg) were analyzed as another set of data. Significant differences in the survival of mice over time were determined using the Kaplan-Meier survival analysis with a log rank test to compare the survival curves. Significant differences in the incidence of tumor failure in mice were determined using the exact Fisher test, meaningful values ​​of p &lt;0.05 were considered significant.

CAS-024 had a dose-dependent inhibitory effect on the growth of DOHH2 tumors. With the exception of the low-dose treatment group (3 μg), the average tumor volume in each treatment group of CAS-024 was significantly lower than in the human IgG treatment group already on day 5 and remained lower to day 12. Mice treated with huIgG , subjected to euthanasia, starting at day 12; Thus, comparison of tumor volumes in the treatment groups CAS-024 and in the huIgG group did not occur at later times. In dose-response terms, significant differences in mean tumor volumes in the two highest-dose groups were not observed at any time in the study. In contrast, the mean tumor volumes in these two groups differed significantly from the average tumor volumes in each of the three groups with lower doses from day 12 to day 16 (day 16 was the last time estimated for the low dose group). Similarly, the average tumor volumes in mice from the 30 μg and 10 μg dosing groups differed from each other and from the low dose group over a given period of time.

Tumors in mice treated with huIgG grew rapidly and all mice in this group were euthanized at day 19. As summarized in Tables 9 and 10 below, the survival rate of mice treated with any dose of CAS-024 was prolonged with respect to the huIgG treatment group (p &lt;0.0001 in all cases). In terms of dose-effect, there were no significant differences in the survival curves of mice treated with the highest-dose regimens (200 and 100 μg) (p = 0.7091). With the exception of this group comparison, there were significant differences between the survival curve for each dosing group and the survival curve for each group treated in a lower dose regimen (p values ​​ranging from 0.0132 to &lt;0.0001).

<tables num="23"><table frame="all"><tgroup cols="6" rowsep="1" colsep="1"><colspec colname="c0" colwidth="19mm" /><colspec colname="c1" colwidth="28mm" /><colspec colname="c2" colwidth="36mm" /><colspec colname="c3" colwidth="25mm" /><colspec colname="c4" colwidth="38mm" /><colspec colname="c5" colwidth="31mm" /><tbody><row><entry align="right" namest="c0" nameend="c5" rowsep="1" colsep="0">Table 9 Average survival time and frequency of tumor absence in mice</entry></row><row><entry align="center" rowsep="1" colsep="1">Group ID^a</entry><entry align="center" rowsep="1" colsep="1">Total dose</entry><entry align="center" rowsep="1" colsep="1">Average survival time (day)^b</entry><entry align="center" rowsep="1" colsep="1">Death (not because of the large volume of the tumor)</entry><entry align="center" rowsep="1" colsep="1">Frequency of absence of tumors at the end of the study^c</entry><entry align="center" rowsep="1" colsep="0">The value of p for the exact Fisher test (comparison of the frequency of the absence of tumors)^d</entry></row><row><entry align="center" rowsep="1" colsep="1">huIgG200</entry><entry align="center" rowsep="1" colsep="1">600 μg</entry><entry align="center" rowsep="1" colsep="1">14</entry><entry align="center" rowsep="1" colsep="1">0/14</entry><entry align="center" rowsep="1" colsep="1">0/14 (0%)</entry><entry align="center" rowsep="1" colsep="0">N.p.</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-024200</entry><entry align="center" rowsep="1" colsep="1">600 μg</entry><entry align="center" rowsep="1" colsep="1"><b>Undefined</b>^ef</entry><entry align="center" rowsep="1" colsep="1">0/14</entry><entry align="center" rowsep="1" colsep="1"><b>11/14 (79%)</b>^g</entry><entry align="center" rowsep="1" colsep="0">&lt;0.0001</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-024100</entry><entry align="center" rowsep="1" colsep="1">300 μg</entry><entry align="center" rowsep="1" colsep="1"><b>Undefined</b></entry><entry align="center" rowsep="1" colsep="1"><b>1/14</b>^h</entry><entry align="center" rowsep="1" colsep="1"><b>11/14 (79%)</b></entry><entry align="center" rowsep="1" colsep="0">&lt;0.0001</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-02430</entry><entry align="center" rowsep="1" colsep="1">90 μg</entry><entry align="center" rowsep="1" colsep="1"><b>35</b></entry><entry align="center" rowsep="1" colsep="1">0/14</entry><entry align="center" rowsep="1" colsep="1"><b>5/14 (36%)</b></entry><entry align="center" rowsep="1" colsep="0">0.0407</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-02410</entry><entry align="center" rowsep="1" colsep="1">30 μg</entry><entry align="center" rowsep="1" colsep="1"><b>28</b></entry><entry align="center" rowsep="1" colsep="1">0/14</entry><entry align="center" rowsep="1" colsep="1">0/14 (0%)</entry><entry align="center" rowsep="1" colsep="0">N.p.</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-0243</entry><entry align="center" rowsep="1" colsep="1">9 μg</entry><entry align="center" rowsep="1" colsep="1"><b>19</b></entry><entry align="center" rowsep="1" colsep="1">0/14</entry><entry align="center" rowsep="1" colsep="1">0/14 (0%)</entry><entry align="center" rowsep="1" colsep="0">N.p.</entry></row><row><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" namest="c0" nameend="c5" rowsep="0" colsep="0">^a The mice were treated with the indicated protein via IV injections on days 0, 4 and 8. The numbers indicate the amount of protein (μg) injected per day.^b "Survival" of the mouse was determined by those days, when it was euthanized due to tumor growth. One mouse in the CAS-024 dosing group 100 μg was euthanized on day 35 due to a weight loss&gt; 20%. At this point, the tumor volume of the mouse was 266 mm³, and it was treated as censored data (the tumor volume did not reach a predetermined limit on day 35) for Kaplan-Meier analysis. No other mouse was euthanized for reasons other than reaching a predetermined limit of tumor volume.^c In mice without tumors, there were no palpable subcutaneous tumors.</entry></row></tbody></tgroup></table></tables>

