EA044601B1 - ANTIBODIES AND RESEARCH FOR CD37 DETECTION - Google Patents

Description

Cross reference to related applications

This invention claims priority to U.S. Provisional Patent Application No. 62/211,455, filed Aug. 28, 2015, and U.S. Provisional Patent Application No. 62/212,183, filed Aug. 31, 2015, each of which is incorporated herein. by reference in its entirety.

Field of technology

The present invention generally relates to diagnostic tests and kits for CD37-targeted treatments, as well as antibodies that bind to human CD37.

State of the art

Cancer is a leading cause of death in developed countries, with more than one million people diagnosed with cancer in the United States alone and causing 500,000 deaths per year. Overall, it is estimated that more than 1 in 3 people will develop some form of cancer during their lifetime.

Leukocyte antigen CD37 (CD37), also known as GP52-40, tetraspanin-26, or TSPAN26, is a transmembrane protein of the tetraspanin superfamily (Maecker et al., 1997, FASEB J. 11:428-442). The protein is a highly glycosylated protein with four transmembrane domains that is expressed on the surface of B cells from pre-B cells to mature peripheral B cells, but is absent during the final stage of differentiation into plasma cells (Link et al., 1987, J Pathol 152:12-21). The CD37 antigen is expressed only to a small extent on T cells, myeloid cells and granulocytes (Schwartz-Albiez et al., 1988, J. Immunol., 140(3):905-914). However, CD37 is also expressed on malignant B cells, such as the B cells found in non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) (Moore et al., 1986, J. Immunol. 137(9):3013-8 ). This expression profile suggests that CD37 represents a promising therapeutic target for the treatment of CD37-expressing B-cell malignancies. However, for these treatments to be as effective as possible, it is necessary to be able to sensitively and specifically detect CD37 over a dynamic range.

Brief description of the invention

Provided herein are anti-CD37 antibodies and antigen-binding fragments thereof, as well as methods for detecting CD37, diagnosing CD37-associated diseases and disorders (such as cancer), monitoring the effectiveness of CD37-targeted treatments, and stratifying patients. Antibodies to CD37 and antigen-binding fragments thereof are particularly preferred because they allow more specific and robust staining, as well as staining without a nuclear background. Antibodies to CD37 and antigen binding fragments thereof can bind to an epitope that contains one or more of amino acids 110-137 of CD37. Antibodies to CD37 and antigen-binding fragments thereof can also bind to a polypeptide containing amino acids 107-242 of CD37 (SEQ ID NO: 20) and/or a polypeptide containing amino acids 107-235 of CD37 (SEQ ID NO: 19), wherein said antibodies to CD37 and their antigen-binding fragments do not bind to the polypeptide consisting of amino acids 138-235 of CD37.

In one embodiment, an antibody or antigen binding fragment thereof as described herein specifically binds to the same CD37 epitope as an antibody comprising the polypeptide represented by SEQ ID NO: 9 and the polypeptide represented by SEQ ID NO: 10.

In another embodiment, an antibody or antigen binding fragment thereof as described herein specifically binds to CD37 and competitively inhibits the binding to CD37 of an antibody comprising the polypeptide represented by SEQ ID NO: 9 and the polypeptide represented by SEQ ID NO: 10.

In another embodiment, an antibody or antigen binding fragment thereof as described herein comprises the heavy chain variable region sequences CDR1, CDR2 and CDR3 represented by SEQ ID NO: 3-5, respectively, and the light chain variable region sequences CDR1, CDR2 and CDR3 represented by SEQ ID NO : 6-8 respectively.

In another embodiment, an antibody or antigen binding fragment thereof as described herein specifically binds to CD37, and said antibody or fragment thereof comprises the heavy chain variable region sequences CDR1, CDR2 and CDR3 represented by SEQ ID NO: 3-5 and the light chain variable region sequences CDR1, CDR2 and CDR3 represented by SEQ ID NO: 6-8 respectively.

In another embodiment, an antibody or antigen binding fragment thereof as described herein comprises polypeptide sequences that are at least 90% identical to the polypeptide sequences set forth in SEQ ID NOs: 9 and 10. In another embodiment, said polypeptide sequences are at least 95% identical to polypeptide sequences. sequences set forth in SEQ ID NOs: 9 and 10. In another embodiment, said polypeptide sequences are at least 99% identical to the polypeptide sequences set forth in SEQ ID NOs: 9 and 10. In another embodiment, said polypeptide sequences comprise the amino acids of SEQ ID NOs. : 9 and 10.

- 1 044601

In another embodiment, an antibody or antigen binding fragment thereof as described herein specifically binds to CD37, wherein said antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 9.

In another embodiment, an antibody or antigen binding fragment thereof as described herein specifically binds to CD37, wherein said antibody or fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 10.

In another embodiment, an antibody or antigen binding fragment thereof as described herein is produced by a recombinant method. In another embodiment, the antibody or antigen binding fragment thereof is murine, non-human, humanized, chimeric, or human. In another embodiment, the antibody or antigen-binding fragment thereof has an altered surface. In another embodiment, the antibody or antigen-binding fragment thereof is a full-length antibody. In another embodiment, the antibody or antigen binding fragment thereof is an antigen binding fragment. In another embodiment, the antibody or antigen binding fragment thereof comprises a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide bond stabilized Fv, V-NAR domain, IgNar, intra-antibody, IgGACH2, mini-antibody, F (ab') ₃ , tetrabody, tribody, diabody, single domain antibody, DVD-Ig, Fcab, _mAb2 , (scFv)2 or scFv-Fc. In another embodiment, the antibody or antigen binding fragment thereof comprises a Fab, Fab', F(ab') ₂ , single chain Fv or scFv, disulfide bond stabilized Fv, intra-antibody, IgGACH2, mini-antibody, F(ab') ₃ , tetrabody, tribody , diabody, DVD-Ig, mAb ₂ , (scFv) ₂ or scFv-Fc. In another embodiment, the antibody or antigen binding fragment is produced by a cell. In another embodiment, said cell is highlighted. In another embodiment, a method for producing an antibody or an antigen-binding fragment thereof is provided, comprising (a) culturing a cell and (b) isolating the antibody or an antigen-binding fragment thereof from the cultured cell.

In another embodiment, the antibody or antigen binding fragment thereof binds to human CD37 with a Kd value of from about 0.5 to about 10 nM. In another embodiment, the antibody or antigen binding fragment thereof binds to human CD37 with a Kd value of 1.0 nM or better.

In another embodiment, the antibody or antigen binding fragment thereof binds to a polypeptide comprising, essentially consisting of, or consisting of amino acids 107-242 of CD37 (SEQ ID NO: 20). In another embodiment, the antibody or antigen binding fragment thereof binds to a polypeptide comprising, essentially consisting of, or consisting of amino acids 107-235 of CD37 (SEQ ID NO: 19). In another embodiment, the antibody or antigen-binding fragment thereof binds to the sequence represented by SEQ ID NO: 15. In another embodiment, the antibody or antigen-binding fragment thereof binds to the sequence represented by SEQ ID NO: 16. In another embodiment, the antibody or antigen-binding fragment thereof binds with the sequence represented by SEQ ID NO: 17. In another embodiment, the antibody or antigen binding fragment thereof binds to the polypeptide comprising CD37 amino acids 107242 (SEQ ID NO: 20) but does not bind to the polypeptide consisting of CD37 amino acids 138-235. In another embodiment, the antibody or antigen binding fragment thereof binds to a polypeptide comprising amino acids 107-235 of CD37 (SEQ ID NO: 19), but does not bind to a polypeptide consisting of amino acids 138-235 of CD37.

In another embodiment, an antibody or antigen binding fragment of the present invention binds to the polypeptide represented by SEQ ID NO: 15, but does not bind to the polypeptide represented by SEQ ID NO: 18. In another embodiment, the antibody or antigen binding fragment thereof binds to the polypeptide represented by SEQ ID NO: 16, but does not bind to the polypeptide represented by SEQ ID NO: 18. In another embodiment, the antibody or antigen binding fragment thereof binds to the polypeptide represented by SEQ ID NO: 17, but does not bind to the polypeptide represented by SEQ ID NO: 18 In another embodiment, the antibody or antigen binding fragment thereof binds to at least one amino acid from amino acids 110-137 of CD37.

In another embodiment, the binding affinity of an antibody or antigen-binding fragment thereof is measured using flow cytometry, Biacore, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay.

In another embodiment, the antibody or antigen-binding fragment thereof has a detectable label. In another embodiment, the label is selected from the group consisting of an immunofluorescent label, a chemiluminescent label, a phosphorescent label, an enzyme label, a radioactive label, avidin/biotin, a colloidal gold particle, a colored particle, and a magnetic particle. In another embodiment, a composition is provided comprising an antibody or an antigen-binding fragment thereof and a buffer selected from the group consisting of an immunohistochemistry (IHC) buffer, an enzyme-linked immunosorbent assay (ELISA) buffer, and a fluorescent cell sorting assay buffer. (FACS).

In another embodiment, a method for detecting CD37 expression in a sample is provided, comprising contacting said sample with an antibody or antigen binding fragment or composition thereof as described herein.

In another embodiment, the antibody or antigen-binding fragment thereof has a detectable label. In another embodiment, the label is selected from the group consisting of an immunofluorescent label, a chemiluminescent label, a phosphorescent label, an enzyme label, a radioactive label, avidin/biotin, a colloidal gold particle, a colored particle, and a magnetic particle. In another embodiment, the label is an enzyme label.

In another embodiment, CD37 expression is determined by radioimmunoassay, Western blot analysis, cytometry, immunofluorescence assay, enzyme-linked immunosorbent assay, immunoprecipitation assay, chemiluminescent assay, or immunohistochemical assay. In another embodiment, CD37 expression is determined using immunohistochemical analysis.

In another embodiment, a method is provided for increasing the effectiveness of cancer treatment using a therapeutically active agent containing an anti-CD37 antibody or an antigen-binding fragment thereof, comprising administering said therapeutically active agent to a subject having cancer, wherein in a cancer sample from said subject using the antibody or antigen-binding fragment thereof fragment or composition according to the present description, increased expression of CD37 was detected.

In another embodiment, a method is provided for identifying a cancer that is likely to respond to a therapeutically active agent comprising an anti-CD37 antibody or an antigen-binding fragment thereof, comprising (a) contacting a biological sample containing cells of said cancer with the antibody or an antigen-binding fragment thereof or composition; (b) detecting binding of an antibody or an antigen binding fragment thereof to CD37 in a biological sample (a); (c) assigning a value to the binding detected in step (b), where the value is assigned based on comparison with one or more reference samples; and (d) comparing the value obtained in step (c) with the value for a reference tissue or cell, where the value for the CD37 level of the specified cancer is greater than the value for the reference sample with normal or low CD37 expression, or the value for the CD37 level of the specified cancer, equal to or greater than the reference sample with high CD37 expression, indicates a cancer that is likely to respond to anti-CD37 antibody.

In another embodiment, a method of treating a patient having cancer is provided, comprising (a) determining a CD37 expression level value based on detection of CD37 expression in a cancer sample obtained from said patient, wherein said detection is performed using an antibody or an antigen binding fragment or composition thereof; and (b) administering a therapeutically active agent comprising an anti-CD37 antibody or an antigen-binding fragment thereof to the patient if said value indicates that the patient will benefit from administration of the therapeutically active agent.

In another embodiment, a method of treating a patient having cancer includes (a) determining a CD37 expression level value based on detection of CD37 expression in a cancer sample obtained from said patient, wherein said detection is performed using an antibody or an antigen binding fragment or composition thereof; and (b) instructing the health care provider to administer a therapeutically active agent comprising an anti-CD37 antibody or an antigen-binding fragment thereof to a patient if the indicated value indicates that the patient will benefit from administration of the therapeutically active agent.

In another embodiment, a method of treating a patient having cancer includes (a) providing a cancer sample obtained from the patient having cancer to determine a CD37 expression level value based on detection of CD37 expression using an antibody or antigen binding fragment or composition thereof and (b) administering a therapeutically active agent comprising an anti-CD37 antibody or an antigen-binding fragment thereof to a patient if the indicated value indicates that the patient will benefit from administration of the therapeutically active agent.

In another embodiment, a method of treating a patient having cancer includes (a) detecting CD37 expression in a cancer sample obtained from the patient using an antibody or antigen binding fragment or composition thereof; (b) determining a CD37 expression level value of the cancer sample; and (c) administering a therapeutically active agent comprising an anti-CD37 antibody or an antigen-binding fragment thereof to the patient if the value indicates that the patient will benefit from administration of the therapeutically active agent.

In another embodiment, a method for identifying a cancer as responsive to treatment with an anti-CD37 active agent comprises (a) determining the level of CD37 expression in a cancer sample of said cancer using an antibody or antigen-binding fragment or composition thereof, including using a method that detects differences in intensity the intensity or uniformity of staining in a CD37 expressing cancer sample compared to the intensity or uniformity of staining in one or more reference samples; (b) determining a CD37 staining intensity or uniformity value for said cancer sample; and (c) comparing a CD37 staining intensity or uniformity value determined in step (b) with a relative value determined by measuring CD37 protein expression in at least one a reference sample being a sample of tissue, cell or cell pellet that is not sensitive to treatment with a therapeutically active agent containing an anti-CD37 antibody or antigen-binding fragment thereof, and wherein the CD37 staining intensity value for said cancer sample is determined at stage (b ), exceeding the specified relative value, identifies the specified cancer as sensitive to treatment using the specified therapeutically active agent.

In another embodiment, a method for identifying a cancer as being responsive to treatment using a therapeutically active agent comprising an anti-CD37 antibody or antigen-binding fragment thereof comprises (a) determining the level of CD37 expression in a cancer sample of said cancer using the antibody or antigen-binding fragment or composition thereof, comprising: using a method that detects differences in staining intensity or staining uniformity in a CD37 expressing cancer sample compared to staining intensity or staining uniformity in one or more reference samples; (b) determining a CD37 staining intensity or uniformity value for said cancer sample; and (c) comparing a CD37 staining intensity or uniformity value determined in step (b) with a relative value determined by measuring CD37 protein expression in at least one a reference sample, which is a sample of tissue, cell or cell pellet that is sensitive to treatment with a therapeutically active agent containing an anti-CD37 antibody or antigen-binding fragment thereof, and wherein the CD37 staining intensity value for said cancer sample is determined in step (b) , greater than or equal to the specified relative value, identifies the specified cancer as sensitive to treatment using the specified therapeutically active agent. In another embodiment, a method is provided that further comprises administering a therapeutically active agent comprising an anti-CD37 antibody or an antigen-binding fragment thereof to the subject from which the cancer or biological sample was obtained. In another embodiment, a patient's CD37 level is determined in a cancer sample or biological sample obtained from the patient. In another embodiment, the cancer sample or biological sample is a liquid extract, blood, plasma, serum, cerebrospinal fluid, lymph fluid, or spleen preparation. In another embodiment, detection is performed by immunohistochemistry (IHC). In another embodiment, the IHC method is a calibrated IHC method that can distinguish between different levels of CD37 expression. In another embodiment, IHC provides a range of color intensities for samples with low CD37 expression, intermediate CD37 expression, or high CD37 expression. In another embodiment, IHC detects differences in staining intensity or uniformity of staining in a CD37-expressing cancer sample or biological sample compared to a reference sample. In another embodiment, IHC is performed manually. In another embodiment, IHC is performed using an automated system. In another embodiment, CD37 is determined based on IHC. In another embodiment, detection is performed using an enzyme-linked immunosorbent assay (ELISA). In another embodiment, the reference sample is a positive reference sample or a negative reference sample. In another embodiment, the reference sample includes cells, a cell pellet, or tissue.

In another embodiment, the antibody or antigen-binding fragment thereof further comprises a detection reagent selected from the group consisting of an immunofluorescent label, a chemiluminescent label, a phosphorescent label, an enzyme, a radioactive label, avidin/biotin, a colloidal gold particle, a colored particle, and a magnetic particle. In another embodiment, the detection reagent is an enzyme.

In another embodiment, the cancer is a CD37-positive cancer. In another embodiment, the cancer is leukemia or lymphoma. In another embodiment, the cancer is selected from the group consisting of B-cell lymphomas, NHL, B-cell lymphoblastic leukemia/progenitor cell lymphoma and mature B-cell neoplasms, B-cell chronic lymphocytic leukemia (CLL)/lymphoma from small lymphocytes (SML), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), low-grade, moderate-grade and high-grade FL, cutaneous follicular center cell lymphoma, B-cell lymphoma marginal zone, MALT-type marginal zone B-cell lymphoma, nodal type marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disease, Wahl macroglobulinemia

- 4 044601 denstrom and anaplastic large cell lymphoma (ALCL). In another embodiment, the CD37-positive cancer is diffuse large B-cell lymphoma, follicular lymphoma, or mantle cell lymphoma. In another embodiment, CD37 expression is detected using at least one additional anti-CD37 antibody or antigen-binding fragment thereof. In another embodiment, CD37 expression is measured using two anti-CD37 antibodies or antigen-binding fragments thereof. In another embodiment, the at least one additional antibody or antigen binding fragment thereof comprises a detection agent. In another embodiment, the detection agent is a chromogenic detection agent, a fluorogenic detection agent, an enzymatic detection agent, or an electrochemiluminescent detection agent. In another embodiment, the detection agent is horseradish peroxidase (HRP).

In another embodiment, the at least one additional antibody or antigen binding fragment thereof is/is bound to a solid carrier. In another embodiment, the at least one additional antibody or antigen binding fragment thereof is/is bound to a microtiter plate.

In another embodiment, the ELISA is a sandwich ELISA.

In another embodiment, the therapeutically active agent comprises an anti-CD37 antibody, huCD37-3. In another embodiment, the therapeutically active agent is an antibody-maytansinoid conjugate comprising the anti-CD37 antibody huCD37-3, the maytansinoid DM1, and a non-cleavable N-succinimidyl-4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) linker (IMGN529).

In another embodiment, a combination diagnostic and pharmaceutical kit is provided that contains an antibody or antigen binding fragment or composition as described herein for diagnostic use and a therapeutically active agent comprising an anti-CD37 antibody or antigen binding fragment thereof for therapeutic use.

In another embodiment, the detection antibody allows CD37 expression to be detected by IHC. In another embodiment, the detection antibody detects CD37 expression using ELISA.

In another embodiment, the anti-CD37 antibody in the therapeutically active agent is conjugated to a cytotoxin.

In another embodiment, a diagnostic kit as described herein comprises an antibody, an antigen binding fragment thereof as described herein, an IHC reagent, and one or more standardized reference samples comprising cells, a cell pellet, or formalin-fixed, paraffin-embedded tissue samples, and one or more more standardized reference samples are derived from non-CD37-expressing, low-CD37-expressing, or high-CD37-expressing cells, cell pellets, or tissues.

Brief description of drawings/figures

In fig. 1A and 1B show the results of an ELISA screen for antibodies obtained by immunizing mice using the hCD37-LEL (large extracellular loop) protein (SEQ ID NO: 15) containing amino acids 107 to 242 of human CD37 fused to a histidine tag (His- tag).

In fig. 1A shows that the supernatant of hybridoma clone 1B11 binds to various variants of the CD37 antigen, hCD37-LEL, hCD37-Fc-LAGA (LAGA refers to the Fc mutations G236A and P238A), SEQ ID NO: 17, containing amino acids 107 to 235 of human CD37, fused to the Fc domain of human IgG1) and hCD37-ECD-Fc (extracellular domain, SEQ ID NO: 16, containing amino acids 107 to 235 of human CD37 fused to the Fc domain of mouse IgG2a) under both native and denaturing conditions.

In fig. Figure 1B shows that supernatants of two hybridoma subclones, 1B11-2 and 1B11-20, bind to hCD37-Fc-LAGA and hCD37-LEL under both native and denaturing conditions.

In fig. 2 shows that antibody 1B11-2 binds to the native proteins hCD37-Fc-LAGA and hCD37-LEL with significantly improved affinity compared to the reference CD37 antibody, NCL-CD37 (clone CT1, Leica Biosystems).

In fig. 3 shows that antibody 1B11-2 binds to the denatured hCD37-LEL protein with significantly improved affinity compared to NCL-CD37.

In fig. 4 shows that antibody 1B11-2 does not bind to the processed protein hCD37-ECDS2-Fc (S2 refers to the second segment of the large extracellular domain of CD37, containing amino acids 138 to 176, SEQ ID NO: 18), while NCL-CD37 retains the ability to binding to this protein CD37.

In fig. Figure 5 shows immunohistochemical images of a normal human tonsil stained with either the mouse monoclonal antibody (Mab) NCL-CD37 (left) or the mouse mAb CD37 1B11-2 (right). Arrows indicate germinal centers, mantle zones (both CD37 positive), and interfollicular regions (CD37 negative), respectively. Black

- 5 044601 arrows point to germinal centers; gray arrows indicate mantle areas (both CD37 positive) and uncolored arrows indicate interfollicular areas (CD37 negative). When using the NCL-CD37 antibody, nuclear staining is present in both the germinal center and the mantle zone. When using the 1B11-2 antibody, there is no staining of the nuclei.

