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

Abstract

CD37-specific bispecific antibody molecules that bind to different epitopes of a human CD37 antigen, the bispecific antibody molecules having enhanced Fc-Fc interaction after binding to CD37 on the cell surface. The invention also relates to a monoclonal parent antibody obtaining the first or second binding region of the bispecific antibody molecule. The invention also relates to pharmaceutical compositions containing these molecules and to the use of these compositions in the treatment of cancer and other diseases.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application date of April 3, 2018, priority date of March 31, 2017, application number 201880032561.6, and invention name “Bispecific anti-CD37 antibodies, monoclonal anti-CD37 antibodies and methods of use thereof”. Field of the Invention

[0002] The present invention relates to bispecific antibodies that specifically bind to the human CD37 antigen. In particular, the present invention relates to CD37-specific bispecific antibody molecules that bind to different epitopes of the human CD37 antigen, wherein the bispecific antibody molecules have enhanced Fc-Fc interactions after binding to CD37 on the cell surface, thereby having enhanced effector function. The present invention also relates to new monoclonal parent antibodies for obtaining the first or second antigen binding region of the bispecific antibody molecule. The present invention also relates to pharmaceutical compositions containing these molecules and the use of these compositions to treat cancer and other diseases. Background of the Invention

[0004] Leukocyte antigen CD37 ("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). In normal physiology, CD37 is expressed on B cells during the pre-B to peripheral mature B cell stages, but is reported to be absent on plasma cells (Link et al., J Pathol. 1987; 152: 12-21). 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). CD37 is widely expressed on malignant cells in a variety of B-cell leukemias and lymphomas, including non-Hodgkin's lymphoma (NHL) and chronic lymphoid leukemia (CLL) (Moore et al. J Immunol. 1986; 137(9):3013).

[0005] Several antibody-based CD37-targeting agents are being evaluated as potential therapeutics for B-cell malignancies and other malignancies. These include, for example, radioimmunoconjugates such as 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 the above forms and in other forms (see, for example, WO 2012 / 135740, WO 2012 / 007576, WO 2011 / 112978, WO 2009 / 126944, WO 2011 / 112978 and EP 2 241 577).

[0006] Betalutin is a mouse anti-CD37 antibody lilotomab (formerly HH1 / tetulomab) conjugated to 177-lutetium. Betalutin is rapidly internalized, inhibits B cell growth in vitro, and prolongs survival in the ivDaudi-SCID model (Dahle et al 2013, Anticancer Res 33:85-96).

[0007] IMGN529 is an ADC consisting of the K7153A antibody conjugated to the maytansinoid DM1 via an SMCC linker. It has been reported that the K7153 antibody induces apoptosis in CD37-expressing Ramos cells without cross-linking. It can also induce CDC and ADCC in Burkitt's lymphoma cell lines, although the ability to induce CDC is 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 antibodies conjugated with DM-1.

[0008] 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).

[0009] Otlertuzumab (originally called TRU-016) is a SMIP (small modular immunopharmaceutical; SMIPS are disulfide-linked dimers of single-chain proteins that contain an antigen-binding VH / VL, a connecting hinge region, and an Fc (fragment, crystallizable) region (CH2-CH3). Its mechanism of action is to induce apoptosis and ADCC, rather than CDC (Zhao et al 2007, Blood 110 (7), 2569-2577).

[0010] mAb37.1 / BI 836826 is a chimeric antibody engineered to bind with high affinity to FcγRIIIa (CD16a) (Heider et al 2011, Blood 118:4159-4168). It has pro-apoptotic activity that is independent of IgG Fc cross-linking, although pro-apoptotic activity is increased by cross-linking. It shows potent ADCC of CD37+ B cell lines and primary CLL cells.

[0011] 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.

[0012] Therefore, it is an object of the present invention to provide anti-CD37 antibodies that can be used to treat cancer and / or other diseases. It is an object of the present invention to provide anti-CD37 antibodies that are improved in terms of CDC of human complement to human cells compared to antibodies of the prior art. Another object is to provide a bispecific antibody having a binding arm obtained from two parent antibodies that bind to different epitopes on CD37, and the bispecific antibody has increased CDC and / or ADCC compared to a combination of two parent monoclonal antibodies that bind to the different epitopes and / or any parent monoclonal antibody itself. A further object is to provide a new monoclonal antibody that binds to different epitopes on CD37, in particular, an anti-CD37 antibody that binds to a new epitope of CD37. Another object of the present invention is to provide a new monoclonal antibody that binds to different epitopes on CD37, which can serve as a parent antibody of the bispecific antibody of the present invention. A further object is to provide a bispecific antibody that binds to two different epitopes on CD37 and which has enhanced Fc-Fc interaction upon binding to CD37 on the plasma membrane compared to a bispecific antibody of the same isotype and having the same binding arms as the bispecific antibody of the invention. SUMMARY OF THE INVENTION

[0014] Surprisingly, the inventors of the present invention have found that a bispecific antibody having binding specificity for two different epitopes on CD37 and having a mutation that increases Fc-Fc interaction after binding to CD37 on the plasma membrane is more potent in inducing CDC than a combination of two anti-CD37 antibodies each having binding specificity for one of the two different epitopes on CD37 and having the same mutation that enhances Fc-Fc interaction or any antibody by itself having the same mutation that enhances Fc-Fc interaction. In addition, a bispecific antibody having binding specificity for two different epitopes on CD37 and having a mutation that increases Fc-Fc interaction is more potent in inducing ADCC than a combination of two anti-CD37 antibodies each having binding specificity for one of the two different epitopes on CD37 and having the same mutation that enhances Fc-Fc interaction.

[0015] Thus, the present invention relates to novel bispecific antibodies that bind to human CD37 and have advantageous properties with respect to their antigen binding properties, their ability to induce CDC and ADCC, their Fc-Fc interactions upon binding to membrane-bound targets, their cytotoxic effects on cells expressing CD37 and other properties, as described herein.

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

[0017] Thus, in one aspect, the bispecific antibody comprises first and second antigen binding regions that bind human CD37 having a 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 different epitopes on CD37, and wherein the first and second Fc regions comprise one or more amino acid mutations that enhance Fc-Fc interactions between the bispecific antibodies after binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies without the mutations.

[0018] In a second aspect, the invention relates to an anti-CD37 antibody that binds to the same epitope on human CD37 as an anti-CD37 antibody comprising:

[0019] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 16, a CDR2 sequence as shown in SEQ ID NO: 17, and a CDR3 sequence as shown in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 20, a CDR2 sequence: KAS, and a CDR3 sequence as shown in SEQ ID NO: 21

[010] ; or

[0020] (ii) a VH region comprising the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10 and the CDR3 sequence shown in SEQ ID NO:11, and a VL region comprising the CDR1 sequence and CDR2 sequence: AAS shown in SEQ ID NO:113 and the CDR3 sequence shown in SEQ ID NO:14

[005] .

[0021] In a third aspect, the present invention relates to an anti-CD37 antibody that binds to human CD37, the antibody comprising:

[0022] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 23, a CDR2 sequence as shown in SEQ ID NO: 24, and a CDR3 sequence as shown in SEQ ID NO: 25, and a VL region comprising a CDR1 sequence and a CDR2 sequence as shown in SEQ ID NO: 27: YAS and a CDR3 sequence as shown in SEQ ID NO: 28; or

[0023] (ii) a VH region comprising the CDR1 sequence shown in SEQ ID NO:2, the CDR2 sequence shown in SEQ ID NO:3 and the CDR3 sequence shown in SEQ ID NO:4, and a VL region comprising the CDR1 sequence and the CDR2 sequence: EAS shown in SEQ ID NO:6, and the CDR3 sequence shown in SEQ ID NO:7

[004] .

[0024] In a fourth aspect, the present invention relates to a pharmaceutical composition comprising the bispecific antibody or antibody of the present invention and a pharmaceutically acceptable carrier.

[0025] In a fifth aspect, the invention relates to a bispecific antibody or an antibody or a composition of the invention for use as a medicament. In a particular aspect, they are used for the treatment of cancer or autoimmune or inflammatory diseases, in particular for the treatment of B-cell malignancies.

[0026] In other aspects, the present invention relates to methods of treatment, combination therapies, nucleic acid sequences encoding the antibodies of the present invention, vectors and host cells expressing the antibodies, and methods of detecting the presence of CD37 antigen or cells expressing CD37 antigen in a sample or subject.

[0027] The present invention further relates to the following embodiments:

[0028] 1. A bispecific antibody comprising first and second antigen binding regions that bind to human CD37 having a sequence of SEQ ID NO: 62 and first and second Fc regions of human immunoglobulins, wherein the first and second antigen binding regions bind to different epitopes on CD37, and wherein the first and second Fc regions comprise one or more amino acid mutations that enhance Fc-Fc interactions between the bispecific antibodies after binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies without the mutations.

[0029] 2. The bispecific antibody of embodiment 1, wherein the first antigen-binding region is obtained from an antibody that competes for binding to human CD37 with a CD37 antibody comprising the following CDR sequences:

[0030] The VH CDR1 sequence shown in SEQ ID NO: 16,

[0031] The VH CDR2 sequence shown in SEQ ID NO: 17,

[0032] The VH CDR3 sequence shown in SEQ ID NO: 18,

[0033] The VL CDR1 sequence shown in SEQ ID NO: 20,

[0034] VL CDR2 sequence: KAS, and

[0035] The VL CDR3 sequence shown in SEQ ID NO:21

[010] .

[0036] 3. The bispecific antibody of any one of the preceding embodiments, wherein the first antigen binding region binds to the same epitope on human CD37 as a CD37 antibody comprising the following CDR sequences:

[0037] The VH CDR1 sequence shown in SEQ ID NO: 16,

[0038] The VH CDR2 sequence shown in SEQ ID NO: 17,

[0039] The VH CDR3 sequence shown in SEQ ID NO: 18,

[0040] The VL CDR1 sequence shown in SEQ ID NO: 20,

[0041] VL CDR2 sequence: KAS, and

[0042] The VL CDR3 sequence is shown in SEQ ID NO:21

[010] .

[0043] 4. The bispecific antibody according to any one of the preceding embodiments, wherein the first antigen binding region comprises the following CDR sequences:

[0044] The VH CDR1 sequence shown in SEQ ID NO: 16,

[0045] The VH CDR2 sequence shown in SEQ ID NO: 17,

[0046] The VH CDR3 sequence shown in SEQ ID NO: 18,

[0047] The VL CDR1 sequence shown in SEQ ID NO: 20,

[0048] VL CDR2 sequence: KAS, and

[0049] The VL CDR3 sequence is shown in SEQ ID NO:21

[010] .

[0050] 5. The bispecific antibody of any one of the preceding embodiments, wherein the first antigen binding region comprises the following VH and VL sequences:

[0051] (i) a VH sequence as shown in SEQ ID NO: 15 and a VL sequence as shown in SEQ ID NO: 19; or

[0052] (ii) a VH sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VH sequence of SEQ ID NO: 15 and a VL sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VL sequence of SEQ ID NO: 19.

[0053] 6. The bispecific antibody of any one of embodiments 1 to 6, wherein the first antigen binding region binds to a functional epitope comprising one or more of amino acids selected from the group consisting of Y182, D189, T191, I192, D194, K195, V196, I197 and P199 of SEQ ID No: 62 (CD37).

[0054] 7. The bispecific antibody of embodiment 1, wherein the first antigen-binding region is obtained from an antibody that competes for binding to human CD37 with a CD37 antibody comprising the following CDR sequences:

[0055] The VH CDR1 sequence shown in SEQ ID NO:9,

[0056] The VH CDR2 sequence shown in SEQ ID NO: 10,

[0057] The VH CDR3 sequence shown in SEQ ID NO: 11,

[0058] The VL CDR1 sequence shown in SEQ ID NO: 13,

[0059] VL CDR2 sequence: AAS, and

[0060] The VL CDR3 sequence shown in SEQ ID NO: 14

[005] .

[0061] 8. The bispecific antibody of embodiment 1 or 7, wherein the first antigen binding region binds to the same epitope on human CD37 as a CD37 antibody comprising the following CDR sequences:

[0062] The VH CDR1 sequence shown in SEQ ID NO:9,

[0063] The VH CDR2 sequence shown in SEQ ID NO: 10,

[0064] The VH CDR3 sequence shown in SEQ ID NO: 11,

[0065] The VL CDR1 sequence shown in SEQ ID NO: 13,

[0066] VL CDR2 sequence: AAS, and

[0067] The VL CDR3 sequence shown in SEQ ID NO: 14

[005] .

[0068] 9. The bispecific antibody of embodiment 1, 7 or 8, wherein the first antigen binding region comprises the following CDR sequences:

[0069] The VH CDR1 sequence shown in SEQ ID NO:9,

[0070] The VH CDR2 sequence shown in SEQ ID NO: 10,

[0071] The VH CDR3 sequence shown in SEQ ID NO: 11,

[0072] The VL CDR1 sequence shown in SEQ ID NO: 13,

[0073] VL CDR2 sequence: AAS, and

[0074] The VL CDR3 sequence shown in SEQ ID NO: 14

[005] .

[0075] 10. The bispecific antibody of any one of embodiments 1 or 7 to 9, wherein the first antigen binding region comprises the following VH and VL sequences:

[0076] (i) a VH sequence as shown in SEQ ID NO: 8 and a VL sequence as shown in SEQ ID NO: 12; or

[0077] (ii) a VH sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VH sequence of SEQ ID NO:8 and a VL sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VL sequence of SEQ ID NO:12.

[0078] 11. The bispecific antibody of any one of the preceding embodiments, wherein the second antigen binding region 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:

[0079] (i) the VH CDR1 sequence shown in SEQ ID NO: 23,

[0080] The VH CDR2 sequence shown in SEQ ID NO: 24,

[0081] The VH CDR3 sequence shown in SEQ ID NO: 25,

[0082] The VL CDR1 sequence shown in SEQ ID NO: 27,

[0083] VL CDR2 sequence: YAS, and

[0084] The VL CDR3 sequence shown in SEQ ID NO: 28;

[0085] (ii) the VH CDR1 sequence shown in SEQ ID NO: 2,

[0086] The VH CDR2 sequence shown in SEQ ID NO: 3,

[0087] The VH CDR3 sequence shown in SEQ ID NO:4,

[0088] The VL CDR1 sequence shown in SEQ ID NO:6,

[0089] VL CDR2 sequences: EAS and

[0090] The VL CDR3 sequence shown in SEQ ID NO:7;

[0091] (iii) the VH CDR1 sequence shown in SEQ ID NO:40,

[0092] The VH CDR2 sequence shown in SEQ ID NO:41,

[0093] The VH CDR3 sequence shown in SEQ ID NO:42,

[0094] The VL CDR1 sequence shown in SEQ ID NO:44,

[0095] VL CDR2 sequence: FAK, and

[0096] The VL CDR3 sequence shown in SEQ ID NO: 45; [G28.1] and

[0097] (iv) the VH CDR1 sequence shown in SEQ ID NO:47,

[0098] The VH CDR2 sequence shown in SEQ ID NO:48,

[0099] The VH CDR3 sequence shown in SEQ ID NO:49,

[0100] The VL CDR1 sequence shown in SEQ ID NO:51,

[0101] VL CDR2 sequence: VAT, and

[0102] The VL CDR3 sequence [37.3] is shown in SEQ ID NO:52.

[0103] 12. The bispecific antibody of any of the preceding embodiments, wherein the second antigen binding region binds to the same epitope on human CD37 as a CD37 antibody comprising a CDR sequence selected from the group consisting of:

[0104] (i) the VH CDR1 sequence shown in SEQ ID NO: 23,

[0105] The VH CDR2 sequence shown in SEQ ID NO: 24,

[0106] The VH CDR3 sequence shown in SEQ ID NO: 25,

[0107] The VL CDR1 sequence shown in SEQ ID NO: 27,

[0108] VL CDR2 sequence: YAS, and

[0109] The VL CDR3 sequence shown in SEQ ID NO: 28;

[0110] (ii) the VH CDR1 sequence shown in SEQ ID NO: 2,

[0111] The VH CDR2 sequence shown in SEQ ID NO: 3,

[0112] The VH CDR3 sequence shown in SEQ ID NO:4,

[0113] The VL CDR1 sequence shown in SEQ ID NO:6,

[0114] VL CDR2 sequence: EAS, and

[0115] The VL CDR3 sequence shown in SEQ ID NO:7;

[0116] (iii) the VH CDR1 sequence shown in SEQ ID NO:40,

[0117] The VH CDR2 sequence shown in SEQ ID NO:41,

[0118] The VH CDR3 sequence shown in SEQ ID NO:42,

[0119] The VL CDR1 sequence shown in SEQ ID NO:44,

[0120] VL CDR2 sequences: FAK, and

[0121] The VL CDR3 sequence shown in SEQ ID NO: 45; [G28.1] and

[0122] (iv) the VH CDR1 sequence shown in SEQ ID NO:47,

[0123] The VH CDR2 sequence shown in SEQ ID NO:48,

[0124] The VH CDR3 sequence shown in SEQ ID NO:49,

[0125] The VL CDR1 sequence shown in SEQ ID NO:51,

[0126] VL CDR2 sequences: VAT and

[0127] The VL CDR3 sequence [37.3] is shown in SEQ ID NO:52.

[0128] 13. The bispecific antibody of any of the preceding embodiments, wherein the second antigen binding region binds to a functional epitope comprising one or more of the amino acids selected from the group consisting of E124, F162, Q163, V164, L165 and H175 of SEQ ID No: 62 (CD37).

[0129] 14. The bispecific antibody of any one of the preceding embodiments, wherein the second antigen binding region comprises a CDR sequence selected from the group consisting of:

[0130] (i) the VH CDR1 sequence shown in SEQ ID NO: 23,

[0131] The VH CDR2 sequence shown in SEQ ID NO: 24,

[0132] The VH CDR3 sequence shown in SEQ ID NO: 25,

[0133] The VL CDR1 sequence shown in SEQ ID NO: 27,

[0134] VL CDR2 sequence: YAS, and

[0135] The VL CDR3 sequence shown in SEQ ID NO: 28;

[0136] (ii) the VH CDR1 sequence shown in SEQ ID NO: 2,

[0137] The VH CDR2 sequence shown in SEQ ID NO: 3,

[0138] The VH CDR3 sequence shown in SEQ ID NO:4,

[0139] The VL CDR1 sequence shown in SEQ ID NO:6,

[0140] VL CDR2 sequence: EAS, and

[0141] The VL CDR3 sequence shown in SEQ ID NO:7;

[0142] (iii) the VH CDR1 sequence shown in SEQ ID NO:40,

[0143] The VH CDR2 sequence shown in SEQ ID NO:41,

[0144] The VH CDR3 sequence shown in SEQ ID NO:42,

[0145] The VL CDR1 sequence shown in SEQ ID NO:44,

[0146] VL CDR2 sequence: FAK, and

[0147] The VL CDR3 sequence shown in SEQ ID NO: 45; [G28.1] and

[0148] (iv) the VH CDR1 sequence shown in SEQ ID NO:47,

[0149] The VH CDR2 sequence shown in SEQ ID NO:48,

[0150] The VH CDR3 sequence shown in SEQ ID NO:49,

[0151] The VL CDR1 sequence shown in SEQ ID NO:51,

[0152] VL CDR2 sequence: VAT, and

[0153] The VL CDR3 sequence [37.3] is shown in SEQ ID NO:52.

[0154] 15. The bispecific antibody of any one of the preceding embodiments, wherein the second antigen binding region comprises a VH and a VL sequence selected from the group consisting of:

[0155] (i) a VH sequence as shown in SEQ ID NO: 22 and a VL sequence as shown in SEQ ID NO: 26; or

[0156] (ii) a VH sequence as shown in SEQ ID NO: 1 and a VL sequence as shown in SEQ ID NO: 5; or

[0157] (iii) a VH sequence as shown in SEQ ID NO: 39 and a VL sequence as shown in SEQ ID NO: 43; or

[0158] (iv) a VH sequence as shown in SEQ ID NO: 46 and a VL sequence as shown in SEQ ID NO: 50; or

[0159] (v) a VH sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical to the VH sequence shown in any one of (i) to (iv) and a VL sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical to the VL sequence shown in any one of (i) to (iv).

[0160] 16. The bispecific antibody according to any of the preceding embodiments, wherein one or more Fc-Fc interaction enhancing mutations in said first and second Fc regions are amino acid substitutions.

[0161] 17. The bispecific antibody of any of the preceding embodiments, 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 of human IgG1 using the EU numbering system.

[0162] 18. The bispecific antibody of any of the preceding embodiments, comprising at least one substitution in said first and second Fc regions selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W.

[0163] 19. The bispecific antibody according to any of the preceding embodiments, comprising at least one substitution in said first and second Fc region, said substitution being selected from E430G or E345K, preferably E430G.

[0164] 20. The bispecific antibody according to any of the preceding embodiments, wherein said Fc-Fc interaction enhancing mutations in said first and second Fc regions are the same substitutions in said first and second Fc regions.

[0165] 21. The bispecific antibody according to any of the preceding embodiments, wherein said bispecific antibody is of IgG1, IgG2, IgG3 or IgG4 isotype or a combination thereof, preferably it is of IgG1 isotype.

[0166] 22. The bispecific antibody according to any of the preceding embodiments, wherein said bispecific antibody is a full length antibody.

[0167] 23. The bispecific antibody according to any of the preceding embodiments, wherein said bispecific antibody is human, humanized or chimeric, or a combination thereof.

[0168] 24. The bispecific antibody according to any of the preceding embodiments, wherein said bispecific antibody binds to human and cynomolgus CD37 having the sequences shown in SEQ ID No 62 and 63, respectively.

[0169] 25. The bispecific antibody of any one of the preceding embodiments, wherein when EU numbering is used,

[0170] A) said first Fc region comprises a further mutation corresponding to F405L in human IgG1, and said second Fc region comprises a further mutation corresponding to K409R in human IgG1, or

[0171] B) said second Fc-region comprises a further mutation corresponding to F405L in human IgG1 and said first Fc-region comprises a further mutation corresponding to K409R in human IgG1.

[0172] 26. The bispecific antibody according to any of the preceding embodiments, which has increased CDC or increased CDC and ADCC effector function compared to the same bispecific antibody but without said Fc-Fc interaction enhancing mutations.

[0173] 27. An anti-CD37 antibody that binds to the same epitope on human CD37 as an anti-CD37 antibody comprising:

[0174] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 16, a CDR2 sequence as shown in SEQ ID NO: 17, and a CDR3 sequence as shown in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 20, a CDR2 sequence: KAS, and a CDR3 sequence as shown in SEQ ID NO: 21

[010] ; or

[0175] (ii) a VH region comprising the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10 and the CDR3 sequence shown in SEQ ID NO:11, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:113, the CDR2 sequence: AAS and the CDR3 sequence shown in SEQ ID NO:14

[005] .

[0176] 28. The anti-CD37 antibody of embodiment 27, comprising:

[0177] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 16, a CDR2 sequence as shown in SEQ ID NO: 17, and a CDR3 sequence as shown in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 20, a CDR2 sequence: KAS, and a CDR3 sequence as shown in SEQ ID NO: 21

[010] ; or

[0178] (ii) a VH region comprising the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10 and the CDR3 sequence shown in SEQ ID NO:11, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:113, the CDR2 sequence: AAS and the CDR3 sequence shown in SEQ ID NO:14

[005] .

[0179] 29. An anti-CD37 antibody that binds to human CD37, the antibody comprising:

[0180] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 23, a CDR2 sequence as shown in SEQ ID NO: 24, and a CDR3 sequence as shown in SEQ ID NO: 25, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 27, a CDR2 sequence: YAS, and a CDR3 sequence as shown in SEQ ID NO: 28;

[016] or

[0181] (ii) a VH region comprising the CDR1 sequence shown in SEQ ID NO:2, the CDR2 sequence shown in SEQ ID NO:3 and the CDR3 sequence shown in SEQ ID NO:4, and a VL region comprising the CDR1 sequence and CDR2 sequence:EAS shown in SEQ ID NO:6 and the CDR3 sequence shown in SEQ ID NO:7

[004] .

[0182] 30. The antibody of any one of embodiments 27-29, wherein the antibody comprises an Fc region comprising one or more amino acid mutations, which enhance the Fc-Fc interaction between the antibodies after target binding compared to the Fc-Fc interaction between the antibodies without the mutations.

[0183] 31. The antibody of embodiment 30, wherein 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 using the EU numbering system.

[0184] 32. The antibody of embodiment 31, comprising at least one amino acid substitution in the Fc region selected from the group consisting of E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W.

[0185] 33. The antibody of embodiment 32, comprising at least one substitution in the Fc region selected from E430G or E345K, preferably E430G.

[0186] 34. The antibody of any one of embodiments 27-33, comprising a mutation corresponding to F405L or K409R in human IgG1.

[0187] 35. An anti-CD37 antibody that binds to human CD37, the antibody comprising:

[0188] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 40, a CDR2 sequence as shown in SEQ ID NO: 41, and a CDR3 sequence as shown in SEQ ID NO: 42, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 44, a CDR2 sequence: FAK, and a CDR3 sequence as shown in SEQ ID NO: 45 [G28.1] or

[0189] (ii) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 47, a CDR2 sequence as shown in SEQ ID NO: 48, and a CDR3 sequence as shown in SEQ ID NO: 49, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 51, a CDR2 sequence: VAT, and a CDR3 sequence as shown in SEQ ID NO: 52 [37.3];

[0190] (iii) and wherein 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; and

[0191] (iv) wherein optionally the Fc region further comprises a mutation of K409R or F405L.

[0192] 36. The antibody of any one of embodiments 27-35, wherein the antibody is of IgG1, IgG2, IgG3 or IgG4 isotype.

[0193] 37. The antibody of any one of embodiments 27-36, wherein the antibody is human, humanized or chimeric.

[0194] 38. The antibody of any one of embodiments 27-37, wherein the antibody binds to human and cynomolgus monkey CD37.

[0195] 39. A pharmaceutical composition comprising the bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38 and a pharmaceutically acceptable carrier.

