Anti-CD79b antibody and its uses

Abstract

The present disclosure relates to anti-CD79b (cluster of differentiation 79B) antibodies, antigen-binding fragments thereof, antibody-drug conjugates (ADCs) derived therefrom, and uses thereof.

Description

Technical Field

[0001] The present invention relates to anti-CD79b (cluster of differentiation 79B) antibodies, antigen-binding fragments thereof, antibody-drug conjugates (ADCs) derived therefrom, and uses thereof. Background Art

[0002] CD79 is a signaling component of the B-cell receptor and functions as a covalent heterodimer containing CD79a and CD79b. CD79b contains an extracellular immunoglobulin (Ig) domain, a transmembrane domain, and an intracellular signaling domain, an immunoreceptor tyrosine-based activation motif (ITAM) domain. CD79b expression is detected on the surface of almost all patients with non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0003] For multiple reasons, CD79b is an attractive therapeutic target for B-cell malignancies. First, CD79b is not only widely expressed in B-cell malignancies, but its expression remains unchanged after CD19 or CD20 deletion, making this receptor an attractive alternative targeted therapy in addition to CD19- or CD20-targeted therapies. Second, when the B-cell receptor is cross-linked, it targets the major histocompatibility complex class II compartment, a lysosome-like compartment, as part of the presentation of class II antigens by B cells.

[0004] Given the important role of CD79b in cancer, there is a need to develop therapeutic agents targeting CD79b. Summary of the Invention

[0005] The present invention relates to anti-CD79b antibodies, antigen-binding fragments thereof, and uses thereof.

[0006] In one aspect, the present invention relates to an antibody or an antigen-binding fragment thereof that binds to CD79b (cluster of differentiation 79B), comprising:

[0007] a heavy-chain variable region (VH) comprising complementary determining regions (CDRs) 1, 2, and 3, wherein the VH CDR1 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR1 amino acid sequence, the VH CDR2 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR2, and the VH CDR3 region comprises an amino acid sequence that is at least 80% identical to a selected VH CDR3; and

[0008] The variable light chain (VL) regions containing CDR 1, 2, and 3, wherein the VL CDR1 region contains an amino acid sequence that is at least 80% identical to the selected VL CDR1, the VL CDR2 region contains an amino acid sequence that is at least 80% identical to the selected VL CDR2, and the VL CDR3 region contains an amino acid sequence that is at least 80% identical to the selected VL CDR3,

[0009] wherein the amino acid sequences of the selected VH CDR 1, 2, and 3 and the amino acid sequences of the selected VL CDR 1, 2, and 3 are one of the following:

[0010] (1) The amino acid sequences of the selected VH CDR 1, 2, 3 are listed in SEQ ID NO: 9, 11, 13 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are listed in SEQ ID NO: 14 - 16 respectively;

[0011] (2) The amino acid sequences of the selected VH CDR 1, 2, 3 are listed in SEQ ID NO: 19, 21, 23 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are listed in SEQ ID NO: 24 - 26 respectively;

[0012] (3) The amino acid sequences of the selected VH CDR 1, 2, 3 are as shown in SEQ ID NO: 29, 31, 33 respectively, and the amino acid sequences of the selected VL CDR 1, 2, 3 are as shown in SEQ ID NO: 24 - 26 respectively;

[0013] (4) The amino acid sequences of the selected VH CDR 1, 2, 3 are listed in SEQ ID NO: 39, 41, 43 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are listed in SEQ ID NO: 44 - 46 respectively;

[0014] (5) The amino acid sequences of the selected VH CDR 1, 2, 3 are listed in SEQ ID NO: 49, 51, 53 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are listed in SEQ ID NO: 54 - 56 respectively;

[0015] (6) The amino acid sequences of the selected VH CDR 1, 2, 3 are listed in SEQ ID NO: 59, 61, 63 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are listed in SEQ ID NO: 64 - 66 respectively;

[0016] (7) The amino acid sequences of the selected VH CDR 1, 2, 3 are listed in SEQ ID NO: 10, 12, 13 respectively, and the amino acid sequences of the selected VL CDR1, 2, 3 are listed in SEQ ID NO: 14 - 16 respectively;

[0017] (8) The amino acid sequences of the selected VH CDR 1, 2, and 3 are listed in SEQ ID NO: 20, 22, and 23 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are listed in SEQ ID NO: 24 - 26 respectively;

[0018] (9) The amino acid sequences of the selected VH CDR 1, 2, and 3 are listed in SEQ ID NO: 30, 32, and 33 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are listed in SEQ ID NO: 34 - 36 respectively;

[0019] (10) The amino acid sequences of the selected VH CDR 1, 2, and 3 are listed in SEQ ID NO: 40, 42, and 43 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are listed in SEQ ID NO: 44 - 46 respectively;

[0020] (11) The amino acid sequences of the selected VH CDR 1, 2, and 3 are listed in SEQ ID NO: 50, 52, and 53 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are listed in SEQ ID NO: 54 - 56 respectively;

[0021] (12) The amino acid sequences of the selected VH CDR 1, 2, and 3 are listed in SEQ ID NO: 60, 62, and 63 respectively, and the amino acid sequences of the selected VL CDR1, 2, and 3 are listed in SEQ ID NO: 64 - 66 respectively.

[0022] In some embodiments, according to the Kabat definition, the amino acid sequences of VH containing CDR 1, 2, and 3 are listed in SEQ ID NO: 9, 11, and 13 respectively, and the amino acid sequences of VL containing CDR 1, 2, and 3 are listed in SEQ ID NO: 14 - 16 respectively.

[0023] In some embodiments, according to the Kabat definition, the amino acid sequences of VH containing CDR 1, 2, and 3 are listed in SEQ ID NO: 19, 21, and 23 respectively, and the amino acid sequences of VL containing CDR 1, 2, and 3 are listed in SEQ ID NO: 24 - 26 respectively.

[0024] In some embodiments, according to the Kabat definition, the amino acid sequences of VH containing CDR 1, 2, and 3 are listed in SEQ ID NO: 29, 31, and 33 respectively, and the amino acid sequences of VL containing CDR 1, 2, and 3 are listed in SEQ ID NO: 34 - 36 respectively.

[0025] In some embodiments, according to the Kabat definition, the amino acid sequences of CDR 1, 2, and 3 of VH are listed in SEQ ID NO: 39, 41, and 43 respectively, and the amino acid sequences of CDR 1, 2, and 3 of VL are listed in SEQ ID NO: 44 - 46 respectively.

[0026] In some embodiments, according to the Kabat definition, the amino acid sequences of CDR 1, 2, and 3 of VH are listed in SEQ ID NO: 49, 51, and 53 respectively, and the amino acid sequences of CDR 1, 2, and 3 of VL are listed in SEQ ID NO: 54 - 56 respectively.

[0027] In some embodiments, according to the Kabat definition, the amino acid sequences of CDR 1, 2, and 3 of VH are listed in SEQ ID NO: 59, 61, and 63 respectively, and the amino acid sequences of CDR 1, 2, and 3 of VL are listed in SEQ ID NO: 64 - 66 respectively.

[0028] In some embodiments, according to the Chothia definition, the amino acid sequences of CDR 1, 2, and 3 of VH are listed in SEQ ID NO: 10, 12, and 13 respectively, and the amino acid sequences of CDR 1, 2, and 3 of VL are listed in SEQ ID NO: 14 - 16 respectively.

[0029] In some embodiments, according to the Chothia definition, the amino acid sequences of CDR 1, 2, and 3 of VH are listed in SEQ ID NO: 20, 22, and 23 respectively, and the amino acid sequences of CDR 1, 2, and 3 of VL are listed in SEQ ID NO: 24 - 26 respectively.

[0030] In some embodiments, according to the Chothia definition, the amino acid sequences of CDR 1, 2, and 3 of VH are listed in SEQ ID NO: 30, 32, and 33 respectively, and the amino acid sequences of CDR 1, 2, and 3 of VL are listed in SEQ ID NO: 34 - 36 respectively.

[0031] In some embodiments, according to the Chothia definition, the amino acid sequences of CDR 1, 2, and 3 of VH are listed in SEQ ID NO: 40, 42, and 43 respectively, and the amino acid sequences of CDR 1, 2, and 3 of VL are listed in SEQ ID NO: 44 - 46 respectively.

[0032] In some embodiments, according to the Chothia definition, the amino acid sequences of CDR 1, 2, and 3 of VH are listed in SEQ ID NO: 50, 52, and 53 respectively, and the amino acid sequences of CDR 1, 2, and 3 of VL are listed in SEQ ID NO: 54 - 56 respectively.

[0033] In some embodiments, according to the Chothia definition, the amino acid sequences of CDR 1, 2, and 3 of VH are listed in SEQ ID NO: 60, 62, and 63, respectively, and the amino acid sequences of CDR 1, 2, and 3 of VL are listed in SEQ ID NO: 64 - 66, respectively.

[0034] In some embodiments, the antibody or its antigen - binding fragment specifically binds to CD79b of human, mouse, monkey, or dog.

[0035] In some embodiments, the antibody or its antigen - binding fragment is a humanized antibody or its antigen - binding fragment, a single - chain variable fragment (scFv), a single - arm antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

[0036] In some embodiments, the antibody or its antigen - binding fragment comprises a human IgG1 constant region, a human IgG2 constant region, or a human IgG4 constant region.

[0037] In one aspect, the present disclosure relates to a nucleic acid comprising a polynucleotide encoding a polypeptide, the polypeptide comprising:

[0038] (1) an immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises amino acid sequences of complementary - determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 9, 11, and 13, respectively, and wherein the VH binds to CD79b when paired with a light - chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8;

[0039] (2) an immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises amino acid sequences of complementary - determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 14 - 16, respectively, and wherein the VL binds to CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 7;

[0040] (3) an immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises amino acid sequences of complementary - determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 19, 21, and 23, respectively, and wherein the VH binds to CD79b when paired with a light - chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 18;

[0041] (4) an immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises amino acid sequences of complementary - determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 24 - 26, respectively, and wherein the VL binds to CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 17;

[0042] (5) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 29, 31, and 33, respectively, and wherein the VH binds CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 28;

[0043] (6) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VL are set forth in SEQ ID NOs: 34 - 36, respectively, and wherein the VL binds CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 27;

[0044] (7) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 39, 41, and 43, respectively, and wherein the VH binds CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 38;

[0045] (8) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VL are set forth in SEQ ID NOs: 44 - 46, respectively, and wherein the VL binds CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 37;

[0046] (9) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 49, 51, and 53, respectively, and wherein the VH binds CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 48;

[0047] (10) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VL are set forth in SEQ ID NOs: 54 - 56, respectively, and wherein the VL binds CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 47;

[0048] (11) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 59, 61, and 63, respectively, and wherein the VH binds CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 58;

[0049] (12) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VL are set forth in SEQ ID NOs: 64 - 66, respectively, and wherein the VL binds to CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 57;

[0050] (13) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 10, 12, 13, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8;

[0051] (14) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 20, 22, 23, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 18;

[0052] (15) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 30, 32, 33, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 28;

[0053] (16) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 40, 42, 43, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 38;

[0054] (17) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 50, 52, 53, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 48;

[0055] (18) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementary determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 60, 62, 63, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 58;

[0056] (19) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 19, 21, and 23, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 70;

[0057] (20) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 24 - 26, respectively, and wherein the VL binds to CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 69;

[0058] (21) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 20, 22, and 23, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 70;

[0059] (22) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 39, 41, and 43, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 72;

[0060] (23) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the VL comprises amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 44 - 46, respectively, and wherein the VL binds to CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 71;

[0061] (24) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 40, 42, and 43, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 72;

[0062] (25) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the VH comprises amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 listed in SEQ ID NO: 49, 51, and 53, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 73;

[0063] (26) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 of the VL are set forth in SEQ ID NOs: 54 - 56, respectively, and wherein the VL binds to CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 74;

[0064] (27) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 50, 52, 53, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 73;

[0065] (28) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 59, 61, 63, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 76;

[0066] (29) An immunoglobulin light chain or a fragment thereof comprising VL, wherein the amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 of the VL are set forth in SEQ ID NOs: 64 - 66, respectively, and wherein the VL binds to CD79b when paired with a VH comprising the amino acid sequence of SEQ ID NO: 75;

[0067] (30) An immunoglobulin heavy chain or a fragment thereof comprising VH, wherein the amino acid sequences of complementarity-determining regions (CDRs) 1, 2, and 3 of the VH are set forth in SEQ ID NOs: 60, 62, 63, respectively, and wherein the VH binds to CD79b when paired with a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 76;

[0068] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the polypeptide comprising VL of CDRs 1, 2, and 3, and the amino acid sequences of CDRs 1, 2, and 3 are set forth in SEQ ID NOs: 14 - 16, respectively.

[0069] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the polypeptide comprising VL of CDRs 1, 2, and 3, and the amino acid sequences of CDRs 1, 2, and 3 are set forth in SEQ ID NOs: 24 - 26, respectively.

[0070] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the polypeptide comprising VL of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NOs: 34-36, respectively.

[0071] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the polypeptide comprising VL of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NOs: 44-46, respectively.

[0072] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the polypeptide comprising VL of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NOs: 54-56, respectively.

[0073] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin light chain or a fragment thereof, the polypeptide comprising VL of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NOs: 64-66, respectively.

[0074] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NOs: 9, 11, 13, respectively.

[0075] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NOs: 19, 21, 23, respectively.

[0076] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NOs: 29, 31, 33, respectively.

[0077] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NOs: 39, 41, 43, respectively.

[0078] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising a VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NO: 49, 51, and 53, respectively.

[0079] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising a VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NO: 59, 61, and 63, respectively.

[0080] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising a VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NO: 10, 12, and 13, respectively.

[0081] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising a VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NO: 20, 22, and 23, respectively.

[0082] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising a VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NO: 30, 32, and 33, respectively.

[0083] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising a VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NO: 40, 42, and 43, respectively.

[0084] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising a VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NO: 50, 52, and 53, respectively.

[0085] In some embodiments, the nucleic acid comprises a polynucleotide that encodes a polypeptide comprising an immunoglobulin heavy chain or a fragment thereof, the polypeptide comprising VH of CDR 1, 2, and 3, and the amino acid sequences of CDR 1, 2, and 3 are set forth in SEQ ID NO: 60, 62, and 63, respectively.

[0086] In some embodiments, VH specifically binds to CD79b of human, mouse, monkey, or dog when paired with VL, or VL specifically binds to CD79b of human, mouse, monkey, or dog when paired with VH.

[0087] In some embodiments, the immunoglobulin heavy chain or a fragment thereof comprises a human immunoglobulin heavy chain fragment (e.g., human IgGl heavy chain CH1, CH2, and / or CH3, human IgG2 heavy chain CH1, CH2, and / or CH3, or human IgG4 heavy chain CH1, CH2, and / or CH3), and the immunoglobulin light chain or a fragment thereof comprises a human immunoglobulin light chain constant region.

