Anti-CD79B x CD3 bispecific antibodies and uses thereof

Abstract

The present invention relates to antibodies that specifically bind to CD79b, bispecific antibodies that specifically bind to CD79b and CD3, nucleic acids encoding anti-CD79b antibodies and anti-CD79b x CD3 bispecific antibodies, vectors containing the nucleic acids, host cells containing the nucleic acids or vectors, and pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof.

Description

[Technical field]

[0001] The present invention relates generally to the fields of immunology and antibody engineering. In particular, the present invention relates to CD79b monospecific antibodies and novel bispecific antibodies that specifically bind to CD79b and CD3. Furthermore, the present invention relates to nucleic acids encoding the antibodies, vectors comprising the nucleic acids, host cells comprising the nucleic acids or vectors, and pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof. Furthermore, the present invention relates to the use of these antibodies, pharmaceutical compositions, etc. in the immunotherapy, prevention, and / or diagnosis of disease. [Background technology]

[0002] CD79b belongs to the immunoglobulin superfamily and forms a heterodimer with CD79a, and the two and surface globulin constitute the BCR receptor complex. The BCR itself does not have a signaling domain, and when an antigen binds to the BCR, the CD79a / b heterodimer is involved in downstream signaling, which is important for maintaining the function of the entire B cell. Studies have shown that after CD79b is knocked out, B cells are restricted to the pre-B stage and cannot further develop and mature.

[0003] B-cell non-Hodgkin's lymphoma accounts for approximately 85% of NHL cases. The relapse rate after first-line treatment for B-cell NHL is high (the relapse rate after first-line treatment for DLBCL is 30-40%), and relapsed patients face poor prognosis (OS for DLBCL is 9-12 months on average) and limited late-stage treatment. Meanwhile, the number of new cases as well as relapsed and refractory cases is huge every year (about 80,000 new cases annually in both China and the United States), and the annual death toll in China in particular reaches about 50,000, which is more than twice that of the United States. Although antibody-drug conjugates against CD79b have shown certain clinical efficacy in NHL patients, some patients have developed resistance to them, and there remains a great unmet need for improved therapies for NHL.

[0004] In recent years, bispecific antibody-based immunotherapy has developed rapidly, and they can simultaneously bind to cytotoxic cells and surface antigens on tumor cells, thus mediating the killing of tumor cells by cytotoxic cells. Compared with antibody-drug conjugates, they have certain advantages in terms of efficacy and safety.

[0005] The present invention fulfills this need by providing bispecific antibodies that bind to CD79b and CD3 with high target specificity and high affinity, in particular bispecific antibodies that recruit T cells to the vicinity of tumor cells by binding to CD79b expressed on the surface of tumor cells. Summary of the Invention

[0006] In one aspect, the present invention relates to novel antibodies or antigen-binding fragments thereof that bind to CD79b.

[0007] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CD79b, comprising a heavy chain variable region, VH, and / or a light chain variable region, VL, 1) the VH comprises three complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the VH depicted in SEQ ID NO:4, and the VL comprises LCDR1, LCDR2, and LCDR3 contained in the VL depicted in SEQ ID NO:9; or 2) VH comprises three complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in VH shown in SEQ ID NO: 14, and VL comprises LCDR1, LCDR2, and LCDR3 contained in VL shown in SEQ ID NO: 19; Antibodies or antigen-binding fragments thereof are provided.

[0008] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CD79b, comprising a heavy chain variable region, VH, and / or a light chain variable region, VL, (i) VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:11, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:12, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:13; and / or (ii) the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:16, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:7 or SEQ ID NO:17, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:8 or SEQ ID NO:18; Antibodies or antigen-binding fragments thereof are provided.

[0009] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CD79b, comprising a heavy chain variable region, VH, and / or a light chain variable region, VL, 1) VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, where HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:1, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:3; VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, where LCDR1 comprises the amino acid sequence set forth in SEQ ID NO:6, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO:7, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:8; 2) VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 11, HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 12, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 13; VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 16, LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 17, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18; Antibodies or antigen-binding fragments thereof are provided.

[0010] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CD79b, comprising a heavy chain variable region, VH, and / or a light chain variable region, VL, (a) the heavy chain variable region VH is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:14; or (ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 14, and preferably the amino acid changes do not occur in the CDRs; and / or (b) the light chain variable region VL is (i) comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:19; or (ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 19; or (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 19, and preferably the amino acid changes do not occur in the CDRs; Antibodies or antigen-binding fragments thereof are provided.