[0196] 40. The bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38 or the composition of embodiment 39 for use as a medicament.

[0197] 41. The bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38 or the composition of embodiment 39 for use in treating cancer or an autoimmune disease or an inflammatory disease.

[0198] 42. The bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38 or the composition of embodiment 39 for use in treating allergy, 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).

[0199] 43. The bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38 or the composition of embodiment 39, for use according to any one of embodiments 40-42 in combination with one or more further therapeutic agents.

[0200] 44. The bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38 or the composition of embodiment 39, for use according to any one of embodiments 40-43, wherein the one or more further therapeutic agents are selected from the group consisting of doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib and an anti-CD 20 antibody such as rituximab or ofatumumab.

[0201] 45. The bispecific antibody of any one of embodiments 43-44 or the antibody of any one of embodiments 43-44 or the composition of embodiments 43-44 for use according to embodiments 43-44, wherein the further therapeutic agent is an anti-CD20 antibody capable of binding to human CD20 comprising a CDR sequence selected from the group consisting of:

[0202] i) the VH CDR1 sequence shown in SEQ ID NO: 75,

[0203] The VH CDR2 sequence shown in SEQ ID NO: 76,

[0204] The VH CDR3 sequence shown in SEQ ID NO: 77,

[0205] The VL CDR1 sequence shown in SEQ ID NO: 79,

[0206] VL CDR2 sequence DAS, and

[0207] The VL CDR3 sequence shown in SEQ ID NO:80;

[0208] ii) the VH CDR1 sequence shown in SEQ ID NO: 82,

[0209] The VH CDR2 sequence shown in SEQ ID NO: 83,

[0210] The VH CDR3 sequence shown in SEQ ID NO: 84,

[0211] The VL CDR1 sequence shown in SEQ ID NO: 85,

[0212] VL CDR2 sequence DAS, and

[0213] The VL CDR3 sequence shown in SEQ ID NO:86;

[0214] iii) the VH CDR1 sequence shown in SEQ ID NO:95,

[0215] The VH CDR2 sequence shown in SEQ ID NO:96,

[0216] The VH CDR3 sequence shown in SEQ ID NO:97,

[0217] The VL CDR1 sequence shown in SEQ ID NO:99,

[0218] VL CDR2 sequence ATS, and

[0219] The VL CDR3 sequence shown in SEQ ID NO: 100;

[0220] iv) the VH CDR1 sequence shown in SEQ ID NO: 88,

[0221] The VH CDR2 sequence shown in SEQ ID NO: 89,

[0222] The VH CDR3 sequence shown in SEQ ID NO:90,

[0223] The VL CDR1 sequence shown in SEQ ID NO:92

[0224] VL CDR2 sequence DAS, and

[0225] The VL CDR3 sequence shown in SEQ ID NO:93; and

[0226] v) the VH CDR1 sequence shown in SEQ ID NO: 102,

[0227] The VH CDR2 sequence shown in SEQ ID NO: 103,

[0228] The VH CDR3 sequence shown in SEQ ID NO: 104,

[0229] The VL CDR1 sequence shown in SEQ ID NO: 106,

[0230] VL CDR2 sequence QMS, and

[0231] The VL CDR3 sequence is shown in SEQ ID NO:107.

[0232] 46. ​​Use of the bispecific antibody of any one of embodiments 1-26, or the antibody of any one of embodiments 27-38, or the composition of embodiment 39 for the preparation of a medicament.

[0233] 47. The use according to embodiment 46 for the preparation of a medicament for treating cancer, an autoimmune disease or an inflammatory disease.

[0234] 48. The use of embodiment 47 for the preparation of a medicament for treating 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).

[0235] 49. The use according to any one of embodiments 46-48, in combination with one or more further therapeutic agents.

[0236] 50. The use of embodiment 49, wherein the one or more further therapeutic agents are selected from the group consisting of doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib and anti-CD 20 antibodies such as rituximab or ofatumumab.

[0237] 51. A method of inducing cell death of tumor cells expressing CD37 or inhibiting the growth and / or proliferation of said tumor cells, the method comprising administering to an individual in need thereof an effective amount of the bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38 or the composition of embodiment 39.

[0238] 52. A method of treating an individual having an allergy, an autoimmune disease, an inflammatory disease, 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), comprising administering to the individual an effective amount of the bispecific antibody of any one of embodiments 1-25, or the antibody of any one of embodiments 27-38, or the composition of embodiment 39.

[0239] 53. The method of any one of embodiments 51-52, comprising administering one or more further therapeutic agents in combination with the antibody or the bispecific antibody.

[0240] 54. The method of embodiment 53, wherein the one or more further therapeutic agents are selected from the group consisting of doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and an anti-CD 20 antibody such as rituximab or ofatumumab.

[0241] 55. A nucleic acid construct encoding one or more sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 6a, 7, 8, 9, 10, 11, 12, 13, 27a, 28, 29, 30, 30a and 31.

[0244] 56. A nucleic acid construct encoding the bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38.

[0245] 57. An expression vector comprising one or more nucleic acid constructs of embodiments 55-56.

[0246] 58. A host cell comprising an expression vector according to embodiment 57.

[0247] 59. The host cell of embodiment 58, wherein the host cell is a recombinant host cell, such as a recombinant prokaryotic cell, a recombinant eukaryotic cell, or a recombinant microbial host cell.

[0248] 60. An anti-idiotypic antibody which binds the antibody of any one of embodiments 27-38.

[0249] 61. An in vitro method for detecting human CD37 antigen or cells expressing human CD37 in a sample, the method comprising:

[0250] (i) contacting the sample with the bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38 under conditions that allow formation of a complex between the antibody or the bispecific antibody and CD37; and

[0251] (ii) Detecting complex formation.

[0252] 62. An in vivo method for detecting human CD37 antigen or cells expressing human CD37 in a subject, the method comprising:

[0253] (i) administering the bispecific antibody of any one of embodiments 1-26 or the antibody of any one of embodiments 27-38 under conditions that allow formation of a complex between the antibody or the bispecific antibody and CD37; and

[0254] (ii) detecting the formed complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0256] Figure 1 : CDC mediated by G28.1 variants on primary CLL tumor cells. 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 on primary CLL tumor cells was determined in vitro. The data shown are the lysis % determined by flow cytometry measuring the percentage of dead cells (corresponding to PI-positive cells).

[0257] 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 (newly diagnosed / untreated patient VM-PB0005) were determined by flow cytometry. Antigen amounts are shown as molecules / cell. mIgG1 is a mouse IgG1, kappa isotype control.

[0258] Figure 3 : Binding of humanized CD37 antibodies and variants thereof 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 determined by flow cytometry. The data shown are mean fluorescence intensity (MFI) values ​​for one representative experiment.

[0259] 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 determined by flow cytometry. The data shown are mean fluorescence intensity (MFI) values ​​for one representative experiment.

[0260] Figure 5 : Binding of variants of 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 determined by flow cytometry. The data shown are the mean fluorescence intensity (MFI) values ​​of one representative experiment.

[0261] 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 determined by flow cytometry. The data shown are mean fluorescence intensity (MFI) values ​​for one representative experiment.

[0262] Figure 7 : Determination of binding competition between CD37 antibodies and CDC mediated by humanized CD37 antibodies, variants thereof, and combinations of CD37 antibodies on Raji cells. (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 determined by flow cytometry. Raji cells were incubated with unlabeled antibodies for primary binding and then incubated with Alexa Fluor 488-labeled detection antibodies. The loss of binding of the A488-labeled detection antibody after pre-incubation with the unlabeled antibody compared to the binding of the A488-labeled antibody alone indicates binding competition between the A488-labeled antibody and the unlabeled antibody. Data shown are replicates of equivalent soluble fluorescent dye molecules (MESF) of one representative experiment. (BG) 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 or combinations of these to induce CDC on Raji cells was determined in vitro. Data shown are % lysis determined by flow cytometry measuring the percentage of dead cells (corresponding to PI-positive cells).

[0263] Figure 8 : Schematic overview of binding competition between CD37 antibodies. The 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 on Raji cells was determined by flow cytometry using unlabeled antibodies for primary binding and Alexa Fluor 488-labeled detection antibodies to detect subsequent binding of competing antibodies. Color indication: black; simultaneous binding, white; binding competition, gray; associated antibodies.

[0264] Fig. 9: CDC mediated by humanized CD37 antibodies and variants thereof on 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 on Daudi cells was determined in vitro. The data shown are the lysis % determined by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry.

[0265] Fig.10 : CDC mediated by G28.1 and 37.3 and their variants on Daudi cells, and CDC in Daudi cells mediated by humanized CD37 antibodies with different Fc-Fc interaction enhancing mutations. (A) The ability of IgG1-G28.1, IgG1-G28.1-E430G, IgG1-37.3 and IgG1-37.3-E430G to induce CDC on Daudi cells was determined in vitro. The data shown are the lysis % determined by flow cytometry measuring the percentage of dead cells (corresponding to PI-positive cells). (BC) 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 were determined in vitro. Data shown are % lysis (maximal killing at an antibody concentration of 10 μg / mL) determined by flow cytometry measuring the percentage of dead cells (corresponding to PI-positive cells) of one representative experiment. Error bars indicate variation within experiments (performed in duplicate).

[0266] Fig.11: CDC mediated by variants of humanized antibody IgG1-016-H5L2 on 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 on Daudi cells was determined in vitro. The data shown are the lysis % determined by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry.

[0267] Fig.12: CDC on Daudi cells mediated by a bispecific CD37 antibody with an Fc-Fc interaction enhancing mutation, a (combination of) a CD37 antibody with an Fc-Fc interaction enhancing mutation, and a monovalent CD37 binding antibody with an Fc-Fc interaction enhancing mutation; and CDC activity of CD37 antibody variants with Fc-Fc interaction enhancing mutations and their combination on OCI-Ly-7 cells. (A) In vitro determination of bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G, IgG1-005-H1L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G, bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G (B) Ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G to induce CDC on Daudi cells. Data shown are % lysis determined by flow cytometry measuring the percentage of dead cells (corresponding to PI-positive cells).(C) In vitro assays of the CD37 bispecific antibody bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, the CD37 monospecific bivalent (monoclonal) antibody IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, and the monovalent CD37 antibody bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G. L2-LC90S-F405L-E430Gxb12-K409R-E430G, bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plus bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G combination induces the ability of OCI-Ly-7 cells. The data shown are the percentage of dead cells (corresponding to PI-positive cells) measured by flow cytometry. (CDC induction of DbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plus bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G (E) EC50 values ​​for CDC induction of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, as determined in 3 independent experiments.

[0268] Fig.13: CDC mediated by bispecific CD37 antibodies and bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations on Daudi cells. (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 were measured in vitro for the ability to induce CDC on Daudi cells. The data shown are the lysis % determined by flow cytometry measuring the percentage of dead cells (corresponding to PI positive cells).

[0269] Fig.14: CDC mediated by bispecific CD37 with Fc-Fc interaction enhancing mutations, CD37 antibodies with Fc-Fc interaction enhancing mutations (combinations) and monovalent binding CD37 antibodies with Fc-Fc interaction enhancing mutations on primary CLL tumor cells. (a) In vitro assay (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 Combination of E430G plus IgG1-016-H5L2-F405L-E430G, bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and (B) bsIgG The ability of 1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, the combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G to induce CDC against primary CLL tumor cells (patient: VM-BM0091 newly diagnosed / untreated (BM=bone marrow derived)). Data shown are % lysis determined by measuring the percentage of dead cells (corresponding to PI-positive cells) by flow cytometry.

[0270] Fig.15: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations on B-cell lymphoma cell lines. The ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G at a concentration of 10 μg / mL was determined in vitro to induce CDC on a panel of B-cell lymphoma cell lines. Expression levels of CD37 were determined by quantitative flow cytometry and are shown as molecules / cell, mean ± SD of 2 experiments. White columns indicate susceptibility to CDC mediated by bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G (>10% lysis, average of 2 experiments), black columns indicate insusceptibility to CDC mediated by bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G (lysis <10%, average of 2 experiments).

[0271] Fig.16: ADCC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations, CD37 antibodies with Fc-Fc interaction enhancing mutations (combinations) and monovalent binding CD37 antibodies with Fc-Fc interaction enhancing mutations on 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 combination of Fc-Fc interaction enhancing mutations were determined in vitro on Daudi cells using a chromium release assay. The combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G and on Daudi cells (B) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G and IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G, and on Raji cells (C) bsIgG1-016- H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, IgG1-010-H5L2-E430G, IgG1-016-H5L2-E430G, IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G combination, bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G ability to induce ADCC. Data shown are specific lysis %; Error bars indicate variation within the assay, 5 replicates (A, B) or 6 replicates (C) per data point.

[0272] Fig.17 : Quantification of CD37, CD46, CD55 and CD59 expression levels on (A) CLL, (B) FL, (C) MCL or (D) DLBCL tumor cells. Expression levels on tumor cells were determined by flow cytometry. Antigen amount is shown as antibody binding capacity.

[0273] Fig.18: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations on primary tumor cells of patients with CLL, FL, MCL, DLBCL or B-NHL (not further specified). The ability of bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G to induce CDC of tumor cells derived from patients with (A) CLL, (B) FL and (C) MCL, DLBCL or B-NHL (not further specified) was determined by flow cytometry. CDC induction is expressed as the percentage of lysis determined by the fraction of 7-AAD positive tumor cells using 100 μg / mL (A and B) or 10 μg / mL (C) bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G.

[0274] Fig.19 :The combination of bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations and B cells in human or cynomolgus monkey blood.By flow cytometry, the combination of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G labeled with Alexa-488 and B cells in (A) human or (B) cynomolgus monkey blood was determined.IgG1-b12 labeled with Alexa-488 was used as a negative control antibody.Data are shown as the geometric mean A488 fluorescence intensity values ​​of a representative donor / animal.Error bars show the variation (repeated measurements) within the experiment.

[0275] Fig. 20 :The cytotoxicity of bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations and monoclonal CD37-specific antibodies with enhanced FcγR interactions to B cells in human or cynomolgus monkey blood. (A) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-CD37-B2-S239D-I332E to B cells in human blood and (B) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to 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 a representative donor / animal. Error bars show changes within the experiment (repeated measurements).

[0276] Fig.21: CDC mediated by bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations, CD20-specific antibodies or their combinations. (AD) Ex vivo determination of the ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G, Ofatumumab or their combinations to induce CDC on tumor cells derived from 2 CLL patients at the specified concentrations. Data are shown as % of viable B cells.

[0277] Fig. 22 : Dose-effect relationship of three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G in the JVM-3 model. (A) Tumor growth of JVM-3 xenografts after treatment with different doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or isotype control antibody (IgG1-b12). Each time point shows the mean and SEM of each group (n=10). (B) Tumor size of each mouse at day 25. Each treatment group indicates the mean and SEM. Differences were analyzed by Mann Whitney test. Statistically significant differences are indicated as follows: **: p<0.01; ***: p<0.001.

[0278] Fig.23 : Dose-effect relationship of three weekly doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G in the Daudi-luc model. (A) Tumor growth (measured by luciferase activity, bioluminescence) of Daudi-luc xenografts treated with different doses of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or isotype control antibody (IgG1-b12). Each time point shows the mean and SEM of each group (n=9). (B) Luciferase activity of each mouse at day 36. Each treatment group indicates the mean and SEM. Differences are analyzed by one-way Anova, uncorrected Fisher LSD. Statistically significant differences are indicated as follows: **: p<0.01; ***: p<0.001.

[0279] Fig.24 : Plasma concentrations of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-b12 after intravenous injection in SCID mice.

[0280] SCID mice were injected with a single iv dose of (AB) 100 μg (5 mg / kg) or (CD) 500 μg (25 mg / kg) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12.

[0281] Fig.25 .Binding analysis of CD37 antibodies to CD37 variants with alanine mutations in the extracellular domain. The Z score (Zscore) (fold change) is 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 mutants. Residues with a z score lower than -1.5 (indicated by the dotted line) are considered "loss of binding mutants". The numbers above the x-axis refer to the amino acid position. Note that the x-axis is discontinuous: the left part of the axis (up to the strip 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 represents a z score (fold change) of -1.5.

[0282] Fig.26 CDC on Raji cells mediated by a cocktail of CD37 antibodies with Fc-Fc interaction enhancing mutations plus a clinically established CD20 antibody product. CDC-mediated killing of Raji cells (% lysis expressed as fraction of PI-positive cells as determined by flow cytometry) of an antibody concentration dilution series of 1:0, 3:1, 1:1, 3:1, and 0:1 antibody mixtures (10 μg / mL final concentration) of CD37 antibodies with Fc-Fc interaction enhancing mutations plus the standard of care CD20 antibody products MabThera (rituximab), Arzerra (ofatumumab), and Gazyva (obinutuzumab, GA101): (A) mixture with IgG1-37.3-E430G, (B) mixture with IgG1-G28.1-E430G, (C) mixture with IgG1-004-E430G, (D) mixture with IgG1-005-E430G, (E) mixture with IgG1-010-E430G, and (F) mixture with IgG1-016-E430G. DETAILED DESCRIPTION OF THE INVENTION

[0284] definition

[0285] 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 (TMs) and a small extracellular domain and a large extracellular domain. Homo sapiens (i.e., human) CD37 protein is encoded by a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 62 (human CD37 protein: UniprotKB / Swissprot P11049). In this amino acid sequence, residues 112 to 241 correspond to the large extracellular domain, residues 39 to 59 correspond to the small extracellular domain, and the remaining residues correspond to the transmembrane and cytoplasmic domains. Macaca mulatta (i.e., cynomolgus monkey) CD37 protein is encoded by a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 63 (cynomolgus monkey CD37 protein: Genbank accession number XP_005589942). Unless the context is contradictory, the term "CD37" refers to "human CD37". The term "CD37" includes any variants, isoforms, and species homologs of CD37 that are naturally expressed by cells (including tumor cells) or expressed on cells transfected with a CD37 gene or cDNA.

[0286] The term "human CD20" or "CD20" refers to human CD20 (UniProtKB / Swiss-Prot No P11836), and includes any variants, isoforms, and species homologs of CD20 that are naturally expressed by cells (including tumor cells) or expressed on cells transfected with a CD20 gene or cDNA. Species homologs include rhesus monkey CD20 (Macaca mulatta; UniProtKB / Swiss-Prot No H9YXP1) and cynomolgus monkey CD20 (Macaca mulatta).

[0287] The terms "antibody that binds CD37," "anti-CD37 antibody," "CD37 binding antibody," "CD37-specific antibody," and "CD37 antibody" are used interchangeably herein to refer to any antibody that binds to an epitope on the extracellular portion of CD37.

[0288] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any of them having the ability to specifically bind to an antigen for a relatively long period of time under typical physiological conditions, such as 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, 4, 5, 6, 7 or more days, etc., or any other relevant functionally defined period (e.g., sufficient to induce, promote, enhance and / or regulate the time of physiological responses associated with the binding of the antibody to the antigen and / or sufficient to allow the antibody to recruit effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. The constant region of the 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 (e.g., C1q), i.e., the first component in the classical pathway of complement activation. As mentioned above, unless otherwise specified or clearly contradictory to the context, the term antibody herein includes as an antigen binding fragment, that is, an antibody fragment that retains the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by a fragment of a full-length antibody. Examples of antigen binding fragments encompassed in the term "antibody" include (i) Fab' or Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains or monovalent antibodies as described in WO2007059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments connected by a disulfide bond at the hinge region; (iii) Fd fragments consisting essentially of VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody, (v) consisting essentially of the VH domain, also known as domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90) dAb fragment (Ward et al., Nature 341 , 544-546 (1989)); (vi) camelid antibodies or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1): 111-24) and (vii) separate complementarity determining regions (CDRs). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be linked using recombinant methods via a synthetic linker that enables them to be prepared as a single protein chain in which the VL region and the VH region pair to form a monovalent molecule (called a single-chain antibody or single-chain Fv (scFv), see, for example, Bird et al., Science242 ,423-426(1988) and Huston et al.,PNAS USA 85 , 5879-5883 (1988)). Unless otherwise specified or the context clearly indicates, such single-chain antibodies are encompassed within the term antibody. Although such fragments are generally included in the meaning of antibodies, they are collectively and independently unique features of the present invention, exhibiting different biological properties and utilities. In the context of the present invention, these and other useful antibody fragments and bispecific forms of such fragments are further discussed herein. For the bispecific antibodies of the present invention, such fragments are connected to the Fc domain. It should also be understood that, unless otherwise specified, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen that can be provided by any known technology, such as enzymatic cleavage, peptide synthesis, and recombinant technology. The antibodies produced can have any isotype.

[0289] The term "bispecific antibody" refers to an antibody that is specific for at least two different, usually non-overlapping epitopes. Such epitopes can be on the same or different targets. For the present invention, the epitope is on the first attack target, i.e., CD37. Examples of different classes of bispecific antibodies comprising an Fc region include, but are not limited to: asymmetric bispecific molecules, such as IgG-like molecules with complementary CH3 domains; and symmetric bispecific molecules, such as recombinant IgG-like dual targeting molecules, in which each antigen binding region of the molecule binds to at least two different epitopes.

[0290] Examples of bispecific molecules include, but are not limited to (Trion Pharma / Fresenius Biotech, WO / 2002 / 020039), protrusion-into-cavity (Genentech, WO 1998 / 50431), CrossMAbs (Roche, WO 2009 / 080251, WO 2009 / 080252, WO 2009 / 080253), electrostatically matched Fc-heterodimer molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO 2010 / 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, WO 2011 / 143545), Azymetric scaffold (Zymeworks / Merck, WO2012058768), mAb-Fv (Xencor, WO 2011 / 028952), XmAb (Xencor), bivalent bispecific antibody (Roche, WO 2009 / 080254), bispecific IgG (Eli Lilly), molecules (Genmab A / S, WO2011 / 131746), DuetMab (Medimmune, US2014 / 0348839), Biclonics (Merus, WO 2013 / 157953), NovImmune (κλBodies, WO 2012 / 023053), FcΔAdp (Regeneron, WO 2010 / 151792), (DT)-Ig (GSK / Domantis), two-in-one antibodies or dual-acting Fab (Genentech, Adimab), mAb2 (F-Star, WO 2008 / 003116), Zybody TMmolecules (Zyngenia), CovX-body (CovX / Pfizer), FynomAbs (Covagen / Janssen Cilag), DutaMab (Dutalys / Roche), iMab (MedImmune), Dual variable domain (DVD)-IgTM (Abbott), Dual domain dual head antibody (Unilever; Sanofi Aventis, WO 2010 / 0226923), Ts2Ab (MedImmune / AZ), BsAb (Zymogenetics), HERCULES (Biogen Idec, US 7,951,918), scFv-fusion (Genentech / Roche, Novartis, Immunomedics, Changzhou Adam Biotech Inc, CN 102250246), TvAb (Roche, WO2012 / 025525, WO2012 / 025530), ScFv / Fc fusion, SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS). Interceptor (Emergent). Dual Affinity Retargeting Technology (Fc-DARTTM) (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).

[0291] As used herein, the term "full length antibody" refers to an antibody (eg, a parent or variant antibody) that contains all heavy and light chain constant and variable domains corresponding to those domains normally found in a wild-type antibody of that class or isotype.

[0292] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the 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 for different types of modifications of antibodies, and is a method well known to technicians. In particular, chimeric antibodies can be produced by using standard DNA techniques described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Chapter 15. Therefore, chimeric antibodies can be genetic or enzyme engineered recombinant antibodies. Producing chimeric antibodies is within the knowledge of technicians, and therefore, the production of chimeric antibodies according to the present invention can be carried out by methods other than those described herein. Chimeric monoclonal antibodies for therapeutic applications are developed to reduce the immunogenicity of antibodies. They can generally include non-human (e.g., mouse) variable regions specific for the target antigen and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" as used in the context of chimeric antibodies refers to the region comprising the CDRs and framework regions of the heavy and light chains of an immunoglobulin.

[0293] As used herein, the term "oligomer" refers to a molecule consisting of more than one but limited number of monomeric units (e.g., antibodies), as opposed to a polymer that is, at least in principle, composed of at least an infinite number of monomers. Exemplary oligomers are dimers, trimers, tetramers, pentamers, and hexamers. Similarly, as used herein, "oligomerization" such as, for example, "hexamerization" refers to an increase in the distribution of antibodies and / or other dimeric proteins comprising a target binding region according to the present invention to oligomers, such as hexamers. The increased formation of oligomers such as hexamers is due to an increase in Fc-Fc interactions after binding to the membrane-bound target.

[0294] As used herein, the terms "antigen-binding region", "antigen binding region", "binding region" or antigen binding domain refer to the region of an antibody that is capable of binding to an antigen. This binding region is generally defined by the VH and VL domains of the antibody, which can be further subdivided into regions of hypervariability (or hypervariable regions, which are hypervariable in sequence and / or form 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, such as a polypeptide, present, for example, on a cell, bacterium or virion, or in solution. Unless the context is contradictory, the terms "antigen" and "target" can be used interchangeably in the context of the present invention.

[0295] 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 generated. With respect to antibodies, the terms "antigen" and "target" may be used interchangeably and constitute the same meaning and purpose with respect to any aspect or embodiment of the invention.

[0296] 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 modified to contain a high level of sequence homology with a human variable domain. This can be achieved by transplanting 6 non-human antibody complementary determining regions (CDRs) that form an antigen binding site together to a homologous human receptor framework region (FR) (see WO92 / 22653 and EP0629240). In order to completely rebuild the binding affinity and specificity of the parent antibody, it may be necessary to replace the framework residues of the parent antibody (i.e., non-human antibody) with human framework regions (reverse mutations). Structural homology modeling can help identify amino acid residues in the framework region that are important for the binding properties of antibodies. Therefore, humanized antibodies may include non-human CDR sequences, mainly human framework regions (which optionally include one or more amino acid reverse mutations to non-human amino acid sequences), and completely human constant regions. Optionally, other amino acid modifications that are not necessarily reverse mutations may be applied to obtain humanized antibodies with preferred properties, such as affinity and biochemical properties.