In fig. 6 shows immunohistochemical images of normal human small intestine (top panel) and normal human pancreas (bottom panel) using either the mouse monoclonal antibody (MAT) NCL-CD37 (left panel) or mouse MAT 1B11-2 (right panel). Arrows in the panels on the left indicate pink staining of the cytoplasm of Paneth cells (top panel, indicated by black arrows) and islet cells (lower panel, indicated by unstained arrows) obtained using the mouse MAT NCL-CD37. The arrows in the panels on the right indicate that this staining is not observed in the corresponding areas when the 1B11-2 antibody is used.

In fig. Figure 7 shows immunohistochemical images of tissue from a patient with diffuse large B-cell lymphoma (top panel) and tissue from a patient with follicular lymphoma (bottom panel) stained using either mouse MAT NCL-CD37 (left panel) or mouse MAT 1B11-2 (right panel). panel). Stronger, more distinct staining is observed for both types of cancer when using murine mAb 1B11-2.

In fig. 8 shows the results of Western blotting after denaturing DNS-PAGE of Fc fusion products with CD37 extracellular domain (ECD) antigens, comparing the mouse monoclonal antibody 1B11-2 with a commercially available mouse monoclonal anti-CD37 antibody (Leica). Molecular mass markers in kilodaltons (kDa) are shown in their original form and as a schematic representation on the right.

In fig. 9 shows the results of Western blotting after native PAGE of Fc fusion products with CD37 extracellular domain (ECD) antigens detected using the mouse monoclonal antibody 1B11-2.

Detailed Description of the Invention

Provided herein are methods for detecting human CD37.

These methods allow the identification of cancers characterized by CD37 expression to improve the effectiveness of treatment using CD37-targeted therapeutics. Detection methods can be used to stratify patients, monitor or determine therapeutic efficacy, or determine the likelihood that a cancer will respond to a CD37-targeted therapeutic agent. Detection methods allow detection of clinically relevant dynamic range of CD37. Also disclosed are novel CD37-binding polypeptides, such as antibodies and antigen-binding fragments thereof, suitable for use in detection methods (eg, immunohistochemical (IHC) assays for CD37). A CD37-binding agent (eg, an antibody or an antigen-binding fragment thereof) provided herein allows for more specific and robust staining without nuclear background. The CD37 binding agent binds to an epitope that contains one or more of amino acids 110-137 of CD37. The CD37-binding agent also binds to a polypeptide containing amino acids 107-242 of CD37 (SEQ ID NO: 20) or a polypeptide containing amino acids 107-235 of CD37 (SEQ ID NO: 19), but does not bind to a polypeptide consisting of amino acids 138-235 CD37. Also provided are related polypeptides and polynucleotides, compositions containing CD37 binding agents and methods for producing CD37 binding agents.

I. Definitions.

To facilitate understanding of the present invention, a number of terms and phrases are defined below.

The term CD37 as used herein refers to any native CD37 unless otherwise noted. CD37 is also related to GP52-40, leukocyte antigen CD37 and tetraspanin-26. The term CD37 covers full-length full-length CD37 as well as any form of CD37 that is produced by cellular processing. The term also covers natural variants of CD37, such as splice variants, allelic variants and isoforms. The CD37 polypeptides described herein can be isolated from a variety of sources, such as human biological samples or other sources, or produced by recombinant or synthetic methods.

The term CD37 overexpression or overexpression refers to a pattern that exhibits increased levels of CD37 expression. CD37 may be increased or overexpressed compared to a negative or low CD37 control or compared to a sample of healthy or unaffected tissue or cells of the same type. Such increased expression or overexpression may be caused, for example, by mutation, gene amplification, increased transcription, increased translation, or increased protein stability. In one example, CD37 expression is measured using immunohistochemistry (IHC) and determining a staining intensity value or staining uniformity value by comparison with calibrated controls having established values (e.g., a test sample is assigned an intensity value of 3 if its intensity is comparable with a calibrated level 3 control, or the test sample is assigned an intensity value of 2 if its intensity is comparable to the calibrated level 2 control). Uniformity of staining that is heterogeneous or homogeneous also indicates increased CD37 expression. The color intensity and color uniformity values can be applied alone or in combination (eg, 2 homo, 2 hetero, 3 homo, 3 hetero, etc.). In another example, an increase in CD37 expression may be determined by detecting an increase of at least 2-fold, at least 3-fold, or at least 5-fold relative to control values (e.g., the level of expression in a biological sample, tissue, or cell from a subject without cancer or with cancer not characterized by elevated CD37 values). In another example, CD37 expression is assigned an H value. The values combine color intensity values with uniformity values using the calculation proposed herein. In another example, CD37 expression is measured by determining the percentage of cells that have at least a particular level of staining (e.g., at least 25% of cells have a value of at least 2, or at least 75% of cells have a value of at least 2).

The reference sample can be used to compare and contrast the results obtained by the methods proposed herein with the test sample. The reference samples may be cells (eg, cell lines, cell pellets) or tissue. The levels of CD37 in a reference sample may be an absolute or relative amount, a range of amounts, a minimum and/or maximum amount, an average amount, and/or a median amount of CD37. Diagnostic methods proposed herein involve comparing CD37 expression levels in a test sample with a reference value. In some embodiments, the reference value is the level of CD37 expression in the reference sample. The reference value may be a predetermined value and may also be determined using reference samples (eg, control biological samples or reference samples) tested in parallel with the test samples. The reference value may be an individual cutoff such as a median or mean, or a range of values such as a confidence interval. Reference values may be pre-established for various subgroups of individuals, such as individuals predisposed to cancer, individuals with early or late stage cancer, males and/or females, or individuals who have undergone cancer therapy. Examples of negative reference samples or values and positive reference samples or values are described herein.

In some embodiments, the reference sample is a sample from healthy tissue, particularly corresponding tissue free of cancer or corresponding tissue free of cancer characterized by CD37 overexpression. These types of reference samples are called negative control samples or reference samples. In other embodiments, the reference sample is a sample from a tumor that expresses CD37. These types of reference samples are referred to as positive controls. Positive controls can also be used as a comparative indicator of type (hetero or homo), grade (0, 1, 2, 3) of staining intensity, H-value, and/or percentage of cells having at least a particular staining level that correlates with the level of CD37 expression. Positive control comparative samples are referred to as calibrated reference samples. Reference samples with low or absent CD37 are described in the examples herein and also include splenic red pulp (eg, monocytes and red blood cells), T cells, and the interfollicular region of human tonsils. CD37-expressing reference samples are described in the examples herein and also include splenic white pulp (eg, B cells and splenic marginal zone), human tonsil germinal center, and human tonsil mantle zone. Examples of non-expressing cell lines include 30019 cells, and examples of expressing cell lines include Daudi, Ramos, Namalwa and RL non-Hodgkin's lymphoma B cells. Another high CD37 positive reference sample is a cell line stably or transiently transfected with CD37 (e.g., 300.19/CD37, as described in US Pub. No. 2015/0093397 and PCT Pub. WO 2013/149171, each of which is incorporated herein by links). Appropriate positive and negative CD37 reference levels for a particular cancer can be determined by measuring CD37 levels in one or more suitable subjects, and such reference levels can be tailored to specific groups of subjects (for example, the reference level can be age-matched so that comparisons have been made between CD37 levels in samples from subjects of a certain age and reference levels for a specific disease course, phenotype, or absence thereof in a specific age group). As used herein, immunohistochemistry refers to histochemical and immunological techniques used to analyze, for example, cells or tissues. Thus, the terms immunohistochemistry, immunocytochemistry, and immunochemistry are used interchangeably.

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The sample or biological sample of the present invention is of biological origin, in particular embodiments such as being from eukaryotic organisms. In some embodiments, the sample is a human sample, but animal samples may also be used in the practice of the present invention. Non-limiting sample sources for use in the context of the present invention include, for example, solid tissues, biopsy aspirates, liquid extracts, blood, plasma, serum, cerebrospinal fluid, lymph fluid, external sections of skin, respiratory, intestinal and genitourinary tracts, tears, saliva, milk , tumors, organs, cell cultures and/or components of cell cultures. A cancer sample (cancer sample) is a sample that contains a cancer cell. The methods proposed herein may be suitable for use with solid tissue samples where the amount of material available is low. The methods provided herein can be used to examine an aspect of CD37 expression or the condition of a sample, including, but not limited to, comparing different cell or tissue types and detecting or determining the presence and/or type of disease or abnormality.

For purposes of this description, a tissue sample section refers to a single portion or fragment of a tissue sample, such as a thin layer of tissue or cells cut from a tissue sample. It will be understood that multiple sections of tissue samples can be taken and analyzed in accordance with the present invention. In some cases, the selected portion or section of tissue contains a homogeneous population of cells. In other cases, the selected portion comprises an area of tissue, such as a lumen, as a non-limiting example. The selected portion may be as small as one cell or two cells, or may be many thousands of cells, for example. In most cases, collection of cells is important, and although the present invention has been described for use in the detection of cellular components, the method can also be applied to the detection of non-cellular components of the body (eg, soluble components in the blood as a non-limiting example).

As used herein, the term capture reagent refers to a reagent capable of binding and capturing a target molecule in a sample such that, under suitable conditions, the capture reagent-target molecule complex can be separated from the rest of the sample. In one embodiment, the capture reagent is immobilized. In one embodiment, the capture reagent in a sandwich immunoassay is an antibody or an antigen-binding fragment thereof or a mixture of different antibodies or antigen-binding fragments thereof to a target antigen.

As used herein, the term detectable antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof that can be detected either directly through a label enhanced by a detection agent or indirectly through, for example, another antibody or antigen-binding fragment thereof, being labeled. For direct labeling, an antibody or antigen-binding fragment thereof is typically conjugated to a moiety that can be detected by some means. In one embodiment, the detectable antibody or antigen binding fragment thereof is a biotinylated antibody or antigen binding fragment thereof. As used herein, the term detection agent refers to a moiety or technique used to detect the presence of a detectable antibody or antigen binding fragment thereof and includes detection agents that enhance an immobilized label, such as a label immobilized on a microtiter plate. In one embodiment, the detection agent is a fluorimetric detection agent such as avidin or streptavidin.

Typically, a sandwich ELISA involves the following steps: (1) a microtiter plate is coated with a capture antibody or antigen-binding fragment thereof; (2) a sample is added and any antigen present is bound to the capture antibody or antigen-binding fragment thereof; (3) adding a detection antibody or an antigen-binding fragment thereof that binds to the antigen; (4) adding an enzyme-linked secondary antibody or antigen-binding fragment thereof that binds to the detection antibody or antigen-binding fragment thereof; and (5) add a substrate that is converted by the enzyme to a detectable form.

The term label as used herein refers to a detectable compound or composition directly or indirectly conjugated to an antibody to produce a labeled antibody. The label may be detectable in itself (eg, radioisotope labels or fluorescent labels) or, in the case of an enzyme label, may catalyze a chemical change in the substrate compound or composition that is detectable.

The terms correlate or correlate mean comparison in any way of the characteristics and/or results of the first analysis with the characteristics and/or results of the second analysis. For example, the results of the first analysis may be used in conducting a second analysis, and/or the results of the first analysis may be used to determine whether a second analysis should be performed, and/or the results of the first analysis may be compared with the results of the second analysis. In one of

- 8 044601 embodiments, increased expression of CD37 correlates with an increased likelihood of effectiveness of CD37-targeted anticancer therapy.

The term antibody means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof, through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term antibody includes intact polyclonal antibodies, intact monoclonal antibodies, multispecific antibodies such as bispecific antibodies derived from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, chimeric proteins containing an antibody, and any other a modified immunoglobulin molecule, provided that said antibodies exhibit the desired biological activity. An antibody may belong to any of the five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains , denoted alpha, delta, epsilon, gamma and mu, respectively. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be unconjugated or conjugated to other molecules such as toxins, radioisotopes, etc.

The term antibody fragment refers to a portion of an intact antibody.

An antigen-binding fragment refers to the part of an intact antibody that binds to an antigen. The antigen binding fragment may contain variable regions of the intact antibody corresponding to the antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, linear antibodies and single chain antibodies. Examples of antibody fragments encompassed by the term antigen-binding antibody fragment include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains (e.g., an antibody cleaved by papain to produce three fragments: two antigen-binding Fab fragments and one Fc fragment that does not bind antigen); (ii) F(ab')2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region (e.g., an antibody cleaved by pepsin to produce two fragments: a bivalent antigen-binding fragment F(ab')2 and a pFc' fragment , which does not bind antigen) and its related monovalent unit F(ab'); (iii) an Fd fragment consisting of the VH and CH1 domains (ie, that part of the heavy chain that is included in the Fab); (iv) an Fv fragment consisting of the VL and VH domains of one arm of the antibody and a related disulfide bond-stabilized Fv; and (v) a dAb (domain antibody) or sdAb (single domain antibody) fragment (Ward et al., Nature 341:544-546 , 1989), which consists of the VH domain.

In some embodiments, the antibody or antigen-binding fragment thereof is a non-naturally occurring antibody. In some embodiments, the antibody or antigen-binding fragment thereof is obtained by purification from natural components. In some embodiments, the antibody or antigen-binding fragment thereof is produced recombinantly. In some embodiments, the antibody or antigen-binding fragment thereof is produced using a hybridoma.

A blocking antibody or antagonist antibody is an antibody that inhibits or reduces the biological activity of its associated antigen, such as CD37. In a specific embodiment, blocking antibodies or antagonist antibodies significantly or completely inhibit the biological activity of the antigen. Preferably, the biological activity is reduced by 10, 20, 30, 50, 70, 80, 90, 95 or even 100%.

The term anti-CD37 antibody or antibody that binds to CD37 refers to an antibody capable of binding to CD37 with sufficient affinity such that the antibody is suitable for use as a diagnostic and/or therapeutic agent when targeting CD37. The extent of binding of an anti-CD37 antibody to a non-CD37 foreign protein is less than about 10% of the extent of binding of an anti-CD37 antibody as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that binds to CD37 has a dissociation constant (Kd) of <1 μM, <100 nM, <10 nM, <1 nM, or <0.1 nM. Examples of anti-CD37 antibodies are known in the art and are disclosed, for example, in US Published Application No. 2011/0256153, which is incorporated herein by reference.

A monoclonal antibody or antigen-binding fragment thereof refers to a homogeneous population of antibodies or antigen-binding fragments involved in highly specific recognition and binding of a single antigenic determinant or epitope. This distinguishes them from polyclonal antibodies, which typically comprise different antibodies directed against different antigenic determinants. The term monoclonal antibody or antigen-binding fragment thereof covers intact and full-length monoclonal antibodies, as well as antibody fragments (such as Fab, Fab', F(ab') ₂ , Fv), single chain (scFv) mutants, chimeric proteins containing the antibody region, and any other modified immunoglobulin molecule containing an antigen recognition site. In addition, a monoclonal antibody or antigen-binding fragment thereof refers to such antibodies and antigen-binding fragments thereof produced by any number of methods, including, but not limited to, hybridoma, phage display, recombinant gene expression, and the use of transgenic animals.

The term humanized antibody or antigen-binding fragment thereof refers to non-human (e.g., murine) forms of antibodies or antigen-binding fragments that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. . Typically, humanized antibodies or antigen-binding fragments thereof are human immunoglobulins in which residues in the complementarity determining regions (CDRs) are replaced by CDR residues from a non-human species (eg, mouse, rat, rabbit, hamster) having the desired specificity, affinity and binding capacity (Jones et al., 1986, Nature, 321:522-525, Riechmann et al., 1988, Nature, 332:323-327, Verhoeyen et al., 1988, Science, 239:1534- 1536). In some cases, human immunoglobulin Fv framework region (FR) residues are replaced with corresponding antibody or fragment residues from other non-human species that have the desired specificity, affinity and binding ability. The humanized antibody or antigen binding fragment thereof may be further modified by substitution of additional residues in either the Fv framework region and/or substituted non-human residues to adjust and optimize the specificity, affinity and/or binding ability of the antibody or antigen binding fragment thereof. In general, a humanized antibody or antigen binding fragment thereof will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to a non-human immunoglobulin, while all or essentially all FR regions are FR regions of the human immunoglobulin consensus sequence. The humanized antibody or antigen binding fragment thereof may also comprise at least a portion of a constant region or domain (Fc) of an immunoglobulin, typically a human immunoglobulin. Examples of methods used to produce humanized antibodies are described in US Pat. No. 5,225,539 or 5,639,641.

The term primary antibody or antigen-binding fragment thereof as used herein refers to an antibody or antigen-binding fragment thereof that specifically binds to a target protein antigen in a sample. The primary antibody or antigen-binding fragment thereof is typically the first antibody or antigen-binding fragment thereof used in performing an immunohistochemical assay. In one embodiment, the primary antibody or antigen-binding fragment thereof is the only antibody or antigen-binding fragment thereof used in performing the immunohistochemical analysis.

The term secondary antibody or antigen-binding fragment thereof as used herein refers to an antibody or antigen-binding fragment thereof that specifically binds to a primary antibody or antigen-binding fragment thereof, thereby forming a bridge between the primary antibody or antigen-binding fragment thereof and the subsequent reagent, if any. A secondary antibody or antigen-binding fragment thereof is typically a second antibody or antigen-binding fragment thereof used in performing an immunohistochemical assay. The term tertiary antibody as used herein refers to an antibody or antigen binding fragment thereof that specifically binds to a secondary antibody or antigen binding fragment thereof, thereby forming a bridge between the secondary antibody or antigen binding fragment thereof and the downstream reagent, if any.

The variable region of an antibody or antigen-binding fragment thereof refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDR regions of each chain are held together in close proximity to the FR regions and, together with the CDR regions of the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two ways to determine CDR regions: (1) an approach based on interspecies sequence variation (e.g., Kabat et al.? Sequences of Proteins of Immunological Interest, ( ^5th ed., 1991, National Institutes of Health, Bethesda Md. .)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Alazikani et al. (1997), J. Molec. Biol. 273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDR.

The Kabat numbering system is commonly used when indicating residues in the variable domain (residues approximately 1-107 of the light chain and approximately 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. ^5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Kabat amino acid position numbering refers to the numbering system used for heavy chain variable domains or light chain variable domains in antibodies, described in Kabat et al., Sequences of Proteins of Immunological Interest, ^5th Ed.

- 10 044601

Public Health Service, National Institutes of Health, Bethesda, Md. (1991). When using this numbering system, the actual linear amino acid sequence may contain fewer amino acids or additional amino acids in accordance with the truncation or insertion in the FR or CDR regions of the variable domain. For example, a heavy chain variable domain may include one amino acid insertion (Kabat residue 52a) after residue 52 in H2 and inserted residues (eg, Kabat residues 82a, 82b and 82c, etc.) after heavy chain FR residue 82. The Kabat numbering of residues for a given antibody can be determined by comparing regions of homology between the antibody sequence and a standard Kabat numbered sequence. Instead, Chothia refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The end of the Chotia CDR-H1 loop when numbered using Kabat numbering varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places inserts at positions H35A and H35B; if neither 35A nor 35B is present , then the cycle ends at position 32, if only 35A is present, the cycle ends at position 33; if both 35A and 35B are present, then the cycle ends at position 34). AbM hypervariable regions represent a compromise between Kabat's CDRs and Chotia's structural loops and are used by Oxford Molecular's AbM antibody modeling program.

A loop Kabat ABM Chotia T.I. I.24-I.34 T.24-1.34 1.24-1.34 L2 L50-L56 L50-L56 L5O-L56 L3 L89-L97 L89-L97 L89-L97 H1 Н31-Н35В Н26-Н35В (Numbering N26-N32..34 according to Kabat) w ΙΙ31-Π35 II26-II35 ΙΙ26-Π32 (Numbering according to Chotia) H2 H50-H65 H50-H58 H52-H56 NZ N95-N102 N95-N102 N95-N102

The term human antibody (human antibody) or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof produced by a human, or an antibody or antigen-binding fragment thereof having an amino acid sequence corresponding to an antibody or antigen-binding fragment thereof produced by a human, obtained using any method known in the art. this field of technology. This definition of a human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

The term chimeric antibodies or antigen-binding fragments thereof refers to antibodies or antigen-binding fragments thereof in which the amino acid sequence is derived from two or more species. Typically, the variable region of both the light and heavy chains corresponds to the variable region of antibodies or antigen-binding fragments thereof with the desired specificity, affinity and binding ability obtained from the same mammalian species (eg, mouse, rat, rabbit, etc.), then as constant regions are homologous to sequences in antibodies or antigen-binding fragments thereof derived from another species (usually humans), to avoid eliciting an immune response in those species.