[0011] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CD79b, 1) a heavy chain variable region VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 4, and a light chain variable region VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 9; 2) a heavy chain variable region VH comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region VL comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO: 19; The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0012] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CD79b, 1) a heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 4 and a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 14; 2) A heavy chain variable region VH comprising the amino acid sequence shown in SEQ ID NO: 9 and a light chain variable region VL comprising the amino acid sequence shown in SEQ ID NO: 19 The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0013] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CD79b, comprising a heavy chain and / or a light chain, (a) the heavy chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:15; or (ii) comprises or consists of the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:15; or (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 15, preferably no amino acid changes occur in the CDRs of the heavy chain, more preferably no amino acid changes occur in the heavy chain variable region; and / or (b) the light chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:10 or SEQ ID NO:20; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 20; or (iii) comprises an amino acid sequence having one or more (preferably 20 or less than 10, more preferably 5, 4, 3, 2, or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 20, preferably no amino acid changes occur in the CDRs of the light chain, more preferably no amino acid changes occur in the light chain variable region; Antibodies or antigen-binding fragments thereof are provided.

[0014] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CD79b, 1) a heavy chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:5, and a light chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID NO:10; 2) a heavy chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15, and a light chain comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 20. The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0015] In some embodiments, the invention provides an antibody or antigen-binding fragment thereof that binds to CD79b, 1) a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:5 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:10; 2) A heavy chain comprising the amino acid sequence shown in SEQ ID NO: 15 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 20 The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0016] In some embodiments, the invention provides isolated nucleic acids encoding antibodies or antigen-binding fragments thereof that bind CD79b of the invention, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors.

[0017] In some embodiments, the invention provides methods for preparing an antibody or antigen-binding fragment thereof that binds CD79b of the invention, comprising culturing a host cell as described herein under conditions suitable for expressing a nucleic acid as described herein.

[0018] In some embodiments, the invention provides immunoconjugates and pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof that binds CD79b of the invention.

[0019] In some embodiments, the present invention also provides use of an antibody or antigen-binding fragment thereof, immunoconjugate, or pharmaceutical composition that binds CD79b of the present invention in the preparation of a medicament for the prevention and / or treatment of a tumor.

[0020] In some embodiments, the present invention also provides a method for preventing and / or treating a tumor, comprising administering to a subject an effective amount of an antibody or antigen-binding fragment thereof, immunoconjugate, or pharmaceutical composition that binds CD79b of the present invention.

[0021] The present invention also relates to a method for detecting CD79b in a sample, the method comprising: (a) contacting with an antibody or antigen-binding fragment thereof described herein; and (b) detecting a complex formed by the antibody or antigen-binding fragment thereof and CD79b.

[0022] In another aspect, the present invention also discloses novel bispecific antibodies targeting both CD79b and CD3, polynucleotides encoding the bispecific antibodies, vectors comprising the polynucleotides, host cells comprising the polynucleotides or the vectors, and uses of the bispecific antibodies in the treatment, prevention, and / or diagnosis of diseases associated with CD79b activity in individuals.

[0023] Thus, in one aspect, the invention provides a bispecific antibody that specifically binds both CD79b and CD3 (anti-CD79bxCD3 bispecific antibody), comprising (i) an anti-CD79b antibody or a fragment thereof and (ii) an anti-CD3 antibody or a fragment thereof. In one embodiment, the invention provides an anti-CD79bxCD3 bispecific antibody in a 1+1 format consisting of four polypeptide chains that are symmetrical, the left half consisting of a first light chain and a first heavy chain, the right half consisting of a second heavy chain and a second light chain, the first light chain and the first heavy chain being the heavy and light chains of an antibody targeting CD79b or CD3, and the second heavy chain and the second light chain being the heavy and light chains of an antibody targeting CD3 or CD79b.

[0024] In one embodiment, the invention provides an anti-CD79bxCD3 bispecific antibody in a 1+1 format, where a first light chain and a first heavy chain specifically bind CD79b, and a second heavy chain and a second light chain specifically bind CD3. In a specific embodiment, the anti-CD79bxCD3 bispecific antibody provided herein comprises a first light chain, a first heavy chain, a second heavy chain, and a second light chain, where the first light chain comprises the three light chain CDRs comprised in SEQ ID NO: 9, 19, or 29, the first heavy chain comprises the three heavy chain CDRs comprised in SEQ ID NO: 4, 14, or 24, the second heavy chain comprises the three heavy chain CDRs comprised in SEQ ID NO: 34 or 44, and the second light chain comprises the three light chain CDRs comprised in SEQ ID NO: 39 or 49.