[0088] In some embodiments, the nucleic acid encodes a single-chain variable fragment (scFv), a single-arm antibody, a multispecific antibody (e.g., a bispecific antibody), or a chimeric antigen receptor (CAR).

[0089] In some embodiments, the nucleic acid is cDNA.

[0090] In one aspect, the present disclosure relates to a vector comprising one or more of the nucleic acids described herein.

[0091] In one aspect, the present disclosure relates to a vector comprising two of the nucleic acids described herein, wherein the vector encodes a VL region and a VH region that bind to CD79b together.

[0092] In one aspect, the present disclosure relates to a pair of vectors, wherein each vector comprises one of the nucleic acids described herein, and the pair of vectors jointly encode and bind to a VL region and a VH region of CD79b.

[0093] In one aspect, the present disclosure relates to a cell comprising the vector described herein or the pair of vectors described herein.

[0094] In some embodiments, the cell is a CHO cell.

[0095] In one aspect, the present disclosure relates to a cell comprising one or more of the nucleic acids described herein.

[0096] In one aspect, the present disclosure relates to a cell comprising two of the nucleic acids described herein.

[0097] In some embodiments, the two nucleic acids jointly encode and bind to a VL region and a VH region of CD79b.

[0098] In one aspect, the present disclosure relates to a method for generating an antibody or an antigen-binding fragment thereof, the method comprising

[0099] (a) culturing the cells described herein under conditions sufficient for the cells to produce an antibody or an antigen-binding fragment; and

[0100] (b) collecting the antibody or antigen-binding fragment produced by the cells.

[0101] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to CD79b, which comprises

[0102] a heavy chain variable region (VH) comprising an amino acid sequence that is at least 90% identical to a selected VH sequence, and a light chain variable region (VL) comprising an amino acid sequence that is at least 90% identical to a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0103] (1) the selected VH sequence is SEQ ID NO: 7 and the selected VL sequence is SEQ ID NO: 8;

[0104] (2) the selected VH sequence is SEQ ID NO: 17 or 69 and the selected VL sequence is SEQ ID NO: 18 or 70;

[0105] (3) the selected VH sequence is SEQ ID NO: 27 and the selected VL sequence is SEQ ID NO: 28;

[0106] (4) the selected VH sequence is SEQ ID NO: 37 or 71 and the selected VL sequence is SEQ ID NO: 38 or 72;

[0107] (5) the selected VH sequence is SEQ ID NO: 47 or 73 and the selected VL sequence is SEQ ID NO: 48 or 74;

[0108] (6) the selected VH sequence is SEQ ID NO: 57 or 75 and the selected VL sequence is SEQ ID NO: 58 or 76.

[0109] In some embodiments, VH comprises the sequence of SEQ ID NO: 7 and VL comprises the sequence of SEQ ID NO: 8.

[0110] In some embodiments, VH comprises the sequence of SEQ ID NO: 17 and VL comprises the sequence of SEQ ID NO: 18.

[0111] In some embodiments, VH comprises the sequence of SEQ ID NO: 27 and VL comprises the sequence of SEQ ID NO: 28.

[0112] In some embodiments, VH comprises the sequence of SEQ ID NO:37 and VL comprises the sequence of SEQ ID NO:38.

[0113] In some embodiments, VH comprises the sequence of SEQ ID NO:47 and VL comprises the sequence of SEQ ID NO:48.

[0114] In some embodiments, VH comprises the sequence of SEQ ID NO:57 and VL comprises the sequence of SEQ ID NO:58.

[0115] In some embodiments, VH comprises the sequence of SEQ ID NO:69 and VL comprises the sequence of SEQ ID NO:70.

[0116] In some embodiments, VH comprises the sequence of SEQ ID NO:71 and VL comprises the sequence of SEQ ID NO:72.

[0117] In some embodiments, VH comprises the sequence of SEQ ID NO:73 and VL comprises the sequence of SEQ ID NO:74.

[0118] In some embodiments, VH comprises the sequence of SEQ ID NO:75 and VL comprises the sequence of SEQ ID NO:76.

[0119] In one aspect, the present disclosure relates to an antibody or an antigen-binding fragment thereof that binds to CD79b and comprises

[0120] a heavy chain variable region (VH) that comprises VH CDR1, VH CDR2, and VH CDR3 that are identical to the VH CDR1, VH CDR2, and VH CDR3 of a selected VH sequence; and a light chain variable region (VL) that comprises VL CDR1, VL CDR2, and VL CDR3 that are identical to the VL CDR1, VL CDR2, and VL CDR3 of a selected VL sequence, wherein the selected VH sequence and the selected VL sequence are one of the following:

[0121] (1) the selected VH sequence is SEQ ID NO:7 and the selected VL sequence is SEQ ID NO:8;

[0122] (2) the selected VH sequence is SEQ ID NO:17 or 69 and the selected VL sequence is SEQ ID NO:18 or 70;

[0123] (3) the selected VH sequence is SEQ ID NO:27 and the selected VL sequence is SEQ ID NO:28;

[0124] (4) The selected VH sequence is SEQ ID NO: 37 or 71, and the selected VL sequence is SEQ ID NO: 38 or 72;

[0125] (5) The selected VH sequence is SEQ ID NO: 47 or 73, and the selected VL sequence is SEQ ID NO: 48 or 74; and

[0126] (6) The selected VH sequence is SEQ ID NO: 57 or 75, and the selected VL sequence is SEQ ID NO: 58 or 76.

[0127] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to CD79b of human, mouse, monkey or dog.

[0128] In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof, a chimeric antibody, a single-chain variable fragment (scFv), a single-arm antibody, and / or a multispecific antibody. Antibody (e.g., bispecific antibody).

[0129] In some embodiments, the antibody or antigen-binding fragment comprises a human IgG1 Fc, a human IgG2 Fc, or a human IgG4 Fc.

[0130] In one aspect, the present disclosure relates to an antibody or antigen-binding fragment thereof that cross-competes with the antibody or antigen-binding fragment thereof described herein.

[0131] In some embodiments, the antibody or antigen-binding fragment thereof comprises a crystallizable fragment (Fc region).

[0132] In one aspect, the present disclosure relates to a chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof described herein.

[0133] In one aspect, the present disclosure relates to an antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof described herein covalently bound to a therapeutic agent.

[0134] In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent.

[0135] In one aspect, the present disclosure relates to a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the antibody or antigen-binding fragment thereof described herein, the CAR described herein, or the antibody-drug conjugate described herein to the subject.

[0136] In some embodiments, the cancer is lymphoma, leukemia, breast cancer, gastric cancer, pancreatic cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, or urothelial cancer.

[0137] In some embodiments, the cancer is non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia (PLL), splenic lymphoma with villous lymphocytes (SLVL), hairy cell leukemia (HCL), follicular lymphoma (FL), or mantle cell (MCL) lymphoma.

[0138] In some embodiments, the subject is further treated with an effective amount of an anti-4-1BB antibody, an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-CD40 antibody, a BTK inhibitor, and a BCL2 inhibitor.

[0139] In one aspect, the present disclosure relates to a method of reducing a tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof described herein, a CAR described herein, or an antibody-drug conjugate described herein.

[0140] In one aspect, the present disclosure relates to a method of killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof described herein, a CAR described herein, or an antibody-drug conjugate described herein.

[0141] In one aspect, the present disclosure relates to a method of increasing an immune response in a subject, the method comprising administering to the subject an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof described herein, a CAR described herein, or an antibody-drug conjugate described herein.

[0142] In one aspect, the present disclosure relates to a method of treating a subject having an autoimmune disease, the method comprising administering a therapeutically effective amount of a composition comprising an antibody or an antigen-binding fragment thereof described herein, a CAR described herein, or an antibody-drug conjugate described herein.

[0143] In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, psoriasis, multiple sclerosis, immune thrombocytopenic purpura, myasthenia gravis, neuromyelitis optica, IgG4-related disease, systemic lupus erythematosus, lupus nephritis, giant cell arteritis, Takayasu disease, cold agglutinin disease, warm autoimmune hemolytic anemia, and antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis) or microscopic polyangiitis (MPA), inflammatory bowel disease (IBD), and autoimmune pancreatitis.

[0144] In some embodiments, the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease (IBD), or autoimmune pancreatitis.

[0145] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof described herein and a pharmaceutically acceptable carrier.

[0146] In one aspect, the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable carrier.

[0147] As used herein, the term "cancer" refers to cells having the ability to grow autonomously. Examples of such cells include cells having an abnormal state or condition characterized by rapid proliferative cell growth. The term is intended to include cancerous growths, such as tumors; carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasion. The term also includes malignancies of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematopoietic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, including malignancies such as most colon cancers, renal cell carcinomas, prostate cancers, and / or testicular tumors, non-small cell carcinomas of the lung, and small intestine cancers. "Naturally occurring" cancers include any cancer not experimentally induced by implanting cancer cells into a subject, including, for example, spontaneously occurring cancers, cancers caused by a patient's exposure to carcinogens, cancers caused by the insertion of a transgenic oncogene or the knockout of a tumor suppressor gene, and cancers caused by infection, such as viral infection. The term "carcinoma" is well recognized in the art and refers to a malignancy of epithelial or endocrine tissue. The term also includes carcinosarcomas, which include malignancies composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to a carcinoma that originates from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is well recognized in the art and refers to a malignancy of mesenchymal origin. The term "hematopoietic neoplastic disease" includes diseases involving proliferative / neoplastic cells of hematopoietic origin. Hematopoietic neoplastic diseases can be caused by myeloid, lymphoid, or erythroid lineages or their progenitor cells.

[0148] As used herein, the term "antibody" refers to any antigen-binding molecule that comprises at least one (e.g., one, two, three, four, five, or six) complementarity-determining region (CDR) (e.g., any one of the three CDRs from an immunoglobulin light chain or any one of the three CDRs from an immunoglobulin heavy chain) and is capable of specifically binding an epitope. Non-limiting examples of antibodies include: monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, chimeric antibodies, human antibodies, and humanized antibodies. In some embodiments, an antibody may comprise the Fc region of a human antibody. The term antibody also includes derivatives such as bispecific antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments.

[0149] As used herein, the term "antigen-binding fragment" refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding an antigen. In some embodiments, the antigen-binding fragment comprises at least one variable domain (e.g., a heavy-chain variable domain or a light-chain variable domain). Non-limiting examples of antibody fragments include, for example, Fab, Fab’, F(ab’)2, and Fv fragments.

[0150] As used herein, the term "chimeric antibody" refers to an antibody that comprises sequences present in at least two different antibodies (e.g., antibodies from two different mammalian species, e.g., a human antibody and a non-human antibody). Non-limiting examples of chimeric antibodies are antibodies that comprise the variable domain sequences (e.g., all or part of the light-chain and / or heavy-chain variable domain sequences) of a non-human (e.g., mouse, rabbit) antibody and the constant domains of a human antibody. Other examples of chimeric antibodies are described herein and are known in the art.

[0151] As used herein, the term "humanized antibody" refers to a non-human antibody that contains a minimal sequence derived from a non-human (e.g., murine, rabbit) immunoglobulin and contains a sequence derived from a human immunoglobulin. In a non-limiting example, a humanized antibody is a human antibody (recipient antibody) in which the residues of the hypervariable regions (e.g., CDRs) of the recipient antibody are replaced with the residues of the hypervariable regions (e.g., CDRs) from a non-human antibody (e.g., donor antibody) such as a murine, rat, or rabbit antibody, having the desired specificity, affinity, and potency. In some embodiments, the Fv framework residues of the human immunoglobulin are replaced with the corresponding non-human (e.g., murine, rabbit) immunoglobulin residues. In some embodiments, a humanized antibody may contain residues not found in the recipient antibody or the donor antibody. These modifications may further improve antibody performance. In some embodiments, a humanized antibody contains substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops (CDRs) correspond to the hypervariable loops of a non-human (CDR) immunoglobulin, and all or substantially all of the framework regions are those of a human immunoglobulin. A humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), usually the constant region of a human immunoglobulin. Humanized antibodies can be produced using molecular biology methods known in the art. Non-limiting examples of methods for producing humanized antibodies are described herein.

[0152] As used herein, the terms "subject" and "patient" are used interchangeably throughout the specification and describe an animal, human or non-human, to which a treatment according to the methods of the present invention is administered. The present invention encompasses veterinary and non-veterinary applications. A human patient can be an adult or a minor (e.g., a person under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Patients also include, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), equines, canines, felines, bovines, and other domestic, farm, and zoo animals.

[0153] As used herein, when referring to an antibody, the phrases "specifically binds" and "specifically binding" mean that the antibody interacts preferably with its target molecule (e.g., CD79b) as opposed to other molecules because the interaction depends on a particular structure (i.e., antigenic determinant or epitope) on the target molecule; in other words, the reagent recognizes and binds to a molecule containing a particular structure, rather than all molecules in general. An antibody that specifically binds to a target molecule can be referred to as a target-specific antibody. For example, an antibody that specifically binds to the CD79b molecule can be referred to as a CD79b-specific antibody or an anti-CD79b antibody.

[0154] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to an amino acid polymer of any length of at least two amino acids.

[0155] As used herein, the terms "polynucleotide", "nucleic acid molecule" and "nucleic acid sequence" are used interchangeably herein and refer to a nucleotide polymer of any length of at least two nucleotides, including but not limited to DNA, RNA, DNA / RNA hybrids and their modifications.

[0156] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art may also be used. The materials, methods and examples are illustrative only and not limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification (including definitions) shall prevail.

[0157] Other features and advantages of the present invention will be apparent from the following detailed description, the drawings and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0158] Figure 1 Showing the ELISA binding affinity of an anti-CD79b antibody for a human CD79b recombinant protein.

[0159] Figure 2 Showing the ELISA binding affinity of an anti-CD79b antibody for a cynomolgus monkey CD79b recombinant protein.

[0160] Figure 3 Showing the BLI binding affinity of an anti-CD79b antibody for a human CD79b recombinant protein.

[0161] Figure 4 Showing the relative internalization of an anti-CD79b antibody from the cell surface of Ramos cells.

[0162] Figure 5 Showing the quantification of anti-CD79b antigen density on the surfaces of malignant B tumor cells and normal B cells

[0163] Figure 6 Showing the binding affinity of an anti-CD79b antibody for BJAB cells.

[0164] Figure 7 Showing the binding affinity of an anti-CD79b antibody for Ramos cells.

[0165] Figure 8 Showing the binding affinity of an anti-CD79b antibody for Daudi cells.

[0166] Figure 9 Show the binding affinity of the anti-CD79b antibody to SU-DHL-4 cells.

[0167] Figure 10 Show the binding affinity of the anti-CD79b antibody to Nalm-6 cells.

[0168] Figure 11 Show the binding affinity of the anti-CD79b antibody to donor 2890.

[0169] Figure 12 Show the binding affinity of the anti-CD79b antibody to donor 2235.

[0170] Figure 13 Show the binding affinity of the anti-CD79b antibody to donor 889.

[0171] Figure 14 Show the binding affinity of the anti-CD79b antibody to donor 356.

[0172] Figure 15 Show the binding affinity of the anti-CD79b antibody to CLL donor 5716.