[0297] Can use immunized rabbit, use germline humanization (CDR implantation) technology to make rabbit antibody humanization, and if necessary, by the residue (as identified in structural modeling) that may be important for antibody binding properties back mutation to rabbit residue to produce humanized antibody.Can apply the screening of potential T cell epitopes.

[0298] As used herein, the term "human antibody" refers to an antibody with a variable region and a constant region derived from a human germline immunoglobulin sequence. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or in vivo somatic mutations). However, as used herein, the term "human antibody" is not intended to include antibodies in which the CDR sequences of the germline of another mammalian species, such as mice, have been implanted into human framework sequences. The human monoclonal antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, for example, Kohler and Milstein, Nature 256:495 (1975) standard somatic cell hybridization techniques. Although somatic cell hybridization procedures are preferred, in principle other techniques for producing monoclonal antibodies can be used, for example, using viral or oncogenic transformation or phage display techniques of B lymphocytes of human antibody gene libraries.

[0299] A suitable animal system for preparing hybridomas secreting human monoclonal antibodies is the murine system. Producing hybridomas in mice is a very well established procedure. Immunization protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (e.g., murine myeloma cells) and fusion procedures are also known.

[0300] Thus, human monoclonal antibodies can be generated, for example, using transgenic or transchromosomic mice or rats that carry parts of the human immune system rather than the mouse or rabbit system.

[0301] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains (all four of which are interconnected by disulfide bonds). The structure of immunoglobulins has been well characterized. See, for example, Chapter 7 of Fundamental Immunology (Paul, W., ed., 2nd edition Raven Press, NY (1989)). Briefly, each heavy chain is generally composed of a heavy chain variable region (abbreviated herein as V H or VH) and the heavy chain constant region (abbreviated herein as C H The heavy chain constant region usually consists of three domains: H 1, C H 2 and C H 3. Each light chain is usually composed of a light chain variable region (abbreviated as V L or VL) and the light chain constant region (abbreviated herein as C L The light chain constant region usually consists of a domain C L Composition. H and V L The V domains can be further subdivided into regions of hypervariability (or hypervariable regions, which are hypervariable in sequence and / or form of the structurally defined loops), also called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V L It is usually composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Unless otherwise specified or contradictory to the context, the 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; see also the Internet http address http: / / www.imgt.org / ). See Unless otherwise specified or contradictory to the context, references to amino acid positions in the constant region in the present invention are according to 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).

[0302] As used herein, unless the context is contradictory, the term "Fab-arm" or "arm" refers to a heavy chain-light chain pair and is used interchangeably herein with "half molecule". Thus, a "Fab arm" includes the variable regions of the heavy and light chains and the constant regions of the light chains and the constant regions of the heavy chains, which include the CH1 region, hinge, CH2 region and CH3 region of an immunoglobulin. "CH1 region" refers to, for example, the region of a human IgG1 antibody corresponding to amino acids 118-215 according to EU numbering. Thus, a Fab fragment contains the binding region of an immunoglobulin.

[0303] The terms "fragment crystallizable region", "Fc region", "Fc fragment" or "Fc domain", which are used interchangeably herein, refer to a region of an antibody comprising at least a hinge region, a CH2 domain and a CH3 domain arranged from the amino terminus to the carboxyl terminus. The Fc region of an IgG1 antibody can be produced, for example, by digesting an IgG1 antibody with papain. The Fc region of an antibody can mediate the binding of an 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 complement activation pathway. As used herein, the term "hinge region" 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 216-230 according to EU numbering.

[0304] As used herein, the term "core hinge" or "core hinge region" refers to the four amino acids corresponding to positions 226-229 of a human IgG1 antibody.

[0305] 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 may also be any other isotype or allotype as described herein.

[0306] 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 may also be any other isotype or allotype as described herein.

[0307] As used herein, the term "isotype" refers to the immunoglobulin class (eg, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by the heavy chain constant region genes.

[0308] In the context of the present invention, the term "monovalent antibody" refers to a single molecule of an antibody molecule that is capable of binding to an antigen and is therefore incapable of antigen cross-linking.

[0309] "CD37 antibody" or "anti-CD37 antibody" is an antibody as described above that binds specifically to the antigen CD37.

[0310] A "CD37xCD37 antibody" or "anti-CD37xCD37 antibody" is a bispecific antibody comprising two different antigen binding regions, one of which specifically binds to a first epitope on the antigen CD37, and the second of which specifically binds to a different epitope on CD37.

[0311] In one embodiment, the bispecific antibodies of the invention are isolated. As used herein, an "isolated bispecific antibody" refers to a bispecific antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated bispecific antibody that specifically binds to CD37 is substantially free of a monospecific antibody that specifically binds to CD37).

[0312] The term "epitope" refers to a protein determinant that is capable of binding to the antigen binding region ("paratope") of an antibody. An epitope is usually composed of surface groupings of molecules such as amino acids or sugar side chains, and usually has specific three-dimensional structural characteristics and specific charge characteristics. The difference between conformational and non-conformational epitopes is that, in the presence of denaturing solvents, the binding to the former but not the latter is lost. Epitope mapping techniques can determine "structural epitopes" or "functional epitopes". Structural epitopes are defined as those residues within the structure that is in direct contact with the antibody, and can be evaluated, for example, by structure-based methods such as X-ray crystallography. Structural epitopes can include amino acid residues that are directly involved in antibody binding and other amino acid residues that are not directly involved in binding, such as amino acid residues that are effectively blocked or covered by the antibody (in other words, the amino acid residue is within the footprint of the antibody). Functional epitopes are defined as those residues that contribute strongly to the antigen-antibody binding interaction and can be evaluated, for example, by site-directed mutagenesis such as alanine scanning (Cunningham, BC, & Wells, JA (1993) Journal of Molecular Biology; Clackson, T., & Wells, J. (1995) Science, 267 (5196), 383-386). Functional epitopes may 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 in the positions of residues involved in direct interactions (Greenspan, NS, & Di Cera, E. (1999) Nature Biotechnology, 17 (10), 936-937). In the case of antibody-antigen interactions, functional epitopes can be used to distinguish antibody molecules from each other. Functional epitopes can be determined by using an alanine scanning method as described in Example 17. Thus, amino acids in a protein can be substituted with alanine, thereby producing a series of mutant proteins, wherein the antigen binding region of the antibody has reduced binding to the mutant proteins compared to the wild-type protein; as described in Example 17, reduced binding is determined as a normalized log (fold change) of binding of the antibody (expressed as a z-score) of less than -1.5.

[0313] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules having essentially a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity having a variable region and a constant region derived from a human germline immunoglobulin sequence. Human monoclonal antibodies can be produced by hybridomas comprising B cells having a genome comprising a human heavy chain transgene and a light chain transgene obtained from a transgenic or transchromosomal non-human animal (e.g., a transgenic mouse) fused to an immortalized cell.

[0314] As used herein, the term "binding" in the context of antibody binding to a predetermined antigen is generally measured as a percentage corresponding to about 10 -6 M or smaller, e.g. 10 -7 M or less, for example about 10 -8 M or less, for example about 10 -9 M or smaller, about 10 -10 M or smaller, or about 10 -11 M or even smaller K D and wherein the antibody binds with an affinity corresponding to K D The affinity of the binding site to the predetermined antigen is D than its K for binding to nonspecific antigens (e.g. BSA, casein) or closely related antigens other than the predetermined antigen. D 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 lower. D The lower amount depends on the K of the antibody. D Therefore, when the antibody K D When the K of antigen binding is very low, D Lower than the K for binding to nonspecific antigens D The amount can be at least 10,000-fold greater (ie, the antibody is highly specific).

[0315] As used herein, the term "K D ” (M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction.

[0316] As used herein, "affinity" and "K D " is inversely correlated, i.e., higher affinity is intended to indicate lower K D , while lower affinity is intended to indicate a higher K D .

[0317] As used herein, an antibody that "competes" or "cross-competes" is used interchangeably with an antibody that "blocks" or "cross-blocks" another antibody, a reference antibody, and refers to an antibody that competes with the reference antibody for binding to human CD37, e.g., as determined in an assay as 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.

[0318] 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, e.g., as determined in the assay described in Example 7 herein. For several pairs of antibodies and reference antibodies, non-competition in the assay of Example 7 was observed only when one antibody bound to the antigen on the cell and the other was used to compete, and vice versa. When used herein, the term "non-competitive" or "non-competitive" or "non-blocking" is also intended to cover such combinations of antibodies. In one embodiment, in the presence of the reference antibody, the antibody binds at least 75%, e.g., at least 80%, e.g., at least 85% of its maximum binding.

[0319] As used herein, the term "Fc-Fc interaction enhancing mutation" refers to a mutation in an IgG antibody that enhances the Fc-Fc interaction between adjacent IgG antibodies that bind to a cell surface target. This can result in enhanced oligomer formation, such as, for example, hexamerization of target-binding antibodies, while the antibody molecules remain monomers in solution, as described in WO 2013 / 004842; WO 2014 / 108198, both of which are incorporated herein by reference.

[0320] As used herein, the term "Fc effector function" or "Fc-mediated effector function" is intended to refer to the function resulting from the binding of a polypeptide or antibody to its target (e.g., antigen) on the cell membrane and the subsequent interaction of the IgG Fc domain with molecules of the innate immune system (e.g., soluble molecules or membrane-bound molecules). Examples of Fc effector functions include (i) C1q binding, (ii) complement activation, (iii) complement-dependent cytotoxicity (CDC), (iv) antibody-dependent cell-mediated cytotoxicity (ADCC), (v) Fc-gamma receptor binding, (vi) antibody-dependent cellular phagocytosis (ADCP), (vii) complement-dependent cellular cytotoxicity (CDCC), (viii) complement-enhanced cytotoxicity, (ix) antibody-mediated binding of opsonized cells to complement receptors, (x) opsonisation, and (xi) a combination of any one of (i) to (x).

[0321] As used herein, the term "heterodimer interaction between the first and second CH3 regions" refers to the interaction between the first CH3 region of the first Fc-region and the second CH3 region of the second Fc-region in a first-CH3 / second-CH3 heterodimeric protein. Bispecific antibodies are examples of heterodimeric proteins.

[0322] When used herein, the term "homodimer interaction of the first and second CH3 regions" refers to the interaction between the first CH3 region and another first CH3 region in the first-CH3 / first-CH3 homodimeric protein and the interaction between the second CH3 region and another second CH3 region in the second CH3 / second CH3 homodimeric protein. Monoclonal antibodies are examples of homodimeric proteins.

[0323] The term "reducing conditions" or "reducing environment" refers to conditions or an environment in which a substrate, such as, for example, a cysteine ​​residue in an antibody hinge region, is more likely to be reduced than oxidized.

[0324] The present invention also provides a bispecific antibody, which comprises V of the bispecific antibody of the embodiment. L Area, V H V regions or functional variants of one or more CDRs. L Area, V H Functional variants of regions or CDRs still allow each arm of the bispecific antibody to retain at least a considerable 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 can associate with greater affinity, selectivity and / or specificity than the parent bispecific antibody. Such functional variants generally maintain significant sequence identity with the parent bispecific antibody. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), wherein the number of gaps and the length of each gap are taken into account, and the gaps need to be introduced to achieve the best alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences can be, for example, using the algorithm of E.Meyers and W.Miller, Comput.Appl.Biosci 4, 11-17 (1988), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table. A gap length penalty of 12 and a gap penalty of 4 were determined. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970).

[0325] 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 substitutions in the variant are conservative amino acid residue substitutions. Preferably, the variant contains up to 10 amino acid substitutions, e.g., up to 9, 8, 7, 6, 5, 4, 3, 2 or up to 1 amino acid substitutions in the VH and / or VL regions of the parent antibody. Preferably, such substitutions are conservative substitutions, particularly if the substitutions are in the CDR sequences.

[0326] In the context of the present invention, conservative substitutions can be defined by substitutions within the amino acid classes as reflected in the following table:

[0327] Conservatively substituted amino acid residue categories

[0328]

[0329] In the context of the present invention, unless otherwise indicated, the following notation is used to describe mutations; i) amino acid substitutions at a given position are written as, for example, K409R, which means substitution of lysine at position 409 with arginine; and ii) for a particular variant, specific three-letter or one-letter codes are used, including the codes Xaa and X to represent any amino acid residue. Thus, substitution of lysine at position 409 with arginine is referred to as: K409R, and substitution of lysine at position 409 with any amino acid residue is referred to as K409X. If lysine is missing in position 409, it is represented by K409*.

[0330] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which an expression vector, e.g., an expression vector encoding an antibody of the invention, has been introduced. Recombinant host cells include, e.g., transfectomas, e.g., CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6 or NS0 cells, and lymphocytes.

[0331] The term "treat / treat" refers to the administration of an effective amount of a therapeutically active bispecific antibody of the invention to alleviate, ameliorate, prevent or eliminate (cure) symptoms or disease states.

[0332] The term "effective amount" or "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic result, at dosages and for periods of time necessary. A therapeutically effective amount of a bispecific antibody can vary with factors such as the disease state, 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 an amount in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0333] The term "anti-idiotypic antibody" refers to an antibody that recognizes unique determinants typically associated with the antigen binding site of an antibody.

[0334] Embodiments of the present invention

[0335] In the first major embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region that binds to human CD37 having a sequence of SEQ ID NO: 62 and a first and a second Fc region of a human immunoglobulin, wherein the first and second antigen binding regions bind to different epitopes on CD37, and wherein the first and second Fc regions comprise one or more amino acid mutations that enhance the Fc-Fc interaction between the bispecific antibodies after binding to a membrane-bound target compared to the Fc-Fc interaction between bispecific antibodies without the mutations. Thus, a bispecific anti-CD37 antibody is provided that binds to two different epitopes on CD37. Preferably, the two epitopes allow the two binding arms to bind to the same protein, and thus each binding arm does not block the binding of the other arm and / or does not compete with the other binding arm of the bispecific molecule for binding. The bispecific antibody also comprises a mutation that enhances the Fc-Fc interaction between two or more bispecific antibodies of the present invention. This has the effect that the bispecific antibody forms oligomers after binding to CD37 expressed on the plasma membrane of a target cell. Compared to the same molecule except for the mutation, the Fc-Fc interaction is enhanced. 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. Preferably, it is a symmetrical substitution, meaning that both half molecules (parent antibodies) have mutations. Another advantage of the present bispecific antibody is that it has enhanced CDC and / or ADCC effector functions compared to the same bispecific molecule without Fc-Fc interaction enhancing mutations. Surprisingly, the bispecific molecule also has improved CDC and / or ADCC compared to a combination of two parental monoclonal anti-CD37 antibodies mutated to have enhanced Fc-Fc interactions, and the bispecific molecule has improved CDC and / or ADCC compared to any parental monoclonal anti-CD37 antibody itself mutated to have enhanced Fc-Fc interactions. Thus, the bispecific antibodies of the invention induce CDC and / or ADCC more potently than a combination of an antibody having a first antigen binding region and a second antibody having a second antigen binding region, wherein both antibodies comprise Fc-Fc interactions, or compared to a single monoclonal anti-CD37 antibody having a first or second antigen binding region and comprising an Fc-Fc interaction enhancing mutation.

[0336] In one embodiment of the invention, the first antigen-binding region of the bispecific antibody is obtained from an antibody that competes for binding to human CD37 with a CD37 antibody comprising the following CDR sequences:

[0337] The VH CDR1 sequence shown in SEQ ID NO: 16,

[0338] The VH CDR2 sequence shown in SEQ ID NO: 17,

[0339] The VH CDR3 sequence shown in SEQ ID NO: 18,

[0340] The VL CDR1 sequence shown in SEQ ID NO: 20,

[0341] VL CDR2 sequence KAS, and

[0342] The VL CDR3 sequence shown in SEQ ID NO: 21.

[0343] Preferably, competition for binding is determined according to Example 7.

[0344] In another embodiment, the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as a CD37 antibody comprising the following CDR sequences:

[0345] The VH CDR1 sequence shown in SEQ ID NO: 16,

[0346] The VH CDR2 sequence shown in SEQ ID NO: 17,

[0347] The VH CDR3 sequence shown in SEQ ID NO: 18,

[0348] The VL CDR1 sequence shown in SEQ ID NO: 20,

[0349] VL CDR2 sequence KAS, and

[0350] The VL CDR3 sequence shown in SEQ ID NO: 21.

[0351] In another embodiment of the present invention, the first antigen-binding region of the bispecific antibody comprises the following CDR sequences:

[0352] The VH CDR1 sequence shown in SEQ ID NO: 16,

[0353] The VH CDR2 sequence shown in SEQ ID NO: 17,

[0354] The VH CDR3 sequence shown in SEQ ID NO: 18,

[0355] The VL CDR1 sequence shown in SEQ ID NO: 20,

[0356] VL CDR2 sequence KAS, and

[0357] The VL CDR3 sequence shown in SEQ ID NO: 21.

[0358] In another embodiment of the present invention, the first antigen-binding region of the bispecific antibody of the present invention comprises the following VH and VL sequences:

[0359] (i) a VH sequence as shown in SEQ ID NO: 15 and a VL sequence as shown in SEQ ID NO: 19; or

[0360] (ii) a VH sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VH sequence of SEQ ID NO: 15 and a VL sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VL sequence of SEQ ID NO: 19.

[0361] In another embodiment of the invention, the first antigen-binding region of the bispecific antibody is obtained from an antibody that competes for binding to human CD37 with a CD37 antibody comprising the following CDR sequences:

[0362] The VH CDR1 sequence shown in SEQ ID NO:9,

[0363] The VH CDR2 sequence shown in SEQ ID NO: 10,

[0364] The VH CDR3 sequence shown in SEQ ID NO: 11,

[0365] The VL CDR1 sequence shown in SEQ ID NO: 13,

[0366] VL CDR2 sequence: AAS, and

[0367] The VL CDR3 sequence shown in SEQ ID NO: 14

[005] .

[0368] In another embodiment of the invention, the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as a CD37 antibody comprising the following CDR sequences:

[0369] The VH CDR1 sequence shown in SEQ ID NO:9,

[0370] The VH CDR2 sequence shown in SEQ ID NO: 10,

[0371] The VH CDR3 sequence shown in SEQ ID NO: 11,

[0372] The VL CDR1 sequence shown in SEQ ID NO: 13,

[0373] VL CDR2 sequence: AAS, and

[0374] The VL CDR3 sequence shown in SEQ ID NO: 14

[005] .

[0375] 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, I192, D194, K195, V196, I197 and P199 of SEQ ID NO:62 (CD37).

[0376] 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 Y182, D189, T191, I192, D194, K195, V196, I197 and P199 of SEQ ID No: 62 (CD37).

[0377] In one embodiment of the invention, the first antigen binding region of the bispecific antibody binds to a functional epitope on CD37, wherein the binding to a mutant CD37 is reduced compared to wild-type CD37 having the amino acid sequence shown in SEQ ID NO:62, in which any one or more of the amino acid residues at the positions corresponding to positions Y182, D189, T191, I192, D194, K195, V196, I197 and P199 of SEQ ID no 62 (CD37) have been substituted with alanine; reduced binding is determined as a z-score (fold change) for binding of the antibody of less than -1.5, wherein the z-score (fold change) for binding is calculated as described in Example 17.

[0378] In another embodiment of the present invention, the first antigen-binding region of the bispecific antibody comprises the following CDR sequences:

[0379] The VH CDR1 sequence shown in SEQ ID NO:9,

[0380] The VH CDR2 sequence shown in SEQ ID NO: 10,

[0381] The VH CDR3 sequence shown in SEQ ID NO: 11,

[0382] The VL CDR1 sequence shown in SEQ ID NO: 13,

[0383] VL CDR2 sequence: AAS, and

[0384] The VL CDR3 sequence shown in SEQ ID NO: 14

[005] .

[0385] In another embodiment of the present invention, the first antigen-binding region of the bispecific antibody comprises the following VH and VL sequences:

[0386] (i) a VH sequence as shown in SEQ ID NO: 8 and a VL sequence as shown in SEQ ID NO: 12; or

[0387] (ii) a VH sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VH sequence of SEQ ID NO:8 and a VL sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VL sequence of SEQ ID NO:12.

[0388] In another embodiment of the invention, the second antigen binding region of the bispecific antibody is obtained from competing for antibody binding to human CD37 with a CD37 antibody comprising a CDR sequence selected from the group consisting of:

[0389] (i) the VH CDR1 sequence shown in SEQ ID NO: 23,

[0390] The VH CDR2 sequence shown in SEQ ID NO: 24,

[0391] The VH CDR3 sequence shown in SEQ ID NO: 25,

[0392] The VL CDR1 sequence shown in SEQ ID NO: 27,

[0393] VL CDR2 sequence: YAS, and

[0394] The VL CDR3 sequence shown in SEQ ID NO: 28;

[0395] (ii) the VH CDR1 sequence shown in SEQ ID NO: 2,

[0396] The VH CDR2 sequence shown in SEQ ID NO: 3,

[0397] The VH CDR3 sequence shown in SEQ ID NO:4

[0398] The VL CDR1 sequence shown in SEQ ID NO:6

[0399] VL CDR2 sequences: EAS and

[0400] The VL CDR3 sequence shown in SEQ ID NO:7;

[0401] (iii) the VH CDR1 sequence shown in SEQ ID NO:40,

[0402] The VH CDR2 sequence shown in SEQ ID NO:41,

[0403] The VH CDR3 sequence is shown in SEQ ID NO:42.

[0404] The VL CDR1 sequence shown in SEQ ID NO:44

[0405] VL CDR2 sequences: FAK, and

[0406] The VL CDR3 sequence shown in SEQ ID NO: 45; [G28.1] and

[0407] (iv) the VH CDR1 sequence shown in SEQ ID NO:47,

[0408] The VH CDR2 sequence shown in SEQ ID NO:48,

[0409] The VH CDR3 sequence shown in SEQ ID NO:49,

[0410] The VL CDR1 sequence shown in SEQ ID NO:51,

[0411] VL CDR2 sequence: VAT, and

[0412] The VL CDR3 sequence [37.3] is shown in SEQ ID NO:52.

[0413] In another embodiment of the invention, the second antigen-binding region of the bispecific 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:

[0414] (i) the VH CDR1 sequence shown in SEQ ID NO: 23,

[0415] The VH CDR2 sequence shown in SEQ ID NO: 24,

[0416] The VH CDR3 sequence shown in SEQ ID NO: 25,

[0417] The VL CDR1 sequence shown in SEQ ID NO: 27,

[0418] VL CDR2 sequence: YAS, and

[0419] The VL CDR3 sequence shown in SEQ ID NO: 28;

[0420] (ii) the VH CDR1 sequence shown in SEQ ID NO: 2,

[0421] The VH CDR2 sequence shown in SEQ ID NO: 3,

[0422] The VH CDR3 sequence shown in SEQ ID NO:4

[0423] The VL CDR1 sequence shown in SEQ ID NO:6

[0424] VL CDR2 sequences: EAS and

[0425] The VL CDR3 sequence shown in SEQ ID NO:7;

[0426] (iii) the VH CDR1 sequence shown in SEQ ID NO:40,

[0427] The VH CDR2 sequence shown in SEQ ID NO:41,

[0428] The VH CDR3 sequence is shown in SEQ ID NO:42.

[0429] The VL CDR1 sequence shown in SEQ ID NO:44

[0430] VL CDR2 sequences: FAK, and

[0431] The VL CDR3 sequence shown in SEQ ID NO: 45; [G28.1] and

[0432] (iv) the VH CDR1 sequence shown in SEQ ID NO:47,

[0433] The VH CDR2 sequence shown in SEQ ID NO:48,

[0434] The VH CDR3 sequence shown in SEQ ID NO:49,

[0435] The VL CDR1 sequence shown in SEQ ID NO:51,

[0436] VL CDR2 sequence: VAT, and

[0437] The VL CDR3 sequence [37.3] is shown in SEQ ID NO:52.

[0438] Thus, bispecific antibodies are provided in which the first and second antigen binding regions bind to different epitopes on human CD37. The inventors of the present invention have found that antibodies having the CDR sequences of antibody 005 (SEQ ID NOs 9, 10, 11 and 13, 13a, 14) and antibody 010 (SEQ ID NOs 16, 17, 18 and 20, 20a, 21) compete for binding to human CD37, but do not compete for binding to human CD37 with any antibody having the CDR sequences of antibody 016 (SEQ ID NOs 23, 24, 25 and 27, 27a, 28), 004 (SEQ ID NOs 2, 3, 4 and 6, 6a, 7), G28.1 (SEQ ID NOs 40, 41, 42 and 44, 44a, 45) and 37.3 (SEQ ID NOs 47, 48, 49 and 51, 51a, 52). However, it has been found that 016, 004, G28.1 and 37.3 antibodies compete with each other for binding to human CD37. Therefore, a bispecific antibody comprising a first binding arm obtained from an antibody that competes for binding with either or both of the 005 or 010 antibodies and a second binding arm obtained from an antibody that competes for binding with either or all of 016, 004, G28.1 and 37.3 is a bispecific antibody specific for two different epitopes on CD37. Surprisingly, the inventors found that such bispecific antibodies have favorable CDC efficacy on CD37-expressing cells compared to treatment of such cells with a combination of two monoclonal antibodies that do not compete for binding to CD37. In addition, the inventors surprisingly found that such bispecific antibodies have favorable ADCC efficacy on such cells compared to treatment of CD37-expressing cells with a combination of two monoclonal antibodies that do not compete for binding to CD37.

[0439] In one embodiment of the present invention, the bispecific antibody comprises a first antigen binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of antibody 010 and a second binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of 016.

[0440] In another embodiment of the present invention, the bispecific antibody comprises a first antigen binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of antibody 010 and a second binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of 004.

[0441] In another embodiment of the present invention, the bispecific antibody comprises a first antigen binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of antibody 010 and a second binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of G28.1.

[0442] In another embodiment of the present invention, the bispecific antibody comprises a first antigen binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of antibody 010 and a second binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of 37.3.