[0025] In certain embodiments, an anti-CD79bxCD3 bispecific antibody provided herein comprises a first light chain, a first heavy chain, a second heavy chain, and a second light chain, wherein the first light chain and the first heavy chain bind to CD79b, and the second heavy chain and the second light chain bind to CD3; 1) the first light chain comprises the three light chain CDRs contained in SEQ ID NO:9, the first heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO:4, the second heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO:34, and the second light chain comprises the three light chain CDRs contained in SEQ ID NO:39; 2) the first light chain comprises the three light chain CDRs contained in SEQ ID NO:9, the first heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO:4, the second heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO:44, and the second light chain comprises the three light chain CDRs contained in SEQ ID NO:49; 3) the first light chain comprises the three light chain CDRs contained in SEQ ID NO: 19, the first heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO: 14, the second heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO: 34, and the second light chain comprises the three light chain CDRs contained in SEQ ID NO: 39; 4) the first light chain comprises the three light chain CDRs contained in SEQ ID NO: 19, the first heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO: 14, the second heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO: 44, and the second light chain comprises the three light chain CDRs contained in SEQ ID NO: 49; 5) the first light chain comprises the three light chain CDRs contained in SEQ ID NO: 29, the first heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO: 24, the second heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO: 34, and the second light chain comprises the three light chain CDRs contained in SEQ ID NO: 39; or 6) the first light chain comprises the three light chain CDRs contained in SEQ ID NO:29, the first heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO:24, the second heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO:44, and the second light chain comprises the three light chain CDRs contained in SEQ ID NO:49.

[0026] In certain embodiments, an anti-CD79bxCD3 bispecific antibody provided herein comprises a first light chain, a first heavy chain, a second heavy chain, and a second light chain, wherein the first light chain comprises the three light chain CDRs contained in SEQ ID NO: 9, 19, or 29 and comprises a VL having at least 90% identity to SEQ ID NO: 9, 19, or 29; the first heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO: 4, 14, or 24 and comprises a VH having at least 90% identity to SEQ ID NO: 4, 14, or 24; the second heavy chain comprises the three heavy chain CDRs contained in SEQ ID NO: 34 or 44 and comprises a VH having at least 90% identity to SEQ ID NO: 34 or 44; and the second light chain comprises the three light chain CDRs contained in SEQ ID NO: 39 or 49 and comprises a VL having at least 90% identity to SEQ ID NO: 39 or 49.

