Bispecific Anti-CD37 antibodies, monoclonal Anti-CD37 antibodies, and methods of use thereof

Abstract

To provide anti-CD37 antibodies which may be useful in treatment of cancer and / or other diseases.SOLUTION: The invention also provides CD37-specific bispecific antibody molecules binding to different epitopes of the human CD37 antigen, the bispecific antibody molecules having enhanced Fc-Fc interactions upon binding to CD37 on a cell surface. The invention also provides monoclonal parental antibodies from which a first or second binding region of the bispecific antibody molecules is obtained. The invention also provides pharmaceutical compositions containing the molecules, and treatment of cancer and other diseases using the compositions.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to bispecific antibodies that specifically bind to the human CD37 antigen. The present invention particularly relates to CD37-specific bispecific antibody molecules that bind to different epitopes on the human CD37 antigen and have enhanced Fc-Fc interactions upon binding to CD37 on the cell surface, thereby having enhanced effector function. The present invention also relates to novel monoclonal parent antibodies from which the first or second antigen-binding regions of the bispecific antibody molecules are derived. The present invention also relates to pharmaceutical compositions containing these molecules and to the treatment of cancer and other diseases using such compositions. [Background technology]

[0002] Background of the Invention Leukocyte antigen CD37 (also known as GP52-40, tetraspanin-26, or TSPAN26) is a transmembrane protein of the tetraspanin superfamily (Maecker et al., FASEB J. 1997;11:428-442 (Non-Patent Document 1)). In normal physiology, CD37 is reportedly expressed on B cells from the pre-B to peripheral mature B cell stages, but is absent on plasma cells (Link et al., J Pathol. 1987;152:12-21 (Non-Patent Document 2)). The CD37 antigen is only weakly expressed on T cells and myeloid cells, such as monocytes, macrophages, dendritic cells, and granulocytes (Schwartz-Albiez et al., J. Immunol 1988;140(3):905-914 (Non-Patent Document 3)). CD37 is widely expressed on malignant cells in a variety of B-cell leukemias and lymphomas, including non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) (Moore et al. J Immunol. 1986;137(9):3013).

[0003] Several antibody-based CD37 targeting agents are being evaluated as potential therapeutic agents for B cell malignancies and other malignancies. These include, for example, radioimmunoconjugates such as Betalutin®, antibody-drug conjugates such as IMGN529 and AGS-67E, and reshaped or Fc-engineered antibodies such as otlertuzumab and BI 836826 (Robak and Robak, Expert Opin Biol Ther 2014;14(5):651-61). Anti-CD37 antibodies have been proposed for use as therapeutic agents in these and other formats (see, for example, WO2012 / 135740 (Patent Document 1), WO2012 / 007576 (Patent Document 2), WO2011 / 112978 (Patent Document 3), WO2009 / 126944 (Patent Document 4), WO2011 / 112978 (Patent Document 5), and EP2241577 (Patent Document 6)).

[0004] Betalutin is the murine anti-CD37 antibody rilotomab (formerly HH1 / tetulomab) conjugated to lutetium-177. Betalutin is rapidly internalized, inhibits B cell growth in vitro, and prolongs survival in the intravenous Daudi-SCID model (Dahle et al. 2013, Anticancer Res 33:85-96).

[0005] IMGN529 is an ADC consisting of the K7153A antibody conjugated to the maytansinoid DM1 via an SMCC linker. The K7153 antibody has been reported to induce apoptosis in CD37-expressing Ramos cells in the absence of cross-linking. It also induced CDC and ADCC in Burkitt's lymphoma cell lines, although its ability to induce CDC was much lower than that of rituximab (Deckert et al., Blood 2013; 122(20):3500-10). These Fc-mediated effector functions of K7153A are retained in the DM1-conjugated antibody.

[0006] Agensys is developing AGS-67E, a human anti-CD37 IgG2 mAb conjugated to monomethyl auristatin E. AGS67E induces potent cytotoxicity and apoptosis (Pereira et al, Mol Cancer Ther 2015; 14(7): 1650-1660).

[0007] Otlertuzumab (formerly known as TRU-016) is a small modular immunopharmaceutical product (SMIP). SMIPs are disulfide-linked single-chain protein dimers consisting of one antigen-binding VH / VL domain, a connecting hinge region, and an Fc (fragment crystallizable) domain (CH2-CH3). Its mechanism of action is the induction of apoptosis and ADCC, but not CDC (Zhao et al. 2007, Blood 110(7), 2569-2577).

[0008] mAb37.1 / BI 836826 is a chimeric antibody engineered for high affinity binding to FcγRIIIa (CD16a) (Heider et al 2011, Blood 118: 4159-4168). It has proapoptotic activity independent of IgG Fc cross-linking, but the proapoptotic activity is enhanced by cross-linking. It binds to CD37 + It exhibits potent ADCC in B cell lines and primary CLL cells.

[0009] However, despite these and other advances in the art, there remains a need for improved anti-CD37 antibodies for the treatment of cancer and other diseases.

[0010] Accordingly, it is an object of the present invention to provide anti-CD37 antibodies that may be useful in the treatment of cancer and / or other diseases. It is an object of the present invention to provide anti-CD37 antibodies that have improved CDC of human cells by human complement compared to prior art antibodies. A further object is to provide bispecific antibodies having binding arms derived from two parent antibodies that bind to different epitopes on CD37, which have enhanced CDC and / or ADCC compared to the combination of the two parent monoclonal antibodies that bind to the different epitopes and / or either parent monoclonal antibody alone. A further object is to provide new monoclonal antibodies that bind to different epitopes on CD37, and in particular, to provide anti-CD37 antibodies that bind to new epitopes of CD37. It is a further object of the present invention to provide new monoclonal antibodies that bind to different epitopes on CD37, which can serve as parent antibodies for the bispecific antibodies of the present invention. A further object is to provide a bispecific antibody that binds to two different epitopes on CD37 and has enhanced Fc-Fc interactions upon binding to CD37 on the cell membrane compared to a bispecific antibody of the same isotype that has the same binding arms as the bispecific antibody of the present invention. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] WO2012 / 135740 [Patent Document 2] WO2012 / 007576 [Patent Document 3] WO2011 / 112978 [Patent Document 4] WO2009 / 126944 [Patent Document 5] WO2011 / 112978 [Patent Document 6] EP2241577

Non-Patent Literature

[0012]

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Summary of the Invention

[0013] The present inventors surprisingly found that a bispecific antibody having binding specificities for two different epitopes on CD37 and containing mutations that enhance Fc-Fc interactions upon binding to CD37 on the cell membrane was more potent in inducing CDC than a combination of two anti-CD37 antibodies, each having binding specificities for one of two different epitopes on CD37 and containing the same mutations that enhance Fc-Fc interactions, or either parent antibody alone having the same mutations that enhance Fc-Fc interactions. In addition, a bispecific antibody having binding specificities for two different epitopes on CD37 and containing mutations that enhance Fc-Fc interactions was more potent in inducing ADCC than a combination of two anti-CD37 antibodies, each having binding specificities for one of two different epitopes on CD37 and containing the same mutations that enhance Fc-Fc interactions.

[0014] Thus, the present invention relates to novel bispecific antibodies that bind to human CD37, which have advantageous properties with regard to antigen binding properties, ability to induce CDC and ADCC, Fc-Fc interactions upon binding to membrane-bound targets, cytotoxic effect on CD37-expressing cells, and other properties as described herein.

[0015] Thus, in a first aspect, the present invention relates to a bispecific antibody comprising first and second antigen-binding regions that bind to human CD37 having the sequence of SEQ ID NO: 62, and first and second Fc regions of a human immunoglobulin, wherein the first and second antigen-binding regions bind to different epitopes on CD37, and the first and second Fc regions comprise one or more amino acid mutations that enhance the Fc-Fc interaction between the bispecific antibody upon binding to membrane-bound CD37 compared to the Fc-Fc interaction between the bispecific antibody without said mutations.

[0016] Thus, in a first aspect, there is provided a bispecific antibody comprising first and second antigen-binding regions that bind to human CD37 having the sequence of SEQ ID NO: 62, and first and second Fc regions of a human immunoglobulin, wherein the first and second antigen-binding regions bind to different epitopes on CD37, and the first and second Fc regions comprise one or more amino acid mutations that enhance the Fc-Fc interaction between the bispecific antibodies upon binding to a membrane-bound target compared to the Fc-Fc interaction between the bispecific antibodies without said mutations.

[0017] In a second aspect, the present invention provides a method for producing a medicament for a medicament comprising: (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 16, the CDR2 sequence set forth in SEQ ID NO: 17, and the CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 20, a CDR2 sequence that is a KAS, and the CDR3 sequence set forth in SEQ ID NO: 21; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 9, the CDR2 sequence set forth in SEQ ID NO: 10, and the CDR3 sequence set forth in SEQ ID NO: 11; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 113, a CDR2 sequence that is an AAS, and the CDR3 sequence set forth in SEQ ID NO: 14. The present invention relates to an anti-CD37 antibody that binds to the same epitope on human CD37 as an anti-CD37 antibody comprising:

[0018] In a third aspect, the present invention provides a method for producing a medicament for a medicament comprising: (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 23, the CDR2 sequence set forth in SEQ ID NO: 24, and the CDR3 sequence set forth in SEQ ID NO: 25, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 27, a CDR2 sequence that is YAS, and the CDR3 sequence set forth in SEQ ID NO: 28; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 2, the CDR2 sequence set forth in SEQ ID NO: 3, and the CDR3 sequence set forth in SEQ ID NO: 4; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 6, a CDR2 sequence that is an EAS, and the CDR3 sequence set forth in SEQ ID NO: 7. The present invention relates to an anti-CD37 antibody that binds to human CD37, comprising:

[0019] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising a bispecific antibody or antibody of the invention and a pharmaceutically acceptable carrier.

[0020] In a fifth aspect, the present invention relates to a bispecific antibody or antibody or composition of the invention for use as a medicament, in a particular aspect for the treatment of cancer or an autoimmune disease or an inflammatory disorder, in particular for the treatment of a B-cell malignancy.

[0021] In other aspects, the invention relates to methods of treatment, combination therapies, nucleic acid sequences encoding the antibodies of the invention, vectors for their expression, and host cells, as well as methods of detecting the presence of the CD37 antigen or cells expressing the CD37 antigen in a sample or a subject. [The present invention 1001] 1. A bispecific antibody comprising first and second antigen-binding regions that bind to human CD37 having the sequence of SEQ ID NO: 62, and first and second Fc regions of a human immunoglobulin, wherein the first and second antigen-binding regions bind to different epitopes on CD37, and the first and second Fc regions comprise one or more amino acid mutations that enhance Fc-Fc interaction between the bispecific antibodies upon binding to a membrane-bound target compared to Fc-Fc interaction between bispecific antibodies without the mutations. [The present invention 1002] The first antigen-binding region comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 16, the VH CDR2 sequence set forth in SEQ ID NO: 17; VH CDR3 sequence set forth in SEQ ID NO: 18; the VL CDR1 sequence set forth in SEQ ID NO: 20; a VL CDR2 sequence that is a KAS, and VL CDR3 sequence set forth in SEQ ID NO: 21 1001. A bispecific antibody of the present invention, obtained from a CD37 antibody comprising the compound and an antibody which competes for binding to human CD37. [The present invention 1003] The first antigen-binding region comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 16, the VH CDR2 sequence set forth in SEQ ID NO: 17; VH CDR3 sequence set forth in SEQ ID NO: 18; the VL CDR1 sequence set forth in SEQ ID NO: 20; a VL CDR2 sequence that is a KAS, and VL CDR3 sequence set forth in SEQ ID NO: 21 any of the bispecific antibodies of the present invention, which bind to the same epitope on human CD37 as an antibody comprising: [The present invention 1004] The first antigen-binding region comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 16, the VH CDR2 sequence set forth in SEQ ID NO: 17; VH CDR3 sequence set forth in SEQ ID NO: 18; the VL CDR1 sequence set forth in SEQ ID NO: 20; a VL CDR2 sequence that is a KAS, and VL CDR3 sequence set forth in SEQ ID NO: 21 any of the bispecific antibodies of the present invention, comprising: [The present invention 1005] The first antigen-binding region comprises the VH and VL sequences: (i) the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 19; or (ii) a VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, for example at least 99% identity, and a VL sequence having at least 90% identity, such as at least 95% identity, for example at least 98% identity, for example at least 99% identity, to the VH and VL sequences of SEQ ID NOs: 15 and 19. any of the bispecific antibodies of the present invention, comprising: [The present invention 1006] 1006. The bispecific antibody of any of claims 1001 to 1006, wherein the first antigen-binding region binds to a functional epitope comprising one or more amino acids selected from the group consisting of Y182, D189, T191, I192, D194, K195, V196, I197, and P199 of SEQ ID NO: 62 (CD37). [The present invention 1007] The first antigen-binding region comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 9, VH CDR2 sequence set forth in SEQ ID NO: 10, VH CDR3 sequence set forth in SEQ ID NO: 11, the VL CDR1 sequence set forth in SEQ ID NO: 13; A VL CDR2 sequence that is an AAS, and VL CDR3 sequence set forth in SEQ ID NO: 14 1001. A bispecific antibody of the present invention, obtained from a CD37 antibody comprising the compound and an antibody which competes for binding to human CD37. [The present invention 1008] The first antigen-binding region comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 9, VH CDR2 sequence set forth in SEQ ID NO: 10, VH CDR3 sequence set forth in SEQ ID NO: 11, the VL CDR1 sequence set forth in SEQ ID NO: 13; A VL CDR2 sequence that is an AAS, and VL CDR3 sequence set forth in SEQ ID NO: 14 The bispecific antibody of the present invention 1001 or 1007, which binds to the same epitope on human CD37 as the CD37 antibody comprising: [The present invention 1009] The first antigen-binding region comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 9, VH CDR2 sequence set forth in SEQ ID NO: 10, VH CDR3 sequence set forth in SEQ ID NO: 11, the VL CDR1 sequence set forth in SEQ ID NO: 13; A VL CDR2 sequence that is an AAS, and VL CDR3 sequence set forth in SEQ ID NO: 14 1001, 1007 or 1008. A bispecific antibody of the present invention comprising: [The present invention 1010] The first antigen-binding region comprises the VH and VL sequences: (i) the VH sequence set forth in SEQ ID NO: 8 and the VL sequence set forth in SEQ ID NO: 12; or (ii) a VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, for example at least 99% identity, and a VL sequence having at least 90% identity, such as at least 95% identity, for example at least 98% identity, for example at least 99% identity, to the VH and VL sequences of SEQ ID NOs: 8 and 12. The bispecific antibody of any one of 1001 and 1007 to 1009 of the present invention, comprising: [The present invention 1011] the second antigen-binding region (i) the VH CDR1 sequence set forth in SEQ ID NO: 23; the VH CDR2 sequence set forth in SEQ ID NO: 24; VH CDR3 sequence set forth in SEQ ID NO: 25; the VL CDR1 sequence set forth in SEQ ID NO: 27; a VL CDR2 sequence that is YAS, and VL CDR3 sequence set forth in SEQ ID NO: 28

[0016] ; (ii) the VH CDR1 sequence set forth in SEQ ID NO: 2; the VH CDR2 sequence set forth in SEQ ID NO: 3; VH CDR3 sequence set forth in SEQ ID NO: 4, VL CDR1 sequence set forth in SEQ ID NO: 6, a VL CDR2 sequence that is an EAS, and VL CDR3 sequence set forth in SEQ ID NO: 7

[0004] ; (iii) the VH CDR1 sequence set forth in SEQ ID NO: 40; VH CDR2 sequence set forth in SEQ ID NO: 41, VH CDR3 sequence set forth in SEQ ID NO: 42; the VL CDR1 sequence set forth in SEQ ID NO: 44; a VL CDR2 sequence that is FAK, and The VL CDR3 sequence [G28.1] set forth in SEQ ID NO: 45; and (iv) the VH CDR1 sequence set forth in SEQ ID NO: 47; VH CDR2 sequence set forth in SEQ ID NO: 48; VH CDR3 sequence set forth in SEQ ID NO: 49; the VL CDR1 sequence set forth in SEQ ID NO: 51; a VL CDR2 sequence that is VAT, and VL CDR3 sequence set forth in SEQ ID NO: 52 [37.3] any of the bispecific antibodies of the present invention, which are obtained from a CD37 antibody comprising a CDR sequence selected from the group comprising: [The present invention 1012] the second antigen-binding region (i) the VH CDR1 sequence set forth in SEQ ID NO: 23; the VH CDR2 sequence set forth in SEQ ID NO: 24; VH CDR3 sequence set forth in SEQ ID NO: 25; the VL CDR1 sequence set forth in SEQ ID NO: 27; a VL CDR2 sequence that is YAS, and VL CDR3 sequence set forth in SEQ ID NO: 28

[0016] ; (ii) the VH CDR1 sequence set forth in SEQ ID NO: 2; the VH CDR2 sequence set forth in SEQ ID NO: 3; VH CDR3 sequence set forth in SEQ ID NO: 4, VL CDR1 sequence set forth in SEQ ID NO: 6, a VL CDR2 sequence that is an EAS, and VL CDR3 sequence set forth in SEQ ID NO: 7

[0004] ; (iii) the VH CDR1 sequence set forth in SEQ ID NO: 40; VH CDR2 sequence set forth in SEQ ID NO: 41, VH CDR3 sequence set forth in SEQ ID NO: 42; the VL CDR1 sequence set forth in SEQ ID NO: 44; a VL CDR2 sequence that is FAK, and The VL CDR3 sequence [G28.1] set forth in SEQ ID NO: 45; and (iv) the VH CDR1 sequence set forth in SEQ ID NO: 47; VH CDR2 sequence set forth in SEQ ID NO: 48; VH CDR3 sequence set forth in SEQ ID NO: 49; the VL CDR1 sequence set forth in SEQ ID NO: 51; a VL CDR2 sequence that is VAT, and VL CDR3 sequence set forth in SEQ ID NO: 52 [37.3] any of the bispecific antibodies of the present invention, which binds to the same epitope on human CD37 as a CD37 antibody comprising a CDR sequence selected from the group comprising: [The present invention 1013] Any of the bispecific antibodies of the invention, wherein the second antigen-binding region binds to a functional epitope comprising one or more amino acids selected from the group consisting of E124, F162, Q163, V164, L165, and H175 of SEQ ID NO: 62 (CD37). [The present invention 1014] the second antigen-binding region (i) the VH CDR1 sequence set forth in SEQ ID NO: 23; the VH CDR2 sequence set forth in SEQ ID NO: 24; VH CDR3 sequence set forth in SEQ ID NO: 25; the VL CDR1 sequence set forth in SEQ ID NO: 27; a VL CDR2 sequence that is YAS, and VL CDR3 sequence set forth in SEQ ID NO: 28

[0016] ; (ii) the VH CDR1 sequence set forth in SEQ ID NO: 2; the VH CDR2 sequence set forth in SEQ ID NO: 3; VH CDR3 sequence set forth in SEQ ID NO: 4, VL CDR1 sequence set forth in SEQ ID NO: 6, a VL CDR2 sequence that is an EAS, and VL CDR3 sequence set forth in SEQ ID NO: 7

[0004] ; (iii) the VH CDR1 sequence set forth in SEQ ID NO: 40; VH CDR2 sequence set forth in SEQ ID NO: 41, VH CDR3 sequence set forth in SEQ ID NO: 42; the VL CDR1 sequence set forth in SEQ ID NO: 44; a VL CDR2 sequence that is FAK, and The VL CDR3 sequence [G28.1] set forth in SEQ ID NO: 45; and (iv) the VH CDR1 sequence set forth in SEQ ID NO: 47; VH CDR2 sequence set forth in SEQ ID NO: 48; VH CDR3 sequence set forth in SEQ ID NO: 49; the VL CDR1 sequence set forth in SEQ ID NO: 51; a VL CDR2 sequence that is VAT, and VL CDR3 sequence set forth in SEQ ID NO: 52 [37.3] any of the bispecific antibodies of the present invention, comprising a CDR sequence selected from the group comprising: [The present invention 1015] the second antigen-binding region (i) the VH sequence set forth in SEQ ID NO: 22 and the VL sequence set forth in SEQ ID NO: 26; or (ii) the VH sequence set forth in SEQ ID NO: 1 and the VL sequence set forth in SEQ ID NO: 5; or (iii) the VH sequence set forth in SEQ ID NO: 39 and the VL sequence set forth in SEQ ID NO: 43; or (iv) the VH sequence set forth in SEQ ID NO: 46 and the VL sequence set forth in SEQ ID NO: 50; or (v) A VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, for example at least 99% identity, and a VL sequence having at least 90% identity, such as at least 95% identity, for example at least 98% identity, for example at least 99% identity, to the VH sequence and VL sequence set forth in any one of (i) to (iv), respectively. any of the aforementioned bispecific antibodies of the invention, comprising a VH and VL sequence selected from the group comprising: [The present invention 1016] Any of the bispecific antibodies of the invention, wherein one or more Fc-Fc interaction-enhancing mutations in the first and second Fc regions are amino acid substitutions. [The present invention 1017] Any of the bispecific antibodies of the invention, wherein the one or more Fc-Fc interaction-enhancing mutations in the first and second Fc regions are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440, and 345 in human IgG1 when using the EU numbering system. [The present invention 1018] Any of the aforementioned bispecific antibodies of the invention, comprising at least one substitution in the first and second Fc regions selected from the group comprising E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. [The present invention 1019] Any of the bispecific antibodies of the invention, comprising at least one substitution in said first and second Fc regions selected from E430G or E345K, preferably E430G. [The present invention 1020] Any of the bispecific antibodies of the present invention, wherein the Fc-Fc interaction-enhancing mutations in the first and second Fc regions are the same substitutions in the first and second Fc regions. [The present invention 1021] Any of the bispecific antibodies of the present invention, which are of the IgG1, IgG2, IgG3 or IgG4 isotype or a combination thereof, preferably of the IgG1 isotype. [The present invention 1022] Any of the bispecific antibodies of the present invention which are full-length antibodies. [The present invention 1023] Any of the bispecific antibodies of the present invention, which are human antibodies, humanized antibodies, chimeric antibodies, or combinations thereof. [The present invention 1024] Any of the bispecific antibodies of the invention which bind to human and cynomolgus CD37 having the sequences set forth in SEQ ID NOs: 62 and 63, respectively. [The present invention 1025] If EU numbering is used, A) the first Fc region comprises an additional mutation corresponding to F405L in human IgG1 and the second Fc region comprises an additional mutation corresponding to K409R in human IgG1; or B) the second Fc region comprises an additional mutation corresponding to F405L in human IgG1, and the first Fc region comprises an additional mutation corresponding to K409R in human IgG1; Any of the bispecific antibodies of the present invention. [The present invention 1026] Any of the bispecific antibodies of the invention which have enhanced CDC, or enhanced CDC and ADCC effector function, compared to the same bispecific molecule without the Fc-Fc interaction enhancing mutation. [The present invention 1027] (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 16, the CDR2 sequence set forth in SEQ ID NO: 17, and the CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 20, a CDR2 sequence that is a KAS, and the CDR3 sequence set forth in SEQ ID NO: 21; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 9, the CDR2 sequence set forth in SEQ ID NO: 10, and the CDR3 sequence set forth in SEQ ID NO: 11; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 113, a CDR2 sequence that is an AAS, and the CDR3 sequence set forth in SEQ ID NO: 14. An anti-CD37 antibody that binds to the same epitope on human CD37 as the anti-CD37 antibody comprising [The present invention 1028] (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 16, the CDR2 sequence set forth in SEQ ID NO: 17, and the CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 20, a CDR2 sequence that is a KAS, and the CDR3 sequence set forth in SEQ ID NO: 21; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 9, the CDR2 sequence set forth in SEQ ID NO: 10, and the CDR3 sequence set forth in SEQ ID NO: 11; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 113, a CDR2 sequence that is an AAS, and the CDR3 sequence set forth in SEQ ID NO: 14. 1027. The anti-CD37 antibody of the present invention, comprising: [The present invention 1029] (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 23, the CDR2 sequence set forth in SEQ ID NO: 24, and the CDR3 sequence set forth in SEQ ID NO: 25, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 27, a CDR2 sequence that is YAS, and the CDR3 sequence set forth in SEQ ID NO: 28; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 2, the CDR2 sequence set forth in SEQ ID NO: 3, and the CDR3 sequence set forth in SEQ ID NO: 4; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 6, a CDR2 sequence that is an EAS, and the CDR3 sequence set forth in SEQ ID NO: 7. An anti-CD37 antibody that binds to human CD37, comprising: [The present invention 1030] Any of the antibodies of the present inventions 1027 to 1029, wherein the antibody comprises an Fc region containing one or more amino acid mutations, and the mutations enhance the Fc-Fc interaction between the antibodies upon target binding compared to the Fc-Fc interaction between antibodies not having the mutations. [The present invention 1031] The antibody of the present invention, wherein the one or more amino acid mutations in the Fc region of said antibody are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440 and 345 in human IgG1 when using the EU numbering system. [The present invention 1032] 1031. The antibody of the invention, comprising at least one amino acid substitution in the Fc region selected from the group comprising E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. [The present invention 1033] 1032. The antibody of the invention, comprising at least one substitution in said Fc region selected from E430G or E345K, preferably E430G. [The present invention 1034] The antibody of any one of 1027 to 1033 of the present invention, which comprises a mutation corresponding to F405L or K409R in human IgG1. [This invention 1035] An anti-CD37 antibody that binds to human CD37, (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 40, the CDR2 sequence set forth in SEQ ID NO: 41, and the CDR3 sequence set forth in SEQ ID NO: 42, and a VL region [G28.1] comprising the CDR1 sequence set forth in SEQ ID NO: 44, a CDR2 sequence which is FAK, and the CDR3 sequence set forth in SEQ ID NO: 45; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 47, the CDR2 sequence set forth in SEQ ID NO: 48, and the CDR3 sequence set forth in SEQ ID NO: 49; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 51, a CDR2 sequence that is VAT, and the CDR3 sequence set forth in SEQ ID NO: 52 [37.3] Includes; (iii) the antibody of (i) or (ii) comprises an Fc region comprising at least one amino acid substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W; (iv) optionally, the Fc region further comprises the mutation K409R or F405L. [The present invention 1036] The antibody of any one of claims 1027 to 1035 of the present invention, which is of the IgG1, IgG2, IgG3, or IgG4 isotype. [This invention 1037] The antibody of any one of claims 1027 to 1036, which is a human antibody, a humanized antibody, or a chimeric antibody. [The present invention 1038] Any of the antibodies 1027 to 1037 of the present invention, which bind to both human and cynomolgus monkey CD37. [This invention 1039] A pharmaceutical composition comprising any one of the bispecific antibodies of the present inventions 1001 to 1026 or any one of the antibodies of the present inventions 1027 to 1038, and a pharmaceutically acceptable carrier. [The present invention 1040] The bispecific antibody of any one of 1001 to 1026, the antibody of any one of 1027 to 1038, or the composition of 1039, for use as a pharmaceutical. [This invention 1041] A bispecific antibody of any of the inventions 1001 to 1026, or any of the inventions 1027 to 1038, or the composition of the invention 1039, for use in treating cancer or an autoimmune disease or an inflammatory disorder. [The present invention 1042] A bispecific antibody of any of claims 1001 to 1026, or an antibody of any of claims 1027 to 1038, or a composition of claim 1039, for use in treating allergy, graft rejection, or a B-cell malignancy, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL). [This invention 1043] A bispecific antibody of any of the inventions 1001 to 1026 or an antibody of any of the inventions 1027 to 1038 or a composition of the invention 1039 for use according to any of the inventions 1040 to 1042 in combination with one or more further therapeutic agents. [This invention 1044] The bispecific antibody of any of inventions 1001 to 1026 or the antibody of any of inventions 1027 to 1038 or the composition of invention 1039, for use in any of inventions 1040 to 1043, wherein said one or more further therapeutic agents are selected from the group comprising doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti-CD20 antibodies, such as rituximab or ofatumumab. [This invention 1045] the additional therapeutic agent is i) the VH CDR1 sequence set forth in SEQ ID NO: 75; VH CDR2 sequence set forth in SEQ ID NO: 76; the VH CDR3 sequence set forth in SEQ ID NO: 77; the VL CDR1 sequence set forth in SEQ ID NO: 79; a VL CDR2 sequence that is a DAS, and VL CDR3 sequence set forth in SEQ ID NO: 80; ii) the VH CDR1 sequence set forth in SEQ ID NO: 82; VH CDR2 sequence set forth in SEQ ID NO: 83; VH CDR3 sequence set forth in SEQ ID NO: 84; the VL CDR1 sequence set forth in SEQ ID NO: 85; a VL CDR2 sequence that is a DAS, and VL CDR3 sequence set forth in SEQ ID NO: 86; iii) the VH CDR1 sequence set forth in SEQ ID NO: 95; VH CDR2 sequence set forth in SEQ ID NO: 96; VH CDR3 sequence set forth in SEQ ID NO: 97; the VL CDR1 sequence set forth in SEQ ID NO: 99; a VL CDR2 sequence that is an ATS, and VL CDR3 sequence set forth in SEQ ID NO: 100; iv) the VH CDR1 sequence set forth in SEQ ID NO: 88; VH CDR2 sequence set forth in SEQ ID NO: 89; VH CDR3 sequence set forth in SEQ ID NO: 90; the VL CDR1 sequence set forth in SEQ ID NO: 92; a VL CDR2 sequence that is a DAS, and The VL CDR3 sequence set forth in SEQ ID NO: 93; and v) the VH CDR1 sequence set forth in SEQ ID NO: 102; the VH CDR2 sequence set forth in SEQ ID NO: 103; the VH CDR3 sequence set forth in SEQ ID NO: 104; the VL CDR1 sequence set forth in SEQ ID NO: 106; a VL CDR2 sequence that is a QMS, and VL CDR3 sequence set forth in SEQ ID NO: 107 The bispecific antibody of the present invention 1043 or 1044, or the antibody of the present invention 1043 or 1044, or the composition of the present invention 1043 or 1044, for use in the present invention 1043 or 1044, which is an anti-CD20 antibody capable of binding to human CD20, comprising a CDR sequence selected from the group consisting of: [The present invention 1046] Use of the bispecific antibody of any of the inventions 1001 to 1026, or any of the antibody of the inventions 1027 to 1038, or the composition of the invention 1039 for the manufacture of a medicine. [This invention 1047] Use of the compound of the present invention 1046 for the manufacture of a medicament for the treatment of cancer, an autoimmune disease, or an inflammatory disease. [This invention 1048] Use of 1047 of the present invention for the manufacture of a medicament for the treatment of allergies, transplant rejection, or B-cell malignancies, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL) or acute lymphoblastic leukemia (ALL). [This invention 1049] The use of any of 1046-1048 in combination with one or more additional therapeutic agents. [The present invention 1050] The use of 1049 of the present invention, wherein said one or more further therapeutic agents are selected from the group comprising doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib and anti-CD20 antibodies, such as rituximab or ofatumumab. [This invention 1051] A method for inducing cell death of tumor cells expressing CD37 or inhibiting the growth and / or proliferation of said cells, the method comprising the step of administering to an individual in need thereof an effective amount of any of the bispecific antibodies of the present inventions 1001 to 1026, any of the antibodies of the present inventions 1027 to 1038, or the composition of the present invention 1039. [This invention 1052] A method for treating an individual having allergy, an autoimmune disease, an inflammatory disease, graft rejection, or a B-cell malignancy, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL), the method comprising the step of administering to the individual an effective amount of the bispecific antibody of any of the bispecific antibodies of the present inventions 1001 to 1025, or any of the antibodies of the present inventions 1027 to 1038, or the composition of the present invention 1039. [This invention 1053] 1053. The method of claim 1051 or 1052, comprising administering one or more additional therapeutic agents in combination with said antibody or said bispecific antibody. [This invention 1054] 1053. The method of claim 1053, wherein said one or more further therapeutic agents are selected from the group comprising doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti-CD20 antibodies, such as rituximab or ofatumumab. [This invention 1055] A nucleic acid construct encoding one or more sequences selected from the group including SEQ ID NOs: 1, 2, 3, 4, 5, 6, 6a, 7, 8, 9, 10, 11, 12, 13, 13a, 14, 15, 16, 17, 18, 19, 20, 20a, 21, 22, 23, 24, 25, 26, 27, 27a, 28, 29, 30, 30a and 31 presented in Table 1. [The present invention 1056] A nucleic acid construct encoding any one of the bispecific antibodies of the present inventions 1001 to 1026 or any one of the antibodies of the present inventions 1027 to 1038. [This invention 1057] An expression vector comprising one or more nucleic acid constructs of the invention 1055 or 1056. [This invention 1058] A host cell containing an expression vector of the present invention. [This invention 1059] A host cell of the invention 1058 that is a recombinant host cell, for example, a recombinant prokaryotic cell, a recombinant eukaryotic cell, or a recombinant microbial host cell. [The present invention 1060] An anti-idiotype antibody that binds to any one of the antibodies 1027 to 1038 of the present invention. [The present invention 1061] 1. An in vitro method for detecting the presence of human CD37 antigen or cells expressing human CD37 in a sample, comprising: (i) contacting the sample with any of the bispecific antibodies of the present inventions 1001 to 1026 or any of the antibodies of the present inventions 1027 to 1038 under conditions that allow the formation of a complex between the antibody or the bispecific antibody and CD37; and (ii) detecting the formation of a complex The method comprising: [The present invention 1062] 1. An in vivo method for detecting the presence of human CD37 antigen or cells expressing human CD37 in a subject, comprising: (i) administering any one of the bispecific antibodies of the present inventions 1001 to 1026 or any one of the antibodies of the present inventions 1027 to 1038 under conditions that allow the formation of a complex between the antibody or the bispecific antibody and CD37; and (ii) detecting the formed complex The method comprising: [Brief explanation of the drawings]