<tables num="24"><table frame="none"><tgroup cols="2" rowsep="0" colsep="0"><colspec colname="c0" colwidth="175mm" /><colspec colname="c1" colwidth="5mm" /><tbody><row><entry align="justify" rowsep="0" colsep="0">The absence of tumor cells was not confirmed by histological examination. The study was completed on day 61.^d Each group was compared to a control group that was treated with huIgG.^e The mean survival time was not determined when&gt; 50% of the mice were alive by the end of the observation period.^f Values ​​in bold indicate that the survival curves for this group were significantly different from the survival curves for control with huIgG (p &lt;0.0001 in each case, the log-rank test).^g Values ​​in bold are significantly different from the control group treated with huIgG.^h One mouse was euthanized on day 35 due to a mass loss&gt; 20%. At this point, the tumor volume in this mouse was 266 mm³ and it was treated as censored data for the Kaplan-Meier analysis.</entry><entry align="left" rowsep="0" colsep="0"> </entry></row></tbody></tgroup></table></tables>

<tables num="25"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="37mm" /><colspec colname="c1" colwidth="73mm" /><colspec colname="c2" colwidth="68mm" /><tbody><row><entry align="right" namest="c0" nameend="c2" rowsep="1" colsep="0">Table 10 Values ​​of p for comparison of survival curves and the incidence of tumors among treatment groups CAS-024</entry></row><row><entry align="center" morerows="1" rowsep="1" colsep="1">Comparison of groups^a</entry><entry align="center" namest="c1" nameend="c2" rowsep="1" colsep="0">The values ​​of p for these comparisons</entry></row><row><entry align="center" rowsep="1" colsep="1">Logarithmic rank test (comparison of survival curves)</entry><entry align="center" rowsep="1" colsep="0">Fisher's exact test (comparison of the frequency of the absence of tumors)</entry></row><row><entry align="justify" rowsep="1" colsep="1">200 in comparison with 100</entry><entry align="center" rowsep="1" colsep="1">0,7091</entry><entry align="center" rowsep="1" colsep="0">1,0000</entry></row><row><entry align="justify" rowsep="1" colsep="1">200 in comparison with 30</entry><entry align="center" rowsep="1" colsep="1"><b>0,0132</b>^b</entry><entry align="center" rowsep="1" colsep="0">0.0542</entry></row><row><entry align="justify" rowsep="1" colsep="1">200 in comparison with 10</entry><entry align="center" rowsep="1" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="1" colsep="0"><b>&lt;0.0001</b></entry></row><row><entry align="justify" rowsep="1" colsep="1">200 in comparison with 3</entry><entry align="center" rowsep="1" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="1" colsep="0"><b>&lt;0.0001</b></entry></row><row><entry align="justify" rowsep="1" colsep="1">100 compared to 30</entry><entry align="center" rowsep="1" colsep="1"><b>0,0035</b></entry><entry align="center" rowsep="1" colsep="0">0.0542</entry></row><row><entry align="justify" rowsep="0" colsep="1">100 versus 10</entry><entry align="center" rowsep="0" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="0" colsep="0"><b>&lt;0.0001</b></entry></row></tbody></tgroup></table></tables>

<tables num="26"><table frame="all"><tgroup cols="3" rowsep="1" colsep="1"><colspec colname="c0" colwidth="37mm" /><colspec colname="c1" colwidth="73mm" /><colspec colname="c2" colwidth="68mm" /><tbody><row><entry align="justify" rowsep="1" colsep="1">100 versus 3</entry><entry align="center" rowsep="1" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="1" colsep="0"><b>&lt;0.0001</b></entry></row><row><entry align="justify" rowsep="1" colsep="1">30 versus 10</entry><entry align="center" rowsep="1" colsep="1"><b>0.0002</b></entry><entry align="center" rowsep="1" colsep="0"><b>0.0407</b></entry></row><row><entry align="justify" rowsep="1" colsep="1">30 in comparison with 3</entry><entry align="center" rowsep="1" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="1" colsep="0"><b>0.0407</b></entry></row><row><entry align="justify" rowsep="1" colsep="1">10 in comparison with 3</entry><entry align="center" rowsep="1" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="1" colsep="0">N.p.</entry></row><row><entry align="justify" namest="c0" nameend="c2" rowsep="0" colsep="0">^a For information about the groups, see the labels in Table 7.^b Values ​​of p &lt;0.05 are highlighted in bold for selection.</entry></row></tbody></tgroup></table></tables>

All mice in the huIgG treatment group and in the two groups with the lowest CAS-024 doses (10 and 3 μg) were euthanized due to the growth of their tumors. In contrast, most tumors in groups of mice treated with 200 or 100 μg of CAS-024 regressed to a condition in which palpable tumors were absent. At the end of the study, 11/14 (79%) of mice in each of the two highest-dose groups and 5/14 (36%) of mice in the dosing group of 30 μg remained without tumors (p &lt;0.0001 and 0.0407, in comparison with the group huIgG).