[0027] In certain embodiments, the anti-CD79bxCD3 bispecific antibody provided herein comprises a first light chain, a first heavy chain, a second heavy chain, and a second light chain; 1) the first light chain comprises three light chain CDRs contained in SEQ ID NO:9 and comprises a VL having at least 90% identity with SEQ ID NO:9; the first heavy chain comprises three heavy chain CDRs contained in SEQ ID NO:4 and comprises a VH having at least 90% identity with SEQ ID NO:4; the second heavy chain comprises three heavy chain CDRs contained in SEQ ID NO:34 and comprises a VH having at least 90% identity with SEQ ID NO:34; and the second light chain comprises three light chain CDRs contained in SEQ ID NO:39 and comprises a VL having at least 90% identity with SEQ ID NO:39; 2) the first light chain comprises three light chain CDRs contained in SEQ ID NO:9 and comprises a VL having at least 90% identity with SEQ ID NO:9; the first heavy chain comprises three heavy chain CDRs contained in SEQ ID NO:4 and comprises a VH having at least 90% identity with SEQ ID NO:4; the second heavy chain comprises three heavy chain CDRs contained in SEQ ID NO:44 and comprises a VH having at least 90% identity with SEQ ID NO:44; and the second light chain comprises three light chain CDRs contained in SEQ ID NO:49 and comprises a VL having at least 90% identity with SEQ ID NO:49; 3) the first light chain comprises three light chain CDRs contained in SEQ ID NO: 19 and comprises a VL having at least 90% identity with SEQ ID NO: 19, the first heavy chain comprises three heavy chain CDRs contained in SEQ ID NO: 14 and comprises a VH having at least 90% identity with SEQ ID NO: 14, the second heavy chain comprises three heavy chain CDRs contained in SEQ ID NO: 34 and comprises a VH having at least 90% identity with SEQ ID NO: 34, and the second light chain comprises three light chain CDRs contained in SEQ ID NO: 39 and comprises a VL having at least 90% identity with SEQ ID NO: 39; 4) the first light chain comprises three light chain CDRs contained in SEQ ID NO: 19 and comprises a VL having at least 90% identity with SEQ ID NO: 19, the first heavy chain comprises three heavy chain CDRs contained in SEQ ID NO: 14 and comprises a VH having at least 90% identity with SEQ ID NO: 14, the second heavy chain comprises three heavy chain CDRs contained in SEQ ID NO: 44 and comprises a VH having at least 90% identity with SEQ ID NO: 44, and the second light chain comprises three light chain CDRs contained in SEQ ID NO: 49 and comprises a VL having at least 90% identity with SEQ ID NO: 49; 5) the first light chain comprises a VL comprising the three light chain CDRs contained in SEQ ID NO:29 and having at least 90% identity to SEQ ID NO:29, the first heavy chain comprises a VH comprising the three heavy chain CDRs contained in SEQ ID NO:24 and having at least 90% identity to SEQ ID NO:24, the second heavy chain comprises a VH comprising the three heavy chain CDRs contained in SEQ ID NO:34 and having at least 90% identity to SEQ ID NO:34, and the second light chain comprises a VL comprising the three light chain CDRs contained in SEQ ID NO:39 and having at least 90% identity to SEQ ID NO:39, or 6) the first light chain comprises a VL that comprises the three light chain CDRs contained in SEQ ID NO:29 and has at least 90% identity to SEQ ID NO:29, the first heavy chain comprises a VH that comprises the three heavy chain CDRs contained in SEQ ID NO:24 and has at least 90% identity to SEQ ID NO:24, the second heavy chain comprises a VH that comprises the three heavy chain CDRs contained in SEQ ID NO:44 and has at least 90% identity to SEQ ID NO:44, and the second light chain comprises a VL that comprises the three light chain CDRs contained in SEQ ID NO:49 and has at least 90% identity to SEQ ID NO:49.

[0028] In certain embodiments, the anti-CD79bxCD3 bispecific antibody provided herein comprises a first light chain, a first heavy chain, a second heavy chain, and a second light chain; 1) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 6, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 8; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; the heavy chain comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 31, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 32 and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 33; the second light chain comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37 and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38; 2) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 6, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 8; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; the heavy chain comprises HCDR1, HCDR2 and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 41, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 42 and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 43; the second light chain comprises LCDR1, LCDR2 and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 46, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 47 and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 48; 3) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 18; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 31, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 32, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 33; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38; 4) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 18; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13; the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 41, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 42, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 43; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 46, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 47, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 48; 5) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 28; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23; the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 31, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 32, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 33; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38; or 6) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 28; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 23; the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 41, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 42, and HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 43; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 46, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 47, and LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 48.

[0029] In another embodiment, an anti-CD79bxCD3 bispecific antibody provided herein comprises a first light chain, a first heavy chain, a second heavy chain, and a second light chain, wherein the first light chain comprises a VL set forth in SEQ ID NO: 9, 19, or 29, the first heavy chain comprises a VH set forth in SEQ ID NO: 4, 14, or 24, the second heavy chain comprises a VH set forth in SEQ ID NO: 34 or 44, and the second light chain comprises a VL set forth in SEQ ID NO: 39 or 49.

[0030] In certain embodiments, the anti-CD79bxCD3 bispecific antibody provided herein comprises a first light chain, a first heavy chain, a second heavy chain, and a second light chain; 1) the first light chain comprises a VL set forth in SEQ ID NO: 9, the first heavy chain comprises a VH set forth in SEQ ID NO: 4, the second heavy chain comprises a VH set forth in SEQ ID NO: 34, and the second light chain comprises a VL set forth in SEQ ID NO: 39; 2) the first light chain comprises a VL set forth in SEQ ID NO: 9, the first heavy chain comprises a VH set forth in SEQ ID NO: 4, the second heavy chain comprises a VH set forth in SEQ ID NO: 44, and the second light chain comprises a VL set forth in SEQ ID NO: 49; 3) the first light chain comprises a VL set forth in SEQ ID NO: 19, the first heavy chain comprises a VH set forth in SEQ ID NO: 14, the second heavy chain comprises a VH set forth in SEQ ID NO: 34, and the second light chain comprises a VL set forth in SEQ ID NO: 39; 4) the first light chain comprises a VL set forth in SEQ ID NO: 19, the first heavy chain comprises a VH set forth in SEQ ID NO: 14, the second heavy chain comprises a VH set forth in SEQ ID NO: 44, and the second light chain comprises a VL set forth in SEQ ID NO: 49; 5) the first light chain comprises a VL set forth in SEQ ID NO: 29, the first heavy chain comprises a VH set forth in SEQ ID NO: 24, the second heavy chain comprises a VH set forth in SEQ ID NO: 34, and the second light chain comprises a VL set forth in SEQ ID NO: 39; or 6) the first light chain comprises the VL set forth in SEQ ID NO: 29, the first heavy chain comprises the VH set forth in SEQ ID NO: 24, the second heavy chain comprises the VH set forth in SEQ ID NO: 44, and the second light chain comprises the VL set forth in SEQ ID NO: 49.