[0173] Figure 16 Show the binding affinity of the anti-CD79b antibody to CLL donor 0255.

[0174] Figures 17A - 17B Show the binding of the anti-CD79b antibody to the long and short isoforms of CD79b.

[0175] Figure 18 Show the ELISA binding of the humanized anti-CD79b antibody to recombinant human CD79b ECD.

[0176] Figure 19 Show the binding of the humanized anti-CD79b antibody to cell lines expressing endogenous CD79b.

[0177] Figure 20 List the CDR sequences of the anti-CD79b antibody defined according to the Kabat definition.

[0178] Figure 21 List the CDR sequences of the anti-CD79b antibody defined according to the Chothia definition. Detailed implementation

[0179] The B lymphocyte antigen receptor is a multimeric complex that includes an antigen-specific component, surface immunoglobulin (Ig). The surface immunoglobulin is non-covalently associated with two other proteins, CD79a and CD79b, which are required for the expression and function of the B cell antigen receptor. CD79 is the signaling component of the B cell receptor and functions as a covalent heterodimer that includes CD79a (i.e., Ig-α or MB1) and CD79b (i.e., Ig-β or B29). CD79b contains an extracellular immunoglobulin (Ig) domain, a transmembrane domain, and an intracellular signaling domain, an immunoreceptor tyrosine-based activation motif (ITAM) domain. CD79b expression is detected on the surface of almost all patients with non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL).

[0180] The present disclosure provides examples of antibodies and antigen-binding fragments thereof that bind CD79b.

[0181] CD79b

[0182] CD79 is composed of CD79a and CD79b components that are expressed almost exclusively on B cells and B cell tumors. During B cell ontogeny, CD79a and CD79b expression precedes immunoglobulin (Ig) heavy chain gene rearrangement and CD20 expression, and is lost later than CD20 at the late (plasma cell) stage of B cell differentiation. Thus, antibodies against CD79a and CD79b can be used for differential diagnosis of B cell tumors from T cell tumors or myeloid tumors, or lymphocyte-predominant Hodgkin lymphoma of the L and H types from classical Hodgkin lymphoma. In addition, anti-CD79a and anti-CD79b antibodies are useful markers for the diagnosis of precursor B acute lymphoblastic leukemia (pre-B-ALL) because many of these tumors are negative for other B cell markers such as CD20 and CD45RA.

[0183] CD79 is considered an interesting therapeutic target for antibodies because it is physiologically and specifically expressed in mature B cells and the vast majority of B cell NHLs, including DLBCL (from 90% to 100%), as well as B acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia (PLL), splenic lymphoma with villous lymphocytes (SLVL), hairy cell leukemia (HCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL).

[0184] CD79 binds to cell surface immunoglobulin (sIg) for antigen recognition, forming the B cell antigen receptor (BCR) complex, which plays a crucial role in B cell maturation and activation. CD79 consists of an α (CD79a) and a β (CD79b) heterodimer, and its function is the signal transduction component of BCR. Both subunits of CD79 contain an extracellular Ig domain, a transmembrane domain, and an intracellular signaling domain, which initiate BCR signaling after antigen binding, ultimately leading to B cell activation, antigen presentation, cytokine production, and cell proliferation and differentiation.

[0185] Antigen binding by BCR induces its internalization and processing into major histocompatibility complex class II (MHCII) compartments, i.e., lysosome-like compartments, for class II antigen presentation by B cells. This intracellular trafficking is of particular interest because drugs are directly delivered to target cells into lysosomal compartments, enhancing cytotoxic activity, and it allows the use of more stable linkers that are cleaved in the MHCII compartment.

[0186] A detailed review of CD79b and its functions can be found in Chu, Peiguo G., and Daniel A. Arber. "CD79: a review." Applied Immunohistochemistry & Molecular Morphology 9.2 (2001): 97 - 106; Bourbon, Estelle, and Gilles Salles. "Polatuzumab vedotin: an investigational anti-CD79b antibody drug conjugate for the treatment of diffuse large B-cell lymphoma." Expert Opinion on Investigational Drugs 29.10 (2020): 1079 - 1088. Each of these references is hereby incorporated by reference in its entirety.

[0187] The present disclosure provides several anti-CD79b antibodies, antigen-binding fragments thereof, and methods of using these anti-CD79b antibodies and antigen-binding fragments to inhibit tumor growth, treat cancer, and treat autoimmune diseases.

[0188] Antibodies and antigen-binding fragments

[0189] The present invention provides anti-CD79b antibodies and antigen-binding fragments thereof. Generally, an antibody (also known as an immunoglobulin) consists of two types of polypeptide chains, a light chain and a heavy chain. The non-limiting antibodies of the present invention can be intact four-immunoglobulin chain antibodies comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype, including IgM, IgG, IgE, IgA or IgD, or isosubtype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a κ light chain or a λ light chain. An antibody can contain two identical copies of the light chain and two identical copies of the heavy chain. Each heavy chain contains a variable domain (or variable region, VH) and multiple constant domains (or constant regions), which are bound to each other by disulfide bonds within its constant domain to form the "backbone" of the antibody. Each light chain contains a variable domain (or variable region, VL) and a constant domain (or constant region), and each light chain is bound to a heavy chain by a disulfide bond. The variable region of each light chain is aligned with the variable region of the heavy chain to which it binds. The variable regions of the light and heavy chains contain three hypervariable regions, which are sandwiched between more conserved framework regions (FRs).

[0190] These hypervariable regions, called complementarity-determining regions (CDRs), form loops that constitute the major antigen-binding surface of the antibody. The four framework regions mainly adopt a β-sheet conformation, and the CDRs form loops connecting the β-sheet structures and, in some cases, form part of the β-sheet structure. The CDRs in each chain are in close proximity to the framework regions and, together with the CDRs in the other chain, contribute to the formation of the antigen-binding region.

[0191] Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known and typically use multiple definitions of CDRs. The Kabat definition is based on sequence variability, while the Chothia definition is based on the location of structural loop regions. These methods and definitions are described in Martin, "Protein sequence and structure analysis of antibody variable domains," Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. "Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains," Molecular immunology 45.14 (2008): 3832-3839; Wu, T.T. and Kabat, E.A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203: 121-53 (1991); Morea et al., Biophys Chem. 68(1-3): 9-16 (Oct. 1997); Morea et al., J Mol Biol. 275(2): 269-94 (Jan. 1998); Chothia et al., Nature 342(6252): 877-83 (Dec. 1989); Ponomarenko and Bourne, BMC Structural Biology 7: 64 (2007), each of which is incorporated herein by reference in its entirety.

[0192] CDRs are important for recognizing epitopes. As used herein, an "epitope" is the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum number of epitopes can be about three, four, five, six, or seven amino acids, but these amino acids do not need to be in a continuous linear sequence in the primary structure of the antigen, as the epitope may depend on the three-dimensional conformation of the antigen based on its secondary and tertiary structures.

[0193] In some embodiments, the antibody is a complete immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, and their constant regions differ, particularly in the hinge and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art and are described in Vidarsson, et al., IgG subclasses and allotypes: from structure to effector functions. Frontiers in immunology 5 (2014); Irani, et al. Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases. Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

[0194] The antibody can also be an immunoglobulin molecule derived from any species (e.g., human, rodent, mouse, camel, rabbit). The antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies, and chimeric antibodies, which include immunoglobulin binding domains fused to another polypeptide. The term "antigen-binding domain" or "antigen-binding fragment" is a part of an antibody that retains the specific binding activity of the intact antibody, i.e., any part of the antibody that can specifically bind to an epitope on the target molecule of the intact antibody. It includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment can be, for example, a single-chain antibody, Fv, Fd, bispecific antibody, single-chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising a binding domain that is homologous to or binds to an antibody-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy-chain and / or light-chain CDRs of an intact antibody, the heavy-chain and / or light-chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or a single CDR in the heavy or light chain of an intact antibody.

[0195] In some embodiments, the antigen-binding fragment can form part of a chimeric antigen receptor (CAR). In some embodiments, the scFv has one heavy-chain variable domain and one light-chain variable domain. In some embodiments, the scFv has two heavy-chain variable domains and two light-chain variable domains.

[0196] Anti-CD79b antibodies and antigen-binding fragments

[0197] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to CD79b (e.g., human CD79b). The antibodies and antigen-binding fragments described herein are capable of binding to CD79b. These antibodies can be agonists or antagonists of CD79b-mediated BCR signaling. In some embodiments, the antibodies and antigen-binding fragments can bind to the extracellular domain of human CD79b.

[0198] The present disclosure provides, for example, anti-CD79b antibodies 22D10, 23D8, 29C3, 44G2, 48H10, 57B9, their chimeric antibodies, and their humanized antibodies.

[0199] According to the Kabat definition, the CDR sequences of 22D10 and 22D10-derived antibodies (e.g., humanized antibodies) include the CDR amino acid sequences of the heavy chain variable region listed in SEQ ID NO: 9, 11, 13, and the CDR amino acid sequences of the light chain variable region listed in SEQ ID NO: 14-16. The CDRs can also be defined by Chothia. According to the Chothia definition, the CDR amino acid sequences of the heavy chain variable domain are listed in SEQ ID NO: 10, 12, 13, and the CDR amino acid sequences of the light chain variable domain are listed in SEQ ID NO: 14-16.

[0200] Similarly, according to the Kabat definition, the CDR sequences of 23D8 and 23D8-derived antibodies include the CDR amino acid sequences of the heavy chain variable region listed in SEQ ID NO: 19, 21, 23, and the CDR amino acid sequences of the light chain variable region listed in SEQ ID NO: 24-26. According to the Chothia definition, the CDR amino acid sequences of the heavy chain variable region are listed in SEQ ID NO: 20, 22, 23, and the CDR amino acid sequences of the light chain variable region are listed in SEQ ID NO: 24-26.

[0201] According to the Kabat definition, the CDR sequences of 29C3 and 29C3-derived antibodies include the CDR amino acid sequences of the heavy chain variable region listed in SEQ ID NO: 29, 31, 33, and the CDR amino acid sequences of the light chain variable region listed in SEQ ID NO: 34-36. According to the Chothia definition, the CDR amino acid sequences of the heavy chain variable region are listed in SEQ ID NO: 30, 32, 33, and the CDR amino acid sequences of the light chain variable region are listed in SEQ ID NO: 34-36.

[0202] According to the Kabat definition, the CDR sequences of 44G2 and 44G2-derived antibodies include the CDR amino acid sequences of the heavy chain variable region listed in SEQ ID NO: 39, 41, 43, and the CDR amino acid sequences of the light chain variable region listed in SEQ ID NO: 44-46. According to the Chothia definition, the CDR amino acid sequences of the heavy chain variable region are listed in SEQ ID NO: 40, 42, 43, and the CDR amino acid sequences of the light chain variable region are listed in SEQ ID NO: 44-46.

[0203] According to the Kabat definition, the CDR sequences of 48H10 and 48H10-derived antibodies include the CDR amino acid sequences of the heavy chain variable region listed in SEQ ID NO: 49, 51, 53, and the CDR amino acid sequences of the light chain variable region listed in SEQ ID NO: 54 - 56. According to the Chothia definition, the CDR amino acid sequences of the heavy chain variable region are listed in SEQ ID NO: 50, 52, 53, and the CDR amino acid sequences of the light chain variable region are listed in SEQ ID NO: 54 - 56.

[0204] According to the Kabat definition, the CDR sequences of 57B9 and 57B9-derived antibodies include the CDR amino acids of the heavy chain variable region listed in SEQ ID NO: 59, 61, 63, and the CDR amino acids of the light chain variable region listed in SEQ ID NO: 64 - 66. According to the Chothia definition, the CDR amino acid sequences of the heavy chain variable region are listed in SEQ ID NO: 60, 62, 63, and the CDR amino acid sequences of the light chain variable region are listed in SEQ ID NO: 64 - 66.

[0205] The amino acid sequence of the heavy chain variable region of the 22D10 antibody is listed in SEQ ID NO: 7. The amino acid sequence of the light chain variable region of the 22D10 antibody is listed in SEQ ID NO: 8.

[0206] The amino acid sequence of the heavy chain variable region of the 23D8 antibody is listed in SEQ ID NO: 17. The amino acid sequence of the light chain variable region of the 23D8 antibody is listed in SEQ ID NO: 18.

[0207] The amino acid sequence of the heavy chain variable region of the 29C3 antibody is listed in SEQ ID NO: 27. The amino acid sequence of the light chain variable region of the 29C3 antibody is listed in SEQ ID NO: 28.

[0208] The amino acid sequence of the heavy chain variable region of the 44G2 antibody is listed in SEQ ID NO: 37. The amino acid sequence of the light chain variable region of the 44G2 antibody is listed in SEQ ID NO: 38.

[0209] The amino acid sequence of the heavy chain variable region of the 48H10 antibody is listed in SEQ ID NO: 47. The amino acid sequence of the light chain variable region of the 48H10 antibody is listed in SEQ ID NO: 48.

[0210] The amino acid sequence of the heavy chain variable region of the 57B9 antibody is listed in SEQ ID NO: 57. The amino acid sequence of the light chain variable region of the 57B9 antibody is listed in SEQ ID NO: 58.

[0211] In some embodiments, the anti-CD79b antibody is a humanized antibody. In some embodiments, the anti-CD79b antibody is humanized 23D8, 44G2, 48H10 or 57B9.

[0212] The amino acid sequence of the heavy chain variable region of the humanized 23D8 antibody is set forth in SEQ ID NO: 69. The amino acid sequence of the light chain variable region of the 23D8 antibody is set forth in SEQ ID NO: 70.

[0213] The amino acid sequence of the heavy chain variable region of the humanized 44G2 antibody is set forth in SEQ ID NO: 71. The amino acid sequence of the light chain variable region of the 44G2 antibody is set forth in SEQ ID NO: 72.

[0214] The amino acid sequence of the heavy chain variable region of the humanized 48H10 antibody is set forth in SEQ ID NO: 73. The amino acid sequence of the light chain variable region of the 48H10 antibody is set forth in SEQ ID NO: 74.

[0215] The amino acid sequence of the heavy chain variable region of the humanized 57B9 antibody is set forth in SEQ ID NO: 75. The amino acid sequence of the light chain variable region of the 57B9 antibody is set forth in SEQ ID NO: 76.

[0216] Also provided are amino acid sequences that modify the heavy chain variable region and the light chain variable region of the antibody. In some embodiments, the heavy chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 7, 17, 27, 37, 47 and 57. In some embodiments, the light chain variable region is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 8, 18, 28, 38, 48 and 58. The heavy chain variable region sequence can pair with the corresponding light chain variable region sequence and jointly bind to CD79b.