Thus, CAS-024 exerted a dose-dependent inhibitory effect on the growth of injected human subcutaneous tumor xenografts (DOHH2) in SCID mice. Two regimens with the highest doses (100 or 200 μg in one intraperitoneal injection, a total dose of 300 or 600 μg, corresponding to approximately 16.7 or 33 mg / kg, respectively) had a similar inhibitory effect and were most effective among the tested regimens from the point of view vision inhibition of tumor growth, prolongation of survival and induction of complete tumor regression.

EXAMPLE 8

EFFECTIVENESS OF CAS-024 AND RITUXANE® AS SEPARATE PRODUCTS IN THE MODEL OF INTRODUCED XENOTRANSPLANTATE OF HUMAN TUMOR (DOHH2) ON SCID MICE

The aim of this study was to study the efficacy of CAS-024 and rituxan as separate agents in the model of injected human tumor xenograft (DOHH2) on SCID mice. As indicated above, DOHH2 is a CD20⁺CD37⁺ B-human lymphoblastoid cell line obtained from a patient with follicular lymphoma.

Five million DOHH2 cells were injected subcutaneously in the side of female CB-17SCID mice (Harlan, Somerville, NJ) at the age of 6.5 weeks. At day 8 after tumor inoculation, palpable tumors in most mice had a distinguishable tumor. The tumor bearing mice were sorted into four groups (n = 15 in the group, 3 cells of 5 mice for each group) with equal average tumor volumes. The day, when sorting was performed, was determined as the day of study 0. Diameters of tumors were determined with a pair of compasses, and the tumor volumes were calculated using the formula: V = 1/2 [length × (width)²]₃. The initial mean tumor volume was 228 mm³; the average initial tumor size was 227 mm³, and the range was from 181 to 272 mm³. Mice (15 per treatment group) were treated on days 0, 4 and 8 by intraperitoneal injection of 0.2 ml of PBS containing 200 μg of human IgG, CAS-024 or rituxan® (total 600 μg after three administrations). For groups with intraperitoneal treatment using huIgG, CAS-024 and rituxan® intraperitoneally, similar volumes of solutions were obtained and the contents of the tubes indicated on the removed labels. A researcher who did not participate in the treatment or evaluation of mice placed a color code on each tube and indicated the code and the corresponding contents of the tube in the laboratory log.

The mice were daily monitored by visual inspection. The mass was determined weekly, and the diameter of the tumors was determined at least 3 times a week (Mon, Wed, Fri) by an observer who did not know the information about the treatment groups (see above). The tumor volume was calculated as described above. The volumes of tumors on the last day, when all mice were alive, in each group were also expressed in terms of tumor volume relative to day 0, using the following formula:

Relative tumor volume in the days of interest = (volume in the days of interest - volume per day 0) / volume per day 0

The mice were euthanized if the tumor volume was more than 1500 mm³ (or 1200 mm³ On Friday). Death was not the endpoint in the protocols of tumor progression and, unless indicated otherwise, the "survival" of the mouse was determined by the time it was euthanized due to a tumor exceeding the volume of predetermined limits. (The authors' protocol required the euthanasia mice to be exposed if the tumor volumes were higher than the above values, the tumor was tumor ulcerated, the tumor interfered with the mobility of the mouse, or the weight loss exceeded 20% of body weight.)

All statistical studies were conducted using the GraphPad Prism software. Significant differences in mean tumor volumes and mean relative tumor volumes were determined using a single-factor variance analysis for nonparametric data (the Kruskal-Wallis criterion) with the a posteriori Dunn's multiple comparison test. Significant differences in the survival of mice over time were determined using the Kaplan-Meier survival analysis with a log rank test to compare the survival curves. Significant differences in the incidence of tumor failure in mice were determined using the exact Fisher test (significant values ​​were p &lt;0.05).

When tumor volumes in mice reached the above limits, they were euthanized. One mouse in the treatment group CAS-024 was euthanized on day 45 due to a weight loss&gt; 20%. This mouse did not have an obvious subcutaneous tumor at this point in time, and it was treated as censored data for survival analysis (the mouse was not euthanized on day 45 due to tumor growth) and was not included in the comparison of tumor-free frequencies at the end of the study. No other mouse was found dead and none were euthanized due to weight loss, tumor ulceration, or decreased mobility. No clear signs of toxicity or weight loss were observed in any treatment group (data not shown).

Mice treated with CAS-024 and rituxan showed a quick response to treatment. The average tumor volumes in the CAS-024 and rituxan® treatment groups were significantly lower than the average tumor volumes in the human IgG treatment group, already on day 4 (after one injection of the drug) and remained lower to day 11. Significant differences in mean volumes tumor or the average relative tumor volumes between the treatment groups CAS-024 and Rituxan® to day 11 were absent. Mice treated with huIgG were euthanized from day 11; Thus, at a later time, comparison of tumor volumes was not performed.