[0031] In another embodiment, an anti-CD79bxCD3 bispecific antibody provided herein comprises a first light chain, a first heavy chain, a second heavy chain, and a second light chain, wherein the first heavy chain and the second heavy chain comprise the same or different Fc regions. In another preferred embodiment, the Fc regions comprised in the first heavy chain and the second heavy chain have "knob" and "hole" structures, respectively, that interact to stabilize the spatial structure of the bispecific antibody.

[0032] In certain embodiments, in the anti-CD79bxCD3 bispecific antibodies described herein, the variable regions in the first and second heavy chains are either homologous or heterologous to the Fc region. In another embodiment, the variable regions in the first and second heavy chains are linked to the Fc region directly or via a linker. In certain embodiments, the linker is a flexible linker commonly used in the art. In another embodiment, the linker is (G4S)n, where n=1-6, preferably n=1, 2, 3, or 4.

[0033] In another aspect, the invention provides an anti-CD79bxCD3 bispecific antibody comprising a first light chain, a first heavy chain, a second heavy chain, and a second light chain, 1) the first light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consists of, SEQ ID NO: 10, and the first heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consists of, SEQ ID NO: 5; the second heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO: 35; and the second light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO: 40. 2) the first light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10 or consists of SEQ ID NO:10, and the first heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:5 or consists of SEQ ID NO:5; the second heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO: 45; and the second light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO: 50. 3) the first light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20 or consists of SEQ ID NO:20, and the first heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:15 or consists of SEQ ID NO:15. the second heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO: 35; and the second light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO: 40. 4) the first light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20 or consists of SEQ ID NO:20, and the first heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:15 or consists of SEQ ID NO:15. wherein the second heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consists of, SEQ ID NO: 45, and the second light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consists of, SEQ ID NO: 50. 5) the first light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 30 or consists of SEQ ID NO: 30, and the first heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 25 or consists of SEQ ID NO: 25. the second heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO: 35; and the second light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO: 40. or 6) the first light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 30 or consists of SEQ ID NO: 30, and the first heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 25 or consists of SEQ ID NO: 25. the second heavy chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO:45; and the second light chain comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to, or consisting of, SEQ ID NO:50; Anti-CD79b x CD3 bispecific antibodies are provided.

[0034] In one embodiment, the light chain variable domain of the first light chain and the heavy chain variable domain of the first heavy chain pair to form an antigen recognition site for CD79b, and the heavy chain variable domain of the second heavy chain and the light chain variable domain of the second light chain pair to form an antigen recognition site for CD3. The Fc domains of each of the first heavy chain and the second heavy chain interact to form an Fc region. In a particular embodiment, the Fc domains of each of the first heavy chain and the second heavy chain comprise mutations that stabilize the interaction, e.g., "knobs-in-holes" mutations.

[0035] In one aspect, the present invention also provides polynucleotides (nucleic acids) encoding the anti-CD79bxCD3 bispecific antibodies of the invention, and vectors comprising the polynucleotides, which vectors are preferably expression vectors.

[0036] In another aspect, the invention provides a host cell comprising a polynucleotide or vector of the invention.The invention also provides a method of producing an anti-CD79bxCD3 bispecific antibody of the invention, comprising the steps of (i) culturing a host cell of the invention under conditions suitable for expression of the anti-CD79bxCD3 bispecific antibody of the invention, and (ii) recovering the anti-CD79bxCD3 bispecific antibody of the invention.

[0037] In one aspect, the invention provides diagnostic kits and pharmaceutical compositions comprising an anti-CD79bxCD3 bispecific antibody of the invention. Further provided is the use of an anti-CD79bxCD3 bispecific antibody, a diagnostic kit or a pharmaceutical composition of the invention in the treatment, prevention and / or diagnosis of a disease associated with CD79b activity, in particular in the treatment, prevention and / or diagnosis of non-Hodgkin's lymphoma.