[0443] In one embodiment of the present invention, the bispecific antibody comprises a first antigen binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of antibody 005 and a second binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of 016.

[0444] In another embodiment of the present invention, the bispecific antibody comprises a first antigen binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of antibody 005 and a second binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of 004.

[0445] In another embodiment of the present invention, the bispecific antibody comprises a first antigen binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of antibody 005 and a second binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of G28.1.

[0446] In another embodiment of the present invention, the bispecific antibody comprises a first antigen binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of antibody 005 and a second binding region obtained from an antibody that competes for binding to human CD37 with an antibody having the CDR sequence of 37.3.

[0447] In a further embodiment, such bispecific antibodies described herein may 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), wherein the substitution corresponds to E430G in IgG1 when EU numbering is used, and the substitution enhances the Fc-Fc interaction of two or more bispecific antibodies of the invention after binding to a membrane-bound target. In another embodiment, the Fc-Fc interaction enhancing substitution corresponds to E345K in IgG1 when EU numbering is used.

[0448] In another embodiment of the invention, the bispecific antibody comprises 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:

[0449] (i) the VH CDR1 sequence shown in SEQ ID NO: 23,

[0450] The VH CDR2 sequence shown in SEQ ID NO: 24,

[0451] The VH CDR3 sequence shown in SEQ ID NO: 25,

[0452] The VL CDR1 sequence shown in SEQ ID NO: 27,

[0453] VL CDR2 sequence: YAS, and

[0454] The VL CDR3 sequence shown in SEQ ID NO: 28;

[0455] (ii) the VH CDR1 sequence shown in SEQ ID NO: 2,

[0456] The VH CDR2 sequence shown in SEQ ID NO: 3,

[0457] The VH CDR3 sequence shown in SEQ ID NO:4,

[0458] The VL CDR1 sequence shown in SEQ ID NO:6,

[0459] VL CDR2 sequence: EAS, and

[0460] The VL CDR3 sequence shown in SEQ ID NO:7;

[0461] (iii) the VH CDR1 sequence shown in SEQ ID NO:40,

[0462] The VH CDR2 sequence shown in SEQ ID NO:41,

[0463] The VH CDR3 sequence shown in SEQ ID NO:42,

[0464] The VL CDR1 sequence shown in SEQ ID NO:44,

[0465] VL CDR2 sequences: FAK, and

[0466] The VL CDR3 sequence shown in SEQ ID NO: 45; [G28.1] and

[0467] (iv) the VH CDR1 sequence shown in SEQ ID NO:47,

[0468] The VH CDR2 sequence shown in SEQ ID NO:48,

[0469] The VH CDR3 sequence shown in SEQ ID NO:49,

[0470] The VL CDR1 sequence shown in SEQ ID NO:51,

[0471] VL CDR2 sequences: VAT and

[0472] The VL CDR3 sequence [37.3] is shown in SEQ ID NO:52.

[0473] In another embodiment of the invention, the bispecific antibody comprises 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:

[0474] (i) the VH CDR1 sequence shown in SEQ ID NO: 23,

[0475] The VH CDR2 sequence shown in SEQ ID NO: 24,

[0476] The VH CDR3 sequence shown in SEQ ID NO: 25,

[0477] The VL CDR1 sequence shown in SEQ ID NO: 27,

[0478] VL CDR2 sequence: YAS, and

[0479] The VL CDR3 sequence shown in SEQ ID NO: 28;

[0480] (ii) the VH CDR1 sequence shown in SEQ ID NO: 2,

[0481] The VH CDR2 sequence shown in SEQ ID NO: 3,

[0482] The VH CDR3 sequence shown in SEQ ID NO:4,

[0483] The VL CDR1 sequence shown in SEQ ID NO:6,

[0484] VL CDR2 sequence: EAS, and

[0485] The VL CDR3 sequence shown in SEQ ID NO:7;

[0486] (iii) the VH CDR1 sequence shown in SEQ ID NO:40,

[0487] The VH CDR2 sequence shown in SEQ ID NO:41,

[0488] The VH CDR3 sequence shown in SEQ ID NO:42,

[0489] The VL CDR1 sequence shown in SEQ ID NO:44,

[0490] VL CDR2 sequence: FAK, and

[0491] The VL CDR3 sequence shown in SEQ ID NO: 45; [G28.1] and

[0492] (iv) the VH CDR1 sequence shown in SEQ ID NO:47,

[0493] The VH CDR2 sequence shown in SEQ ID NO:48,

[0494] The VH CDR3 sequence shown in SEQ ID NO:49,

[0495] The VL CDR1 sequence shown in SEQ ID NO:51,

[0496] VL CDR2 sequences: VAT and

[0497] The VL CDR3 sequence [37.3] is shown in SEQ ID NO:52.

[0498] In one embodiment of the present 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).

[0499] 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).

[0500] In one embodiment of the invention, the second antigen binding region of the bispecific antibody binds to a functional epitope on CD37, wherein the binding to a mutant CD37 is reduced compared to wild-type CD37 having the amino acid sequence shown in SEQ ID NO: 62, in which any one or more of the amino acid residues at the positions corresponding to positions E124, F162, Q163, V164, L165 and H175 of SEQ ID no 62 (CD37) have been substituted with alanine; the reduced binding is determined as a z-score (fold change) of the binding of the antibody below -1.5, wherein the z-score (fold change) of binding is calculated as described in Example 17

[0501] Therefore, in one embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010, and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 016.

[0502] In another embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequence of antibody 010, and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequence of antibody 004.

[0503] In another embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010, and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody G28.1.

[0504] In another embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 010, and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 37.3.

[0505] In another embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequence of antibody 005, and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequence of antibody 016.

[0506] In another embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequence of antibody 005, and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequence of antibody 004.

[0507] In another embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody 005, and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequences of antibody G28.1.

[0508] In another embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequence of antibody 005, and wherein the second antigen binding region of the bispecific antibody binds to the same epitope on human CD37 as an anti-CD37 antibody comprising the CDR sequence of antibody 37.3.

[0509] In another embodiment of the present invention, the second antigen-binding region of the bispecific antibody comprises a CDR sequence selected from the group consisting of:

[0510] (i) the VH CDR1 sequence shown in SEQ ID NO: 23,

[0511] The VH CDR2 sequence shown in SEQ ID NO: 24,

[0512] The VH CDR3 sequence shown in SEQ ID NO: 25,

[0513] The VL CDR1 sequence shown in SEQ ID NO: 27,

[0514] VL CDR2 sequence: YAS, and

[0515] The VL CDR3 sequence shown in SEQ ID NO: 28;

[0516] (ii) the VH CDR1 sequence shown in SEQ ID NO: 2,

[0517] The VH CDR2 sequence shown in SEQ ID NO: 3,

[0518] The VH CDR3 sequence shown in SEQ ID NO:4,

[0519] The VL CDR1 sequence shown in SEQ ID NO:6,

[0520] VL CDR2 sequence: EAS, and

[0521] The VL CDR3 sequence shown in SEQ ID NO:7;

[0522] (iii) the VH CDR1 sequence shown in SEQ ID NO:40,

[0523] The VH CDR2 sequence shown in SEQ ID NO:41,

[0524] The VH CDR3 sequence shown in SEQ ID NO:42,

[0525] The VL CDR1 sequence shown in SEQ ID NO:44,

[0526] VL CDR2 sequence: FAK, and

[0527] The VL CDR3 sequence shown in SEQ ID NO: 45; [G28.1] and

[0528] (iv) the VH CDR1 sequence shown in SEQ ID NO:47,

[0529] The VH CDR2 sequence shown in SEQ ID NO:48,

[0530] The VH CDR3 sequence shown in SEQ ID NO:49,

[0531] The VL CDR1 sequence shown in SEQ ID NO:51,

[0532] VL CDR2 sequence: VAT, and

[0533] The VL CDR3 sequence [37.3] is shown in SEQ ID NO:52.

[0534] In another embodiment of the present invention, the second antigen-binding region of the bispecific antibody comprises a CDR sequence selected from the group consisting of:

[0535] (i) the VH CDR1 sequence shown in SEQ ID NO: 23,

[0536] The VH CDR2 sequence shown in SEQ ID NO: 24,

[0537] The VH CDR3 sequence shown in SEQ ID NO: 25,

[0538] The VL CDR1 sequence shown in SEQ ID NO: 27,

[0539] VL CDR2 sequence: YAS, and

[0540] The VL CDR3 sequence shown in SEQ ID NO: 28;

[0541] (ii) the VH CDR1 sequence shown in SEQ ID NO: 2,

[0542] The VH CDR2 sequence shown in SEQ ID NO: 3,

[0543] The VH CDR3 sequence shown in SEQ ID NO:4,

[0544] The VL CDR1 sequence shown in SEQ ID NO:6,

[0545] VL CDR2 sequence: EAS, and

[0546] The VL CDR3 sequence shown in SEQ ID NO:7;

[0547] (iii) the VH CDR1 sequence shown in SEQ ID NO:40,

[0548] The VH CDR2 sequence shown in SEQ ID NO:41,

[0549] The VH CDR3 sequence shown in SEQ ID NO:42,

[0550] The VL CDR1 sequence shown in SEQ ID NO:44,

[0551] VL CDR2 sequences: FAK, and

[0552] The VL CDR3 sequence shown in SEQ ID NO: 45; [G28.1] and

[0553] (iv) the VH CDR1 sequence shown in SEQ ID NO:47,

[0554] The VH CDR2 sequence shown in SEQ ID NO:48,

[0555] The VH CDR3 sequence shown in SEQ ID NO:49,

[0556] The VL CDR1 sequence shown in SEQ ID NO:51,

[0557] VL CDR2 sequence: VAT, and

[0558] The VL CDR3 sequence [37.3] is shown in SEQ ID NO:52.

[0559] Therefore, the present invention also provides in another embodiment a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody comprises the CDR sequence of antibody 010 (i.e., SEQ ID NOs 16-18 and 20-21), and wherein the second antigen binding region of the bispecific antibody comprises the CDR sequence of antibody 016 (i.e., SEQ ID NOs 23-25 ​​and 27-28). As also described above, such bispecific antibodies of the present invention further comprise an Fc-Fc interaction enhancing mutation in the Fc region of the antibody. In one embodiment, the mutation corresponds to a mutation at position E430 or E345 in IgG1 when the EU numbering system is used. In one embodiment, the mutation is an E430G substitution. In another embodiment, it is an E345K substitution.

[0560] 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 of the bispecific antibody comprises the CDR sequence of antibody 010 (i.e., SEQ ID NOs 16-18 and 20-21), and wherein the second antigen binding region of the bispecific antibody comprises the CDR sequence of antibody 004 (i.e., SEQ ID NOs 2-4 and 6-7).

[0561] 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 of the bispecific antibody comprises the CDR sequence of antibody 010 (i.e., SEQ ID NOs 16-18 and 20-21) and wherein the second antigen binding region of the bispecific antibody comprises the CDR sequence of antibody G28.1 (i.e., SEQ ID NOs 40-42 and 44-45).

[0562] 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 of the bispecific antibody comprises the CDR sequence of antibody 010 (i.e., SEQ ID NOs 16-18 and 20-21) and wherein the second antigen binding region of the bispecific antibody comprises the CDR sequence of antibody 37.3 (i.e., SEQ ID NOs 47-49 and 51-52).

[0563] In another embodiment of the present invention, the second antigen-binding region of the bispecific antibody comprises a VH and a VL sequence selected from the group consisting of:

[0564] (i) a VH sequence as shown in SEQ ID NO: 22 and a VL sequence as shown in SEQ ID NO: 26

[016] or

[0565] (ii) a VH sequence as shown in SEQ ID NO: 1 and a VL sequence as shown in SEQ ID NO: 5

[004] or

[0566] (iii) the VH sequence shown in SEQ ID NO: 39 and the VL sequence shown in SEQ ID NO: 43 [G28.1] or

[0567] (iv) a VH sequence as shown in SEQ ID NO: 46 and a VL sequence as shown in SEQ ID NO: 50 [37.3] or

[0568] A VH sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, for example at least 99% identical to the VH sequence shown in any one of (i) to (iv) and a VL sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, for example at least 99% identical to the VL sequence shown in any one of (i) to (iv).

[0569] In another embodiment of the present invention, the second antigen-binding region of the bispecific antibody comprises a VH and a VL sequence selected from the group consisting of:

[0570] (i) the VH sequence shown in SEQ ID NO: 22 and the VL sequence shown in SEQ ID NO: 26

[016]

[0571] (ii) the VH sequence shown in SEQ ID NO: 1 and the VL sequence shown in SEQ ID NO: 5

[004]

[0572] (iii) the VH sequence shown in SEQ ID NO:39 and the VL sequence shown in SEQ ID NO:43 [G28.1]

[0573] (iv) the VH sequence shown in SEQ ID NO: 46 and the VL sequence shown in SEQ ID NO: 50 [37.3] and

[0574] A VH sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, for example at least 99% identical to the VH sequence shown in any one of (i) to (iv) and a VL sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, for example at least 99% identical to the VL sequence shown in any one of (i) to (iv).

[0575] Therefore, 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 of the bispecific antibody comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID NOs 15 and 19) and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 016 (i.e., SEQ ID NOs 22 and 26).

[0576] 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 of the bispecific antibody comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID Nos 15 and 19), and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 004 (i.e., SEQ ID NOs 1 and 5).

[0577] 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 of the specific antibody comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID NOs 15 and 19), and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody G28.1 (i.e., SEQ ID NOs 39 and 43).

[0578] 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 of the bispecific antibody comprises the VH and VL sequences of antibody 010 (i.e., SEQ ID Nos 15 and 19), and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 37.3 (i.e., SEQ ID Nos 46 and 50).

[0579] In another embodiment, the present invention provides a bispecific antibody comprising a first and a second antigen binding region, wherein the first antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 005 (i.e., SEQ ID NOs 8 and 12), and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 016 (i.e., SEQ ID NOs 22 and 26).

[0580] 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 of the bispecific antibody comprises the VH and VL sequences of antibody 005 (i.e., SEQ ID NOs 8 and 12), and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 004 (i.e., SEQ ID NOs 1 and 5).

[0581] 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 of the bispecific antibody comprises the VH and VL sequences of antibody 005 (i.e., SEQ ID NOs 8 and 12), and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody G28.1 (i.e., SEQ ID NOs 39 and 43).

[0582] 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 of the bispecific antibody comprises the VH and VL sequences of antibody 005 (i.e., SEQ ID NOs 8 and 12), and wherein the second antigen binding region of the bispecific antibody comprises the VH and VL sequences of antibody 37.3 (i.e., SEQ ID NOs 46 and 50).

[0583] In other embodiments of the invention, the VH and VL sequences disclosed above may vary within 90% sequence identity.

[0584] In another embodiment, the invention provides a CD37 binding molecule comprising an antigen binding region as described herein, wherein the CDR sequence is a CDR sequence of one of antibodies 004, 005, 010, 016, 28.1 or 37.3. In one embodiment, such molecules have only one antigen binding region. Thus, the binding molecule has monovalent binding to CD37. Preferably, such molecules comprise a complete Fc region of an immunoglobulin. In one embodiment, the CD37 binding molecule comprises a second antigen binding region for an unrelated target, which target may be, for example, b12.

[0585] Fc-Fc enhancing mutations

[0586] In one embodiment of the invention, one or more Fc-Fc interaction enhancing mutations in the first and second Fc regions of the bispecific antibody are amino acid substitutions. It can be said that the Fc region of the bispecific antibody comprises two different Fc regions, one from each parent anti-CD37 antibody. Alternatively, the bispecific antibody may 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 symmetrical, i.e., the same mutations are produced in the two Fc regions.

[0587] In one embodiment, the invention provides a bispecific antibody, 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 the EU numbering system is used. In one embodiment, the invention provides a bispecific antibody, 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 the EU numbering system is used, with the proviso that the substitution in 440 is 440Y or 440W.

[0588] In another embodiment, the present invention provides a bispecific antibody comprising at least one substitution in said first and second Fc regions selected from: 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 said first and second Fc regions selected from E430G or E345K, preferably E430G. Thus, a bispecific antibody is provided which will have enhanced Fc-Fc interactions between different antibodies having said mutations. It is believed that this mutation causes the antibody to form oligomers on target cells, thereby enhancing CDC.

[0589] In another embodiment, the bispecific antibody comprises a further mutation in the Fc region selected from K439E, S440K and S440R. The bispecific antibody with the additional mutation of K439E and the second different antibody or bispecific antibody with the additional mutation of S440K or S440R will form oligomers in an alternating pattern of the first antibody and the second antibody. It is believed that this is because the additional mutation will result in a preference for interaction between the first antibody and the second antibody rather than the interaction between the first antibody and the first antibody or the second antibody and the second antibody due to the non-covalent binding between the Fc regions.

[0590] Preferably, the Fc-Fc interaction enhancing mutations in the first and second Fc regions are the same substitutions in the first and second Fc regions. Thus, in a preferred embodiment, the bispecific antibody has the same Fc-Fc interaction enhancing mutations in both Fc regions. The Fc region can also be described as an Fc chain, such that the antibody has two Fc chains, which constitute the common Fc region of the antibody. Thus, in a preferred embodiment, the two Fc chains each comprise a substitution at a position selected from the group consisting of amino acid positions 430, 440 and 345 in human IgG1 when the EU numbering system is used. In one embodiment, the two Fc chains each comprise an E430G substitution, such that the bispecific antibody of the invention comprises two E430G substitutions. In another embodiment, the two Fc chains each comprise an E345K substitution, such that the bispecific antibody of the invention comprises two E345K substitutions.

[0591] In one embodiment of the invention, the bispecific antibody is of IgG1 isotype.

[0592] In one embodiment of the invention, the bispecific antibody is of the IgG2 isotype.

[0593] In one embodiment of the invention, the bispecific antibody is of the IgG3 isotype.

[0594] In one embodiment of the invention, the bispecific antibody is of the IgG4 isotype.

[0595] In one embodiment of the invention, the bispecific antibody is of the IgG isotype.

[0596] In one embodiment of the invention, the bispecific antibody is a combination of isotypes IgG1, IgG2, IgG3 and IgG4. For example, the first half antibody obtained from the first parent antibody can be an IgG1 isotype, and the second half antibody obtained from the second parent antibody can be an IgG4 isotype, so that the bispecific antibody is a combination of IgG1 and IgG4. In another embodiment, it is a combination of IgG1 and IgG2. In another embodiment, it is a combination of IgG1 and IgG3. In another embodiment, it is a combination of IgG2 and IgG3. In another embodiment, it is a combination of IgG2 and IgG4. In another embodiment, it is a combination of IgG3 and IgG4. Typically, the core hinge will be an IgG1-type core hinge with the sequence CPPC, but it can also be other hinges that are stable and do not allow Fab arm exchange in vivo, which is the case for an IgG4 core hinge with the sequence CPSC.

[0597] In a preferred embodiment, the bispecific antibodies of the invention are full-length antibodies.

[0598] In another embodiment of the invention, the bispecific antibody is a human antibody. In another embodiment of the invention, the bispecific antibody is a humanized antibody. In another embodiment of the invention, the bispecific antibody is a chimeric antibody. In one embodiment of the invention, the bispecific antibody is a combination of human, humanized and chimeric antibodies. For example, the first half antibody obtained from the first parent antibody can be a human antibody, and the second half antibody obtained from the second parent antibody can be a humanized antibody, so that the bispecific antibody is a combination of human and humanized antibodies. In a preferred embodiment of the invention, the bispecific antibody binds to human and cynomolgus monkey CD37, which has the sequences shown in SEQ ID No 62 and 63, respectively. This is an advantage because it will allow the same bispecific molecule to be used in cynomolgus monkeys for preclinical toxicology studies, which will then be tested in humans. In the case where antibodies against human targets do not also bind to targets in animal models, it is difficult to conduct preclinical toxicology studies and nonclinical safety profiling of molecules, which is a requirement of regulatory agencies.

[0599] Bispecific antibody formats

[0600] The present invention provides bispecific CD37×CD37 antibodies that effectively promote CDC and / or ADCC-mediated killing of tumor cells expressing CD37, such as tumors of B cell origin. Depending on the desired functional properties of a specific use, a specific antigen binding region can be selected from the antibody group or antigen binding region provided by the present invention. Many different forms and uses of bispecific antibodies are known in the art and have been reviewed by Kontermann; Drug Discov Today, 2015 Jul; 20(7): 838-47 and MAbs, 2012 Mar-Apr; 4(2): 182-97.

[0601] The bispecific antibodies according to the present invention are not limited to any particular bispecific format or method of producing it, however the antibodies of the present invention should have a complete Fc domain to induce enhanced Fc-Fc interactions.

[0602] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody having two arms comprising different antigen binding regions; and (ii) a dual variable domain antibody (DVD-Ig), in which each light chain and heavy chain contains two variable domains connected in series by a short peptide (Wu et al., Generation and Characterization of a DualVariable 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.

[0603] In one embodiment, the bispecific antibody of the invention is a cross-body or bispecific antibody obtained by controlled Fab arm exchange (eg as described in WO2011131746 (Genmab)).

[0604] 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 the molecule contains Fab fragments or portions of Fab fragments of at least two different antibodies on either side; (iii) IgG fusion molecules in which a full-length IgG antibody is fused to an additional Fab fragment or a portion of a 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, an Fc region or a portion thereof; (v) Fab fusion molecules in which different Fab fragments are fused together to a heavy chain constant domain, an Fc region or a portion thereof; and (vi) heavy chain antibodies (e.g., domain antibodies, nanobodies) based on scFv and diabodies in which different single-chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to Fc.

[0605] Examples of IgG class molecules with complementary CH3 domain molecules include, but are not limited to, Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), so-called protrusion-into-hole molecules (Genentech, WO9850431), CrossMAbs (Roche, WO2011117329), and electrostatic manipulation 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), chain 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 molecule (Genmab A / S, WO2011131746).

[0606] Examples of recombinant IgG-based dual targeting molecules include, but are not limited to, 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), methods using common light chains (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.).

[0607] Examples of IgG fusion molecules include, but are not limited to, dual variable domain (DVD)-Ig molecules (Abbott, US7,612,181), dual domain bi-headed antibodies (Unilever; Sanofi Aventis, WO20100226923), IgG class 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, WO2010111625), HERCULES molecules (Biogen Idec, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246) and TvAb molecules (Roche, WO2012025525, WO2012025530).

[0608] 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).

[0609] Examples of Fab fusion 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-acting or Bis-Fab molecules (Genentech, Bostrom, et al 2009. Science 323, 1610-1614.), docking-lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), bivalent bispecific molecules (Biotecnol, Schoonjans, J Immunol. 2000 Dec 15; 165(12): 7050-7.) and Fab-Fv molecules (UCB-Celltech, WO 2009040562A1).

[0610] Examples of scFv-based, 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).

[0611] On the one hand, the bispecific antibody of the present 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 are different and make the heterodimer interaction between the first and second CH3 regions stronger than each homodimer interaction of the first and second CH3 regions. More details about these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0612] As further described herein, based on a homodimeric starting CD37 antibody and another homodimeric starting CD37 antibody containing only a few fairly conservative asymmetric mutations in the CH3 region, a stable CD37xCD37 bispecific antibody can be obtained with high yield using a specific method. Asymmetric mutations refer to the sequences of the first and second CH3 regions containing amino acid substitutions at different positions, so that the first and second CH3 regions have different amino acid sequences.

[0613] In one aspect, the bispecific antibody as defined in any embodiment disclosed herein comprises a first and a second Fc region, wherein said first and second Fc region each comprises at least a hinge region, a CH2 and a CH3 region, wherein in said first Fc region, at least one of the amino acids in the positions corresponding to the positions selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and in said second Fc region, at least one of the amino acids in the positions corresponding to the positions selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 in a human IgG1 heavy chain has been substituted, and wherein said first and said second Fc region are not substituted in the same position.

[0614] Therefore, in a preferred embodiment of the present invention, the first Fc region of the bispecific antibody comprises a mutation corresponding to the amino acid at position F405 in human IgG1, and the second Fc region of the bispecific antibody comprises a further mutation corresponding to the amino acid at position K409 in human IgG1. Therefore, these mutations are asymmetric compared to the Fc-Fc interaction enhancing mutations mentioned above.

[0615] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 366, and the 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 substitution at position 366 is selected from Ala, Asp, Glu, His, Asn, Val or Gln.

[0616] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 368 and the second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 370, 399, 405, 407 and 409.

[0617] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 370 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 399, 405, 407 and 409.

[0618] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 399 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 405, 407 and 409.

[0619] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 405 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 407 and 409.

[0620] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 407 and the second Fc region has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 409.

[0621] In one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has an amino acid substitution at position 409 and the second Fc region has an amino acid substitution at a position selected from the group consisting of: 366, 368, 370, 399, 405 and 407.

[0622] Thus, in one embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the sequences of said first and second Fc regions contain asymmetric mutations, i.e. mutations at different positions in the two Fc regions, e.g. a mutation at position 405 in one of the Fc regions and a mutation at position 409 in the other Fc region.

[0623] In one embodiment of the bispecific antibody as 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, such as 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 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 further embodiments 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 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.

[0624] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region comprises Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409 and the second Fc region comprises an amino acid other than Phe, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met or Cys, at position 405 and Lys at position 409. In further embodiments herein, the first Fc region comprises Phe at position 405 and an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, 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, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, at position 405 and Lys at position 409.

[0625] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the 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 the second Fc region comprises Leu at position 405 and Lys at position 409. In a further embodiment herein, the first CH3 region comprises Phe at position 405 and Arg at position 409 and the second Fc region comprises an amino acid other than Phe, Arg or Gly at position 405, e.g., Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys and Lys at position 409. In another embodiment, said first Fc region comprises Phe at position 405 and Arg at position 409 and said second CH3 region comprises Leu at position 405 and Lys at position 409.

[0626] In another embodiment of the bispecific antibody as 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, such as 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 another 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.

[0627] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc region comprises Lys at position 370, Phe at position 405 and Arg at position 409, and said second Fc region comprises Lys at position 409, Thr at position 370 and Leu at position 405.