In mice treated with huIgG, the tumors grew rapidly and all mice in this group were euthanized at day 15. In contrast, most tumors in the treatment groups CAS-024 and rituxan on day 15 regressed to a state in which there were no palpable tumor. It is noteworthy that the response to treatment was long-term only in the treatment group CAS-024. By the end of the study, all mice treated with rituxan were euthanized due to growth of their tumors, whereas 10/14 (71%) of mice in the CAS-024 treatment group remained without tumors. See Table 9. Thus, at the end of the study, the survival curves and tumor absence rate in mice in the CAS-024 treatment group differed significantly from the control group huIgG and the rituxan® treatment group. In Fig. 6 shows,<i>in vivo</i>.

<tables num="27"><table frame="all"><tgroup cols="7" rowsep="1" colsep="1"><colspec colname="c0" colwidth="8mm" /><colspec colname="c1" colwidth="42mm" /><colspec colname="c2" colwidth="27mm" /><colspec colname="c3" colwidth="32mm" /><colspec colname="c4" colwidth="24mm" /><colspec colname="c5" colwidth="19mm" /><colspec colname="c6" colwidth="25mm" /><tbody><row><entry align="right" namest="c0" nameend="c6" rowsep="1" colsep="0">Table 11 Average survival time and frequency of tumor absence in mice</entry></row><row><entry align="center" rowsep="1" colsep="1">Treatment group</entry><entry align="center" rowsep="1" colsep="1">Day of treatment and total dose</entry><entry align="center" rowsep="1" colsep="1">Average survival time (day)^a</entry><entry align="center" rowsep="1" colsep="1">The value of p in the log rank criterion^b</entry><entry align="center" rowsep="1" colsep="1">Death (other than euthanasia due to the size of the tumor)</entry><entry align="center" rowsep="1" colsep="1">Mice without tumors per day 81^c</entry><entry align="center" rowsep="1" colsep="0">Fisher's exact test (comparison of the frequency of the absence of tumors)^b</entry></row><row><entry align="center" rowsep="1" colsep="1">huIgG</entry><entry align="center" rowsep="1" colsep="1">day 0, 4 and 8 day600 μg</entry><entry align="center" rowsep="1" colsep="1">13</entry><entry align="center" rowsep="1" colsep="1">-</entry><entry align="center" rowsep="1" colsep="1">0/15</entry><entry align="center" rowsep="1" colsep="1">0/15 (0%)</entry><entry align="center" rowsep="1" colsep="0">n.p.</entry></row><row><entry align="center" rowsep="1" colsep="1">CAS-024POI</entry><entry align="center" rowsep="1" colsep="1">day 0, 4 and 8600 μg</entry><entry align="center" rowsep="1" colsep="1"><b>Undefined</b>^{d, e}</entry><entry align="center" rowsep="1" colsep="1">&lt;0.0001</entry><entry align="center" rowsep="1" colsep="1">1/15^f</entry><entry align="center" rowsep="1" colsep="1"><b>10/14 (71%)</b>^f</entry><entry align="center" rowsep="1" colsep="0">&lt;0.0001</entry></row><row><entry align="center" rowsep="1" colsep="1">Rituxan® POI</entry><entry align="center" rowsep="1" colsep="1">day 0, 4 and 8600 μg</entry><entry align="center" rowsep="1" colsep="1"><b>43</b></entry><entry align="center" rowsep="1" colsep="1">&lt;0.0001</entry><entry align="center" rowsep="1" colsep="1">0/15</entry><entry align="center" rowsep="1" colsep="1">0/15 (0%)</entry><entry align="center" rowsep="1" colsep="0">n.p.</entry></row><row><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry><entry align="left" rowsep="0" colsep="0"> </entry></row><row><entry align="justify" namest="c0" nameend="c6" rowsep="0" colsep="0">^a "Survival" was determined by those days when the mouse was euthanized due to tumor growth. With the exception of one mouse in the dosing group CAS-024 (see (f)), no mouse was euthanized for reasons other than that the tumor volume had previously reached</entry></row></tbody></tgroup></table></tables>

<tables num="28"><table frame="none"><tgroup cols="2" rowsep="0" colsep="0"><colspec colname="c0" colwidth="175mm" /><colspec colname="c1" colwidth="5mm" /><tbody><row><entry align="justify" rowsep="0" colsep="0">given limit.^b Each group was compared to a control group that was treated with huIgG.^c Mice without tumors had no palpable subcutaneous tumors; the absence of tumor cells was not confirmed by histological examination.^d The mean survival time was not determined when&gt; 50% of the mice remained alive at the end of the observation period.^e The values ​​in bold are significantly different from the values ​​for huIgG control.^f One mouse was euthanized on day 45 due to a loss of mass&gt; 20%. This mouse did not have an obvious subcutaneous tumor at this point and was excluded from the group for comparison of mice without tumors per day 81.</entry><entry align="left" rowsep="0" colsep="0"> </entry></row></tbody></tgroup></table></tables>

In conclusion, CAS-024 and rituxan demonstrated efficacy as separate agents in the human tumor xenograft model (DOHH2) on SCID mice. Although both agents caused tumor regression in most mice, prolonged tumor regression was observed only in the group of mice treated with CAS-024, as the tumors recurred after optimal treatment against CD20. Thus, CAS-024, a humanized SMIP to CD37, demonstrates significant efficacy in preclinical tumor xenograft models, including models that demonstrate that treatment with rituxan® over time is unsuccessful. Thus, these results indicate that,