[0038] In another aspect, the present invention provides a method for treating a disease associated with CD79b activity comprising administering to a patient in need thereof a therapeutically effective amount of an anti-CD79bxCD3 bispecific antibody of the invention or a pharmaceutical composition of the invention. Preferably, the disease is a cancer that overexpresses CD79b, more preferably the disease is non-Hodgkin's lymphoma.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples described herein are for illustrative purposes only and are not intended to be limiting. Other features, objects, and advantages of the present invention will become apparent from the specification, drawings, and appended claims. [Brief description of the drawings]

[0040] BRIEF DESCRIPTION OF THE DRAWINGS Preferred embodiments of the invention described in detail below will be better understood when read in conjunction with the following drawings: For the purpose of illustrating the invention, there are shown in the drawings preferred embodiments, it being understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0041] [Figure 1] FIG. 1 shows the binding ability of chimeric anti-CD79b antibodies to Ramos cells. [Diagram 2] FIG. 2 shows the binding ability of chimeric anti-CD79b antibodies to BJAB cells. [Diagram 3] FIG. 3 shows the binding ability of humanized anti-CD79b antibodies to Ramos cells. [Figure 4] FIG. 4 shows the binding ability of humanized anti-CD79b antibodies to BJAB cells. [Diagram 5] FIG. 5 shows detection of ADCC activity of humanized anti-CD79b antibodies. [Figure 6] FIG. 6 shows the binding ability of exemplary antibodies to BJAB cells. [Figure 7] FIG. 7 shows the binding ability of exemplary antibodies to WSU-DLCL2 cells. [Figure 8] FIG. 8 shows activation of NFAT signaling mediated by an exemplary antibody. [Figure 9] FIG. 9 shows exemplary antibody-mediated tumor cell killing by CD8+ T cells. [Figure 10] FIG. 10 shows a flow cytometry assay of human CD8+ T cell activation by exemplary antibodies. [Figure 11] FIG. 11 shows a flow cytometry assay of the amount of the cytokine IFN-γ released upon exemplary antibody-induced killing of tumor cells by human CD8+ T cells. [Figure 12] FIG. 12 shows a flow cytometry assay of the amount of the cytokine TNFα released upon exemplary antibody-induced killing of tumor cells by human CD8+ T cells. [Figure 13]FIG. 13 shows a flow cytometry assay of human CD4+ T cell activation by exemplary antibodies. [Figure 14] FIG. 14 shows the proliferation of CD8+ T cells promoted by exemplary antibodies. [Figure 15] FIG. 15 shows CD4+ T cell proliferation promoted by exemplary antibodies. [Figure 16] FIG. 16 shows the tumor inhibitory effects of exemplary antibodies in a WSU-DLCL2 tumor-bearing humanized mouse model. [Figure 17] FIG. 17 shows the tumor inhibitory effects of exemplary antibodies in a Ramos tumor-bearing humanized mouse model. [Figure 18] FIG. 18 shows the PK of bispecific antibodies in mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] I. Definition Before the present invention is described in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, which may be modified. It should also be understood that the terms used herein are not intended to limit the scope of the present invention, but are intended only to describe specific embodiments, and the scope of the present invention is limited only by the appended claims. Unless otherwise defined, any technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0043] For the purpose of describing the present specification, the following definitions are used, however, where appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0044] The term "about" when used in conjunction with a numerical value is intended to encompass numerical values ​​ranging from a lower limit of 5% less than the specified numerical value to an upper limit of 5% greater than the specified numerical value.

[0045] It is meant that the term "and / or," when used in conjunction with two or more options, should be understood to refer to any one of the options or to more than one of the options.

[0046] The term "comprise" or "include" means the inclusion of the recited elements, integers, or steps, but does not exclude any other elements, integers, or steps. As used herein, the term "comprise" or "include" also encompasses situations in which the entirety of a recited element, integer, or step is comprised, unless otherwise specified. For example, reference to an antibody variable region "comprising" a particular sequence is intended to encompass an antibody variable region consisting of the particular sequence.

[0047] The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, intact antibodies, or antibody fragments thereof that exhibit the desired antigen-binding activity. Intact antibodies generally comprise at least two full-length heavy chains and two full-length light chains, although in some cases may comprise fewer chains, for example, native antibodies from camelids may comprise only heavy chains.