[0022] [Figure 1]CDC in primary CLL tumor cells mediated by the G28.1 variant. The ability of (A) IgG1-G28.1-K409R-delK, IgG1-G28.1-E345R, or IgG1-b12-E345R (cells: patient-derived, newly diagnosed / untreated (PB = peripheral blood-derived)) and (B) IgG1-G28.1, IgG1-G28.1-E430G, or IgG1-b12 (cells: patient-derived, newly diagnosed / untreated (BM = bone marrow-derived)) to induce CDC in primary CLL cells was measured in vitro. Data shown are % lysis rates measured by flow cytometry counting the percentage of dead cells (corresponding to PI-positive cells). [Figure 2] Quantification of CD37, CD46, CD55, and CD59 expression levels on CLL tumor cells. Expression levels of CD37, CD46, CD55, and CD59 on CLL cells from one patient (patient VM-PB0005, newly diagnosed / untreated) were measured by flow cytometry. Antigen amounts are shown as molecules / cell. mIgG1 is the murine IgG1κ isotype control. [Figure 3] Binding of humanized CD37 antibodies and their variants to Daudi cells. Binding of IgG1-004-H5L2, IgG1-004-H5L2-E430G, IgG1-005-H1L2, IgG1-005-H1L2-E430G, IgG1-010-H5L2, IgG1-010-H5L2-E430G, IgG1-016-H5L2, and IgG1-016-H5L2-E430G to Daudi cells was measured by flow cytometry. Data shown are mean fluorescence intensity (MFI) values from one representative experiment. [Figure 4] Binding of G28.1 and 37.3 and their variants to Daudi cells. Binding of IgG1-G28.1, IgG1-G28.1-E430G, IgG1-37.3, and IgG1-37.3-E430G to Daudi cells was measured by flow cytometry. Data shown are mean fluorescence intensity (MFI) values from one representative experiment. [Figure 5]Binding of variants of the humanized CD37 antibody IgG1-016-H5L2 to Daudi cells. Binding of IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G, and IgG1-016-H5L2-LC90S-F405L-E430G to Daudi cells was measured by flow cytometry. Data shown are mean fluorescence intensity (MFI) values from one representative experiment. [Figure 6] Binding of CD37 antibody variants to CHO cells expressing cynomolgus monkey CD37. Binding of IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, IgG1-G28.1, and IgG1-G28.1-E430G was measured by flow cytometry. Data shown are mean fluorescence intensity (MFI) values from one representative experiment. [Figure 7A]Figure 7: Measurement of binding competition between CD37 antibodies and CDC on Raji cells mediated by humanized CD37 antibodies, their variants, and combinations of CD37 antibodies. (A) Binding competition between IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G, and IgG1-016-H5L2-E434G was measured by flow cytometry. Raji cells were incubated with unlabeled antibody for primary binding, followed by incubation with Alexa Fluor 488-labeled probe antibody. Loss of binding of the A488-labeled probe antibody after preincubation with unlabeled antibody compared to binding of the A488-labeled antibody alone indicates binding competition between the A488-labeled antibody and the unlabeled antibody. Data shown are duplicate MESF (number of soluble fluorescent molecules) values from one representative experiment. (B-G) The ability of IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2, IgG1-016-H5L2, and IgG1-37.3 (with or without the E430G mutation) and their combinations to induce CDC in Raji cells was measured in vitro. Data shown are the % lysis rate measured by flow cytometry counting the percentage of dead cells (corresponding to PI-positive cells). [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 7F] See legend to Figure 7A. [Figure 7G] See legend to Figure 7A. [Figure 8]Overview of binding competition between CD37 antibodies. Binding competition between IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G, and IgG1-016-H5L2-E4340G to Raji cells was measured by flow cytometry using unlabeled antibodies for primary binding and an Alexa Fluor 488-labeled probe antibody to detect subsequent binding of the competing antibodies. Color representation: black; simultaneous binding, white; competing for binding, gray; same antibody. [Figure 9] CDC mediated by humanized CD37 antibodies and their variants in Daudi cells. The ability of IgG1-004-H5L2, IgG1-004-H5L2-E430G, IgG1-005-H1L2, IgG1-005-H1L2-E430G, IgG1-010-H5L2, IgG1-010-H5L2-E430G, IgG1-016-H5L2, and IgG1-016-H5L2-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are the % lysis rates measured by flow cytometry to measure the percentage of dead cells (corresponding to PI-positive cells). [Figure 10A]Figure 10: CDC mediated by G28.1 and 37.3 and their variants, and by a humanized CD37 antibody with an Fc-Fc interaction-enhancing mutation, in Daudi cells. (A) The ability of IgG1-G28.1, IgG1-G28.1-E430G, IgG1-37.3, and IgG1-37.3-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are the % lysis rates measured by flow cytometry counting the percentage of dead cells (corresponding to PI-positive cells). (B–C) The ability of (A) IgG1-010-H5L2-K409R-E430G, IgG1-010-H5L2-E345R-K409R, IgG1-010-H5L2-E345K-K409R, IgG1-010-H5L2-K409R-E430S, IgG1-010-H5L2-RRGY, and (B) IgG1-016-H5L2-LC90S-F405L-E430G, IgG1-016-H5L2-E345K-F405L, IgG1-016-H5L2-F405L-E430S, and IgG1-016-H5L2-E345R-F405L to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis (maximum killing at an antibody concentration of 10 μg / mL) measured by flow cytometry counting the percentage of dead cells (corresponding to PI-positive cells) from one representative experiment. Error bars indicate variability within experiments (performed in duplicate). [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 11]CDC mediated by variants of the humanized antibody IgG1-016-H5L2 in Daudi cells. The ability of IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G, and IgG1-016-H5L2-LC90S-F405L-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis determined by flow cytometry counting the percentage of dead cells (corresponding to PI-positive cells). [Figure 12A]Figure 12: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations, (combinations of) CD37 antibodies with Fc-Fc interaction-enhancing mutations, and monovalent CD37-binding antibodies with Fc-Fc interaction-enhancing mutations in Daudi cells; and CDC activity of CD37 antibody variants with Fc-Fc interaction-enhancing mutations and their combinations in OCI-Ly-7 cells. (A) bsIgG1-016-H5L2-LC90S-F405L-E430G×005-H1L2-K409R-E430G, IgG1-005-H1L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-005-H1L2-K409R-E430G and IgG1-016-H5L2-F405L-E430G, bsIgG1-b12-F405L-E430G×005-H1L2-K409R-E430G, and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G, and (B) bsIgG1 The ability of bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G, and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis determined by flow cytometric counting of the percentage of dead cells (corresponding to PI-positive cells).(C) CD37 bispecific antibody bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, CD37 monospecific bivalent (monoclonal) antibodies IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, a combination of IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G, and the monovalent CD37 antibody bsIgG1-016-H5L2-LC90S The ability of bsIgG1-F405L-E430G×b12-K409R-E430G, bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G, and the combination of bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G to induce lysis of OCI-Ly-7 cells in vitro was measured. Data shown are % lysis determined by flow cytometry counting the percentage of dead cells (corresponding to PI-positive cells). (D) EC50 values for CDC induction by bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G, and IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G, measured in two independent experiments. (E) EC50 values for CDC induction by bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, and IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G, determined in three independent experiments. [Figure 12B] See legend to Figure 12A. [Figure 12C] See legend to Figure 12A. [Figure 12D] See legend to Figure 12A. [Figure 12E] See legend to Figure 12A. [Figure 13A]Figure 13: CDC mediated by bispecific CD37 antibodies and bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations in Daudi cells. The ability of (A) bsIgG1-016-H5L2-F405Lx005-H1L2-K409R, and bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, and (B) bsIgG1-016-H5L2-F405Lx010-H5L2-K409R and bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to induce CDC in Daudi cells was measured in vitro. Data shown are % lysis determined by flow cytometric counting of the percentage of dead cells (corresponding to PI-positive cells). [Figure 13B] See legend to Figure 13A. [Figure 14A]Figure 14: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations, (combinations of) CD37 antibodies with Fc-Fc interaction-enhancing mutations, and monovalent CD37 antibodies with Fc interaction-enhancing mutations in primary CLL tumor cells. (A) bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, IgG1-005-H1L2-K409R-E430G, IgG1-016-H5L2-F405L-E430G, IgG1-005-H1L2-K409R-E430G and IgG1-016- (B) bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G, bsIgG1-b12-F405L-E430G×005-H1L2-K409R-E430G, and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G, and (C) bsIgG1-016-H5L2-LC The ability of 90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G, and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G to induce CDC in primary CLL tumor cells (patient: VM-BM0091, newly diagnosed / untreated (BM = bone marrow derived)) was measured in vitro. Data shown are % lysis determined by flow cytometric counting of the percentage of dead cells (corresponding to PI-positive cells). [Figure 14B] See legend to Figure 14A. [Figure 15]CDC mediated by a bispecific CD37 antibody with Fc-Fc interaction-enhancing mutations in B-cell lymphoma cell lines. The ability of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G (at a concentration of 10 μg / mL) to induce CDC in various B-cell lymphoma cell lines was measured in vitro. CD37 expression levels were measured by quantitative flow cytometry and are shown as molecules / cell (mean ± SD of two experiments). White bars indicate susceptibility to CDC mediated by bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G (>10% lysis, average of two experiments), and black bars indicate insusceptibility to CDC mediated by bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G (<10% lysis, average of two experiments). [Figure 16A]Figure 16: ADCC mediated by bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations, (combinations of) CD37 antibodies with Fc-Fc interaction-enhancing mutations, and monovalent binding CD37 antibodies with Fc interaction-enhancing mutations in Daudi and Raji cells. (A) bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, IgG1-005-H1L2-K409R-E430G, IgG1-016-H5L2-F405L-E430G, and IgG1-005-H1L2-K409R-E430G with IgG1-016-H5L2-F405L-E430G. Ability of the combinations to induce ADCC in Daudi cells. (B) bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, and IgG1-010-H5L2-E430G with IgG1-016-H5L2-E (C) Ability of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, IgG1-010-H5L2-E430G and IgG1-016 to induce ADCC in Daudi cells. The ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G, and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G to induce ADCC in Raji cells was measured in vitro using a chromium release assay. Data shown are % specific lysis; error bars indicate intra-assay variability (5 replicates (A, B) or 6 replicates (C) per data point). [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 17A]Figure 17: Quantification of CD37, CD46, CD55, and CD59 expression levels in (A) CLL, (B) FL, (C) MCL, or (D) DLBCL tumor cells. Expression levels in tumor cells were measured by flow cytometry. Antigen amounts are shown as antibody binding capacity. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 17D] See legend to Figure 17A. [Figure 18A] Figure 18: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations in primary tumor cells from CLL, FL, MCL, DLBCL, or B-NHL (not further specified) patients. The ability of bsIgG1-016-H5L2-LC90S-F405L×010-H5L2-K409R-E430G to induce CDC in tumor cells derived from (A) CLL, (B) FL, and (C) MCL, DLBCL, or B-NHL (not further specified) patients was measured by flow cytometry. CDC induction is presented as lysis rate (%) measured by the fraction of 7-AAD-positive tumor cells using bsIgG1-016-H5L2-LC90S-F405L×010-H5L2-K409R-E430G at 100 μg / mL (A and B) or 10 μg / mL (C). [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 19]Binding of bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations to B cells in human or cynomolgus monkey blood. Binding of Alexa-488-labeled bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G to B cells in (A) human or (B) cynomolgus monkey blood was measured by flow cytometry. Alexa-488-labeled IgG1-b12 was used as a negative control antibody. Data are shown as geometric mean A488 fluorescence intensity values from one representative donor / animal. Error bars indicate within-experiment (duplicate measurements). [Figure 20] Cytotoxicity of bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations and monoclonal CD37-specific antibodies with enhanced FcγR interaction against B cells in human or cynomolgus monkey blood. (A) The cytotoxicity of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G and IgG1-CD37-B2-S239D-I332E against B cells in human blood, and (B) the cytotoxicity of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G against B cells in cynomolgus monkey blood was measured in a whole blood cytotoxicity assay. IgG1-b12 was used as a negative control antibody. Data are shown as % B cell depletion for one representative donor / animal, error bars indicate within-experiment variability (duplicate measurements). [Figure 21A] Figure 21: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations, CD20-specific antibodies, or their combination. (A-D) The ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, ofatumumab, or their combination (at the indicated concentrations) to induce CDC in tumor cells from two CLL patients was measured ex vivo. Data are shown as the percentage of surviving B cells. [Figure 21B] See legend to Figure 21A. [Figure 21C] See legend to Figure 21A. [Figure 21D] See legend to Figure 21A. [Figure 22] Dose-effect relationship of three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G in the JVM-3 model. (A) Tumor growth in JVM-3 xenografts after treatment with various doses of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G or an isotype control antibody (IgG1-b12). Mean and SEM values are shown for each group (n=10) at each time point. (B) Tumor size per mouse at day 25. Mean and SEM values are shown for each treatment group. Differences were analyzed by the Mann-Whitney test. Statistically significant differences were indicated as follows: **: p<0.01; ***: p<0.001. [Figure 23] Dose-effect relationship of three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G in the Daudi-luc model. (A) Tumor growth (measured by luciferase activity, bioluminescence) of Daudi-luc xenografts after treatment with various doses of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G or an isotype control antibody (IgG1-b12). Mean and SEM are shown for each group (n=9) at each time point. (B) Luciferase activity per mouse at day 36. Mean and SEM are shown for each treatment group. Differences were analyzed by one-way ANOVA with Fisher's LSD uncorrected. Statistically significant differences were indicated as follows: **: p<0.01; ***: p<0.001. [Figure 24-1]Figure 24: Plasma concentrations of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G and IgG1-b12 after intravenous injection in SCID mice. SCID mice were injected intravenously once with (A-B) 100 μg (5 mg / kg) or (C-D) 500 μg (25 mg / kg) of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G or IgG1-b12. [Figure 24-2] See description of Figure 24-1. [Figure 25] Analysis of binding of CD37 antibodies to CD37 variants with alanine mutations in the extracellular domain. The z-score (fold change) was defined as (normalized gMFI [aa position] - μ) / σ, where μ and σ are the mean and standard deviation (SD) of the normalized gMFI of all variants. Residues with a z-score below -1.5 (indicated by the dotted line) were considered "loss-of-binding mutants." The numbers on the x-axis refer to amino acid positions. Note that the x-axis is discontinuous. The left part of the axis (up to the striped line) represents aa residues in the small extracellular loop of human CD37 that are not alanine or cysteine; the right part of the axis represents aa residues in the large extracellular loop of human CD37 that are not alanine or cysteine. The dotted line indicates a z-score (fold change) of -1.5. [Figure 26A]Figure 26: CDC mediated by a mixture of CD37 antibodies with Fc-Fc interaction enhancing mutations plus a clinically established CD20 antibody product on Raji cells. CDC-mediated Raji cell killing (% lysis expressed as PI-positive cell fraction measured by flow cytometry) for a dilution series (final concentration 10 μg / mL) of 1:0, 3:1, 1:1, 3:1, and 0:1 antibody mixtures of CD37 antibodies with Fc-Fc interaction-enhancing mutations plus standard-of-care CD20 antibody products MabThera (rituximab), Arzerra (ofatumumab), and Gazyva (obinutuzumab, GA101): (A) with IgG1-37.3-E430G, (B) with IgG1-G28.1-E430G, (C) with IgG1-004-E430G, (D) with IgG1-005-E430G, (E) with IgG1-010-E430G, and (F) with IgG1-016-E430G. [Figure 26B] See legend to Figure 26A. [Figure 26C] See legend to Figure 26A. [Figure 26D] See legend to Figure 26A. [Figure 26E] See legend to Figure 26A. [Figure 26F] See legend to Figure 26A. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention definition As used herein, the term "CD37" refers to the leukocyte antigen CD37, also known as GP52-40, tetraspanin-26, and TSPAN26, which is a heavily glycosylated transmembrane protein with four transmembrane domains (TM), one small extracellular domain, and one large extracellular domain. Homo sapiens, or human, CD37 protein is encoded by a nucleic acid sequence that encodes the amino acid sequence set forth in SEQ ID NO: 62 (human CD37 protein: UniprotKB / Swissprot P11049). In this amino acid sequence, residues 112-241 correspond to the large extracellular domain, residues 39-59 correspond to the small extracellular domain, and the remaining residues correspond to the transmembrane and cytoplasmic domains. Macaca fascicularis, or cynomolgus monkey, CD37 protein is encoded by a nucleic acid sequence that encodes the amino acid sequence set forth in SEQ ID NO: 63 (cynomolgus monkey CD37 protein; Genbank Accession No. XP_005589942). Unless contradicted by context, the term "CD37" means "human CD37." The term "CD37" includes any variants, isoforms, and orthologues of CD37 that are naturally expressed by cells, including tumor cells, or that are expressed on cells transfected with the CD37 gene or cDNA.

[0024] The term "human CD20" or "CD20" refers to human CD20 (UniProtKB / Swiss-Prot No. P11836) and includes any variants, isoforms, and orthologues of CD20 that are naturally expressed by cells, including tumor cells, or expressed on cells transfected with the CD20 gene or cDNA. Orthologues include rhesus monkey CD20 (macaca mulatta; UniProtKB / Swiss-Prot No. H9YXP1) and cynomolgus monkey CD20 (Macaca fascicularis).

[0025] The terms "antibody that binds to CD37," "anti-CD37 antibody," "CD37-binding antibody," "CD37-specific antibody," and "CD37 antibody," which may be used interchangeably herein, refer to any antibody that binds to an epitope on the extracellular portion of CD37.

[0026] The term "antibody (Ab)" in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, that has the ability to specifically bind to an antigen under typical physiological conditions for a significant half-life, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3 days, 4 days, 5 days, 6 days, 7 days, or more, or any other relevant functionally determined period (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody binding to the antigen and / or a period sufficient for the antibody to mobilize effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domains that interact with the antigen. The constant region of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q (the first component in the classical pathway of complement activation). As indicated above, the term "antibody" as used herein includes antigen-binding fragments, i.e., fragments of antibodies that retain the ability to specifically bind to antigens, unless otherwise stated or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed by the term "antibody" include: (i) V L , V H , C L , and C H (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a monovalent antibody comprising essentially a VH and C H (iv) a Fd fragment consisting of only one domain; (iv) a V fragment consisting essentially of a single arm of an antibody L and V H (v) an Fv fragment consisting essentially of the V domain; H They consist of only domains and are also called domain antibodies (Holt et al. Trends Biotechnol. 2003 Nov; 21 (11):484-90) dAb fragments (Ward et al., Nature 341 , 544-546 (1989));(vi) Camelidae or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24) and (vii) isolated complementarity-determining regions (CDRs). Furthermore, the two domains V of the Fv fragment L and V H are encoded by separate genes, they can be synthesized using recombinant methods to produce a single protein chain (V L and V H The domains may be joined by synthetic linkers that allow them to be made as single-chain antibodies (known as single-chain Fvs (scFvs) - see, e.g., Bird et al., Science 242 , 423-426 (1988) and Huston et al., PNAS USA 85, pp. 5879-5883 (1988)). Such single-chain antibodies are encompassed by the term "antibody" unless otherwise specified or clearly indicated by the context. While such fragments are generally included within the meaning of antibody, they collectively, and each individually, are unique features of the present invention and exhibit various biological properties and utilities. These and other useful antibody fragments and bispecific formats of such fragments are further described herein. In the case of the bispecific antibodies of the present invention, such fragments are linked to the Fc domain. The term "antibody" should also be understood to include, unless otherwise specified, polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen, provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant technology. The antibodies generated can have any isotype.

[0027] The term "bispecific antibody" refers to an antibody having specificity for at least two different, generally non-overlapping, epitopes. Such epitopes may be on the same target or on different targets. In the context of the present invention, the epitopes are on the same target (i.e., CD37). Examples of various classes of bispecific antibodies that contain Fc regions include, but are not limited to, asymmetric bispecific molecules, e.g., IgG-like molecules with complementary CH3 domains; and symmetric bispecific molecules, e.g., recombinant IgG-like dual targeting molecules, in which each antigen-binding region of the molecule binds to at least two different epitopes.

[0028] Examples of bispecific molecules include Triomab® (Trion Pharma / Fresenius Biotech, WO / 2002 / 020039), Knobs-into-Holes (Genentech, WO1998 / 50431), CrossMAbs (Roche, WO2009 / 080251, WO2009 / 080252, WO2009 / 080253), electrostatically matched Fc heterodimer molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133;Oncomed, WO2010 / 129304), LUZ-Y (Genentech), DIG-body, PIG-body and TIG-body (Pharmabcine), Strand Exchange Engineered Domain body (SEEDbody) (EMD Serono, WO2007110205), bispecific IgG1 and IgG2 (Pfizer / Rinat, WO2011 / 143545), Azymetric scaffolds (Zymeworks / Merck, WO2012058768), mAb-Fv (Xencor, WO2011 / 028952), XmAb (Xencor), bivalent bispecific antibodies (Roche, WO2009 / 080254), bispecific IgG (Eli Lilly), DuoBody® molecules (Genmab A / S, WO2011 / 131746), DuetMab (Medimmune, US2014 / 0348839), Biclonics (Merus, WO2013 / 157953), NovImmune (κλBodies, WO2012 / 023053), FcΔAdp (Regeneron, WO2010 / 151792), (DT)-Ig (GSK / Domantis), Two-in-one Antibody or Dual Action Fabs (Genentech, Adimab), mAb2 (F-Star, WO2008 / 003116), Zybody™ molecules (Zyngenia), CovX-body (CovX / Pfizer), FynomAbs (Covagen / Janssen) Cilag), DutaMab (Dutalys / Roche), iMab (MedImmune), Dual Variable Domain (DVD)-Ig™ (Abbott), Dual Domain Double-Head Antibody (Unilever;Sanofi Aventis, WO2010 / 0226923), Ts2Ab (MedImmune / AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec, US7,951,918), scFv fusion (Genentech / Roche, Novartis, Immunomedics, Changzhou Adam Biotech Inc, CN102250246), TvAb (Roche, WO2012 / 025525, WO2012 / 025530), ScFv / Fc fusion, SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), Interceptor (Emergent), Dual Affinity Retargeting Technology (Fc-DART™) (MacroGenics, WO2008 / 157379, WO2010 / 080538), BEAT (Glenmark), Di-Diabody (Imclone / Eli Lilly), and chemically cross-linked mAbs (Karmanos Cancer Center) and covalently fused mAbs (AIMM therapeutics), but are not limited to these.

[0029] As used herein, the term "full-length antibody" refers to an antibody (e.g., a parent or variant antibody) that contains all heavy and light chain constant and variable domains corresponding to those normally found in wild-type antibodies of that class or isotype.