EXAMPLE 9

ASSESSMENT OF CAS-024 COMBINED WITH CHEMOTHERAPEUTIC AGENTS, <i>IN VITRO</i>

It was previously shown that CAS-006 in combination with a chemotherapeutic agent fludarabine acts synergistically, killing cells of chronic lymphocytic leukemia (CLL) <i>in vitro</i> (see, for example, US Patent Application Publication No. 2007/0059306). Because CLL cells do not actively divide in cell culture<i>in vitro</i>, these data indicate that cell proliferation is not necessary for the proapoptotic effect of CAS-006 or CAS-024 for their synergistic interaction with chemotherapeutic agents. Thus, the aim of this study was to determine the efficacy of CAS-024 and various chemotherapeutic agents for the lymphoma cell line from the cells of the mantle zone (LKMZ), Rec-1, which are actively growing and dividing in cell culture<i>in vitro</i>, and determining whether the combination of CAS-024 and the chemotherapeutic agent (drug) will reduce sensitivity or enhance the response of lymphoma cells from the cells of the mantle zone to various chemotherapeutic agents. The chemotherapeutic agents doxorubicin, vincristine and fludarabine have been tested, which are used to treat non-Hodgkin's lymphoma and other lymphoid malignancies.

Cells Rec-1, CD37⁺ The human B cell line obtained from a patient with lymphoma from mantle cell cells was tested for growth inhibition in response to crosslinked CAS-024 in the presence or absence of doxorubicin, vincristine or fludarabine (see Figure 7). CAS-024 was preincubated with F (ab) '₂ to human IgG for cross-linking proteins. Cells were cultured only in medium or in medium containing different concentrations of cross-linked CAS-024 protein, in the presence or absence of different concentrations of doxorubicin, vincristine, or fludarabine. The cultures were incubated for 96 hours, and the growth inhibition was evaluated using the ATP detection system for viable cells (i.e., the number of viable cells was determined by ATP release).

For the data analysis, the mean effect / combination index (CI) method Chou and Talalay (Adv., Enzyme Regul., 22:27, 1984) was used. The numerical value assigned to each combination of drugs with predetermined dose levels allows for quantitative comparisons of drug interactions between different drug combinations. The results were expressed as combination indices (CI), depending on the effect level, where the level of effect was the percentage inhibition of cell growth. The mean CI ± standard error of the mean for each level of effect was averaged over three experiments. CI &lt;1.0 was considered as synergism, CI = 1.0 as additive action and CI&gt; 1.0 as antagonism. The values ​​given are the mean ± standard error of the mean for each effect level,

The combination of CAS-024 with vincristine or fludarabine was synergistic (CI &lt;1.0), and the combination of CAS-024 and doxorubicin was additive (CI slightly differed from 1.0). None of the combinations of CAS-024 and the chemotherapeutic agent was antagonistic (CI&gt; 1.0) at all levels of the effect. Thus, the tested CAS-024 combinations with each of the three chemotherapeutic agents did not reduce the sensitivity of the target cells to inhibition of growth induced by the drug, but instead provided a synergistic or additive inhibitory effect on the growth of target cells. A preferred embodiment is the combination of CAS-024 (SEQ ID NO: 253) with vincristine or fludarabine. These data indicate that,

EXAMPLE 10

RESULTS OF THE PRELIMINARY PHASE 1/2 CLINICAL STUDIES

As indicated in this paper, preclinical studies have shown that SMIP molecules to CD37 mediate a much better direct and mediated by natural killer (NK) destruction of cells of chronic lymphocytic leukemia (CLL) compared to other therapeutic antibodies used in CLL. Consequently, in patients with recurrence of chronic lymphocytic leukemia (CLL), they proceeded to phase 1/2, an open-label study with increasing doses.

Patients with recurrent / resistant CLL or small cell lymphoma (SLL) with adequate organ function were selected, platelets&gt; 30000 / mm³. Six doses and two different regimens (groups 1-10) were examined or examined. Planned doses range from 0.03 mg / kg to 10 mg / kg iv once a week for 4 doses (groups 1-6 and 9). In the second scheme (groups 7, 8 and 10), 3.0, 6.0 or 10.0 mg / kg for days 1, 3 and 5 of the first week are tested, followed by 3 weekly doses. Dose increase and reduction is based on Common Toxicity Criteria Adverse Events (CTC AE) toxicity. Patients can undergo 2 additional cycles in the case of a positive biological effect after the first cycle.