The terms epitope or antigenic determinant are used interchangeably herein and refer to that portion of an antigen that can be recognized and specifically bound by a particular antibody or antigen-binding fragment thereof. In the case when the antigen is a polypeptide, epitopes can be formed both by adjacent amino acids and by non-adjacent amino acids that come closer together when the protein is folded into the tertiary structure. Epitopes formed from contiguous amino acids are usually retained when the protein is denatured, whereas epitopes formed when the protein is folded into a tertiary structure are usually lost when the protein denatures. An epitope typically comprises at least 3, and typically at least 5 or 8-10 amino acids, in a unique spatial conformation.

Binding affinity generally refers to the strength of the total number of noncovalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, binding affinity refers to intrinsic binding affinity, which reflects the interaction between members of a binding pair (eg, antibody and antigen) in a 1:1 ratio. The affinity of a molecule X for its partner Y can usually be characterized by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art, including those described herein. Low-affinity antibodies tend to bind antigen slowly and tend to dissociate easily, whereas high-affinity antibodies tend to bind antigen more quickly and tend to remain bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. Specific illustrative embodiments are described below.

Or better as used herein, binding affinity refers to a stronger binding between a molecule and its binding partner. Or better in the context of the present description refers to a stronger binding characterized by a lower numerical value of Kd. For example, for an antibody that has an affinity for an antigen of 0.6 nM or better, the affinity of the antibody for the antigen is <0.6 nM, i.e. 0.59 nM, 0.58 nM, 0.57 nM, etc. or any value less than 0.6 nM.

The phrase substantially similar or substantially the same as used herein means a sufficiently high degree of similarity between two numerical values (typically one associated with an antibody of the present invention and the other associated with a reference/comparison antibody) such that one skilled in the art It would be clear in the art that the difference between these two values has little or no biological and/or statistical significance in the context of the biological characteristics defined by the values (eg, Kd values). The difference between the two values is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% depending on the value for the reference/comparison antibody.

An isolated polypeptide, antibody, polynucleotide, vector, cell or composition is a polypeptide, antibody, polynucleotide, vector, cell or composition that is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells or compositions include those that have been purified to the extent that they are no longer in the form in which they are found in nature. In some embodiments, the isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

As used herein, substantially pure refers to a substance that is at least 50% pure (i.e. free from impurities), at least 90% pure, at least 95% pure, at least 98% pure or at least 99% pure.

The term immunoconjugate or conjugate as used herein refers to a compound or derivative thereof that is associated with a cell-binding agent (i.e., an anti-CD37 antibody or an antigen-binding fragment thereof) and is defined by the general formula: C-L-A, where C = cytotoxin, L = linker and A = cell binding agent, anti-CD37 antibody, or antigen-binding fragment thereof. Immunoconjugates can also be defined by the general formula in reverse order: A-L-C.

A linker is any chemical moiety capable of linking a compound, typically a drug such as a maytansinoid, to a cell-binding agent, such as an anti-CD37 antibody or fragment thereof, in a stable, covalent manner. Linkers may be susceptible to or substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions under which the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioester groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers and hydrophilic forms thereof, as described herein and known in the art.

The terms cancer and cancerous refer to or describe a physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific examples of cancer or tumorigenic diseases include B-cell lymphomas, including NHL, B-cell lymphoblastic leukemia/progenitor cell lymphoma, and mature B-cell neoplasms, such as B-cell chronic lymphocytic leukemia (CLL)/small cell lymphoma. lymphocyte lymphoma (LML), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), including low-grade, moderate-grade and high-grade FL, cutaneous follicular center cell lymphoma, B-cell lymphoma marginal zone (MALT type, nodular type and splenic lymphoma), hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma (BL), plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disease and anaplastic large cell lymphoma (ALCL). In some embodiments, said cancer is leukemia or lymphoma.

Tumor and neoplasm refer to any formation that is the result of excess growth or proliferation of cells, either benign (noncancerous) or malignant (cancerous), including precancerous lesions.

The terms cancer cell, tumor cell, and grammatical equivalents refer to the general population of cells derived from a tumor or precancerous lesion, including both nontumorigenic cells, which constitute the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term tumor cell will be appended to the term non-tumorigenic when it refers solely to those tumor cells that lack the ability to renew and differentiate to distinguish these tumor cells from cancer stem cells.

The term subject refers to any animal (eg, mammal), including, but not limited to, humans, non-human primates, rodents, and the like, that are intended to be recipients of a particular treatment. Generally, the terms subject and patient are used interchangeably herein to refer to a human being.

A chemotherapeutic agent is a chemical compound useful for the treatment of cancer, regardless of its mechanism of action. Classes of chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, plant alkaloids, spindle poisons, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in targeted therapy and conventional chemotherapy. Administration in combination with one or more additional therapeutic agents includes simultaneous and sequential administration in any order.

The term pharmaceutical composition refers to a preparation that is in such a form as to provide effective biological activity of the active ingredient, and which does not contain additional components that are unacceptably toxic to the subject to whom the drug will be administered. Such a composition may be sterile.

An effective amount of an antibody, antigen-binding fragment or immunoconjugate thereof disclosed herein is an amount sufficient to achieve the specifically stated purpose. The effective amount can be determined empirically and routinely in relation to the stated purpose.

The term therapeutically effective amount refers to the amount of an antibody, antigen binding fragment thereof, or other drug effective to treat a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; suppress (ie, to some extent slow down and in a certain embodiment stop) the infiltration of cancer cells into peripheral organs; suppress (i.e., to some extent slow down and in a certain embodiment stop) tumor metastasis; suppress tumor growth to some extent; relieve to some extent one or more symptoms associated with cancer; and/or result in a positive response such as improved progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR) or in some cases stable disease (SD) , reduction in disease progression (PD), reduction in time to progression (TTP), or any combination thereof. See definition of treatment herein. Depending on the extent to which a drug can prevent the growth and/or destroy existing cancer cells, it may be cytostatic and/or cytotoxic. In some embodiments, detection of elevated CD37 levels allows administration of reduced amounts of the CD37-targeting therapeutic agent to achieve the same therapeutic effect as at higher doses. A prophylactically effective amount refers to an amount effective in doses and for periods of time necessary to achieve the desired prophylactic result. Typically, but not necessarily, since the prophylactic dose is used in subjects prior to the onset of the disease or early in the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

The term responding positively generally refers to conditioning a favorable state in a subject. In relation to cancer treatment, the term refers to the delivery of a therapeutic effect to a subject. Beneficial therapeutic effects in cancer can be measured in several ways (See W.A. Weber, J. Nucl. Med. 50:1S-10S (2009)). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can be used to evaluate the therapeutic efficacy of an anticancer therapeutic agent. In terms of tumor suppression, NCI T/C standards <42% correspond to the minimum level of antitumor activity. T/C <10% is considered a high level of antitumor activity, where T/C (%) = mean tumor volume in the case / mean tumor volume in the control x 100. A positive response can be expressed, for example, in increased progression-free survival (PFS ), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR) or in some cases, stable disease (SD), reduction in disease progression (PD), reduction in time to progression (TTP) or any of them

- 13 044601 combinations.

PFS, DFS, and OS can be measured according to standards established by the National Cancer Institute and the US Food and Drug Administration for the approval of new drugs. See Johnson et al., (2003), J. Clin. Oncol. 21(7):1404-1411.

Progression-free survival (PFS) refers to the period of time from registration to disease progression or death. For patients with NHL, PFS can be measured using the standards presented in the revised International Harmonization Project Outcome Criteria for Malignant Lymphomas (Cheson et al., J. Clin. Oncol. 25:579-86 (2007), incorporated herein by reference in its entirety). For patients with CLL, PFS can be measured using the standards presented by the International Working Group on CLL Response Criteria of the National Cancer Institute (Hallek et al., Blood, 777:5446-56 (2008), incorporated herein by reference in its entirety ). For patients with Hodgkin's lymphoma and non-Hodgkin's lymphoma, PFS can be measured using the Lugano classification standards (Cheson et al., J. Clin. Oncol. 32:3059-68 (2014)). In general, progression-free survival refers to the situation in which a patient remains alive without the cancer getting worse. Time to tumor progression (TTP) is defined as the period of time from registration to disease progression. For patients with NHL, TTP can be measured using the standards presented in the revised International Harmonization Project Outcome Criteria for Malignant Lymphomas (Cheson et al., J. Clin. Oncol. 25:579-86 (2007), incorporated herein by reference in its entirety). For patients with CLL, TTP can be measured using the standards presented by the International Working Group on CLL Response Criteria of the National Cancer Institute (Hallek et al., Blood, 111:5446-56 (2008), incorporated herein by reference in its entirety ). For patients with Hodgkin's lymphoma and non-Hodgkin's lymphoma, PFS can be measured using the Lugano classification standards (Cheson et al., J. Clin. Oncol. 32:3059-68 (2014)).

Complete response or complete remission or CR indicates the disappearance of all signs of the tumor or cancer in response to treatment. This does not always mean that the cancer has been cured.

Partial response or PR refers to a decrease in the size or volume of one or more tumors or lesions or the spread of cancer in the body in response to treatment.

Stable disease refers to disease without progression or relapse. In stable disease, there is not a sufficient decrease in tumor size to meet the criteria for a partial response or a sufficient increase in tumor size to meet the criteria for disease progression.

Disease progression refers to the appearance of another new lesion or tumor and/or clear progression of existing non-target lesions. Disease progression can also refer to tumor growth of more than 20% since the start of treatment due to either increased mass or spread of the tumor.

Disease-free survival (DFS) refers to the period of time during and after treatment during which the patient shows no signs of disease.

Overall survival (OS) refers to the time period from patient enrollment to death or date of last presentation. OS includes an increase in life expectancy compared to control or untreated individuals or patients. Overall survival refers to the situation where a patient remains alive for a certain period of time, such as a year, five years, etc., such as from the time of diagnosis or treatment.

Terms such as treating or treating or treating or alleviating or alleviating refer to therapeutic measures that cure, slow, reduce symptoms and/or stop the progression of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those already diagnosed or suspected of having a disorder. In some embodiments, a subject is successfully cured of cancer in accordance with the methods of the present invention if the patient demonstrates one or more of the following: a decrease in the number or complete absence of cancer cells; reduction in tumor size; suppression or absence of infiltration of cancer cells into peripheral organs, including, for example, spread of cancer into soft tissue and bone; suppression or absence of tumor metastasis; suppression or absence of tumor growth; relief of one or more symptoms associated with a specific cancer; reduction in morbidity and mortality; improving quality of life; reducing tumorigenicity, incidence of tumorigenic conditions, or tumorigenic ability of a tumor; reducing the number or frequency of cancer stem cells in the tumor; differentiation of tumor cells into a non-tumorigenic state; improved progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), decreased disease progression (PD), decreased time to progression ( TTP) or any combination thereof.

Prophylactic or preventive measures refer to therapeutic measures that prevent and/or slow the development of the target pathological condition or disorder. Thus, those in need of prophylactic or preventive measures include those who are susceptible to the disorder and those who have the disorder that is preventable.

As used herein, the term health care provider refers to individuals or institutions that directly interact with and deliver exposure to living subjects, such as human patients. Non-limiting examples of health care providers include physicians, nurses, laboratory technicians, physicians, pharmacists, counselors, alternative medicine practitioners, medical facilities, doctors' offices, hospitals, emergency departments, clinics, urgent care centers, alternative medicine clinics/facilities, and any other organizations providing general and/or specialized treatment, supervision, support, therapy, remedy and/or advice relating to all or any part of the patient's health condition, including, but not limited to, general medical, specialized medical, surgical and/ or any other type of treatment, supervision, support, therapy, treatment and/or advice. In some aspects, a health care provider may administer or instruct another health care provider to administer therapy to treat cancer. Providing therapy as used herein includes administering therapy to a subject, as well as delivering, administering, or providing therapy to a subject. A health care provider may provide or direct another health care provider or patient to do the following: obtain a sample, process a sample, provide a sample, receive a sample, transmit a sample, analyze or measure a sample, quantify a sample, provide results , obtained after analysis/measurement/quantification of a sample, obtain results obtained after analysis/measurement/quantification of a sample, compare/evaluate results obtained after analysis/measurement/quantification of one or more samples, provide comparison/evaluation of one or more samples , obtain a comparison/evaluation of one or more samples, prescribe therapy or a therapeutic agent (eg, CD37 binding agent), initiate therapy, discontinue therapy, continue therapy, temporarily interrupt therapy, increase the amount of therapeutic agent administered, decrease the amount administered therapeutic agent, continue administration of the amount of therapeutic agent administered, increase the frequency of administration of the therapeutic agent, decrease the frequency of administration of the therapeutic agent, maintain the same frequency of administration of the therapeutic agent, replace the therapy or therapeutic agent with at least another therapy or therapeutic agent, combine the therapy or therapeutic agent by at least with another therapy or additional therapeutic agent. These actions may be performed automatically by the health care provider using a computerized method (eg, through a web service or stand-alone computer system).

The terms polynucleotide or nucleic acid, used interchangeably herein, refer to polymers of nucleotides of any length and include DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and analogs thereof. The modification, if present, can be introduced into the nucleotide structure before or after polymer assembly. The nucleotide sequence can be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example, by conjugation with a labeled moiety. Other types of modifications include, for example, caps, replacement of one or more natural nucleotides with analogues, internucleotide modifications, such as, for example, modifications with uncharged bonds (for example, methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g. phosphorothioates, phosphorodithioates, etc.) containing terminal fragments, such as, for example, proteins (e.g. nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), with intercalators (for example, acridine, psoralen, etc.), containing chelators (for example, metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, with modified bonds (for example, alpha anomeric nucleic acids etc.), as well as unmodified forms of polynucleotide (polynucleotides). Additionally, any of the hydroxyl groups typically present in sugars may be substituted, for example, with phosphonate groups, phosphate groups, protected with standard protecting groups, or activated to provide additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5' and 3' OH can be phosphorylated or replaced with amines or organic capping moieties containing from 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups. The polynucleotides may also contain sugar analogues derived from ribose or deoxyribose, which are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido- ribose, carbocyclic sugar analogues, alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogues and nucleoside analogues lacking nitrogenous bases such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments where the phosphate is replaced by P(O)S (thioate), P(S)S (dithioate), (O)NR2 (amidate), P(O)R, P(O)OR', CO or CH2 (formacetal), in which each R or R' independently represents H or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all bonds in a polynucleotide need to be the same. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

The term vector means a construct that is capable of delivering and expressing one or more desired genes or sequences into a host cell. Examples of vectors include, but are not limited to, viral vectors, deproteinized DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors coupled to cationic condensing agents, DNA or RNA expression vectors in liposomal dosage forms, and certain eukaryotic cells, such as producer cells.

The terms polypeptide, peptide and protein are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids and its chain may include non-amino acids. The terms also cover a polymer of amino acids that has been modified naturally or through intervention; for example, the formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a tag component. Also included in the definition are, for example, polypeptides containing one or more amino acid analogues (including, for example, non-naturally occurring amino acids, etc.), as well as other modifications known in the art. It is understood that, since the polypeptides of the present invention are antibody based, in some embodiments the polypeptides may occur as single chains or linked chains. In some embodiments, the polypeptide, peptide, or protein is not naturally occurring. In some embodiments, the polypeptide, peptide, or protein is obtained by purification from natural components. In some embodiments, the polypeptide, peptide, or protein is produced recombinantly.

The terms identical or percent identity, in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or contain a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned (and gapping, if necessary) for maximum matching without considering any conservative amino acid substitutions in the context of sequence identity. Percent identity can be determined using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to align amino acid or nucleotide sequences. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-2268, modified from Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-5877 and included in the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In some embodiments, the Gapped BLAST program may be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389–3402. Other publicly available programs that can be used for sequence alignment are BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR). In some embodiments, the percent identity of two nucleotide sequences is determined using the GAP program included in the GCG software (e.g., using the matrix NWSgapdna.CMP and a gap penalty value of 40, 50, 60, 70, or 90 and a prolongation penalty value gap equal to 1, 2, 3, 4, 5 or 6). Alternatively, in some embodiments, the GAP program of the GCG software package including the Needleman-Wunsch algorithm (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity of two amino acid sequences ( for example, using a Blossum 62 matrix or a PAM250 matrix and a skip penalty value of 16, 14, 12, 10, 8, 6 or 4 and a skip penalty value of 1, 2, 3, 4, 5). Alternatively, in some embodiments, the percent identity of two nucleotide or amino acid sequences is determined using the Miller-Myers algorithm (CABIOS, 4:11-17 (1989)). For example, percent identity can be determined using the ALIGN program (version 2.0) and using PAM120 with a residual table, a pass-over penalty value of 12, and a skip-penalty value of 4. Suitable

- 16 044601 parameters for maximum alignment by means of specific alignment software can be determined by one skilled in the art. In some embodiments, the default parameters of the alignment software are used. In some embodiments, the percent identity X of the first amino acid sequence with respect to the second amino acid sequence is calculated as 100x(Y/Z), where Y is the number of amino acid residues marked as identical matches at alignment of the first and second sequences (according to visual inspection or a specific sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is greater than the length of the second sequence, the percentage identity of the first sequence with respect to the second sequence will be greater than the percentage identity of the second sequence with respect to the first sequence. By way of non-limiting example, having a certain percentage of sequence identity to a particular polynucleotide (e.g., at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95, 96, 97, 98, or 99% identical) to the reference sequence in certain embodiments can be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison , WI 53711). Bestfit uses the local homology algorithm according to Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percent identity is calculated over the entire length of the reference nucleotide sequence and gaps are allowed in homology up to 5% of the total number of nucleotides in the reference sequence.

In some embodiments, two nucleic acids or two polypeptides of the present invention are substantially identical, that is, have at least 70%, at least 75%, at least 80%, at least 85% identity in nucleotides or amino acid residues. at least 90%, and in some embodiments, at least 9, 96, 97, 98, 99% when compared and aligned for best fit, determined using a sequence comparison algorithm or visual inspection. In some embodiments, the identity occurs in a sequence region that is at least about 10, about 20, about 40-60 residues in length, or any integer value therebetween, or in a region that is greater than 60-80 residues in length by at least about 90-100 residues, or sequences, are substantially identical throughout the entire length of the compared sequences, such as, for example, the coding region of a nucleotide sequence.

A conservative amino acid substitution is an amino acid substitution in which one amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). For example, replacing phenylalanine with tyrosine is a conservative substitution. In some embodiments, conservative substitutions in the polypeptide and antibody sequences of the present invention do not abolish the binding of the polypeptide or antibody comprising the amino acid sequence to the antigen(s), i.e., CD37, to which the polypeptide or antibody binds. Methods for identifying nucleotide and amino acid conservative substitutions that do not interfere with antigen binding are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12 (10):879-884 (1999) and Burks et al. Proc. Natl. Acad. Sci. USA, 94:412-417 (1997)).

As used herein and in the claims, the indefinite and definite singular forms include the plural forms unless the context clearly dictates otherwise.

It is understood that whenever embodiments are described herein using the language including/comprising, other similar embodiments described in terms of consisting of and/or essentially consisting of are also provided.

The term and/or as used herein in a phrase such as A and/or B is intended to include A and B, A or B, A and B. Similarly, the term and/or used in a phrase such as A, B and /or C is intended to cover each of the following implementations: A, B and C; A,

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B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (separately); B (separately) and C (separately).

II. CD37 binding agents.

Provided herein are agents that specifically bind to human CD37. These agents are referred to herein as CD37 binding agents. In some embodiments, the CD37 binding agents are antibodies, antigen binding fragments thereof, immunoconjugates, or polypeptides. Full-length amino acid sequences for human CD37 are known in the art and are also set forth herein as set forth in SEQ ID NO: 1, respectively. Human CD37:

MSAQESCLSLIKYFLFVFNLFFFVLGSLIFCFGIWILIDKTSFVSFVGLAFVPLQIWSKVLAI SGIFTMGIALLGCVGALKELRCLLGLYFGMLLLLFATQITLGILISTQRAQLERSLRDVVE KTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCY NL SATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQKWLHNNL ISIVGICLGVGLLELGFMTLSIFLCRNLDHVYNRLARYR (SEQ ID NO:1)

Human CD37 nucleic acid sequence:

atgtcagcccaggagagctgcctcagcctcatcaagtacttcctcttcgttttcaacctcttcttcttcgtcctcggcagcctgatcttctgcttcg gcatctggatcctcattgacaagaccagcttcgtgtcctttgtgggcttggccttcgtgcctctgcagatctggtccaaagtcctggccat ctca ggaatcttcaccatgggcatcgccctcctgggttgtgtgggggccctcaaggagctccgctgcctcctgggcctgtattttgggatgctgctg ctcctgtttgccacacagatcaccctgggaatcctcatctccactcagcgggcccagctggagcgaagcttgcgggacgtcgtagagaaaa ccatccaaa agtacggcaccaaccccgaggagaccgcggccgaggagagctgggactatgtgcagttccagctgcgctgctgcggctg gcactaccccgcaggactggttccaagtcctcatcctgagaggtaacgggtcggaggcgcaccgcgtgccctgctcctgctacaacttgtcg gcgaccaacgactccacaatcctagataaggtgatctt gccccagctcagcaggcttggacacctggcgcggtccagacacagtgcagac atctgcgctgtccctgcagagagccacatctaccgcgagggctgcgcgcagggcctccagaag1ggc1gcacaacaaccttatttccatag tgggcatttgcctgggcgtcggcctactcgagctcgggttcatgacgctctcgatatt cctgtgcagaaacctggaccacgtctacaaccggc tcgctcgataccgt (SEQ ID NO:2)

Thus, in some embodiments, CD37 binding agents may specifically bind to the epitope represented by SEQ ID NO: 1 or to the epitope of the polypeptide encoded by SEQ ID NO: 2.