[0030] As used herein, the term "chimeric antibody" refers to an antibody whose variable region is derived from a non-human species (e.g., rodent) and whose constant region is derived from a different species (e.g., human). Chimeric antibodies can be produced by antibody engineering. "Antibody engineering" is a general term used for various types of antibody modification, a process well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques, such as those described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, chimeric antibodies can be genetically or enzymatically modified recombinant antibodies. Producing chimeric antibodies is within the knowledge of those skilled in the art, and therefore, production of the chimeric antibodies of the present invention may be performed by methods other than those described herein. Chimeric monoclonal antibodies have been developed for therapeutic use to reduce the immunogenicity of antibodies. Such antibodies generally comprise a non-human (e.g., murine) variable region specific for an antigen of interest and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" as used in reference to a chimeric antibody refers to the region comprising the CDR and framework regions of both the heavy and light immunoglobulin chains.

[0031] As used herein, the term "oligomer" refers to a molecule consisting of more than one, but a limited number of, monomer units (e.g., antibodies), as opposed to a polymer, which, at least in principle, consists of an infinite number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Similarly, as used herein, "oligomerization," e.g., "hexamerization," refers to the increased distribution of antibodies and / or other dimeric proteins comprising the target binding region of the present invention into oligomers, e.g., hexamers. The increased formation of oligomers, such as hexamers, is due to increased Fc-Fc interactions after binding to membrane-bound targets.

[0032] As used herein, the terms "antigen-binding region," "binding region," or "antigen-binding domain" refer to the region of an antibody capable of binding to an antigen. This binding region is generally defined by the VH and VL domains of an antibody, which can be further subdivided into regions of hypervariability (or hypervariable regions that may be hypervariable in the sequence and / or shape of structurally defined loops), also called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). An antigen can be any molecule, e.g., a polypeptide, present, for example, on cells, bacteria, or virions, or in solution. The terms "antigen" and "target" can be used interchangeably in the context of the present invention, unless the context indicates otherwise.

[0033] As used herein, the term "target" refers to a molecule to which the antigen-binding region of an antibody binds. Targets include any antigen against which an antibody is raised. The terms "antigen" and "target" are used interchangeably with respect to antibodies and may have the same meaning and intent with respect to any aspect or embodiment of the present invention.

[0034] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs) that together form the antigen-binding site into homologous human acceptor framework regions (FRs) (see WO92 / 22653 and EP0629240). To fully reconstitute the binding affinity and specificity of the parent antibody, it may be necessary to substitute framework residues from the parent antibody (i.e., non-human antibody) into human framework regions (backmutations). Structural homology modeling can assist in identifying amino acid residues in the framework regions that are important for antibody binding. Thus, a humanized antibody can contain primarily human framework regions containing non-human CDR sequences, optionally with one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, further amino acid modifications, not necessarily back mutations, may be applied to obtain humanized antibodies with preferred properties, such as affinity and biochemical properties.

[0035] Humanized antibodies can be produced by immunizing rabbits, humanizing the rabbit antibodies using germline humanization (CDR grafting) techniques, and, if necessary, backmutating residues that may be critical for antibody binding (identified in structural modeling) to rabbit residues. Screening for potential T-cell epitopes can be applied.

[0036] As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human monoclonal antibodies of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256: 495 (1975). While somatic cell hybridization procedures are preferred, other techniques for producing monoclonal antibodies, such as viral or oncogenic transformation of B lymphocytes or phage display techniques using libraries of human antibody genes, can also be employed in principle.

[0037] A suitable animal system for preparing hybridomas secreting human monoclonal antibodies is the murine system. Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolation of immunized spleen cells for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0038] Human monoclonal antibodies can be generated, for example, using transgenic or transchromosomal mice or rabbits carrying parts of the human immune system rather than the mouse or rabbit system.

[0039] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, a pair of low molecular weight light (L) chains and a pair of heavy (H) chains, with all four chains interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain generally contains a heavy chain variable region (herein referred to as V H or VH) and a heavy chain constant region (herein referred to as C H The heavy chain constant region is generally composed of three domains: C H 1. C H 2 and C H Each light chain is generally composed of a light chain variable region (referred to herein as V L or VL) and a light chain constant region (herein referred to as C L The light chain constant region generally consists of one domain, C L It is composed of: V H and V L The regions can be further subdivided into regions of hypervariability (or hypervariability that may be hypervariable in the sequence and / or configuration of structurally defined loops), also called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is generally composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise stated or contradicted by context, CDR sequences herein are identified according to the IMGT rules (Brochet X., Nucl Acids Res. 2008;36:W503-508 and Lefranc MP., Nucleic Acids Research 1999;27:209-212; also see internet http address http: / / www.imgt.org / ). Unless otherwise stated or contradicted by context, references to amino acid positions in the constant region herein are in accordance with EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242).

[0040] Unless otherwise indicated, the term "Fab arm" or "arm" as used herein refers to a single heavy-light chain pair and is used interchangeably with "half molecule" herein. Thus, a "Fab arm" includes the variable regions of the heavy and light chains, as well as the constant region of the light chain and the constant region of the heavy chain, which includes the CH1, hinge, CH2, and CH3 regions of an immunoglobulin. The "CH1 region" refers, for example, to the region corresponding to amino acids 118-215 of a human IgG1 antibody according to EU numbering. Thus, a Fab fragment includes the binding region of an immunoglobulin.

[0041] The terms "fragment crystallizable region," "Fc region," "Fc fragment," or "Fc domain," which may be used interchangeably herein, refer to an antibody region comprising at least the hinge region, CH2 domain, and CH3 domain arranged from the amino terminus to the carboxy terminus. The Fc region of an IgG1 antibody can be generated, for example, by digesting the IgG1 antibody with papain. The Fc region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q (the first component in the classical pathway of complement activation). The term "hinge region," as used herein, is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216 to 230 according to EU numbering.

[0042] As used herein, the term "core hinge" or "core hinge region" refers to the four amino acids corresponding to positions 226 to 229 of a human IgG1 antibody.

[0043] As used herein, the term "CH2 region" or "CH2 domain" is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region can also be of any of the other isotypes or allotypes described herein.

[0044] As used herein, the term "CH3 region" or "CH3 domain" is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region can also be of any of the other isotypes or allotypes described herein.

[0045] As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by heavy chain constant region genes.

[0046] In the context of the present invention, the term "monovalent antibody" means that the antibody molecule can only bind to a single antigen molecule and is therefore incapable of antigen cross-linking.

[0047] A "CD37 antibody" or "anti-CD37 antibody" is an antibody as described above that specifically binds to the antigen CD37.

[0048] A "CD37xCD37 antibody" or "anti-CD37xCD37 antibody" is a bispecific antibody that contains two different antigen-binding regions, one that specifically binds to a first epitope on the antigen CD37 and the other that specifically binds to a different epitope on CD37.

[0049] In one embodiment, a bispecific antibody of the invention is isolated. As used herein, an "isolated bispecific antibody" is intended to refer to a bispecific antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds to CD37 is substantially free of monospecific antibodies that specifically bind to CD37).

[0050] The term "epitope" refers to a protein determinant capable of binding to the antigen-binding region ("paratope") of an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural features and specific charge characteristics. Conformational epitopes are distinguished from nonconformational epitopes in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents. "Structural epitopes" or "functional epitopes" can be determined by epitope mapping techniques. A structural epitope is defined as residues in the structure that are in direct contact with the antibody and can be evaluated, for example, by structure-based methods such as X-ray crystallography. A structural epitope can include amino acid residues directly involved in antibody binding as well as other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked or hidden by the antibody (in other words, those amino acid residues within the antibody footprint). A functional epitope is defined as a residue that contributes energetically to the antigen-antibody binding interaction and can be evaluated, for example, by site-directed mutagenesis such as alanine scanning (Cunningham, B. C, & Wells, JA (1993) Journal of Molecular Biology; Clackson, T., & Wells, J. (1995) Science, 267(5196), 383-386). A functional epitope can include amino acid residues that are directly involved in antibody binding, as well as other amino acid residues that are not directly involved in binding, such as amino acid residues that cause conformational changes at the positions of residues involved in direct interactions (Greenspan, NS, & Di Cera, E. (1999) Nature Biotechnology, 17(10), 936-937). In the case of antigen-antibody interactions, functional epitopes can be used to distinguish antibody molecules from each other. Functional epitopes can be determined using the alanine scanning method described in Example 17.Thus, amino acids in a protein can be substituted with alanine to generate a series of mutant proteins, which reduce binding of the antigen-binding region of an antibody to the mutant proteins compared to the wild-type protein; reduced binding is determined as a normalized log(fold change) in binding of the antibody (expressed as a z-score) of less than -1.5, as described in Example 17.

[0051] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules of essentially uniform molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas, which comprise B cells obtained from a transgenic or transchromosomal non-human animal, e.g., a transgenic mouse, whose genome contains human heavy chain and light chain transgenes, fused to an immortalized cell.

[0052] As used herein, the term "binding" with respect to the binding of an antibody to a given antigen generally refers to a binding of about 10, as determined by BioLayer Interferometry (BLI) technology on an Octet HTX instrument using, for example, the antibody as the ligand and the antigen as the analyte. -6 M or less, for example, about 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 K below M D and antibodies bind with an affinity equivalent to the K for binding to nonspecific antigens other than the designated antigen or closely related antigens (e.g., BSA, casein). D a K that is at least 10 times lower, such as at least 100 times lower, such as at least 1,000 times lower, such as at least 10,000 times lower, such as at least 100,000 times lowerD binds to a given antigen with an affinity equivalent to the K D How low is the K D and therefore the K D If the K of binding to the antigen is very low, D is the K for nonspecific antigen binding D The antibody may be at least 10,000 times less specific than the antibody itself (i.e., the antibody is highly specific).

[0053] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0054] As used herein, the terms "affinity" and "K D " is inversely correlated with K D A lower affinity is intended to refer to a higher K D is intended to refer to.

[0055] As used herein, an antibody that "competes" or "cross-competes" with another antibody (i.e., a reference antibody) is used interchangeably with an antibody that "blocks" or "cross-blocks," and means that the antibody and the reference antibody compete for binding to human CD37, e.g., as determined in the assay described herein in Example 7. In one embodiment, the antibody binds at less than 50%, e.g., less than 20%, e.g., less than 15%, of its maximal binding in the presence of the competing reference antibody.

[0056] As used herein, an antibody that "does not compete" or "does not cross-compete" or "does not block" another antibody (i.e., a reference antibody) means that the antibody and the reference antibody do not compete for binding to human CD37, as determined in the assay described in Example 7 herein. For some pairs of antibodies and reference antibodies, non-competition in the assay of Example 7 is observed only when one antibody binds to an antigen on a cell and the other antibody is used for competition, but not vice versa. The terms "does not compete with" or "non-competing" or "non-blocking" as used herein are also intended to encompass such combinations of antibodies. In one embodiment, the antibody binds at least 75%, e.g., at least 80%, e.g., at least 85% of its maximum binding in the presence of the reference antibody.

[0057] As used herein, the term "Fc-Fc interaction enhancing mutation" refers to a mutation in an IgG antibody that strengthens the Fc-Fc interaction between adjacent IgG antibodies that bind to a cell surface target. This can result in enhanced oligomerization (e.g., hexamerization) of the antibody upon binding to a target, although the antibody molecule remains a monomer in solution, as described in WO2013 / 004842; WO2014 / 108198, both of which are incorporated herein by reference.

[0058] As used herein, the term "Fc effector function" or "Fc-mediated effector function" is intended to refer to a function that results from the binding of a polypeptide or antibody to its target (e.g., an antigen) on a cell membrane and the subsequent interaction of the IgG Fc domain with molecules of the innate immune system (e.g., soluble or membrane-bound molecules). Examples of Fc effector functions include: (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cellular cytotoxicity (ADCC), (v) Fc-γ receptor binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) antibody-mediated opsonization (binding of an antibody to a complement receptor), (x) opsonization, and (xi) any combination of (i)-(x).

[0059] As used herein, the term "heterodimeric interaction between the first and second CH3 regions" refers to the interaction between the first CH3 region of a first Fc domain and the second CH3 region of a second Fc domain in a first CH3 / second CH3 heterodimeric protein. Bispecific antibodies are an example of heterodimeric proteins.

[0060] As used herein, the term "homodimeric interaction of a first and second CH3 region" refers to the interaction between a first CH3 region and another first CH3 region in a first CH3 / first CH3 homodimeric protein, and the interaction between a second CH3 region and another second CH3 region in a second CH3 / second CH3 homodimeric protein. Monoclonal antibodies are an example of homodimeric proteins.

[0061] The term "reducing conditions" or "reducing environment" refers to conditions or circumstances in which a substrate, for example, a cysteine residue in the hinge region of an antibody, is more likely to be reduced than oxidized.

[0062] The present invention also relates to the V of the bispecific antibodies of the examples. L area, V HThe present invention provides bispecific antibodies comprising functional variants of one or more CDRs of the V domain. L , V H or functional variants of the CDRs still allow each arm of the bispecific antibody to retain at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or more) of the affinity and / or specificity / selectivity of the parent bispecific antibody, and in some cases, such bispecific antibodies may be associated with higher affinity, selectivity, and / or specificity than the parent bispecific antibody. Such functional variants generally retain significant sequence identity with the parent bispecific antibody. The percent identity between two sequences is a function of the number of identical positions shared by both sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., percent homology = number of identical positions / total number of positions × 100). The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988), incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences may be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0063] Exemplary variants include those that differ from the VH and / or VL and / or CDR regions of the parent bispecific antibody sequence primarily by conservative substitutions. For example, 10, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the substitutions in the variant are conservative amino acid residue substitutions. Preferably, the variant contains at most 10 amino acid substitutions, e.g., at most 9, 8, 7, 6, 5, 4, 3, 2, or at most 1 amino acid substitution, in the VH and / or VL regions of the parent antibody. Preferably, such substitutions are conservative substitutions, especially when they occur in the CDR sequences.

[0064] In the context of the present invention, conservative substitutions may be defined by substitutions within the amino acid classes shown in the table below. Conservatively substituted amino acid residue classes TIFF2025121994000001.tif52136

[0065] In the context of the present invention, unless otherwise indicated, the following notation is used to describe mutations: i) substitution of an amino acid at a given position is noted, for example, K409R (meaning substitution of lysine at position 409 with arginine); ii) in the case of a particular variant, a designated three- or one-letter code is used to indicate any amino acid residue, for example, the codes Xaa and X. For example, substitution of lysine at position 409 with arginine is designated K409R; substitution of lysine at position 409 with any amino acid residue is designated K409X. In the case of deletion of lysine at position 409, it is designated by K409*.

[0066] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which an expression vector has been introduced, such as an expression vector encoding an antibody of the invention. Recombinant host cells include, for example, transfectomas, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NS0 cells, and lymphocytic cells.

[0067] The term "treatment" refers to the administration of an effective amount of a therapeutically active bispecific antibody of the invention with the intent to alleviate, ameliorate, suppress, or eradicate (cure) the symptoms or disease state.

[0068] The term "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic result, at dosages and for periods of time necessary. A therapeutically effective amount of a bispecific antibody may vary according to factors such as the disease state, the age, sex, and weight of the individual, and the ability of the bispecific antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0069] The term "anti-idiotypic antibody" refers to an antibody that recognizes unique determinants generally associated with the antigen-binding site of an antibody.

[0070] Aspects of the invention In a first major aspect, the present invention provides a bispecific antibody comprising first and second antigen-binding regions that bind to human CD37 having the sequence of SEQ ID NO: 62 and first and second Fc regions of a human immunoglobulin, wherein the first and second antigen-binding regions bind to different epitopes on CD37, and the first and second Fc regions contain one or more amino acid mutations that enhance the Fc-Fc interaction between the bispecific antibody upon binding to a membrane-bound target compared to the Fc-Fc interaction between the bispecific antibody without the mutations. Also provided herein are bispecific anti-CD37 antibodies that bind to two different epitopes on CD37. Preferably, the two epitopes are such that both binding arms can bind to the same protein, and thus each binding arm does not block binding of the other arm of the bispecific molecule and / or does not compete for binding with the other binding arm of the bispecific molecule. The bispecific antibody also contains mutations that enhance the Fc-Fc interaction between two or more bispecific antibodies of the present invention. This has the effect of causing the bispecific molecule to form oligomers when bound to CD37 expressed on the plasma membrane of target cells. Fc-Fc interaction is enhanced compared to an otherwise identical molecule. Preferably, the mutation is in the Fc region of the bispecific molecule. In one embodiment, it is a single amino acid substitution in the Fc region of the bispecific molecule. It is preferably a symmetric substitution, meaning that both half-molecules (parent antibodies) have the mutation. A further advantage of this bispecific antibody is that it has enhanced CDC and / or ADCC effector function compared to an identical bispecific molecule without the Fc-Fc interaction-enhancing mutation. Surprisingly, the bispecific molecule also has improved CDC and / or ADCC compared to the combination of two parent monoclonal anti-CD37 antibodies mutated to have enhanced Fc-Fc interaction, and also compared to either parent monoclonal anti-CD37 antibody alone mutated to have enhanced Fc-Fc interaction.Thus, the bispecific antibodies of the present invention are more potent in inducing CDC and / or ADCC than a combination of an antibody having a first antigen-binding region and a second antibody having a second antigen-binding region (both antibodies containing Fc-Fc interaction-enhancing mutations), or than a single monoclonal anti-CD37 antibody having either the first or second antigen-binding region and containing Fc-Fc interaction-enhancing mutations.

[0071] In one embodiment of the invention, the first antigen-binding region of the bispecific antibody comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 16, the VH CDR2 sequence set forth in SEQ ID NO: 17; VH CDR3 sequence set forth in SEQ ID NO: 18; the VL CDR1 sequence set forth in SEQ ID NO: 20; a VL CDR2 sequence that is a KAS, and VL CDR3 sequence set forth in SEQ ID NO: 21 The antibody is derived from an antibody that competes with a CD37 antibody comprising the compound of formula (I) for binding to human CD37.

[0072] Preferably, competition for binding is determined according to Example 7.

[0073] In another embodiment, the first antigen-binding region of the bispecific antibody comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 16, the VH CDR2 sequence set forth in SEQ ID NO: 17; VH CDR3 sequence set forth in SEQ ID NO: 18; the VL CDR1 sequence set forth in SEQ ID NO: 20; a VL CDR2 sequence that is a KAS, and VL CDR3 sequence set forth in SEQ ID NO: 21 It binds to the same epitope on human CD37 as the CD37 antibody comprising

[0074] In a further embodiment of the invention, the first antigen-binding region of the bispecific antibody comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 16, the VH CDR2 sequence set forth in SEQ ID NO: 17; VH CDR3 sequence set forth in SEQ ID NO: 18; the VL CDR1 sequence set forth in SEQ ID NO: 20; a VL CDR2 sequence that is a KAS, and VL CDR3 sequence set forth in SEQ ID NO: 21 Includes.

[0075] In a further embodiment of the invention, the first antigen-binding region of the bispecific antibody of the invention comprises the VH and VL sequences: (i) the VH sequence set forth in SEQ ID NO: 15 and the VL sequence set forth in SEQ ID NO: 19, or (ii) a VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, for example at least 99% identity, and a VL sequence having at least 90% identity, such as at least 95% identity, for example at least 98% identity, for example at least 99% identity, to the VH and VL sequences of SEQ ID NOs: 15 and 19. Includes.

[0076] In a further embodiment of the invention, the first antigen-binding region of the bispecific antibody comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 9, VH CDR2 sequence set forth in SEQ ID NO: 10, VH CDR3 sequence set forth in SEQ ID NO: 11, the VL CDR1 sequence set forth in SEQ ID NO: 13; A VL CDR2 sequence that is an AAS, and VL CDR3 sequence set forth in SEQ ID NO: 14 The antibody is derived from an antibody that competes with a CD37 antibody comprising the compound of formula (I) for binding to human CD37.

[0077] In a further embodiment of the invention, the first antigen-binding region of the bispecific antibody comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 9, VH CDR2 sequence set forth in SEQ ID NO: 10, VH CDR3 sequence set forth in SEQ ID NO: 11, the VL CDR1 sequence set forth in SEQ ID NO: 13; A VL CDR2 sequence that is an AAS, and VL CDR3 sequence set forth in SEQ ID NO: 14 It binds to the same epitope on human CD37 as the CD37 antibody comprising

[0078] In one embodiment of the invention, the first antigen-binding region of the bispecific antibody has a functional epitope comprising one or more of amino acids Y182, D189, T191, 1192, D194, K195, V196, 1197, and P199 of SEQ ID NO: 62 (CD37).

[0079] In one embodiment of the present invention, the first antigen-binding region binds to a functional epitope comprising one or more amino acids selected from the group consisting of Y182, D189, T191, I192, D194, K195, V196, I197, and P199 of SEQ ID NO: 62 (CD37).

[0080] In one embodiment of the invention, the first antigen-binding region of the bispecific antibody binds to a functional epitope on CD37, and binding to a mutant CD37 in which any one or more of the amino acid residues at positions corresponding to positions Y182, D189, T191, 1192, D194, K195, V196, 1197, and P199 of SEQ ID NO: 62 (CD37) have been substituted with alanine is reduced compared to wild-type CD37 having the amino acid sequence set forth in SEQ ID NO: 62, wherein the reduced binding is determined as a z-score (fold change) in binding of the antibody being less than −1.5, wherein the z-score (fold change) in binding is calculated as described in Example 17.

[0081] In a further embodiment of the invention, the first antigen-binding region of the bispecific antibody comprises the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 9, VH CDR2 sequence set forth in SEQ ID NO: 10, VH CDR3 sequence set forth in SEQ ID NO: 11, the VL CDR1 sequence set forth in SEQ ID NO: 13; A VL CDR2 sequence that is an AAS, and VL CDR3 sequence set forth in SEQ ID NO: 14 Includes.

[0082] In a further embodiment of the invention, the first antigen-binding region of the bispecific antibody comprises the VH and VL sequences: (i) the VH sequence set forth in SEQ ID NO: 8 and the VL sequence set forth in SEQ ID NO: 12, or (ii) a VH sequence having at least 90% identity, such as at least 95% identity, such as at least 98% identity, for example at least 99% identity, and a VL sequence having at least 90% identity, such as at least 95% identity, for example at least 98% identity, for example at least 99% identity, to the VH and VL sequences of SEQ ID NOs: 8 and 12. Includes.

[0083] In a further embodiment of the invention, the second antigen-binding region of the bispecific antibody comprises (i) the VH CDR1 sequence set forth in SEQ ID NO: 23; the VH CDR2 sequence set forth in SEQ ID NO: 24; VH CDR3 sequence set forth in SEQ ID NO: 25; the VL CDR1 sequence set forth in SEQ ID NO: 27; a VL CDR2 sequence that is YAS, and VL CDR3 sequence set forth in SEQ ID NO: 28

[0016] ; (ii) the VH CDR1 sequence set forth in SEQ ID NO: 2; the VH CDR2 sequence set forth in SEQ ID NO: 3; VH CDR3 sequence set forth in SEQ ID NO: 4, VL CDR1 sequence set forth in SEQ ID NO: 6, a VL CDR2 sequence that is an EAS, and VL CDR3 sequence set forth in SEQ ID NO: 7

[0004] ; (iii) the VH CDR1 sequence set forth in SEQ ID NO: 40; VH CDR2 sequence set forth in SEQ ID NO: 41, VH CDR3 sequence set forth in SEQ ID NO: 42; the VL CDR1 sequence set forth in SEQ ID NO: 44; a VL CDR2 sequence that is FAK, and The VL CDR3 sequence [G28.1] set forth in SEQ ID NO: 45; and (iv) the VH CDR1 sequence set forth in SEQ ID NO: 47; VH CDR2 sequence set forth in SEQ ID NO: 48; VH CDR3 sequence set forth in SEQ ID NO: 49; the VL CDR1 sequence set forth in SEQ ID NO: 51; a VL CDR2 sequence that is VAT, and VL CDR3 sequence set forth in SEQ ID NO: 52 [37.3] The antibody is obtained from an antibody that competes for binding to human CD37 with a CD37 antibody comprising a CDR sequence selected from the group comprising:

[0084] In a further embodiment of the invention, the second antigen-binding region of the bispecific antibody comprises (i) the VH CDR1 sequence set forth in SEQ ID NO: 23; the VH CDR2 sequence set forth in SEQ ID NO: 24; VH CDR3 sequence set forth in SEQ ID NO: 25; the VL CDR1 sequence set forth in SEQ ID NO: 27; a VL CDR2 sequence that is YAS, and VL CDR3 sequence set forth in SEQ ID NO: 28

[0016] ; (ii) the VH CDR1 sequence set forth in SEQ ID NO: 2; the VH CDR2 sequence set forth in SEQ ID NO: 3; VH CDR3 sequence set forth in SEQ ID NO: 4, VL CDR1 sequence set forth in SEQ ID NO: 6, a VL CDR2 sequence that is an EAS, and VL CDR3 sequence set forth in SEQ ID NO: 7

[0004] ; (iii) the VH CDR1 sequence set forth in SEQ ID NO: 40; VH CDR2 sequence set forth in SEQ ID NO: 41, VH CDR3 sequence set forth in SEQ ID NO: 42; the VL CDR1 sequence set forth in SEQ ID NO: 44; a VL CDR2 sequence that is FAK, and The VL CDR3 sequence [G28.1] set forth in SEQ ID NO: 45; and (iv) the VH CDR1 sequence set forth in SEQ ID NO: 47; VH CDR2 sequence set forth in SEQ ID NO: 48; VH CDR3 sequence set forth in SEQ ID NO: 49; the VL CDR1 sequence set forth in SEQ ID NO: 51; a VL CDR2 sequence that is VAT, and VL CDR3 sequence set forth in SEQ ID NO: 52 [37.3] The antibody is obtained from an antibody that competes for binding to human CD37 with a CD37 antibody comprising a CDR sequence selected from the group consisting of:

[0085] Provided herein are bispecific antibodies in which the first and second antigen-binding regions bind to different epitopes on human CD37. The inventors have found that antibodies having the CDR sequences of antibody 005 (SEQ ID NOs: 9, 10, 11, and 13, 13a, 14) and antibodies having the CDR sequences of antibody 010 (SEQ ID NOs: 16, 17, 18, and 20, 20a, 21) compete for binding to human CD37, and that antibodies having the CDR sequences of antibody 016 (SEQ ID NOs: 23, 24, 25, and 27, 27a, 28), antibodies having the CDR sequences of antibody 004 (SEQ ID NOs: 2, 3, 4, and 6, 6a, 7), antibodies having the CDR sequences of antibody G28.1 (SEQ ID NOs: 40, 41, 42, and 44, 44a, 45), and antibodies having the CDR sequences of antibody 37.3 (SEQ ID NOs: We found that the 016, 004, G28.1, and 37.3 antibodies did not compete with any of the antibodies with the 005 or 010 sequences (47, 48, 49, and 51, 51a, and 52). However, we found that the 016, 004, G28.1, and 37.3 antibodies compete with each other for binding to human CD37. Thus, a bispecific antibody comprising a first binding arm derived from an antibody that competes for binding with either or both of the 005 or 010 antibodies and a second binding arm derived from an antibody that competes for binding with any or all of 016, 004, G28.1, and 37.3 is a bispecific antibody with specificity for two different epitopes on CD37. We surprisingly found that such antibodies have advantageous CDC efficacy in CD37-expressing cells compared to treatment of the cells with a combination of two monoclonal antibodies that do not compete for binding to CD37. In addition, the inventors have surprisingly found that such bispecific antibodies have advantageous ADCC efficacy in CD37-expressing cells compared to treatment of the cells with a combination of two monoclonal antibodies that do not compete for binding to CD37.

[0086] In one embodiment of the invention, the bispecific antibody comprises a first antigen-binding region obtained from an antibody that competes with an antibody having the CDR sequences of antibody 010 for binding to human CD37, and a second binding region obtained from an antibody that competes with an antibody having the CDR sequences of 016 for binding to human CD37.

[0087] In another embodiment of the invention, the bispecific antibody comprises a first antigen-binding region obtained from an antibody that competes with an antibody having the CDR sequences of antibody 010 for binding to human CD37, and a second binding region obtained from an antibody that competes with an antibody having the CDR sequences of 004 for binding to human CD37.