[0628] In another embodiment of the bispecific antibody as 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, such as 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 and: a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

[0629] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc region comprises Arg at position 409 and said second Fc region comprises Lys at position 409 and: a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

[0630] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region comprises Thr at position 350, Lys at position 370, Phe at position 405 and Arg at position 409 and the second Fc region comprises Lys at position 409 and: a) Ile at position 350 and Leu at position 405, or b) Thr at position 370 and Leu at position 405.

[0631] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region comprises Thr at position 350, Lys at position 370, Phe at position 405 and Arg at position 409, and the second Fc region comprises Ile at position 350, Thr at position 370, Leu at position 405 and Lys at position 409.

[0632] In one embodiment of the bispecific antibody as 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 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.

[0633] In one embodiment, the bispecific antibody as 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.

[0634] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first CH3 region having Tyr at position 407 and an amino acid other than Lys, Leu or Met at position 409 and a second CH3 region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and Lys at position 409.

[0635] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region having Tyr at position 407 and Arg at position 409 and a second Fc region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr at position 407 and Lys at position 409.

[0636] In another embodiment, the first Fc region has an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, at position 409 and the second Fc region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser or Thr, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp or Cys, at position 407. In another embodiment, the first Fc region has an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys at position 409 and the second Fc region has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407.

[0637] In another embodiment of the bispecific antibody as 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, such as 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.

[0638] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys 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 Lys at position 409.

[0639] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys at position 409, and the second Fc region has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at position 409.

[0640] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has Tyr at position 407 and an amino acid other than Lys, Leu or Met, e.g. Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys at position 409, and the second Fc region has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409.

[0641] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, the first Fc region has Tyr at position 407 and 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, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp or Cys and Lys at position 409.

[0642] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc region has Tyr at position 407 and Arg at position 409 and said second Fc region has Ala, Gly, His, Ile, Leu, Met, Asn, Val or Trp at position 407 and Lys at position 409.

[0643] In another embodiment of the bispecific antibody as defined in any of the embodiments disclosed herein, said first Fc region has Tyr at position 407 and Arg at position 409, and said second Fc region has Gly, Leu, Met, Asn or Trp at position 407 and Lys at position 409.

[0644] In another embodiment of the bispecific antibody as 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, such as Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys and the second Fc region has

[0645] (i) an amino acid other than Phe, Leu and Met at position 368, such as Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr or Cys, or

[0646] (ii) Trp at position 370, or

[0647] (iii) 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

[0648] (iv) an amino acid other than Lys, Arg, Ser, Thr or Trp at position 366, e.g., Phe, Leu, Met, Ala, Val, Gly, He, Asn, His, Asp, Glu, Gln, Pro, Tyr or Cys.

[0649] In one embodiment, the first Fc region has Arg, Ala, His or Gly at position 409 and the second Fc region has

[0650] (i) Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp at position 368, or

[0651] (ii) Trp at position 370, or

[0652] (iii) Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg or Tyr at position 399, or

[0653] (iv) Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, He, Leu, Met, or Tyr at position 366.

[0654] In one embodiment, the first Fc region has Arg at position 409 and the second Fc region has:

[0655] (i) Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val or Trp at position 368 or

[0656] (ii) Trp at position 370, or

[0657] (iii) Phe, His, Lys, Arg or Tyr at position 399, or

[0658] (iv) Ala, Asp, Glu, His, Asn, Val, Gln at position 366.

[0659] In addition to the amino acid substitutions specified above, the first and second Fc regions may further contain amino acid substitutions, deletions or insertions relative to the wild-type Fc sequence.

[0660] In a preferred embodiment of the invention, 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 when EU numbering is used.

[0661] In one embodiment, the mutations at positions F405 and K409 are substitutions. In a specific embodiment, the substitution at position F405 is a F405L substitution. In another embodiment, the substitution at position K409 is a K409R substitution.

[0662] 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 such embodiments, the second Fc region is not substituted at either of the 405 and 409 amino acid positions.

[0663] It should be understood that, unless explicitly stated otherwise, all references to amino acid mutations at disclosed positions are mutations relative to human IgG1 and are numbered using the EU numbering system using human IgG1.

[0664] In one embodiment, neither said first Fc region nor said second Fc region comprises a Cys-Pro-Ser-Cys sequence in the core hinge region.

[0665] In another embodiment, both said first and second Fc regions comprise a Cys-Pro-Pro-Cys sequence in the core hinge region.

[0666] Thus, bispecific antibodies are provided which can be produced in high yield and are stable in vivo.

[0667] In another embodiment, the bispecific antibodies of the invention have increased CDC and / or ADCC effector function compared to the same bispecific antibodies without Fc-Fc interaction enhancing mutations. In another embodiment, the bispecific antibodies of the invention have increased CDC and / or ADCC effector function compared to a monoclonal parent antibody having the binding region of the first or second binding region of the bispecific antibody and having the same Fc-Fc enhancing mutation as the bispecific antibodies of the invention.

[0668] Method for preparing the bispecific antibody of the present invention

[0669] Conventional methods such as hybrid hybridoma and chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin 26: 649) can be used to prepare the bispecific antibodies of the present invention. In addition to the desired bispecific antibodies, co-expression of two antibodies (which are composed of different heavy and light chains) in host cells leads to a mixture of possible antibody products, which can then be separated by, for example, affinity chromatography or similar methods.

[0670] When co-expressing different antibody constructs, strategies that are conducive to the formation of functional bispecific products can also be used, for example, the method described by Lindhofer et al. (1995 J Immunol 155: 219). The fusion of rat and mouse hybridomas that produce different antibodies leads to a limited number of heterodimeric proteins, which is due to the preferred species-restricted heavy / light chain pairing. Another strategy to promote heterodimer formation relative to homodimers is the "protrusion-into-cavity" strategy, wherein a protrusion is introduced in the first heavy chain polypeptide, and a corresponding cavity is introduced in the second heavy chain polypeptide so that the protrusion can be located in the cavity at the interface of the two heavy chains, thereby promoting the formation of heterodimers and hindering the formation of homodimers. "Protrusion" is constructed by replacing the small amino acid side chains from the interface of the first polypeptide with larger side chains. Compensation "cavities" of the same or similar size as protrusions are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller amino acid side chains (U.S. Patent No. 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies that are conducive to heterodimer formation after co-expression of different antibody domains in host cells. In these methods, one or more residues constituting the CH3-CH3 interface in these two CH3 domains are replaced with charged amino acids, making the formation of homodimers electrostatically unfavorable, while heterodimerization is electrostatically favorable. WO2007110205 (Merck) describes another strategy in which the differences between IgA and IgG CH3 domains are utilized to promote heterodimerization.

[0671] Another in vitro method for producing bispecific antibodies has been described in WO2008119353 (Genmab), in which the bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (exchange of heavy chain and attached light chain) between two monospecific antibodies IgG4- or IgG4-like antibodies after incubation under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences.

[0672] Preferred methods for preparing the bispecific CD37xCD37 antibodies of the present invention include the methods described in WO2011131746 and WO2013060867 (Genmab), comprising the following steps:

[0673] a) providing a first antibody comprising an Fc region, wherein the Fc region comprises a first CH3 region;

[0674] b) providing a second antibody comprising a second Fc region, said Fc region comprising a second CH3 region, wherein said first antibody is a CD37 antibody and said second antibody is a different CD37 antibody;

[0675] wherein the sequences of the first and second CH3 regions are different and such that a heterodimeric interaction between the first and second CH3 regions is stronger than each homodimeric interaction between the first and second CH3 regions;

[0676] c) incubating the first antibody with the second antibody under reducing conditions; and

[0677] d) obtaining the bispecific antibody.

[0678] In one embodiment, the first antibody is incubated with the second antibody under reducing conditions sufficient to cause the cysteines in the hinge region to undergo disulfide bond isomerization, wherein the heterodimeric interaction between the first antibody and the second antibody in the resulting heterodimeric antibody is such that no Fab arm exchange occurs in 0.5 mM GSH after 24 hours at 37°C.

[0679] Without being limited by theory, in step c), the heavy chain disulfide bonds in the hinge region of the parent antibody are reduced, and the resulting cysteine ​​is then able to form inter-heavy chain disulfide bonds with cysteine ​​residues of another parent antibody molecule (initially with a different specificity). In one embodiment of this method, the reducing conditions in step c) comprise adding a reducing agent, such as a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithiobutanetetrol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine ​​and β-mercapto-ethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol and tris(2-carboxyethyl)phosphine. In another embodiment, step c) comprises restoring the conditions to become non-reducing or less reducing, such as by removing the reducing agent, such as by desalting.

[0680] For this method, any CD37 antibody described herein can be used, including first and second CD37 antibodies comprising a first and / or second Fc region. Examples of such first and second Fc regions (including combinations of such first and second Fc regions) can include any of those described herein.

[0681] In one embodiment of this method, the first and / or second antibody is a full length antibody.

[0682] The Fc region of the first and second antibodies can be any isotype, including but not limited to IgG1, IgG2, IgG3 or IgG4. In one embodiment of this method, the Fc region of both the first and second antibodies is an IgG1 isotype. In another embodiment, one of the Fc regions of the antibodies is an IgG1 isotype, and the other is an IgG4 isotype. In the latter embodiment, the resulting bispecific antibody comprises an Fc region of IgG1 and an Fc region of IgG4, and therefore can have interesting intermediate properties with respect to the activation of effector functions.

[0683] In another embodiment, one of the antibody starting proteins has been engineered to not bind Protein A, thereby allowing the heterodimeric protein to be separated from the homodimeric starting protein by passing the product through a Protein A column.

[0684] As mentioned above, the sequences of the first and second CH3 regions of the homodimer starting antibody (parent antibody) are different, and make the heterodimer interaction between the first and second CH3 regions stronger than each homodimer interaction of the first and second CH3 regions. More details about these interactions and how they can be achieved have been provided in WO2011131746 and WO2013060867 (Genmab) (which are fully incorporated by reference at this).

[0685] In particular, stable bispecific CD37xCD37 antibodies can be obtained in high yield using the above-described method of the invention based on two homodimeric starting antibodies that bind to different epitopes of CD37 and contain only a few rather conservative asymmetric mutations in the CH3 region. Asymmetric mutations refer to sequences of the first and second CH3 regions containing amino acid substitutions at different positions.

[0686] The bispecific antibodies of the present invention can also be obtained by co-expressing a construct encoding a first and a second polypeptide in a single cell. Therefore, in another aspect, the present invention relates to a method for producing a bispecific antibody, the method comprising the following steps:

[0687] a) providing a first nucleic acid construct encoding a first polypeptide comprising a first Fc region of a first antibody heavy chain and a first antigen binding region, wherein the first Fc region comprises a first CH3 region,

[0688] b) providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc region of a second antibody heavy chain and a second antigen binding region, wherein the second Fc region comprises a second CH3 region,

[0689] wherein the sequences of the first and second CH3 regions are different and such that the heterodimeric interaction between the first and second CH3 regions is stronger than each homodimeric interaction between the first and second CH3 regions,

[0690] Optionally, wherein the first and second nucleic acid constructs encode the light chain sequences of the first and second antibodies

[0691] c) co-expressing the first and second nucleic acid constructs in a host cell, and

[0692] d) obtaining the heterodimeric protein from cell culture.

[0693] Therefore, the present invention also relates to recombinant eukaryotic or prokaryotic host cells producing the bispecific antibodies of the present invention.

[0694] In one embodiment of the invention, the bispecific antibody is obtained by any of the methods according to the invention.

[0695] Suitable 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, bacteria and mammalian cells, such as CHO or HEK cells.

[0696] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein both said first Fc region and said second Fc region do not comprise a Cys-Pro-Ser-Cys sequence in the hinge region.

[0697] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein both said first Fc region and said second Fc region comprise a Cys-Pro-Pro-Cys sequence in the hinge region.

[0698] In one embodiment, the bispecific antibody as defined in any of the embodiments disclosed herein comprises a first Fc region and a second Fc region, wherein said first Fc region and second Fc region are human antibody Fc regions.

[0699] In one embodiment, the 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.

[0700] In one embodiment, the 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 are from a heavy chain antibody.

[0701] In one embodiment, the 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 first and a second light chain.

[0702] In other embodiments, the co-expression method according to the invention comprises any other features described under the in vitro methods above.

[0703] Parental antibody

[0704] In another embodiment, the invention relates to parent antibodies for use in preparing the bispecific antibodies of the invention.

[0705] Thus, in one embodiment, the invention relates to an anti-CD37 antibody that binds to the same epitope on human CD37 as an anti-CD37 antibody comprising:

[0706] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 16, a CDR2 sequence as shown in SEQ ID NO: 17, and a CDR3 sequence as shown in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 20, a CDR2 sequence: KAS, and a CDR3 sequence as shown in SEQ ID NO: 21

[010] ; or

[0707] (ii) a VH region comprising the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10 and the CDR3 sequence shown in SEQ ID NO:11, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:113, the CDR2 sequence: AAS and the CDR3 sequence shown in SEQ ID NO:14

[005] .

[0708] In another embodiment, the invention relates to an anti-CD37 antibody that competes for binding with an anti-CD37 antibody comprising:

[0709] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 16, a CDR2 sequence as shown in SEQ ID NO: 17, and a CDR3 sequence as shown in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 20, a CDR2 sequence: KAS, and a CDR3 sequence as shown in SEQ ID NO: 21

[010] ; or

[0710] (ii) a VH region comprising the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10 and the CDR3 sequence shown in SEQ ID NO:11, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:113, the CDR2 sequence: AAS and the CDR3 sequence shown in SEQ ID NO:14

[005] .

[0711] In another embodiment, the present invention relates to an anti-CD37 antibody comprising:

[0712] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 16, a CDR2 sequence as shown in SEQ ID NO: 17, and a CDR3 sequence as shown in SEQ ID NO: 18, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 20, a CDR2 sequence: KAS, and a CDR3 sequence as shown in SEQ ID NO: 21

[010] ; or

[0713] (ii) a VH region comprising the CDR1 sequence shown in SEQ ID NO:9, the CDR2 sequence shown in SEQ ID NO:10 and the CDR3 sequence shown in SEQ ID NO:11, and a VL region comprising the CDR1 sequence shown in SEQ ID NO:113, the CDR2 sequence: AAS and the CDR3 sequence shown in SEQ ID NO:14

[005] .

[0714] In another embodiment, the present invention relates to an anti-CD37 antibody comprising:

[0715] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 23, a CDR2 sequence as shown in SEQ ID NO: 24, and a CDR3 sequence as shown in SEQ ID NO: 25, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 27, a CDR2 sequence: YAS, and a CDR3 sequence as shown in SEQ ID NO: 28;

[016] or

[0716] (ii) a VH region comprising the CDR1 sequence shown in SEQ ID NO:2, the CDR2 sequence shown in SEQ ID NO:3 and the CDR3 sequence shown in SEQ ID NO:4, and a VL region comprising the CDR1 sequence and CDR2 sequence:EAS shown in SEQ ID NO:6 and the CDR3 sequence shown in SEQ ID NO:7

[004] .

[0717] In another embodiment, the invention relates to an antibody as defined above, comprising one or more amino acid mutations in the Fc region of the antibody, which mutations enhance the Fc-Fc interaction between antibodies upon target binding compared to the Fc-Fc interaction between antibodies without said mutations.

[0718] It is believed that the enhanced Fc-Fc interaction has the following effect: the antibody forms oligomers, such as, for example, hexamer, on the target cell, and the oligomer forms the effect of causing enhanced CDC. In a preferred embodiment, the one or more amino acid mutations in the Fc region of the antibody are amino acid substitutions corresponding to one or more positions of amino acid positions 430, 440 and 345 in human IgG1 when using the EU numbering system and replacing the amino acid sequence relative to human IgG1. In one embodiment, the one or more amino acid mutations in the Fc region of the antibody are amino acid substitutions corresponding to one or more positions of amino acid positions 430, 345 and 440 in human IgG1 when using the EU numbering system, with the condition that the substitution in 440 is 440Y or 440W. In one embodiment, 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.

[0719] 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 both Fc chains of the antibody have the same mutation / substitution.

[0720] In another embodiment, the antibody may further comprise substitutions at positions corresponding to 366, 368, 370, 399, 405, 407, and 409 in human IgG1. An antibody having substitutions in one of these amino acid positions is a stable antibody. However, it can form bispecific antibodies with antibodies having substitutions in another of these amino acid positions and having different antigen-binding regions in a so-called "fab arm exchange" reaction under reducing conditions. Under reducing conditions sufficient to cause the cysteine ​​in the hinge region to undergo disulfide isomerization, the antibodies of the present invention will form half molecules, each half molecule comprising a single antigen-binding site and an Fc region. Substitutions at different positions corresponding to any of 366, 368, 370, 399, 405, 407, and 409 in human IgG1 will result in half molecules of the first antibody being favorable for dimerization with half molecules of the second antibody, thereby forming a bispecific (heterodimer) antibody when the reducing conditions are removed and the disulfide bonds in the hinge region are reformed.

[0721] Thus, two of the following antibodies of the present invention, which have different antigen binding regions and bind to different epitopes on CD37 and contain substitutions in any of the amino acid positions corresponding to 366, 368, 370, 399, 405, 407 and 409 in human IgG1 but in different positions in both Fc chains (Fc regions), may be suitable for preparing the bispecific antibodies of the present invention.

[0722] In one embodiment, the first antibody has an amino acid substitution at position 366, and the 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.

[0723] In one embodiment, the first antibody has an amino acid substitution at position 368 and the second antibody has an amino acid substitution at a position selected from the group consisting of 366, 370, 399, 405, 407 and 409.

[0724] In one embodiment, the first antibody has an amino acid substitution at position 370 and the second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 399, 405, 407 and 409.

[0725] In one embodiment, the first antibody has an amino acid substitution at position 399 and the second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 405, 407 and 409.

[0726] In one embodiment, the first antibody has an amino acid substitution at position 405 and the second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 407 and 409.

[0727] In one embodiment, the first antibody has an amino acid substitution at position 407 and the second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 409.

[0728] In one embodiment, the first antibody has an amino acid substitution at position 409 and the second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405 and 407.

[0729] In one embodiment, the first antibody has an amino acid other than Lys, Leu or Met at position 409, e.g., an amino acid at position 409 selected from the group consisting of Gly, Ala, Val, Ile, Ser, Thr, Arg, His, Asp, Asn, Glu, Gln, Trp, Phe, or Tyr, and the second antibody has an amino acid substitution at a position selected from the group consisting of 366, 368, 370, 399, 405, and 407.

[0730] 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 consisting of Gly, Ala, Val, Ile, Ser, Thr, Arg, His, Asp, Asn, Glu, Gln, Trp, Phe, or Tyr at position 409, and the second antibody has an amino acid other than Phe at position 405, e.g., an amino acid selected from the group consisting of Gly, Ala, Val, Leu, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Trp, Met, or Tyr at a position corresponding to 405 of IgG1. In another embodiment of the invention, 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.

[0731] In another embodiment, the first antibody comprises 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 Lys at position 409.

[0732] In another embodiment, the first antibody comprises Phe at position 405, Arg at position 409, and the second antibody comprises Leu at position 405 and Lys at position 409. In embodiments where the antibodies are of IgG1, IgG2 or IgG3 isotypes, the first antibody may comprise a F405L substitution and the second antibody may comprise a K409R substitution, or vice versa. However, in embodiments where the antibodies are both of IgG4 isotypes, the amino acid at position 409 is naturally Arg (R). Therefore, in such embodiments, the first antibody is not substituted at position 409, but naturally has R409, and the second antibody comprises F405L and R405K substitutions, or vice versa; the second antibody is not substituted at position 409, but naturally has R409, and the first antibody comprises F405L and R405K substitutions. In embodiments where one or both of the first and second antibodies are of IgG4 isotypes.

[0733] Thus, in one embodiment, an antibody of the invention may comprise a substitution corresponding to F405L in human IgG1. In another embodiment, an antibody of the invention may comprise a substitution corresponding to K409R in human IgG1. These two different antibodies are suitable for preparing the bispecific antibodies of the invention. In a particularly preferred embodiment, the first antibody of the invention comprises F405L and E430G substitutions, and the second antibody of the invention comprises K409R and E430G substitutions. Thus, antibodies are provided that can form the bispecific antibodies of the invention, which, when numbered using IgG1, contain a first half molecule comprising F405L+E430G substitutions and a second half molecule comprising K409R+E430G. In an embodiment where the isotype is IgG4, when numbered using IgG4, the first half molecule comprises F405L+R409K+E430G substitutions, and the second half molecule comprises E430G. 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 having a CPSC core hinge.

[0734] In one embodiment of the present invention, the first antibody comprises a VH region and a VL region, the VH region comprising a CDR1 sequence as shown in SEQ ID NO: 16, a CDR2 sequence as shown in SEQ ID NO: 17 and a CDR3 sequence as shown in SEQ ID NO: 18, the VL region comprising a CDR1 sequence and a CDR2 sequence as shown in SEQ ID NO: 20: KAS and a CDR3 sequence as shown in SEQ ID NO: 21

[010] ; and an Fc region, the Fc region comprising a F405L substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment, it is E430G.

[0735] In one embodiment of the present invention, the first antibody comprises a VH region and a VL region, the VH region comprising a CDR1 sequence as shown in SEQ ID NO: 16, a CDR2 sequence as shown in SEQ ID NO: 17 and a CDR3 sequence as shown in SEQ ID NO: 18, the VL region comprising a CDR1 sequence and a CDR2 sequence as shown in SEQ ID NO: 20: KAS and a CDR3 sequence as shown in SEQ ID NO: 21

[010] ; and an Fc region, the Fc region comprising a K409R substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment, it is E430G.

[0736] In another embodiment of the present invention, the first antibody comprises a VH region and a VL region, the VH region comprising a CDR1 sequence as shown in SEQ ID NO: 9, a CDR2 sequence as shown in SEQ ID NO: 10 and a CDR3 sequence as shown in SEQ ID NO: 11, the VL region comprising a CDR1 sequence as shown in SEQ ID NO: 113, and a CDR2 sequence: AAS and a CDR3 sequence as shown in SEQ ID NO: 14

[005] ; and an Fc region, the Fc region comprising a F405L substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment, it is E430G.

[0737] In another embodiment of the present invention, the first antibody comprises a VH region and a VL region, the VH region comprising a CDR1 sequence as shown in SEQ ID NO: 9, a CDR2 sequence as shown in SEQ ID NO: 10 and a CDR3 sequence as shown in SEQ ID NO: 11, the VL region comprising a CDR1 sequence as shown in SEQ ID NO: 113, and a CDR2 sequence: AAS and a CDR3 sequence as shown in SEQ ID NO: 14

[005] ; and an Fc region, the Fc region comprising a K409R substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment, it is E430G.

[0738] In one embodiment of the invention, the second antibody comprises a VH region and a VL region, the VH region comprising a CDR1 sequence as shown in SEQ ID NO: 23, a CDR2 sequence as shown in SEQ ID NO: 24 and a CDR3 sequence as shown in SEQ ID NO: 25, the VL region comprising a CDR1 sequence and a CDR2 sequence as shown in SEQ ID NO: 27: YAS and a CDR3 sequence as shown in SEQ ID NO: 28

[016] ; an Fc region, the Fc region comprising a F405L substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment, it is E430G.

[0739] In another embodiment of the present invention, the second antibody comprises a VH region and a VL region, wherein the VH region comprises a CDR1 sequence as shown in SEQ ID NO: 23, a CDR2 sequence as shown in SEQ ID NO: 24, and a CDR3 sequence as shown in SEQ ID NO: 25, wherein the VL region comprises a CDR1 sequence and a CDR2 sequence as shown in SEQ ID NO: 27: YAS and a CDR3 sequence as shown in SEQ ID NO: 28

[016] ; an Fc region, wherein the Fc region comprises a K409R substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345, and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, it is E430G.

[0740] In another embodiment of the present invention, the second antibody comprises a VH region and a VL region, the VH region comprising a CDR1 sequence as shown in SEQ ID NO: 2, a CDR2 sequence as shown in SEQ ID NO: 3 and a CDR3 sequence as shown in SEQ ID NO: 4, the VL region comprising a CDR1 sequence and a CDR2 sequence as shown in SEQ ID NO: 6: EAS and a CDR3 sequence as shown in SEQ ID NO: 7

[004] ; and an Fc region, the Fc region comprising a F405L substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution of one or more amino acid positions selected from the group consisting of: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment, it is E430G.

[0741] In another embodiment of the present invention, the second antibody comprises a VH region and a VL region, the VH region comprising a CDR1 sequence as shown in SEQ ID NO: 2, a CDR2 sequence as shown in SEQ ID NO: 3 and a CDR3 sequence as shown in SEQ ID NO: 4, the VL region comprising a CDR1 sequence and a CDR2 sequence as shown in SEQ ID NO: 6: EAS and a CDR3 sequence as shown in SEQ ID NO: 7

[004] ; and an Fc region, the Fc region comprising a K409R substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution of one or more amino acid positions selected from the group consisting of: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment, it is E430G.

[0742] In another embodiment of the present invention, the second antibody comprises a VH region and a VL region, wherein the VH region comprises a CDR1 sequence as shown in SEQ ID NO: 40, a CDR2 sequence as shown in SEQ ID NO: 41, and a CDR3 sequence as shown in SEQ ID NO: 42, and the VL region comprises a CDR1 sequence as shown in SEQ ID NO: 44, a CDR2 sequence: FAK, and a CDR3 sequence [G28.1] as shown in SEQ ID NO: 45; an Fc region, wherein the Fc region comprises a F405L substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345, and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, it is E430G.

[0743] In another embodiment of the present invention, the second antibody comprises a VH region and a VL region, wherein the VH region comprises a CDR1 sequence as shown in SEQ ID NO: 40, a CDR2 sequence as shown in SEQ ID NO: 41, and a CDR3 sequence as shown in SEQ ID NO: 42, and the VL region comprises a CDR1 sequence as shown in SEQ ID NO: 44, a CDR2 sequence: FAK, and a CDR3 sequence [G28.1] as shown in SEQ ID NO: 45; an Fc region, wherein the Fc region comprises a K409R substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345, and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, it is E430G.