CD37 binding agents also include CD37 binding agents with the 1B11-2 antibody sequences set forth below.

muCD37-lBl 1-2 VH-CDR1

GYFMN (SEQ ID NO:3) muCD37-lBl 1-2 VH-CDR2 (Kabat)

RINPYNGDTFYNQKFKr (SEQ ID NO:4) muCD37-lBll-2 VH-CDR2 (AbM)

RINPYNGDTF (SEQ ID NO:34) muCD37-lBl 1-2 VH -CDR3

RGIVASSRFFDV (SEQ ID NO:5)

- 18 044601 muCD37-lBl 1-2 VL-CDR1

KASQGVSNDVD (SEQ ID NO:6) muCD37-lBl 1-2 VL-CDR2

YASNRYT (SEQ ID NO:7) muCD37-lBl 1-2 VL-CDR3

CHQDYTSPT (SEQ ID NO:8) muCD37-lBl 1-2 heavy chain variable region (VH)

EVQLLQSGPELVKPGASVKISCKASGYSFTGYFMNWVIQSHGKFLEWIGRINPYNGDTFY NQKFKrKATLTVDKSSTTAHMELLSLTSEDSAVYYCGSRGIVASSRFFDVWGAGTSVIVS S(SEQIDNO:9) muCD37-lBl 1-2 light chain variable region (VL)

SIVMFQTPKFLLVSAGDRVTITCKASQGVSNDVDWYQQKPGQSPKLLIYYASNRYTGVP DRFTGSGYGTDFTFS1STVQAEDLAVYFCHQDYTSPTFGGGTKLE1KR (SEQ ID NO: 10) muCD37- lBl 1-2 full-length heavy chain (HC)

EVQLLQSGPELVKPGASVKISCKASGYSFTGYFMNWVIQSHGKFLEW1GRINPYNGDTFY NQKFKFKATLTVDKSSTTAHMELLSLTSEDSAVYYCGSRGIVASSRFFDVWGAGTSVIVS SAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKTYFPEPVTVTWNSGSLSSGVHTFPAVLES DLYTLSSSVTVPSSPRPSETVTCN VAHPASSFKVDKKIVPRDCGCKPCICTVPEVSSVFIFP PKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDEVHTAQTQPREEQFNSTFRSVSE LP1MHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKFRPKAPQVYTIPPPKEQMAKDKVSL TCMITDFFPEDITVEWQWNGQPAEN YKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTF TCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO:11) muCD37-lBl 1-2 full length light chain

SIVMTQTPKFLLVSAGDRVTITCKASQGVSNDVDWYQQKPGQSPKLLIYYASNRYTGVP DRFTGSGYGTDFTFSISTVQAEDLAVYFCHQDYTSPTFGGGTKLEIKRADAAPTVSIFPPS SEQLTSGGASWCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT LTKDEYERH NSYTCEATHKTSTSP1VKSFNRNEC (SEQ ID NO: 12)

CD37 binding agents include CD37 binding agents (eg, antibodies and antigen binding fragments thereof) that contain the six CDR sequences of the 1B11-2 antibody represented by SEQ ID NOs: 3-8. CD37-binding molecules may be CD37-binding agents (eg, antibodies and antigen-binding fragments thereof) that contain antibody 1B11-2 CDR regions (i.e., SEQ ID NO: 3-8) containing up to four (i.e. 0, 1, 2, 3 or 4) conservative amino acid substitutions per CDR. CD37-binding molecules may be CD37-binding agents (eg, antibodies and antigen-binding fragments thereof) that contain antibody 1B11-2 CDR regions (i.e., SEQ ID NO: 3-8) containing up to four (i.e. 0, 1, 2, 3 or 4) conservative amino acid substitutions. CD37-binding molecules may be CD37-binding agents (e.g., antibodies and antigen-binding fragments thereof) that contain (i) CDR regions represented by SEQ ID NOs: 3, 4, 6 and 7, containing up to four (i.e. 0, 1, 2, 3 or 4) conservative amino acid substitutions on the CDRs and (ii) the CDR regions represented by SEQ ID NOs: 5 and 8. The CD37-binding molecules may be CD37-binding agents (e.g., antibodies and antigen-binding fragments thereof ), which contain (i) CDR regions represented by SEQ ID NOs: 3, 4, 6 and 7, containing up to four (i.e. 0, 1, 2, 3 or 4) conservative amino acid substitutions and (ii) CDR regions represented by SEQ ID NOs: 5 and 8. The CD37-binding molecules may be CD37-binding agents (eg, antibodies and antigen-binding fragments thereof) that contain (i) the CDR regions represented by SEQ ID NOs: 3, 4, and 6- 8, containing up to four (i.e., 0, 1, 2, 3 or 4) conservative amino acid substitutions per CDR and (ii) the CDR region represented by SEQ ID NO: 5. CD37-binding molecules may be CD37-binding agents (e.g., antibodies and antigen-binding fragments thereof) that contain (i) CDR regions represented by SEQ ID NOs: 3, 4 and 6-8, containing up to four (i.e. 0, 1, 2, 3 or 4) conservative amino acid substitutions and (ii) the CDR region represented by SEQ ID NO: 5.

The polypeptides, antibodies, and antigen binding fragments thereof may comprise a light chain variable region, a heavy chain variable region, or both light and heavy chain variable regions of the 1B11-2 antibody. Also provided are polypeptides that comprise (a) a polypeptide having a sequence at least 90% identical to SEQ ID NO: 9 and/or (b) a polypeptide having a sequence at least 90% identical to SEQ ID NO: 10. In some embodiments, the polypeptide is an antibody and/or polypeptide that specifically binds to CD37. In some embodiments, the polypeptide is murine, chimeric, or

- 19 044601 humanized antibody that specifically binds to CD37. In some embodiments, a polypeptide having a certain percentage identity to SEQ ID NO: 9 or 10 differs from SEQ ID NO: 9 or 10 only by the presence of conservative amino acid substitutions.

In some embodiments, the polypeptide, antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 9. In some embodiments, the polypeptide, antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain, comprising a sequence at least 96% identical to SEQ ID NO: 9. In some embodiments, the polypeptide, antibody, or antigen binding fragment thereof comprises a heavy chain variable domain comprising a sequence at least 97% identical to SEQ ID NO: 9. B In some embodiments, the polypeptide, antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising a sequence at least 98% identical to SEQ ID NO: 9. In some embodiments, the polypeptide, antibody, or antigen-binding fragment thereof comprises a heavy chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 9. In some embodiments, the polypeptide is an antibody and/or polypeptide that specifically binds to CD37. In some embodiments, the polypeptide is a murine, chimeric, or humanized antibody that specifically binds to CD37. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 9 differs from SEQ ID NO: 9 only by the presence of conservative amino acid substitutions. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 9 differs from SEQ ID NO: 9 only in amino acids outside the CDR regions. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 9 differs from SEQ ID NO: 9 only by conservative amino acid substitutions outside the CDR regions.

In some embodiments, the polypeptide, antibody, or antigen-binding fragment thereof comprises a light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the polypeptide, antibody, or antigen-binding fragment thereof comprises a light chain variable domain, comprising a sequence at least 96% identical to SEQ ID NO: 10. In some embodiments, the polypeptide, antibody, or antigen binding fragment thereof comprises a light chain variable domain comprising a sequence at least 97% identical to SEQ ID NO: 10. B In some embodiments, the polypeptide, antibody, or antigen-binding fragment thereof comprises a light chain variable domain comprising a sequence at least 98% identical to SEQ ID NO: 10. In some embodiments, the polypeptide, antibody, or antigen-binding fragment thereof comprises a light chain variable domain comprising a sequence that is at least 99% identical to SEQ ID NO: 10. In some embodiments, the polypeptide is an antibody and/or polypeptide that specifically binds to CD37. In some embodiments, the polypeptide is a murine, chimeric, or humanized antibody that specifically binds to CD37. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 10 differs from SEQ ID NO: 10 only by the presence of conservative amino acid substitutions. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 10 differs from SEQ ID NO: 10 only in amino acids outside the CDR regions. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 10 differs from SEQ ID NO: 10 only by conservative amino acid substitutions outside the CDR regions.

In some embodiments, the polypeptide, antibody, or antigen binding fragment thereof comprises a heavy chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 9 and a light chain variable domain comprising a sequence at least 95% identical to SEQ ID NO: 10. In some embodiments, the polypeptide, antibody, or antigen binding fragment thereof comprises a heavy chain variable domain comprising a sequence at least 96% identical to SEQ ID NO: 9 and a light chain variable domain comprising a sequence of at least 96% identical to SEQ ID NO: 10. In some embodiments, the polypeptide, antibody, or antigen binding fragment thereof comprises a heavy chain variable domain comprising a sequence at least 97% identical to SEQ ID NO: 9, and a light chain variable domain comprising a sequence at least 97% identical to SEQ ID NO: 10. In some embodiments, the polypeptide, antibody, or antigen binding fragment thereof comprises a heavy chain variable domain comprising a sequence at least 98% identical to SEQ ID NO: 9, and a variable a light chain domain comprising a sequence at least 98% identical to SEQ ID NO: 10. In some embodiments, the polypeptide, antibody, or antigen binding fragment thereof comprises a heavy chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: : 9, and a light chain variable domain comprising a sequence at least 99% identical to SEQ ID NO: 10. In some embodiments, the polypeptide is an antibody and/or a polypeptide that specifically binds to CD37. In some embodiments, the polypeptide is a murine, chimeric, or humanized antibody that specifically binds to CD37. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 9 and/or 10 differs from SEQ ID NO: 9 and/or 10 only by the presence of conservative amino acid substitutions. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 9 and/or 10 differs from SEQ ID NO: 9 and/or 10 only by amino acids outside the CDR regions. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 9 and/or 10 differs from SEQ ID NO: 9 and/or 10 only by conservative amino acid substitutions outside the CDR regions.

In some embodiments, the polypeptide, antibody, or antigen binding fragment thereof comprises a heavy chain variable domain comprising the sequence represented by SEQ ID NO: 9 and a light chain variable domain comprising the sequence represented by SEQ ID NO: 10.

Polypeptides may contain one of the individual light chains or heavy chains described herein. Antibodies and polypeptides may also contain both a light chain and a heavy chain. The light chain and heavy chain sequences of antibody 1B11-2 are shown in SEQ ID NOs: 11 and 12.

Also provided are polypeptides that comprise (a) a polypeptide having at least about 90% sequence identity to SEQ ID NO: 11, and/or (b) a polypeptide having at least about 90% sequence identity to SEQ ID NO: 12. B In some embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 11, or 12. Thus, in some embodiments, the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID NO: 11, and/or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NO: 11 NO: 12. In some embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence represented by SEQ ID NO: 11; and/or (b) a polypeptide having the amino acid sequence represented by SEQ ID NO: 12. In some embodiments, the polypeptide is an antibody and/or a polypeptide that specifically binds CD37. In some embodiments, the polypeptide is a murine, chimeric, or humanized antibody that specifically binds to CD37. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 11 and/or 12 differs from SEQ ID NO: 11 and/or 12 only by the presence of conservative amino acid substitutions. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 11 and/or 12 differs from SEQ ID NO: 11 and/or 12 only by amino acids outside the CDR regions. In some embodiments, a polypeptide having a certain percentage sequence identity to SEQ ID NO: 11 and/or 12 differs from SEQ ID NO: 11 and/or 12 only by conservative amino acid substitutions outside the CDR regions.

In some embodiments, the CD37 binding agent (e.g., an antibody or antigen binding fragment thereof) binds to the same epitope as the antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising the sequence represented by SEQ ID NO: 9 and a variable region light chain comprising the sequence represented by SEQ ID NO: 10. In some embodiments, a CD37 binding agent (e.g., an antibody or an antigen binding fragment thereof) competitively inhibits the binding of an antibody or antigen binding fragment thereof comprising a heavy chain variable region comprising the sequence represented by SEQ ID NO: 9, and a light chain variable region containing the sequence represented by SEQ ID NO: 10 with CD37. In some embodiments, a CD37 binding agent (e.g., an antibody or an antigen binding fragment thereof) competitively inhibits the binding of an antibody or an antigen binding fragment thereof comprising a heavy chain variable region comprising the sequence represented by SEQ ID NO: 9 and a light chain variable region comprising the sequence represented by SEQ ID NO: 10, with CD37, and binds to an epitope that overlaps with an epitope to which an antibody or antigen binding fragment thereof binds, containing a heavy chain variable region containing the sequence represented by SEQ ID NO: 9, and a light chain variable region chain comprising the sequence represented by SEQ ID NO: 10. In some embodiments, the CD37 binding agent (eg, an antibody or antigen binding fragment thereof) binds and/or does not bind to the CD37 antigenic sequences set forth herein below.

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CD37 antigenic sequences:

hCD37-LEL (AK 107-242 CD37 is underlined)

TMELLISTQRAQLERSLRDVVEKT1OKYGTNPEETAAEESWDYVQFOLRCCGWHYPQD WFOVLILRNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVP AESHIYREGCAOGLOKWLHNNLSFLEOKLISEEDLNSAVDHHHHHHfSEO GO NO: 15) hCD37-ECD- Fc (underlined AK 107-235 hCD37)

GPEFL1STORAOLERSLRDVVEKTIOKYGTNPEETAAEESWDYVOFOLRCCGWHYPQD WFQVLILRGNGSEAHRVPCSCYNl·SATNDSTILDKVILPOLSRLGHLARSRHSADICAVP AESHIYREGCAOGLOGSEPRGPTIKPCPPCKCPAPNLLGGPSVF1FPPKIKDVLMISLSPIVT CVVVDVSEDDPDVQI SWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKE FKCKVNNKDLPAPIERTISKPKFSVRAPQVYVLPPPEEEMTKKQVTLTLTCMVTDFMPEDIY VEWTWGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNH HTTKSFSRTPGK (SEQ GO NO: 1 6) hCD37-Fc-LAGA (underlined AK 107-235 hCD37)

GPEFLISTORAOLERSLRDVVEKTIOKYGTNPEETAAEESWDYVOFOLRCCGWHYPOD WFOVLILRGNGSEAHRVPCSCYNLSATNDST1L·DKVIL·POLSRLGHLARSRHSADICAVP AESHIYREGCAQGLQGSDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKFQPREPQVYTLPPSRDELTKNQVSLTCLVKrFYPSDTAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG (SEQ ID NO:17) hCD 37-ECD-S2-Fc (AK 107-109 hCD37 is underlined twice and AK 138-235 is underlined)

GPEFLISAAEESWDYVOFOLRCCGWHYPODWFOVLILRGNGSEAHRVPCSCYNl·SATN DSTILDKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQGSEPRGPTIKPCPPC KCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQ TQTHREDYNSTLRV VSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKrSVRAPQ VYVLPPPEEEMTKKQVTLTCMVTDFMPEDTYVEWTNNGKTELNYKNTEPVLDSDGSYF MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK (SEQ ID NO: 18) hCD37 (AK 107-235)

LISTQRAQLERSLRDVVEKTIQKYGTNPEETAAEESWDYVQFQLRCCGWHYPQDWFQV LILRGNGSEAHRVPCSCYNLSATNDSTILDKVILPQLSRLGHLARSRHSADICAVPAESHI YREGCAQGLQ (SEQ ID NO: 19) hCD37 (AK 107-242)

ListqraqlerslrdvvekkkygtnpeetnpeeswhyvqfqlrclrclrgwhypcdwfQv LilrGngseahrvpskvilsrSrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhsrhlhpaeshi (SEQ ID NO: 20) HCD37 (AK 107-109 and 138-235)

LISAAEESWDYVQFQLRCCGWHYPQDWFQVLILRGNGSEAHRVPCSCYNLSATNDSTIL DKVILPQLSRLGHLARSRHSADICAVPAESHIYREGCAQGLQ (SEQ ID NO :21)

In some embodiments, the CD37 binding agent (eg, an antibody or antigen binding fragment thereof) binds to amino acids 107-242 of CD37 (SEQ ID NO: 20). In some embodiments, the CD37 binding agent (eg, an antibody or antigen binding fragment thereof) binds to amino acids 107-235 of CD37 (SEQ ID NO: 19).

In some embodiments, a CD37 binding agent (e.g., an antibody or antigen binding fragment thereof) binds to the sequence represented by SEQ ID NO: 15. In some embodiments, a CD37 binding agent (e.g., an antibody or antigen binding fragment thereof) binds to the sequence represented by SEQ ID NO: 16. In some embodiments, the CD37 binding agent (e.g., an antibody or antigen binding fragment thereof) binds to the sequence represented by SEQ ID NO: 17.

In some embodiments, the CD37 binding agent (eg, an antibody or antigen binding fragment thereof) binds to amino acids 107-242 of CD37 (SEQ ID NO: 20) but does not bind to amino acids 138-235 of CD37. In some embodiments, the CD37 binding agent (eg, an antibody or antigen binding fragment thereof) binds to amino acids 107235 of CD37 (SEQ ID NO: 19) but does not bind to amino acids 138-235 of CD37.

In some embodiments, the CD37-binding agent (e.g., an antibody or antigen-binding fragment thereof) binds to the polypeptide represented by SEQ ID NO: 15, but does not bind to the polypeptide represented by SEQ ID NO: 18. In some embodiments, the CD37-binding agent ( e.g., an antibody or antigen-binding fragment thereof) binds to the polypeptide represented by SEQ ID NO: 16, but does not bind to the polypeptide represented by

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SEQ ID NO: 18. In some embodiments, the CD37 binding agent (e.g., an antibody or antigen binding fragment thereof) binds to the polypeptide represented by SEQ ID NO: 17, but does not bind to the polypeptide represented by SEQ ID NO: 18.

In some embodiments, a CD37-binding agent (e.g., an antibody or an antigen-binding fragment thereof) binds to an epitope that contains at least one amino acid from SEQ ID NO: 110-137. Amino acids 110-137 represent the first segment (S1) of the large extracellular domain of CD37.

The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method well known in the art, for example, flow cytometry, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) or kinetics (eg, BIACORE™ assay). Direct binding assay formats as well as competitive binding assay formats can be easily used. (See, for example, Berzofsky, et al., Berzofsky, et al., Antibody-Antigen Interactions, In Fundamental Immunology, Paul, W.E., Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, WH Freeman and Company: New York, NY (1992) and the methods described herein.The measured affinity of an antibody-antigen interaction may vary when measured under different conditions (eg, salt concentration, pH, temperature).Thus, affinity measurements and other antigen binding parameters (eg, K _D or K _d , K _on , K _off ) are performed with standardized antibody and antigen solutions and a standardized buffer, such as those known in the art and such as the buffer described herein.

In one aspect, the binding assay can be performed by flow cytometry on cells expressing the CD37 antigen on the surface. For example, CD37-positive cells, such as 300-19 cells, can be incubated with various concentrations of anti-CD37 antibodies using 1x105 cells per sample in 100 μl FACS buffer (RPMI-1640 medium supplemented with 2% normal goat serum).

Cells can then be pelleted, washed, and incubated for 1 hour with 100 μl of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or goat anti-human IgG (such as those available from Jackson Laboratory, 6 μg/ml in buffer for FACS). Cells are then pelleted again, washed with FACS buffer, and resuspended in 200 μl of phosphate-buffered saline (PBS) containing 1% formaldehyde. Samples can be obtained, for example, using a FACS Calibur flow cytometer with an HTS multiwell sampler and analyzed using CellQuest Pro (all BD Biosciences instruments, San Diego, USA). For each sample, the mean FL1 fluorescence intensity (MFI) can be exported and plotted against antibody concentration on a semi-log scale to obtain a binding curve. Binding curves are plotted with a sigmoidal dose-response curve and EC50 values are calculated using programs such as GraphPad Prism v4 with default parameters (GraphPad Software, San Diego, CA). EC ₅₀ values can be used as a measure of the apparent dissociation constant Kd or _KD for each antibody.