[0088] In another embodiment of the invention, the bispecific antibody comprises a first antigen-binding region obtained from an antibody that competes with an antibody having the CDR sequences of antibody 010 for binding to human CD37, and a second binding region obtained from an antibody that competes with an antibody having the CDR sequences of G28.1 for binding to human CD37.

[0089] In another embodiment of the invention, the bispecific antibody comprises a first antigen-binding region obtained from an antibody that competes with an antibody having the CDR sequences of antibody 010 for binding to human CD37, and a second binding region obtained from an antibody that competes with an antibody having the CDR sequences of 37.3 for binding to human CD37.

[0090] In one embodiment of the invention, the bispecific antibody comprises a first antigen-binding region obtained from an antibody that competes with an antibody having the CDR sequences of antibody 005 for binding to human CD37, and a second binding region obtained from an antibody that competes with an antibody having the CDR sequences of 016 for binding to human CD37.

[0091] In another embodiment of the invention, the bispecific antibody comprises a first antigen-binding region obtained from an antibody that competes with an antibody having the CDR sequences of antibody 005 for binding to human CD37, and a second binding region obtained from an antibody that competes with an antibody having the CDR sequences of 004 for binding to human CD37.

[0092] In another embodiment of the invention, the bispecific antibody comprises a first antigen-binding region obtained from an antibody that competes with an antibody having the CDR sequences of antibody 005 for binding to human CD37, and a second binding region obtained from an antibody that competes with an antibody having the CDR sequences of G28.1 for binding to human CD37.

[0093] In another embodiment of the invention, the bispecific antibody comprises a first antigen-binding region obtained from an antibody that competes with an antibody having the CDR sequences of antibody 005 for binding to human CD37, and a second binding region obtained from an antibody that competes with an antibody having the CDR sequences of 37.3 for binding to human CD37.

[0094] Such bispecific antibodies described herein may, in a further embodiment, comprise an Fc-Fc interaction-enhancing substitution in both Fc regions of the bispecific antibody (i.e., the Fc regions obtained from the first and second parent antibodies), which substitution corresponds to E430G in IgG1 when using EU numbering, and which enhances the Fc-Fc interaction of two or more bispecific antibodies of the invention upon binding to a membrane-bound target. In another embodiment, the Fc-Fc interaction-enhancing substitution corresponds to E345K in IgG1 when using EU numbering.

[0095] In another embodiment of the invention, the bispecific antibody comprises: (i) the VH CDR1 sequence set forth in SEQ ID NO: 23; the VH CDR2 sequence set forth in SEQ ID NO: 24; VH CDR3 sequence set forth in SEQ ID NO: 25; the VL CDR1 sequence set forth in SEQ ID NO: 27; a VL CDR2 sequence that is YAS, and VL CDR3 sequence set forth in SEQ ID NO: 28

[0016] ; (ii) the VH CDR1 sequence set forth in SEQ ID NO: 2; the VH CDR2 sequence set forth in SEQ ID NO: 3; VH CDR3 sequence set forth in SEQ ID NO: 4, VL CDR1 sequence set forth in SEQ ID NO: 6, a VL CDR2 sequence that is an EAS, and VL CDR3 sequence set forth in SEQ ID NO: 7

[0004] ; (iii) the VH CDR1 sequence set forth in SEQ ID NO: 40; VH CDR2 sequence set forth in SEQ ID NO: 41, VH CDR3 sequence set forth in SEQ ID NO: 42; the VL CDR1 sequence set forth in SEQ ID NO: 44; a VL CDR2 sequence that is FAK, and The VL CDR3 sequence [G28.1] set forth in SEQ ID NO: 45; and (iv) the VH CDR1 sequence set forth in SEQ ID NO: 47; VH CDR2 sequence set forth in SEQ ID NO: 48; VH CDR3 sequence set forth in SEQ ID NO: 49; the VL CDR1 sequence set forth in SEQ ID NO: 51; a VL CDR2 sequence that is VAT, and VL CDR3 sequence set forth in SEQ ID NO: 52 [37.3] and a second antigen-binding region that binds to the same epitope on human CD37 as a CD37 antibody comprising a CDR sequence selected from the group comprising:

[0096] In another embodiment of the invention, the bispecific antibody comprises: a. The VH CDR1 sequence set forth in SEQ ID NO: 23; the VH CDR2 sequence set forth in SEQ ID NO: 24; VH CDR3 sequence set forth in SEQ ID NO: 25; the VL CDR1 sequence set forth in SEQ ID NO: 27; a VL CDR2 sequence that is YAS, and VL CDR3 sequence set forth in SEQ ID NO: 28

[0016] ; b. The VH CDR1 sequence set forth in SEQ ID NO: 2; the VH CDR2 sequence set forth in SEQ ID NO: 3; VH CDR3 sequence set forth in SEQ ID NO: 4, VL CDR1 sequence set forth in SEQ ID NO: 6, a VL CDR2 sequence that is an EAS, and VL CDR3 sequence set forth in SEQ ID NO: 7

[0004] ; c. The VH CDR1 sequence set forth in SEQ ID NO: 40; VH CDR2 sequence set forth in SEQ ID NO: 41, VH CDR3 sequence set forth in SEQ ID NO: 42; the VL CDR1 sequence set forth in SEQ ID NO: 44; a VL CDR2 sequence that is FAK, and The VL CDR3 sequence [G28.1] set forth in SEQ ID NO: 45; and d. The VH CDR1 sequence set forth in SEQ ID NO: 47; VH CDR2 sequence set forth in SEQ ID NO: 48; VH CDR3 sequence set forth in SEQ ID NO: 49; the VL CDR1 sequence set forth in SEQ ID NO: 51; a VL CDR2 sequence that is VAT, and VL CDR3 sequence set forth in SEQ ID NO: 52 [37.3] and a second antigen-binding region that binds to the same epitope on human CD37 as a CD37 antibody comprising a CDR sequence selected from the group consisting of:

[0097] In one embodiment of the invention, the second antigen-binding region of the bispecific antibody has a functional epitope comprising one or more of amino acids E124, F162, Q163, V164, L165, and H175 of SEQ ID NO: 62 (CD37).

[0098] In one embodiment of the present invention, the second antigen-binding region binds to a functional epitope comprising one or more amino acids selected from the group consisting of E124, F162, Q163, V164, L165, and H175 of SEQ ID NO: 62 (CD37).

[0099] In one embodiment of the invention, the second antigen-binding region of the bispecific antibody binds to a functional epitope on CD37 and has reduced binding to a mutant CD37 in which any one or more of the amino acid residues at positions corresponding to positions E124, F162, Q163, V164, L165, and H175 of SEQ ID NO: 62 (CD37) have been substituted with alanine compared to wild-type CD37 having the amino acid sequence set forth in SEQ ID NO: 62; reduced binding is determined as a z-score (fold change) in binding of the antibody of less than −1.5, where the z-score (fold change) in binding is calculated as described in Example 17.

[0100] Thus, in one aspect, the present invention provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010, and the second antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 016.

[0101] In another aspect, the present invention provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010, and the second antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 004.

[0102] In another aspect, the present invention provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010, and the second antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody G28.1.

[0103] In another aspect, the present invention provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010, and the second antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 37.3.

[0104] In another aspect, the present invention provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 005, and the second antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 016.

[0105] In yet another aspect, the present invention provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 005, and the second antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 004.

[0106] In another aspect, the present invention provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 005, and the second antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody G28.1.

[0107] In another aspect, the present invention provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 005, and the second antigen-binding region binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 37.3.

[0108] In a further embodiment of the invention, the second antigen-binding region of the bispecific antibody comprises: (i) the VH CDR1 sequence set forth in SEQ ID NO: 23; the VH CDR2 sequence set forth in SEQ ID NO: 24; VH CDR3 sequence set forth in SEQ ID NO: 25; the VL CDR1 sequence set forth in SEQ ID NO: 27; a VL CDR2 sequence that is YAS, and VL CDR3 sequence set forth in SEQ ID NO: 28

[0016] ; (ii) the VH CDR1 sequence set forth in SEQ ID NO: 2; the VH CDR2 sequence set forth in SEQ ID NO: 3; VH CDR3 sequence set forth in SEQ ID NO: 4, VL CDR1 sequence set forth in SEQ ID NO: 6, a VL CDR2 sequence that is an EAS, and VL CDR3 sequence set forth in SEQ ID NO: 7

[0004] ; (iii) the VH CDR1 sequence set forth in SEQ ID NO: 40; VH CDR2 sequence set forth in SEQ ID NO: 41, VH CDR3 sequence set forth in SEQ ID NO: 42; the VL CDR1 sequence set forth in SEQ ID NO: 44; a VL CDR2 sequence that is FAK, and The VL CDR3 sequence [G28.1] set forth in SEQ ID NO: 45; and (v) the VH CDR1 sequence set forth in SEQ ID NO: 47; VH CDR2 sequence set forth in SEQ ID NO: 48; VH CDR3 sequence set forth in SEQ ID NO: 49; the VL CDR1 sequence set forth in SEQ ID NO: 51; a VL CDR2 sequence that is VAT, and VL CDR3 sequence set forth in SEQ ID NO: 52 [37.3] The CDR sequences are selected from the group comprising:

[0109] In a further embodiment of the invention, the second antigen-binding region of the bispecific antibody comprises: (i) the VH CDR1 sequence set forth in SEQ ID NO: 23; the VH CDR2 sequence set forth in SEQ ID NO: 24; VH CDR3 sequence set forth in SEQ ID NO: 25; the VL CDR1 sequence set forth in SEQ ID NO: 27; a VL CDR2 sequence that is YAS, and VL CDR3 sequence set forth in SEQ ID NO: 28

[0016] ; (ii) the VH CDR1 sequence set forth in SEQ ID NO: 2; the VH CDR2 sequence set forth in SEQ ID NO: 3; VH CDR3 sequence set forth in SEQ ID NO: 4, VL CDR1 sequence set forth in SEQ ID NO: 6, a VL CDR2 sequence that is an EAS, and VL CDR3 sequence set forth in SEQ ID NO: 7

[0004] ; (iii) the VH CDR1 sequence set forth in SEQ ID NO: 40; VH CDR2 sequence set forth in SEQ ID NO: 41, VH CDR3 sequence set forth in SEQ ID NO: 42; the VL CDR1 sequence set forth in SEQ ID NO: 44; a VL CDR2 sequence that is FAK, and The VL CDR3 sequence [G28.1] set forth in SEQ ID NO: 45; and (vi) the VH CDR1 sequence set forth in SEQ ID NO: 47; VH CDR2 sequence set forth in SEQ ID NO: 48; VH CDR3 sequence set forth in SEQ ID NO: 49; the VL CDR1 sequence set forth in SEQ ID NO: 51; a VL CDR2 sequence that is VAT, and VL CDR3 sequence set forth in SEQ ID NO: 52 [37.3] The CDR sequences are selected from the group consisting of:

[0110] Accordingly, in another embodiment, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR sequence of antibody 010 (i.e., SEQ ID NOs: 16-18 and 20-21) and the second antigen-binding region comprises the CDR sequence of antibody 016 (i.e., SEQ ID NOs: 23-25 and 27-28). Also as described above, such a bispecific antibody of the present invention further comprises an Fc-Fc interaction-enhancing mutation in the Fc region of the antibody. In one embodiment, this mutation corresponds to a mutation at position E430 or E345 in IgG1 when using the EU numbering system. In one embodiment, the mutation is an E430G substitution. In another embodiment, the mutation is an E345K substitution.

[0111] In another aspect, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR sequence of antibody 010 (i.e., SEQ ID NOs: 16-18 and 20-21) and the second antigen-binding region comprises the CDR sequence of antibody 004 (i.e., SEQ ID NOs: 2-4 and 6-7).

[0112] In another aspect, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR sequence of antibody 010 (i.e., SEQ ID NOs: 16-18 and 20-21) and the second antigen-binding region comprises the CDR sequence of antibody G28.1 (i.e., SEQ ID NOs: 40-42 and 44-45).

[0113] In another aspect, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the CDR sequence of antibody 010 (i.e., SEQ ID NOs: 16-18 and 20-21) and the second antigen-binding region comprises the CDR sequence of antibody 37.3 (i.e., SEQ ID NOs: 47-49 and 51-52).

[0114] In a further embodiment of the invention, the second antigen-binding region of the bispecific antibody comprises: (i) the VH sequence set forth in SEQ ID NO: 22 and the VL sequence set forth in SEQ ID NO: 26; or (ii) the VH sequence set forth in SEQ ID NO: 1 and the VL sequence set forth in SEQ ID NO: 5

[0004] ; or (iii) the VH sequence set forth in SEQ ID NO: 39 and the VL sequence set forth in SEQ ID NO: 43 [G28.1]; or (iv) the VH sequence set forth in SEQ ID NO: 46 and the VL sequence set forth in SEQ ID NO: 50 [37.3]; or A VH sequence having at least 90% identity, for example at least 95% identity, for example at least 98% identity, for example at least 99% identity, and a VL sequence having at least 90% identity, for example at least 95% identity, for example at least 98% identity, for example at least 99% identity, to the VH sequence and VL sequence set forth in any one of (i) to (iv), respectively. The VH and VL sequences are selected from the group comprising:

[0115] In a further embodiment of the invention, the second antigen-binding region of the bispecific antibody comprises: (i) the VH sequence set forth in SEQ ID NO: 22 and the VL sequence set forth in SEQ ID NO: 26; or (ii) the VH sequence set forth in SEQ ID NO: 1 and the VL sequence set forth in SEQ ID NO: 5

[0004] ; or (iii) the VH sequence set forth in SEQ ID NO: 39 and the VL sequence set forth in SEQ ID NO: 43 [G28.1]; or (iv) the VH sequence set forth in SEQ ID NO: 46 and the VL sequence set forth in SEQ ID NO: 50 [37.3]; or A VH sequence having at least 90% identity, for example at least 95% identity, for example at least 98% identity, for example at least 99% identity, and a VL sequence having at least 90% identity, for example at least 95% identity, for example at least 98% identity, for example at least 99% identity, to the VH sequence and VL sequence set forth in any one of (i) to (iv), respectively. The VH and VL sequences are selected from the group consisting of:

[0116] Accordingly, in another aspect, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID NOs: 15 and 19) and the second antigen-binding region comprises the VH and VL sequences of antibody 016 (i.e., SEQ ID NOs: 22 and 26).

[0117] In another embodiment, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID NOs: 15 and 19) and the second antigen-binding region comprises the VH and VL sequences of antibody 004 (i.e., SEQ ID NOs: 1 and 5).

[0118] In another embodiment, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID NOs: 15 and 19) and the second antigen-binding region comprises the VH and VL sequences of antibody G28.1 (i.e., SEQ ID NOs: 39 and 43).

[0119] In another embodiment, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID NOs: 15 and 19) and the second antigen-binding region comprises the VH and VL sequences of antibody 37.3 (i.e., SEQ ID NOs: 46 and 50).

[0120] In yet another embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the VH and VL sequences of antibody 005 (i.e., SEQ ID NOs: 8 and 12) and the second antigen-binding region comprises the VH and VL sequences of antibody 016 (i.e., SEQ ID NOs: 22 and 26).

[0121] In another embodiment, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the VH and VL sequences of antibody 005 (i.e., SEQ ID NOs: 8 and 12) and the second antigen-binding region comprises the VH and VL sequences of antibody 004 (i.e., SEQ ID NOs: 1 and 5).

[0122] In another embodiment, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the VH and VL sequences of antibody 005 (i.e., SEQ ID NOs: 8 and 12) and the second antigen-binding region comprises the VH and VL sequences of antibody G28.1 (i.e., SEQ ID NOs: 39 and 43).

[0123] In another embodiment, the present invention also provides a bispecific antibody comprising a first and a second antigen-binding region, wherein the first antigen-binding region comprises the VH and VL sequences of antibody 005 (i.e., SEQ ID NOs: 8 and 12) and the second antigen-binding region comprises the VH and VL sequences of antibody 37.3 (i.e., SEQ ID NOs: 46 and 50).

[0124] In yet another embodiment of the present invention, the VH and VL sequences disclosed above may differ within 90% sequence identity.

[0125] In yet another embodiment, the present invention provides a CD37-binding molecule comprising one antigen-binding region described herein, wherein the CDR sequences are those of one of antibodies 004, 005, 010, 016, 28.1, or 37.3. In one embodiment, the molecule comprises only one antigen-binding region, wherein the binding molecule has monovalent binding to CD37. Preferably, the molecule comprises an intact Fc region of an immunoglobulin. In one embodiment, the CD37-binding molecule comprises a second antigen-binding region against an unrelated target, which may be, for example, b12.

[0126] Fc-Fc enhancing mutations In one embodiment of the present invention, one or more Fc-Fc interaction-enhancing mutations in the first and second Fc regions of a bispecific antibody are amino acid substitutions. The Fc regions of a bispecific antibody can be said to comprise two different Fc regions (one from each parent anti-CD37 antibody). Alternatively, a bispecific antibody can comprise one or more Fc-Fc interaction-enhancing mutations in each half molecule. It should be understood that the Fc-Fc interaction-enhancing mutations are symmetric, i.e., the same mutations occur in the two Fc regions.

[0127] In one embodiment, the present invention provides a bispecific antibody, wherein the one or more Fc-Fc interaction enhancing mutations in said first and second Fc regions are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440, and 345 in human IgG1 when using the EU numbering system. In one embodiment, the present invention provides a bispecific antibody, wherein the one or more Fc-Fc interaction enhancing mutations in said first and second Fc regions are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440, and 345 in human IgG1 when using the EU numbering system, with the proviso that the substitution at 440 is 440Y or 440W.

[0128] In another embodiment, the present invention provides a bispecific antibody comprising at least one substitution in the first and second Fc regions selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a particularly preferred embodiment, the bispecific antibody comprises at least one substitution in the first and second Fc regions selected from E430G or E345K, preferably E430G. The present disclosure provides a bispecific antibody having enhanced Fc-Fc interactions between different antibodies having the mutation. It is believed that this mutation allows antibody oligomerization on target cells, thereby enhancing CDC.

[0129] In another embodiment, the bispecific antibody comprises one additional mutation in the Fc region selected from K439E, S440K, and S440R. A bispecific antibody having the K439E additional mutation and a second, different, or bispecific antibody having the additional S440K or S440R mutation form oligomers in an alternating pattern of the first and second antibodies. This is believed to be because the additional mutation favors interactions between the first and second antibodies over interactions between the first antibodies themselves or between the second antibodies themselves due to the non-covalent bond between the Fc regions.

[0130] Preferably, the Fc-Fc interaction enhancing mutations in the first and second Fc regions are identical substitutions. Thus, in a preferred embodiment, a bispecific antibody has the same Fc-Fc interaction enhancing mutations in both Fc regions. An Fc region can also be referred to as an Fc chain, and an antibody has two Fc chains that constitute the antibody's common Fc region. Thus, in a preferred embodiment, each of the two Fc chains contains a substitution at a position selected from the group of positions corresponding to amino acid positions 430, 440, and 345 in human IgG1 when using the EU numbering system. In one embodiment, each of the two Fc chains contains an E430G substitution, such that the bispecific antibody of the present invention contains two E430G substitutions. In another embodiment, each of the two Fc chains contains an E345K substitution, such that the bispecific antibody of the present invention contains two E345K substitutions.

[0131] In an embodiment of the invention, the bispecific antibody is of the IgG1 isotype.

[0132] In an embodiment of the invention, the bispecific antibody is of the IgG2 isotype.

[0133] In an embodiment of the invention, the bispecific antibody is of the IgG3 isotype.

[0134] In an embodiment of the invention, the bispecific antibody is of the IgG4 isotype.

[0135] In an embodiment of the invention, the bispecific antibody is of the IgG isotype.

[0136] In one embodiment of the present invention, the bispecific antibody is a combination of isotypes IgG1, IgG2, IgG3, and IgG4. For example, a first half antibody derived from a first parent antibody can be of the IgG1 isotype, and a second half antibody derived from a second parent antibody can be of the IgG4 isotype, such that the bispecific antibody is a combination of IgG1 and IgG4. In another embodiment, the bispecific antibody is a combination of IgG1 and IgG2. In another embodiment, the bispecific antibody is a combination of IgG1 and IgG3. In another embodiment, the bispecific antibody is a combination of IgG2 and IgG3. In another embodiment, the bispecific antibody is a combination of IgG2 and IgG4. In another embodiment, the bispecific antibody is a combination of IgG3 and IgG4. Typically, the core hinge is an IgG1-type core hinge having the sequence CPPC, but can also be other stable hinges that do not allow Fab arms to exchange in vivo (as is the case for an IgG4 core hinge having the sequence CPSC).

[0137] In a preferred embodiment, the bispecific antibodies of the invention are full-length antibodies.

[0138] In yet another embodiment of the present invention, the bispecific antibody is a human antibody. In yet another embodiment of the present invention, the bispecific antibody is a humanized antibody. In yet another embodiment of the present invention, the bispecific antibody is a chimeric antibody. In an embodiment of the present invention, the bispecific antibody is a combination of human, humanized, and chimeric. For example, a first half antibody derived from a first parent antibody can be a human antibody, and a second half antibody derived from a second parent antibody can be a humanized antibody, such that the bispecific antibody is a combination of human and humanized. In a preferred embodiment of the present invention, the bispecific antibody binds to both human and cynomolgus monkey CD37, having the sequences set forth in SEQ ID NOs: 62 and 63, respectively. This is advantageous because it allows preclinical toxicity testing to be performed in cynomolgus monkeys using the same bispecific molecule, which can then be tested in humans. If an antibody against a human target does not bind to the target in animal models, it can be very difficult to perform preclinical toxicity testing and nonclinical safety profiles of the molecule required by regulatory authorities.

[0139] Bispecific antibody format The present invention provides bispecific CD37xCD37 antibodies that efficiently promote CDC- and / or ADCC-mediated killing of CD37-expressing tumor cells, such as tumors of B cell origin. A specific antigen-binding region can be selected from the set of antibodies or antigen-binding regions provided by the present invention depending on the functional properties desired for a particular use. Many different formats and uses of bispecific antibodies are known in the art and are discussed by Kontermann; Drug Discov Today, 2015 Jul;20(7):838-47 and MAbs, 2012 Mar-Apr;4(2):182-97.

[0140] Although the bispecific antibodies of the invention are not limited to any particular bispecific format or method of production thereof, they should have an intact Fc domain to induce enhanced Fc-Fc interactions.

[0141] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody having two arms containing different antigen-binding regions; (ii) a dual variable domain antibody (DVD-Ig) in which each light and heavy chain contains two variable domains in tandem connected via a short peptide bond (Wu et al., "Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig)"). TM ) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (iii) so-called “dock and lock” molecules based on the “dimerization and docking domain” in protein kinase A.

[0142] In one embodiment, the bispecific antibody of the invention is a crossbody, i.e. a bispecific antibody obtained via controlled Fab arm exchange (e.g. as described in WO2011131746 (Genmab)).

[0143] Examples of different classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules with complementary CH3 domains to force heterodimerization; (ii) recombinant IgG-like dual-targeting molecules, in which each of the two halves of the molecule contains an Fab fragment or a portion of an Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, in which a full-length IgG antibody is fused to an extra Fab fragment or a portion of an Fab fragment; (iv) Fc fusion molecules, in which a single-chain Fv molecule or a stabilized diabody is fused to a heavy-chain constant domain, Fc region, or a portion thereof; (v) Fab fusion molecules, in which different Fab fragments are fused to each other and to a heavy-chain constant domain, Fc region, or a portion thereof; and (vi) scFv and diabody-based heavy-chain antibodies (e.g., domain antibodies, nanobodies, etc.), in which different single-chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, nanobodies) are fused to the Fc-.

[0144] Examples of IgG-like molecules with complementary CH3 domains are Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), so-called Knobs-into-Holes molecules (Genentech, WO9850431), CrossMAb (Roche, WO2011117329) and electrostatically steered molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y molecules (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi:10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG-body and PIG-body molecules (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body (SEEDbody) molecules (EMD Serono, WO2007110205), Biclonics molecules (Merus, WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), hinge-engineered bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks / Merck, WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254) and DuoBody® molecules (Genmab A / S, WO2011131746).

[0145] Examples of recombinant IgG-like dual targeting molecules include dual targeting (DT)-Ig molecules (WO2009058383), two-in-one antibodies (Genentech; Bostrom, et al. 2009. Science 323, 1610-1614), cross-linked MAbs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), common light chain approaches (Crucell / Merus, US7,262,028), κλ Bodies (NovImmune, WO2012023053) and CovX-body (CovX / Pfizer; Doppalapudi, VR, et al. 2007. Bioorg. Med. Chem. Lett. 17,501-506).

[0146] Examples of IgG fusion molecules include Dual Variable Domain (DVD)-Ig molecules (Abbott, US 7,612,181), Dual Domain Double-Headed Antibodies (Unilever; Sanofi Aventis, WO 2010 / 0226923), IgG-like Bispecific Molecules (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92) and BsAb molecules (Zymogenetics, WO 2010111625), HERCULES molecules (Biogen Idec, US 007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc., CN102250246) as well as TvAb molecules (Roche, WO2012025525, WO2012025530).

[0147] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc Fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), SCORPION molecules (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract #5465); Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538), and Dual(ScFv)2-Fab molecules (National Research Center for Antibody Medicine—China).

[0148] Examples of Fab-fused bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al. J. Immunol. 1998 Feb. 15;160(4):1677-86), Dual-Action or Bis-Fab molecules (Genentech, Bostrom, et al. 2009. Science 323, 1610-1614), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), Bivalent Bispecific molecules (Biotechnol, Schoonjans, J. Immunol. 2000 Dec. 15;165(12):7050-7), and Fab-Fv molecules (UCB-Celltech, WO2009040562A1).

[0149] Examples of scFv-based antibodies, diabody-based antibodies, and domain antibodies include, but are not limited to, Dual Affinity Retargeting Technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75), and dual-targeting nanobodies (Ablynx, Hmila et al., FASEB J. 2010).

[0150] In one aspect, a bispecific antibody of the invention comprises a first Fc region comprising a first CH3 region and a second Fc region comprising a second CH3 region, wherein the sequences of the first and second CH3 regions differ such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction of the first and second CH3 regions, respectively. Further details regarding these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0151] As further described herein, stable bispecific CD37xCD37 antibodies can be obtained in high yields using a particular method based on one homodimeric starting CD37 antibody and another homodimeric starting CD37 antibody that contains a small number of fairly conservative asymmetric mutations in its CH3 region, meaning that the sequences of the first and second CH3 regions contain amino acid substitutions at non-identical positions, such that the first and second CH3 regions have different amino acid sequences.

[0152] In one aspect, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first and a second Fc region, each of the first and second Fc regions comprising at least a hinge region, a CH2 region, and a CH3 region, wherein the first Fc region has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, and the second Fc region has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain, and wherein the first and second Fc regions are not substituted at the same positions.

[0153] Thus, in a preferred embodiment of the invention, the first Fc region of the bispecific antibody comprises a mutation at an amino acid corresponding to position F405 in human IgG1, and the second Fc region of the bispecific antibody comprises an additional mutation at an amino acid corresponding to position K409 in human IgG1, and these mutations are therefore asymmetric compared to the Fc-Fc interaction enhancing mutations described above.

[0154] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 366, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 368, 370, 399, 405, 407, and 409. In one embodiment, the amino acid at position 366 is selected from Ala, Asp, Glu, His, Asn, Val, or Gln.

[0155] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 368, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 370, 399, 405, 407, and 409.

[0156] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 370, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 399, 405, 407, and 409.

[0157] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 399, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 405, 407, and 409.

[0158] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 405, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 407, and 409.

[0159] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 407, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 409.

[0160] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 409, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407.

[0161] Thus, in one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the sequences of the first and second Fc regions comprise asymmetric mutations, i.e., mutations at different positions in the two Fc regions, for example a mutation at position 405 in one Fc region and a mutation at position 409 in the other Fc region.

[0162]

[0023] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and said second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407. In one such embodiment, the first Fc region has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second Fc region has an amino acid other than Phe at position 405, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys, or Leu. In a further embodiment thereof, the first Fc region has an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and the second Fc region has an amino acid other than Phe, Arg or Gly at position 405, e.g., Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys.