[0217] The percentage of humanization refers to the percentage of identity of the variable region sequence of the heavy or light chain compared to the human antibody sequence in the International Immunogenetics Information System (IMGT) database. In some embodiments, the percentage of humanization is greater than 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95%. A detailed description of how to determine the percentage of humanization and how to determine hot spot hits is known in the art and is described in Jones, et al. The INNs and outs of antibody nonproprietary names. MAbs. Vol. 8. No. 1. Taylor & Francis, 2016, which is incorporated herein by reference in its entirety. High percentages of humanization generally have various advantages, such as being safer and more effective for humans, being more likely to be tolerated by human subjects, and / or being less likely to produce side effects. In some embodiments, the variable region is fully human, for example, derived from the human heavy chain immunoglobulin locus sequence (e.g., recombination of human IGHV, human IGHD, and human IGHJ genes) and / or the human κ chain immunoglobulin locus sequence (e.g., recombination of human IGKV and human IGKJ genes).

[0218] In addition, in some embodiments, the antibody or its antigen-binding fragment described herein may further contain one, two, or three heavy chain variable region CDRs selected from SEQ ID NO: 9, 11, 13; SEQ ID NO: 19, 21, 23; SEQ ID NO: 29, 31, 33; SEQ ID NO: 39, 41, 43; SEQ ID NO: 49, 51, 53; SEQ ID NO: 59, 61, 63; SEQ ID NO: 10, 12, 13; SEQ ID NO: 20, 22, 23; SEQ ID NO: 30, 32, 33; SEQ ID NO: 40, 42, 43; SEQ ID NO: 50, 52, 53; SEQ ID NO: 60, 62, 63, and / or one, two, or three light chain variable region CDRs selected from SEQ ID NO: 14 - 16, SEQ ID NO: 24 - 26, SEQ ID NO: 34 - 36, SEQs ID NO: 44 - 46, SEQs ID NO: 54 - 56, and SEQ ID NO: 64 - 66.

[0219] In some embodiments, an antibody may have a heavy chain variable region (VH) that includes complementarity determining regions (CDR) 1, 2, and 3, where the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH CDR3 amino acid sequence. In some embodiments, an antibody may have a light chain variable region (VL) that includes complementarity determining regions (CDR) 1, 2, and 3, where the CDR1 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR1 amino acid sequence, the CDR2 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR2 amino acid sequence, and the CDR3 region comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL CDR3 amino acid sequence. Figure 20 (Kabat CDR) and Figure 21 (Chathia CDR) show the selected VH CDR 1, 2, 3 amino acid sequences and the selected VL CDR, 1, 2, 3 amino acid sequences.

[0220] In some embodiments, an antibody or antigen-binding fragment described herein may contain a heavy chain variable region that contains one, two, or three CDRs and has SEQ ID NO: 9 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 11 with zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 13 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0221] In some embodiments, an antibody or antigen-binding fragment described herein may contain a heavy chain variable region that contains one, two, or three CDRs and has SEQ ID NO: 19 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 21 with zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 23 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0222] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable region that comprises one, two, or three CDRs and has SEQ ID NO: 29 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 31 with zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 33 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0223] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable region that comprises one, two, or three CDRs and has SEQ ID NO: 39 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 41 with zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 43 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0224] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable region that comprises one, two, or three CDRs and has SEQ ID NO: 49 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 51 with zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 53 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0225] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable region that comprises one, two, or three CDRs and has SEQ ID NO: 59 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 61 with zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 63 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0226] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a heavy chain variable region that comprises one, two, or three CDRs and has SEQ ID NO: 10 with zero, one, or two amino acid insertions, deletions, or substitutions; SEQ ID NO: 12 with zero, one, or two amino acid insertions, deletions, or substitutions; and SEQ ID NO: 13 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0227] In some embodiments, the antibodies or antigen-binding fragments described herein can comprise a heavy chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 20 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 22 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 23 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0228] In some embodiments, the antibodies or antigen-binding fragments described herein can comprise a heavy chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 30 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 32 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 33 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0229] In some embodiments, the antibodies or antigen-binding fragments described herein can comprise a heavy chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 40 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 42 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 43 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0230] In some embodiments, the antibodies or antigen-binding fragments described herein can comprise a heavy chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 50 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 52 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 53 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0231] In some embodiments, the antibodies or antigen-binding fragments described herein can comprise a heavy chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 60 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 62 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 63 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0232] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 14 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 15 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 16 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0233] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 24 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 25 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 26 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0234] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 34 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 35 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 36 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0235] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 44 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 45 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 46 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0236] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable region that comprises one, two, or three CDRs, having SEQ ID NO: 54 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 55 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 56 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0237] In some embodiments, the antibodies or antigen-binding fragments described herein may comprise a light chain variable region, said variable region comprising one, two, or three CDRs, having SEQ ID NO: 64 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 65 with zero, one, or two amino acid insertions, deletions, or substitutions; having SEQ ID NO: 66 with zero, one, or two amino acid insertions, deletions, or substitutions.

[0238] Insertions, deletions, and substitutions may be within the CDR sequences or at one or both termini of the CDR sequences. In some embodiments, the CDRs are determined based on the Kabat definition. In some embodiments, the CDRs are determined based on the Chothia definition. In some embodiments, the CDRs are determined based on a combination of the Kabat definition and the Chothia definition.

[0239] The present disclosure also provides antibodies or antigen-binding fragments thereof that bind to CD79b. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VH sequence, and a light chain variable region (VL) that comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to a selected VL sequence. In some embodiments, the selected VH sequence is SEQ ID NO: 7, and the selected VL sequence is SEQ ID NO: 8. In some embodiments, the selected VH sequence is SEQ ID NO: 17, and the selected VL sequence is SEQ ID NO: 18. In some embodiments, the selected VH sequence is SEQ ID NO: 27, and the selected VL sequence is SEQ ID NO: 28. In some embodiments, the selected VH sequence is SEQ ID NO: 37, and the selected VL sequence is SEQ ID NO: 38. In some embodiments, the selected VH sequence is SEQ ID NO: 47, and the selected VL sequence is SEQ ID NO: 48. In some embodiments, the selected VH sequence is SEQ ID NO: 57, and the selected VL sequence is SEQ ID NO: 58. In some embodiments, the selected VH sequence is SEQ ID NO: 69, and the selected VL sequence is SEQ ID NO: 70. In some embodiments, the selected VH sequence is SEQ ID NO: 71, and the selected VL sequence is SEQ ID NO: 72. In some embodiments, the selected VH sequence is SEQ ID NO: 73, and the selected VL sequence is SEQ ID NO: 74. In some embodiments, the selected VH sequence is SEQ ID NO: 75, and the selected VL sequence is SEQ ID NO: 76.

[0240] The present disclosure also provides antibodies or antigen-binding fragments thereof that can compete with the antibodies described herein. In some aspects, the antibody or antigen-binding fragment can bind to the same epitope as the antibodies described herein.

[0241] The present disclosure also provides antibodies or antigen-binding fragments thereof that cross-compete with any of the antibodies or antigen-binding fragments described herein. Cross-competition assays are known in the art and are described in Moore et al., "Antibody cross-competition analysis of the human immunodeficiency virus type 1 gp120 exterior envelope glycoprotein." Journal of virology 70.3 (1996): 1863-1872, which is incorporated herein by reference in its entirety. In one aspect, the present disclosure also provides antibodies or antigen-binding fragments thereof that bind to the same epitope or region as any of the antibodies or antigen-binding fragments described herein. Epitope binning assays are known in the art and are described in Estep et al. "High throughput solution-based measurement of antibody-antigen affinity and epitope binning." MAbs. Vol. 5. No. 2. Taylor & Francis, 2013, which is incorporated herein by reference in its entirety.

[0242] To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for comparison purposes, and optimal alignment and non-homologous sequences can be disregarded). Then the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to achieve optimal alignment of the two sequences. For example, comparison of sequences and determination of the percent identity between two sequences can be accomplished using the Blossum 62 scoring matrix, a gap of 12, a gap extension of 4, and a frameshift gap of 5.

[0243] The present disclosure also provides nucleic acids comprising polynucleotides encoding polypeptides that comprise an immunoglobulin heavy chain or an immunoglobulin light chain. The CDRs comprised by the immunoglobulin heavy chain or immunoglobulin light chain are asFigure 20 as shown in SEQ ID NO: 21 or as shown in Table 4. When the polypeptide pairs with a corresponding polypeptide (e.g., a corresponding heavy chain variable region or a corresponding light chain variable region), the paired polypeptides bind to CD79b (e.g., human CD79b).

[0244] The anti-CD79b antibodies and antigen-binding fragments can also be antibody variants (including derivatives and conjugates) of an antibody or an antibody fragment, as well as multispecific (e.g., bispecific) antibodies or antibody fragments. Other antibodies provided herein are polyclonal, monoclonal, multimeric, multispecific (e.g., bispecific), humanized antibodies, chimeric antibodies (e.g., human-mouse chimeras), single-chain antibodies, intracellularly produced antibodies (i.e., intrabodies), and antigen-binding fragments thereof. The antibody or its antigen-binding fragment can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0245] Antibody fragments are suitable for the provided methods as long as they retain the affinity and specificity required for the full-length antibody. Thus, an antibody fragment that binds to CD79b will retain the ability to bind to CD79b. An Fv fragment is an antibody fragment that contains the complete antigen recognition and binding site. The region consists of a dimer of one heavy chain variable region and one light chain variable region tightly bound together, which can be covalently bonded in nature, such as in scFv. It is in this configuration that the three CDRs of each variable region interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs or a subset thereof confer antigen-binding specificity to the antibody. However, even a single variable region (or half of the Fv containing only three CDRs specific for the antigen) can have the ability to recognize and bind the antigen, although the affinity is generally lower than that of the entire binding site.

[0246] A single-chain Fv or (scFv) antibody fragment contains the VH and VL domains (or regions) of an antibody, where these domains are present in a single polypeptide chain. Typically, the scFv polypeptide also contains a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure required for antigen binding.

[0247] A Fab fragment contains the variable domain and the constant domain of the light chain, as well as the variable domain and the first constant domain (CH1) of the heavy chain. An F(ab')2 antibody fragment contains a pair of Fab fragments that are typically covalently linked near their carboxyl termini by a hinge cysteine. Other chemical conjugations of antibody fragments are also known in the art.

[0248] The antibodies and antibody fragments of the present invention can be modified in the Fc region to provide desired effector functions or serum half-lives. In some embodiments, the Fc region can be modified to silence or reduce complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC).

[0249] In some embodiments, the multispecific antibody is a bispecific antibody. Bispecific antibodies can be made by engineering the interface between a pair of antibody molecules to maximize the percentage of heterodimers recovered from recombinant cell culture. For example, the interface can comprise at least a portion of the CH3 domain of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end products, such as homodimers. This method is described, for example, in WO96 / 27011, which is incorporated herein by reference in its entirety.

[0250] Any antibody or antigen-binding fragment described herein can be conjugated to a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or in solution). Non-limiting examples of stabilizing molecules include: polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation of a stabilizing molecule can increase the half-life or extend the biological activity of the antibody or antigen-binding fragment in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0251] In some embodiments, the antibodies or antigen-binding fragments described herein can be conjugated to a therapeutic agent. Antibody-drug conjugates comprising an antibody or its antigen-binding fragment can bind the therapeutic agent covalently or non-covalently. In some embodiments, the therapeutic agent is a cytotoxic or cytostatic agent (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracene, maytansine alkaloids, such as DM-1 and DM-4, diketopiperazines, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and the like).

[0252] In some embodiments, the antibodies or their antigen-binding fragments described herein recognize endogenous CD79b or recombinant CD79b. In some embodiments, the antibodies or their antigen-binding fragments described herein recognize human CD79b (e.g., the extracellular region of human CD79b).

[0253] Antibody-drug conjugate (ADC)

[0254] The antibodies, antigen-binding fragments or antigen-binding protein constructs (e.g., bispecific antibodies) described herein can be conjugated to a therapeutic agent (drug). The therapeutic agent can be covalently or non-covalently bound to the antibody or antigen-binding fragment or antigen-binding protein construct (e.g., bispecific antibody).

[0255] In some embodiments, the therapeutic agent is a cytotoxic agent or cytostatic agent (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dithranol, maytansine alkaloids such as DM-1 and DM-4, dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs). Useful cytotoxic agents, cytostatic agents or immunomodulators include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors and alkylating agents.

[0256] In some embodiments, the therapeutic agent can include, but is not limited to, cytotoxic reagents such as chemotherapeutic agents, immunotherapeutic agents, etc., antiviral agents or antimicrobial agents. In some embodiments, the therapeutic agent to be conjugated can be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0257] In some embodiments, the therapeutic agent is an auristatin, such as auristatin E (a derivative also known in the art as dolastatin - 10) or a derivative thereof. An auristatin can be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can react with p - aminobenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatins are described in U.S. Patent Application Publication No. 2003 - 0083263; International Patent Publication No. WO 04 / 010957, International Patent Publication No. WO 02 / 088172, and U.S. Patent No. 5,990,910. Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; 4,486,414 and 4,486,414, each of which is incorporated herein by reference in its entirety for all purposes.

[0258] Auristatin has been shown to interfere with microtubule dynamics and nuclear and cell division and has anticancer activity. Auristatin binds to tubulin and can exert a cytotoxic or cytostatic effect on cancer cells. There are a variety of different assays known in the art that can be used to determine whether an auristatin or the resulting antibody - drug conjugate exerts a cytostatic or cytotoxic effect on the desired cells.

[0259] In some embodiments, the therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN TM) alkyl sulfonates such as busulfan, isopropyl busulfan, and piposulfan; aziridines such as bendopa, carboquone, medopa, and urodopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; nitrogen mustards such as chlorambucil, chloronaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride, melphalan, novantrone, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclarubicin, actinomycin, erythromycin, azaserine, bleomycin, actinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, actinomycin, daunorubicin, destomycin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, clarithromycin, rodorubicin, streptozocin, streptomycin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as demethylfolate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thioguanine, tioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, didoxuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, drostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folic acid; acetylacetone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defosfamide; demecolcine; diaziquone; elfomithine; emitefur; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazine; procarbazine; PSK7; razoxane; sizofiran; spirogermanium; tinzaparin; triaziquone; 2’,2’,2’-trichloroethylamine; polyurethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; cytarabine; arabinoside (“Ara-C”); cyclophosphamide; taxanes such as paclitaxel( Bristol-Myers Squibb Oncology, Princeton, New Jersey) and docetaxel( Rhone-Poulenc Rorer, Anthony, France); chlorambucil; gemcitabine; 6-thioguanine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; vinca rosea; novantrone; teniposide; daunomycin; aminopterin; capecitabine; ibandronate sodium; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. The definition also includes antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens including, for example, tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing. A detailed description of chemotherapeutic agents can be found, for example, in US20180193477A1, which is incorporated herein by reference in its entirety.