Monoclonal antibodies can be produced using hybridoma methods, such as those described by Kohler and Milstein (1975), Nature, 256:495. In the hybridoma method, a mouse, hamster, or other suitable host animal is immunized to induce the lymphocytes to produce antibodies that will specifically bind to the immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells, which can then be separated from the unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies specifically directed against a selected antigen, as determined, for example, by immunoprecipitation, immunoblotting or in vitro binding assays (eg, radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)), can then be propagated in or in culture in vitro using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986), or in vivo in the form of ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid. Alternatively, monoclonal antibodies can also be produced by methods using recombinant DNA, as described in US Pat. No. 4,816,567. Polynucleotides encoding a monoclonal antibody are isolated from mature B cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers, which specifically amplify the genes encoding the heavy and light chains of an antibody, and their sequence is determined using conventional methods. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which are transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that are not otherwise produce immunoglobulin protein, and monoclonal antibodies are produced by host cells. Also, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries expressing CDR regions

- 23 044601 desired species as described (McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991, Nature, 352:624-628 and Marks et al., 1991, J. Mol. Biol., 222:581-597). The polynucleotide(s) encoding a monoclonal antibody can be further modified in various ways using recombinant DNA technology to produce alternative antibodies. In some embodiments, the heavy and light chain constant domains of, for example, a mouse monoclonal antibody can be replaced with 1) the same regions, for example, a human antibody to produce a chimeric antibody or 2) a non-immunoglobulin polypeptide to produce a fusion antibody. In some embodiments, constant regions are truncated or removed to produce the desired monoclonal antibody fragment. To optimize specificity, affinity, etc. of a monoclonal antibody, site-directed or high-density mutagenesis of the variable region can be used.

In some embodiments, the anti-human CD37 monoclonal antibody is a humanized antibody. In some embodiments, the humanized antibody is a surface-edited antibody.

Human antibodies can be obtained directly using various methods known in the art. Immortalized human B lymphocytes that produce antibody directed against a target antigen can be prepared, immunized in vitro, or isolated from an immunized individual (see, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147(1):86-95 and US patent 5750373). Also, the human antibody may be selected from a phage library if said phage library expresses human antibodies, as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al., 1991, J. Mol. Biol., 222:581). Methods for obtaining and using phage antibody libraries are also described in US patents No. 5969108, 6172197, 5885793, 6521404; 6544731; 6555313; 6582915; 6593081; 6300064; 6653068; 6706484 and 7264963 and Rothe et al., 2007, J. Mol. Bio., doi:10.1016/j.jmb.2007.12.018 (each of which is incorporated by reference in its entirety). Affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio/Technology 10:779-783, incorporated by reference in their entirety) are known in the art and can be used to produce high-affinity human antibodies.

Humanized antibodies can also be produced in transgenic mice carrying human immunoglobulin loci, capable of producing the full range of human antibodies after immunization in the absence of endogenous immunoglobulin production. This approach is described in US patents No. 5545807;5545806;5569825;5625126;5633425 and 5661016.

In some embodiments, an antibody fragment is provided to, for example, enhance tumor penetration. Various methods are known for producing antibody fragments. Traditionally, these fragments are obtained by proteolytic cleavage of intact antibodies (eg, Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods, 24:107-117; Brennan et al., 1985, Science, 229:81). In some embodiments, the antibody fragments are produced recombinantly. Fab, Fv and scFv antibody fragments can be expressed and secreted by E. coli or other host cells, thereby allowing the production of large quantities of these fragments. Such antibody fragments can also be isolated from the phage antibody libraries discussed above. The antibody fragment may also be a linear antibody, as described, for example, in US Pat. No. 5,641,870, and may be monospecific or bispecific. Other methods for producing antibody fragments will be apparent to one skilled in the art.

In accordance with the present invention, the methods can be adapted to produce single chain antibodies specific for CD37 (see US patent No. 4946778). In addition, the methods can be adapted to construct Fab expression libraries (Huse, et al., Science, 246:1275-1281 (1989)) to provide rapid and efficient identification of monoclonal Fab fragments with the desired CD37 specificity or derivative fragments thereof, analogues or homologues. Antibody fragments can be produced by methods in the art, including, but not limited to: (a) an F(ab') ₂ fragment obtained by digestion of an antibody molecule with pepsin; (b) a Fab fragment obtained by reducing the disulfide bridges of the F(ab') ₂ fragment; (c) a Fab fragment obtained by treating the antibody molecule with papain and a reducing agent; and (d) Fv fragments.

For the purposes of this description, it should be understood that the modified antibodies may contain any type of variable region that allows the antibody to bind to human CD37 polypeptides. In this regard, the variable region may comprise a region of any type of mammal or be derived from any type of mammal. Thus, the variable region of the modified antibodies may be derived from, for example, humans, mice, non-human primates (eg, cynomolgus monkeys, macaques, etc.), or wolves. In some embodiments, both the variable and constant regions of the modified immunoglobulins are human. In other embodiments, the variable regions of compatible antibodies (usually derived from a non-human source) can be engineered

- 24 044601 baths or specially adapted to improve the binding properties of the molecule. Accordingly, variable regions useful in the present invention may be humanized or otherwise modified to include other (imported) amino acid sequences. In some embodiments, the variable regions of the modified antibodies may be non-human (eg, murine), and the constant regions of the modified antibodies may be human.

In some embodiments, the variable domains in both the heavy and light chains are altered by at least partially replacing one or more CDR regions and, if necessary, partially replacing the framework region and changing the sequence. Although the CDR regions may be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is contemplated that the CDR regions will be derived from an antibody of a different class and possibly from a different species of antibody. It is not always necessary to replace all CDR regions with complete CDR regions from the donor variable region in order to transfer the antigen binding capacity of one variable domain to another. Rather, in some cases it is necessary to transfer only those residues that are necessary to maintain the activity of the antigen binding site.

Notwithstanding the changes in the variable region, those skilled in the art will appreciate that the modified antibodies of the present invention will comprise antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least a portion of one or more constant region domains have been deleted or otherwise modified to provide the desired biochemical characteristics. In some embodiments, the constant region of the modified antibodies will comprise a human constant region. Modifications to the constant region may include insertions, deletions, or substitutions of one or more amino acids in one or more domains. Thus, the modified antibodies disclosed herein may contain changes or modifications in one or more of the three heavy chain constant domains (CH1, CH2 or CH3) and/or the light chain constant domain (CL). In some embodiments, modified constant regions are contemplated where one or more domains are partially or completely removed. In some embodiments, the modified antibodies will contain domain deleted constructs or variants where the entire CH2 domain has been deleted (ACH2 constructs). In some embodiments, the missing constant region domain will be replaced by a short amino acid spacer (eg, 10 residues) that provides some of the molecular flexibility typically provided by the missing constant region. In some embodiments, modifications to the constant region may be used to remove disulfide bonds or oligosaccharide moieties, allowing for improved positioning due to increased antigen specificity or flexibility of the antibody. Modifications to the constant region can be readily accomplished using well-known biochemical or molecular engineering techniques within the skill of one skilled in the art.

It should be noted that, in some embodiments, the modified antibodies can be designed to fuse the CH3 domain directly to the hinge region of the corresponding modified antibodies. In other designs, it may be desirable to provide a peptide spacer between the hinge region and the modified CH2 and/or CH3 domains. For example, compatible constructs can be expressed where the CH2 domain has been removed and the remaining CH3 domain (modified or unmodified) is attached to the hinge region via a 5-20 amino acid spacer. Such a spacer may be added, for example, to ensure that the constant domain regulatory elements remain free and accessible or that the hinge region remains flexible.

The present description further covers variants and equivalents that are substantially homologous to the murine, chimeric, humanized and human antibodies or fragments thereof provided herein. They may include, for example, mutations - conservative substitutions, i.e. replacing one or more amino acids with similar amino acids. For example, a conservative substitution refers to the replacement of one amino acid with another within the same general class, such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. It is well known in the art what is meant by a conservative amino acid substitution.

The polypeptides herein may be recombinant polypeptides, naturally occurring polypeptides, or synthetic polypeptides comprising an anti-human CD37 antibody or fragment thereof. Those skilled in the art will appreciate that certain amino acid sequences of the present invention may be altered without significantly affecting the structure or function of the protein. Thus, the present invention further includes variations of polypeptides that exhibit significant activity or that contain regions of an antibody to the CD37 protein or a fragment thereof. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.

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The isolated polypeptides described herein can be prepared by any suitable method known in the art. Such methods range from direct methods of protein synthesis to the design of DNA sequences encoding isolated polypeptide sequences and expression of these sequences in a suitable transformed host. In some embodiments, the DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. The specified sequence may optionally be subjected to mutagenesis using site-directed mutagenesis to obtain functional analogues thereof. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA, 81:5662-5066 (1984) and US Patent No. 4588585.

In some embodiments, the DNA sequence encoding the polypeptide of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and the selection of those codons that are preferred by the host cell in which the recombinant polypeptide of interest will be produced.

Standard methods can be used to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, the complete amino acid sequence can be used to construct a gene with the sequence reconstructed from the polypeptide. Additionally, a DNA oligomer may be synthesized containing a nucleotide sequence encoding the specific isolated polypeptide. For example, several small oligonucleotides encoding parts of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or other means), polynucleotide sequences encoding the particular isolated polypeptide of interest will be inserted into an expression vector and operably linked to an expression control sequence suitable for expression of the protein in the desired host. Correct assembly can be confirmed by nucleic acid sequencing, restriction mapping and expression of the biologically active polypeptide in a suitable host. As is well known in the art, to obtain high levels of expression of a transfected gene in a host, the gene must be operably linked to transcriptional and translational expression control sequences that are functionally active in the chosen expression host.

In some embodiments, recombinant expression vectors are used to amplify and express DNA encoding anti-human CD37 antibodies or fragments thereof. Recombinant expression vectors are replicable DNA constructs that contain synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an anti-CD37 antibody or fragment thereof, operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. . A transcription unit typically contains a collection of (1) a genetic element or elements having regulatory significance in gene expression, such as transcriptional promoters or enhancers, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) suitable sequences initiation and termination of transcription and translation, as detailed below. Such regulatory elements may include an operator sequence to control transcription. Additionally, the ability to replicate in the host, usually provided by the origin of replication, and a selection gene may be introduced to facilitate recognition of transformants. Regions of DNA are functionally related when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader sequence) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned to allow translation. Structural elements intended for use in yeast expression systems contain a leader sequence that ensures extracellular secretion of the translated protein by the host cell. Alternatively, when the recombinant protein is expressed without a leader or transport sequence, it may contain an N-terminal methionine residue. This residue may subsequently be optionally cleaved from the expressed recombinant protein to obtain the final product.

The choice of expression control sequence and expression vector will depend on the choice of host. A wide range of host/expression vector combinations can be used. Suitable expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus,

- 26 044601 adenovirus and cytomegalovirus. Suitable expression vectors for bacterial hosts include known bacterial plasmids, such as those from Escherichia coli, including pCR 1, pBR322, pMB9 and derivatives thereof, wider host range plasmids such as M13 and filamentous phages containing single-stranded DNA. Suitable host cells for expressing a CD37-binding polypeptide or antibody (or CD37 protein for use as an antigen) include prokaryotic, yeast, insect, or higher eukaryotic cells under the control of suitable promoters. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or rod-shaped bacteria. Higher eukaryotic cells include stable mammalian cell lines, as described below. Cell-free translation systems can also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian host cells are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevant description of which is incorporated herein by reference. Additional information regarding methods for producing protein, including antibody production, is provided, for example, in US Patent Publication No. 2008/0187954, US Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO 04009823, each of which is hereby incorporated herein by reference. in its entirety.

Various mammalian or insect cell culture systems are also effectively used for recombinant protein expression. Expression of recombinant proteins in mammalian cells can be accomplished because such proteins are usually correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the COS-7 monkey kidney cell lines described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing a suitable vector, including, for example, L cells, C127, 3T3 cells Chinese hamster ovary (CHO), HeLa and BHK cell lines. Mammalian expression vectors may contain non-transcribed elements, such as an origin of replication, a suitable promoter and enhancer associated with the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences and 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for producing heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology, 6:47 (1988).

Proteins produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (eg, ion exchange, affinity and size distribution column chromatography), centrifugation, differential solubility methods, or any other standard protein purification method. Affinity tags such as hexahistidine, maltose binding domain, influenza virus envelope protein sequence, and glutathione S-transferase can be attached to the protein to allow easy purification through a suitable affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

For example, supernatants from systems that secrete recombinant protein into the culture medium may first be concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a suitable matrix for purification. Alternatively, an anion exchange resin may be used, such as a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices may be acrylamide, agarose, dextran, cellulose, or other types commonly used for protein purification. Alternatively, a cation exchange step may be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reverse-phase high-performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel having pendant methyl or other aliphatic groups, can be used to further purify the CD37 binding agent. Some or all of the above purification steps in various combinations can also be used to obtain a homogeneous recombinant protein.

The recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets followed by one or more steps of concentration, salting out, aqueous ion exchange or size exclusion chromatography. High performance liquid chromatography (HPLC) can be used in the final stages of purification.

The microbial cells used to express the recombinant protein may be disrupted by any convenient means, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysing agents.

- 27 044601

Methods known in the art for purifying antibodies and other proteins also include, for example, those described in US Patent Publications No. 2008/0312425, 2008/0177048 and

2009/0187005, each of which is hereby incorporated by reference in its entirety.

III. Polynucleotides.

In some embodiments, the present invention includes polynucleotides comprising polynucleotides encoding a CD37-specific binding polypeptide or a fragment of such a polypeptide. For example, the present invention provides a polynucleotide comprising a nucleic acid sequence encoding an anti-human CD37 antibody or encoding a fragment of such an antibody. The polynucleotides of the present invention may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA and synthetic DNA and can be double-stranded or single-stranded and, in the case of single-stranded, can be a coding strand or a non-coding (antisense) strand. In some embodiments, the polynucleotide is cDNA or DNA lacking one or more endogenous introns.

In some embodiments, the polynucleotides provided herein are not naturally occurring. In some embodiments, the polynucleotides provided herein are produced by a recombinant process. In some embodiments, the polynucleotides are isolated. In some embodiments, the polynucleotides are substantially pure.

Provided herein is a polynucleotide encoding a polypeptide containing a sequence selected from the group consisting of SEQ ID NOs: 3-11.

In some embodiments, the polynucleotide comprises sequences encoding the polypeptides represented by SEQ ID NOs: 3-5. In some embodiments, the polynucleotide comprises sequences encoding the polypeptides represented by SEQ ID NOs: 6-8. In some embodiments, the polynucleotide comprises sequences encoding the polypeptides represented by SEQ ID NOs: 3-5 and SEQ ID NOs: 6-8. In some embodiments, the vector contains polynucleotides encoding the polypeptides represented by SEQ ID NOs: 3-8. In some embodiments, the composition comprises a polynucleotide encoding the polypeptides represented by SEQ ID NOs: 3-5 and a polynucleotide encoding the polypeptides represented by SEQ ID NOs: 6-8. In some embodiments, the composition comprises a vector containing polynucleotides encoding the polypeptides represented by SEQ ID NOs: 3-5, and a vector containing polynucleotides encoding the polypeptides represented by SEQ ID NOs: 6-8.

In some embodiments, the composition comprises a polynucleotide encoding the polypeptide represented by SEQ ID NO: 9 and a polynucleotide encoding the polypeptide represented by SEQ ID NO: 10. In some embodiments, the composition contains a vector containing a polynucleotide encoding the polypeptide represented by SEQ ID NO: 9, and a vector comprising a polynucleotide encoding the polypeptide represented by SEQ ID NO: 10. In some embodiments, the vector contains a polynucleotide encoding the polypeptide represented by SEQ ID NO: 9 and a polynucleotide encoding the polypeptide represented by SEQ ID NO: 10. B The present description further provides a polynucleotide containing a sequence selected from the sequences represented by SEQ ID NOs: 13 and 14.

Nucleic acid sequence VH muCD37-lBl 1-2 gaggttcaactgctgcagtctggacctgagctggtgaagcctggggcttcagtgaagatatcctgcaaggcttctggttactcatttactggct actttatgaactgggtgatacagagccatggaaagggccttgagtggattggacgtattaatccttacaatggtgat accttctacaaccagaa gttcaagggcaaggccacattgactgtagacaaatcctctaccacagcccacatggagctcctgagcctgacatctgaggactctgccgtct attattgtggatcccgggggatagtggcttcctctaggttcttcgatgtctggggcgcagggacctcggtcatcgtctcctcagccaaaacga cac (SEQ ID NO: 13)

Nucleic acid sequence VL muCD37-lBl 1-2 agtattgtgatgacccagactcccaaattcctgcttgtatcagcaggagacagggttaccataacctgcaaggccagtcagggtgtgagtaat gatgtagattggtaccaacagaagccagggcagtctcctaaactgctgatatactatgcatccaatcgctacactggagtccctgat cgcttca ctggcagtggatatgggacggatttcactttcagcatcagcactgtgcaggctgaagacctggcagtttatttctgtcaccaggattatacctct ccgacgttcggtggaggcaccaagctggaaatcaaacgggctgat (SEQ ID NO: 14)

Also provided is a polynucleotide having a sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to one of SEQ ID NOs: 13 and 14. Also provided is a composition comprising a polynucleotide that is at least approximately 90% identical to SEQ ID NO: 13, and a polynucleotide

- 28 044601 at least about 90% identical to SEQ ID NO: 14. Also provided is a composition comprising a polynucleotide at least about 95% identical to SEQ ID NO: 13 and a polynucleotide at least about 95% identical to SEQ ID NO: 14. Also provided is a composition containing a polynucleotide at least about 96% identical to SEQ ID NO: 13 and a polynucleotide at least about 96% identical to SEQ ID NO: 14. Also provided is a composition containing a polynucleotide at least about 97% identical to SEQ ID NO: 13, and a polynucleotide at least about 97% identical to SEQ ID NO: 14. Also provided is a composition comprising a polynucleotide at least about 98% identical to SEQ ID NO: 14. 13, and a polynucleotide that is at least about 98% identical to SEQ ID NO: 14. Also provided is a composition comprising a polynucleotide that is at least about 99% identical to SEQ ID NO: 13, and a polynucleotide that is at least about 99% identical identical to SEQ ID NO: 14.

Also provided is a composition containing a vector or vectors containing a polynucleotide at least about 90% identical to SEQ ID NO: 13 and a polynucleotide at least about 90% identical to SEQ ID NO: 14. Also provided is a composition containing a vector or vectors containing a polynucleotide at least about 95% identical to SEQ ID NO: 13, and a polynucleotide at least about 95% identical to SEQ ID NO: 14. Also provided is a composition containing a vector or vectors containing a polynucleotide at least at least about 96% identical to SEQ ID NO: 13, and a polynucleotide at least about 96% identical to SEQ ID NO: 14. Also provided is a composition containing a vector or vectors containing a polynucleotide at least about 97% identical to SEQ ID NO: 13, and a polynucleotide at least about 97% identical to SEQ ID NO: 14.

Also provided is a composition containing a vector or vectors containing a polynucleotide at least about 98% identical to SEQ ID NO: 13 and a polynucleotide at least about 98% identical to SEQ ID NO: 14. Also provided is a composition containing a vector or vectors containing a polynucleotide at least about 99% identical to SEQ ID NO: 13, and a polynucleotide at least about 99% identical to SEQ ID NO: 14. Also provided is a composition containing a vector or vectors containing the polynucleotide sequence represented by SEQ ID NO: 13, and the polynucleotide sequence represented by SEQ ID NO: 14. In some embodiments, the polynucleotides comprise a coding sequence for a mature polypeptide fused in frame to a polynucleotide that promotes, for example, expression and secretion of the polypeptide from a cell. host (eg, a leader sequence that acts as a secretory sequence to regulate transport of the polypeptide out of the cell). A polypeptide having a leader sequence is a precursor protein, and the leader sequence can be cleaved by a host cell to produce the mature form of the polypeptide. Polynucleotides can also encode a proprotein, which is the mature protein plus additional 5' amino acid residues. The mature protein, which has a prosequence, is a proprotein and is an inactive form of the protein. When the prosequence is cleaved, the active mature protein remains.

In some embodiments, the polynucleotides comprise a coding sequence for a mature polypeptide fused in frame to a marker sequence that allows, for example, purification of the coding polypeptide. For example, the marker sequence may be a hexahistidine tag provided by the pQE-9 vector to allow purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence may be a hemagglutinin (HA) tag derived from the influenza virus hemagglutinin protein. in the case of using mammalian host cells (eg COS-7 cells). The present description also relates to variants of the polynucleotides described above, encoding, for example, fragments, analogs and derivatives.

Polynucleotide variants may contain changes in coding regions, non-coding regions, or both. In some embodiments, the polynucleotide variants contain changes that result in silent substitutions, insertions, or deletions, but do not change the properties or activities of the encoded polypeptide. In some embodiments, polynucleotide variants arise from silent substitutions due to degeneracy of the genetic code. Polynucleotide variants can be generated for a number of reasons, for example to optimize codon expression for a particular host (changing codons in human mRNA to those preferred by a bacterial host such as E. coli).