[0163]

[0023] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region comprises a Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second Fc region comprises an amino acid other than Phe at position 405, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met, or Cys, and Lys at position 409. In a further embodiment thereof, said first Fc region comprises Phe at position 405 and an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and said second Fc region comprises an amino acid other than Phe, Arg or Gly at position 405, e.g., Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and Lys at position 409.

[0164]

[0023] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, said first Fc region comprises Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and said second Fc region comprises Leu at position 405 and Lys at position 409. In a further embodiment thereof, the first Fc region has a Phe at position 405 and comprises an Arg at position 409, and the second Fc region comprises an amino acid other than Phe, Arg, or Gly, e.g., Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405 and comprises a Lys at position 409. In another embodiment, the first Fc region comprises a Phe at position 405 and an Arg at position 409, and the second Fc region comprises a Leu at position 405 and a Lys at position 409.

[0165] In a further embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region comprises an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second Fc region comprises Lys at position 409, Thr at position 370, and Leu at position 405. In a further embodiment, the first Fc region comprises Arg at position 409, and the second Fc region comprises Lys at position 409, Thr at position 370, and Leu at position 405.

[0166] In still further embodiments of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region comprises Lys at position 370, Phe at position 405, and Arg at position 409, and the second Fc region comprises Lys at position 409, Thr at position 370, and Leu at position 405.

[0167]

[0023] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, said first Fc region comprises an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and said second Fc region comprises Lys at position 409, a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

[0168] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, said first Fc region comprises an Arg at position 409 and said second Fc region comprises a Lys at position 409, a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.

[0169] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region comprises a Thr at position 350, a Lys at position 370, a Phe at position 405, and an Arg at position 409, and the second Fc region comprises Lys at position 409, and a) an Ile at position 350 and a Leu at position 405, or b) a Thr at position 370 and a Leu at position 405.

[0170] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region comprises a Thr at position 350, a Lys at position 370, a Phe at position 405, and an Arg at position 409; and the second Fc region comprises an Ile at position 350, a Thr at position 370, a Leu at position 405, and a Lys at position 409.

[0171]

[0023] In one embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid other than Lys, Leu, or Met at position 409 and the second Fc region has an amino acid other than Phe, such as an amino acid other than Phe, Arg, or Gly, at position 405; or, the first CH3 region has an amino acid other than Lys, Leu, or Met at position 409 and the second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407.

[0172] In one embodiment, a bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region having an amino acid at position 409 other than Lys, Leu or Met, and a second Fc region having an amino acid at position 407 other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr.

[0173] In one embodiment, a bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region having a Tyr at position 407 and an amino acid at position 409 other than Lys, Leu or Met, and a second Fc region having an amino acid at position 407 other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr and Lys at position 409.

[0174] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region having a Tyr at position 407 and an Arg at position 409, and a second Fc region having an amino acid at position 407 other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr and a Lys at position 409.

[0175] In another embodiment, the first Fc region has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second Fc region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys. In another embodiment, the first Fc region has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second Fc region has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407.

[0176]

[0039] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second Fc region has Gly, Leu, Met, Asn, or Trp at position 407.

[0177]

[0039] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has a Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and the second Fc region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and a Lys at position 409.

[0178]

[0039] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has a Tyr at 407 and an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and the second Fc region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and a Lys at 409.

[0179]

[0039] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has a Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second Fc region has a Gly, Leu, Met, Asn, or Trp at position 407 and a Lys at position 409.

[0180]

[0039] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has a Tyr at position 407 and an Arg at position 409, and the second Fc region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and an Lys at position 409, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys.

[0181]

[0023] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has a Tyr at position 407 and an Arg at position 409, and the second Fc region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and a Lys at position 409.

[0182]

[0023] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has a Tyr at position 407 and an Arg at position 409, and the second Fc region has a Gly, Leu, Met, Asn, or Trp at position 407 and a Lys at position 409.

[0183]

[0039] In another embodiment of the bispecific antibody defined in any of the embodiments disclosed herein, the first Fc region has an amino acid other than Lys, Leu or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, and the second Fc region comprises (i) an amino acid other than Phe, Leu, and Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 368; or (ii) Trp at position 370, or (iii) has an amino acid other than Asp, Cys, Pro, Glu, or Gln at position 399, e.g., Phe, Leu, Met, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asn, Trp, Tyr, or Cys; or (iv) has an amino acid other than Lys, Arg, Ser, Thr, or Trp at position 366, e.g., Phe, Leu, Met, Ala, Val, Gly, Ile, Asn, His, Asp, Glu, Gln, Pro, Tyr, or Cys.

[0184] In one embodiment, the first Fc region has an Arg, Ala, His, or Gly at position 409 and the second Fc region has (i) Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368; or (ii) Trp at position 370, or (iii) Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg, or Tyr at position 399; or (iv) Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr at position 366.

[0185] In one embodiment, the first Fc region has an Arg at position 409 and the second Fc region has (i) Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp at position 368; or (ii) Trp at position 370, or (iii) Phe, His, Lys, Arg, or Tyr at position 399; or (iv) Ala, Asp, Glu, His, Asn, Val, Gln at position 366.

[0186] In addition to the amino acid substitutions specified above, the first and second Fc regions may comprise further amino acid substitutions, deletions or insertions relative to the wild-type Fc sequence.

[0187] In a preferred embodiment of the present invention, when using EU numbering, the second Fc region of the bispecific antibody comprises a mutation corresponding to F405 in human IgG1, and the first Fc region comprises a mutation corresponding to K409 in human IgG1.

[0188] In one embodiment, the mutations at positions F405 and K409 are substitutions. In a particular embodiment, the substitution at position F405 is a F405L substitution. In another embodiment, the substitution at position K409 is a K409R substitution.

[0189] In embodiments where the bispecific antibody is of the IgG4 isotype, the first Fc region may further comprise a F405L substitution and a R409K substitution, in which the second Fc region has no substitutions at either amino acid position 405 or 409.

[0190] Unless expressly stated otherwise, it will be understood that all amino acid mutations at the disclosed positions are mutations relative to human IgG1, using human IgG1 for numbering using the EU numbering system.

[0191] In one embodiment, neither the first nor the second Fc region comprises a Cys-Pro-Ser-Cys sequence in the core hinge region.

[0192] In a further embodiment, the first and second Fc regions both comprise a Cys-Pro-Pro-Cys sequence in the core hinge region.

[0193] Provided herein are bispecific antibodies that can be produced in high yield and are stable in vivo.

[0194] In another embodiment, a bispecific antibody of the invention has enhanced CDC and / or ADCC effector function compared to the same bispecific molecule without the Fc-Fc interaction enhancing mutations. In another embodiment, a bispecific molecule of the invention has enhanced CDC and / or ADCC effector function compared to a parent monoclonal antibody having either the first or second binding domain of the bispecific antibody and the same Fc-Fc interaction enhancing mutations as the bispecific antibody of the invention.

[0195] Methods for preparing bispecific antibodies of the present invention Conventional methods, such as hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649), can be used to prepare bispecific antibodies of the invention. Co-expression in a host cell of two antibodies consisting of different heavy and light chains will result in a mixture of possible antibody products in addition to the desired bispecific antibody, which can be isolated, for example, by affinity chromatography or similar methods.

[0196] Alternatively, a convenient strategy for forming functional bispecific products by coexpression of different antibody constructs can be used, such as the method described by Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse hybridomas producing different antibodies results in only a limited number of heterodimeric proteins due to preferential species-restricted heavy / light chain pairing. Another strategy for promoting heterodimer formation over homodimers is the "knob-into-hole" strategy, in which a protrusion is introduced into a first heavy chain polypeptide and a corresponding cavity is introduced into a second heavy chain polypeptide, such that the protrusion is positioned within the cavity at the interface of these two heavy chains, promoting heterodimer formation and preventing homodimer formation. The "knob" is constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains. A complementary "void" of the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (U.S. Pat. No. 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies for favoring heterodimer formation by co-expression of different antibody domains in host cells. In these methods, one or more residues constituting the CH3-CH3 interface in both CH3 domains are substituted with charged amino acids, making homodimer formation electrostatically unfavorable and heterodimerization electrostatically favorable. WO2007110205 (Merck) describes yet another strategy that exploits the differences in the CH3 domains of IgA and IgG to promote heterodimerization.

[0197] Another in vitro method for producing bispecific antibodies is described in WO2008119353 (Genmab), in which bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (exchange of heavy chains and associated light chains) between two monospecific IgG4 or IgG4-like antibodies by incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences.

[0198] A preferred method for preparing the bispecific CD37xCD37 antibodies of the invention is the method described in WO2011131746 and WO2013060867 (Genmab), comprising the following steps: a) providing a first antibody comprising an Fc region comprising a first CH3 region; b) providing a second antibody comprising a second Fc region comprising a second CH3 region, wherein the first antibody is a CD37 antibody and the second antibody is a different CD37 antibody; the sequences of the first and second CH3 regions are different, such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction of each of the first and second CH3 regions; c) incubating the first antibody with the second antibody under reducing conditions; and d) obtaining the bispecific antibody The method includes:

[0199] In one embodiment, the first antibody and the second antibody are incubated under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization, and the heterodimeric interaction between the first and second antibodies in the resulting heterodimeric antibody is such that no Fab arm exchange occurs after 24 hours at 37°C with 0.5 mM GSH.

[0200] Without being bound by theory, in step c), heavy chain disulfide bonds in the hinge region of the parent antibody are reduced, and the resulting cysteines can form inter-heavy chain disulfide bonds with cysteine residues of another parent antibody molecule (originally with different specificity). In one embodiment of this method, the reducing conditions in step c) comprise the addition of a reducing agent, e.g., a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and beta-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a further embodiment, step c) comprises returning the conditions to non-reducing or less reducing, e.g., by removing the reducing agent, e.g., by desalting.

[0201] For this method, any of the CD37 antibodies described herein may be used, including first and second CD37 antibodies comprising a first and / or second Fc region. Examples of such first and second Fc regions may include any of those described herein, including combinations of such first and second Fc regions.

[0202] In one embodiment of this method, the first and / or second antibody is a full-length antibody.

[0203] The Fc regions of the first and second antibodies can be of any isotype, including IgG1, IgG2, IgG3, or IgG4. In one embodiment of this method, the Fc regions of both the first and second antibodies are of the IgG1 isotype. In another embodiment, one of the antibody Fc regions is of the IgG1 isotype and the other is of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody contains an IgG1 Fc region and an IgG4 Fc region and may therefore have interesting intermediate properties with respect to activation of effector functions.

[0204] In a further embodiment, one of the starting antibody proteins is engineered not to bind to Protein A, thus allowing the heterodimeric protein to be separated from the homodimeric starting protein by passing the product over a Protein A column.

[0205] As described above, the sequences of the first and second CH3 regions of the homodimeric starting antibody (parent antibody) are different, such that the heterodimeric interaction between the first and second CH3 regions is stronger than the homodimeric interaction of each of the first and second CH3 regions. Further details regarding these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference in their entirety.

[0206] In particular, stable bispecific CD37xCD37 antibodies can be obtained in high yields using the above method of the invention, which is based on two homodimeric starting antibodies that bind to different epitopes of CD37 and contain a small number of fairly conservative asymmetric mutations in their CH3 regions, meaning that the sequences of the first and second CH3 regions contain amino acid substitutions at non-identical positions.

[0207] The bispecific antibody of the present invention may also be obtained by co-expression of constructs encoding the first and second polypeptides in a single cell. Thus, in a further aspect, the present invention provides a method for producing a bispecific antibody comprising the steps of: a) providing a first nucleic acid construct encoding a first polypeptide comprising a first Fc region (including a first CH3 region) and a first antigen-binding region of a first antibody heavy chain; b) providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc region (including a second CH3 region) and a second antigen-binding region of a second antibody heavy chain, wherein the sequences of the first and second CH3 regions are different such that a heterodimeric interaction between the first and second CH3 regions is stronger than a homodimeric interaction between the first and second CH3 regions, respectively, and optionally, the first and second nucleic acid constructs encode light chain sequences of the first and second antibodies; c) co-expressing the first and second nucleic acid constructs in a host cell; and d) obtaining the heterodimeric protein from the cell culture. The present invention relates to a method for producing a bispecific antibody, comprising:

[0208] Therefore, the present invention also relates to recombinant eukaryotic or prokaryotic host cells producing the bispecific antibodies of the present invention.

[0209] In one aspect of the invention, a bispecific antibody is obtainable by any of the methods according to the invention.

[0210] Expression vectors (including promoters, enhancers, etc.) and host cells suitable for producing antibodies are well known in the art. Examples of host cells include yeast cells, bacterial cells, and mammalian cells (e.g., CHO cells or HEK cells).

[0211] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein neither the first nor the second Fc region comprises a Cys-Pro-Ser-Cys sequence in the hinge region.

[0212] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein said first and said second Fc region both comprise a Cys-Pro-Pro-Cys sequence in the hinge region.

[0213] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein the first and second Fc regions are human antibody Fc regions.

[0214] In one embodiment, a bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein the first and second antigen-binding regions comprise a human antibody VH sequence and optionally a human antibody VL sequence.

[0215] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein the first and second antigen-binding regions are derived from a heavy chain antibody.

[0216] In one embodiment, the bispecific antibody defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, and the first and second antigen-binding regions comprise first and second light chains.

[0217] In a further aspect, the co-expression method of the invention comprises any of the additional features described above under the in vitro method.

[0218] Parent antibody In another aspect, the present invention relates to the parent antibodies used to prepare the bispecific antibodies of the present invention.

[0219] Thus, in one aspect, the present invention provides a method for producing a composition comprising: (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 16, the CDR2 sequence set forth in SEQ ID NO: 17, and the CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 20, a CDR2 sequence that is a KAS, and the CDR3 sequence set forth in SEQ ID NO: 21; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 9, the CDR2 sequence set forth in SEQ ID NO: 10, and the CDR3 sequence set forth in SEQ ID NO: 11; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 113, a CDR2 sequence that is an AAS, and the CDR3 sequence set forth in SEQ ID NO: 14. The present invention relates to an anti-CD37 antibody that binds to the same epitope on human CD37 as an anti-CD37 antibody comprising:

[0220] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 16, the CDR2 sequence set forth in SEQ ID NO: 17, and the CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 20, a CDR2 sequence that is a KAS, and the CDR3 sequence set forth in SEQ ID NO: 21; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 9, the CDR2 sequence set forth in SEQ ID NO: 10, and the CDR3 sequence set forth in SEQ ID NO: 11; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 113, a CDR2 sequence that is an AAS, and the CDR3 sequence set forth in SEQ ID NO: 14. The present invention relates to an anti-CD37 antibody that competes for binding with an anti-CD37 antibody comprising the compound

[0221] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 16, the CDR2 sequence set forth in SEQ ID NO: 17, and the CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 20, a CDR2 sequence that is a KAS, and the CDR3 sequence set forth in SEQ ID NO: 21; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 9, the CDR2 sequence set forth in SEQ ID NO: 10, and the CDR3 sequence set forth in SEQ ID NO: 11; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 113, a CDR2 sequence that is an AAS, and the CDR3 sequence set forth in SEQ ID NO: 14. The present invention relates to an anti-CD37 antibody comprising:

[0222] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 23, the CDR2 sequence set forth in SEQ ID NO: 24, and the CDR3 sequence set forth in SEQ ID NO: 25, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 27, a CDR2 sequence that is YAS, and the CDR3 sequence set forth in SEQ ID NO: 28; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 2, the CDR2 sequence set forth in SEQ ID NO: 3, and the CDR3 sequence set forth in SEQ ID NO: 4; and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 6, a CDR2 sequence that is an EAS, and the CDR3 sequence set forth in SEQ ID NO: 7. The present invention relates to an anti-CD37 antibody comprising:

[0223] In another aspect, the invention relates to an antibody as defined above comprising one or more amino acid mutations in the Fc region, wherein the mutations enhance the Fc-Fc interaction between the antibodies upon target binding compared to the Fc-Fc interaction between antibodies not carrying said mutations.

[0224] The enhanced Fc-Fc interaction is believed to have the effect of causing the antibody to form oligomers (e.g., hexamerization) on target cells, and this oligomerization is thought to result in an enhanced CDC effect. In a preferred embodiment, the one or more amino acid mutations in the Fc region of the antibody are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 440, and 345 in human IgG1 when using the EU numbering system, where the substitutions are relative to the amino acid sequence of human IgG1. In one embodiment, the one or more amino acid mutations in the Fc region of the antibody are amino acid substitutions at one or more positions corresponding to amino acid positions 430, 345, and 440 in human IgG1 when using the EU numbering system, where the substitution at 440 is 440Y or 440W. In one embodiment, the at least one amino acid substitution in the Fc region is selected from the group comprising E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W.

[0225] In a preferred embodiment, at least one substitution in the Fc region is selected from E430G or E345K, preferably E430G. These Fc-Fc interaction-enhancing mutations are symmetric mutations such that the two Fc chains of the antibody have the same mutation / substitution.

[0226] In further embodiments, the antibody may further contain substitutions at positions corresponding to 366, 368, 370, 399, 405, 407, and 409 in human IgG1. Antibodies with substitutions at one of these amino acid positions are stable, but can form bispecific antibodies with antibodies with substitutions at other of these amino acid positions and different antigen-binding regions in a so-called "fab arm exchange" reaction under reducing conditions. Under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization, the antibodies of the present invention form half molecules, each containing a single antigen-binding site and an Fc region. Substitutions at any of the non-identical positions corresponding to 366, 368, 370, 399, 405, 407, and 409 in human IgG1 favorably dimerize half molecules of a first antibody with half molecules of a second antibody, forming a bispecific (heterodimeric) antibody when reducing conditions are lifted and the disulfide bonds in the hinge region reform.

[0227] Thus, two antibodies of the present invention that have different antigen-binding regions, bind to different epitopes on CD37, and contain substitutions in both Fc chains (Fc regions) at any of the amino acid positions corresponding to 366, 368, 370, 399, 405, 407, and 409 in human IgG1 (but at non-identical positions) may be suitable for preparing bispecific antibodies of the present invention.

[0228] In one embodiment, the first antibody has an amino acid substitution at position 366 and said second homodimeric protein has an amino acid substitution at a position selected from the group consisting of 368, 370, 399, 405, 407, and 409. In one embodiment, the amino acid at position 366 is selected from Arg, Lys, Asn, Gln, Tyr, Glu, and Gly.

[0229] In one embodiment, the first antibody has an amino acid substitution at position 368 and said second antibody has an amino acid substitution at a position selected from the group consisting of 366, 370, 399, 405, 407, and 409.

[0230] In one embodiment, the first antibody has an amino acid substitution at position 370 and said second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 399, 405, 407, and 409.

[0231] In one embodiment, the first antibody has an amino acid substitution at position 399 and said second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 405, 407, and 409.

[0232] In one embodiment, the first antibody has an amino acid substitution at position 405 and said second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 407, and 409.

[0233] In one embodiment, the first antibody has an amino acid substitution at position 407 and said second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 409.

[0234] In one embodiment, the first antibody has an amino acid substitution at position 409 and said second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407.

[0235] In one embodiment, the first antibody has an amino acid at position 409 selected from the group comprising an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Arg, His, Asp, Asn, Glu, Gln, Trp, Phe or Tyr, and said second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407.

[0236] In one such embodiment, the first antibody has an amino acid other than Lys, Leu, or Met at position 409, e.g., an amino acid selected from the group including Gly, Ala, Val, Ile, Ser, Thr, Arg, His, Asp, Asn, Glu, Gln, Trp, Phe, or Tyr, and the second antibody has an amino acid other than Phe, e.g., an amino acid selected from the group including Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Trp, Met, or Tyr, at the position corresponding to 405 in IgG1. In a further embodiment thereof, the first antibody has an amino acid other than Lys, Leu, or Met at position 409, and the second antibody has an amino acid other than Phe, Arg, or Gly at position 405.

[0237] In another embodiment, the first antibody comprises a Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, and the second antibody comprises an amino acid other than Phe at position 405 and a Lys at position 409.

[0238] In another embodiment, the first antibody comprises a Phe at position 405 and an Arg at position 409, and the second antibody comprises a Leu at position 405 and a Lys at position 409. In embodiments in which the antibodies are of an IgG1, IgG2, or IgG3 isotype, the first antibody may comprise a F405L substitution and the second antibody may comprise a K409R substitution, or vice versa. However, in embodiments in which both antibodies are of an IgG4 isotype, the amino acid at position 409 is naturally Arg (R). Thus, in such embodiments, the first antibody is unsubstituted at position 409 and naturally has an R409, and the second antibody comprises an F405L and an R405K substitution, or vice versa; the second antibody is unsubstituted at position 409 and naturally has an R409, and the first antibody comprises an F405L and an R405K substitution. In embodiments in which one or both of the first and second antibodies are of an IgG4 isotype.

[0239] Thus, in one embodiment, an antibody of the present invention may contain a substitution corresponding to F405L in human IgG1. In another embodiment, an antibody of the present invention may contain a substitution corresponding to K409R in human IgG1. Two such different antibodies are suitable for preparing a bispecific antibody of the present invention. In a particularly preferred embodiment, a first antibody of the present invention contains F405L and E430G substitutions, and a second antibody of the present invention contains K409R and E430G substitutions. Provided herein are antibodies capable of forming a bispecific antibody of the present invention, comprising a first half molecule containing F405L+E430G substitutions and a second half molecule containing K409R+E430G substitutions when IgG1 is used for numbering. In an embodiment in which the isotype is IgG4, the first half molecule contains F405L+R409K+E430G substitutions and the second half molecule contains E430G when IgG4 is used for numbering. Preferably, in such IgG4 embodiments, the core hinge region is substituted from a "CPSC" amino acid sequence to a "CPPC" sequence to render the bispecific antibody more stable in vivo and / or in vitro compared to an IgG4 antibody with a CPSC core hinge.

[0240] In one embodiment of the present invention, the first antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 16, the CDR2 sequence set forth in SEQ ID NO: 17, and the CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 20, a CDR2 sequence that is a KAS, and the CDR3 sequence set forth in SEQ ID NO: 21; and an Fc region comprising an F405L substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0241] In another embodiment of the invention, the first antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 16, the CDR2 sequence set forth in SEQ ID NO: 17, and the CDR3 sequence set forth in SEQ ID NO: 18, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 20, a CDR2 sequence that is KAS, and the CDR3 sequence set forth in SEQ ID NO: 21; and an Fc region comprising a K409R substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0242] In another embodiment of the invention, the first antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 9, the CDR2 sequence set forth in SEQ ID NO: 10, and the CDR3 sequence set forth in SEQ ID NO: 11, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 113, a CDR2 sequence that is an AAS, and the CDR3 sequence set forth in SEQ ID NO: 14; and an Fc region comprising an F405L substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0243] In yet another embodiment of the present invention, the first antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 9, the CDR2 sequence set forth in SEQ ID NO: 10, and the CDR3 sequence set forth in SEQ ID NO: 11, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 113, a CDR2 sequence that is an AAS, and the CDR3 sequence set forth in SEQ ID NO: 14; and an Fc region comprising a K409R substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0244] In one embodiment of the present invention, the second antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 23, the CDR2 sequence set forth in SEQ ID NO: 24, and the CDR3 sequence set forth in SEQ ID NO: 25, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 27, a CDR2 sequence which is YAS, and the CDR3 sequence set forth in SEQ ID NO: 28; and an Fc region comprising an F405L substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0245] In another embodiment of the invention, the second antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 23, the CDR2 sequence set forth in SEQ ID NO: 24, and the CDR3 sequence set forth in SEQ ID NO: 25, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 27, a CDR2 sequence which is YAS, and the CDR3 sequence set forth in SEQ ID NO: 28; and an Fc region comprising a K409R substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0246] In another embodiment of the invention, the second antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 2, the CDR2 sequence set forth in SEQ ID NO: 3, and the CDR3 sequence set forth in SEQ ID NO: 4, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 6, a CDR2 sequence that is an EAS, and the CDR3 sequence set forth in SEQ ID NO: 7; and an Fc region comprising an F405L substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0247] In yet another embodiment of the present invention, the second antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 2, the CDR2 sequence set forth in SEQ ID NO: 3, and the CDR3 sequence set forth in SEQ ID NO: 4, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 6, a CDR2 sequence that is an EAS, and the CDR3 sequence set forth in SEQ ID NO: 7; and an Fc region comprising a K409R substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0248] In another embodiment of the invention, the second antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 40, the CDR2 sequence set forth in SEQ ID NO: 41, and the CDR3 sequence set forth in SEQ ID NO: 42, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 44 and a CDR2 sequence that is FAK and the CDR3 sequence set forth in SEQ ID NO: 45 [G28.1]; and an Fc region comprising an F405L substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0249] In yet another embodiment of the invention, the second antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 40, the CDR2 sequence set forth in SEQ ID NO: 41, and the CDR3 sequence set forth in SEQ ID NO: 42, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 44 and a CDR2 sequence which is FAK and the CDR3 sequence set forth in SEQ ID NO: 45 [G28.1]; and an Fc region comprising a K409R substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0250] In another embodiment of the invention, the second antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 47, the CDR2 sequence set forth in SEQ ID NO: 48, and the CDR3 sequence set forth in SEQ ID NO: 49, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 51 and a CDR2 sequence that is VAT and the CDR3 sequence set forth in SEQ ID NO: 52 [37.3]; and an Fc region comprising an F405L substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0251] In yet another embodiment of the invention, the second antibody comprises a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 47, the CDR2 sequence set forth in SEQ ID NO: 48, and the CDR3 sequence set forth in SEQ ID NO: 49, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 51 and a CDR2 sequence that is VAT and the CDR3 sequence set forth in SEQ ID NO: 52 [37.3]; and an Fc region comprising a K409R substitution and one or more Fc-Fc interaction-enhancing mutations. In a preferred embodiment, the Fc-Fc interaction-enhancing mutations are substitutions at one or more amino acid positions selected from the group consisting of 430, 345, and 440, e.g., E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, the substitution is E430G.

[0252] Therefore, the present invention also provides an anti-CD37 antibody that binds to human CD37, (i) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 40, the CDR2 sequence set forth in SEQ ID NO: 41, and the CDR3 sequence set forth in SEQ ID NO: 42, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 44, a CDR2 sequence that is FAK, and the CDR3 sequence set forth in SEQ ID NO: 45 [G28.1]; or (ii) a VH region comprising the CDR1 sequence set forth in SEQ ID NO: 47, the CDR2 sequence set forth in SEQ ID NO: 48, and the CDR3 sequence set forth in SEQ ID NO: 49, and a VL region comprising the CDR1 sequence set forth in SEQ ID NO: 51, a CDR2 sequence that is VAT, and the CDR3 sequence set forth in SEQ ID NO: 52 [37.3] Includes; (iii) the antibody of (i) or (ii) comprises an Fc region comprising at least one amino acid substitution selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W; (iv) Optionally, the Fc region further comprises the mutation K409R or F405L.

[0253] As noted above, the anti-CD37 antibodies of the present invention may be of the IgG1, IgG2, IgG3, or IgG4 isotype. In one embodiment, the anti-CD37 antibodies of the present invention are of the IgG isotype.

[0254] In one embodiment, an antibody of the invention is a human antibody, a humanized antibody, or a chimeric antibody.

[0255] In one embodiment, the antibodies of the invention bind to both human and cynomolgus CD37 antigens.

[0256] Further aspects of the invention In another aspect, the invention relates to a composition comprising a bispecific antibody of the invention and further comprising a monospecific anti-CD37 antibody, preferably an anti-CD37 antibody having an antigen-binding region that is either the first or second antigen-binding region of the bispecific antibody.

[0257] In one aspect, the present invention relates to a pharmaceutical composition comprising a bispecific antibody of the invention or an anti-CD37 antibody of the invention and a pharmaceutically acceptable carrier.

[0258] In another aspect, the present invention relates to the bispecific antibody herein or the antibody of the present invention or the composition of the present invention for use as a medicament.

[0259] In one aspect of the invention, the bispecific antibody of the invention is for use in the treatment of cancer, an autoimmune disease, or an inflammatory disorder.

[0260] In one embodiment of the invention, the anti-CD37 antibodies of the invention are for use in the treatment of cancer, an autoimmune disease, or an inflammatory disorder.