[0048] The term "antigen-binding fragment" (used interchangeably herein with "antibody fragment" and "antigen-binding portion") refers to a molecule distinct from an intact antibody that comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, antigen-binding fragments of Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies (dAbs), linear antibodies, single-chain antibodies (e.g., scFv), single domain antibodies, bivalent or bispecific antibodies, camelid antibodies, and other fragments that exhibit the desired ability to bind an antigen (e.g., CD79b and / or CD3).

[0049] As used herein, the terms "binding" and "specific binding" mean that the binding effect of the antibody is selective for the antigen and can be distinguished from undesired or non-specific interactions. The ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), or biolayer interferometry (ForteBio), or conventional binding assays known in the art. For example, an antibody may bind to a specific antigen at a concentration of about 1×10 in SPR. -7 KD below 1×10 -8 KD below 1×10 -9 KD below 1×10 -10 KD less than or equal to 1 x 10 -11 An antibody is an antibody that "specifically binds to CD79b and / or CD3" if it binds to CD79b and / or CD3 with the following KD. However, an antibody that specifically binds to CD79b and / or CD3 may have cross-reactivity with CD79b and / or CD3 proteins from other species. For example, an antibody specific for human CD79b and / or CD3 may, in some embodiments, cross-react with cynomolgus monkey CD79b and / or CD3. Methods for determining cross-reactivity include those described in the Examples and standard assays known in the art, such as biological optical interferometry or flow cytometry.

[0050] The term "single-chain variable fragment" or "scFv" refers to a genetically engineered small molecule antibody. It is a small molecule recombinant antibody obtained by genetic engineering at the DNA level by linking the heavy chain variable region (VH) and the light chain variable region (VL) of a natural antibody (usually via a synthetic linker peptide (or linker)). Compared to an intact antibody molecule, a single-chain scFv antibody has the following advantages: it has the variable region of an intact antibody, and therefore retains the antigen specificity and antigen binding activity of the original antibody; it does not have an Fc region, and therefore is weakly immunogenic and hardly causes an immune response when used in humans; it is easy to manipulate and suitable for use as a genetically engineered component in preparing other antigen-specific binding molecules with new properties, such as full-length antibodies, scFv-Fc antibodies, etc.

[0051] The term "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In certain embodiments, a human IgG heavy chain Fc region generally extends from Cys226 or Pro230 to the carbonyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise indicated, the amino acid residues of the Fc region or constant region are as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. Numbered according to the EU numbering system, also referred to as the EU index, as described in 1991.

[0052] The term "knob-in-hole" means that a "knob" structure is formed on one Fc chain of the bispecific antibody molecule described herein and a "hole" structure is formed on the other chain. Thus, the hole and knob are of the same or similar size and are suitably positioned such that upon interaction of the two Fcs, the knob of one Fc is placed into the corresponding hole of the other Fc, thereby stabilizing the heteromultimeric structure. (See, e.g., U.S. Pat. No. 5,731,168).

[0053] In some embodiments, the knobs can be constructed by replacing small amino acid side chains with larger side chains, in accordance with techniques known in the art, while in some embodiments the holes may be constructed by replacing large amino acid side chains with smaller side chains.

[0054] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies typically have a similar structure, with each domain containing four conserved framework regions (FRs) and three complementarity determining regions (see, for example, Kindt et al., Kuby Immunology, 6 th ed., WH Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to provide antigen-binding specificity.

[0055] "Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are regions in antibody variable domains that are highly variable in sequence and that form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of heavy and light chains are numbered sequentially from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. The CDRs located in the heavy chain variable domain of an antibody are also referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located in the light chain variable domain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. For a given amino acid sequence of a light chain variable region or a heavy chain variable region, the CDR sequences can be determined according to various schemes well known in the art, such as the Chothia scheme based on the three-dimensional structure of an antibody and the topology of the CDR loops (Chothia et al., (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273:927-948 (1997)), which is based on the variation of antibody sequences (Kabat et al., Sequences of Proteins of Immunological Interest, 4 thedition, USDepartment of Health and Human Services, National Institutes of Health (1987), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), as well as the North CDR definition, which is based on affinity propagation clustering using multiple crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256, (2011)).

[0056] For example, the Kabat and Chothia numbered CDR regions have different defined ranges. [Table 1]

[0057] Unless otherwise stated, the term "CDR" or "CDR sequence" as used herein encompasses CDR sequences determined by any one of the above schemes.