Vectors and cells containing the polynucleotides described herein are also provided.

IV. Biological samples.

Biological samples are often fixed using a fixative. Aldehyde fixatives such as formalin (formaldehyde) and glutaraldehyde are commonly used. Tissue specimens fixed using other fixation methods such as alcohol immersion (Battifora and Kopinski, J.

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Histochem. Cytochem. (1986) 34:1095) are also suitable. The samples used can also be embedded in paraffin. In one embodiment, the samples are formalin fixed and paraffin embedded (FFPE). In another embodiment, the FFPE block is stained with hematoxylin and eosin before selecting one or more portions for analysis to select a specific sample area(s) from the central portion of the FFPE block. Methods for obtaining tissue blocks from these particulate samples have been used in previous IHC studies of various prognostic factors and/or are well known to those skilled in the art (see, e.g., Abbondanzo et al., Am. J. Clin. Pathol 1990 May;93(5):698-702;Allred et al., Arch Surg 1990 Jan;125(1):107-13).

Briefly, any intact organ or tissue can be cut into fairly small pieces and incubated in various fixatives (eg formaldehyde, alcohol, etc.) for varying periods of time until the tissue is fixed. The samples can be virtually any intact tissue that has been surgically removed from the body. Specimens can be cut into pieces small enough to accommodate equipment commonly used in histopathology laboratories. The size of the cut pieces usually ranges from a few millimeters to several centimeters. The biological sample may also be liquid extracts, blood, plasma, serum, cerebrospinal fluid, lymph fluid, and/or a spleen preparation.

V. Correlation of CD37 expression and therapeutic efficacy.

CD37 immunoconjugates are disclosed in US Published Application No. 2011/0256153, which is incorporated herein by reference in its entirety. In some embodiments, the CD37 immunoconjugate is IMGN529. IMGN529 contains the antibody huCD37-3 (containing the CDR regions represented by SEQ ID NO: 22-27, VH represented by SEQ ID NO: 28, and VL represented by SEQ ID NO: 29), the SMCC linker and the maytansinoid DM1. The sequences of the huCD37-3 antibody are given below.

huCD37-3 VH-CDR1: TSGVS (SEQ ID NO:22) huCD37-3 VH-CDR2: V1WGDGSTN (SEQ ID NO:23) huCD37-3 VH-CDR3: GGYSLAH (SEQ ID NO:24) huCD37-3 VL- CDR1; RASENIRSNLA (SEQ ID NO:25) huCD37-3 VL-CDR2: VATNLAD (SEQ ID NO 26) huCD37-3 VL-CDR3: QHYWGTTWT (SEQ JD NO:27) huCD37-3 VHv. 1.0:

QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKrLEWLGVIWGDGSTNYHPSLKS

RLSIKKDHSKSQVFLKLNSLTAADTATYYCAKrGYSLAHWGQGTLVTVSS (SEQ ID NO 28) huCD37-3 VHv. 1.1:

QVQVQESGPGLVAPSQTLSITCTVSGFSLTTSGVSWVRQPPGKrLEWLGVIWGDGSTNYHSSLKS

RLSIKKDHSKSQVFLKLNSLTAADTATYYCAKFGYSLAHWGQGTLVTVSS (SEQ ID NO:32) huCD37-3 VL:

DIQMTQSPSSLSVSVGERVTITCRASENIRSNLAWYQQKPGKSPKLLVNVATNLADGVPSRFSGS

GSGTDYSLK1NSLQPEDFGTYYCQHYWGTTWTFGQGTKLEIKR (SEQ ID NO 29)

Heavy chain (HC) huCD37-3 v. 1.0:

QVQVQESGPGLVAPSQTLS1TCTVSGFSLTTSGVSWVRQPPGKFLEWLGVIWGDGSTNYHPSLKS RLSIKKDHSKSQVFLKLNSLTAADTATYYCAKFGYSLAHWGQGTLVTVSSASTKrPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQT

- 30 044601

YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV

VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

KALPAPIEKTISKAKFQPREPQVYTLPPSRDELTKNQVSLTCLVKFFYPSD1AVEWESNGQPENNY

KTTPPVLDSDr.SFFLYSKITX'DKSRWQQC.NVFSCSVMHEAEHA'H>'TOKSLSISPG (SEQ ID

NO:30)

Heavy chain (HC) huCD37-3 v. I, I:

QVQVQESGPGLVAPSQTLS1TCTVSGFSLTTSGVSWVRQPPGKFLEWLGV1WGDGSTNYHSSLK.S

RL·S1KKDHSKSQVFLKLNSLTAADTATYYCAKΓGYSLAHWGQGTLVTVSSASTKΓPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKFQPREPQVYTLPPSRDELTKNQVSLTCLVKFFYPSD1AVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG (SEQ ID

NO:33)

Light chain (LC) of huCD37-3:

DIQMTQSPSSLSVSVGERVTITCRASENIRSNLAWYQQKPGKSPKLLVNVATNLADGVPSRFSGS

GSGTDYSLKINSLQPEDFGTYYCQHYWGTTWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTAS

VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC

EVTHQGLSSPVTKSFNRGEC (SEQ ID NO:31)

In some embodiments, the immunoconjugate may comprise an anti-CD37 antibody obtained from a hybridoma with ATCC deposit designation PTA-10664 deposited with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, Virginia 20110 on February 18, 2010, or its antigen binding fragment. In some embodiments, the antibody or antigen binding fragment thereof comprises the VH-CDR and VL-CDR regions of an antibody derived from hybridoma PTA-10664.

IMGN529 is currently in clinical trials for the treatment of leukemia and lymphoma. Methods of administration of IMGN529 are suggested in US Application No. 14/710354 (Publication No. 2015/0343077) and Stathis, et al., Preliminary Findings from a phase I, multi-center, open-label study of the anti-CD37 antibody-drug conjugate (ADC), IMGN529, in adult patients with relapsed or refractory non-Hodgkin Lymphoma (NHL), Abstract Number 8526, ASCO Annual Meeting (2014)), each of which is incorporated herein by reference.

In some embodiments, the present disclosure provides a method for identifying subjects who may respond favorably to CD37-targeted therapy due to increased levels of CD37 expression in the subject, particularly using antibodies and antigen-binding fragments thereof provided herein that can detect dynamic range of CD37 expression levels, for example by IHC. Evaluation of patient samples and correlation with in vivo efficacy using xenograft models demonstrates the power of expression analysis to select subjects who are more likely to respond to treatment. Using IHC, the CD37 expression value in tumor cells is obtained: from 0 (no expression) to 3 (very high levels of expression). Samples with CD37 expression scores of 1, 2 or 3 (or 2 or 3) are characterized by an increased likelihood of responding to CD37-targeted anticancer therapy at clinically relevant doses of CD37 immunoconjugates (see, for example, WO 2013/149171, which is included in this description by reference). Thus, identifying individuals who have elevated CD37 values will help identify individuals who may respond to a clinically relevant dose. Moreover, expression of more uniform levels of CD37 provides a better correlation with therapeutic benefit. Thus, homogeneous staining uniformity or a combination of higher staining intensity with heterogeneous staining uniformity may indicate increased CD37 expression. For example, values greater than 2 hetero may be used as a criterion for selecting a patient for treatment with a CD37-targeted therapeutic agent. In addition, patient selection criteria may be based on the percentage of cells in the sample expressing membranous CD37 at a particular level, which reflects both staining intensity (eg, 1, 2, or 3) and uniformity (eg, heterogeneous or homogeneous (see Table 1). Table 3)). For example, a sample may be characterized as containing, for example, at least 25% of cells staining positive for CD37 with a score of at least 2 or more, or at least 75% of cells staining positive for CD37 with a score of at least 2 or more. In another example, the sample may be characterized as containing, for example, at least 25% of cells that stain positive for CD37 with

- 31 044601 with a value of at least 3, or at least 75% of cells stain positive for CD37 with a value of 3. In another example, CD37 expression is assigned an H value. In addition, immunological detection (using immunohistochemistry) of CD37 can be assessed using H-scores. H-values combine color intensity values with uniformity values using the calculation proposed herein. As described in more detail below, H-values can range from 1 to 300.

CD37 expression analysis can also be used to identify patients in whom reduced levels of CD37-targeted anticancer therapy (low-dose therapy) may be effective in producing antitumor responses. As will be understood in the art, compounds are typically administered at the lowest dose that produces the desired therapeutic response. This is especially important for therapeutic agents that cause clinical and often unwanted side effects. The ability to recognize such subjects with elevated levels of CD37 expression allows one to potentially minimize the dose of a CD37-targeted therapeutic, thereby reducing potential side effects while maintaining therapeutic efficacy.

VI. Immunodetection methods.

In some embodiments, CD37-binding agents (eg, antibodies and antigen-binding fragments thereof) can be used in immunodetection methods to identify samples and/or subjects with elevated or overexpressed CD37. Immunodetection methods include, for example, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry, and fluorescence-activated cell sorting (FACS), to mention just a few.

Typically, immunodetection methods involve obtaining a sample suspected of containing CD37 and contacting said sample with a first CD37-binding agent (eg, an anti-CD37 antibody or antigen-binding fragment thereof) under conditions suitable to promote the formation of immunocomplexes.

With respect to antigen detection, the biological sample analyzed may be any sample in which CD37 is desired to be detected, such as liquid extract, blood, plasma, serum, cerebrospinal fluid, lymph fluid, tissue section or section, homogenized tissue extract, biopsy aspirates, cell, separated and/or purified forms of CD37-containing compositions or any biological fluid. In some embodiments, blood, plasma or lymph samples or extracts are used.

Contacting a selected biological sample with a CD37-binding agent (e.g., an anti-CD37 antibody or an antigen-binding fragment thereof) under suitable conditions and for a period of time sufficient to allow formation of immune complexes (primary immune complexes) typically involves the addition of a CD37-binding agent. agent (eg, an anti-CD37 antibody or an antigen-binding fragment thereof) to the sample and incubating the mixture for a period of time sufficient to allow the CD37-binding agent (eg, an anti-CD37 antibody or an antigen-binding fragment thereof) to form complexes, i.e. contacted any CD37 present. After this time, the sample, such as a tissue section or liquid extract, ELISA plate, or Western blot, is typically washed to remove any nonspecifically bound CD37 binding agent (e.g., anti-CD37 antibody or antigen-binding fragment thereof), ensuring that only those binding to CD37 are detected. with CD37 agents (eg, anti-CD37 antibodies or antigen-binding fragments thereof) that are specifically bound in primary immune complexes.

In general, detection of immunocomplex formation is well known in the art and can be achieved using numerous approaches. The CD37-binding agent (eg, an anti-CD37 antibody or antigen-binding fragment thereof) used for detection may itself be associated with a detectable label such that the label can be easily detected, thereby determining the amount of primary immune complexes in the composition to be analyzed. Alternatively, a first CD37 binding agent (e.g., an anti-CD37 antibody or an antigen-binding fragment thereof) that binds in primary immune complexes can be detected by a second binding agent (e.g., an antibody or an antigen-binding fragment thereof) that has binding ability relative to the first CD37-binding agent (eg, an anti-CD37 antibody or an antigen-binding fragment thereof). In these cases, the second binding agent may be associated with the detectable label. When the second binding agent is itself an antibody or an antigen-binding fragment thereof, it may be referred to as a secondary antibody or an antigen-binding fragment thereof. The primary immune complexes are brought into contact with a labeled secondary binding agent under suitable conditions and for a period of time sufficient to allow formation of secondary immune complexes. The secondary immune complexes are then typically washed to remove any nonspecifically bound labeled secondary binders, and the remaining label in the secondary immune complexes is then detected. Additionally, methods include detection of primary immune cells

- 32,044601 plex using a two-stage approach. A second binding agent (eg, an antibody or an antigen-binding fragment thereof) that has binding activity to a first CD37-binding agent (eg, an anti-CD37 antibody or an antigen-binding fragment thereof) is used to form secondary immune complexes as described herein. After washing, the secondary immune complexes are brought into contact with a third binding agent that has binding activity to the second binding agent (e.g., an antibody or an antigen-binding fragment thereof), also under suitable conditions and for a period of time sufficient to allow formation of immune complexes ( tertiary immune complexes). The third binding agent is bound to a detectable label, allowing the detection of tertiary immune complexes formed. This system can provide signal amplification if desired.

In another embodiment, a biotinylated CD37 binding agent (eg, an anti-CD37 antibody or an antigen-binding fragment thereof) is used to detect CD37, and a second binding agent (eg, an antibody or an antigen-binding fragment thereof) is then used to detect biotin. In this method, the test sample is first incubated in a solution containing a biotinylated CD37 binding agent (eg, an anti-CD37 antibody or antigen-binding fragment thereof). If CD37 is present, the binding agent moiety binds to CD37 to form a biotinylated CD37 binding agent-CD37 complex. The complex is then amplified by sequential incubation in solutions of streptavidin (or avidin), biotinylated DNA and/or complementary biotinylated DNA, adding additional biotin sites to the antibody/antigen complex at each step. The amplification steps are repeated until a suitable level of amplification is achieved, after which the sample is incubated in a solution containing a second binding agent (eg, an antibody or an antigen-binding fragment thereof) that binds to biotin. This second binding agent is labeled, for example, with an enzyme that can be used to detect the presence of an antibody/antigen complex by histoenzymology using a chromogenic substrate. With suitable amplification, a conjugate can be obtained that is macroscopically visible.

In one embodiment, immunohistochemistry (IHC) is used for immunological detection. When using IHC, detection of CD37 in a sample can be achieved by targeting the sample with a probe, such as an anti-CD37 antibody or antigen-binding fragment thereof. The probe may be either directly or indirectly associated with the detectable label or may be detected by another probe that is either directly or indirectly associated with the detectable label.

In some embodiments, IHC can detect differences between different levels of protein expression, such as in a calibrated IHC method. In some embodiments, IHC can distinguish staining intensities for samples characterized by low, intermediate, or high expression of CD37.

In one embodiment, immunological detection (using immunohistochemistry) of CD37 is assessed for both intensity and uniformity (percentage of membrane-only cells stained). Comparative scales for the intensity of CD37 expression are related as 0 - negative, 0-1 - very weak, 1 - weak, 1-2 - weak to moderate, 2 - moderate, 2-3 - moderate to strong, 3 - strong to very strong. Quantitatively, a value of 0 means that no staining is observed or membrane staining is observed in less than 10% of tumor cells. A value of 1 or 1+ indicates that weak/barely noticeable membrane staining is detected in more than 10% of tumor cells. Cells are stained only in part of their membranes. The values 1 and 1+ are used interchangeably. At a value of 2, weak to moderate complete membrane staining is observed in more than 10% of tumor cells. Finally, a value of 3 indicates that moderate to strong complete membrane staining is observed in more than 10% of tumor cells. Samples with a score of 0 or 1 for CD37 expression may be characterized as having no overexpression of CD37, while samples with scores of 2 or 3 may be characterized as having overexpression or increased levels of CD37. In another embodiment, using antibodies, antigen binding fragments thereof, or polypeptides provided herein, samples with a CD37 expression score of 0 may be characterized as having no CD37 overexpression, samples with a score of 1 may be characterized as having CD37 overexpression, and samples with scores of 2 or 3 can be characterized as having overexpression or increased levels of CD37. In some embodiments, a value of at least 2 in a sample obtained from a subject identifies the subject as a candidate for treatment with a CD37-targeted treatment regimen (eg, IMGN529). In some embodiments, a value of at least 3 in a sample obtained from a subject identifies the subject as a candidate for treatment with a CD37-targeted treatment regimen (eg, IMGN529). Specimens overexpressing CD37 can also be assessed by immunohistochemistry, corresponding to the copy number of the molecules expressed

- 33 044601

CD37 per cell or antibody-associated per cell (ABC), and can be biochemically determined.

Comparative scales for CD37 uniformity (percentage of cell membrane staining) are as follows: negative = 0%; focal = <25%; heterogeneous (hetero) = 25-75% and homogeneous (homo) = >75%.

In some embodiments, a value of at least 2 hetero in a sample obtained from a subject identifies the subject as a candidate for treatment with a CD37-targeted treatment regimen (eg, IMGN529). In some embodiments, a value of at least 2 homo in a sample obtained from a subject identifies the subject as a candidate for treatment with a CD37-targeted treatment regimen (eg, IMGN529). In some embodiments, a value of at least 3 hetero in a sample obtained from a subject identifies the subject as a candidate for treatment with a CD37-targeted treatment regimen (eg, IMGN529). In some embodiments, a value of at least 3 homo in a sample obtained from a subject identifies the subject as a candidate for treatment with a CD37-targeted treatment regimen (eg, IMGN529). In one embodiment, immunological detection (using immunohistochemistry) of CD37 is assessed using H-scores. H-scores combine staining intensity values (eg, values from 0 to 3, where 0 means no staining and 3 means strong staining) with the percentage of cells that are positive for membrane staining (i.e., uniformity). The H-value can be calculated as follows: H-value = [0*(percentage of cells stained with intensity 0)] + [1*(percentage of cells stained with intensity 1)] + [2*(percentage of cells , stained with intensity 2)] + [3*(percentage of cells stained with intensity 3)]. Accordingly, the H-value can vary from 0 (no staining of cell membranes) to 300 (all cell membranes are stained with intensity 3).

As an example, the H-value in a subject having cancer may be as follows:

H-value = (75% with intensity 0) + (0% with intensity 1) + (0% with intensity 2) + (25% with intensity 3) = 75 or

H-value = (0% with intensity 0) + (75% with intensity 1) + (0% with intensity 2) + (25% with intensity 3) = 150.

In another example, the H-value in a subject having cancer may be:

H-value = (75% with intensity 0) + (0% with intensity 1) + (25% with intensity 2) + (0% with intensity 3) = 50 or

H-value = (0% with intensity 0) + (75% with intensity 1) + (25% with intensity 2) + (0% with intensity 3) = 125.

In one embodiment, immunological detection (using immunohistochemistry) of CD37 is assessed using percent positive staining and intensity per sample. In this embodiment, selection for treatment using a CD37-targeted treatment regimen is based on the percentage of cells in the sample expressing membranous CD37 at a particular level, which reflects both the intensity of staining (e.g., 1, 2, or 3) and the uniformity ( for example, heterogeneous or homogeneous (see Table 3)). For example, a sample containing at least 25% (i.e., 25-75% or >75%) of cells staining positive for CD37 with a score of 3 may be characterized as 3 hetero and 3 homo or, together, as at least At least 25% are positive with a value of 3.

IHC can be performed manually or using an automated system (eg, using an automated filter). IHC can be performed on cells, cell pellets, tissues, blood preparations, plasma, serum or lymph fluid, etc. In some embodiments, the samples are fixed patterns. In some embodiments, the samples are paraffin-embedded samples. In some embodiments, the samples are formalin-fixed, paraffin-embedded samples.

In one embodiment, an enzyme-linked immunosorbent assay (ELISA) is used for immunological detection. The methodology underlying ELISA is well known in the art. See, for example, Lequin R, Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA), Clin. Chem. 57:2415-2418 (2005), incorporated herein by reference in its entirety. In one exemplary ELISA, CD37-binding agents (eg, antibodies or antigen-binding fragments thereof) are immobilized on a selected surface exhibiting affinity for the protein, such as a well in a polystyrene microtiter plate. A test sample, such as a clinical sample containing or suspected to contain CD37, is then added to the wells. After binding and washing to remove nonspecifically bound immunocomplexes, bound CD37 can be detected. Detection is usually achieved by adding a second antibody specific for CD37, or an antigen-binding fragment thereof, that is linked to the detectable label. This type of ELISA is a sandwich ELISA. Detection may also be achieved by adding a second antibody or antigen-binding fragment thereof, followed by the addition of a third antibody or antigen-binding fragment thereof that has binding activity to the second antibody or antigen-binding fragment thereof, wherein the third antibody or antigen-binding fragment thereof is associated with the detectable tag. The appearance of color can be detected spectrophotometrically and correlated with CD37 concentration by calibration using a calibration curve. In one exemplary ELISA, test samples are immobilized on the surface of a well and then brought into contact with CD37-binding agents (eg, antibodies or antigen-binding fragments thereof). After binding and washing to remove nonspecifically bound immunocomplexes, bound CD37 is detected. If the original CD37-binding agents (eg, antibodies or antigen-binding fragments thereof) are associated with a detectable label, the immunocomplexes can be detected directly. Again, immunocomplexes can be detected using a second binding agent that has binding properties to the first binding agent, the second binding agent being bound to a detectable label. Another ELISA in which the test samples are immobilized involves the use of competition in detection. In this ELISA, labeled CD37-binding agents (eg, antibodies or antigen-binding fragments thereof) are added to wells, allowed to bind to CD37, and detected by their label. The amount of CD37 in a sample is then determined by mixing said sample with labeled CD37-binding agents (eg, antibodies or antigen-binding fragments thereof) before or during incubation in coated wells. The presence of CD37 in a sample reduces the amount of CD37-binding agents (eg, antibodies or antigen-binding fragments thereof) available to bind to the well and thus reduces the final signal.