[0261] In one aspect of the invention, the compositions of the invention are for use in the treatment of cancer, an autoimmune disease, or an inflammatory disorder.

[0262] In another embodiment, the invention relates to a bispecific antibody of the invention for use in the treatment of allergy, graft rejection, or a B-cell malignancy, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL) or acute lymphoblastic leukemia (ALL).

[0263] In another aspect, the present invention relates to a method for treating rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune-mediated arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis and juvenile rheumatoid arthritis, osteoarthritis, progressive chronic arthritis, osteoarthritis, primary chronic polyarthritis, reactive arthritis and ankylosing spondylitis, systemic lupus erythematosus (SLE), such as cutaneous SLE or The present invention relates to a bispecific antibody of the present invention for use in the treatment of subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus erythematosus, multiple sclerosis, inflammatory bowel diseases (IBD) including ulcerative colitis and Crohn's disease, chronic obstructive pulmonary disease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, diabetes mellitus, Raynaud's syndrome and glomerulonephritis, palmoplantar pustulosis (PPP), erosive lichen planus, bullous pemphigus, epidermolysis bullosa, contact dermatitis and atopic dermatitis, polyneuritis including Guillain-Barré syndrome.

[0264] In another embodiment, the invention relates to an anti-CD37 antibody of the invention for use in the treatment of allergy, transplant rejection, or B-cell malignancies.

[0265] In another aspect, the invention relates to an anti-CD37 antibody of the invention for use in the treatment of allergy, transplant rejection, or a B-cell malignancy, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL), or acute lymphoblastic leukemia (ALL).

[0266] In another aspect, the present invention relates to a method for treating rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune-mediated arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis and juvenile rheumatoid arthritis, osteoarthritis, progressive chronic arthritis, osteoarthritis, primary chronic polyarthritis, reactive arthritis and ankylosing spondylitis, systemic lupus erythematosus (SLE), such as cutaneous SLE or The present invention relates to an anti-CD37 antibody of the present invention for use in the treatment of subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus erythematosus, multiple sclerosis, inflammatory bowel diseases (IBD) including ulcerative colitis and Crohn's disease, chronic obstructive pulmonary disease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, diabetes mellitus, Raynaud's syndrome and glomerulonephritis, palmoplantar pustulosis (PPP), erosive lichen planus, bullous pemphigus, epidermolysis bullosa, contact dermatitis and atopic dermatitis, and polyneuropathies including Guillain-Barré syndrome.

[0267] In another embodiment, the present invention relates to a bispecific antibody of the present invention for use in combination with one or more additional therapeutic agents. In another embodiment of the present invention, the anti-CD37 antibody of the present invention is for use in combination with one or more additional therapeutic agents. The one or more additional therapeutic agents may be selected from the group including, for example, doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti-CD20 antibodies (e.g., rituximab, ofatumumab, obinutuzumab, veltuzumab, ocaratuzumab, ocrelizumab, or TRU-015).

[0268] In a preferred embodiment of the invention, the additional therapeutic agent is an anti-CD20 antibody.

[0269] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to human CD20 having the sequence set forth in SEQ ID NO:72.

[0270] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to cynomolgus monkey CD20 having the sequence set forth in SEQ ID NO:73.

[0271] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to human and cynomolgus CD20 having the sequences set forth in SEQ ID NOs: 72 and 73, respectively.

[0272] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to an epitope on human CD20 that does not include or require amino acid residues alanine at position 170 or proline at position 172, but does include or require amino acid residues asparagine at position 163 and asparagine at position 166 of SEQ ID NO: 72. Examples of such antibodies are the antibodies designated 2F2 and 7D8, disclosed in WO2004035607 (Genmab), and the antibody designated 2C6, disclosed in WO2005103081 (Genmab). The CDR sequences of 7D8 are disclosed in Table 1.

[0273] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to an epitope on human CD20 that does not include or require amino acid residues alanine at position 170 or proline at position 172 of SEQ ID NO: 72. An example of such an antibody is 11B8, disclosed in WO2004035607 (Genmab). The CDR sequences of 11B8 are disclosed in Table 1.

[0274] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to a discontinuous epitope on human CD20, wherein the epitope comprises part of the first small extracellular loop and part of the second extracellular loop.

[0275] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to a discontinuous epitope on human CD20, the epitope having residues AGIYAP in the first small extracellular loop and residues MESLNFIRAHTPY in the second extracellular loop.

[0276] Anti-CD20 antibodies can be characterized as type I and type II anti-CD20 antibodies. Type I anti-CD20 antibodies have high CDC and ADCC activity but low apoptotic activity (e.g., ofatumumab (2F2) and rituximab), while type II anti-CD20 antibodies have low or no CDC activity but high ADCC and apoptotic activity (e.g., obinutuzumab and 11B8). Type I antibodies also induce CD20 to redistribute into large detergent-resistant microdomains (rafts), whereas type II antibodies do not.

[0277] In one embodiment of the invention, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) and variable light chain (VL) sequences set forth in SEQ ID NO: 74 and SEQ ID NO: 78, respectively.

[0278] In one embodiment of the invention, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) and variable light chain (VL) sequences set forth in SEQ ID NO: 81 and SEQ ID NO: 109, respectively.

[0279] In one embodiment of the invention, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) sequences and variable light chain (VL) sequences set forth in SEQ ID NO: 94 and SEQ ID NO: 98, respectively.

[0280] In one embodiment of the invention, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) sequences and variable light chain (VL) sequences set forth in SEQ ID NO: 87 and SEQ ID NO: 91, respectively.

[0281] In one embodiment of the invention, the anti-CD20 antibody comprises an antigen-binding region capable of binding to human CD20, and the antigen-binding region competes for binding to human CD20 with an anti-CD20 antibody comprising the variable heavy chain (VH) sequences and variable light chain (VL) sequences set forth in SEQ ID NO: 101 and SEQ ID NO: 105, respectively.

[0282] In one embodiment of the invention, the anti-CD20 antibody has the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 75; VH CDR2 sequence set forth in SEQ ID NO: 76; the VH CDR3 sequence set forth in SEQ ID NO: 77; the VL CDR1 sequence set forth in SEQ ID NO: 79; a VL CDR2 sequence that is a DAS, and VL CDR3 sequence set forth in SEQ ID NO: 80 [7D8] The antigen-binding region comprises a region capable of binding to human CD20, the region comprising:

[0283] In one embodiment of the invention, the anti-CD20 antibody has the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 82; VH CDR2 sequence set forth in SEQ ID NO: 83; VH CDR3 sequence set forth in SEQ ID NO: 84; the VL CDR1 sequence set forth in SEQ ID NO: 85; a VL CDR2 sequence that is a DAS, and VL CDR3 sequence set forth in SEQ ID NO: 86 [118B] The antigen-binding region comprises a region capable of binding to human CD20, the region comprising:

[0284] In one embodiment of the invention, the anti-CD20 antibody has the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 95; VH CDR2 sequence set forth in SEQ ID NO: 96; VH CDR3 sequence set forth in SEQ ID NO: 97; the VL CDR1 sequence set forth in SEQ ID NO: 99; a VL CDR2 sequence that is an ATS, and VL CDR3 sequence set forth in SEQ ID NO: 100 Rituximab contains an antigen-binding region capable of binding to human CD20.

[0285] In one embodiment of the invention, the anti-CD20 antibody has the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 88; VH CDR2 sequence set forth in SEQ ID NO: 89; VH CDR3 sequence set forth in SEQ ID NO: 90; the VL CDR1 sequence set forth in SEQ ID NO: 92; a VL CDR2 sequence that is a DAS, and VL CDR3 sequence set forth in SEQ ID NO: 93 It contains an antigen-binding region capable of binding to human CD20. [Ofatumumab]

[0286] In one embodiment of the invention, the anti-CD20 antibody has the CDR sequence: VH CDR1 sequence set forth in SEQ ID NO: 102; the VH CDR2 sequence set forth in SEQ ID NO: 103; the VH CDR3 sequence set forth in SEQ ID NO: 104; the VL CDR1 sequence set forth in SEQ ID NO: 106; a VL CDR2 sequence that is a QMS, and VL CDR3 sequence set forth in SEQ ID NO: 107 It contains an antigen-binding region capable of binding to human CD20, including the following: [obinutuzumab]

[0287] In one embodiment of the invention, the anti-CD20 antibody i) the VH CDR1 sequence set forth in SEQ ID NO: 75; VH CDR2 sequence set forth in SEQ ID NO: 76; the VH CDR3 sequence set forth in SEQ ID NO: 77; the VL CDR1 sequence set forth in SEQ ID NO: 79; a VL CDR2 sequence that is a DAS, and VL CDR3 sequence set forth in SEQ ID NO: 80 [7D8]; ii) the VH CDR1 sequence set forth in SEQ ID NO: 82; VH CDR2 sequence set forth in SEQ ID NO: 83; VH CDR3 sequence set forth in SEQ ID NO: 84; the VL CDR1 sequence set forth in SEQ ID NO: 85; a VL CDR2 sequence that is a DAS, and VL CDR3 sequence set forth in SEQ ID NO: 86 [118B]; iii) the VH CDR1 sequence set forth in SEQ ID NO: 95; VH CDR2 sequence set forth in SEQ ID NO: 96; VH CDR3 sequence set forth in SEQ ID NO: 97; the VL CDR1 sequence set forth in SEQ ID NO: 99; a VL CDR2 sequence that is an ATS, and VL CDR3 sequence set forth in SEQ ID NO: 100 [rituximab]; iv) the VH CDR1 sequence set forth in SEQ ID NO: 88; VH CDR2 sequence set forth in SEQ ID NO: 89; VH CDR3 sequence set forth in SEQ ID NO: 90; the VL CDR1 sequence set forth in SEQ ID NO: 92; a VL CDR2 sequence that is a DAS, and The VL CDR3 sequence set forth in SEQ ID NO: 93 [ofatumumab]; and v) the VH CDR1 sequence set forth in SEQ ID NO: 102; the VH CDR2 sequence set forth in SEQ ID NO: 103; the VH CDR3 sequence set forth in SEQ ID NO: 104; the VL CDR1 sequence set forth in SEQ ID NO: 106; a VL CDR2 sequence that is a QMS, and VL CDR3 sequence set forth in SEQ ID NO: 107 [obinutuzumab] The antibody comprises an antigen-binding region capable of binding to human CD20, the antigen-binding region comprising a CDR sequence selected from the group consisting of:

[0288] In another embodiment, the invention relates to the use of a bispecific antibody of the invention or an anti-CD37 antibody of the invention for the manufacture of a medicament. In that further embodiment, the use is in the treatment of cancer, an autoimmune disease, or an inflammatory disorder, such as allergy, graft rejection, or B-cell malignancies, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL) or acute lymphoblastic leukemia (ALL), rheumatoid arthritis, such as acute arthritis, rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune-mediated arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and juvenile rheumatoid arthritis. and ankylosing spondylitis; systemic lupus erythematosus (SLE), such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE) and disseminated lupus erythematosus; multiple sclerosis; inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn's disease; chronic obstructive pulmonary disease (COPD); psoriasis; IgA nephropathy; IgM polyneuropathy; myasthenia gravis; diabetes; Raynaud's syndrome and glomerulonephritis; palmoplantar pustulosis (PPP); erosive lichen planus; bullous pemphigus; epidermolysis bullosa; contact dermatitis and atopic dermatitis; and polyradiculoneuritis, including Guillain-Barré syndrome.

[0289] In another embodiment, the invention relates to a method for inducing cell death or inhibiting the growth and / or proliferation of tumor cells expressing CD37, comprising administering to an individual in need thereof an effective amount of a bispecific antibody of the invention or an anti-CD37 antibody of the invention. In a particular embodiment, the method is a method for treating an individual with allergy, graft rejection, or a B-cell malignancy, such as non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), diffuse large B-cell lymphoma (DLBCL) or acute lymphoblastic leukemia (ALL), comprising administering to said individual an effective amount of a bispecific antibody of the invention or an anti-CD37 antibody of the invention. In certain embodiments, the method comprises administering one or more additional therapeutic agents, such as doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, or an anti-CD20 antibody (e.g., rituximab, ofatumumab, obinutuzumab, veltuzumab, ocralizumab, or TRU-015), in combination with the antibody or bispecific antibody.

[0290] In one embodiment of the invention, the additional therapeutic agent is cyclophosphamide, chlorambucil, bendamustine, ifosfamide, cisplatin, carboplatin, oxaliplatin, carmustine, prednisone, dexamethasone, fludarabine, pentostatin, cladribine, fluorouracil, gemcitabine, cytarabine, methotrexate, pralatrexate, gemcitabine, vincristine, paclitaxel, docetaxel, doxorubicin, mitoxantrone, etoposide, topotecan, irinotecan, bleomycin, CD20-specific rituximab, obinutuzumab, and ofatumumab, CD52-specific alemtuzumab, CD30-specific brentuximab, the antibody or antibodies specific for other immunomodulatory targets, brentuximab vedotin, HuMax-TAC-ADC, interferon, thalidomide, lenalidomide, axicabtagene ciloleucel, bortezomib, romidepsin, belinstat, vorinostat, ibrutinib, acalabrutinib, idelalisib, copanlisib, sorafenib, sunitinib, everolimus, recombinant human TRAIL, birinapant, and venetoclax.

[0291] In one embodiment of the invention, the further therapeutic agent is selected from the group comprising ibrutinib, rituximab, venetoclax, CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), bendamustine, fludarabine, cyclophosphamide, and chlorambucil.

[0292] In one aspect of the invention, the further therapeutic agent is selected from the group comprising ibrutinib, rituximab, and venetoclax.

[0293] In a further aspect, the present invention relates to a nucleic acid construct encoding one or more sequences presented in Table 1. In a further aspect, the present invention relates to a nucleic acid construct encoding one or more sequences selected from the group comprising SEQ ID NOs: 1, 2, 3, 4, 5, 6, 6a, 7, 8, 9, 10, 11, 12, 13, 13a, 14, 15, 16, 17, 18, 19, 20, 20a, 21, 22, 23, 24, 25, 26, 27, 27a, 28, 29, 30, 30a, and 31.

[0294] The present invention further relates to nucleic acid constructs encoding the VH and / or VL regions of the bispecific antibody or anti-CD37 antibody of any of the embodiments herein.

[0295] The present invention further relates to nucleic acid constructs encoding the bispecific antibodies or anti-CD37 antibodies of any of the embodiments herein.

[0296] In a further aspect, the present invention relates to an expression vector comprising one or more of the nucleic acid constructs described above. In another aspect, the present invention relates to a host cell comprising the expression vector described above.

[0297] Expression vectors in the context of the present invention can be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (nucleic acid sequences comprising an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the CD37 antibody is contained within, for example, a naked DNA or RNA vector comprising a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355 59 (1997)), a compacted nucleic acid vector (e.g., as described in US 6,077,835 and / or WO 00 / 70087), a plasmid vector, e.g., pBR322, pUC19 / 18, or pUC118 / 119, a "midge" minimal size nucleic acid vector (e.g., as described in Schakowski et al., Mol Ther 3, 793 800 (2001)), or a precipitated nucleic acid vector construct, e.g., a CaPO4 precipitated construct (e.g., as described in WO 200046147, Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and their uses are well known in the art (see, e.g., US Pat. No. 5,589,466 and US Pat. No. 5,973,972).

[0298] In one embodiment, the vector is suitable for expressing a CD37 antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264, 5503-5509 (1989)), pET vectors (Novagen, Madison WI), and the like.

[0299] Similarly or alternatively, the expression vector may be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH (discussed in F. Ausubel et al., ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153, 516-544 (1987)).

[0300] Also or alternatively, the expression vector may be a vector suitable for expression in mammalian cells, such as a vector containing glutamine synthetase as a selectable marker, such as the vector described in Bebbington (1992) Biotechnology (NY) 10:169-175.

[0301] The nucleic acid and / or vector may also include a nucleic acid sequence encoding a secretion / localization sequence capable of directing a polypeptide (e.g., a nascent polypeptide chain) into the periplasmic space or into the cell medium. Such sequences are known in the art and include secretory leaders or signal peptides.

[0302] The expression vector may contain or be associated with any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter / enhancer and the RSV, SV40, SL33, MMTV, and HIV LTR promoters), an efficient poly(A) termination sequence, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also contain an inducible promoter, as opposed to a constitutive promoter such as CMV IE.

[0303] In one embodiment, an expression vector encoding a CD37 antibody can be placed into and / or delivered to a host cell or animal via a viral vector.

[0304] Therefore, the present invention also relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, which produce the bispecific antibodies of the present invention.

[0305] The present invention further relates to anti-idiotypic antibodies that bind to the antigen-binding region of an antibody or bispecific antibody of the invention.

[0306] 1. An in vitro method for detecting the presence of human CD37 antigen or cells expressing human CD37 in a sample, comprising: (i) contacting a sample with the bispecific antibody of any of the above embodiments or the antibody of any of the embodiments herein under conditions that allow for the formation of a complex between the antibody or bispecific antibody and CD37; and (ii) detecting the formation of a complex A method comprising:

[0307] 1. An in vivo method for detecting the presence of human CD37 antigen or cells expressing human CD37 in a subject, comprising: (i) administering the bispecific antibody of any of the above embodiments or any of the antibodies of the embodiments herein under conditions that allow for the formation of a complex between the antibody or bispecific antibody and CD37; and (ii) detecting the formed complex A method comprising:

[0308] array (Table 1) TIFF2025121994000002.tif35170TIFF2025121994000003.tif207170TIFF20251219940 00004.tif206170TIFF2025121994000005.tif204170TIFF2025121994000006.tif206170 TIFF2025121994000007.tif204170TIFF2025121994000008.tif207170TIFF20251219940 00009.tif207170TIFF2025121994000010.tif203170TIFF2025121994000011.tif203170 [Example]

[0309] Example 1: Generation of CD37-specific antibodies in rabbits Expression construct for CD37 Codon-optimized constructs were generated for the expression of full-length CD37 variants: human (Homo sapiens) CD37 (Genbank accession number NP_001765) (SEQ ID NO: 62), cynomolgus monkey (Macaca fascicularis) CD37 (mfCD37) (SEQ ID NO: 63), and various CD37 ECD variants: a signal peptide coding sequence followed by the second extracellular domain (EC2) (aa 112-241) of human CD37 fused to the Fc (CH2-CH3) domain of human IgG with a C-terminal His tag (CD37EC2-FcHis, SEQ ID NO: 64), and a similar construct for mfCD37 (CD37mfEC2-FcHis, SEQ ID NO: 65). The constructs contained appropriate restriction sites for cloning and the optimal Kozak (GCCGCCACC) sequence [Kozak et al. (1999) Gene 234: 187-208]. The constructs were cloned into the mammalian expression vector pcDNA3.3 (Invitrogen) or an equivalent vector.

[0310] Transient expression in CHO and HEK cells Membrane proteins were transiently expressed in Freestyle 293-F (HEK293F) cells (Life technologies, USA) using 293fectin (Life technologies) essentially as described by the manufacturer, or in Freestyle CHO-S cells (CHO) (Life technologies) using Freestyle Max reagent (Life technologies) essentially as described by the manufacturer. Soluble proteins were transiently expressed in Expi293 cells (Life technologies) using ExpiFectamine 293 reagent (Life technologies) essentially as described by the manufacturer.

[0311] Fc fusion proteins (CD37mfEC2-FcHis and CD37EC2-FcHis) were purified from cell culture supernatants using protein A affinity chromatography.

[0312] Immunization of rabbits Rabbit immunizations were performed at MAB Discovery GmbH (Neuried, Germany). Rabbits were repeatedly immunized with a mixture of CD37EC2-FcHis and CD37mfEC2-FcHis or HEK293F cells transiently expressing human or mfCD37. Blood was collected from these rabbits, and B lymphocytes were isolated. Using a proprietary MAB Discovery process, single B cells were sorted into wells of microtiter plates and further expanded. Supernatants from these single B cells were analyzed for specific binding to CHO-S cells transiently expressing CD37 (CHO-CD37) and CHO-S cells transiently expressing mfCD37 (CHO-mfCD37).

[0313] Recombinant antibody production After analyzing the primary screening results, primary hits were selected for sequencing, recombinant mAb production, and purification. Unique variable heavy (VH) and light (VL) chain coding regions were gene synthesized and cloned into a mammalian expression vector containing human IgG1 constant region coding sequences (IgG1 allotype G1m(f) containing an Ig kappa chain and an E430G mutation (EU numbering) in the heavy chain). During this process, undesired unpaired cysteines in some antibody light chains were replaced with serine.

[0314] Recombinant chimeric antibodies were produced in HEK293 cells by transient cotransfection of heavy chain (HC) and light chain (LC)-encoding expression vectors using an automated procedure on a Tecan Freedom Evo platform. Immunoglobulins were purified from cell supernatants using affinity purification (Protein A) on a Dionex Ultimate 3000 HPLC system.

[0315] The reactivity of the produced chimeric (VH rabbit, Fc human) monoclonal antibody (mAb) containing the mutation E430G was again analyzed for binding to CHO-CD37 or CHO-mfCD37 cells, as well as for binding to the human lymphoma cell line Daudi and functionality in a CDC assay on Daudi cells.

[0316] Example 2: Humanization of rabbit chimeric antibodies Generation of humanized antibody sequences Humanized antibody sequences were generated from rabbit antibodies, rabbit anti-CD37-004, -005, -010, and -016, at Antitope (Cambridge, UK). The humanized antibody sequences were generated using germline humanization (CDR grafting) technology. Humanized V-region genes were designed based on human germline sequences with the greatest homology to the VH and Vκ amino acid sequences of rabbit and mouse antibodies. For each rabbit antibody, a set of four to six VH and a set of four or five Vκ (VL) germline humanized V-region genes were designed.

[0317] A structural model of the rabbit antibody V region was generated using the Swiss PDB and analyzed to identify amino acids in the V region framework that may be important for antibody binding. These amino acids were noted for incorporation into one or more variant CDR-grafted antibodies.

[0318] The heavy and light chain V region amino acid sequences were compared against a database of human germline V and J segment sequences to identify heavy and light chain human sequences with the greatest degree of homology for use as human variable domain frameworks. The germline sequences used as the basis for humanization design are shown in Table 2.

[0319] Table 2. Best-matched human germline V and J segment sequences TIFF2025121994000012.tif60165

[0320] Next, a series of humanized heavy and light chain V regions were designed by grafting CDRs onto the framework and, if necessary, backmutating residues that may be important for antibody binding (identified by structural modeling) to rabbit residues. Antitope's proprietary in silico technology, iTope™, and TCED™ (T Cell Epitope Database) (Perry, LCA, Jones, TD, and Baker, MP. New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development (2008). Drugs in R&D 9(6): 385-396; Bryson, CJ, Jones, TD, and Baker, MP. Prediction of Immunogenicity of Therapeutic Proteins (2010). Biodrugs 24(1): 1-8) were then used to select variant sequences with the lowest occurrence of potential T cell epitopes. Finally, the nucleotide sequences of the designed variants were codon-optimized.

[0321] For antibody IgG1-016-H5L2, a variant with point mutations in the variable domain to replace the free cysteines was generated: IgG1-016-H5L2-LC90S (also generated with additional F405L and E430G mutations). This variant was generated by gene synthesis (Geneart).

[0322] The variable region sequences of the humanized CD37 antibodies are shown in the sequence listing herein and in Table 1 above.

[0323] Example 3: Generation of bispecific antibodies Bispecific IgG1 antibodies were generated by Fab arm exchange under controlled reducing conditions. The basis of this method is the use of complementary CH3 domains to promote heterodimer formation under specific assay conditions described in WO2011 / 131746. F405L and K409R (EU numbering) mutations were introduced into the CD37 antibody to create an antibody pair with complementary CH3 domains. In certain cases, F405L and K409R were combined with the E430G mutation.

[0324] To generate bispecific antibodies, two parental complementary antibodies (each at a final concentration of 0.5 mg / mL) were incubated with 75 mM 2-mercaptoethylamine HCl (2-MEA) in a total volume of 100 μL of TE for 5 h at 31°C. The reduction reaction was stopped by removing the reducing agent 2-MEA using a spin column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol.

[0325] Example 4: Expression constructs for antibodies, transient expression and purification For antibody expression, the VH and VL sequences were cloned into an expression vector (pcDNA3.3) containing the appropriate constant heavy chain (HC) region in the case of VH, in certain cases containing F405L or K409R mutations and / or E345R or E430G mutations, and the light chain (LC) region in the case of VL.

[0326] The antibody was expressed as IgG1κ. Plasmid DNA mixtures encoding both the heavy and light chains of the antibody were transiently transfected into Expi293F cells (Life Technologies, USA) using 293fectin (Life Technologies) essentially as described by Vink et al. (Vink et al., Methods, 65(1), 5-10, 2014). The antibody was then purified by immobilized protein G chromatography.

[0327] The following antibodies were used in the examples:

[0328] Wild type IgG1 antibody: IgG1-004-H5L2 (having the VH and VL sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 5) IgG1-005-H1L2 (having the VH and VL sequences set forth in SEQ ID NO: 8 and SEQ ID NO: 12) IgG1-010-H5L2 (having the VH and VL sequences set forth in SEQ ID NO: 15 and SEQ ID NO: 19) IgG1-016-H5L2 (having the VH and VL sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 26) IgG1-G28.1 (having the VH and VL sequences set out in SEQ ID NO: 39 and SEQ ID NO: 43 - based on SEQ ID NOs: 1 and 3 of EP2241577) IgG1-G28.1-K409R-delK (also contains the C-terminal heavy chain mutation 445-PG-446) IgG1-37.3 (having the VH and VL sequences set forth in SEQ ID NO: 46 and SEQ ID NO: 50 - based on SEQ ID NOs: 55 and 72 of WO2011 / 112978) IgG1-b12 (having the VH and VL sequences set forth in SEQ ID NO: 32 and SEQ ID NO: 36 - based on the gp120-specific antibody b12 [Barbas, CF. J Mol Biol. 1993 Apr 5;230(3):812-23])

[0329] IgG1 antibody with the Fc-Fc interaction-enhancing mutation E430G: IgG1-004-H5L2-E430G IgG1-005-H1L2-E430G IgG1-010-H5L2-E430G IgG1-016-H5L2-E430G IgG1-G28.1-E430G IgG1-37.3-E430G IgG1-b12-E430G IgG1-005-H1L2-K409R-E430G IgG1-010-H5L2-K409R-E430G IgG1-016-H5L2-F405L-E430G IgG1-016-H5L2-LC90S-F405L-E430G IgG1-004-E430G IgG1-005-E430G IgG1-010-E430G IgG1-016-E430G

[0330] IgG1 antibody with the Fc-Fc interaction-enhancing mutation E430S: IgG1-010-H5L2-K409R-E430S IgG1-016-H5L2-F405L-E430S

[0331] IgG1 antibody with the Fc-Fc interaction-enhancing mutation E345K: IgG1-010-H5L2-K409R-E345K IgG1-016-H5L2-F405L-E345K

[0332] IgG1 antibody with the Fc-Fc interaction enhancing mutation E345R: IgG1-G28.1-E345R IgG1-b12-E345R IgG1-010-H5L2-K409R-E345R IgG1-016-H5L2-F405L-E345R

[0333] bispecific antibody bsIgG1-016-H5L2-F405L×IgG1-IgG1-005-H1L2-K409R bsIgG1-016-H5L2-F405L×IgG1-010-H5L2-K409R

[0334] Bispecific antibody with Fc-Fc interaction enhancing mutation E430G: bsIgG1-016-H5L2-LC90S-F405L-E430G×005-H1L2-K409R-E430G bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G bsIgG1-b12-F405L-E430G×005-H1L2-K409R-E430G

[0335] IgG1 antibody with the FcγR interaction-enhancing mutation S239D-I332E: IgG1-G28.1-S239D-I332E

[0336] Example 5: Introduction of Fc-Fc interaction enhancing mutations into CD37 antibodies results in a novel enhanced ability to induce complement-dependent cytotoxicity (CDC) Determination of complement-dependent cytotoxicity (CDC) In the first experiment, tumor cells (AllCells, California, USA) from an untreated CLL patient were resuspended in RPMI containing 0.2% BSA (bovine serum albumin) and cultured at 0.2 × 10 5Cells were seeded into a polystyrene 96-well round-bottom plate (Greiner Bio-One Cat # 650101) at a density of 1000 cells / well (40 μL / well), and 40 μL of a concentration series of IgG1-G28.1-K409R-delK, IgG1-G28.1-E345R, or IgG1-b12-E345R (0.003-10 μg / mL final antibody concentration) was added. IgG1-b12-E345R (based on the gp120-specific antibody b12 [Barbas, C.F. J. Mol. Biol. 1993 Apr 5;230(3):812-23]) was used as a negative control. It should be noted that in the case of IgG1-G28.1-K409R-delK, the K409R mutation had no effect on binding or CDC induction capacity. Similarly, the delK(445-PG-446) mutation, which was introduced into the antibody to facilitate biochemical analysis, did not affect its ability to bind to the target or induce CDC (see below).