<u>results</u>: At present, 22 patients (groups 1-7 and 9) have been registered and completed treatment (all previously treated with fludarabine and rituximab). Six patients started the second cycle and two patients started the third cycle. Patients undergoing treatment underwent a number of previous regimens (for example, patients from group 4 passed from 6 to 10 (median 6), and from group 5 from 5 to 13 (median 9.5) of previous regimens). In eight out of ten, there was a risk of genomic lesions [del (17p13.1), n ​​= 5 and del (11q22.3), n = 3]. Toxic effects that limit the dose, or serious undesirable consequences, did not develop. Three patients had an average infusion toxicity (1-2 degrees). Starting at a dose of 0.3 mg / kg, all eight patients showed signs of biological activity, including patients with del (17p13.1). Two patients had a partial disappearance of hematodermatosis, and the average decrease in the number of peripheral lymphocytes was 64% (see Figure 5). One patient demonstrated a 99% reduction in the number of peripheral lymphocytes without serious adverse events and continued to respond after 3 months of treatment (see Figure 6). One patient experienced an increase in hemoglobin by 40% and a decrease in the size of the lymph nodes by 36%, which was determined by CT scan, and he continues to respond after 3 months of treatment (see Figure 7). Two patients had a significant increase in the number of platelets. One patient demonstrated a 99% reduction in the number of peripheral lymphocytes without serious adverse events and continued to respond after 3 months of treatment (see Figure 6). One patient experienced an increase in hemoglobin by 40% and a decrease in the size of the lymph nodes by 36%, which was determined by CT scan, and he continues to respond after 3 months of treatment (see Figure 7). Two patients had a significant increase in the number of platelets. One patient demonstrated a 99% reduction in the number of peripheral lymphocytes without serious adverse events and continued to respond after 3 months of treatment (see Figure 6). One patient experienced an increase in hemoglobin by 40% and a decrease in the size of the lymph nodes by 36%, which was determined by CT scan, and he continues to respond after 3 months of treatment (see Figure 7). Two patients had a significant increase in the number of platelets.

<u>Conclusion</u>: To date, this SMIP molecule to CD37 is a well tolerated drug with minimal infusion toxicity and without the detected dose limiting toxicity. Also, it seems that complement is involved to some degree, as patients with a strong decrease in the number of lymphocytes do not show signs of tumor lysis syndrome. An encouraging decrease in the number of lymphocytes in the blood, a decrease in the size of the lymph nodes / spleen, the disappearance of hematodermatosis, and / or a partial disappearance of bone marrow disease and / or an improvement in normal hematopoietic function in patients at high risk of genomic CLL was observed already at low unsaturated doses of the SMIP molecule to CD37.

EXAMPLE 11

EFFECTIVENESS OF CAS-024 IN THE COMBINATION WITH BENDAMUSTIN <i>IN VITRO</i>

This study was conducted to determine the effects of CAS-024, bendamustine, and the combination of CAS-024 and bendamustine on Rec-1 cells (the cell line of lymphoma from cells in the mantle zone) and SU-DHL-6 (diffuse large cell cell lymphoma cell line).

The following human cell lines expressing CD37: Rec-1 and SU-DHL-6 (both from DSMZ, Braunschweig, Germany) were used. Bendamustine (TREENDA®) was purchased at the University of Washington Pharmacy (Seattle, WA), dissolved in PBS and stored at -20 ° C until use.

The Rec-1 and SU-DHL-6 cells were plated at a concentration of 1 × 10⁴ cells / well in 100 μl medium in 96-well plates with black walls and a bottom. The cells were treated with various concentrations of CAS-024 that had been preincubated with F (ab) '2 to human IgG and the plates were incubated for 96 hours at 37 ° C, 5% CO₂ in the presence of serial dilutions of bendamustine. The final volume in each well was 150 μl. After incubation, the plates were cooled to room temperature and labeled using a detection reagent ATPlite at a concentration of 100 μl / well (Perkin Elmer, Boston, MA). In this test, cellular ATP was changed as a marker of viable cells. Samples were analyzed by detection of luminescence using a Topcount NXT scanner (Perkin Elmer, Waltham, MA). The data was converted using the interpolation of the 4-parameter curve in Prism (version 4.0, Graphpad Software, San Diego, CA) and determined IC₅₀ as a concentration leading to 50% inhibition compared to untreated cultures.

To determine the synergy, a mean effect / combination index (CI) (Chou and Talalay) was used to analyze the data. The numerical value assigned to each drug combination at predetermined dose levels makes it possible to quantitatively compare drug interactions for various drug combinations. The values ​​of CI interactions were assigned one of three categories: synergism, additivity and antagonism (CI &lt;1.0, = 1 or&gt; 1.0, respectively). After labeling and data conversion, the Raman Index values ​​(CI) were determined using the Calcusyn software package (Biosoft, Cambridge, UK). The results of two separate experiments show, that the combination of CAS-024 with bendamustine had a synergistic inhibitory effect on the growth of target cells (see Figure 11). Similar results were obtained that show that the combination of CAS-024 with bendamustine also synergistically inhibited SU-DHL-6 cell growth.

Also, using the method described above, the effect of the combination CAS-024 with another alkylating agent, chlorambucil was determined, and the concentrations are shown in FIG. 12. Unlike bendamustine, chlorambucil in combination with CAS-024 did not exert a synergistic inhibitory effect on the growth of SU-DHL-6 cells (see Figure 13)

EXAMPLE 12

EFFECTIVENESS OF CAS-024 IN THE COMBINATION WITH BENDUMUSTIN ON THE MODEL OF XENOTRANSPLANTATE OF THE HUMAN TUMOR

This study was conducted to compare the efficacy of the combination of CAS-024 and bendamustine with each agent administered alone against subcutaneous human tumor DOHH2 xenografts in SCID mice.

<i>Introduction of tumor xenografts and sorting into treatment groups</i>

As described above, DOHH2 is CD20⁺CD37⁺ B-lymphoblastoid cell line of a person obtained from a patient with follicular lymphoma. Five million DOHH2 cells were subcutaneously injected into the side of the female CB-17SCID mice. On day 8 after tumor inoculation, palpable tumors were distinguishable in most mice. The tumor bearing mice were sorted into five groups with equal average tumor volumes (n = 15 in the group, 3 cells of 5 mice for each group). The day of sorting was determined as day 0. The diameter of the tumors was determined using a pair of compasses, and the tumor volumes were calculated using the formula: V = 1/2 [length × (width)²]. The initial mean tumor volume was 231 mm³, the average baseline tumor size was 229 mm³, and the range was from 201 to 261 mm³.