In one embodiment, Western blotting is used for immunological detection. For Western blotting, proteins are extracted from a cell sample and subjected to electrophoresis (eg, DNS-PAGE) and imprinted on a membrane (eg, nitrocellulose or polyvinylidene fluoride (PVDF)). The membrane is then contacted with a CD37-binding agent (eg, an antibody or an antigen-binding fragment thereof), which can either be directly labeled or subsequently exposed to a labeled secondary binding agent. Detection can be carried out, for example, using autoradiography, colorimetric reaction or chemiluminescence. This method provides both the ability to quantify substrate content and determine its identity by relative position on the membrane, which indicates the path length in the acrylamide gel during electrophoresis.

In one embodiment, flow cytometry is used for immunological detection. For example, the amount of antibody bound per cell (ABC) can be assessed using flow cytometry. A high number of bound anti-CD37 antibodies per cell may indicate high levels of CD37 expression and a high likelihood of sensitivity to treatment with an anti-CD37 antibody or its immunoconjugate.

In one embodiment, FACS is used for immunological detection. FACS analysis detects CD37 on cell membranes. Briefly, CD37-binding agents (eg, antibodies or antigen-binding fragments thereof) are coupled to fluorophores, and detection is accomplished using a cell sorter that reads the wavelength of light emitted by each cell as it passes through a beam of light. In this method, two or more antibodies can be used simultaneously.

VII. Detection agents.

The CD37 binding agents provided herein may be coupled to at least one agent to form a detection conjugate. In addition, the CD37 binding agents provided herein can be detected using a detection agent that is coupled to at least one agent to form a detection conjugate. Molecules and moieties for detection are well known in the art. To increase the effectiveness of antibody molecules as diagnostic molecules, it is common to attach, covalently link, or complex with at least one desired molecule or fragment. Such a molecule or fragment may be at least one reporter molecule, but is not limited to it. A reporter molecule is defined as any fragment that can be detected using an assay. Non-limiting examples of reporter molecules that have been conjugated to antibodies include enzymes, radiolabels, haptens, fluorescent tags, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles and/or ligands such as biotin.

Antibody or antigen binding moiety detection conjugates provided by the present invention include detection conjugates for in vitro use wherein the antibody or moiety is bound to a secondary binding ligand and/or enzyme (enzyme tag) that will form a colored product upon contact with chromogenic substrate. The anti-CD37 antibodies and antigen-binding fragments thereof provided herein are particularly suitable for use in conjugate methods because, for example, they allow detection

- 35 044601 enhance the dynamic range of CD37. Examples of suitable enzymes include urease, alkaline phosphatase, horseradish peroxidase and/or glucose oxidase. In some embodiments, the secondary binding ligands are biotin and/or avidin and streptavidin compounds. In the case of enzymes, the intensity of staining developed and measured will represent a direct measurement of the amount of CD37 present. If HRP is a label, coloring can be detected using the substrate 3,3',5,5'-tetramethylbenzidine (TMB), for example by absorbance at 450 nm. Alternatively, other chromogenic HRP substrates such as 3,3'-diaminobenzidine (DAB) or 2,2'-azino-bis(3ethylbenzthiazoline-6-sulfonic acid) (ABTS) can be used. Substrates such as TMB, DAB and ABTS can be used, for example, in immunodetection methods using ELISA.

3,3'-Diaminobenzidine (DAB) is a substrate for enzymes such as HRP, producing a brown end product that is very slightly soluble in alcohol and other organic solvents. Oxidation of DAB also causes polymerization, resulting in the ability to react with osmium tetroxide and thus increase its color intensity and electron density. Of the several metals and methods used to enhance the optical density of polymerized DAB, gold chloride in combination with silver sulfide appears to be the most effective.

3-Amino-9-ethylcarbazole (AEC) is a substrate for enzymes such as HRP and upon oxidation produces a pink-red end product that is soluble in alcohol. Therefore, AEC-treated specimens should not be immersed in alcohol or alcohol solutions (eg Harris hematoxylin). Instead, an aqueous medium should be used for counterstaining and mounting.

4-Chloro-1-naphthol (CN) is a substrate for enzymes such as HRP, which precipitates as a blue end product. Because CN is soluble in alcohol and other organic solvents, the sample should not be dehydrated, exposed to alcohol counterstains, or mounted under a coverslip using mounting media containing organic solvents. Unlike DAB, CN tends to diffuse away from the site of deposition.

p-Phenylenediamine dihydrochloride/pyrocatechol (Hanker-Yates reagent) is a substrate for enzymes such as HRP, which produces a blue-black reaction product that is insoluble in alcohol and other organic solvents. Like polymerized DAB, this reaction product can be treated with osmic acid. Various results have been obtained using the Hanker-Yate reagent in the immunoperoxidase method.

Chemiluminescent substrates such as ECL can be used. Substrates such as ECL can be used, for example, in immunodetection methods using Western blotting.

Exemplary fluorescent tags for use in binding agent (e.g., antibody) conjugates include, for example, Alexa 350, Alexa 430, Alexa 488, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Dylight 488, fluorescein isothiocyanate (FITC), green fluorescent protein (GFP), HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, Phycoerythrin, REG, rhodamine green, rhodamine red, tetramethylrhodamine (TMR) renographine, ROX, TAMRA, TET, tetramethylrhodamine, Texas red and derivatives of these labels (i.e., halogenated analogues modified with isothiocyanate or other linkers to conjugation, etc.). An example of a radioactive tracer is tritium.

Molecules containing azido groups can also be used to form covalent bonds with proteins through reactive nitrene intermediates, which are formed by exposure to low-intensity ultraviolet radiation (Potter & Haley, 1983). In particular, 2- and 8-azido analogs of purine nucleotides have been used as site-directed photoprobes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987; Atherton et al., 1985). 2- and 8-azidonucleotides have also been used to map the nucleotide-binding domains of purified proteins (Khatoon et al., 1989; King et al., 1989 and Dholakia et al., 1989) and can be used as antibody binding agents.

In other embodiments of the present invention, the CD37 binding agents (eg, antibodies and antigen binding fragments thereof) or secondary binding agents are radiolabeled nuclides, such as tritium. Additional embodiments employ gold nanoparticles (such as those ranging in size from about 0.5 to 40 nm) and/or quantum dots (Hayward, Calif.).

In some embodiments, CD37 is detected using the Optiview DAB IHC Detection reagent (Ventana catalog #760-700).

In some embodiments, CD37 is detected using the BenchMark Ultra staining system.

In some embodiments, CD37 is detected using the BenchMark Ultra staining system using the Optiview DAB IHC Detection reagent (Ventana catalog

- 36 044601 # 760-700).

VIII. Compositions and sets.

Also provided herein are compositions and kits for use in the practice of the methods disclosed herein. Such kits may contain containers, each of which contains one or more different reagents (usually in concentrated form) used in these methods, including, for example, one or more CD37 binding agents (for example, antibodies or antigen-binding fragments thereof), buffers and /or CD37 detection reagents and equipment to enable the practical application of the present invention. The kit may further comprise a second binding agent that binds to the CD37 binding agent, and optionally a third binding agent that binds to the second binding agent. The CD37 binding agent, the second binding agent, or the third binding agent may be coupled to the detection reagent, or the kit may contain reagents for coupling the detection reagent to the CD37 binding agent, the second binding agent, or the third binding agent. A description of the label or indicator or a set of instructions for use of the kit components in the ligand detection method of the present invention will also typically be included, which instructions may be contained in the package insert and/or on the packaging of the kit or its components.

In some embodiments, the kit comprises a first antibody or antigen binding fragment that binds to CD37, a second antibody or antigen binding fragment that binds to the first antibody or antigen binding fragment, a third antibody or antigen binding fragment that binds to the second antibody or antigen binding fragment, wherein said third an antibody or antigen binding fragment is/is bound to a detection reagent (e.g., an enzyme tag), optionally a substrate for a detection reagent (e.g., TMB, DAB, or ABTS), and optionally a CD37 protein or a cell sample containing CD37 (e.g., a paraffin-embedded sample) . The kits may further contain a therapeutic agent for the treatment of cancer, such as an anti-CD37 immunoconjugate.

In one embodiment, the CD37 binding agent is 1B11-2 or an antigen binding fragment thereof. In one embodiment, the CD37 binding agent is an antibody or antigen binding fragment that binds to the same CD37 epitope as 1B11-2. In one embodiment, the CD37 binding agent is an antibody or antigen binding fragment that contains six CDR 1B11-2 regions.

In one embodiment, the CD37 binding agent is an antibody or antigen binding fragment that contains VH and VL 1B11-2.

In one embodiment, the CD37-specific antibody or antigen-binding fragment thereof is included at a concentration of about 0.1 to about 20 μg/ml, about 0.1 to about 15 μg/ml, about 0.1 to about 10 μg/ml ml, from about 0.5 to about 20 μg/ml, from about 0.5 to about 15 μg/ml, from about 0.5 to about 10 μg/ml, from about 1 to about 20 μg/ml, from about 1 to about 15 μg/ml, about 1 to about 10 μg/ml, about 2 to about 20 μg/ml, about 2 to about 15 μg/ml, or about 2 to about 10 μg/ml.

In another embodiment, the CD37-specific antibody or antigen binding fragment thereof is included at a concentration of about 1.5 μg/ml, about 2 μg/ml, about 3 μg/ml, about 4 μg/ml, about 5 μg/ml, about 6 μg /ml, approximately 7 μg/ml, approximately 8 μg/ml, approximately 9 μg/ml, or approximately 10 μg/ml. In another embodiment, the CD37-specific antibody or antigen binding fragment thereof is included at a concentration of approximately 2 μg/ml. In another embodiment, the CD37-specific antibody or antigen binding fragment thereof is included at a concentration of approximately 10 μg/ml.

In another embodiment, the antibody or antigen binding fragment thereof is included in a concentrated solution with dilution instructions to achieve a final concentration of about 1 to about 20 μg/ml, about 1 to about 15 μg/ml, about 1 to about 10 μg/ml , from about 2 to about 20 μg/ml, from about 2 to about 15 μg/ml, or from about 2 to about 10 μg/ml.

In another embodiment, the antibody or antigen binding fragment thereof is included in a concentrated solution with dilution instructions to achieve a final concentration of about 1.5 μg/ml, about 2 μg/ml, about 3 μg/ml, about 4 μg/ml, about 5 μg /ml, approximately 6 μg/ml, approximately 7 μg/ml, approximately 8 μg/ml, approximately 9 μg/ml, or approximately 10 μg/ml. In another embodiment, the antibody or antigen binding fragment thereof is included in a concentrated solution with dilution instructions to achieve a final concentration of approximately 2 μg/ml. In another embodiment, the antibody or antigen-binding fragment thereof is included in a concentrated solution with instructions

- 37 044601 dilutions to achieve a final concentration of approximately 2.1 µg/ml. In another embodiment, the antibody or antigen binding fragment thereof is included in a concentrated solution with dilution instructions to achieve a final concentration of approximately 4.2 μg/ml. In another embodiment, the antibody or antigen binding fragment thereof is included in a concentrated solution with dilution instructions to achieve a final concentration of approximately 8.4 μg/ml. In another embodiment, the antibody or antigen binding fragment thereof is included in a concentrated solution with dilution instructions to achieve a final concentration of approximately 10 μg/ml. The kit may also contain instructions for detecting and assessing CD37 expression. The kit may also contain control or reference samples. Non-limiting examples of control or reference samples include cell pellets or tissue culture cell lines derived from normal (normal control) or tumor (positive control) samples. Exemplary cell lines include cell lines stably or transiently transfected with an expression vector that expresses CD37. Additional examples include cell pellets and tissue samples described in the examples. Positive cell lines include Daudi cells (high expression), Ramos cells (moderate expression), and Namalwa cells (moderate to low expression). The human amygdala serves as both a positive and negative control tissue, with germinal centers and pallial zones showing high expression and the interfollicular region showing no expression. In some embodiments, the control or reference samples are paraffin-embedded samples.

In some embodiments, the kit is a packaged combination comprising the core elements of: (a) capture reagents consisting of anti-human CD37 monoclonal antibodies; and (b) reagents, which may also contain monoclonal antibodies to CD37, but may also contain detectable (labeled or unlabeled) antibodies that bind to CD37. These basic elements are defined herein.

In one embodiment, the kit further comprises a solid carrier for the capture reagents, which may be provided as a separate element or on which the capture reagents are already immobilized. Therefore, the capture antibodies in the kit may be immobilized on a solid support, or they may be immobilized on such a support that is included in the kit or provided separately from the kit.

In one embodiment, the capture reagent is applied to a microtiter plate. The detection reagent may be labeled antibodies that are detected directly, or unlabeled antibodies that are detected using labeled antibodies directed against unlabeled antibodies obtained from other species. In the case where the label is an enzyme, the kit will typically contain the substrates and cofactors required for the enzyme, and in the case where the label is a fluorophore, a precursor dye that provides the detectable chromophore. In the case where the detection reagent is unlabeled, the kit may further contain an agent for detecting detectable antibodies, such as labeled antibodies directed to unlabeled antibodies, for example, in a fluorimetrically detectable form. In the case where the tag is an enzyme, the kit will typically contain the substrates and cofactors required for the enzyme, when the tag is a dye precursor fluorophore that provides a detectable chromophore, and when the tag is a biotin-avidin such as avidin, streptavidin or streptavidin , conjugated to HRP or α-galactosidase together with 4methylumbelliferyl-beta-O-glucuronide (MUG).

In one embodiment, the capture reagent is an anti-CD37 antibody 1B11-2 or an antibody comprising sequences from the 1B11-2 antibody. In one embodiment, the detection reagent is an anti-CD37 antibody 1B11-2 or an antibody comprising sequences from the 1B11-2 antibody. In another embodiment, the 1B11-2 anti-CD37 detection reagent or an antibody comprising 1B11-2 antibody sequences is biotinylated.

In another embodiment, the kit further comprises a detection reagent selected from the group consisting of an enzyme, a fluorophore, a radiolabel, and a phosphor. In another embodiment, the detection reagent is selected from the group consisting of biotin, digoxigenin, fluorescein, tritium and rhodamine. The kit may also contain assay instructions and/or CD37 protein or fragments thereof (e.g., CD37 extracellular domain or CD37 extracellular domain with all or part of the GPI binding domain) as the antigen standard, as well as other additives such as stabilizers, buffers for washing and incubation, etc. In one embodiment, the CD37 antigen standard is the CD37 protein described in the examples herein. The kit may also contain instructions for detecting and assessing CD37 expression. The components of the kit can be presented in predetermined ratios, with the respective amounts of the various reagents suitably varied to provide concentrations of reagents in solution that significantly enhance the sensitivity of the assay. In particular, the reagents may be provided in the form of dry powders, usually lyophilized, including excipients which, when dissolved, will provide a reagent solution having a suitable

- 38 044601 concentration for connection with the test sample. Also provided are compositions containing antibodies or antigen-binding fragments described herein. In one embodiment, the composition comprises an anti-CD37 antibody or antigen binding fragment as described herein and a buffer, for example a buffer that can be used in a detection assay such as IHC, ELISA or FACS. Such buffers are known to those of ordinary skill in the art and include diluents. By way of example, IHC buffers may contain, for example, casein, whey or albumin (such as bovine serum, goat serum or BSA), Tween or Triton, PBS and/or sodium azide, or any combination thereof. Buffers for IHC are also provided herein and are known to those of ordinary skill in the art. ELISA buffers are also provided herein and are known to those of ordinary skill in the art. ELISA buffers may contain, for example, whey or albumin (such as bovine serum, goat serum or BSA), nonfat dry milk, casein and/or gelatin, or any combination thereof. Some buffers for FACS are proposed herein, for example in the working examples. FACS buffers may also contain, for example, serum or albumin (such as bovine serum, goat serum or BSA) and/or sodium azide. FACS buffers may also contain PBS, EDTA and/or DNase or any combination thereof.

Embodiments according to the present description can be further defined by reference to the following non-limiting examples, which describe in detail the preparation of certain antibodies according to the present description and methods of using the antibodies according to the present description. It will be apparent to those skilled in the art that many modifications to both the materials and methods may be practiced without departing from the scope of the present disclosure.

IX. Examples.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes will be suggested to those skilled in the art in light thereof, and such modifications or changes are intended to be included within the spirit and scope of this application.

Example 1. Preparation of CD37 hybridoma.

Hybridomas producing monoclonal antibodies to human CD37 that are suitable for immunohistochemical (IHC) staining (antibodies as described herein) were selected from approximately 4800 hybridomas. Hybridomas were generated by immunizing wild-type Balb/c mice with the recombinant CD37 antigen hCD37-LEL derived from E. coli. This antigen contains amino acids 107 to 242 of CD37 with a 6χHis tag added to the 3' end to facilitate purification (SEQ ID NO: 15). Immunization with recombinant CD37 protein was performed by subcutaneous injection of the protein emulsified in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant for stimulation (Sigma) or Magic mouse adjuvant (Creative Diagnostics). Typically, mice were immunized five times at two-week intervals before receiving a final challenge by intraperitoneal injection of the immunizing agent three days before fusion. A total of 5 independent fusions were performed using spleen cells that were obtained from immunized wild-type Balb/c mice and P3X63Ag8.653 murine myeloma cells (P3 cells). Cell fusion was performed using the ECM200 electrofusion instrument (BTX Harvard Apparatus) according to standard protocols. Each fusion resulted in the formation of more than 1000 hybridomas.

Antibodies produced by these hybridomas were screened and confirmed by ELISA using different recombinant versions of the CD37 antigen. hCD37-LEL contains amino acids 107 to 242 of human CD37 with a 6xHis tag added to the 3' end to facilitate purification (SEQ ID NO: 15) and was produced in E. coli. hCD37-Fc-LAGA contains amino acids 107 to 235 of human CD37 with the human IgG1 Fc domain added to the 3' end to facilitate purification (SEQ ID NO: 17) and was produced in HEK-293T cells. hCD37-ECD-Fc contains amino acids 107 to 235 of human CD37 with the mouse IgG2a Fc domain added to the 3' end to facilitate purification (SEQ ID NO: 16) and was produced in HEK-293T cells.

To create native conditions, recombinant proteins were diluted directly in 50 mM sodium bicarbonate sorption buffer (Sigma-Aldrich). To create denaturing conditions, recombinant proteins were incubated in 1% sodium dodecyl sulfate (SDS) with 50 mM dithiothreitol (DTT) for 30 min at 65°C, followed by incubation with 100 mM iodoacetamide for 30 min at room temperature and then diluted in 50 mM sodium bicarbonate buffer for sorption. Each recombinant protein was immobilized at a concentration of approximately 25-100 ng/well in microtiter plates by incubating overnight at 4°C.

The plates were washed once with PBS supplemented with 0.05% Tween-20 and blocked with PBS supplemented with 1% bovine serum albumin (BSA). The plates were washed three times with PBS supplemented with 0.05% Tween-20, and hybridoma supernatants were added to the plates. The plates were incubated for 1 hour at room temperature, washed three times as described above, and incubated with labeled HRP

- 39 044601 goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch, dilution 1:5000) for 1 hour at room temperature. The plates were washed three times as described above, and bound HRP-conjugated antibody was detected by adding the HRP substrate TMB (Bio-FX). The plates were incubated for approximately 10 minutes and then color development was stopped by adding stopping solution (Bio-FX). For each plate, absorbance was measured at 450 nm using a microplate spectrophotometer. The hybridoma supernatant from 1B11 produced a strong positive ELISA signal under both native and denaturing conditions (see Fig. 1A) and was selected for subcloning. From clone 1B11, two subclones were obtained: 1B11-2 and 1B11-20. Hybridoma supernatant from both subclones produced a strong positive ELISA signal under both native and denaturing conditions (see Fig. 1B), and clone 1B11-2 was selected for additional analysis.

Example 2 Characterization of anti-CD37 antibodies by ELISA.

Antibody from 1B11-2 was purified using standard Protein A chromatography. An anti-CD37 antibody available from Leica Biosystems (product code NCL-CD37) was used for comparison. The binding of anti-CD37 antibodies was examined by ELISA with recombinant CD37 proteins as the antigen. Each recombinant protein was immobilized at a concentration of approximately 25-100 ng/well in microtiter plates in sodium bicarbonate adsorption buffer (SigmaAldrich) using either native or denaturing conditions as described above. The plates were washed once with PBS supplemented with 0.05% Tween-20 and blocked with PBS supplemented with 1% bovine serum albumin (BSA). The plates were washed three times with PBS supplemented with 0.05% Tween-20, and purified antibodies were added to the plates. The plates were incubated for 1 h at room temperature, washed three times as described above, and incubated with HRP-labeled goat anti-mouse IgG secondary antibody (Jackson ImmunoResearch, dilution 1:5000) for 1 h at room temperature. The plates were washed three times as described above, and bound HRP-conjugated antibody was detected by adding HRP substrate (Bio-FX). The plates were incubated for approximately 10 minutes and then color development was stopped by adding stopping solution (Bio-FX). For each plate, absorbance was measured at 450 nm using a microplate spectrophotometer. Representative binding results with native hCD37-Fc-LAGA and hCD37-LEL are shown in FIG. 2. Antibody 1B11-2 binds to both native CD37 proteins with significantly improved affinity compared to NCL-CD37. The binding results with denatured hCD37-LEL protein are shown in FIG. 3. Antibody 1B11-2 binds to the denatured hCD37-LEL protein with significantly improved affinity compared to NCL-CD37.