[0337] After incubation (10 min at room temperature with shaking), 20 μL of pooled normal human serum (NHS Cat # M0008 Sanquin, Amsterdam, The Netherlands) was added to each well as a complement source, and the plate was incubated at 37°C for 45 min. The reaction was stopped by chilling the plate on ice. Next, propidium iodide (PI; 10 μL of a 10 μg / mL solution; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was added, and lysis was detected by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (FACS Canto II; BD Biosciences). Graphs were generated using the best-fit values of a nonlinear dose-response fit with log-transformed concentrations in GraphPad Prism V6.04 software (GraphPad Software, San Diego, CA, USA).

[0338] In a second experiment, tumor cells from another untreated CLL patient (AllCells, California, USA) were resuspended in RPMI containing 0.2% BSA and diluted to 0.5 × 10 5 Cells were seeded at a density of 1000 cells / well (30 μL / well) into a polystyrene 96-well round-bottom plate (Greiner bio-one Cat # 650101), and 50 μL of a concentration series of IgG1-G28.1, IgG1-G28.1-E430G, or IgG1-b12 (0.003–10 μg / mL final antibody concentrations in 3.33× serial dilutions) was added. After incubation (15 min at room temperature), 20 μL of pooled normal human serum (NHS Cat # M0008, Sanquin, Amsterdam, The Netherlands) was added to each well as a complement source, and the plate was incubated at 37°C for 45 min. The reaction was stopped by chilling the plate on ice. Next, propidium iodide (PI; 20 μL of a 10 μg / mL solution; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was added, and lysis was detected by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (FACS Canto II; BD Biosciences). Graphs were generated using the best-fit values of a nonlinear dose-response fit with log-transformed concentrations in GraphPad Prism V6.04 software (GraphPad Software, San Diego, CA, USA).

[0339] Figures 1A and 1B show that the CD37 antibody G28.1 without the Fc-Fc interaction-enhancing E345R or E430G mutations (IgG1-G28.1 or IgG1-G28.1-K409R-delK) did not induce CDC in primary tumor cells derived from CLL patients, whereas G28.1 with the Fc-Fc interaction-enhancing mutations E345R or E430G (IgG1-G28.1-E345R or IgG1-G28.1-E430G) induced profound dose-dependent CDC in primary CLL cells.

[0340] Quantitative measurement of cell surface antigens by flow cytometry (Qifi) The Human IgG Calibrator Kit (Biocytix Cat # CP010) was used to measure CD37 and membrane complement regulatory protein (mCRP; CD46, CD55, and CD59) expression levels on CLL tumor cells. Briefly, tumor cells from CLL patients (as in the first experiment above) resuspended in RPMI containing 0.2% BSA were cultured at 0.5 x 10 5 Cells were seeded at a density of 30 μL / well into a polystyrene 96-well round-bottom plate (Greiner bio-one Cat. # 650101) and centrifuged. 50 μL of CD37 (Abcam, cat. no. 76522) or control mouse antibody (Purified Mouse IgG1κ Isotype Control, Clone MOPC-21; BD cat. no. 555746) was added. After incubation (30 min at 4°C), 50 μL of calibration beads was added to separate wells. After washing the beads and cells twice (150 μL of FACS buffer, centrifuged at 300 × g for 3 min at 4°C between washes), 50 μL / well of the secondary antibody (FITC-conjugated) dilution provided in the Human IgG Calibrator Kit was added. After incubation in the dark (45 min at 4°C), cells were washed twice with FACS buffer, resuspended in 35 μL of FACS buffer, and analyzed by flow cytometry (Intellicyt iQue™ Screener). Antigen amounts were determined by calculating antibody binding capacity based on a calibration curve according to the manufacturer's guidelines.

[0341] Figure 2 shows that CD37 was highly expressed in primary tumor cells from this CLL patient, who also had normal mCRP expression levels.

[0342] Example 6: Binding of CD37 antibodies and their variants to cell surface-expressed CD37 Binding to cell surface-expressed CD37 (Daudi cells, CHO cells expressing cynomolgus monkey CD37) was measured by flow cytometry. Cells resuspended in RPMI containing 0.2% BSA were seeded at 100,000 cells / well into a polystyrene 96-well round-bottom plate (Greiner Bio-One Cat # 650101) and centrifuged at 300 x g for 3 minutes at 4°C. Serial dilutions of CD37 or control antibodies (0.003–10 μg / mL final antibody concentrations in 3.33x serial dilutions) were added, and the cells were incubated for 30 minutes at 4°C. Plates were washed twice with FACS buffer (PBS / 0.1% BSA / 0.01% sodium azide) and centrifuged. The cells were then incubated with R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; cat #: 109-116-098) diluted 1 / 100 in PBS / 0.1% BSA / 0.01% sodium azide for 30 minutes at 4°C. Cells were washed twice with FACS buffer, centrifuged, resuspended in 30 μL of FACS buffer, and analyzed by measuring mean fluorescence intensity using an Intellicyt iQue™ screener (Westburg). Binding curves were generated using nonlinear regression (sigmoidal dose-response, variable slope) analysis in GraphPad Prism V6.04 software (GraphPad Software, San Diego, CA, USA).

[0343] Binding to Daudi cells Figure 3 shows that the humanized CD37 antibodies IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2, and IgG1-016-H5L2 exhibited dose-dependent binding to Daudi cells. Introduction of the Fc-Fc interaction-enhancing E430G mutation into these antibodies, and in the case of IgG1-005-H1L2, the additional K409R mutation, did not affect binding.

[0344] Figure 4 shows that introduction of the E430G mutation into IgG1-G28.1 or IgG1-37.3 did not affect binding to Daudi cells.

[0345] For antibody IgG1-016-H5L2, a variant with a point mutation in the variable domain to replace a free cysteine in the light chain was generated: IgG1-016-H5L2-LC90S. This variant was also generated with the additional F405L and E430G mutations, which have previously been shown not to affect target binding properties. Figure 5 shows that IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G, and IgG1-016-H5L2-LC90S-F405L-E430G all exhibited equivalent binding to Daudi cells, indicating that the LC90S mutation did not affect binding.

[0346] Binding to CHO cells expressing cynomolgus monkey CD37 Binding to CHO cells expressing cynomolgus monkey CD37 was measured by flow cytometry using the method described above. Figure 6 shows that IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G, and IgG1-016-H5L2-E430G exhibited dose-dependent binding to CHO cells expressing cynomolgus monkey CD37. IgG1-G28.1 and IgG1-28.1-E430G did not bind to CHO cells expressing cynomolgus monkey CD37.

[0347] Example 7: Identification of CD37 antibodies that do not compete for binding to CD37 (Lack of) binding competition - measured by flow cytometry CD37 antibody was labeled with Alexa Fluor 488 NHS Ester (succinimidyl ester). 1 mg of CD37 antibody (dissolved in PBS) was transferred to a 1 ml microcentrifuge vial (reaction vial). The pH was increased by adding 10% by volume of 1 M sodium bicarbonate buffer (pH 9). Immediately prior to use, 1 mg of Alexa Fluor 488 NHS Ester (adjusted to room temperature) was dissolved in 100 μL of DMSO. The labeling reaction was initiated by adding 10 μL of fresh Alexa dye solution per mg of antibody. The reaction vials were capped and gently mixed by inversion. After a 1-hour incubation at room temperature, the reactions were quenched by adding 50 μL of 1 M Tris to each reaction vial. Unreacted dye was removed from the Alexa-labeled antibody by gel filtration using a BioRad PDP10 column equilibrated with borate-buffered saline according to the manufacturer's instructions. The Alexa-labeled antibody was stored at 4°C and protected from light.

[0348] Binding competition between different CD37 antibodies was measured by flow cytometry. Raji cells (ATCC, CCL-86) were cultured at 1 × 10 7The cells were resuspended in Raji medium (RPMI 1640, 10% FBS, 100 U / mL penicillin, 100 μg / mL streptomycin, 10 mM HEPES, and 1 mM pyruvate) at a concentration of 100 cells / mL. A 30 μL aliquot of the cell suspension was then transferred to a FACS tube along with a 30 μL aliquot of unlabeled antibody solution (40 μg / mL final concentration). The mixture was incubated at 37°C for 15 minutes with gentle shaking. A488-labeled antibody dilutions were then prepared, and after incubation, 10 μL of labeled antibody (final concentration 4 μg / mL) was transferred to the FACS tube containing the unlabeled antibody and cells. The mixture was incubated at 37°C for 15 minutes with gentle shaking. After incubation, samples were quenched by adding 4 ml of ice-cold PBS, centrifuged at 2000 rpm for 3 minutes at 4°C, aspirated twice, and then resuspended in 125 μL of PBS. Binding competition was analyzed by measuring mean fluorescence intensity using a BD FACSCalibur (BD Biosciences). For quantification, fluorescence intensity was converted to MESF (molecules of soluble fluorescence).

[0349] Figures 7A and 8 show that pre-incubation of Raji cells with IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G blocked the subsequent binding of IgG1-005-H1L2-E430G and IgG1-010-H5L2E430G, but did not block the subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, and IgG1-016-H5L2-E430G.

[0350] Pre-incubation of Raji cells with IgG1-004-H5L2-E430G substantially reduced the subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, and IgG1-016-H5L2-E430G, but did not reduce the subsequent binding of IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.

[0351] Preincubation of Raji cells with IgG1-016-H5L2-E430G blocked the subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, and IgG1-016-H5L2-E430G, but did not block the subsequent binding of IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.

[0352] Preincubation of cells with IgG1-37.3-E430G blocked subsequent binding of all antibodies tested, but as noted above, preincubation with either IgG1-005-H1L2-E430G or IgG1-010-H5L2-E430G did not block binding of IgG1-37.3-E430G.

[0353] Preincubation of cells with IgG1-G28.1-E430G blocked the subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, and IgG1-016-H5L2-E430G, but did not block the subsequent binding of IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.

[0354] (Lack of) binding competition - measured by functional screening using the CDC assay To determine whether non-cross-blocking CD37 antibodies exhibit enhanced CDC when combined and to confirm the feasibility of functionally combining non-cross-blocking CD37 antibodies, CDC assays were performed using individual CD37 antibodies and their combinations.

[0355] Raji cells resuspended in RPMI containing 0.2% BSA were added to 1 × 10 5Cells were seeded into a polystyrene 96-well round-bottom plate (Greiner bio-one Cat # 650101) at a density of 30 μL / well, and 50 μL of humanized CD37 antibodies, their variants, or combinations thereof, or the control antibody IgG1-b12 was added (final antibody concentration: 10 μg / mL, combination: 5 + 5 μg / mL). After incubation (15 minutes at room temperature with shaking), 20 μL of pooled normal human serum (NHS Cat # M0008 Sanquin, Amsterdam, The Netherlands) was added to each well, and the plate was incubated at 37°C for 45 minutes. The plate was centrifuged (3 minutes, 1200 rpm), and the supernatant was discarded. Propidium iodide (PI; 30 μL of a 1.67 μg / mL solution; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was added, and lysis was detected by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (Intellicyt iQue™ screener, Westburg). Data were analyzed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA).

[0356] Figures 7B and C show that the combinations of IgG1-004-H5L2 + IgG1-010-H5L2 (with or without the E430G mutation) and IgG1-005-H1L2 + IgG1-016-H5L2 (with or without the E430G mutation) induced enhanced CDC compared to their individual counterparts. The combination of IgG1-004-H5L2 + IgG1-016-H5L2 (with or without the E430G mutation) did not induce enhanced CDC compared to their individual counterparts.

[0357] Figures 7D and E show that the combinations of IgG1-004-H5L2 + IgG1-005-H1L2 (with or without the E430G mutation) and IgG1-010-H5L2 + IgG1-016-H5L2 (with or without the E430G mutation) induced enhanced CDC compared to their individual counterparts. The combination of IgG1-005-H1L2 + IgG1-010-H5L2 (with or without the E430G mutation) did not induce enhanced CDC compared to their individual counterparts.

[0358] Figures 7F and G show that the combinations IgG1-37.3 + IgG1-005-H1L2 (with or without the E430G mutation) and IgG1-37.3 + IgG1-010-H5L2 (with or without the E430G mutation) induced enhanced CDC compared to their individual counterparts.

[0359] Thus, functional combination studies confirmed the results of the described binding competition studies for CD37 and demonstrated that non-cross-blocking CD37 antibodies can be functionally combined.

[0360] Example 8: Introduction of Fc-Fc interaction-enhancing mutations into a humanized CD37 antibody results in a novel enhanced ability to induce complement-dependent cytotoxicity (CDC) Daudi cells resuspended in RPMI containing 0.2% BSA were cultured at 1 × 10 5Cells were seeded at a density of 30 μL / well into a polystyrene 96-well round-bottom plate (Greiner Bio-One Cat # 650101), and 50 μL of a concentration series of humanized CD37 antibodies and their variants or the control antibody IgG1-b12 (0.003–10 μg / mL final antibody concentration in a 3.33× serial dilution) was added. After incubation (15 min at room temperature), 20 μL of pooled normal human serum (NHS Cat # M0008, Sanquin, Amsterdam, The Netherlands) was added to each well, and the plate was incubated at 37°C for 45 min. The plate was centrifuged (3 min, 1200 rpm), and the supernatant was discarded. Propidium iodide (PI; 30 μL of a 1.67 μg / mL solution; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was added, and lysis was detected by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry (Intellicyt iQue™ screener, Westburg). Graphs were generated using best-fit values from a nonlinear dose-response fit with log-transformed concentrations in GraphPad Prism V6.04 software (GraphPad Software, San Diego, CA, USA).

[0361] Figure 9 shows that IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2, and IgG1-016-H5L2 did not induce CDC in Daudi cells. Upon introduction of the Fc-Fc interaction-enhancing E430G mutation, these antibodies (IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G, and IgG1-016-H5L2-E430G) induced profound dose-dependent CDC in Daudi cells.

[0362] Figure 10A shows that IgG1-G28.1 and IgG1-37.3 did not induce CDC in Daudi cells. Upon introduction of the Fc-Fc interaction-enhancing E430G mutation, these antibodies (IgG1-G28.1-E430G and IgG1-37.3-E430G) induced profound, dose-dependent CDC in Daudi cells.

[0363] For antibody IgG1-016-H5L2, a variant with a point mutation in the variable domain to replace a free cysteine in the light chain was generated: IgG1-016-H5L2-LC90S. In addition, this variant was also generated with the F405L mutation (previously shown not to affect target binding or CDC) and the Fc-Fc interaction-enhancing E430G mutation. Figure 11 shows that IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G, and IgG1-016-H5L2-LC90S-F405L-E430G all exhibited comparable activity in an in vitro CDC assay, thus demonstrating that the LC90S mutation did not affect the ability to induce CDC. IgG1-016-H5L2 did not induce CDC in Daudi cells.

[0364] Furthermore, introduction of other Fc-Fc interaction-enhancing mutations, E345K, E345R, E430S, and RRGY, into IgG1-010-H5L2 and IgG1-016-H5L2 caused profound CDC in Daudi cells. Figures 10B and 10C show that the maximum lysis of Daudi cells was comparable for all Fc-Fc interaction-enhancing mutations tested.

[0365] Example 9: Bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations are more potent at inducing CDC than monospecific bivalent CD37 antibodies with Fc-Fc interaction-enhancing mutations due to monovalent binding and dual epitope targeting The F405L or K409R mutation was introduced into a humanized CD37 antibody containing the E430G mutation to enable the generation of a bispecific antibody (bsIgG1) with two CD37-specific Fab arms that do not compete for binding to CD37. The ability of the bispecific CD37 antibody containing the E430G mutation to induce CDC was measured as described above and compared with the ability of a CD37 monospecific bivalent antibody containing the E430G mutation, a combination of two CD37 monospecific bivalent antibodies containing the E430G mutation that do not compete for binding to CD37 (the final concentration of the combined antibodies is the same as the concentration of each bispecific antibody), a monovalent CD37 antibody containing the E430G mutation (i.e., a bispecific antibody containing one CD37-specific Fab arm and one non-binding Fab arm derived from IgG1-b12 and containing the E430G mutation), or a combination of two monovalent CD37 antibodies containing the E430G mutation that do not compete for binding to CD37.

[0366] CDC in Daudi cells Figure 12A shows that bsIgG1-016-H5L2-LC90S-F405L-E430G x 005-H1L2-K409R-E430G was more potent than either IgG1-005-H1L2-E430G or IgG1-016-H5L2-E430G in inducing CDC in Daudi cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430G x 005-H1L2-K409R-E430G was also more potent than the combination of IgG1-005-H1L2-K409R-E430G + IgG1-016-H5L2-F405L-E430G. The monovalent CD37-binding antibodies bsIgG1-b12-F405L-E430G×005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G also induced CDC in Daudi cells, but less efficiently than bsIgG1-016-H5L2-LC90S-F405L-E430G×005-H1L2-K409R-E430G.

[0367] Figure 12B shows that bsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G was more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDC in Daudi cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G was also more potent than the combination of IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G. The monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G also induced CDC in Daudi cells, whereas bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G was significantly lower than bsIgG1-0 bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G was less potent than 16-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G was equally potent than bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G.

[0368] The ability to induce CDC by bispecific CD37 antibodies containing the E430G mutation was also compared to that of bispecific CD37 antibodies without the E430G mutation. Figure 13 shows that bsIgG1-016-H5L2-F405Lx005-H1L2-K409R and bsIgG1-016-H5L2-F405Lx010-H5L2-K409R were able to induce CDC in Daudi cells, but were less potent at doing so than their E430G-containing counterparts bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.

[0369] CDC in OCI-Ly-7 cells Figure 12C shows that the monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G were more potent in inducing CDC in OCI-Ly-7 cells than their monospecific bivalent binding counterparts IgG1-016-H5L2-E430G and IgG1-010-H5L2-E430G. The monovalent binding antibody (bsIgG1-016-H5L2-LC90S-F405L-E430G x b12-K409R-E430G + bsIgG1-b12-F405L-E430G x 010-H5L2-K409R-E430G) was more potent than the bivalent antibody combination (IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G), as demonstrated by a consistently lower EC50 in two independent experiments (Figure 12D). Furthermore, bsIgG1-016-H5L2-LC90S-F405L-E430G × 010-H5L2-K409R-E430G was more potent than the bivalent antibody combination (IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G) in inducing CDC in OCI-Ly-7 cells, as demonstrated by a consistently lower EC50 in three independent experiments (Figure 12E).

[0370] The ability of the monovalent binding antibody combination (bsIgG1-016-H5L2-LC90S-F405L-E430G x b12-K409R-E430G + bsIgG1-b12-F405L-E430G x 010-H5L2-K409R-E430G) to induce CDC in OCI-Ly-7 cells was comparable to that of bsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G.

[0371] CDC in primary CLL tumor cells The ability of bispecific CD37 antibodies containing the E430G mutation to induce CDC in tumor cells from CLL patients was measured as described above and compared with the ability of CD37 antibodies containing the E430G mutation or combinations of CD37 antibodies containing the E430G mutation or monovalent CD37 antibodies containing the E430G mutation.

[0372] Figure 14A shows that bsIgG1-016-H5L2-LC90S-F405L-E430G x 005-H1L2-K409R-E430G was more potent than either IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F405L-E430G in inducing CDC in primary CLL tumor cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430G x 005-H1L2-K409R-E430G was also more potent than the combination of IgG1-005-H1L2-K409R-E430G + IgG1-016-H5L2-F405L-E430G. The monovalent binding antibodies bsIgG1-b12-F405L-E430G×005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G also induced CDC in primary CLL tumor cells, but did so less efficiently than bsIgG1-016-H5L2-LC90S-F405L-E430G×005-H1L2-K409R-E430G.

[0373] Figure 14B shows that bsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G was more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDC in primary CLL tumor cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G was also more potent than the combination of IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G. The monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G also induced CDC in primary CLL tumor cells, whereas bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G was significantly less potent than bsIgG1- bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G was less potent than 016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G, and bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G was equally potent than bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G.

[0374] Example 10: Bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations induce CDC in diverse B cell lymphoma cell lines displaying a broad range of CD37 expression The ability of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G at 10 μg / mL to induce CDC in various B-cell lymphoma cell lines derived from various B-cell lymphoma subtypes was measured (as described above). The expression levels of CD37 molecules on the cell surface of these cell lines were measured by quantitative flow cytometry as described above.

[0375] Table 3 provides an overview of the cell lines tested.

[0376] Table 3. B-cell lymphoma cell lines TIFF2025121994000013.tif88149

[0377] Figure 15 shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G induced CDC in a broad range of B cell lymphoma cell lines derived from different B cell lymphoma types.

[0378] Example 11: Bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations are more potent in inducing antibody-dependent cellular cytotoxicity (ADCC) Labeling of target cells The ability of CD37 antibodies to induce ADCC was measured by a chromium release assay. Daudi or Raji cells (5 × 10 cells) were harvested in 1 mL of medium (RPMI 1640 supplemented with 10% donor bovine serum with iron (DBSI; ThermoFischer, Cat. # 10371029) and penicillin / streptomycin mixture (pen / strep) plus 100 μCi of 51Cr (Chromium-51; PerkinElmer, Cat. # NEZ030005MC). 6 The cells were incubated for 1 hour with shaking in a water bath at 37°C. After washing the cells (twice in PBS, 1500 rpm, 5 min), the cells were resuspended in RPMI 1640 / 10% DBSI / pen / strep and counted by trypan blue exclusion. The cells were cultured at 1 x 10 5 The mixture was diluted to a density of 1000 cells / mL.

[0379] Preparation of effector cells Peripheral blood mononuclear cells (Sanquin, Amsterdam, The Netherlands) from healthy volunteers were isolated from 45 mL of freshly drawn heparinized blood (buffy coat) by Ficoll density gradient centrifugation (BioWhittaker; lymphocyte separation medium, cat. 17-829E) according to the manufacturer's instructions. Cells were resuspended in RPMI 1640 / 10% DBSI / pen / strep and counted by trypan blue exclusion. A total of 1 × 10 cells were collected. 7 The mixture was diluted to a density of 1000 cells / mL.

[0380] ADCC assay procedure 51 Fifty microliters of Cr-labeled target cells were pipetted into a 96-well round-bottom microtiter plate (Greiner Bio-One; Cat # 650101) and 50 μL of a concentration series of CD37 or control antibodies (1.5–5,000 ng / mL final concentration in 3-fold dilutions) diluted in RPMI 1640 / 10% DBSI / pen / strep was added. Cells were incubated for 15 min at room temperature (RT), and 50 μL of effector cells were added, resulting in an effector:target ratio of 100:1. Cells were incubated for 4 h at 37°C and 5% CO2. To measure maximum lysis, 50 μL (5,000 cells) of 51Cr-labeled Daudi cells were incubated with 100 μL of 5% Triton-X100; to measure spontaneous lysis (background lysis), 5,000 51Cr-labeled Daudi cells were incubated in 150 μL of medium without any antibody or effector cells. The level of antibody-independent cytolysis was measured by incubating 5,000 Daudi cells with 500,000 PBMCs without antibody. The plate was centrifuged (1200 rpm, 10 min), and 25 μL of the supernatant was transferred to 100 μL of Microscint-40 solution (Packard, Cat # 6013641) in a 96-well plate. The plate was sealed and shaken at 800 rpm for 15 min, and the released 51Cr was counted using a scintillation counter (TopCount®, PerkinElmer). The specific lysis rate (%) was calculated as follows: % specific lysis = (sample cpm - spontaneous lysis cpm) / (maximum lysis cpm - spontaneous lysis cpm) where cpm is counts per minute.

[0381] Figure 16A shows that bsIgG1-016-H5L2-LC90S-F405L-E430G x 005-H1L2-K409R-E430G was more potent than either IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F409L-E430G or the combination of IgG1-005-H1L2-K409R-E430G + IgG1-016-H5L2-F405L-E430G in inducing ADCC in Daudi cells.

[0382] Figure 16B shows that bsIgG1-016-H5L2-LC90S-F405L-E430G x 010-H5L2-K409R-E430G was more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G or the combination of IgG1-010-H5L2-E430G + IgG1-016-H5L2-E430G in inducing ADCC in Daudi cells.

[0383] Figure 16C shows similar results to Figure 16B for PBMCs from a different donor and additionally shows that bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G was more potent than the monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430G×b12-K409R-E430G and bsIgG1-b12-F405L-E430G×010-H5L2-K409R-E430G in inducing ADCC in Raji cells.

[0384] Example 12: Bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations induce potent ex vivo CDC in primary tumor cells derived from patients with various B-cell malignancies The CDC efficacy of bsIgG1-016-H5L2-LC90S-F405L×010-H5L2-K409R-E430G was analyzed using patient-derived primary tumor cells from five different B-cell malignancies: chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and non-Hodgkin's lymphoma (not further specified). All patient samples were obtained and stored after written informed consent using protocols approved by the VUmc Medical Ethics Committee in accordance with the Declaration of Helsinki. Patient bone marrow mononuclear cells (BMNCs) or peripheral blood mononuclear cells (PBMCs) were isolated from patient bone marrow aspirates or peripheral blood samples by density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare). Cells were either used immediately or stored in liquid nitrogen until further use.

[0385] Patient lymph node tissue was dissected into small pieces and collected in α-MEM medium (ThermoFischer Scientific, Waltham, MA) containing 1% penicillin-streptomycin, 0.2% heparin, and 5% platelet lysate, and incubated overnight at 37°C. After incubation, the supernatant (the non-stromal cell compartment containing tumor cells) was collected, and cells were strained using a 70 μM Easy Strainer (Greiner Bio-one). Cells were counted, resuspended in RPMI 1640 medium containing 25% heat-inactivated FBS and 10% DMSO, and frozen in liquid nitrogen until further use.

[0386] We used the QifiKit (DAKO, cat. no. K007811) to measure CD37 and membrane complement regulatory protein (mCRP; CD46, CD55, and CD59) expression levels on isolated patient cells. Cells were incubated with purified antibodies CD37 (BD, cat. no. 555456), CD46 (BioLegend, cat. no. 352404), CD55 (BioLegend, cat. no. 311302), CD59 (BioLegend, cat. no. 304702), and b12 (Genmab) for 30 minutes at 4°C. Following this, we used the method provided by the QifiKit manufacturer. After the final step of the QifiKit procedure, cells were incubated with lymphoma cell-specific markers to enable tumor cell identification. Figure 17 shows expression levels by indication.

[0387] Patient-derived tumor cells were opsonized with bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G at 10 μg / mL or 100 μg / mL in the presence of 20% pooled NHS, and CDC induction was assessed. The following cell markers were used to identify different cell populations: CD45-KO (Beckman Coulter B36294), CD19-PC7 (Beckman Coulter, cat. no. IM3628), CD3-V450 (BD, cat. no. 560365), CD5-APC (BD, cat. no. 345783), CD5-PE (DAKO, cat. no. R084201), CD10-APC-H7 (BD, cat. no. 655404), CD10-PE (DAKO, cat. no. R084201), CD23-FITC (Biolegend, cat. no. 338505), lambda-APC-H7 (BD, cat. no. 656648), kappa-PE (DAKO, cat. no. R043601) and lambda-FITC (Emelca Bioscience CYT-LAMBF). Within the CD45+ cell population, malignant B cells were identified by different markers depending on the indication: CD3- / CD19+ / CD5+ (CLL), CD3- / CD19+ / CD10+ (FL, DLBCL), and CD3- / CD19+ / CD5+ / CD23- (MCL). If malignant B cells could not be identified based on these markers, they were identified based on clonality using kappa / lambda staining. In some samples, malignant B cells could not be identified based on clonality. In such cases, the entire B cell population was evaluated without distinguishing between normal and malignant B cells. Killing was calculated as the fraction (%) of 7-aminoactinomycin D (7-AAD; BD, cat. no. 555816)-positive malignant B cells as measured by an LSRFortessa flow cytometer (BD Biosciences, San Jose, CA).