[0744] In another embodiment of the present invention, the second antibody comprises a VH region and a VL region, wherein the VH region comprises a CDR1 sequence as shown in SEQ ID NO: 47, a CDR2 sequence as shown in SEQ ID NO: 48, and a CDR3 sequence as shown in SEQ ID NO: 49, wherein the VL region comprises a CDR1 sequence as shown in SEQ ID NO: 51 and a CDR2 sequence: VAT and a CDR3 sequence [37.3] as shown in SEQ ID NO: 52; and an Fc region, wherein the Fc region comprises a F405L substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345, and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y, and S440W. In a preferred embodiment, it is E430G.

[0745] In another embodiment of the present invention, the second antibody comprises a VH region and a VL region, the VH region comprising a CDR1 sequence as shown in SEQ ID NO: 47, a CDR2 sequence as shown in SEQ ID NO: 48 and a CDR3 sequence as shown in SEQ ID NO: 49, the VL region comprising a CDR1 sequence as shown in SEQ ID NO: 51 and a CDR2 sequence: VAT and a CDR3 sequence [37.3] as shown in SEQ ID NO: 52; an Fc region, the Fc region comprising a K409R substitution and one or more Fc-Fc interaction enhancing mutations. In a preferred embodiment, the Fc-Fc interaction enhancing mutation is a substitution at one or more amino acid positions selected from the group consisting of: 430, 345 and 440, such as E430G, E345K, E430S, E430F, E430T, E345Q, E345R, E345Y, S440Y and S440W. In a preferred embodiment, it is E430G.

[0746] Therefore, the present invention also relates to an anti-CD37 antibody that binds to human CD37, the antibody comprising:

[0747] (i) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 40, a CDR2 sequence as shown in SEQ ID NO: 41, and a CDR3 sequence as shown in SEQ ID NO: 42, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 44, a CDR2 sequence: FAK, and a CDR3 sequence as shown in SEQ ID NO: 45 [G28.1] or

[0748] (ii) a VH region comprising a CDR1 sequence as shown in SEQ ID NO: 47, a CDR2 sequence as shown in SEQ ID NO: 48, and a CDR3 sequence as shown in SEQ ID NO: 49, and a VL region comprising a CDR1 sequence as shown in SEQ ID NO: 51, a CDR2 sequence: VAT, and a CDR3 sequence as shown in SEQ ID NO: 52 [37.3];

[0749] (iii) and wherein 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; and

[0750] (iv) wherein the Fc region optionally further comprises a mutation of K409R or F405L.

[0751] As described above, the anti-CD37 antibodies of the present invention may be of IgG1, IgG2, IgG3 or IgG4 isotype. In one embodiment, the anti-CD37 antibodies of the present invention are of IgG isotype.

[0752] In one embodiment, the antibodies of the invention are human, humanized or chimeric.

[0753] In one embodiment, the antibodies of the invention bind to both human and cynomolgus CD37 antigens.

[0754] Other embodiments of the present invention

[0755] In another embodiment, the invention relates to a composition comprising the bispecific antibody of the invention and further comprising a monospecific anti-CD37 antibody, preferably an anti-CD37 antibody having the antigen binding region of the first or second antigen binding region of the bispecific antibody.

[0756] In one embodiment, the present invention relates to a pharmaceutical composition comprising a bispecific antibody of the present invention or an anti-CD37 antibody of the present invention and a pharmaceutically acceptable carrier.

[0757] In another embodiment, the invention relates to a bispecific antibody of the invention or an antibody of the invention or a composition of the invention for use as a medicament.

[0758] In one embodiment of the invention, the bispecific antibody of the invention is used to treat cancer, autoimmune diseases or inflammatory diseases.

[0759] In one embodiment of the invention, the anti-CD37 antibodies of the invention are used to treat cancer, autoimmune diseases or inflammatory diseases.

[0760] In one embodiment of the invention, the composition of the invention is used to treat cancer, autoimmune diseases or inflammatory diseases.

[0761] In another embodiment, the invention relates to the bispecific antibody of the invention for use in the treatment of allergy, 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).

[0762] In another embodiment, the present invention relates to a composition for use in the treatment of rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral osteoarthritis and juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis (arthritis chronica progrediente), deforming arthritis, chronic primary arthritis (polyarthritis chronica primaria), reactive 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 disease (IBD), including ulcerative colitis and Crohn's disease, chronic obstructive pulmonary disease (COPD), psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, diabetes, Reynaud's syndrome and glomerulonephritis, pustulosis palmoplantaris (PPP), erosive lichen planus, pemphigus bullosa, epidermolysis bullosa, contact dermatitis and atopic dermatitis, polyradiculitis, including Guillain-Barre syndrome.

[0763] 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.

[0764] In another embodiment, 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).

[0765] In another embodiment, the invention relates to an anti-CD37 antibody of the invention for use in the treatment of rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral osteoarthritis and juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, deforming arthritis, chronic primary arthritis, reactive 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 disease (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, palmar-plantar pustulosis (PPP), erosive lichen planus, bullous pemphigus, epidermolysis bullosa, contact dermatitis and atopic dermatitis, polyradiculitis, including Guillain-Barre syndrome.

[0766] In another embodiment, the present invention relates to a bispecific antibody of the present invention used in combination with one or more further therapeutic agents. In another embodiment of the present invention, the anti-CD37 antibody of the present invention is used in combination with one or more further therapeutic agents. One or more further therapeutic agents can be selected, for example, from the group consisting of doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, and anti-CD20 antibodies such as rituximab, ofatumumab, obinutuzumab, veltuzumab, ocaraatuzumab, ocrelizumab, or tru-015.

[0767] In a preferred embodiment of the invention, the further therapeutic agent is an anti-CD20 antibody.

[0768] In one embodiment of the present invention, the anti-CD20 antibody is capable of binding to human CD20 having the sequence shown in SEQ ID NO:72.

[0769] In one embodiment of the present invention, the anti-CD20 antibody is capable of binding to cynomolgus monkey CD20 having the sequence shown in SEQ ID NO:73.

[0770] In one embodiment of the present invention, the anti-CD20 antibody is capable of binding to human and cynomolgus monkey CD20 having the sequences shown in SEQ ID No 72 and 73, respectively.

[0771] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to an epitope on human CD20 that does not contain or require an amino acid residue alanine at position 170 or a proline at position 172 of SEQ ID No. 72, but contains or requires an amino acid residue asparagine at position 163 and an asparagine at position 166. Examples of such antibodies are antibodies designated 2F2 and 7D8 as disclosed in WO2004035607 (Genmab) and an antibody designated 2C6 as disclosed in WO2005103081 (Genmab). The CDR sequences of 7D8 are disclosed in Table 1.

[0772] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to an epitope on human CD20 that does not contain or require the amino acid residue alanine at position 170 or proline at position 172 of SEQ ID No. 72. An example of such an antibody is 11B8 as disclosed in WO2004035607 (Genmab). The CDR sequences of 11B8 are disclosed in Table 1.

[0773] 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 a portion of a first small extracellular loop and a portion of a second extracellular loop.

[0774] In one embodiment of the invention, the anti-CD20 antibody is capable of binding to a discontinuous epitope on human CD20, wherein the epitope has a small first extracellular loop with residues AGIYAP and a second extracellular loop with residues MESLNFIRAHTPY.

[0775] 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, such as ofatumumab (2F2) and rituximab, while type II anti-CD20 antibodies have low or no CDC activity, but high ADCC and apoptotic activity, such as obinutuzumab and 11B8. Type I antibodies also induce CD20 to redistribute into larger detergent-resistant microdomains (rafts), while type II antibodies do not.

[0776] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) shown in SEQ ID No 74 and SEQ ID No 78, respectively.

[0777] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) shown in SEQ ID No 81 and SEQ ID No 109, respectively.

[0778] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) shown in SEQ ID No 94 and SEQ ID No 98, respectively.

[0779] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) shown in SEQ ID No 87 and SEQ ID No 91, respectively.

[0780] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region competes for binding to human CD20 with an anti-CD20 antibody comprising a variable heavy chain (VH) sequence and a variable light chain (VL) shown in SEQ ID No 101 and SEQ ID No 105, respectively.

[0781] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region comprises the following CDR sequences:

[0782] The VH CDR1 sequence shown in SEQ ID NO:75,

[0783] The VH CDR2 sequence shown in SEQ ID NO: 76,

[0784] The VH CDR3 sequence shown in SEQ ID NO: 77,

[0785] The VL CDR1 sequence shown in SEQ ID NO: 79

[0786] VL CDR2 sequence DAS, and

[0787] The VL CDR3 sequence is shown in SEQ ID NO:80. [7D8]

[0788] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region comprises the following CDR sequences:

[0789] The VH CDR1 sequence shown in SEQ ID NO:82,

[0790] The VH CDR2 sequence shown in SEQ ID NO: 83,

[0791] The VH CDR3 sequence shown in SEQ ID NO: 84,

[0792] The VL CDR1 sequence shown in SEQ ID NO: 85

[0793] VL CDR2 sequence DAS, and

[0794] The VL CDR3 sequence is shown in SEQ ID NO:86. [118B]

[0795] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region comprises the following CDR sequences:

[0796] The VH CDR1 sequence shown in SEQ ID NO:95,

[0797] The VH CDR2 sequence shown in SEQ ID NO:96,

[0798] The VH CDR3 sequence shown in SEQ ID NO:97,

[0799] The VL CDR1 sequence shown in SEQ ID NO:99,

[0800] VL CDR2 sequence ATS, and

[0801] The VL CDR3 sequence is shown in SEQ ID NO: 100. [Rituximab]

[0802] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region comprises the following CDR sequences:

[0803] The VH CDR1 sequence shown in SEQ ID NO: 88,

[0804] The VH CDR2 sequence shown in SEQ ID NO: 89,

[0805] The VH CDR3 sequence shown in SEQ ID NO:90,

[0806] The VL CDR1 sequence shown in SEQ ID NO:92

[0807] VL CDR2 sequence DAS, and

[0808] The VL CDR3 sequence is shown in SEQ ID NO: 93. [Ofatumumab]

[0809] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region comprises the following CDR sequences:

[0810] The VH CDR1 sequence shown in SEQ ID NO: 102,

[0811] The VH CDR2 sequence shown in SEQ ID NO: 103,

[0812] The VH CDR3 sequence shown in SEQ ID NO: 104,

[0813] The VL CDR1 sequence shown in SEQ ID NO: 106,

[0814] VL CDR2 sequence QMS, and

[0815] The VL CDR3 sequence is shown in SEQ ID NO: 107. [Obinutuzumab]

[0816] In one embodiment of the present invention, the anti-CD20 antibody comprises an antigen binding region capable of binding to human CD20, wherein the antigen binding region comprises a CDR sequence selected from the group consisting of:

[0817] i) the VH CDR1 sequence shown in SEQ ID NO: 75,

[0818] The VH CDR2 sequence shown in SEQ ID NO: 76,

[0819] The VH CDR3 sequence shown in SEQ ID NO: 77,

[0820] The VL CDR1 sequence shown in SEQ ID NO: 79,

[0821] VL CDR2 sequence DAS, and

[0822] The VL CDR3 sequence shown in SEQ ID NO:80 [7D8];

[0823] ii) the VH CDR1 sequence shown in SEQ ID NO: 82,

[0824] The VH CDR2 sequence shown in SEQ ID NO: 83,

[0825] The VH CDR3 sequence shown in SEQ ID NO: 84,

[0826] The VL CDR1 sequence shown in SEQ ID NO: 85,

[0827] VL CDR2 sequence DAS, and

[0828] The VL CDR3 sequence shown in SEQ ID NO:86 [118B];

[0829] iii) the VH CDR1 sequence shown in SEQ ID NO:95,

[0830] The VH CDR2 sequence shown in SEQ ID NO:96,

[0831] The VH CDR3 sequence shown in SEQ ID NO:97,

[0832] The VL CDR1 sequence shown in SEQ ID NO:99,

[0833] VL CDR2 sequence ATS, and

[0834] The VL CDR3 sequence shown in SEQ ID NO: 100 [rituximab];

[0835] iv) the VH CDR1 sequence shown in SEQ ID NO: 88,

[0836] The VH CDR2 sequence shown in SEQ ID NO: 89,

[0837] The VH CDR3 sequence shown in SEQ ID NO:90,

[0838] The VL CDR1 sequence shown in SEQ ID NO:92,

[0839] VL CDR2 sequence DAS, and

[0840] The VL CDR3 sequence shown in SEQ ID NO:93 [ofatumumab]; and

[0841] v) the VH CDR1 sequence shown in SEQ ID NO: 102,

[0842] The VH CDR2 sequence shown in SEQ ID NO: 103,

[0843] The VH CDR3 sequence shown in SEQ ID NO: 104,

[0844] The VL CDR1 sequence shown in SEQ ID NO: 106,

[0845] VL CDR2 sequence QMS, and

[0846] The VL CDR3 sequence shown in SEQ ID NO: 107 [Obinutuzumab].

[0847] In another embodiment, the invention relates to the use of the bispecific antibody of the invention or the anti-CD37 antibody of the invention for the preparation of a medicament. In another embodiment herein, the use is for the preparation of a medicament for the treatment of cancer, autoimmune diseases or inflammatory diseases such as 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), rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, hyperplastic arthritis, psoriatic arthritis, stigma arthritis, , spondylotic arthritis, spondylotic arthritis, juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, deforming arthritis, chronic primary arthritis, reactive 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 disease (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, palmar-plantar pustulosis (PPP), erosive lichen planus, bullous pemphigus, epidermolysis bullosa, contact dermatitis and atopic dermatitis, polyradiculitis, including Guillain-Barre syndrome.

[0848] In another embodiment, the present invention relates to a method for inducing cell death of tumor cells expressing CD37 or inhibiting growth and / or proliferation of tumor cells expressing CD37, the method comprising administering an effective amount of a bispecific antibody of the present invention or an anti-CD37 antibody of the present invention to an individual in need thereof. In certain embodiments, the method is used to treat an individual with allergy, 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), comprising administering an effective amount of a bispecific antibody of the present invention or an anti-CD37 antibody of the present invention to the individual. In certain embodiments, the methods comprise using one or more further therapeutic agents in combination with the antibody or the bispecific antibody, such as doxorubicin, cisplatin, bleomycin, carmustine, cyclophosphamide, chlorambucil, bendamustine, vincristine, fludarabine, ibrutinib, or an anti-CD20 antibody, such as rituximab, ofatumumab, obinutuzumab, veltuzumab, ocaratumumab, or TRU-015.

[0849] In one embodiment of the invention, the further therapeutic agent is selected from the group consisting of 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 inhibitors, cytosine ... Tuximab, obinutuzumab and ofatumumab, CD52-specific alemtuzumab, CD30-specific brentuximab, JNJ-63709178, JNJ-64007957, HuMax-IL8, anti-DR5, anti-VEGF, anti-CD38, anti-PD-1, anti-PD-L1, anti-CTLA4, anti-CD40, anti-CD137, anti-GITR, anti-VISTA, antibodies specific for other immunomodulatory targets, brentuximab vedotin, HuMax-TAC-ADC, interferon, thalidomide, lenalidomide, Axicabtagene ciloleucel, bortezomib, romidepsin, belinostat, vorinostat, ibrutinib, acalabrutinib, idelalisib, copanlisib, sorafenib, sunitinib, everolimus, recombinant human TRAIL, birinapant, and venetoclax.

[0850] In one embodiment of the invention, the further therapeutic agent is selected from the group consisting of: ibrutinib, rituximab, vena citrate, CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone), bendamustine, fludarabine, cyclophosphamide and chlorambucil.

[0851] In one embodiment of the invention, the further therapeutic agent is selected from the group consisting of: ibrutinib, rituximab and vena cava.

[0852] In a further aspect, the present invention relates to a nucleic acid construct encoding one or more sequences shown 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 consisting of SEQ ID NO: 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.

[0853] 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 embodiment herein.

[0854] The present invention further relates to a nucleic acid construct encoding a bispecific antibody or an anti-CD37 antibody according to any embodiment herein.

[0855] In a further embodiment, the invention relates to an expression vector comprising one or more of the nucleic acid constructs defined above. In another embodiment, the invention relates to a host cell comprising an expression vector as defined above.

[0856] In the context of the present invention, the expression vector may be any suitable vector, including chromosomal, non-chromosomal and synthetic nucleic acid vectors (containing a nucleic acid sequence of a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination 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 in a naked DNA or RNA vector, which includes, for example, 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 WO00 / 70087), a plasmid vector, such as pBR322, pUC 19 / 18 or pUC 118 / 119, a "midge" minimal size nucleic acid vector (as described in Schakowski et al., Mol Ther 3, 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaPO4 precipitated construct (e.g., WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., 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, for example, US 5,589,466 and US 5,973,972).

[0857] In one embodiment, the vector is suitable for expressing CD37 antibodies 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 vector (Novagen, Madison WI), etc.

[0858] Additionally 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 (reviewed in: F. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol 153 ,516-544(1987)).

[0859] Additionally or alternatively, the expression vector may be a vector suitable for expression in mammalian cells, such as a vector comprising glutamine synthetase as a selectable marker, such as that described in Bebbington (1992) Biotechnology (NY) 10: 169-175.

[0860] Nucleic acid and / or vector can also comprise nucleic acid sequence encoding secretion / localization sequence, and described secretion / localization sequence can target polypeptide such as nascent polypeptide chain to periplasmic space or enter cell culture medium.Such sequence is known in the art, and comprises secretion leader sequence or signal peptide.

[0861] The expression vector may include any suitable promoter, enhancer and other expression promoting elements or may be associated therewith. Examples of such elements include strong expression promoters (e.g., human CMV IE promoter / enhancer and RSV, SV40, SL3-3, MMTV and HIV LTR promoters), effective poly (A) terminator sequences, origins of replication of plasmid products in Escherichia coli, antibiotic resistance genes as selective markers and / or convenient cloning sites (e.g., polylinkers). Contrary to constitutive promoters such as CMV IE, nucleic acid may also include inducible promoters.

[0862] In one embodiment, an expression vector encoding a CD37 antibody can be localized in and / or delivered to a host cell or a host animal via a viral vector.

[0863] Therefore, the present invention also relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, that produce the bispecific antibodies of the present invention.

[0864] The present invention further relates to anti-idiotypic antibodies that bind to the antigen binding region of an antibody or bispecific antibody of the present invention.

[0865] An in vitro method for detecting human CD37 antigen or cells expressing human CD37 in a sample, the method comprising:

[0866] (i) contacting the sample with the bispecific antibody of any of the above embodiments or the antibody of any of the embodiments herein under conditions that allow formation of a complex between the antibody or bispecific antibody and CD37; and

[0867] (ii) Detecting complex formation.

[0868] An in vivo method for detecting human CD37 antigen or cells expressing human CD37 in a subject, the method comprising:

[0869] (i) administering the bispecific antibody of any of the above embodiments or the antibody of any embodiment herein under conditions that allow formation of a complex between the antibody or bispecific antibody and CD37; and

[0870] (ii) Detecting the formed complex.

[0871] sequence

[0872] Table 1:

[0873]

[0874]

[0875]

[0876]

[0877]

[0878]

[0879]

[0880]

[0881]

[0882]

[0883]

[0884] Example

[0885] Example 1: Generation of CD37-specific antibodies in rabbits

[0886] CD37 expression construct

[0887] The following codon-optimized constructs were generated for 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). In addition, the following codon-optimized constructs were generated for expression of various CD37 ECD variants: a signal peptide coding sequence followed by the second extracellular domain (EC2) of human CD37 (aa 112-241), 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 restriction sites suitable for cloning and an optimal Kozak (GCCGCCACC) sequence [Kozak et al. (1999) Gene 234:187-208]. The construct was cloned in the mammalian expression vector pcDNA3.3 (Invitrogen) or an equivalent vector. Transient expression in CHO and HEK cells

[0888] Membrane proteins were transiently transfected 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) by using Freestyle Max reagent (Life technologies) essentially as described by the manufacturer. Soluble proteins were transiently expressed in Expi293 cells (Life Technologies) by using ExpiFectamine 293 reagent (Life technologies) essentially as described by the manufacturer.

[0889] Fc fusion proteins (CD37mfEC2-FcHis and CD37EC2-FcHis) were purified from cell culture supernatants using Protein A affinity chromatography.

[0890] Rabbit immunization

[0891] Rabbit immunization was 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 from these animals was collected and B lymphocytes were isolated. Using a MAB Discovery proprietary process, single B cells were sorted into wells of microtiter plates and further propagated. Supernatants of these single B cells were analyzed for specific binding to CHO-S cells transiently expressing CD37 (CHO-CD37) and mfCD37 (CHO-mfCD37).

[0892] Recombinant Antibody Production

[0893] After analyzing the initial screening results, the main hits were selected for sequencing, recombinant mAb production and purification. Unique variable heavy chain (VH) and light chain (VL) coding regions were synthesized and cloned into a mammalian expression vector containing human IgG1 constant region coding sequences (Ig Kappa chain and IgG1 allotype G1m (f), which contains the E430G mutation (EU numbering) heavy chain). During this process, unfavorable unpaired cysteines in some antibody light chains were replaced with serines.

[0894] Recombinant chimeric antibodies were produced in HEK 293 cells by transient co-transfection of expression vectors encoding heavy chain (HC) and light chain (LC) 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.

[0895] The reactivity of the generated chimeric (VH rabbit, Fc human) monoclonal antibodies (mAbs) containing the mutation E430G was reanalyzed for binding to CHO-CD37 or CHO-mfCD37 cells. In addition, binding to the human lymphoma cell line Daudi and functionality in a CDC assay on Daudi cells were analyzed.

[0896] Example 2: Humanization of rabbit chimeric antibodies

[0897] Generation of humanized antibody sequences

[0898] Humanized antibody sequences from rabbit antibodies rabbit anti-CD37-004, -005, -010 and -016 were generated at Antitope (Cambridge, UK). Humanized antibody sequences were generated using germline humanization (CDR implantation) technology. Humanized V region genes were designed based on human germline sequences with the closest homology to the VH and Vκ amino acid sequences of rabbit and mouse antibodies. A series of 4 to 6 VH and 4 or 5 Vκ (VL) germline humanized V region genes were designed for each rabbit antibody.

[0899] Structural models of rabbit antibody V regions were generated using the Swiss PDB and analyzed to identify amino acids in the V region framework that may be important for the binding properties of the antibody. These amino acids were recorded for incorporation into one or more antibodies implanted with variant CDRs.

[0900] The heavy and light chain V region amino acid sequences were compared to 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.

[0901] Table 2: Closest matching human germline V-segment and J-segment sequences.

[0902]

[0903]

[0904] A series of humanized heavy and light chain V regions were then designed by implanting the CDRs onto the frameworks and, if necessary, by backmutating residues that may be critical for the antibody binding properties (as identified in structural modeling) to rabbit residues. TM and TCED TM(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) select the variant sequence with the lowest occurrence of potential T cell epitopes. Finally, the nucleotide sequence of the designed variant was codon optimized.

[0905] For antibody IgG1-016-H5L2, a variant was generated with point mutations in the variable domain to replace the free cysteine: IgG1-016-H5L2-LC90S (also generated with additional F405L and E430G mutations). This mutant was generated by gene synthesis (Geneart).

[0906] The variable region sequences of the humanized CD37 antibodies are shown in the Sequence Listing herein and in Table 1 above.

[0907] Example 3: Generation of bispecific antibodies

[0908] Bispecific IgG1 antibodies are produced by exchanging Fab arms under controlled reducing conditions. The basis of this method is the use of complementary CH3 domains, which promote the formation of heterodimers under specific assay conditions, as described in WO2011 / 131746. F405L and K409R (EU numbering) mutations are introduced into CD37 antibodies to create antibody pairs with complementary CH3 domains. In some cases, F405L and K409R mutations are combined with E430G mutations.

[0909] To generate bispecific antibodies, two parent complementary antibodies (final concentration of each antibody 0.5 mg / mL) were incubated with 75mM 2-mercaptoethylamine-HCl (2-MEA) for 5 hours at 31 ° C in a total volume of 100 μL TE. 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.

[0910] Example 4: Expression constructs, transient expression and purification of antibodies

[0911] For antibody expression, the VH and VL sequences were cloned in an expression vector (pcDNA3.3) containing the relevant constant heavy chain (HC) in the case of VH, in some cases containing F405L or K409R mutations and / or E345R or E430G mutations, and the light chain (LC) region in the case of VL.

[0912] The antibodies were expressed as IgG1, κ. A mixture of plasmid DNA encoding both the heavy and light chains of the antibodies was transiently transfected in 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). Next, the antibodies were purified by immobilized protein G chromatography.