[0260] In some embodiments, the antigen-binding construct is conjugated to a drug via a cleavable linker such as a SPDB linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the antigen-binding construct is conjugated to a drug via a non-cleavable linker such as an MCC linker formed using SMCC or sulfo-SMCC. A person of ordinary skill in the art, having knowledge of the relevant art and considering relevant factors such as the site of attachment to the antigen-binding construct, any structural constraints and hydrophobicity of the drug, can readily select an appropriate linker for a given ADC (see, e.g., Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer). In certain embodiments, many specific linker-toxin combinations have been described and can be used with the antigen-binding constructs described herein to prepare ADCs. Examples include, but are not limited to, cleavable peptidyl linkers having auristatins such as MMAE and MMAF, camptothecins such as SN-38, duocarmycins, and PBD dimers; non-cleavable MC-based linkers having auristatins MMAF and MMAE; acid-labile hydrazone linkers having calicheamicin and doxorubicin; disulfide-based maytansinoid linkers such as DM1 and DM4, and a maytansinoid DM1 linker based on bismaleimido tri(ethylene glycol) (BMPEO). Some therapeutic agents and linkers are described in Peters & Brown, (2015) Biosci. Rep. e00225; Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65; U.S. Patent Publication Nos. US2015 / 0374847 and US20180193477A1; the entire contents of which are incorporated herein by reference in their entirety.

[0261] Depending on the desired drug and the linker selected, one of ordinary skill in the art can select a suitable method to conjugate them together. For example, some conventional conjugation methods, such as amine conjugation methods, can be used to form the desired drug-linker complex, which still contains a reactive group that can be conjugated to the antibody via a covalent bond. In some embodiments, a drug-maleimide complex (i.e., a maleimide-linked drug) can be used as the payload with a reactive group in the context of the present invention. The most common reactive group capable of binding to a thiol group in ADC preparation is maleimide. In addition, organic bromides and iodides are also often used.

[0262] The preparation of ADCs can be achieved by one of several approaches known in the art, using organic chemical reactions, conditions, and reagents known to those skilled in the art. See Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press) for details. For example, conjugation can be achieved in the following ways: (1) a nucleophilic or electrophilic group of the antibody reacts with a divalent linker reagent to form an antibody-linker intermediate Ab-L through a covalent bond, and then reacts with an activated drug moiety D; or (2) a nucleophilic or electrophilic group of the drug moiety reacts with a linking reagent to form a drug-linker intermediate D-L through a covalent bond, and then reacts with a nucleophilic or electrophilic group of the antibody. Conjugation methods (1) and (2) can be used with a variety of antibodies, drug moieties, and linkers to prepare the ADCs described herein. The various linkers, linker components, and toxins prepared are commercially available or can be prepared using standard synthetic organic chemistry techniques. These methods are described in March’s Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press); US20210379193A1 and US20180193477A1, the entire contents of which are incorporated herein by reference in their entirety. In addition, many preformed drug-linkers suitable for reacting with the selected antigen-binding construct are also commercially available. For example, linker toxins containing DM1, DM4, MMAE, MMAF, or Duocarmycin SA are available from Creative BioLabs (Shirley, N.Y.).

[0263] Several specific examples of ADC preparation methods are known in the art and are described in U.S. Patent No. 4,070,007. U.S. Patent No. 8,624,003 (one-pot method), U.S. Patent No. 8,163,888 (one-step method), and U.S. Patent No. 8,163,888 (one-step method), U.S. Patent No. 5,208,020 (two-step method), and US20180193477A1, the entire contents of which are incorporated herein by reference in their entirety. Other methods are known in the art and include Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer.

[0264] The drug loading is expressed as the number of drug moieties per antibody in the ADC molecule. For some antibody-drug conjugates, the drug loading may be limited by the number of attachment sites on the antibody. For example, when the linker is cysteine thiol, in accordance with certain exemplary embodiments described herein, the drug loading can range from 0 to 8 drug moieties per antibody. In certain embodiments, higher drug loadings, such as p≥5, may result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody-drug conjugates. In certain embodiments, the average drug loading of the antibody-drug conjugate ranges from 1 to about 8; about 2 to about 6; or about 3 to about 5. In fact, it has been shown that for certain antibody-drug conjugates, the optimal ratio of drug moieties per antibody is about 4. In some embodiments, the drug-to-antibody ratio (DAR) is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 3 to about 5, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0265] Antibody and ADC Characteristics

[0266] The antibody or antigen-binding fragment thereof or the ADC derived therefrom as described herein can be an agonist or antagonist. In some embodiments, by binding to CD79b, the antibody can inhibit CD79b-mediated BCR signaling.

[0267] In some embodiments, the antibody (or antigen-binding fragment thereof) or the ADC derived therefrom specifically binds to CD79b (e.g., human CD79b, monkey CD79b (such as rhesus macaque, cynomolgus macaque), dog CD79b, mouse CD79b) with a dissociation rate. The dissociation rate (koff) is less than 0.1 s-1, less than 0.01 s-1, less than 0.001 s-1, less than 0.0001 s-1, less than 0.00001 s-1, less than 0.000001 s-1, or less than 0.0000001 s-1. In some embodiments, the dissociation rate (koff) is greater than 0.01 s-1, greater than 0.001 s-1, greater than 0.0001 s-1, greater than 0.00001 s-1, greater than 0.000001 s-1, greater than 0.0000001 s-1, or greater than 0.00000001 s-1.

[0268] In some embodiments, the kinetic association rate (kon) is greater than 1×102 / Ms, greater than 1×103 / Ms, greater than 1×104 / Ms, greater than 1×105 / Ms, or greater than 1×106 / Ms. In some embodiments, the kinetic association rate (kon) is less than 1×105 / Ms, less than 1×106 / Ms, or less than 1×107 / Ms. In some embodiments, KD is less than 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1×10-7 M, greater than 1×10-8 M, greater than 1×10-9 M, greater than 1×10-10 M, greater than 1×10-11 M, greater than 1×10-12 M, greater than 1×10-13 M, greater than 1×10-14 M.

[0269] General techniques for measuring the affinity of an antibody for an antigen include, for example, BLI, ELISA, RIA, flow cytometry, and surface plasmon resonance (SPR). In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein bind to human CD79b, monkey CD79b, dog CD79b, and / or mouse CD79b. In some embodiments, the antibody does not bind to human CD79b, monkey CD79b, dog CD79b, and / or mouse CD79b.

[0270] In addition, alternative splicing isoforms of CD79b in chronic lymphocytic leukemia have been described. The isoform (also referred to as the "short isoform") encodes 125 amino acids (SEQ ID NO: 67) and differs from the wild-type 229 amino acids (also referred to as the "long isoform") (SEQ ID NO: 68) by deletion of exon 3, which essentially encodes most of the extracellular domain. In some embodiments, the anti-CD79b antibodies described herein can bind to both isoforms of CD79b. In some embodiments, the anti-CD79b antibody binds to the CD79b isoform (e.g., SEQ ID NO: 67). In some embodiments, the anti-CD79b antibody does not bind to the CD79b isoform (e.g., SEQ ID NO: 67). In some embodiments, the anti-CD79b antibody binds to wild-type CD79b (e.g., SEQ ID NO: 68). In some embodiments, the antibodies described herein bind to the extracellular domain of CD79b.

[0271] In some embodiments, the anti-CD79b antibody binds to a CD79b variant (e.g., SEQ ID NO: 2). In some embodiments, the anti-CD79b antibody does not bind to a CD79b variant (e.g., SEQ ID NO: 2). In some embodiments, the anti-CD79b antibody binds to an epitope within amino acids 1-13 of the ECD of CD79b (e.g., SEQ ID NO: 1).

[0272] In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein are added to Ramos cells to detect the internalization rate. In some embodiments, at different time points (e.g., 1 hour, 3 hours, or 6 hours), the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein have an internalization rate of more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, or more than 98%. In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein have a slower internalization rate compared to polatuzumab.

[0273] In some embodiments, the thermal stability is determined. The antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein may have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the Tm is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0274] Since IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, namely the first denaturation temperature Tm1 and the second denaturation temperature Tm2. The presence of these two peaks generally indicates the denaturation of the Fc domain (Tm1) and the Fab domain (Tm2), respectively. Thus, in some embodiments, the antibodies or antigen-binding fragments described herein have a Tm1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C. In some embodiments, the antibodies or antigen-binding fragments described herein have a Tm2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0275] In some embodiments, Tm, Tm1, Tm2 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 °C.

[0276] In some embodiments, as detected by ELISA, the antibodies or their antigen-binding fragments or ADCs derived therefrom described herein can bind to human or monkey CD79b. In some embodiments, the antibodies or antigen-binding fragments or ADCs derived therefrom described herein can bind to human or monkey CD79b with an IC50 less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, or less than 0.25 nM.

[0277] In some embodiments, the antibodies or their antigen-binding fragments or ADCs derived therefrom described herein can bind to the same epitope of CD79b. In some embodiments, the antibodies or their antigen-binding fragments or ADCs derived therefrom described herein can bind to different epitopes of CD79b.

[0278] In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein can bind malignant B cell lines (such as BJAB, Ramos, Daudi, SU-DHL-4, and Nalm-6) with high and low CD79b antigen density as determined by flow cytometry. In some embodiments, the EC50 is less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, or less than 0.25 nM.

[0279] In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein can bind human B cells with a higher cell surface binding affinity than Polatuzumab. In some embodiments, the EC50 is less than 200 nM, less than 100 nM, less than 50 nM, less than 10 nM, less than 5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, or less than 0.25 nM.

[0280] In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein can bind cell surface CD79b on B lymphocytes of patients with chronic lymphocytic leukemia (CLL). In some embodiments, the binding is more effective than polatuzumab.

[0281] In some embodiments, the ADCs described herein have an average drug-to-antibody ratio (DAR) higher than 3, higher than 3.2, higher than 3.4, higher than 3.6, higher than 3.8, higher than 4, higher than 4.2, higher than 4.4, or higher than 4.6 as determined by HPLC. In some embodiments, the ADCs described herein have an average DAR lower than 3, lower than 3.2, lower than 3.4, lower than 3.6, lower than 3.8, lower than 4, lower than 4.2, lower than 4.4, or lower than 4.6 as determined by HPLC. In some embodiments, the DAR is about 4.

[0282] In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein have a tumor growth inhibition percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein have a tumor growth inhibition percentage less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be determined 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula:

[0283] TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100

[0284] Ti is the average tumor volume of the treatment group on day i. T0 is the average tumor volume of the treatment group on day 0. Vi is the average tumor volume of the control group on day i. V0 is the average tumor volume of the control group on day 0.

[0285] In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein can bind to tumor cells expressing CD79b. In some embodiments, the antibodies or antigen-binding fragments or ADCs derived therefrom described herein can induce complement-dependent cytotoxicity (CDC) and / or antibody-dependent cell cytotoxicity (ADCC) and kill tumor cells.

[0286] In some embodiments, the antibodies or antigen-binding fragments thereof or ADCs derived therefrom described herein have a functional Fc region. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector function of the functional Fc region is ADCC and phagocytosis.

[0287] In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the antibody is a humanized IgG1 antibody, optionally having SI mutations, LALA mutations, N297A mutations, YTE mutations and / or FLAA mutations. In some embodiments, the antibody is a humanized IgG4 antibody, optionally having SI mutations, LALA mutations, N297A mutations, YTE mutations and / or FLAA mutations.

[0288] In some embodiments, the antibody or antigen-binding fragment or ADC derived therefrom described herein does not have a functional Fc region. For example, the antibody or antigen-binding fragment is a Fab, Fab', F(ab')2 and Fv fragment. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations according to EU numbering) or LALA-PG mutations (L234A, L235A, P329G mutations according to EU numbering). In some embodiments, the Fc region has FLAA mutations (F234A and L235A according to EU numbering). In some embodiments, Fc has SI mutations (S239D and I332E mutations according to EU numbering). In some embodiments, Fc has an N297A mutation according to EU numbering. In some embodiments, Fc has YTE mutations (M252Y, S254T and T256E according to EU numbering).

[0289] Method for preparing anti-CD79b antibody

[0290] Isolated fragments of human CD79b can be used as immunogens to generate antibodies using standard techniques for preparing polyclonal and monoclonal antibodies. Polyclonal antibodies can be generated in animals by multiple injections (e.g., subcutaneous or intraperitoneal injection) of the antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected together with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to a reagent that is immunogenic in the species to be immunized. The animal can be injected with the antigenic peptide or protein more than once (e.g., two, three or four times).

[0291] A full-length polypeptide or protein (or its extracellular domain) can be used, or, an antigenic peptide fragment thereof can be used as an immunogen. The antigenic peptide of the protein comprises at least 8 (e.g., at least 10, 15, 20 or 30) amino acid residues of the amino acid sequence of CD79b and comprises an epitope of the protein such that an antibody formed against the peptide forms a specific immune complex with the protein. As described above, the full-length sequence of human CD79b is known in the art. In some embodiments, an Fc-tagged or His-tagged human CD79b protein is used as an immunogen. In some embodiments, the extracellular domain (ECD) of human CD79b is used as an immunogen.

[0292] Immunogens are typically used to prepare antibodies by immunizing a suitable subject (e.g., a human or transgenic animal expressing at least one human immunoglobulin locus). Suitable immunogenic preparations can comprise, for example, recombinantly expressed or chemically synthesized polypeptides (e.g., fragments of human CD79b). The preparations can also include adjuvants, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agents.

[0293] Polyclonal antibodies are prepared according to the above by immunizing a suitable subject with a CD79b polypeptide or its antigenic peptide (such as a portion of CD79b) as an immunogen. The antibody titer of the immunized subject can be monitored over time by standard techniques, such as an enzyme-linked immunosorbent assay (ELISA) using immobilized CD79b polypeptide or peptide. If desired, antibody molecules can be isolated from a mammal (such as from blood) and further purified by well-known techniques, such as protein A or protein G chromatography, to obtain the IgG fraction. At an appropriate time after immunization, for example, when the specific antibody titer is highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985) or trioma technique. Techniques for generating hybridomas are well known (generally see Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, NY). Hybridoma cells producing monoclonal antibodies are detected by screening the hybridoma culture supernatant for antibodies that bind to the polypeptide or epitope of interest, for example, using a standard ELISA assay.

[0294] Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody or antigen-binding fragment described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence that constitutes the antigen-binding site or antigen-binding domain of the antibody. Among such variant populations, some antibodies or antigen-binding fragments will have increased affinity for a target protein (such as CD79b). Any combination of deletions, insertions, and / or combinations can be made to obtain an antibody or its antigen-binding fragment with increased binding affinity for the target. Amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications to the antibody or antigen-binding fragment, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation site (e.g., altering the amino acid sequence such that a different sugar is attached by an enzyme present in the cell), or introducing new glycosylation sites.

[0295] The antibodies described herein can be derived from any animal species, including mammals. Non-limiting examples of native antibodies include those derived from humans, primates (such as monkeys and apes), rabbits, cows, pigs, horses, sheep, camelids (such as camels and llamas), chickens, goats, and rodents (such as rats, mice, hamsters, and guinea pigs), including transgenic animals that have been genetically engineered to produce human antibodies.

[0296] Human antibodies and humanized antibodies include antibodies having variable and constant regions that are derived from human germline immunoglobulin sequences (or having the same amino acid sequences as those derived therefrom). A human antibody can include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by in vitro random or site-directed mutagenesis or by in vivo somatic mutation), such as in the CDRs.