<i>Treatment in vivo</i>

Groups of mice were treated with injections of 0.2 ml of PBS containing 10 μg of huIgG (day 0, 4, 8 intravenously), 10 μg of CAS-024 (day 0, 4, 8 intravenously), 10 mg / kg of bendamustine (0, 2, 4, 7, 9 intraperitoneally) or 10 μg of CAS-024 (day 0, 4, 8 intravenously) and 10 mg / kg of bendamustine (0, 2, 4, 7, 9 intraperitoneally).

<i>Surveillance and endpoints</i>

The mice were daily monitored by visual inspection. The mass was determined weekly, and the tumor diameter was determined at least 3 times a week (Mon, Wed, Fri) by an observer who did not know the information about the treatment groups (see above). The tumor volume was calculated as described above.

The mice were euthanized if the tumor volume was more than 1500 mm³ (or 1200 mm³ On Friday). Death was not the endpoint in the protocols of tumor progression and, unless indicated otherwise, the "survival" of the mouse was determined by the time it was euthanized due to a tumor exceeding the volume of predetermined limits. The mice were euthanized, if the tumor volumes were higher than the above values, the tumor was tumor ulcerated, the tumor interfered with the mobility of the mouse, or the weight loss exceeded 20%.

<i>Statistical analysis</i>

All statistical studies were carried out using the GraphPad Prism software. Significant differences in mean tumor volumes and mean relative tumor volumes were determined using a single-factor variance analysis for nonparametric data (the Kruskal-Wallis criterion) with the a posteriori Dunn's multiple comparison test. Significant differences in the survival of mice over time were determined using the Kaplan-Meier survival analysis with a log rank test to compare the survival curves. Significant differences in the incidence of tumor failure in mice were determined using the exact Fisher test, meaningful values ​​of p &lt;0.05 were considered significant.

In untreated wool and diarrhea, patients treated with bendamustine were observed starting at about 6 days. On day 10, one mouse in the bendamustine + CAS-024 treatment group was euthanized due to a loss of more<u>&gt;</u>20% of the mass. This mouse was treated as censored data for the analysis of survival curves. In the treatment group, one CAS-024 clinical signs of toxicity were not observed.

All drugs demonstrated an inhibitory effect on the growth of DOHH2 as compared to huIgG. On day 13 (which was the last day, when all mice were alive), the average tumor volume and the mean relative tumor volume in all treatment groups were statistically significantly different from the control group of mice receiving huIgG (Figures 14A and 14B). Also, between the bendamustine treatment groups and the combination of bendamustine + CAS-024, significant differences in mean tumor volumes and average relative tumor volumes were observed. Between any other two treatment groups, significant differences in mean tumor volumes and mean relative tumor volumes were absent. The dynamics of the average tumor volumes for the four groups is shown in Fig. 15.

In mice treated with huIgG, the tumors grew rapidly, and all mice in this group were euthanized at day 17. As shown in FIG. 16 and summarized in Tables 12 and 13, the survival rate of mice in any treatment group as compared to the huIgG treatment group is increased (p<u>&lt;</u>0.0001 for all groups). There was also a significant difference between the survival curves of all three treatment groups and any other, with the combination of CAS-024 / bendamustine superior to any single agent.

None of the mice treated with huIgG survived (thus, none had a tumor) until the end of the study (day 34) (Figure 17 and Table 12). The incidence of tumor absence in mice in the other groups was 0/15 (0%) in the CAS-024 and bendamustine and 2/14 (14%) treatment groups in the CAS-024 + bendamustine combination. Between any other treatment groups, there were no significant differences in the incidence of tumor absence in mice.

<tables num="29"><table frame="all"><tgroup cols="4" rowsep="1" colsep="1"><colspec colname="c0" colwidth="19mm" /><colspec colname="c1" colwidth="48mm" /><colspec colname="c2" colwidth="53mm" /><colspec colname="c3" colwidth="58mm" /><tbody><row><entry align="right" namest="c0" nameend="c3" rowsep="1" colsep="0">Table 12 Average survival time and frequency of tumor absence in mice at the end of the observation period</entry></row><row><entry align="center" rowsep="1" colsep="1">Treatment group^a</entry><entry align="center" rowsep="1" colsep="1">Day treatment</entry><entry align="center" rowsep="1" colsep="1">Average survival time (days)^a</entry><entry align="center" rowsep="1" colsep="0">Frequency of absence of tumors at the end of the study</entry></row><row><entry align="center" rowsep="1" colsep="1">huIgG</entry><entry align="center" rowsep="1" colsep="1">day 0, 4, 8</entry><entry align="center" rowsep="1" colsep="1">15</entry><entry align="center" rowsep="1" colsep="0">0/15 (0%)</entry></row><row><entry align="center" rowsep="0" colsep="1">CAS-02410 μg</entry><entry align="center" rowsep="0" colsep="1">day 0, 4, 8</entry><entry align="center" rowsep="0" colsep="1"><b>17th</b>^b</entry><entry align="center" rowsep="0" colsep="0">0/15 (0%)</entry></row></tbody></tgroup></table></tables>