[0388] Figure 18 shows that bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G was highly potent (>50% lysis) in inducing CDC in tumor cells from patients with CLL, FL, MCL, DLBCL, or B-NHL (not further specified). In cells from one patient with relapsed / refractory FL, bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G was less able to induce CDC.

[0389] Example 13: Binding of bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations to human or cynomolgus monkey B cells in whole blood and induction of cytotoxicity in B cells in whole blood Binding to human or cynomolgus monkey B cells Binding to human or cynomolgus monkey B cells was measured in a whole blood binding assay. Heparinized human blood from healthy volunteers was obtained from UMC Utrecht (Utrecht, The Netherlands), and hirudin-treated blood from cynomolgus monkeys was obtained from Covance (Münster, Germany). Blood was aliquoted (35 μL / well) into wells of a 96-well round-bottom plate (Greiner Bio-one, cat. no. 65010). Red blood cells (RBCs) were lysed by adding 100 μL of RBC lysis buffer (10 mM KHC03 [Sigma P9144], 0.1 mM EDTA [Fluka 03620], and 0.15 mM NH4CL [Sigma A5666]) and incubated on ice until RBC lysis was complete. After centrifugation at 300 × G for 3 minutes, cells were incubated with Alexa-488-labeled bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G or Alexa-488-labeled control IgG1 (IgG1-b12) and serial dilutions of directly labeled antibodies (0.014–30 μg / mL final antibody concentration in 3× serial dilutions) for 30 minutes at 4°C to identify B cells (in a mixture of antibodies to further identify blood cell subsets).

[0390] For human blood B cells, the following antibodies were used: TIFF2025121994000014.tif20160

[0391] For cynomolgus monkey blood B cells, the following antibodies were used: TIFF2025121994000015.tif26156

[0392] Cells were pelleted, washed twice in 150 μL of FACS buffer, and resuspended in 150 μL of TO-PRO-3 (final concentration 0.2 μM; Molecular Probes, cat no. T3605). Samples were measured by flow cytometry using an LSR Fortessa flow cytometer. Binding was assessed by the quantification of viable TO-PRO-3. - / CD14 - / CD19 + B cells (human) or viable TO-PRO-3 - / CD14 - / CD19 + / CD20 + Expressed as the geometric mean of A488 fluorescence intensity for B cells (cynomolgus monkeys). Log-transformed data were analyzed using best fit values of a nonlinear dose-response fit in GraphPad PRISM.

[0393] Figure 19 shows the concentration-dependent binding of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in blood from (A) human and (B) cynomolgus monkey as one representative donor / animal. Mean EC for binding to human and cynomolgus monkey B cells. 50 The values were within the same range (0.85 μg / mL ± 0.284 [based on binding to B cells in blood from six human donors] and 0.63 μg / mL ± 0.228 [based on binding to B cells in blood from four cynomolgus monkeys], respectively), indicating that bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G exhibits equivalent binding to human and cynomolgus monkey CD37.

[0394] Cytotoxic effect on human or cynomolgus monkey B cells Cytotoxicity against human or cynomolgus monkey B cells was measured in a whole blood cytotoxicity assay. Hirudin-treated human blood from healthy volunteers was obtained from UMC Utrecht (Utrecht, The Netherlands), and hirudin-treated blood from cynomolgus monkeys was obtained from Covance (Munster, Germany). Blood was aliquoted into wells of a 96-well round-bottom plate at 35 μL / well.

[0395] Serial dilutions of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12 (0.0005–10 μg / mL final antibody concentration in 3× serial dilutions; final volume 100 μL / well) were added. For cytotoxicity assays using human whole blood, the monoclonal FcγR interaction-enhanced CD37-specific antibody IgG1-G28.1-S239D-I332E was included as a reference. Samples were incubated at 37°C for 4 hours. Afterward, red blood cells were lysed as described above, and samples were stained to identify B cells as described above. Cells were pelleted, washed twice in 150 μL of FACS buffer, and resuspended in 150 μL of TO-PRO-3 (final concentration 0.2 μM; Molecular Probes, cat no. T3605). Samples were measured by flow cytometry using an LSR Fortessa flow cytometer. After doublet exclusion, viable TO-PRO-3 - / CD14 - / CD19 + B cells (human) or viable TO-PRO-3 - / CD14 - / CD19 + / CD20 + The percentage of B cells (cynomolgus monkeys) was measured. The percentage of B cell depletion was calculated as follows: percentage of B cell depletion = 100 × [(% B cells in no-Ab control - % B cells in sample) / (% B cells in no-Ab control)]. Log-transformed data were analyzed using the best fit value of a nonlinear dose-response fit in GraphPad PRISM.

[0396] Figure 20 shows the concentration-dependent cytotoxic effect of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G against B cells in the blood of (A) a human and (B) a cynomolgus monkey as one representative donor / animal.

[0397] EC 50 Based on the mean EC20 cytotoxicity against human B cells (in blood from six donors), the ability of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G to induce cytotoxicity was comparable between human and cynomolgus B cells. 50 The mean EC for cytotoxicity against cynomolgus monkey B cells (in blood from four animals) was 0.077 μg / mL ± 0.039. 50 was 0.043 μg / mL ± 0.019.

[0398] Figure 20A also shows the cytotoxic effect of the monoclonal CD37 antibody IgG1-G28.1-S239D-I332E, which has enhanced FcγR interaction, on human B cells from a representative responding donor, which showed lower cytotoxicity than bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G. A maximal B cell depletion of 50% was measured with IgG1-G28.1-S239D-I332E in B cells from three responding donors, whereas no cytotoxic effect of this antibody on B cells from the other three donors was measured. bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G induced cytotoxicity in 93–99% of B cells in 6 / 6 donors. Binding of IgG1-G28.1-S239D-I332E to CD37 expressed on Daudi cells was comparable to that of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G (data not shown).

[0399] Example 14: Potent CDC activity by combining a bispecific CD37 antibody with an Fc-Fc interaction-enhancing mutation and a CD20-specific antibody The combination of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and an anti-CD20 antibody (IgG1-CD20-ofa; ofatumumab) was tested for its ability to induce CDC in patient-derived CLL tumor cells obtained from ConversantBio (Huntsville, Alabama, USA). Patient-derived PBMCs were resuspended in RPMI containing 0.2% BSA (bovine serum albumin) and plated at 0.1 × 10 in a polystyrene 96-well round-bottom plate (Greiner bio-one Cat # 650101). 6 Plates were seeded at a density of 1000 cells / well (30 μL / well), and 50 μL of a concentration series of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G (0.0625-0.05 μg / mL) and IgG1-CD20-ofa (1-8 μg / mL) was added in two-fold dilutions. bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G and IgG1-CD20-ofa were combined at antibody concentrations based on the relative potency (EC50 difference) of each antibody by mixing two concentrations that would achieve the same effect on average separately. IgG1-b12 was used as a negative control.

[0400] After incubation (15 min at room temperature with shaking), 20 μL of pooled normal human serum (NHS Cat # M0008, Sanquin, Amsterdam, The Netherlands) was added to each well as a complement source, and the plate was incubated at 37°C for 45 min. The reaction was stopped by chilling the plate on ice. After centrifugation at 300 × g for 3 min, the cells were washed twice with 150 μL of FACS buffer and incubated with R-phycoerythrin (PE)-conjugated mouse anti-human IgG1-CD19 antibody (clone J3-119, Beckman Coulter, cat no. A07769, diluted 1:50 from stock solution) for 30 min at 4°C to measure tumor B cells and TO-PRO-3 (final concentration 0.2 μM; Molecular Probes, cat no. T3605) for identification of dead cells. Cells were pelleted, washed twice in 150 μL of FACS buffer, and counted by flow cytometry using an LSRFortessa flow cytometer. The percentage of viable cells was calculated as follows: viable cells (%) = 100 × (number of TO-PRO-3 negative events) / (total number of events).

[0401] Figures 21A-D show that both bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G and ofatumumab induced CDC in tumor cells from two CLL patients, with CDC activity increasing with increasing dose levels. Combining bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G with ofatumumab resulted in enhanced CDC activity in both CLL patients tested at all concentrations tested, although these effects were less pronounced at higher antibody concentrations where the single agents induced near-complete cell death (Figures 21A and B). These results indicate that the addition of ofatumumab to bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G can improve CDC-mediated tumor cell death in malignant B cells obtained from CLL patients.

[0402] Example 15: Antitumor activity of bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations in xenograft models of B-cell malignancies Antitumor activity in a subcutaneous JVM-3 human chronic B-cell leukemia xenograft model JVM-3 cells (1 x 10 7 CB17.SCID mice were inoculated with 1000 cells / ml of 10 ... 3 Antibody treatment (three weekly intravenous injections at doses of 0.1, 0.3, 1, 3, or 10 mg / kg; IgG1-b12 was used as a negative control and administered at 10 mg / kg) was initiated when tumor volumes reached a mean volume of 100 mm. Tumor volumes were measured twice weekly using calipers in two dimensions and expressed in mm using the formula: V = (L × W × W) / 2. 3 where V is the tumor volume, L is the tumor length (longest tumor dimension), and W is the tumor width (longest tumor dimension perpendicular to L).

[0403] Figure 22A shows tumor volume over time by treatment group, and Figure 22B shows tumor volume per mouse by treatment group at day 25, when all groups were still intact. Three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G at 1, 3, or 10 mg / kg significantly reduced JVM-3 cell tumor growth, whereas dosing at 0.1 or 0.3 mg / kg had no effect on tumor growth (Mann-Whitney test, p<0.01).

[0404] Antitumor activity in an intravenous Daudi-luc Burkitt lymphoma xenograft model On day 0, SCID mice (CB-17 / IcrHan® Hsd-Prkdcscid; Harlan) were inoculated with Daudi-luc (luciferase-transfected Daudi cells, 2.5 × 10 6Mice were intravenously injected with bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G at 0.1, 0.3, 1, 3, or 10 mg / kg on days 14, 21, and 28. IgG1-b12 was used as a negative control antibody and administered at 10 mg / kg. Tumor growth was assessed weekly (starting on day 2) by bioluminescence imaging (BLI). Mice were intraperitoneally injected with 100 μL of firefly D-luciferin (30 mg / mL; Caliper LifeSciences, cat. no. 119222), and bioluminescence (p / s / cm) was measured using a Biospace Bioluminescence Imaging System (PerkinElmer; mice were photographed from the dorsal side) under isoflurane anesthesia. 2 / sr[number of photons / sec / cm 2 The radiance (units of radians per square radian) was measured.

[0405] Figure 23A shows luciferase activity (bioluminescence, as a measure of tumor volume) over time per treatment group. Figure 23B shows luciferase activity per mouse per treatment group at day 36, when all groups were still intact. Three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G at 0.1, 0.3, 1, 3, or 10 mg / kg significantly reduced the in vivo growth of Daudi-luc cells (one-way ANOVA, Fisher's LSD uncorrected).

[0406] Example 16: Evaluation of plasma clearance of bispecific CD37 antibodies with Fc-Fc interaction-enhancing mutations in SCID mice Eleven- to 12-week-old female SCID mice (CB-17 / IcrHan® Hsd-Prkdcscid; Harlan) (3 mice per group) were intravenously (iv) injected once with 100 μg (5 mg / kg) or 500 μg (25 mg / kg) of bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G or IgG1-b12. Because neither bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G nor IgG1-b12 showed cross-reactivity with mice, this experiment was designed to study antibody clearance in the absence of target-mediated clearance.

[0407] At 10 min, 4 h, 24 h, 2 days, 7 or 8 days, 14 days, and 21 days after antibody administration, 50–100 μL blood samples were collected from the saphenous vein. Blood was collected in heparin-containing vials and centrifuged at 10,000 g for 5 min. Plasma samples were diluted 1:50 (20 μL sample in 980 μL PBSA (PBS supplemented with 0.2% bovine serum albumin (BSA))) for mice administered 5 mg / kg and 1:20 (20 μL sample in 380 μL PBSA) for mice administered 25 mg / kg and stored at -20°C until measurement of mAb concentration.

[0408] Human IgG concentrations were measured using a sandwich ELISA. Mouse mAb anti-human IgG kappa clone MH16 (CLB Sanquin, The Netherlands; cat. no. M 1268) was used as the capture antibody at a concentration of 2 μg / mL, with 100 μL coated onto a 96-well Microlon ELISA plate (Greiner, Germany) overnight at 4°C. After blocking the plate with PBSA for 1 hour at room temperature (RT), samples were added, serially diluted in PBSA, and incubated for 1 hour at RT on a plate shaker. The plate was washed three times with 300 μL of PBST (PBS supplemented with 0.05% Tween 20) and then incubated with goat anti-human IgG immunoglobulin (Jackson, West Grace, PA; cat. no. 109-035-098; 1:10,000 in PBST supplemented with 0.2% BSA) for 1 hour at RT. The plate was again washed three times with 300 μL of PBST and then incubated with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany) under light protection. The reaction was stopped by adding 100 μL of 2% oxalic acid. Absorbance was measured at 405 nm in a microplate reader (Biotek, Winooski, VT). Human IgG concentrations were calculated using the injected material as a reference curve. Purified human IgG1 (The Binding Site, cat. no. BP078) was included as a plate control. Human IgG concentrations (in μg / mL) were plotted (Figures 24A and C), and the AUC (area under the curve) was calculated using Graphpad Prism 6.0. IgG clearance was measured by the formula D*1.000 / AUC, where D is the infusion dose (1 mg / kg), up to the last day of blood sampling (day 21) (FIGS. 24B and D).

[0409] There was no substantial difference in plasma clearance rates between bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G and IgG1-b12, demonstrating that bsIgG1-016-H5L2-LC90S-F405L-E430G×010-H5L2-K409R-E430G exhibits a pharmacokinetic profile comparable to wild-type human IgG1 in the absence of target binding.

[0410] Example 17: Determining the contribution of CD37 amino acid residues to binding of CD37 antibodies using alanine scanning Library Design A CD37 single-residue alanine library was synthesized (Geneart) by individually mutating all amino acid (aa) residues in the extracellular domain of human CD37 (Uniprot P11049) to alanine, except for positions that already contained alanine or cysteine. Cysteines were not mutated to minimize potential disruption of the antigen's structure. The library was cloned into the pMAC expression vector, which contains a CMV / TK-polyA expression cassette, an Amp resistance gene, and a pBR322 origin of replication.

[0411] Library construction and screening Wild-type CD37 and alanine mutants were individually expressed in Freestyle HEK293 cells according to the manufacturer's instructions (Thermo Scientific). Cells were harvested one day after transfection. Approximately 100,000 cells were incubated with 20 μL of Alexa488-conjugated bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G (monovalent binding 010) or Alexa488-conjugated bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G (monovalent binding 016) at a concentration of 3 μg / mL in FACS buffer (PBS + 0.1% (w / v) bovine serum albumin (BSA) + 0.02% (w / v) sodium azide). The cells were incubated for 1 hour at room temperature. Afterwards, the cells were washed twice by adding 150 μL of FACS buffer and removing the supernatant after centrifugation. The cells were resuspended in 20 μL of fresh FACS buffer and stored at 4°C until analysis by flow cytometry using an iQue screener (IntelliCyt). The entire experiment was performed twice.

[0412] Data analysis For each sample, the average antibody binding per cell was measured as the geometric mean fluorescence intensity (gMFI) of the ungated cell population. gMFI is affected by the affinity of the antibody for the CD37 variant and the expression level of the CD37 variant per cell. Because specific alanine mutations can affect the surface expression level of variant CD37, and to generally correct for expression differences between CD37 variants, data were normalized to the binding intensity of a non-competing CD37-specific control antibody (in this example, monovalent binding antibody 010 and monovalent binding antibody 016 were non-competing antibodies, and one antibody was used as a control for the other). The following formula was used: TIFF2025121994000016.tif9128 where "aa position" refers to the specific alanine mutation position in CD37 or wild-type (wt) CD37.

[0413] To represent the loss or gain of antibody binding, a standard score was determined according to the following calculation: TIFF2025121994000017.tif8128 where μ and σ are the mean and standard deviation (SD) of the normalized gMFI of all variants.

[0414] Gains in binding in many cases result from loss of binding of the reference antibody to a particular ala variant. Using these calculations, amino acid positions that result in no loss or gain of binding by a particular antibody when the amino acid is substituted with alanine will receive a z-score of "0," gains in binding will receive a "z-score >0," and losses in binding will receive a "z-score <0." To correct for sample-to-sample variability, only CD37 amino acid residues that had a z-score less than -1.5 were considered "loss-of-binding variants." The gMFI of the control antibody for a particular CD37 variant is the average gMFI コントロールAb Data were excluded from analysis if the mean gMFI was below 2.5 × SD (expression levels were considered insufficient for such CD37 variants).

[0415] Figure 25 shows the "z-scores (fold change)" of CD37 antibodies against CD37 variants with ala mutations at positions 42-131 (according to SEQ ID NO: 94). The results show: The binding of antibody 010 is dependent on at least aa Y182, D189, T191, I192, D194, K195, V196, I197, and P199 of human CD37; The binding of antibody 016 is dependent on at least aa E124, F162, Q163, V164, L165, and H175 of human CD37.

[0416] summary In summary, a bispecific antibody composed of two non-competing CD37-specific antibodies with Fc-Fc interaction-enhancing mutations for target binding exhibited the most favorable combination of CDC and ADCC potency in CD37-positive tumor cells. For both effector mechanisms, the bispecific antibody with Fc-Fc interaction-enhancing mutations exhibited superior potency compared with a combination of two non-competing CD37 antibodies containing Fc-Fc interaction-enhancing mutations or a single CD37 antibody with an Fc-Fc interaction-enhancing mutation.

[0417] Example 18: In vitro evaluation of the CDC activity of a mixture of a novel hexamerization-enhanced CD37 antibody and a clinically established CD20 antibody product on Raji cells The CD37 antibodies IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-E430G, IgG1-005-E430G, IgG1-010-E430G, and IgG1-016-E430G (the latter four are chimeric rabbit / human antibodies) with Fc-Fc interaction-enhancing mutations were tested in vitro for CDC activity using Burkitt's lymphoma Raji cells. The latter four are chimeric rabbit / human antibodies, and the clinically established CD20-targeting monoclonal antibody products MabThera (rituximab; Roche, H0124B08), Arzerra (ofatumumab; Novartis, C656294), and Gazyva (obinutuzumab, GA101; Roche, D287-41A GACD20). Raji cells (ATCC, Cat No. CCL-86) were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS, 1 U / mL penicillin, 1 μg / mL streptomycin, and 4 mM L-glutamine. 6Raji cells were preincubated with antibody in a total volume of 80 μL per well in RPMI / 0.2% BSA on a shaker for 15 minutes at room temperature. NHS was then added to the preincubated cells to a final volume of 100 μL (final antibody concentration 10 μg / mL; 20% NHS), and the cells were incubated at 37°C for 45 minutes. For all antibody concentrations tested, different ratios of the two antibodies in the mixture (1:0, 3:1, 1:1, 1:3, 0:1) were tested. The plates were centrifuged, and the cells were resuspended in 30 μL of PI (2 μg / mL). Killing was calculated as the fraction (%) of PI-positive cells, as measured by flow cytometry on an iQue screener (IntelliCyt). Data were analyzed and plotted using GraphPad Prism software.

[0418] Mixtures of the tested CD37 antibodies with Fc-Fc interaction-enhancing mutations and clinically established CD20 antibody products showed enhanced dose-dependent CDC activity against Raji cells compared to the same concentration of either antibody alone (Figure 8). There was little difference in CDC activity at different tested ratios of the two antibodies in the mixture (1:3, 1:1, or 3:1). These data indicate that a mixture of hexamerization-enhanced CD37 antibodies with Fc-Fc interaction-enhancing mutations plus clinically established CD20 antibody products, such as MabThera, Arzerra (type I CD20 antibody), or Gazyva (type II CD20 antibody), may improve therapeutic potential for patients with B-cell malignancies that are often refractory to standard CD20-targeting treatments alone.

[0419] Sequence information SEQUENCE LISTING <110> Genmab Holding BV <120> BISPECIFIC ANTI-CD37 ANTIBODIES, MONOCLONAL ANTI-CD37 ANTIBODIES AND METHODS OF USE THEREOF <150> US 62 / 479,712 <151> 2017-03-31 <150> PCT / EP2018 / 057836 <151> 2018-03-27 <160> 117 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 1 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Thr Tyr 20 25 30 Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ile Ile Tyr Ser Ser Val Gly Ala Tyr Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Tyr Gly Ala Ser Ser Ser Asp Tyr Ile Phe Ser Leu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 2 Gly Phe Ser Leu Ser Thr Tyr Asp 1 5 <210> 3 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 3 Ile Tyr Ser Ser Val Gly Ala 1 5 <210> 4 <211> 15 <212> PRT <213> Oryctolagus cuniculus <400> 4 Ala Arg Glu Tyr Gly Ala Ser Ser Ser Asp Tyr Ile Phe Ser Leu 1 5 10 15 <210> 5 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 5 Ala Gln Val Leu Thr Gln Ser Pro Ser Pro Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Val Tyr Asn Ser 20 25 30 Gln Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Glu Ala Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gly Glu Phe Ser Cys 85 90 95 Ile Ser Ala Asp Cys Thr Ala Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 6 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 6 Gln Ser Val Tyr Asn Ser Gln Asn 1 5 <210> 7 <211> 13 <212> PRT <213> Oryctolagus cuniculus <400> 7 Gln Gly Glu Phe Ser Cys Ile Ser Ala Asp Cys Thr Ala 1 5 10 <210> 8 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 8 Glu Gln Ser Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Phe Ser Leu Ser Ser Asn 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Tyr Ala Ser Gly Asn Thr Asp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Tyr Leu Lys Ile 65 70 75 80 Thr Ser Pro Thr Ala Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Glu 85 90 95 Gly Ser Val Trp Gly Ala Ala Phe Asp Pro Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 9 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 9 Gly Phe Ser Leu Ser Ser Asn Ala 1 5 <210> 10 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 10 Ile Tyr Ala Ser Gly Asn Thr 1 5 <210> 11 <211> 13 <212> PRT <213> Oryctolagus cuniculus <400> 11 Ala Arg Glu Gly Ser Val Trp Gly Ala Ala Phe Asp Pro 1 5 10 <210> 12 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 12 Ala Tyr Asp Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Ser Ile Ser Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Gln Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Asn Ser Asn 85 90 95 Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 13 <211> 6 <212> PRT <213> Oryctolagus cuniculus <400> 13 Gln Ser Ile Ser Asn Trp 1 5 <210> 14 <211> 12 <212> PRT <213> Oryctolagus cuniculus <400> 14 Gln Gln Gly Tyr Ser Asn Ser Asn Ile Asp Asn Thr 1 5 10 <210> 15 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Tyr Asn 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ile Ile Phe Ala Ser Gly Arg Thr Asp Tyr Ala Ser Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Glu Gly Ser Thr Trp Gly Asp Ala Leu Asp Pro Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 16 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 16 Gly Phe Ser Leu Ser Tyr Asn Ala 1 5 <210> 17 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 17 Ile Phe Ala Ser Gly Arg Thr 1 5 <210> 18 <211> 13 <212> PRT <213> Oryctolagus cuniculus <400> 18 Ala Arg Glu Gly Ser Thr Trp Gly Asp Ala Leu Asp Pro 1 5 10 <210> 19 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 19 Ala Tyr Asp Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Ile Asp Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 His Lys Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr Ser Asn Ser Asn 85 90 95 Ile Asp Asn Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 20 <211> 6 <212> PRT <213> oryctolagus cuniculus <400> 20 Gln Asn Ile Ile Asp Tyr 1 5 <210> 21 <211> 12 <212> PRT <213> oryctolagus cuniculus <400> 21 Gln Gln Gly Tyr Ser Asn Ser Asn Ile Asp Asn Thr 1 5 10 <210> 22 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 22 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Asp Ala Ser Gly Thr Thr Tyr Tyr Ala Thr Trp Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Glu Leu Leu Tyr Phe Gly Ser Ser Tyr Tyr Asp Leu Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 23 <211> 8 <212> PRT <213> Oryctolagus cuniculus <400> 23 Gly Phe Ser Leu Ser Asn Tyr Asn 1 5 <210> 24 <211> 7 <212> PRT <213> Oryctolagus cuniculus <400> 24 Ile Asp Ala Ser Gly Thr Thr 1 5 <210> 25 <211> 14 <212> PRT <213> Oryctolagus cuniculus <400> 25 Ala Arg Glu Leu Leu Tyr Phe Gly Ser Ser Tyr Tyr Asp Leu 1 5 10 <210> 26 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 26 Asp Val Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Asp Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Leu Pro Phe Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Cys Ala Asp Val Gly Ser Thr 85 90 95 Tyr Val Ala Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 27 <211> 6 <212> PRT <213> Oryctolagus cuniculus <400> 27 Gln Asn Ile Asp Ser Asn 1 5 <210> 28 <211> 12 <212> PRT <213> Oryctolagus cuniculus <400> 28 Gln Cys Ala Asp Val Gly Ser Thr Tyr Val Ala Ala 1 5 10 <210> 29 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 29 Asp Val Val Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asn Ile Asp Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile 35 40 45 Tyr Tyr Ala Ser Asn Leu Pro Phe Gly Val Pro Ser Arg Phe Lys Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Ser Ala Asp Val Gly Ser Thr 85 90 95 Tyr Val Ala Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 30 <211> 6 <212> PRT <213> Oryctolagus cuniculus <400> 30 Gln Asn Ile Asp Ser Asn 1 5 <210> 31 <211> 12 <212> PRT <213> Oryctolagus cuniculus <400> 31 Gln Ser Ala Asp Val Gly Ser Thr Tyr Val Ala Ala 1 5 10 <210> 32 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Gln Ala Ser Gly Tyr Arg Phe Ser Asn Phe 20 25 30 Val Ile His Trp Val Arg Gln Ala Pro Gly Gln Arg Phe Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Tyr Asn Gly Asn Lys Glu Phe Ser Ala Lys Phe 50 55 60 Gln Asp Arg Val Thr Phe Thr Ala Asp Thr Ser Ala Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Gly Pro Tyr Ser Trp Asp Asp Ser Pro Gln Asp Asn Tyr 100 105 110 Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120 125 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 33 Gly Tyr Arg Phe Ser Asn Phe Val 1 5 <210> 34 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 34 Ile Asn Pro Tyr Asn Gly Asn Lys 1 5 <210> 35 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 35 Ala Arg Val Gly Pro Tyr Ser Trp Asp Asp Ser Pro Gln Asp Asn Tyr 1 5 10 15 Tyr Met Asp Val 20 <210> 36 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 36 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Phe Ser Cys Arg Ser Ser His Ser Ile Arg Ser Arg 20 25 30 Arg Val Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Val 35 40 45 Ile His Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val Glu 65 70 75 80 Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Val Tyr Gly Ala Ser Ser 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys 100 105 <210> 37 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 37 His Ser Ile Arg Ser Arg Arg 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 38 Gln Val Tyr Gly Ala Ser Ser Tyr Thr 1 5 <210> 39 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 39 Ala Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln Asn Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Ile Asp Pro Tyr Tyr Gly Gly Thr Thr Tyr Asn Arg Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Gly Pro Met Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 40 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 40 Gly Tyr Ser Phe Thr Gly Tyr Asn 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 41 Ile Asp Pro Tyr Tyr Gly Gly Thr 1 5 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 42 Ala Arg Ser Val Gly Pro Met Asp Tyr 1 5 <210> 43 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 43 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Val Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Ser Phe Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ser Gly Ser Tyr Phe Cys Gln His His Ser Asp Asn Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys 100 105 <210> 44 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 44 Glu Asn Val Tyr Ser Tyr 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 45 Gln His His Ser Asp Asn Pro Trp Thr 1 5 <210> 46 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> N / A <400> 46 Gln Val Gln Val Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Val Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Asp Gly Ser Thr Asn Tyr His Ser Ala Leu Lys 50 55 60 Ser Arg Leu Ser Ile Lys Lys Asp His Ser Lys Ser Gln Val Phe Leu 65 70 7...

Claims

[Claim 1] The invention described herein.

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