[0297] Humanized antibodies typically have a human framework (FR) grafted with non-human CDRs. Thus, a humanized antibody has one or more amino acid sequences introduced therein from non-human sources. These non-human amino acid residues are commonly referred to as "import" residues and are typically taken from "import" variable domains. Humanization can essentially be carried out, for example, by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. These methods are described in, for example, Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); each of which is incorporated herein by reference in its entirety. Thus, a "humanized" antibody is a chimeric antibody in which substantially less than the entire human V domain has been replaced by the corresponding sequences from a non-human species. In fact, a humanized antibody is typically a non-human antibody in which some CDR residues and some FR residues have been replaced by residues at similar sites in a human antibody.

[0298] The selection of human VH and VL domains for use in preparing humanized antibodies is very important for reducing immunogenicity. According to the so-called "best-fit" method, the V domain sequences of non-human antibodies are screened against a library of known human domain sequences. Then, the human sequence that is closest to the non-human animal sequence is accepted as the human FR for the humanized antibody (Jones et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).

[0299] More importantly, the antibody is humanized while retaining high specificity and affinity for the antigen as well as other favorable biological properties. To achieve this goal, humanized antibodies can be prepared by using three-dimensional models of the parental and humanized sequences to analyze the parental sequence and the process of various conceptual humanized products. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs can be used to illustrate and display the possible three-dimensional conformational structures of the selected candidate immunoglobulin sequences. Examination of these displays can analyze the possible role of residues in the function of the candidate immunoglobulin sequence, i.e., analyze the residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the acceptor and import sequences to achieve the desired antibody characteristics, such as increased affinity for the target antigen.

[0300] Typically, amino acid sequence variants of human, humanized, or chimeric anti-CD79b antibodies will comprise an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence present in the light or heavy chain of the original antibody.

[0301] Identity or homology to the original sequence is generally the percentage of amino acid residues in the candidate sequence that are identical to the sequence present in a human, humanized, or chimeric anti-CD79b antibody or fragment, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity.

[0302] Other modifications can be made to the anti-CD79b antibody or antigen-binding fragment. For example, cysteine residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The resulting homodimeric antibody can have any increased in vitro and / or in vivo half-life. Homodimeric antibodies with an extended in vitro and / or in vivo half-life can also be prepared using heterobifunctional crosslinking agents, e.g., as described by Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, antibodies with a dual Fc region can be designed (see, e.g., Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0303] In some embodiments, the anti-CD79b antibody or its antigen-binding fragment can be covalently modified. These covalent modifications can be carried out by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting the target amino acid residue of the antibody or fragment with an organic derivatization reagent capable of reacting with the selected side chain or N- or C-terminal residue.

[0304] In some embodiments, the provided antibody variants have a carbohydrate structure lacking fucose (directly or indirectly) linked to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the Asn297 glycan, relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn 297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (Eu numbering of Fc region residues; or position 314 in Kabat numbering); however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0305] In some embodiments, to avoid Fab arm exchange to improve production efficiency, the Fc region of the antibody is further engineered to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P). A detailed description of the S228 mutation is provided, for example, in Silva et al. "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation." Journal of Biological Chemistry 290.9 (2015):5462-5469, which is incorporated herein by reference in its entirety.

[0306] Recombinant vector

[0307] The present invention also provides recombinant vectors (e.g., expression vectors) that include the isolated polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cells contain the polynucleotide and / or the vector containing the polynucleotide), and recombinant antibody polypeptides or fragments thereof produced by recombinant techniques.

[0308] As used herein, a "vector" is any construct that is capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by operably linking it to regulatory elements (such as a promoter, enhancer, and / or polyadenylation tail) within the vector or in the genome. The host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell into which the expression vector has been introduced.

[0309] As used herein, a "vector" is any construct that is capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by operably linking it to regulatory elements (such as a promoter, enhancer, and / or polyadenylation tail) within the vector or in the genome. The host cell at or near or flanking the integration site of the polynucleotide of interest such that the polynucleotide of interest will be translated in the host cell into which the expression vector has been introduced.

[0310] A vector can be introduced into a host cell by methods known in the art, such as electroporation, chemical transfection (such as DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with a recombinant virus). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors complexed with cationic condensing agents.

[0311] In some embodiments, viral expression systems (e.g., vaccinia virus or other poxviruses, retroviruses, or adenoviruses) are used to introduce the polynucleotides described herein (e.g., polynucleotides encoding the polypeptides described herein), which may involve the use of non-pathogenic (defective), replication-competent viruses, or may involve the use of replication-defective viruses. In the latter case, viral propagation generally occurs only in complementary virus packaging cells. Suitable systems are described in Fisher-Hoch et al., 1989, Proc. Natl. Acad. Sci. USA 86:317-321; Flexner et al., 1989, Ann. N.Y. Acad Sci. 569:86-103; Flexner et al., 1990, Vaccine, 8:17-21; U.S. Pat. NO. 4,603,112, 4,769,330, and 5,017,487; WO 89 / 01973; U.S. Pat. No. 4,777,127; GB 2,200,651; EP 0,345,242; WO 91 / 02805; Berkovich-Biotechniques, 6:616-627, 1988; Rosenfeld et al., 1991, Science, 252:431-434; Kolls et al., 1994, Proc. Natl. Acad. Sci. USA, 91:215-219; Kass-Eisler et al., 1993, Proc. Natl. Acad. Sci. USA, 90:11498-11502; Guzman et al., 1993, Circulation, 88:2838-2848; and Guzman et al., 1993, Cir. Res., 73:1202-1207. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. DNA can also be "naked", as described in Ulmer et al., 1993, Science, 259:1745-1749, and Cohen, 1993, Science, 259:1691-1692. Uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are capable of efficient transport into cells.

[0312] For expression, the DNA insert containing the polynucleotide encoding the antibody or polypeptide described herein can be operably linked to a suitable promoter, such as a heterologous promoter, for example, the bacteriophage λPL promoter, the E. coli lac, trp, and tac promoters, the SV40 early and late promoters, and the promoters of retroviral LTRs, etc. Other suitable promoters are known to those skilled in the art. The expression construct can further include sites for transcription initiation, termination, and ribosome binding sites for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct can include translation initiation and termination codons (UAA, UGA, or UAG) starting at the end of the polypeptide to be translated.

[0313] As indicated, the expression vector can include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells; insect cells such as Drosophila S2, Spodoptera frugiperda Sf9, etc.; animal cells such as CHO, COS, Bowes melanoma, HK293 cells, etc.; and plant cells. Media and conditions suitable for the host cells described herein are known in the art.

[0314] Non-limiting vectors for bacteria include pQE70, pQE60, and pQE-9, available from Qiagen; pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, available from Stratagene; pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. Other suitable vectors will be apparent to those skilled in the art.

[0315] Suitable non-limiting bacterial promoters include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, λPR and PL promoters, and trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs such as the promoter of Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0316] In Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters can be used, such as alpha factor, alcohol oxidase, and PGH. For relevant descriptions, see Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, and Grant et al., Methods Enzymol., 153:516 - 544 (1997).

[0317] The construct can be introduced into the host cell by calcium phosphate transfection, DEAE - dextran - mediated transfection, cationic lipid - mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as in Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

[0318] Transcription of DNA encoding the antibodies of the present invention in higher eukaryotes can be increased by inserting enhancer sequences into the vector. Enhancers are cis - acting elements of DNA, usually about 10 to 300 bp in length, which function to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer, which is located late at 100 to 270 base pairs from the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer late at the origin of replication, and the adenovirus enhancer.

[0319] To secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signals can be endogenous signals of the polypeptide or they can be heterologous signals.

[0320] Polypeptides (such as antibodies) can be expressed in a modified form, such as a fusion protein (such as a GST - fusion protein) or with a histidine tag, and can include not only secretion signals but also additional heterologous functional regions. For example, an additional amino acid region, especially a charged amino acid, can be added to the N - terminus of the polypeptide to enhance its stability and persistence in the host cell during purification or subsequent processing and storage. In addition, a peptide moiety can be added to the polypeptide to facilitate purification. These regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides to effect secretion or excretion, enhance stability, and facilitate purification, etc. are well - known and conventional techniques in the art.

[0321] Therapeutic methods

[0322] The antibodies or antigen-binding fragments thereof of the present disclosure can be used for various therapeutic purposes.

[0323] In one aspect, the present disclosure provides a method of treating cancer in a subject, a method of reducing the rate of increase in tumor volume over time in a subject, a method of reducing the risk of metastasis, or a method of reducing metastasis. The risk of additional metastasis in a subject. In some embodiments, treatment can halt, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0324] In one aspect, the present disclosure features a method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein to a subject in need thereof (such as a subject having or identified or diagnosed as having cancer), e.g., breast cancer (such as triple-negative breast cancer), carcinoid, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancies. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the cancer is NSCLC, ovarian cancer, melanoma, colorectal cancer, breast cancer, hematological malignancy, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer. In some embodiments, the subject has Hodgkin lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial cancer, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematological malignancy, particularly non-Hodgkin lymphoma, lymphoma, chronic lymphocytic leukemia, or advanced solid tumors. In some embodiments, the cancer is lymphoma, breast cancer, gastric cancer, pancreatic cancer, prostate cancer, cervical cancer, endometrial cancer, ovarian cancer, or urothelial cancer.

[0325] In some embodiments, the compositions and methods described herein can be used to treat patients at risk of cancer. Patients having cancer can be identified by various methods known in the art.

[0326] On the one hand, the present disclosure provides methods for treating, preventing, or reducing the risk of developing a disorder associated with an abnormal or unwanted immune response, such as an autoimmune disorder. These autoimmune diseases include, but are not limited to, alopecia areata, lupus, ankylosing spondylitis, Meniere's disease, antiphospholipid syndrome, mixed connective tissue disease, autoimmune Addison's disease, multiple sclerosis, autoimmune hemolytic anemia, myasthenia gravis, autoimmune hepatitis, pemphigus vulgaris, Behcet's disease, pernicious anemia, bullous pemphigoid, nodular polyarthritis, cardiomyopathy, polychondritis, celiac sprue, polyglandular syndrome, chronic fatigue syndrome (CFIDS), polymyalgia rheumatica, chronic inflammatory demyelination, polymyositis and dermatomyositis, chronic inflammatory polyneuropathy, essential gammopathy, Churg-Strauss syndrome, primary biliary cirrhosis, cicatricial pemphigoid, psoriasis, CREST syndrome, Raynaud's phenomenon, cold agglutinin disease, Reiter's syndrome, Crohn's disease, rheumatic fever, discoid lupus, rheumatoid arthritis, cryoglobulinemia, sarcoidosis, fibromyalgia, scleroderma, Grave's disease, syndrome, Guillain-Barre syndrome, stiff-man syndrome, Hashimoto's thyroiditis, temporal arteritis, idiopathic pulmonary fibrosis, temporal arteritis / giant cell arteritis, idiopathic thrombocytopenic purpura (ITP), ulcerative colitis, IgA nephropathy, uveitis, diabetes (such as type I), vasculitis, lichen planus, and vitiligo. An anti-CD79b antibody or an antigen-binding fragment thereof can also be administered to a subject to treat, prevent, or reduce the risk of developing a disorder associated with an abnormal or unwanted immune response related to cell, tissue, or organ transplantation, such as kidney transplantation, liver, and heart transplantation, such as graft-versus-host disease (GVHD), or to prevent allograft rejection. In some embodiments, the subject has a skin disease, a liver disease (e.g., cirrhosis), hidradenitis suppurativa, experimental autoimmune encephalomyelitis. In some embodiments, the subject has a kidney disease, lupus, Sjogren's syndrome, ulcerative colitis, psoriasis, hidradenitis suppurativa, immune thrombocytopenia (ITP), or other inflammatory arthritis. In some embodiments, the subject has multiple sclerosis or myasthenia gravis. In some embodiments, the subject has Crohn's disease, ulcerative colitis, or type 1 diabetes. In some embodiments, the subject has an autoimmune thyroid disease, Grave's disease, multiple sclerosis, psoriasis, inflammatory bowel disease (e.g., Crohn's disease (CD) and ulcerative colitis), rheumatoid arthritis, Sjogren's syndrome, autoimmune nephritis, or systemic lupus erythematosus. In some embodiments, the method involves administering to the subject an effective amount of a composition comprising an antibody or an antigen-binding fragment thereof as described herein.

[0327] As used herein, "effective amount" refers to an amount or dose sufficient to achieve a beneficial or desired result, including halting, slowing, retarding or inhibiting the progression of a disease such as an autoimmune disease or cancer. The effective amount will vary depending on, for example, the age and weight of the subject to which the antibody, antigen-binding fragment, antibody-encoding polynucleotide, vector comprising the polynucleotide and / or a composition thereof is to be administered, the severity of the symptoms and the route of administration, and thus the dosing can be determined on an individual basis.

[0328] The effective amount can be administered in one or more administrations. For example, an effective amount of an antibody or antigen-binding fragment is an amount sufficient to ameliorate, halt, stabilize, reverse, inhibit, slow down and / or delay the progression of an autoimmune disease or cancer in a patient, or is an amount sufficient to ameliorate, halt, stabilize, reverse, slow down and / or delay the proliferation of cells (e.g., biopsy cells, any cancer cells or cell lines described herein (e.g., cancer cell lines)) in vitro. As will be understood in the art, the effective amount of an antibody or antigen-binding fragment can vary, particularly depending on the patient history and other factors such as the type (and / or dose) of the antibody used.

[0329] The effective amount and schedule for administering the antibodies, polynucleotides encoding antibodies and / or compositions described herein can be determined empirically, and making such determinations is within the skill of the art. Those skilled in the art will understand that the dose to be administered will vary depending on, for example, the mammal to which the antibodies, polynucleotides encoding antibodies and / or compositions described herein are to be administered, the route of administration, the particular type of combination of the antibodies, polynucleotides encoding antibodies, antigen-binding fragments and / or compositions described herein and other drugs being administered to the mammal. Guidance for selecting an appropriate dose for an antibody or antigen-binding fragment can be found in the literature on the therapeutic use of antibodies and antigen-binding fragments, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., 1985, ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York, 1977, pp. 365-389.

[0330] The typical daily dose of the effective amount of the antibody is from 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose can be less than 100 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg or 0.1 mg / kg. In some embodiments, the dose can be greater than 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, 0.1 mg / kg, 0.05 mg / kg or 0.01 mg / kg. In some embodiments, the dose is about 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg or 0.1 mg / kg.

[0331] In any of the methods described herein, at least one antibody, an antigen-binding fragment thereof or a pharmaceutical composition (such as any antibody, antigen-binding fragment or pharmaceutical composition described herein) and optionally at least one additional therapeutic agent can be administered to a subject at least once a week (such as once a week, twice a week, three times a week, four times a week, once a day, twice a day or three times a day). In some embodiments, at least two different antibodies and / or antigen-binding fragments are administered in the same composition (such as a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in the same composition (such as a liquid composition). In some embodiments, at least one antibody or antigen-binding fragment and at least one additional therapeutic agent are administered in two different compositions (such as a liquid composition containing at least one antibody or antigen-binding fragment and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a pill, tablet or capsule. In some embodiments, at least one additional therapeutic agent is administered in a sustained-release oral formulation.