<tables num="30"><table frame="all"><tgroup cols="4" rowsep="1" colsep="1"><colspec colname="c0" colwidth="19mm" /><colspec colname="c1" colwidth="48mm" /><colspec colname="c2" colwidth="53mm" /><colspec colname="c3" colwidth="58mm" /><tbody><row><entry align="justify" rowsep="1" colsep="1">Bendamustine 10 mg / kg</entry><entry align="center" rowsep="1" colsep="1">day 0, 2, 4, 7, 9</entry><entry align="center" rowsep="1" colsep="1"><b>17th</b></entry><entry align="center" rowsep="1" colsep="0">0/15 (0%)</entry></row><row><entry align="justify" rowsep="1" colsep="1">CAS-024 + bendamustine</entry><entry align="center" rowsep="1" colsep="1">day 0, 4, 80, 2, 4, 7, 9</entry><entry align="center" rowsep="1" colsep="1"><b>24</b></entry><entry align="center" rowsep="1" colsep="0">2/14 (14%)^d</entry></row><row><entry align="justify" namest="c0" nameend="c3" rowsep="0" colsep="0">^a "Survival" of the mouse was determined by those days, when it was euthanized due to tumor growth. One mouse in the treatment group with the combination CAS-024 + bendamustine was euthanized at day 10 due to loss<u>&gt;</u>20% of the mass. This mouse was treated as censored data in the calculation of survival curves. No other mouse was euthanized for reasons other than reaching a predetermined limit of tumor volume.^b The values ​​in bold indicate that the survival curves of this group differ significantly from the survival curves for huIgG control (p &lt;0.0001 for all treatment groups, the log rank test).^c Mice that had no tumors had no palpable subcutaneous tumors. The absence of tumor cells was not confirmed by histological examination. The study was completed on day 34.^d In the treatment group with the combination CAS-024 + bendamustine, one mouse was euthanized on day 10 as a result of loss <u>&gt;</u>20% of the mass. No other mouse was euthanized due to toxicity.</entry></row></tbody></tgroup></table></tables>

<tables num="31"><table frame="all"><tgroup cols="5" rowsep="1" colsep="1"><colspec colname="c0" colwidth="45mm" /><colspec colname="c1" colwidth="23mm" /><colspec colname="c2" colwidth="21mm" /><colspec colname="c3" colwidth="30mm" /><colspec colname="c4" colwidth="42mm" /><tbody><row><entry align="right" namest="c0" nameend="c4" rowsep="1" colsep="0">Table 13 Values ​​of p when comparing survival curves between treatment groups</entry></row><row><entry align="center" namest="c0" nameend="c4" rowsep="1" colsep="0">Values ​​of p for comparison of survival curves (logarithmic rank test)</entry></row><row><entry align="left" rowsep="1" colsep="1"> </entry><entry align="center" rowsep="1" colsep="1">huIgG</entry><entry align="center" rowsep="1" colsep="1">TRU-016</entry><entry align="center" rowsep="1" colsep="1">Bendamustine</entry><entry align="center" rowsep="1" colsep="0">TRU-016 + bendamustine</entry></row><row><entry align="center" rowsep="1" colsep="1">huIgG</entry><entry align="center" rowsep="1" colsep="1">n.p.</entry><entry align="center" rowsep="1" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="1" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="1" colsep="0"><b>&lt;0.0001</b></entry></row><row><entry align="center" rowsep="1" colsep="1">TRU-016</entry><entry align="center" rowsep="1" colsep="1"><b>&lt;0.0001</b>^a</entry><entry align="center" rowsep="1" colsep="1">n.p.</entry><entry align="center" rowsep="1" colsep="1"><b>0.0050</b></entry><entry align="center" rowsep="1" colsep="0"><b>0.01</b></entry></row><row><entry align="center" rowsep="1" colsep="1">Bendamustine</entry><entry align="center" rowsep="1" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="1" colsep="1"><b>0.0050</b></entry><entry align="center" rowsep="1" colsep="1">n.p.</entry><entry align="center" rowsep="1" colsep="0"><b>&lt;0.0001</b></entry></row><row><entry align="center" rowsep="0" colsep="1">TRU-016 + bendamustine</entry><entry align="center" rowsep="0" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="0" colsep="1"><b>0.01</b></entry><entry align="center" rowsep="0" colsep="1"><b>&lt;0.0001</b></entry><entry align="center" rowsep="0" colsep="0">n.p.</entry></row></tbody></tgroup></table></tables>

This study shows that CAS-024 in combination with bendamustine had an inhibitory effect on the growth of DOHH2 tumors in SCID mice more than that observed for each agent alone.

The various embodiments described above can be combined to provide additional embodiments. All US patents, US patent applications, US patent applications, foreign patents, foreign patent applications and non-patent publications referred to herein and / or listed in the Application Data Sheet are incorporated herein in their entirety by reference. Aspects of embodiments, if necessary, can be modified to use the concepts of various patents, applications and publications to provide other further embodiments.

These and other changes can be made in embodiments in the light of the above detailed description. Generally, the terms used in the following claims are not to be construed as limiting the claims to the specific embodiments described herein and the claims, but should be considered to include all possible embodiments together with the total amount of equivalents to which such the claims relate. Thus, the claims are not limited to the present disclosure.