[0913] The following antibodies were used in the Examples:

[0914] Wild-type IgG1 antibody:

[0915] IgG1-004-H5L2 (having the VH and VL sequences shown in SEQ ID NO: 1 and SEQ ID NO: 5)

[0916] IgG1-005-H1L2 (having VH and VL sequences shown in SEQ ID NO: 8 and SEQ ID NO: 12)

[0917] IgG1-010-H5L2 (having VH and VL sequences shown in SEQ ID NO: 15 and SEQ ID NO: 19)

[0918] IgG1-016-H5L2 (having VH and VL sequences shown in SEQ ID NO: 22 and SEQ ID NO: 26)

[0919] IgG1-G28.1 (having VH and VL sequences shown in SEQ ID NO:39 and SEQ ID NO:43 - based on SEQ ID No 1 and 3 in EP2241577)

[0920] IgG1-G28.1-K409R-delK (also contains C-terminal heavy chain mutations 445-PG-446)

[0921] IgG1-37.3 (having VH and VL sequences shown as SEQ ID NO: 46 and SEQ ID NO: 50 - based on SEQ ID No 55 and 72 in WO2011 / 112978)

[0922] IgG1-b12 ((having VH and VL sequences shown in SEQ ID NO:32 and SEQ ID NO:36 - based on gp120-specific antibody b12 [Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3):812-23])

[0923] IgG1 antibodies with the Fc-Fc interaction enhancing mutation E430G:

[0924] IgG1-004-H5L2-E430G

[0925] IgG1-005-H1L2-E430G

[0926] IgG1-010-H5L2-E430G

[0927] IgG1-016-H5L2-E430G

[0928] IgG1-G28.1-E430G

[0929] IgG1-37.3-E430G

[0930] IgG1-b12-E430G

[0931] IgG1-005-H1L2-K409R-E430G

[0932] IgG1-010-H5L2-K409R-E430G

[0933] IgG1-016-H5L2-F405L-E430G

[0934] IgG1-016-H5L2-LC90S-F405L-E430G

[0935] IgG1-004-E430G

[0936] IgG1-005-E430G

[0937] IgG1-010-E430G

[0938] IgG1-016-E430G

[0939] IgG1 antibodies with the Fc-Fc interaction enhancing mutation E430S:

[0940] IgG1-010-H5L2-K409R-E430S

[0941] IgG1-016-H5L2-F405L-E430S

[0942] IgG1 antibodies with the Fc-Fc interaction enhancing mutation E345K:

[0943] IgG1-010-H5L2-K409R-E345K

[0944] IgG1-016-H5L2-F405L-E345K

[0945] IgG1 Antibodies with Fc-Fc Interaction Enhancing Mutation E345R:

[0946] IgG1-G28.1-E345R

[0947] IgG1-b12-E345R

[0948] IgG1-010-H5L2-K409R-E345R

[0949] IgG1-016-H5L2-F405L-E345R

[0950] Bispecific Antibodies

[0951] bsIgG1-016-H5L2-F405LxIgG1-IgG1-005-H1L2-K409RbsIgG1-016-H5L2-F405LxIgG1-010-H5L2-K409R

[0952] Bispecific antibodies with Fc-Fc interaction enhancing mutation E430G: bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430GbsIgG1-016-H5L2-LC90S-F4 05L-E430Gx010-H5L2-K409R-E430GbsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G

[0953] bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G

[0954] bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G

[0955] IgG1 Antibodies with FcγR Interaction Enhancing Mutations S239D-I332E:

[0956] IgG1-G28.1-S239D-I332E

[0957] Example 5: Introduction of Fc-Fc interaction enhancing mutations into CD37 antibodies results in enhanced de novo ability to induce complement dependent cytotoxicity (CDC)

[0958] Complement-dependent cytotoxicity (CDC) assay

[0959] In the first experiment, tumor cells from untreated CLL patients (AllCells, California, USA) were resuspended in RPMI containing 0.2% BSA (bovine serum albumin) and then distributed into polystyrene 96-well round-bottom plates (Greiner bio-one Cat#650101) at a density of 0.2×105 cells / well (40 μL / well), and 40 μL of IgG1-G28.1-K409R-delK, IgG1-G28.1-E345R or IgG1-b12-E345R concentration series (final antibody concentration 0.003-10 μg / mL) were added. IgG1-b12-E345R (based on gp120-specific antibody b12 [Barbas, CF. J Mol Biol. 1993 Apr 5; 230(3):812-23]) was used as a negative control. For IgG1-G28.1-K409R-delK, it should be noted that the K409R mutation had no effect on binding capacity or the ability to induce CDC. Similarly, the delK (445-PG-446) mutation, which had been introduced into the antibody to facilitate biochemical analysis, did not affect target binding or the ability to induce CDC (see below).

[0960] After incubation (room temperature, 10 minutes under shaking), 20 μL of normal human serum (NHS Cat#M0008Sanquin, Amsterdam, The Netherlands) merged was added to each well as a complement source, and the plate was incubated at 37°C for 45 minutes. The reaction was terminated by cooling the plate on ice. Next, propidium iodide (PI; 10 μL of 10 μg / mL solution; Sigma-Aldrich Chemie BV, Zwijndrecht, The Netherlands) was added, and the percentage of dead cells (corresponding to PI positive cells) was measured by flow cytometry (FACS Canto II; BD Biosciences) to detect lysis. The best fit value of the nonlinear dose response fit with logarithmic conversion concentration was used to generate a graph in GraphPad Prism V6.04 software (GraphPad Software, San Diego, CA, USA).

[0961] In the second experiment, tumor cells from another untreated CLL patient (AllCells, California, USA) were resuspended in RPMI containing 0.2% BSA and distributed to polystyrene 96-well round bottom plates (Greiner bio-one Cat#650101) at a density of 0.5x105 cells / well (30 μL / well), and 50 μL of IgG1-G28.1, IgG1-G28.1-E430G or IgG1-b12 concentration series (final antibody concentration 0.003-10 μg / mL, with 3.33x serial dilution). After incubation (room temperature, 15 minutes), 20 μL of combined 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 minutes. The reaction was terminated by cooling 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 CantoII; BD Biosciences). Graphs were generated using the best fit values ​​of a nonlinear dose response fit with log-transformed concentrations in GraphPadPrism V6.04 software (GraphPad Software, San Diego, CA, USA).

[0962] Figure 1 A and B show that 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 on primary tumor cells from CLL patients, while 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 on primary CLL cells.

[0963] Quantification of cell surface antigens by flow cytometry (QiFi)

[0964] CD37 and membrane complement regulatory proteins (mCRP; CD46, CD55 and CD59) expression levels on CLL tumor cells were determined using a human IgG calibration kit (Biocytix Cat#CP010). In brief, tumor cells from CLL patients (as in the first experiment described above) were resuspended in RPMI containing 0.2% BSA, then distributed to polystyrene 96-well round-bottom plates (Greiner bio-one Cat#650101) at a density of 0.5x105 cells / well (30 μL / well), centrifuged, and 50 μL CD37 (Abcam, catalog number 76522) or control mouse antibodies (purified mouse IgG1, κ isotype control, clone MOPC-21; BD catalog number 555746) were added. After incubation (4°C, 30 minutes), 50 μL calibration beads were added to separate wells. After washing the beads and cells twice (150 μL FACS buffer, centrifuged at 300xg for 3 minutes at 4°C between washing steps), 50 μL / well secondary antibody (FITC conjugated) diluent as provided in the human IgG calibration kit was added. After incubation in the dark (4°C, 45 minutes), the cells were washed twice with FACS buffer, and the cells were resuspended in 35 μL FACS buffer and analyzed by flow cytometry (Intellicyt iQueTM screener). According to the manufacturer's guidelines, the amount of antigen was determined by calculating the antibody binding capacity according to the calibration curve.

[0965] Figure 2 It was shown that CD37 was highly expressed on primary tumor cells from this CLL patient. The patient showed normal expression levels of mCRP.

[0966] Example 6: Binding of CD37 Antibodies and Variants Thereof to CD37 Expressed on the Cell Surface

[0967] Binding to cell surface expressed CD37 (Daudi cells, CHO cells expressing cynomolgus CD37) was determined by flow cytometry. The cells were resuspended in RPMI containing 0.2% BSA, seeded at 100,000 cells / well in a polystyrene 96-well round bottom plate (Greiner bio-one Cat#650101), and centrifuged at 300xg for 3 minutes at 4°C. Serial dilutions of CD37 or control antibodies (final antibody concentration 0.003-10 μg / mL, serially diluted at 3.33x) were added, and the cells were incubated at 4°C for 30 minutes. The plate was washed / centrifuged twice using FACS buffer (PBS / 0.1% BSA / 0.01% sodium azide). Next, the cells were incubated with goat anti-human IgG F(ab')2 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; cat#: 109-116-098) conjugated to R-phycoerythrin (PE) diluted 1 / 100 in PBS / 0.1% BSA / 0.01% sodium azide at 4°C for 30 minutes. The cells were washed / centrifuged twice with FACS buffer, resuspended in 30 μL FACS buffer, and analyzed by measuring the mean fluorescence intensity using Intellicyt iQueTM Screener (Westburg). Binding curves were generated using nonlinear regression (sigmoidal dose response with variable slope) analysis in GraphPad Prism V6.04 software (GraphPad Software, Sand Diego, CA, USA).

[0968] Binding to Daudi cells

[0969] Figure 3 It was shown that humanized CD37 antibodies IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2 and IgG1-016-H5L2 showed dose-dependent binding to Daudi cells. Introduction of the Fc-Fc interaction enhancing E430G mutation and, for IgG1-005-H1L2, also the K409R mutation into these antibodies did not affect binding.

[0970] Figure 4 It was shown that introduction of the E430G mutation into IgG1-G28.1 or IgG1-37.3 did not affect binding to Daudi cells.

[0971] For antibody IgG1-016-H5L2, a variant with point mutations in the variable domain to replace free cysteine ​​in the light chain was generated: IgG1-016-H5L2-LC90S. This variant was also generated with additional F405L and E430G mutations, which were previously shown not to affect target binding properties. Figure 5 It was shown that IgG1-016-H5L2, IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G and IgG1-016-H5L2-LC90S-F405L-E430G all showed comparable binding to Daudi cells, thus the LC90S mutation did not affect binding.

[0972] Binding to CHO cells expressing cynomolgus CD37

[0973] Binding to CHO cells expressing cynomolgus CD37 was determined by flow cytometry using the method described above. Figure 6 It was shown that IgG1-004-H5L2-E430G, IgG1-005-H1L2-E430G, IgG1-010-H5L2-E430G and IgG1-016-H5L2-E430G showed dose-dependent binding to CHO cells expressing cynomolgus CD37. IgG1-G28.1 and IgG1-28.1-E430G did not bind to CHO cells expressing cynomolgus CD37.

[0974] Example 7: Identification of CD37 Antibodies That Do Not Compete for Binding to CD37 (Lack of) binding competition – by flow cytometry Determination

[0975] CD37 antibody was labeled with Alexa Fluor 488 NHS ester (succinimidyl ester). 1 mg CD37 antibody (dissolved in PBS) was transferred to a 1 ml microcentrifuge vial (reaction vial). The pH was raised by adding 10% volume of 1M sodium bicarbonate buffer (pH 9). Immediately before use, 1 mg Alexa Fluor 488 NHS ester (adjusted to room temperature) was dissolved in 100 μL DMSO. The labeling reaction was started by adding 10 μL of fresh Alexa dye solution per mg of antibody. The reaction vial was capped and gently mixed by inversion. After incubation at room temperature for 1 hour, the reaction was quenched by adding 50 μL 1M Tris to each reaction bottle. Unreacted dye was removed from the Alexa-labeled antibody by gel filtration using a BioRad PDP10 column equilibrated with borate saline buffer according to the manufacturer's instructions. The Alexa-labeled antibody was stored at 4°C in the dark.

[0976] The binding competition between different CD37 antibodies was determined by flow cytometry. Raji cells (ATCC, CCL-86) were resuspended in Raji culture medium (RPMI 1640, 10% FBS, 100U / mL penicillin, 100μg / mL streptomycin, 10mM HEPES and 1mM pyruvic acid) with a concentration of 1x107 cells / mL. Next, 30μL cell suspension aliquots were transferred to FACS tubes together with 30μL aliquots (final concentration 40μg / mL) of unlabeled antibody solution. The mixture was incubated at 37°C for 15 minutes while shaking gently. Next, antibody diluents labeled with A488 were prepared, and after incubation, 10μL of labeled antibodies (final antibody concentration 4μg / mL) were transferred to FACS tubes containing unlabeled antibodies and cells. The mixture was incubated at 37°C for 15 minutes while shaking gently. After incubation, the 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 PBS. The binding competition was analyzed by measuring the mean fluorescence intensity using BD FACSCalibur (BD Biosciences). The fluorescence intensity was converted to equivalent soluble fluorescent molecules (MESF) for quantification.

[0977] Figure 7 A and Figure 8 It is shown that preincubation of Raji cells with IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G blocks subsequent binding of IgG1-005-H1L2-E430G and IgG1-010-H5L2E430G, but not IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G, and IgG1-016-H5L2-E430G.

[0978] Preincubation of Raji cells with IgG1-004-H5L2-E430G greatly reduced the subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G and IgG1-016-H5L2-E430G, but not IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.

[0979] 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 not IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.

[0980] Pre-incubation of cells with IgG1-37.3-E430G blocked subsequent binding of all antibodies tested. However, as discussed above, pre-incubation with either IgG1-005-H1L2-E430G or IgG1-010-H5L2-E430G did not block binding of IgG1-37.3-E430G.

[0981] Preincubation of cells with IgG1-G28.1-E430G blocked subsequent binding of IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-H5L2-E430G and IgG1-016-H5L2-E430G but not IgG1-005-H1L2-E430G and IgG1-010-H5L2-E430G.

[0982] (Lack of) binding competition – functional screening assay using a CDC assay

[0983] To determine whether non-cross-blocking CD37 antibodies show enhanced CDC when combined and to confirm the potential to functionally combine non-cross-blocking CD37 antibodies, CDC assays were performed using individual CD37 antibodies and their combinations.

[0984] Raji cells were resuspended in RPMI containing 0.2% BSA at a concentration of 1×10 5The density of cells / well (30 μL / well) was distributed into a polystyrene 96-well round bottom plate (Greiner bio-one Cat#650101), and 50 μL of humanized CD37 antibody, its variant, its combination or control antibody IgG1-b12 (final antibody concentration 10 μg / mL, combination 5+5 μg / mL) was added. After incubation (room temperature, 15 minutes with shaking), 20 μL of combined 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-AldrichChemie 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).

[0985] Figure 7 B and C show that the combination of IgG1-004-H5L2 plus IgG1-010-H5L2 (with or without E430G mutation) and the combination of IgG1-005-H1L2 plus IgG1-016-H5L2 (with or without E430G mutation) induced enhanced CDC compared to their individual counterparts. The combination of IgG1-004-H5L2 plus IgG1-016-H5L2 (with or without E430G mutation) did not induce enhanced CDC compared to its individual counterparts.

[0986] Figure 7 D and E show that the combination of IgG1-004-H5L2 plus IgG1-005-H1L2 (with or without E430G mutation) and the combination of IgG1-010-H5L2 plus IgG1-016-H5L2 (with or without E430G mutation) induced enhanced CDC compared to their individual counterparts. The combination of IgG1-005-H1L2 plus IgG1-010-H5L2 (with or without E430G mutation) did not induce enhanced CDC compared to its individual counterparts.

[0987] Figure 7F and G show that the combination of IgG1-37.3 plus IgG1-005-H1L2 (with or without E430G mutation) and the combination of IgG1-37.3 plus IgG1-010-H5L2 (with or without E430G mutation) induced enhanced CDC compared to their individual counterparts.

[0988] Thus, the functional combination studies confirmed the results of the binding competition studies of the described CD37 antibodies and showed that non-cross-blocking CD37 antibodies can be functionally combined.

[0989] Example 8: Introduction of Fc-Fc interaction enhancing mutations into humanized CD37 antibodies results in enhanced de novo ability to induce complement dependent cytotoxicity (CDC)

[0990] Raji cells were resuspended in RPMI containing 0.2% BSA at a concentration of 1×10 5 The density of cells / well (30 μL / well) was distributed into a polystyrene 96-well round bottom plate (Greiner bio-one Cat#650101), and 50 μL of a concentration series of humanized CD37 antibody and its variants or control antibody IgG1-b12 was added (final antibody concentration 0.003-10 μg / mL, with 3.33x serial dilution). After incubation (room temperature, 15 minutes), 20 μL of combined 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 (IntellicytiQue™ screener, Westburg). 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).

[0991] Fig. 9It was shown that IgG1-004-H5L2, IgG1-005-H1L2, IgG1-010-H5L2 and IgG1-016-H5L2 did not induce CDC in Daudi cells. After the 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.

[0992] Fig.10 A shows that IgG1-G28.1 and IgG1-37.3 do not induce CDC on Daudi cells. After the 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 on Daudi cells.

[0993] For antibody IgG1-016-H5L2, variants with point mutations in the variable domain were generated to replace the free cysteine ​​in the light chain: IgG1-016-H5L2-LC90S. In addition, variants with the F405L mutation (previously shown not to affect target binding or CDC) and the Fc-Fc interaction enhancing E430G mutation were also generated. Fig.11 It was shown that IgG1-016-H5L2-E430G, IgG1-016-H5L2-F405L-E430G and IgG1-016-H5L2-LC90S-F405L-E430G all showed comparable activity in the in vitro CDC assay, so the LC90S mutation did not affect the ability to induce CDC. IgG1-016-H5L2 did not induce CDC on Daudi cells.

[0994] Introduction of other Fc-Fc interaction enhancing mutations E345K, E345R, E430S and RRGY into IgG1-010-H5L2 and IgG1-016-H5L2 also resulted in profound CDC in Daudi cells. Fig.10 B and C show that maximal lysis of Daudi cells was comparable for all Fc-Fc interaction enhancing mutations tested.

[0995] Example 9: Due to monovalent binding and dual epitope targeting, bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations induce CDC more potently than monospecific bivalent CD37 antibodies with Fc-Fc interaction enhancing mutations.

[0996] F405L or K409R mutations were introduced into humanized CD37 antibodies containing the E430G mutation to allow the generation of bispecific antibodies (bsIgG1) with two CD37-specific Fab arms that do not compete for binding to CD37. The ability of bispecific CD37 antibodies containing the E430G mutation to induce CDC was determined as described above and compared to the ability of two CD37 monospecific bivalent antibodies 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 together equals the concentration of the individual bispecific antibodies), 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.

[0997] CDC on Daudi cells

[0998] Fig.12 A shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G is more potent than either IgG1-005-H1L2-E430G or IgG1-016-H5L2-E430G in inducing CDC on Daudi cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G is also more potent than the combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G. The monovalent CD37 binding antibodies bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G also induced CDC on Daudi cells, but less efficiently than bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G.

[0999] Fig.12B shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G is more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDC on Daudi cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G is also more potent than the combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G. The monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G also induced CDC on Daudi cells, with bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G being less potent than bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G being equally potent.

[1000] The ability of the bispecific CD37 antibody containing the E430G mutation to induce CDC was also compared to the ability of the bispecific CD37 antibody without the E430G mutation to induce CDC. Fig.13 It was shown that bsIgG1-016-H5L2-F405Lx005-H1L2-K409R and bsIgG1-016-H5L2-F405Lx010-H5L2-K409R were able to induce CDC on Daudi cells, but were less potent than their E430G-containing counterparts bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G.

[1001] CDC on OCI-Ly-7 cells

[1002] Fig.12C shows that the monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G are more potent in inducing CDC on OCI-Ly-7 cells compared to their monospecific bivalent binding counterparts IgG1-016-H5L2-E430G and IgG1-010-H5L2-E430G. The combination of monovalent binding antibodies (bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plus bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G) was more potent than the combination of bivalent antibodies (IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G), as demonstrated by the consistently lower EC50 values ​​in two independent experiments ( Fig.12 D). bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G was also more potent than the combination of bivalent antibodies (IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G) in inducing CDC on OCI-Ly-7 cells, as demonstrated by a consistently lower EC50 in three independent experiments ( Fig.12 E).

[1003] The combination of monovalent binding antibodies (bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G plus bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G) and bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G were comparable in their potency in inducing CDC on OCI-Ly-7 cells.

[1004] CDC in primary CLL tumor cells

[1005] The ability of bispecific CD37 antibodies containing the E430G mutation to induce CDC on tumor cells derived from CLL patients was determined as described above and compared to the ability of CD37 antibodies containing the E430G mutation or a combination of CD37 antibodies containing the E430G mutation or a monovalent CD37 antibody comprising the E430G mutation.

[1006] Fig.14A shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G is more potent than IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F405L-E430G in inducing CDC on primary CLL tumor cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G is also more potent than the combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G. The monovalent binding antibodies bsIgG1-b12-F405L-E430Gx005-H1L2-K409R-E430G and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G also induced CDC on primary CLL tumor cells, but were less efficient than bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G in this regard.

[1007] Fig.14 B shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G is more potent than either IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G in inducing CDC on primary CLL tumor cells. The bispecific bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G is also more potent than the combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G. The monovalent conjugated antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G also induced CDC in primary CLL tumor cells, which was similar to bsIgG1-016-H5L2-LC90S- Compared to bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G, bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G was less potent and bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G was equally potent.

[1008] Example 10: Bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations induce CDC on multiple B cell lymphoma cell lines with broad CD37 expression

[1009] The ability of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to induce CDC at a concentration of 10 μg / mL was determined for a panel of B cell lymphoma cell lines derived from various B cell lymphoma subtypes (as described above). As described above, the expression levels of CD37 molecules on the cell surface of these cell lines were determined by quantitative flow cytometry.

[1010] Table 3 gives an overview of the cell lines tested.

[1011] Table 3: B cell lymphoma cell lines.

[1012] Cell Line Lymphoma Type Origin

[1013] JVM-2 MCL DSMZ;ACC 12 JVM-13 MCL ATCC;CRL-3003 Jeko-1 MCL DSMZ;ACC 553 Z-138 MCL ATCC;CRL-3001 Daudi Burkitt's ATCC; CCL-213 Raji Burkitt's ATCC; CCL-86 Vienna-133 Burkitt's BioAnaLab, Oxford, UK SU-DHL-8 DLBCL DSMZ;ACC 573 OCI-Ly19 DLBCL DSMZ;ACC 528 OCI-Ly7 DLBCL DSMZ;ACC 688 SU-DHL-4 DLBCL DSMZ;ACC 495 RC-K8 DLBCL DSMZ;ACC 561 U-2932 DLBCL DSMZ;ACC 633 WIL-2S Plasmablastic ATCC;CRL-8885 RI-1 DLBCL DSMZ;ACC 585 WSU-DLCL2 DLBCL DSMZ;ACC 575

[1014] Fig.15 It was shown that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G induces CDC on a large panel of B-cell lymphoma cell lines derived from various B-cell lymphoma types.

[1015] Example 11: Bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations are more potent in inducing antibody-dependent cell-mediated cytotoxicity (ADCC)

[1016] Target cell labeling

[1017] The ability of CD37 antibodies to induce ADCC was determined by chromium release assay. Daudi or Raji cells (5x10 6 Cells were collected in 1 mL of culture medium (RPMI 1640 supplemented with 10% iron-containing donor bovine serum (DBSI; ThermoFischer, Cat#10371029) and penicillin-streptomycin mixture (pen / strep), to which 100 μCi 51Cr (chromium-51; PerkinElmer, Cat#NEZ030005MC) was added, and the cells were incubated in a 37°C water bath with shaking for 1 hour. After washing the cells (twice in PBS, 1500 rpm, 5 minutes), the cells were resuspended in RPMI 1640 / 10% DBSI / pen / strep and counted by trypan blue exclusion, and the cells were diluted to 1×10 5 The density of cells / mL.

[1018] Preparation of effector cells

[1019] Peripheral blood mononuclear cells from healthy volunteers (Sanquin, Amsterdam, The Netherlands) were isolated from 45 mL of freshly drawn heparinized blood (buffy coat) by Ficoll density centrifugation (Bio Whittaker; Lymphocyte Separation Medium, cat 17-829E) according to the manufacturer's instructions. After resuspending the cells in RPMI 1640 / 10% DBSI / pen / strep, the cells were counted by trypan blue exclusion and diluted to a density of 1×107 cells / mL.

[1020] ADCC Assay Procedure

[1021] 50 μL of 51 Cr-labeled target cells were pipetted into a 96-well round-bottom microtiter plate (Greiner Bio-One; Cat#650101), and then 50 μL of a concentration series of CD37 or control antibodies diluted in RPMI 1640 / 10% DBSI / pen / strep was added (1.5-5,000 ng / mL final concentration, diluted 3 times). The cells were incubated at room temperature (RT) for 15 minutes, and 50 μL of effector cells were added to produce an effector to target ratio of 100:1. The cells were incubated at 37°C and 5% CO2 for 4 hours. To determine the maximum lysis, 50 μL of 51Cr-labeled Daudi cells (5.000 cells) were incubated with 100 μL of 5% Triton-X100; to determine spontaneous lysis (background lysis), 5,000 51Cr-labeled Daudi cells were incubated in 150 μL of culture medium without any antibodies or effector cells. The level of cell lysis independent of antibody was determined by incubating 5,000 Daudi cells with 500,000 PBMCs in the absence of antibody. The plate was centrifuged (1200 rpm, 10 minutes) and 25 μL of 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 minutes and counted using a scintillation counter ( PerkinElmer) counts the released 51Cr. The percentage of specific lysis is calculated as follows:

[1022] % specific lysis = (cpm sample - cpm spontaneous lysis) / (cpm maximum lysis - cpm spontaneous lysis), where cpm is counts per minute.

[1023] Fig.16 A shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx005-H1L2-K409R-E430G is more potent than IgG1-005-H1L2-K409R-E430G or IgG1-016-H5L2-F409L-E430G or than the combination of IgG1-005-H1L2-K409R-E430G plus IgG1-016-H5L2-F405L-E430G in inducing ADCC on Daudi cells.

[1024] Fig.16 B shows that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G is more potent than IgG1-010-H5L2-E430G or IgG1-016-H5L2-E430G or the combination of IgG1-010-H5L2-E430G plus IgG1-016-H5L2-E430G in inducing ADCC on Daudi cells.

[1025] Fig.16 C shows the relationship between PBMCs from different donors and Fig.16 B Similar results, and additionally show that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G is more potent in inducing ADCC on Raji cells than the monovalent binding antibodies bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G and bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G.

[1026] Example 12: Bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations induce potent ex vivo CDC in primary tumor cells from patients with various B-cell malignancies

[1027] The CDC efficacy of bsIgG1-016-H5L2-LC90S-F405Lx010-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 after written informed consent and stored using protocols approved by the VUmc Medical Ethics Committee in accordance with the Declaration of Helsinki. Bone marrow mononuclear cells (BMNC) or peripheral blood mononuclear cells (PBMC) of patients were isolated from bone marrow aspirates or peripheral blood samples by density gradient centrifugation (Ficoll-Paque PLUS, GE Healthcare). Cells were used directly or stored in liquid nitrogen until further use.