[0332] In some embodiments, one or more additional therapeutic agents (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein) can be administered to a subject before or after administering at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein). In some embodiments, the biological activity periods of one or more additional therapeutic agents and at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein) overlap when administered to a subject.

[0333] In some embodiments, at least one antibody, antigen-binding antibody fragment, or pharmaceutical composition (e.g., any antibody, antigen-binding antibody fragment, or pharmaceutical composition described herein) can be administered to a subject over an extended period of time, such as over a period of at least 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, or 5 years. A skilled medical professional can use any method described herein to determine the length of the treatment period to diagnose or track the effectiveness of the treatment (e.g., observing at least one cancer symptom). As described herein, a skilled medical professional can also vary the identity and amount (e.g., increase or decrease) of the antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) administered to the subject, and can also adjust (e.g., increase or decrease) the dose or frequency of administration of at least one antibody or antigen-binding antibody fragment (and / or one or more additional therapeutic agents) to the subject based on an assessment of the treatment effectiveness (e.g., increase or decrease) using any method described herein and known in the art.

[0334] In some embodiments, one or more additional therapeutic agents can be administered to a subject. The additional therapeutic agents can include one or more inhibitors selected from the group consisting of: a B-Raf inhibitor, an EGFR inhibitor, a MEK inhibitor, an ERK inhibitor, a K-Ras inhibitor, a c-Ras inhibitor, Met, an anaplastic lymphoma kinase (ALK) inhibitor, a phosphatidylinositol 3-kinase (PI3K) inhibitor, an Akt inhibitor, an mTOR inhibitor, a dual PI3K / mTOR inhibitor, a Bruton's tyrosine kinase (BTK) inhibitor, and an inhibitor of isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2). In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

[0335] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of: PD-1 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, agents that activate the Hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

[0336] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of: trabectedin, nab-paclitaxel, trebananib, pazopanib, cediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, vinflunine, an Hsp90 inhibitor, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0337] In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF)α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapies targeting CX3CL1, therapies targeting CXCL9, therapies targeting CXCL10, therapies targeting CCL5, LFA-1 agonists, ICAM1 agonists, and PD-1 agonists.

[0338] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI is administered to a subject.

[0339] In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L2 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA4 antibody, an anti-ICOS antibody, an anti-CD27 antibody, an anti-4-1BB antibody, an anti-CD40 antibody, an anti-VEGFR2 antibody, an anti-EGFR antibody, an anti-HER2 antibody, a TIM3 antibody, a CD103 antibody, a TGFBR2 antibody, and / or an anti-GITR antibody.

[0340] On the one hand, the present disclosure provides combination therapies. In some embodiments, an anti-CD79b antibody or an antigen-binding fragment thereof (any antibody as described herein) can be administered together with an anti-CTLA4 antibody.

[0341] Pharmaceutical Compositions and Routes of Administration

[0342] The present disclosure also provides pharmaceutical compositions that comprise at least one (such as one, two, three, or four) antibody or antigen-binding fragment as described herein. Two or more (such as two, three, or four) of any antibody or antigen-binding fragment as described herein can be present in the pharmaceutical composition in any combination. The pharmaceutical composition can be formulated in any manner known in the art.

[0343] The pharmaceutical composition is formulated to be compatible with its intended route of administration (such as intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition can include sterile diluents (such as sterile water or saline), fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents, such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc., antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid, buffers, such as acetate, citrate, or phosphate, and isotonic agents, such as sugars (such as glucose), polyols (such as mannitol or sorbitol), or salts (such as sodium chloride) or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers (see, e.g., U.S. Patent No. 4,522,811). The composition can be formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. When needed (such as in injectable formulations), appropriate fluidity can be maintained by using coatings, such as lecithin or surfactants. Absorption of the antibody or its antigen-binding fragment can be prolonged by including agents that delay absorption, such as aluminum monostearate and gelatin. Alternatively, controlled release can be achieved by implants and microencapsulation delivery systems, which can include biodegradable biocompatible polymers (such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid; Alza Corporation and Nova Pharmaceutical, Inc.).

[0344] Compositions containing one or more of any antibody or antigen-binding fragment as described herein can be formulated in unit dosage form (i.e., physically discrete units containing a predetermined quantity of the active compound, suitable for ease of administration and uniform dosing).

[0345] The pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic and is manufactured under Good Manufacturing Practice (GMP) conditions. The pharmaceutical composition can be provided in unit dose form (i.e., the dose for a single administration). The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients or adjuvants. The formulation depends on the chosen route of administration. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer to reduce discomfort at the injection site. The solution can contain formulating agents such as suspending agents, stabilizers and / or dispersing agents. Alternatively, the antibody can be in lyophilized form for reconstitution with a suitable carrier (such as sterile pyrogen-free water) before use.

[0346] The toxicity and therapeutic efficacy of the composition can be determined by standard pharmacological procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents with a high therapeutic index are preferably exhibited. When a drug exhibits adverse side effects, care should be taken to minimize potential damage (i.e., reduce adverse side effects). Toxicity and therapeutic effects can be determined by other standard pharmaceutical procedures.

[0347] Data obtained from cell culture assays and animal studies can be used to formulate an appropriate dose of any given agent for a subject (such as a human). A therapeutically effective amount of one or more (such as one, two, three or four) antibodies or antigen-binding fragments thereof (such as any antibody or antibody fragment described herein) will be an amount that treats a disease in a subject (such as killing cancer cells) in a subject (such as a human subject identified as having cancer), or a subject identified as being at risk of developing a disease (such as a human who has previously had cancer but is now cured), reducing the severity, frequency and / or duration of one or more disease symptoms in a subject (such as a human). The effectiveness and dose of any antibody or antigen-binding fragment described herein can be determined by a healthcare professional or a veterinary professional using methods known in the art and by observing one or more disease symptoms in a subject (such as a human). Certain factors may affect the dose and time required to effectively treat a subject (such as the severity of the disease or condition, previous treatments, the general health status and / or age of the subject, and the presence of other diseases).

[0348] Exemplary doses include milligram or microgram amounts per kilogram of subject body weight of any antibody or antigen-binding fragment described herein (e.g., from about 1 μg / kg to about 500 mg / kg; from about 100 μg / kg to about 500 mg / kg; from about 100 μg / kg to about 50 mg / kg; from about 10 μg / kg to about 5 mg / kg; from about 10 μg / kg to about 0.5 mg / kg; or from about 1 μg / kg to about 50 μg / kg). While these doses cover a wide range, one of ordinary skill in the art will understand that the potencies of therapeutic agents, including antibodies and their antigen-binding fragments, vary and that an effective amount can be determined by methods known in the art. Generally, a relatively low dose is administered first, and the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still in the development stage) can subsequently increase the dose gradually until an appropriate response is obtained. Additionally, it is understood that the specific dose level for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, the age, weight, general health, sex and diet of the subject, the mode of administration, the route of administration, the rate of excretion, and the half-life of the antibody or antibody fragment in the body.

[0349] The pharmaceutical composition can be contained in a container, package, or dispenser together with instructions for administration. The present disclosure also provides methods of making antibodies or their antigen-binding fragments for the various uses described herein.

[0350] Example

[0351] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure as described in the claims.

[0352] Example 1. Generation of Anti - CD79b Antibody

[0353] Immunize rabbits with CD79b antigen

[0354] To generate monoclonal antibodies against human CD79b, two New Zealand white rabbits were immunized with human CD79b ECD antigen (SEQ ID NO: 1, aa. 29 - 159, with his tag, from AcroBiosystems) using complete Freund's adjuvant or incomplete Freund's adjuvant. Serum titers were monitored by ELISA after the second injection.

[0355] Screen individual B cells that produce CD79b monoclonal antibodies

[0356] After 3 - 4 immunizations at 200 μg / rabbit / injection with a good titer (>1:100,000), rabbits were given a final boost injection and spleens were harvested 7 days after the last injection.

[0357] Isolate splenocytes from rabbits

[0358] All of the following procedures (except centrifugation) are carried out in a biosafety cabinet. Harvest rabbit spleens 7 days after the last booster injection. Prepare splenocytes in a sterile cell strainer placed on the bottom of a 100 mm sterile Petri dish containing 20 mL RPMI + 1% penicillin - streptomycin (P / S). Using sterile forceps, transfer the spleen tissue to the cell strainer. Specifically, while holding the spleen with forceps, cut the spleen into small pieces and then press it through the mesh of the cell strainer. Wash the tissue fragments with 10 mL RPMI + 1% P / S. Transfer the splenocytes from the Petri dish to a new 50 mL conical tube. Add RPMI + 1% P / S to a final volume of 50 ml. Centrifuge the cells at 400×g for 5 minutes and aspirate the supernatant. Add 13 mL ACK buffer (Gibco catalog number A1049201) to resuspend the cells. Incubate the suspended cells at room temperature for 1 minute. Add RPMI + 1% P / S to reach a final volume of 50 ml. Centrifuge the cells at 400×g for 5 minutes and aspirate the supernatant. Resuspend the cell pellet in RPMI + 10% FBS + 1% P / S. Centrifuge the cells at 400×g for 5 minutes and aspirate the supernatant. Resuspend the pellet in 15 ml RPMI + 10% FBS + 1% P / S. Pass the cells through a 100 μm cell strainer into a 50 mL conical tube to remove cell clumps. Then seed the splenocytes at the desired density (e.g., 4E6 cells / ml) with an appropriate medium for sorting. The remaining splenocytes are frozen overnight at -80 °C in 90% serum + 10% DMSO at 6×10 7 cells / vial (~1.8 ml). Transfer the frozen cells to a liquid nitrogen tank for long - term storage.

[0359] Antigen - specific B cell sorting

[0360] For B cell sorting, culture in B cell medium (RPMI-1640, 15% FBS, 1×HEPES, 1×2-ME (2-mercaptoethanol), 1% penicillin / streptomycin) overnight before sorting. Prepare 96-well B cell feeder plates accordingly one day before sorting. On the day of sorting, collect suspended and loosely attached splenocytes by gently pipetting the medium against the culture surface of the flask. Then transfer the cells to a conical tube and centrifuge at 400×g for 3 minutes. Wash the cell pellet twice with fluorescence-activated cell sorting (FACS) buffer (1×PBS + 0.5% BSA). Add biotinylated antigen at 5 μg / ml (final concentration). Incubate the mixture at room temperature (RT) for 20 minutes. Then centrifuge the staining mixture at 400×g for 3 minutes and resuspend the cells in FACS buffer. Transfer the cells to a 1.5 ml amber Eppendorf tube. Then add the stained antibody mixture to the cells. Incubate the staining mixture at 4°C for 15 - 30 minutes, then centrifuge at 400×g for 3 minutes. Wash the cell pellet twice with FACS buffer. Resuspend the washed cell pellet in 1×PBS + 1% FBS at a concentration of approximately 10 7 cells / ml. Using fluorescence-activated cell sorting, sort antigen-specific single B cells into 96-well plates (20 plates per rabbit). Incubate the 96-well B cell culture plates with sorted B cells at 37°C, 5% CO 2 2 for 12 days.

[0361] Screening of single B cell cultures

[0362] On day 8 after sorting, collect 15 μL of B cell culture supernatant from each well for antigen-specific ELISA. Briefly, transfer the B cell culture supernatant to the coated extracellular domain (ECD) of CD79b, then block the 384-well plate. Incubate the cells at room temperature for 1 hour and wash 3 times with PBS plus 0.05% Tween-20. Detect the secreted antibody using goat anti-rabbit IgG HRP + TMB substrate. Then select B cell supernatants that meet the OD450 cut-off value (>0.5 or 3 times the pre-immune serum). Using FACS, back-screen the selected cells (1) for CD79b variants with N-terminal deletions (delete the first 13 amino acid residues from the N-terminus, SEQ ID NO: 2); (2) perform cross-species binding screening against rhesus CD79b (SEQ ID NO: 3), and (3) perform cell surface binding screening against Daudi cells.

[0363] Perform an initial ELISA screen on the supernatants of sorted B cells from 40 plates (96-well plates) to screen for CD79b-specific antibodies. Approximately 230 CD79b antigen-specific positive B cell clones were identified.

[0364] On day 12 after sorting, centrifuge the B cell culture plates at 400 × g for 3 minutes. Collect the supernatants from the positive clones (OD greater than the selected cut-off value for antigen-specific ELISA), and store the cell pellet in 100 μL DNA / RNA shield (Zymo Cat#R1100-250) in a 250 μL PCR tube. The collected supernatants are subjected to additional tests as described below. Screen for CD79b-specific antibodies that bind to CD79b expressed on the cell surface

[0365] CD79b is a member of the B cell receptor complex. Due to epitope accessibility, it is expected that not all antibody clones that bind CD79b in ELISA will also bind CD79b expressed on the cell surface. Perform a cell surface binding assay with Daudi cells for the CD79b-specific B cell clones identified by ELISA by FACS. Briefly, incubate 50 μl of the B cell supernatant (diluted 1:10 in FACS buffer, 1x PBS + 0.5% BSA) with 50,000 Daudi cells (seeded in a 96-well plate) on ice for 1 hour, then wash with 150 μl of ice-cold FACS buffer. Add 100 μl of anti-rabbit-PE secondary antibody (Biolegend), mix and incubate on ice for 30 minutes. Wash the cells 3 times with 150 μl of FACS buffer and finally resuspend in 100 μl of FACS buffer. 5,000 cells from each staining are collected in a flow cytometer for analysis. Among approximately 230 ELISA-positive B cell clones, 12 clones showed binding to CD79b on the cell surface.

[0366] Obtain the VH and VL gene sequences from the selected anti-CD79b clones

[0367] DNA fragments encoding the heavy chain variable domain (VH) and light chain variable domain (VL) from the B cell clones showed high cell surface binding affinity for CD79b by 5' RACE (rapid amplification of cDNA ends), TOPO cloning, and sequencing.

[0368] Example 2. Generation of Chimeric Expression Construct

[0369] To express the rabbit / human chimeric IgG1 antibody, the human IgG1 heavy chain constant region (CH1-CH3, SEQ ID NO: 4) and the human kappa light chain constant region (CL-kappa, SEQ ID NO: 5) were separately synthesized and cloned into the pcDNA3.4 vector (Invitrogen). The heavy chain cloning vector pcDNA3.4-huIgG1-Hc was digested with EcoRI / NheI for the cloning of the VH fragment. The light chain cloning vector pcDNA3.4-huKappa-Lc was digested with EcoRI / BsiWI for the cloning of the VL fragment. A recombinant rabbit / human chimeric antibody construct was generated. Specifically, the VH and VL sequences selected from the rabbit anti-CD79b antibody were obtained by gene synthesis (Integrated DNA Technologies) and ligated with the leader sequence (SEQ ID NO: 6) for Gibson Assembly (NEB HiFi DNA Assembly) to generate the HC and LC expression plasmids. The assembled plasmids were used to transform competent Escherichia coli( 5-alpha). Clones with correct sequences were screened according to the sequencing results (Elim Biopharm), and then cultured in LB containing carbenicillin (100 ug / ml). The plasmids were purified (QIAGEN Plasmid Plus Kits), eluted in nuclease-free H2O (Sigma), and stored at -80 °C.