Example 3 Determination of antigen epitope characteristics.

The binding of anti-CD37 antibodies was further examined by ELISA with the recombinant CD37 protein hCD37-ECD-S2-Fc used as the antigen under native conditions as described above. (S2 refers to the second segment of the large extracellular domain of CD37, containing amino acids 138 to 176). Typical results are shown in Fig. 4. hCD37-ECD-S2-Fc contains amino acids 107 to 109 and 138 to 235 of human CD37 with the mouse IgG2a Fc domain added to the 3' end to facilitate purification (SEQ ID NO: 18) and was prepared in HEK-293T cells. Antibody 1B11-2 does not bind to hCD37-ECD-S2-Fc, while NCL-CD37 retains the ability to bind to this fragment of the CD37 protein. Similar results were observed when analyzed by Western blotting.

Example 4. Immunohistochemical evaluation of antibodies to CD37 FFPE CD37 IHC.

Purified antibody from clone 1B11-2 was analyzed and compared with the mouse monoclonal antibody NCL-CD37 (Leica) by IHC. The analysis was performed using a Leica Bond RX automated stainer and the reagents and conditions listed in Table. 1.

Table 1

IHC reagents and assay conditions

Stage Action/Reagent (Vendor) Time Drying Temperature: 6°C 30 minutes Dewaxing Bond Dewax Solution (Leica), 100% pure ethanol for analysis (Arantik) Installed Antigen unmasking Bond Epitope Retrieval 1 solution (citrate buffer based solution with pH 6.0) 20 minutes Blocking endogenous peroxidase Peroxide (Leica) 5 minutes Test sample obtained by ImmunoGen, Inc. Antibodies in various concentrations obtained by dilution in Leica Antibody Diluent 15 minutes

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Detection Post Primary Regent (Leica) 8 minutes Polymer (Leica) 8 minutes Mixed DAB (Leica) 10 minutes Contrast staining Hematoxylin (Leica) 5 minutes

Slides containing formalin-fixed paraffin-embedded (FFPE) cell samples, normal tissues, tumor biopsies from patients with diffuse large B-cell lymphoma, tumor biopsies from patients with follicular lymphoma, and tumor biopsies from patients with chronic lymphocytic leukemia/small lymphoma lymphocytes were dried at 60°C and dewaxed using Bond Dewax Solution and 100% ethanol. High-temperature unmasking of the epitope was carried out using Bond Epitope Retrieval 1 solution (citrate buffer based on a solution with pH 6.0) for 20 minutes and endogenous peroxidase was blocked with peroxide for 5 minutes. The preparations were incubated with the resulting ImmunoGen, Inc. antibody 1B11-2, antibody NCL-CD37 (clone CT1) (Leica/Novocastra) or control antibody muIgG1 (Leica/Novocastra) at various concentrations for 15 min. Bound antibodies were detected by incubation with a Leica Bond Refine detection system. After application of antibodies, the slides were incubated with Post Primary Reagent (rabbit IgG to mouse IgG) for 8 min, polymer (goat polymer to rabbit IgG) for 8 min, and DAB (3,3-diaminobenzidine tetrahydrochloride) for 10 min. This provided a brown signal. The preparations were counterstained with hematoxylin for 5 minutes.

FFPE samples of normal spleen and tonsil tissue were obtained from human tissue blocks obtained from Mercy Health Systems and Ardais Corporation as follows. FFPE cell samples were obtained from the Daudi and Ramos cell lines supplied by the DSMZ (German Collection of Microorganisms and Cell Cultures), and the Namalwa cell line was provided by the American Tissue Culture Collection. Slides containing sample sections were prepared from FFPE blocks using a microtome set at 5 μm and mounted under positively charged glass slides. These slides were left to air dry overnight before staining. Human normal tissue micropanels were purchased from Pantomics, and non-Hodgkin's lymphoma tissue micropanels were purchased from TriStar Technology Group LLC.

table 2

FFPE test samples

Human tissue type Commercial source Normal spleen (2) Mercy Health Systems Normal tonsil (3) Mercy Health Systems (2) and Ardais Corporation (1) Micropanel of normal tissues Pantomics Micropanel of non-Hodgkin's lymphoma tissues Tri Star Technology Group LLC

CD37 staining intensity and distribution pattern were assessed in comparison with control IgG staining (nonspecific), and 1B11-2 staining was compared with staining observed for the mouse monoclonal antibody NCL-CD37 (Leica). Intensity was graded on a scale from 0 to 3, where 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong staining. Staining uniformity was graded as negative (no positively stained cells), focal (<25% stained cells), heterogeneous (25–75% stained cells), and homogeneous (>75% stained cells). Staining intensity and grading scales are described below. All stains were evaluated by a board-certified pathologist.

Table 3

Color intensity and uniformity

Intensity (amount of membrane staining) 0 Negative 1 Weak 2 Moderate 3 Strong

Uniformity (percentage of positive cells) 0 Negative Focal <25% Heterogeneous (hetero) 25-75% Homogeneous (homo) >75%

Validation of purified antibody 1B11-2 for FFPE CD37 IHC.

Purified antibody from clone 1B11-2, diluted to concentrations of 8.4, 4.2 and 2.1 μg/ml per solution

- 41 044601 Leica Antibody Diluent (Tris-buffered saline, surfactant and protein stabilizer with 0.35% ProClin™ 950) was used to stain CD37 positive control samples (normal human tonsil, Daudi cells, Ramos cells and Namalwa cells) . The antibody was assessed for CD37 specificity, as determined by acceptable membrane staining and specificity in CD37-positive samples. This clone was also compared to the mouse mAb NCL-CD37 (clone CT1) diluted to 4.2 μg/ml in Leica Antibody Diluent using the same CD37-positive human tissue and cells. Antibody NCL-CD37 (Leica) provided acceptable membrane staining in each of the CD37-positive cell pellets and acceptable membrane staining in germinal centers and mantle zones of tonsil tissue (both CD37 positive) along with some low nuclear background and lack of staining in the interfollicular region of the tonsil (negative for CD37). 1B11-2 showed acceptable membrane staining in CD37-positive samples and good specificity with the added benefit of complete absence of nuclear background staining (see Fig. 5 for comparison of images for 1B11-2 and mouse mAb NCL-CD37 (Leica)). 1B11-2 provided membrane staining with a structure similar to that observed with the mouse monoclonal antibody NCL-CD37 (Leica); however, this membrane staining was more specific and clearer than that obtained with the NCL-CD37 antibody (Leica). It is also important to note that 1B11-2 did not stain in the interfollicular region of the tonsil (negative for CD37 expression), demonstrating increased specificity. For 1B11-2, the concentration suitable for staining was experimentally determined to be 4.2 μg/ml.

1B11-2, diluted to 4.2 μg/mL in Leica Antibody Diluent, was also used to stain a micropanel of normal human tissue (purchased from Pantomics) and human tonsil and spleen tissue to assess specificity. 1B11-2 was again compared with the mouse monoclonal antibody NCL-CD37 (Leica), diluted to a concentration of 4.2 μg/ml in Leica Antibody Diluent, on the same normal tonsil and spleen tissues, as well as a micropanel of normal human tissues. In most normal tissues (except tonsil and spleen), the mouse monoclonal antibody NCL-CD37 (Leica) showed staining only in scattered lymphocytic cells, leaving the rest of the tissue negative. Some background pink staining was observed in the cytoplasm of the Paneth cells of the small intestine and in the islet cells of the pancreas (see Fig. 6). Antibody NCL-CD37 (Leica) provided acceptable membrane staining in the germinal centers and pallial zones of normal human amygdala and in the marginal zone in normal human splenic tissues, although with some low-level nuclear background. There was no staining in the interfollicular region of the tonsil or the red pulp of the spleen using the NCL-CD37 antibody (Leica). 1B11-2 showed acceptable membrane staining in the germinal centers and mantle zones of normal human tonsil and in the marginal zone in normal human spleen tissues and staining only in scattered lymphocytes in the remaining normal tissues without any background staining (see Fig. 6). There was no staining in the interfollicular region of the tonsil or the red pulp of the spleen. These results indicate that 1B11-2 is superior in terms of staining sensitivity, specificity, and reduced background, all of which are critical for high-quality performance and analysis of IHC studies.

Example 5: Evaluation of anti-CD37 antibody 1B11-2 by IHC using human tumor samples.

Human tumor specimens representative of diffuse large B-cell lymphoma (n=52), follicular lymphoma (n=20), and chronic lymphocytic leukemia/small lymphocyte lymphoma (n=8) (all included in the non-Hodgkin's lymphoma tissue micropanel purchased from TriStar Technology Group LLC) was assessed for CD37 expression by IHC using the 1B11-2 antibody. The intensity of CD37 staining and the distribution of values are summarized in Table 1. 4 below. In fig. Figure 7 shows an example of tissue staining for diffuse large B-cell lymphoma and follicular lymphoma using the 1B11-2 antibody. These results demonstrate the advantages of 1B11-2 as a more specific and sensitive antibody for use in IHC studies to evaluate CD37 expression in tissues from patients with non-Hodgkin's lymphoma.

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Table 4

Distribution of values (% positive results)

TUMOR TYPE: DIFFUSE LARGE CELL LYMPHOMA c-52 FOLLICULAR LYMPHOMA n=20 CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL LYMPHOCYTE LYMPHOMA n=8 Positive (any intensity): 96% 95% 100% >level 2 intensity when staining at least 25% of tumor cells: 58% 35% 50% >level 3 intensity when staining at least 25% of tumor cells: 60% 100% 100%

Antibody 1B11-2 and mouse monoclonal antibody NCL-CD37 (Leica) were compared using a non-Hodgkin's lymphoma (TMA) tissue micropanel (purchased from TriStar Technology Group LLC). When using the NCL-CD37 antibody in an IHC assay (CD37 assay), 27% of diffuse large B-cell lymphoma samples (14 of 52), 85% of follicular lymphoma samples (17 of 20), and 37% of chronic lymphocytic leukemia/small lymphocyte lymphoma samples (3 of 8) were classified as the highest category (level 3 staining intensity on at least 25% of tumor cells, Table 5). In contrast, antibody 1B11-2 in the IHC assay described above using the Leica Bond Refine Detection Kit on the Leica Bond Rx automated slide stainer provided staining with increased sensitivity and specificity in 59% of diffuse large B-cell lymphoma samples (31 of 52 ), 95% of follicular lymphoma samples (19 of 20) and 100% of chronic lymphocytic leukemia/small lymphocyte lymphoma samples (8 of 8), providing staining that was classified as the highest category (level 3 staining intensity of at least 25% tumor cells, Table 5). Antibody 1B11-2 also detected low-level CD37 expression in three samples of diffuse large B-cell lymphoma, whereas antibody NCL-CD37 (Leica) did not detect this low level of expression. These results indicate that 1B11-2 is superior in terms of staining sensitivity and specificity and provides a greater dynamic range of CD37 staining.

Table 5

Comparison of CD37 prevalence in TMA of non-Hodgkin's lymphoma

Diffuse large B-cell lymphoma (n=52) Follicular lymphoma (n=20) Chronic lymphocytic leukemia/small lymphocyte lymphoma (n-8) Values NCL- CD37 1V11-2 NCLCD37 1V11-2 NCL- CD37 1V11-2 Polar burn (any intensity) 47 (90%) 50 (96%) 19(95%) 19 (95%) 8 (100%) 8(100%) > level 1 intensity when staining at least 25% of tumor cells: 26 (50%) 11 (21%) 0 0 0 0 >level 2 intensity when staining at least 25% of tumor cells: 33 (63%) 30 (58%) 12 (60%) 7 (35%) 7 (88%) 4 (50%) >level 3 intensity when staining at least 25% of tumor cells: 14 (27%) 31 (60%) 17 (85%) 19 (95%) 3 (38%) 8 (100%)

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Example 6: Domain mapping.

Domain mapping using denaturing SDS-PAGE and Western blotting.

The ability to recognize various Fc-fusion CD37-ECD preparations was examined by denaturing SDS-PAGE followed by Western blotting for 1B11-2 (left panel) in direct comparison with a commercially available anti-CD37 antibody (Leica; right panel). Fc-fusion CD37-ECD preparations were denatured by incubation in Laemmli buffer containing 70 mM β-mercaptoethanol at 100°C for 10 min and separated by SDS-polyacrylamide gel electrophoresis, followed by electrophoretic transfer to PVDF membranes. . Membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature. Primary antibodies were applied overnight followed by detection with horseradish peroxidase-conjugated anti-mouse F(ab) ₂ secondary antibody for 1 h at room temperature. Blots were developed using enhanced chemiluminescence detection following standard techniques. The results are shown in Fig. 8. While the Leica antibody recognizes all three Fc-CD37 ECD preparations, the 1B11-2 antibody of the present invention recognizes only hCD37-ECD-Fc and hCD37-Fc-LAGA, but not hCD37-ECD-S2-Fc. The hCD37-ECD-S2-Fc construct does not contain the S1 segment (amino acids 110-137) of the CD37 ECD. Thus, the region of amino acids 110-137 is critical for epitope recognition using 1B11-2, but not the Leica antibody. Domain mapping using native PAGE and Western blotting

The ability to recognize various Fc-fusion CD37-ECD preparations was examined by native PAGE followed by Western blotting for 1B11-2. Fc-fusion CD37-ECD preparations were loaded into native electrophoresis sample buffer and separated by SDS-polyacrylamide gel electrophoresis, followed by electrophoretic transfer to PVDF membranes or staining of the gel with Coomassie brilliant blue. PVDF membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 1 h at room temperature. The primary antibody was applied overnight followed by detection with horseradish peroxidase-conjugated anti-mouse F(ab) ₂ secondary antibody for 1 h at room temperature. The blot was developed using enhanced chemiluminescence detection following standard techniques. The results are shown in Fig. 9. According to denaturing SDS-PAGE data (Fig. 8) and ELISA experiments (see Fig. 4), the 1B11-2 antibody of the present invention recognizes only hCD37-ECD-Fc and hCD37-Fc-LAGA, but not hCD37-ECD-S2-Fc. These data indicate that the S1 segment (amino acids 110-137) of the CD37 ECD or a portion thereof is critical for recognition of the 1B11-2 epitope (left panel of Fig. 9). Coomassie brilliant blue staining of the gel (right panel of Fig. 9) shows that hCD37-ECD-S2-Fc protein was present in sufficient quantities to be detected using 1B11-2.

Example 7: Cloning and sequencing of the VL and VH regions of anti-human CD37 antibodies.

Total cellular RNA was prepared from 5x106 anti-human CD37 hybridoma 1B11-2 cells described above using the RNeasy kit (QIAgen) according to the manufacturer's protocol. cDNA was then synthesized from the first strand of total RNA using the Superscript III cDNA Synthesis Kit (Invitrogen).

PCR procedures for amplification of antibody variable region cDNA obtained from hybridoma cells were based on methods described by Wang et al. ((2000), J. Immunol. Methods. 233:167-77) and Co et al. ((1992), J. Immunol. 148:1149-54). Variable light chain (VL) and variable heavy chain (VH) sequences were amplified using degenerate primers at the 5' end and mouse kappa constant region or IgG1 specific primers at the 3' end. The PCR reaction mixtures were then electrophoresed on a 1% low melting point agarose gel and 300 to 400 bp amplicon regions were excised and purified using Zymo DNA mini columns. Purified amplicons were submitted to Beckman Coulter Genomics for sequencing using the same 5′ and 3′ PCR reaction primers to obtain the variable region cDNA sequence in both directions.

The degenerate primers used to clone the VL and VH cDNA sequences alter the 5′ end, so additional sequencing steps were necessary to verify the complete variable region cDNA sequences. Preliminary sequences were entered into a search query on the NCBI IgBlast website (www.ncbi.nlm.nih.gov/igblast/) to identify the mouse germline sequences from which the antibody sequences were derived. The PCR primers were then designed to anneal to the germline-associated mouse antibody leader sequence so that this new PCR reaction would result in the complete variable region cDNA sequence unaltered by the PCR primers. PCR reactions, zone purification, and sequencing were performed as described above. Variable region cDNA sequences obtained for the anti-human CD37 antibody were combined

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Claims (12)

with germline constant region sequences to obtain full-length antibody cDNA sequences. The molecular weights of the heavy and light chains were then calculated using translations of the cDNA sequences and compared with the molecular weights obtained by LC/MS analysis of purified mouse anti-human CD37 antibody. The observed molecular weights for the light and heavy chains of murine 1B11-2 were consistent with the expected values, confirming the correctness of the cDNA sequences. The CDR sequences of VH and VL are given in table. 1 and 2 respectively. The sequences of VH and VL are given in table. 3 and 4 respectively. The full-length sequences of the heavy chain and light chain are given in table. 5 and 6 respectively. Polynucleotide sequences of VH and VL are given in table. 8. All publications, patents, patent applications, Internet sites and accession numbers/database sequences (including both polynucleotide and polypeptide sequences) cited herein are hereby incorporated by reference in their entirety and in all respects to the same extent , as if each individual publication, patent, patent application, Internet site, or accession number/database sequence were specifically and individually designated as being incorporated by reference. CLAIM 1. An antibody or antigen-binding fragment of said antibody that specifically binds to CD37, wherein said antibody or fragment thereof contains VH CDR1, CDR2 and CDR3 sequences according to SEQ ID NO: 3-5 and VL CDR1, CDR2 and CDR3 sequences according to SEQ ID NO: 6-8 respectively. 2. An antibody or antigen-binding fragment of said antibody according to claim 1, wherein said antibody or fragment thereof comprises polypeptide sequences containing amino acids of the sequences SEQ ID NO: 9 and/or 10. 3. The antibody or antigen binding fragment of said antibody according to any one of claims 1 or 2, wherein said antibody or antigen binding fragment thereof is a mouse antibody or antigen binding fragment thereof, a humanized, chimeric antibody or antigen binding fragment thereof, a human antibody or antigen binding fragment thereof. 4. Antibody according to any one of claims 1-3, which is a full-length antibody. 5. An antigen-binding fragment according to any one of claims 1 to 3, which is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain Fv or scFv, disulfide bond stabilized Fv, intraantibody, IgGACH ₂ , mini-antibody, F(ab') ₃ , tetrabody, tribody, diabody, DVD-Ig, mAb ₂ , (scFv)2 and scFv-Fc. 6. An antibody or antigen-binding fragment of said antibody according to any one of claims 1 to 5, wherein said antibody or antigen-binding fragment thereof is detectably labeled. 7. A method for detecting CD37 expression in a sample in vitro, comprising contacting said sample with an antibody or antigen-binding fragment of said antibody according to any one of claims 1 to 6, and determining the level of CD37 expression in said sample. 8. The method of claim 7, wherein CD37 expression is determined by immunohistochemical (IHC) analysis, radioimmunoassay, Western blotting, cytometry, immunofluorescence assay, enzyme-linked immunosorbent assay, immunoprecipitation assay, or chemiluminescent assay. 9. The use of an antibody or antigen-binding fragment of said antibody according to any one of claims 1 to 6 in a method of treating a patient having cancer by administering a therapeutically active agent that is an antibody-maytansinoid conjugate including an anti-CD37 antibody that contains a heavy chain variable region, comprising SEQ ID NO: 28, and a light chain variable region comprising SEQ ID NO: 29, wherein said use includes detecting increased expression of CD37 in a cancer sample from said patient. 10. Use according to claim 9, where said detection is carried out by immunohistochemistry (IHC). 11. Use according to any one of claims 9 or 10, wherein said cancer is leukemia or lymphoma. 12. Use according to any one of claims 9 or 10, wherein said cancer is selected from the group consisting of B-cell lymphomas, non-Hodgkin's lymphoma (NHL), B-cell lymphoblastic leukemia/progenitor cell lymphoma and neoplasms of mature B cells , B-cell chronic lymphocytic leukemia (CLL)/small lymphocyte lymphoma (SLL), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL), low-grade, intermediate-grade and high-grade FL degree of malignancy, cutaneous lymphoma from follicular center cells, B-cell lymphoma from marginal zone cells, MALT-type B-cell lymphoma from marginal zone cells, nodal type B-cell lymphoma from marginal zone cells, B-cell lymphoma -