[1028] Patient lymph node tissue was cut 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 placed overnight at 37°C. After incubation, the supernatant (including the non-stromal cell compartment of tumor cells) was collected and the cells were filtered using 70 μM EasyStrainer (Greiner Bio-one). The cells were counted, resuspended in RPMI 1640 medium containing 25% heat-inactivated FBS and 10% DMSO, and frozen in liquid nitrogen until further use.

[1029] The expression levels of CD37 and membrane complement regulatory proteins (mCRP; CD46, CD55 and CD59) on isolated patient cells were determined using QifiKit (DAKO, catalog number K007811). The cells were incubated with purified antibodies CD37 (BD, catalog number 555456), CD46 (BioLegend, catalog number 352404), CD55 (BioLegend, catalog number 311302), CD59 (BioLegend, catalog number 304702), and b12 (Genmab) at 4°C for 30 minutes. Thereafter, the method provided by the QifiKit manufacturer was used. After the last step of the Qifi kit procedure, the cells were incubated with lymphoma cell-specific markers to achieve tumor cell identification. Fig.17 The expression levels for each indication are shown.

[1030] Patient-derived tumor cells were conditioned with 10 μg / mL or 100 μg / mL bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G and assessed for CDC induction in the presence of 20% pooled NHS. The following cell markers were used to identify different cell populations: CD45-KO (Beckman Coulter B36294), CD19-PC7 (Beckman Coulter, catalog no. IM3628), CD3-V450 (BD, catalog no. 560365), CD5-APC (BD, catalog no. 345783), CD5-PE (DAKO, cat. no. R084201), CD10-APC-H7 (BD, catalog no. 655404), CD10-PE (DAKO, cat. no. R084201), CD23-FITC (Biolegend, catalog no. 338505), lambda-APC-H7 (BD, catalog no. 656648), kappa-PE (DAKO, cat. no. R043601) and lambda-FITC (Emelca Bioscience CYT-LAMBF). In the CD45+ cell population, malignant B cells were defined by different markers depending on the indication: CD3- / CD19+ / CD5+ (CLL), CD3- / CD19+ / CD10+ (FL, DLBCL), CD3- / CD19+ / CD5+ / CD23- (MCL). In cases where malignant B cells could not be identified based on these markers, kappa / lambda staining could be used to identify malignant cells based on clonality. In several samples, malignant B cells could not be identified based on clonality; in these cases, the total B cell population was evaluated without distinguishing between normal and malignant B cells. Killing was calculated as the fraction (%) of malignant B cells positive for 7-aminoactinomycin D (7-AAD; BD, catalog number 555816) as determined by LSRFortessa flow cytometer (BD Biosciences, San Jose, CA).

[1031] Fig.18It was shown that bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G was highly potent (more than 50% lysis) in inducing CDC in tumor cells from patients with CLL, FL, MCL, DLBCL or B-NHL (not further specified). In cells from a patient with relapsed / refractory FL, the CDC induction ability of bsIgG1-016-H5L2-LC90S-F405Lx010-H5L2-K409R-E430G was weak.

[1032] 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

[1033] Binding to human or cynomolgus monkey B cells was determined 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 sampled in equal portions into wells of 96-well round-bottom plates (Greiner Bio-one, catalog number 65010; 35 μL / well). Red blood cells (RBCs) were lysed by adding 100 μL RBC lysis buffer (10 mM KHCO3 [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 300xg for 3 minutes, cells were incubated at 4°C for 30 minutes with Alexa-488 labeled bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or Alexa-488 labeled control IgG1 (IgG1-b12) and serial dilutions (0.014-30 μg / mL final concentration in 3x serial dilutions) of directly labeled antibodies to identify B cells (wherein a mixture of antibodies further identifies blood cell subsets):

[1034] For human blood B cells, the following antibodies were used

[1035] Target protein clone mark Target cells company Cat.no. CD19 HIB19 BV711 B cells Biolegend 302245

[1036] For cynomolgus monkey blood B cells, the following antibodies were used

[1037]

[1038] The cells were pelleted and washed twice in 150 μL FACS buffer and resuspended in 150 μL TO-PRO-3 (final concentration 0.2 μM; Molecular Probes, catalog number T3605). The samples were measured by flow cytometry using a LSRF ortessa flow cytometer. Binding was indicated as live TO-PRO-3 - / CD 14 - / CD19 + B cells (human) or live TO-PRO-3 - / CD14 - / CD19 + / CD20 + Geometric mean of A488 fluorescence intensity of B cells (cynomolgus monkey). Log-transformed data were analyzed using the best fit of a nonlinear dose response fit in GraphPad PRISM.

[1039] Fig.19 Concentration-dependent binding of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G to B cells in (A) human and (B) cynomolgus monkey blood is shown for one representative donor / animal. Mean EC binding to human and cynomolgus monkey B cells 50 The values ​​were in the same range ([0.85 μg / mL ± 0.284 based on binding to B cells in blood from 6 human donors] and [0.63 μg / mL ± 0.228 based on binding to B cells in blood from 4 animals]), indicating that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G showed comparable binding to human and cynomolgus monkey CD37.

[1040] Cytotoxicity against human or cynomolgus monkey B cells

[1041] Cytotoxicity to human or cynomolgus monkey B cells was determined 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 UMC Utrecht (Utrecht, The Netherlands). Blood was aliquoted into wells of a 96-well round-bottom plate, 35 μL / well.

[1042] Serial dilutions of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12 were added (0.0005-10 μg / mL final antibody concentration, 3x serial dilutions; final volume 100 μL / well). In the cytotoxicity assay using human whole blood, the monoclonal FcγR interaction-enhanced CD37-specific antibody IgG1-G28.1-S239D-I332E was included as a reference. The samples were incubated at 37°C for 4 hours. Thereafter, the red blood cells were lysed as described above, and the samples were stained as described above to identify B cells. The cells were pelleted and washed twice in 150 μL FACS buffer and then resuspended in 150 μL TO-PRO-3 (final concentration 0.2 μM; Molecular Probes, catalog number T3605). Samples were measured by flow cytometry using an LSRFortessa flow cytometer. After exclusion of doublets, live TO-PRO-3 was determined. - / CD14 - / CD19 + B cells (human) or live TO-PRO-3 - / CD14 - / CD19 + / CD20 + Percentage of B cells (cynomolgus monkeys). Percentage of B cell depletion was calculated as follows: % B cell depletion = 100*[(% B cells of no antibody control - % of B cell sample) / (% B cells of no antibody control)]. Log-transformed data were analyzed using the best fit value of a nonlinear dose response fit in GraphPad PRISM.

[1043] Fig. 20 Shown are the concentration-dependent cytotoxicity of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G on B cells in (A) human and (B) cynomolgus monkey blood for one representative donor / animal.

[1044] Based on EC 50 , bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G had comparable ability to induce cytotoxicity in human and cynomolgus monkey B cells: mean EC values ​​for cytotoxicity against human B cells (from blood of 6 donors) 50 The average EC value of cytotoxicity against cynomolgus monkey B cells (from blood of 4 animals) was 0.077 μg / mL ± 0.039. 50 It is 0.043μg / mL±0.019.

[1045] Fig. 20 A also shows the cytotoxicity of the monoclonal CD37 antibody IgG1-G28.1-S239D-I332E with enhanced FcγR interaction on human B cells of representative responding donors, which showed lower cytotoxicity than bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G. In B cells from 3 responding donors, a maximum B cell depletion of 50% was measured for IgG1-G28.1-S239D-I332E, while in the other 3 donors, no cytotoxicity of this antibody on B cells was measured. BsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G induced cytotoxicity in 93-99% of B cells from 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-E430Gx010-H5L2-K409R-E430G (data not shown).

[1046] Example 14: Potent CDC activity of a combination of a bispecific CD37 antibody with Fc-Fc interaction enhancing mutations and a CD20-specific antibody

[1047] The ability of the combination of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and anti-CD20 antibody (IgG1-CD20-ofa; ofatumumab) to induce CDC was tested on 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 incubated at 0.1×10 6The density of cells / well (30 μL / well) was distributed into a polystyrene 96-well round bottom plate (Greiner bio-one Cat#650101), and 50 μL of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G (0.0625-0.05 μg / mL) and IgG1-CD20-ofa (1-8 μg / mL) concentration series were added in 2-fold dilution. BsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-CD20-ofa were combined at an antibody concentration based on the relative potency (EC50 difference) of each antibody by mixing two concentrations that achieved the same effect on average. IgG1-b12 was used as a negative control.

[1048] After incubation (room temperature, shaking for 15 minutes), 20 μL of normal human serum (NHS Cat#M0008Sanquin, Amsterdam, The Netherlands) combined was added to each well as a complement source, and the plate was incubated at 37°C for 45 minutes. The reaction was terminated by cooling the plate on ice. After centrifugation at 300xg for 3 minutes, the cells were washed twice with 150 μL FACS buffer and incubated at 4°C with mouse anti-human IgG1-CD19 antibodies (clone J3-119, Beckman Coulter, catalog number A07769, diluted 1:50 from storage solution) labeled with R-phycoerythrin (PE) for 30 minutes to determine tumor B cells and TO-PRO-3 (final concentration of 0.2 μM; Molecular Probes, catalog number T3605) for dead cell identification. The cells were precipitated and washed twice in 150 μL FACS buffer and measured by flow cytometry using LSRFortessa flow cytometer. The percentage of live cells was calculated as follows: % live cells = 100*(number of TO-PRO-3 negative events) / (total number of events).

[1049] Fig.21AD shows that both bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and ofatumumab induced CDC in tumor cells derived from 2 CLL patients, with CDC activity increasing with increasing dose levels. Combining bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G with ofatumumab resulted in enhanced CDC activity at all concentrations tested for the two CLL patients tested, although these effects were less pronounced at higher antibody concentrations, where single agents induced almost complete cell killing ( Fig.21 A and B). These results indicate that the addition of ofatumumab to bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G can improve CDC-mediated tumor cell killing in malignant B cells obtained from CLL patients.

[1050] Example 15: Antitumor activity of bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations in a xenograft model of B cell malignancies

[1051] Antitumor activity of subcutaneous JVM-3 in a human chronic B-cell leukemia xenograft model

[1052] JVM-3 cells (1x10 7 ) were inoculated into the right flank of CB17.SCID mice, and when the tumors reached approximately 158 mm 3 Antibody treatment was started when the mean volume of the tumor was 1.54 × 10 mm Hg (three weekly doses of 0.1, 0.3, 1, 3 or 10 mg / kg, injected intravenously; IgG1-b12 was used as a negative control, administered at 10 mg / kg). Tumor volumes were measured twice weekly in two dimensions using calipers and expressed in mm using the following formula: 3 Volume is expressed as: V = (L x W x W) / 2, where V is the tumor volume, L is the tumor length (the longest tumor dimension), and W is the tumor width (the longest tumor dimension perpendicular to L).

[1053] Fig. 22 A shows the tumor volume for each dose group over time. Fig. 22 B shows the tumor volume per mouse in each dose group on day 25 when all groups were still intact. Three weekly doses of 1, 3 or 10 mg / kg of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G significantly reduced JVM-3 cell tumor growth, while administration at 0.1 or 0.3 mg / kg did not affect tumor growth (Mann Whitney test, p<0.01).

[1054] Antitumor activity of intravenous Daudi-luc in a Burkitt's lymphoma xenograft model

[1055] On day 0, SCID mice ( Hsd-Prkdcscid; Harlan) intravenously injected Daudi-luc cells (Daudi cells transfected with luciferase, 2.5×10 6 Cells / mouse). On days 14, 21 and 28, mice were injected intraperitoneally with 0.1, 0.3, 1, 3 or 10 mg / kg of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G. IgG1-b12 was used as a negative control antibody and was administered at 10 mg / kg. Tumor growth was assessed weekly (starting from day 2) by bioluminescence imaging (BLI). Mice were injected intraperitoneally with 100 μL of firefly D-luciferin (30 mg / mL; Caliper LifeSciences, catalog number 119222), and bioluminescence (in p / s / cm2) was measured using the Biospace bioluminescence imaging system (PerkinElmer; mice were imaged from the back) under isoflurane anesthesia. 2 / sr [square radian per cm 2 [photons per second] meter's radiance).

[1056] Fig.23 A shows luciferase activity (bioluminescence, as a measure of tumor volume) for each dose group over time, Fig.23 B shows the luciferase activity per mouse in each dose group on day 36 when all groups were still intact. Three weekly doses of 0.1, 0.3, 1, 3, or 10 mg / kg of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G significantly reduced the in vivo growth of Daudi-luc cells (one-way Anova, uncorrected Fisher's LSD).

[1057] Example 16: Evaluation of plasma clearance of bispecific CD37 antibodies with Fc-Fc interaction enhancing mutations in SCID mice

[1058] Female SCID mice (11-12 weeks old) Hsd-Prkdcscid; Harlan) (3 mice per group) were injected intravenously (iv) with a single dose of 100 μg (5 mg / kg) or 500 μg (25 mg / kg) bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G or IgG1-b12. The experiment was set up to study antibody clearance in the absence of target-mediated clearance, as neither bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G nor IgG1-b12 showed cross-reactivity with mice.

[1059] 50-100 μL blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 24 hours, 2 days, 7 or 8 days, 14 days and 21 days after antibody administration. Blood was collected in vials containing heparin and centrifuged at 10,000 g for 5 minutes. Plasma samples were diluted with 1:50 (20 μL samples in 980 μL PBSA (PBS supplemented with 0.2% bovine serum albumin (BSA)) for mice dosed with 5 mg / kg and with 1:20 (20 μL samples in 380 μL PBSA) for mice dosed with 25 mg / kg, and stored at -20°C until the concentration of the monoclonal antibody was determined.

[1060] Human IgG concentration was determined using sandwich ELISA. Mouse monoclonal anti-human IgG-kappa clone MH16 (CLB Sanquin, The Netherlands; catalog number M1268) coated to 96-well Microlon ELISA plates (Greiner, Germany) at a concentration of 2 μg / mL in 100 μL at 4°C overnight was used as capture antibody. After the plate was blocked with PBSA for 1 hour at room temperature (RT), the sample was added, serially diluted in PBSA, and incubated on a plate shaker at room temperature for 1 hour. The plate was washed 3 times with 300 μL PBST (PBS supplemented with 0.05% Tween 20), and then incubated with goat anti-human IgG immunoglobulin (Jackson, West Grace, PA; catalog number 109-035-098; 1:10.000 in PBST supplemented with 0.2% BSA) at room temperature for 1 hour. The plate was washed again with 300 μL PBST 3 times and then incubated in the dark with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS; Roche, Mannheim, Germany). The reaction was terminated by adding 100 μL 2% oxalic acid. The absorbance was measured at 405 nm in a microplate reader (Biotek, Winooski, VT). The human IgG concentration was calculated by using the injected material as a reference curve. As a plate control, purified human IgG1 (The binding site, product catalog number BP078) was included. The human IgG concentration (in μg / mL) was plotted ( Fig.24 A and C), and the area under the curve (AUC) was calculated using Graphpad prism 6.0. IgG clearance until the last day of blood sampling (day 21) was determined using the formula D*1.000 / AUC, where D is the injected dose (1 mg / kg) ( Fig.24 B and D).

[1061] There was no substantial difference between the plasma clearance of bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G and IgG1-b12, demonstrating that bsIgG1-016-H5L2-LC90S-F405L-E430Gx010-H5L2-K409R-E430G displayed a pharmacokinetic profile comparable to wild-type human IgG1 in the absence of target binding.

[1062] Example 17: Determination of the contribution of CD37 amino acid residues to CD37 antibody binding using alanine scanning Library design

[1063] A CD37 single residue alanine library (Geneart) was synthesized in which all amino acid (aa) residues in the extracellular domain of human CD37 (Uniprot P11049) were individually mutated to alanine, except for positions that already contained alanine or cysteine. Cysteines were not mutated to minimize the chance of structural disruption of the antigen. The library was cloned in a pMAC expression vector containing a CMV / TK-polyA expression cassette, an Amp resistance gene, and a pBR322 replication origin.

[1064] Library generation and screening

[1065] Wild-type CD37 and alanine mutants were expressed individually in FreeStyle HEK293 cells according to the manufacturer's instructions (Thermo Scientific). One day after transfection, cells were harvested. About 100,000 cells were incubated with 20 μL 3 μg / mL concentrations of bsIgG1-b12-F405L-E430Gx010-H5L2-K409R-E430G (monovalent binding 010) or bsIgG1-016-H5L2-LC90S-F405L-E430Gxb12-K409R-E430G (monovalent binding 016) conjugated with Alexa488 in FACS buffer (PBS+0.1% (w / v) bovine serum albumin (BSA)+0.02% (w / v) sodium azide). Cells were incubated at room temperature for 1 hour. Subsequently, the cells were washed twice by adding 150 μL FACS buffer and removing the supernatant after centrifugation. The cells were resuspended in 20 μL fresh FACS buffer and stored at 4°C until analyzed by flow cytometry using an iQue filter (IntelliCyt). The entire experiment was performed twice.

[1066] Data analysis

[1067] For each sample, the average antibody binding per cell was determined as the geometric mean (gMFI) of the fluorescence intensity of the ungated cell population. The gMFI is affected by the affinity of the antibody for the CD37 mutant and the expression level of the CD37 mutant per cell. Since specific alanine mutations can affect the surface expression level of mutant CD37, and expression differences of each CD37 mutant are usually corrected, the data are normalized relative to the binding intensity of a non-competitive CD37-specific control antibody using the following equation (in this example, antibodies monovalent binding 010 and monovalent binding 016 are non-competitive antibodies, and one antibody is used as a control for the other antibody):

[1068]

[1069] Wherein "aa position" refers to a specific alanine mutant position in CD37 or wild-type (wt) CD37.

[1070] To express loss or gain of antibody binding, the assay standard score was calculated as follows:

[1071]

[1072] where μ and σ are the mean and standard deviation (SD) of the normalized gMFI of all mutants.

[1073] In most cases, gain of binding will result from loss of binding of the reference antibody to a specific ala mutant. Using these calculations, amino acid positions where there is no loss or gain of binding for a specific antibody upon replacement of the amino acid with alanine will be given a z-score of "0", gain of binding will result in a "z-score > 0" and loss of binding will result in a "z-score < 0". To correct for sample variation, only CD37 amino acid residues with a z-score below –1.5 were considered "loss of binding mutants". In cases where the gMFI of the control antibody against a specific CD37 mutant was below the mean gMFI 对照抗体 In cases where the mean gMFI was less than 2.5xSD, data were excluded from the analysis (for those CD37 mutants, expression levels were assumed to be insufficient).

[1074] Fig.25 The "z-score (fold change)" of CD37 antibodies against CD37 variants with ala mutations at positions 42 to 131 (according to SEQ ID No 94) are shown. The results indicate:

[1075] The binding of antibody 010 is dependent on at least amino acids Y182, D189, T191, I192, D194, K195, V196, I197 and P199 of human CD37,

[1076] The binding of antibody 016 is dependent on at least amino acids E124, F162, Q163, V164, L165 and H175 of human CD37.

[1077] Summary

[1078] In summary, bispecific antibodies composed of two CD37-specific antibodies with Fc-Fc interaction enhancing mutations that do not compete for target binding showed the most favorable combination of CDC efficacy and ADCC efficacy in CD37-positive tumor cells. For both effector mechanisms, bispecific antibodies with Fc-Fc interaction enhancing mutations showed superior efficacy compared to a combination of two non-competitive CD37 antibodies containing Fc-Fc interaction enhancing mutations or to a single CD37 antibody with Fc-Fc interaction enhancing mutations.

[1079] 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.

[1080] The CDC activity of a mixture of CD37 antibodies IgG1-37.3-E430G, IgG1-G28.1-E430G, IgG1-004-E430G, IgG1-005-E430G, IgG1-010-E430G and IgG1-016-E430G (the last four are chimeric rabbit / human) with Fc-Fc interaction enhancing mutations, plus the clinically established CD20-targeting monoclonal antibody products MabThera (rituximab; Roche, H0124B08), Arzerra (ofatumumab; Novartis; C656294) and Gazyva (obituzumab, GA101; Roche, D287-41A GACD20) were tested in vitro using Burkitt's lymphoma Raji cells. Raji cells (ATCC, catalog number 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. 0.1×10 6 Raji cells were incubated with antibodies for 15 minutes in a total volume of 80 μL RPMI / 0.2% BSA per well. Next, NHS was added to the pre-incubated cells in a final volume of 100 μL (final antibody concentration 10 μg / mL; 20% NHS) and incubated at 37°C for 45 minutes. For all total antibody concentrations tested, different ratios of the two antibodies in the mixture were tested (1:0–3:1–1:1–1:3–0:1). The plate was centrifuged and the cells were resuspended in 30 μL PI (2 μg / mL). Killing was calculated by the PI positive cell fraction (%) determined by flow cytometry on an iQue filter (Intellicyt). Data were analyzed and plotted using GraphPad Prism software.

[1081] Cocktails of tested CD37 antibodies with Fc-Fc interaction enhancing mutations and clinically established CD20 antibody products showed enhanced dose-dependent CDC activity on Raji cells compared to the same concentrations of the single antibodies ( Figure 8There 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 mixtures of hexamer-enhanced CD37 antibodies with Fc-Fc interaction enhancing mutations plus clinically established CD20 antibody products such as MabThera, Arzerra (type I CD20 antibodies) or Gazyva (type II CD20 antibodies) may improve the therapeutic potential of patients with B-cell malignancies that often become refractory to standard CD20-targeted therapies alone.

Claims

1. A bispecific antibody comprising first and second antigen binding regions that bind to human CD37 having a sequence of SEQ ID NO: 62 and first and second Fc regions of human immunoglobulins, wherein the first and second antigen binding regions bind to different epitopes on CD37, and wherein the first and second Fc regions comprise one or more amino acid mutations that enhance Fc-Fc interactions between the bispecific antibodies after binding to a membrane-bound target compared to Fc-Fc interactions between bispecific antibodies without the mutations.

2. The bispecific antibody of claim 1, wherein the first antigen binding region is obtained from an antibody that competes for binding to human CD37 with a CD37 antibody comprising the following CDR sequences: The VH CDR1 sequence shown in SEQ ID NO: 16, The VH CDR2 sequence shown in SEQ ID NO: 17, The VH CDR3 sequence shown in SEQ ID NO: 18, The VL CDR1 sequence shown in SEQ ID NO: 20, VL CDR2 sequence: KAS, and The VL CDR3 sequence shown in SEQ ID NO:21 [010].

3. The bispecific antibody of any one of the preceding claims, wherein the first antigen binding region binds to the same epitope on human CD37 as a CD37 antibody comprising the following CDR sequences: The VH CDR1 sequence shown in SEQ ID NO: 16, The VH CDR2 sequence shown in SEQ ID NO: 17, The VH CDR3 sequence shown in SEQ ID NO: 18, The VL CDR1 sequence shown in SEQ ID NO: 20, VL CDR2 sequence: KAS, and The VL CDR3 sequence shown in SEQ ID NO:21 [010].

4. The bispecific antibody of any one of the preceding claims, wherein the first antigen binding region comprises the following CDR sequences: The VH CDR1 sequence shown in SEQ ID NO: 16, The VH CDR2 sequence shown in SEQ ID NO: 17, The VH CDR3 sequence shown in SEQ ID NO: 18, The VL CDR1 sequence shown in SEQ ID NO: 20, VL CDR2 sequence: KAS, and The VL CDR3 sequence shown in SEQ ID NO:21 [010].

5. The bispecific antibody of any one of the preceding claims, wherein the first antigen binding region comprises the following VH and VL sequences: (i) a VH sequence as shown in SEQ ID NO: 15 and a VL sequence as shown in SEQ ID NO: 19; or (ii) a VH sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VH sequence of SEQ ID NO: 15 and a VL sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VL sequence of SEQ ID NO:

19.

6. The bispecific antibody of any one of claims 1 to 5, wherein the first antigen binding region binds to a functional epitope comprising one or more of the amino acids selected from the group consisting of Y182, D189, T191, I192, D194, K195, V196, I197 and P199 of SEQ ID No: 62 (CD37).

7. The bispecific antibody of claim 1, wherein the first antigen binding region is obtained from an antibody that competes for binding to human CD37 with a CD37 antibody comprising the following CDR sequences: The VH CDR1 sequence shown in SEQ ID NO:9, The VH CDR2 sequence shown in SEQ ID NO: 10, The VH CDR3 sequence shown in SEQ ID NO: 11, The VL CDR1 sequence shown in SEQ ID NO: 13, VL CDR2 sequence: AAS, and The VL CDR3 sequence shown in SEQ ID NO: 14 [005].

8. The bispecific antibody of claim 1 or 7, wherein the first antigen binding region binds to the same epitope on human CD37 as a CD37 antibody comprising the following CDR sequences: The VH CDR1 sequence shown in SEQ ID NO:9, The VH CDR2 sequence shown in SEQ ID NO: 10, The VH CDR3 sequence shown in SEQ ID NO: 11, The VL CDR1 sequence shown in SEQ ID NO: 13, VL CDR2 sequence: AAS, and The VL CDR3 sequence shown in SEQ ID NO: 14 [005].

9. The bispecific antibody of claim 1, 7 or 8, wherein the first antigen binding region comprises the following CDR sequences: The VH CDR1 sequence shown in SEQ ID NO:9, The VH CDR2 sequence shown in SEQ ID NO: 10, The VH CDR3 sequence shown in SEQ ID NO: 11, The VL CDR1 sequence shown in SEQ ID NO: 13, VL CDR2 sequence: AAS, and The VL CDR3 sequence shown in SEQ ID NO: 14 [005].

10. The bispecific antibody of any one of claims 1 or 7 to 9, wherein the first antigen binding region comprises the following VH and VL sequences: (i) a VH sequence as shown in SEQ ID NO: 8 and a VL sequence as shown in SEQ ID NO: 12; or (ii) a VH sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VH sequence of SEQ ID NO:8 and a VL sequence that is at least 90% identical, such as at least 95% identical, such as at least 98% identical, such as at least 99% identical, to the VL sequence of SEQ ID NO:12.

Images