[0370] Example 3. Expression and Purification of Chimeric Anti - CD79b Antibody

[0371] The recombinant chimeric antibody was expressed in CHO cells (ExpiCHO TM Expression System, Gibco) by transfecting cells with pcDNA3.4-huIgG1-Hc and pcDNA3.4-huKappa-Lc containing paired VH and VL sequences. ExpiCHO cells were cultured in ExpiCHO expression medium and maintained at 0.3 to 6×10 6 cells / ml, 37 °C, 125 rpm, 5% CO 2 and 80% humidity. In a 125 ml baffled flask, 25 ml of fresh ExpiCHO cells (6×10 6 / ml, viability > 95%, prepared one day before transfection) were inoculated at 3×10 6 / ml. 1 ml of serum-free medium (OptiPRO TM SFM, Gibco) containing pcDNA3.4-huIgG1-Hc and pcDNA3.4-huKappa-Lc (12 ug of each plasmid) was mixed with 1 ml of OptiPRO TMSFM (ExpiFectamine TM CHO reagent, Gibco). Then the transfection mixture was added to 25 ml of ExpiCHO cells and cultured at 37 °C. The next day, after adding 150 ul of ExpiFectamine TM CHO Enhancer, 6 ml of ExpiCHO TM Feed and 1× Penicillin-Streptomycin (Gibco), the transfection culture was transferred to a 32 °C incubator. The cell density and viability of the transfection culture were monitored, and the IgG1 antibody titer in the culture medium was monitored using a Biolayer Interferometry (BLI) instrument with a Protein A biosensor (Gator prime from Gator Bio).

[0372] After 5 days, the culture medium containing the secreted IgG1 antibody was collected (centrifuged at 2000 g for 10 minutes), filtered (ThermoScientific TM Nalgene TM Rapid-Flow TM sterile disposable filter) and loaded onto a gravity flow column (Bio-Rad) packed with Protein A resin (TOYOPEARL AF-rProtein Hc-650F). The IgG1 antibody was eluted with 3.5 ml of Glycine-HCl (100 mM, pH 2.7), immediately neutralized with 1 M Tris-HCl (pH 8.5), dialyzed against dialysis buffer (pH 7.2) in 1× PBS using a Thermo Scientific TM Slide-A-Lyzer TM G2 dialysis cassette (20K MWCO), and stored at 4 °C. The concentration of the purified IgG1 antibody was determined using a NanoDrop TM One / OneC Microvolume UV-Visible Spectrophotometer (Thermo Scientific TM ), and the quality of the IgG1 antibody was examined by SDS-PAGE gel under denaturing and non-denaturing conditions.

[0373] Example 4. Binding Affinity of Anti - CD79b Antibody to Human and Cynomolgus Macaque CD79b Recombinant Proteins Measured by ELISA and BLI Binding Affinity

[0374] The binding affinity of the anti-CD79b antibody to human and cynomolgus monkey CD79b recombinant proteins was tested by ELISA. 1 μg / ml (in PBS buffer) of human or cynomolgus monkey CD79b recombinant protein was coated on the ELISA plate overnight. The ELISA plate was washed and blocked with blocking buffer (PBS + 1% BSA), and then incubated with serially diluted anti-CD79b primary antibody. Anti-human IgG HRP secondary antibody (Biolegend catalog number 410902) and HRP substrate were used to quantify the anti-CD79b antibody binding. The ELISA binding affinities of the anti-CD79b antibody to human and cynomolgus monkey CD79b recombinant proteins are shown in Table 1, Figure 1 and Figure 2 in.

[0375] The binding affinity of the anti-CD79b antibody to human CD79b recombinant protein was tested by BLI. 10 μg / ml of the anti-CD79b antibody was captured by the anti-human IgG Fc probe (Gator Bio Catalog#160024). The binding and dissociation of serially diluted (146 nM to 0 nM, diluted 2-fold in PBS + 0.05% Tween-20) human CD79b recombinant protein were measured. The absolute kD was determined by applying a 1:1 binding model (Global Rmax unlinked). The BLI binding affinities of the anti-CD79b antibody to human CD79b recombinant protein are shown in Table 1 and Figure 3 in. Polatuzumab was used for comparison purposes. For ch44G2, based on the BLI data, Kon was 3.77E+05, but koff could not be detected. This indicates that ch44G2 has a strong binding affinity for CD79b.

[0376] Table 1 ELISA IC50 (nM) binding of anti-CD79b antibody to human and cynomolgus monkey CD79b recombinant proteins, and BLI kD (M) binding of anti-CD79b antibody to human CD79b recombinant protein

[0377]

[0378] Example 5. Internalization of Anti - CD79b Antibody

[0379] The internalization of anti-CD79b antibodies was evaluated. Approximately 50,000 Ramos cells were incubated with 20 μg / ml labeled anti-CD79b antibody and incubated at 37 °C. At different time points, the cells were washed and the presence of anti-CD79b antibody-binding receptors was detected using anti-human IgG PE secondary antibody (Jackson Research Catalogue#109-116-170). For analysis, the mean fluorescence intensity (MFI(PE)) of each antibody was normalized to its respective zero time point (100%) and the percentage decrease in MFI(PE) over time was plotted. As Figure 4 shown, the internalization of all screened anti-CD79b antibodies was slower than that of polatuzumab. Among them, ch23D8, ch44G2, ch48H10, and ch57B9 exhibited a much slower internalization process than other antibodies.

[0380] Example 6. Quantification of CD79b Antigen Density on the Surface of Tumor Cells

[0381] To estimate the CD79b antigen expression levels on the surface of malignant B tumor cells and normal B cells, the malignant B tumor cell line and PBMC from two healthy donors were stained with the anti-CD79b antibody ch44G2 at a saturating concentration on ice for 30 minutes. After washing with PBS, the cells were stained with secondary PE-labeled anti-human IgG (Invitrogen, catalogue number: 12-4998-82) on ice for 30 minutes. Then the cells were washed with PBS and resuspended in FACS buffer for flow cytometry analysis using Cytek northern lights. The results were analyzed and graphed using GraphPad prism software (version 9.4.1; GraphPad Software Inc.). The quantification of CD79b antigen density on the surface of malignant B tumor cells and normal B cells is shown in Table 2 and Figure 5 in.

[0382] Table 2. Quantification of CD79b antigen density on the surface of malignant B tumor cells and normal B cells

[0383]

[0384] Example 7. Malignant B - cell Line Binding Assay

[0385] The binding affinity of anti-CD79b antibodies to malignant B cell lines was tested. Polatuzumab and IgG1 were used as positive and negative controls, respectively. Malignant B cell lines (BJAB, Ramos, Daudi, SU-DHL-4, and Nalm-6) at 50,000 cells / well in 96-well plates were incubated with anti-CD79b antibodies (ch22D10, ch23D8, ch29C3, ch44G2, ch48H10, ch57B9, and polatuzumab) and IgG1 (Biolegend, catalog number: 403502) at different concentrations (serial 1:3 dilutions starting from 40 nM) on ice for 30 minutes and washed twice with 200 μl PBS. The cells were then incubated with 100 μl anti-human IgG1-PE (Invitrogen, cat: 12-4998-82), diluted 1:1000, on ice for 30 minutes and washed 3 times with 200 μl PBS before flow cytometry analysis. As Figures 6 - 10 shown, the anti-CD79b antibodies ch23D8, ch44G2, ch48H10, and ch57B9 showed higher cell surface binding affinity (lower EC50) than Polatuzumab for BJAB ( Figure 6 ), Ramos ( Figure 7 ), Daudi ( Figure 8 ), SU-DHL-4 ( Figure 9 ), and Nalm-6 ( Figure 10 ). The results indicate that ch23D8, ch44G2, ch48H10, and ch57B9 can bind to malignant B cell lines with high and low levels of CD79b antigen density.

[0386] Example 8. Endogenous B - cell Binding Assay

[0387] The binding affinity of anti-CD79b antibodies to endogenous B cells was tested. Polatuzumab and IgG1 control were used as positive and negative controls, respectively. PBMCs from 4 donors (Stanford Blood Center) were incubated with different concentrations (serially diluted 1:5 from 50 nM) of anti-CD79b antibodies (ch23D8, ch44G2, ch48H10, and polatuzumab) and IgG1 (Biolegend, catalog number: 403502) at 200,000 cells / well in a 96-well plate on ice for 30 minutes and washed twice with 200 μl PBS. The cells were then incubated with a mixture of 100 μl of 1:1000 diluted anti-human IgG1-PE (Invitrogen, catalog number: 12-4998-82) and CD19-BV421 (Biolegend, cat#: 302230, 1:400 dilution) on ice bath for 30 min and washed 3 times with 200 ul PBS before flow cytometry analysis. As Figures 11 - 14 shown, ch44G2, ch48H10, and ch23D8 showed higher cell surface binding affinity than Polatuzumab in all 4 different donors.

[0388] Example 9. PBMC Binding Assay in CLL Patients

[0389] To determine the binding affinity of anti-CD79b antibodies to cell surface CD79b on B lymphocytes from chronic lymphocytic leukemia (CLL) patients, a cell-based binding assay was performed. Polatuzumab and IgG1 control were used as positive and negative controls, respectively. PBMCs from CLL patients were purchased from Bioscience. FACS analysis showed that 95% of B lymphocytes (CD19+) in PBMCs were positive. Specifically, 2.5×10 4 PBMC cells in 50 μl of FACS buffer (1×PBS + 2% BSA + 2 mM EDTA) were seeded into each well of a V-bottom 96-well plate. Then, 50 μl of serially diluted anti-CD79b antibodies (ch22D10, ch23D8, ch29C3, ch44G2, ch48H10, ch57B9, or polatuzumab) or IgG1 was added to each well. The mixture was incubated on ice for 30 minutes and then washed twice with FACS buffer. The cells were resuspended in 100 μl of FACS buffer containing PE-conjugated goat anti-human IgG Fc (1:1000) and anti-human CD19 (BV421) and incubated in the dark on ice for 30 minutes. After washing twice with FACS buffer, the cells were resuspended in 100 μl of FACS buffer and analyzed using a Cytek cytometer. As Figures 15 - 16As shown, ch23D8, ch44G2, ch48H10, and ch57B9 showed more effective binding to B lymphocytes of CLL patients than polatuzumab.

[0390] Example 10. Binding of CD79b mAb to Long and Short Forms of CD79b

[0391] Studies on the surface binding of anti-CD79b antibodies to the short and long forms of CD79b were conducted. Polatuzumab was used as a positive control. Approximately 2 million Nalm-6 cells were electroporated with expression plasmids encoding the short form (Nalm-6 + short form) and long form (Nalm-6 + long form) of CD79b, respectively. After 48 hours, the cells were stained with the selected anti-CD79b antibodies (ch23D8, ch44G2, or polatuzumab) and detected using anti-IgM-APCch23D8, ch44G2, or polatuzumab. As Figures 17A - 17B shown, ch23D8 and ch44G2 had strong binding affinities for both the long form and short form of CD79b.

[0392] Example 11. Humanization of Anti - CD79b Antibody

[0393] Four rabbit anti-human CD79b monoclonal antibodies, clones 23D8, 44G2, 48H10, and 57B9, were humanized by transplanting the CDRs of the lead antibodies into selected human germline frameworks that were closest to the rabbit frameworks identified by IgBLAST (https: / / www.ncbi.nlm.nih.gov / igblast / ) and / or IMGT / DomainGapAlig (https: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi). Human germline IGHV, IGKV, IGHJ, and IGKJ were selected based on sequence similarity within the frameworks and CDRs. Some germline framework residues were reverted to the corresponding rabbit residues to maintain canonical loop structures and the light chain / heavy chain interface (Padlan Mol. Immunol., 1994, 31:169; Foote and Winter JMB, 1992, 224:487; Padlan Mol. Immunol., 1994, 31:169). Through humanization, the humanized antibodies hu23D8, hu44G2, hu48H10, and hu57B9 were generated.

[0394] The thermal stability of the humanized antibody clones was evaluated by Nano DSF (Table 3). In ELISA ( Figure 18 ), and cell lines expressing endogenous CD79b ( Figure 19)In it, it was confirmed that the humanized antibody binds to recombinant human CD79b ECD.

[0395] The results showed that the humanized anti-CD79b antibody exhibited the same technical effects as the chimeric anti-CD79b antibody, such as high affinity for CD79b, slow internalization, high binding to malignant B cell lines with high and low CD79b antigen densities, high cell surface binding affinity with endogenous B cells, high binding to B lymphocytes of CLL patients, and strong binding affinity to both the long form and short form of CD79b.

[0396] Table 3. Thermal stability of the humanized anti-CD79b antibody

[0397]

[0398] Other Examples

[0399] It should be understood that although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

[0400] Table 4. Selected amino acid sequences discussed in the present invention.

[0401]

[0402]

[0403]

[0404]

[0405]

Claims

1. An antibody or antigen-binding fragment thereof that binds to CD79b, comprising a heavy-chain variable region (VH) and a light-chain variable region (VL), wherein the selected VH CDR sequences and the selected VL CDR sequences are selected from the following (1) or (2): (1) According to the Kabat definition, the amino acid sequences of the selected VH CDR 1, 2, and 3 are listed in SEQ ID NO:9, 11, and 13 respectively, and the amino acid sequences of the selected VL CDR 1, 2, and 3 are listed in SEQ ID NO:14-16 respectively; (2) According to the Chothia definition, the amino acid sequences of the selected VH CDR 1, 2, and 3 are listed in SEQ ID NO:10, 12, and 13 respectively, and the amino acid sequences of the selected VL CDR 1, 2, and 3 are listed in SEQ ID NO:14-16 respectively.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the selected VH sequence and the selected VL sequence are the following sequences: (1) The selected VH amino acid sequence is listed in SEQ ID NO:7, and the selected VL amino acid sequence is listed in SEQ ID NO:

8.

3. The antibody or antigen-binding fragment thereof according to any one of claims 1 and 2, wherein the antibody or antigen-binding fragment specifically binds to human or monkey CD79b.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 and 2, wherein the antibody or antigen-binding fragment is a humanized antibody or antigen-binding fragment thereof, a single-chain variable fragment (scFv), and / or a single-arm antibody.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 and 2, wherein the antibody or antigen-binding fragment comprises a human IgG1 constant region, a human IgG2 constant region, or a human IgG4 constant region.

6. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof that binds to CD79b according to any one of claims 1-5.

7. A vector comprising one or more of the nucleic acids according to claim 6.

8. A pair of vectors, wherein each vector comprises the nucleic acid according to claim 6, and wherein the pair of vectors jointly encodes the VL region and the VH region of the antibody or antigen-binding fragment thereof that binds to CD79b.

9. A cell comprising the vector according to claim 7, or the pair of vectors according to claim 8.

10. The cell according to claim 9, wherein the cell is a CHO cell.

11. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 and 2, and a pharmaceutically acceptable carrier.

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