[0058] CDRs can also be determined based on having the same Kabat numbering position as the reference CDR sequence. Unless otherwise specified, the residue positions of the antibody variable region (including heavy chain variable region residues and light chain variable region residues) in the present invention are based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0059] However, it should be noted that the boundaries of the CDRs of the variable regions of an antibody based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined by different assignment systems are different. Thus, with respect to the definition of an antibody having a specific CDR sequence defined in the present invention, the scope of the antibody also includes antibodies whose variable region sequences contain the specific CDR sequences, but have CDR boundaries that fall within the scope of the claims that differ from the specific CDR boundaries defined by the present invention due to different protocols applied (e.g., different assignment system rules or a combination thereof).

[0060] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, while CDRs differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. The minimal overlapping region can be determined using at least two of the methods of Kabat, Chothia, AbM, Contact, and North, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit may be a sub-portion of the CDR. As will be apparent to those skilled in the art, the residues of the remaining part of the CDR sequence can be determined by the structure and protein folding of the antibody. Thus, variants of any CDR presented herein are also contemplated. For example, in a variant of one CDR, the amino acid residues of the minimum binding unit remain unchanged, but the remaining CDR residues defined according to Kabat or Chothia may be replaced with conservative amino acid residues.

[0061] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0062] The term "chemotherapeutic agent" includes chemical compounds useful in the treatment of cancer.

[0063] The term "small molecule drug" refers to low molecular weight organic compounds that can modulate biological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, usually less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules penetrate cells better, are less likely to be degraded, and induce less immune response than larger molecules.

[0064] The term "functional Fc region" refers to an Fc region that has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, downregulation of a cell surface receptor (e.g., B cell receptor, or BCR), and the like. Such effector functions generally require that the Fc region is associated with a binding domain (e.g., an antibody variable domain) and can be assessed using a variety of assays, such as those disclosed herein.

[0065] The term "therapeutic agent" as used herein includes any substance effective in the prevention or treatment of tumors (e.g., cancer), including chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infective agents, small molecule drugs, or immunomodulatory agents.

[0066] The term "immunomodulatory agent" as used herein refers to a natural or synthetic active agent or drug that suppresses or modulates the immune response, which may be a humoral response or a cellular response.

[0067] The term "effective amount" refers to the amount or dose of the antibody, fragment thereof, conjugate, or composition of the present invention that produces the expected effect in a patient in need of treatment or prevention after administration to the patient in one or more doses. For therapeutic or prophylactic purposes, the "effective amount" can be divided into a "therapeutically effective amount" and a "prophylactically effective amount". The effective amount can be easily determined by the attending physician, who is skilled in the art, by considering various factors such as the species, size, age, and general health of the mammal, the specific disease involved, the extent or severity of the disease, the response in the individual patient, the specific antibody administered, the mode of administration, the bioavailability profile of the administered formulation, the selected dosing regimen, and the use of any combination therapy.

[0068] In one embodiment, an effective amount of a bispecific antibody of the invention inhibits a measurable parameter (e.g., tumor growth rate, tumor volume, etc.) by preferably at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and even more preferably at least about 80% or 90% compared to a control.

[0069] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the original, primarily transformed cell and its progeny, regardless of the number of passages. The progeny may not be exactly identical to the parent cell in terms of nucleic acid content, and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected from the originally transformed cell are included herein.

[0070] As used herein, the term "multispecific" antibody refers to an antibody having at least two different antigen binding sites, each binding to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody having binding specificity for at least two different epitopes. In one embodiment, a bispecific antibody is provided herein having binding specificity for a first antigen target (CD79b) and a second antigen target (CD3). Given that the antibody construct according to the invention is (at least) bispecific, the antibody construct does not occur in nature and is clearly different from naturally occurring products. Thus, a "bispecific" antibody or immunoglobulin is an artificial hybrid antibody or immunoglobulin having at least two different binding sides with different specificities.

[0071] The term "target" means CD79b or CD3. The terms "primary target and secondary target" means that CD79b is the primary target and CD3 is the secondary target, or vice versa.

[0072] The term "cytokine" is a general term for proteins released by a cell population and acting as intercellular mediators for other cells. Examples of such cytokines are lymphokines; monokines; interleukins (IL), such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, and IL-15; tumor necrosis factors, such as TNF-α or TNF-β; and other polypeptide factors, including LIF and Kit ligand (KL), and gamma-interferon. As used herein, the term "cytokine" includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of native sequence cytokines, including small molecule entities produced by artificial synthesis, and pharma-ceutically acceptable derivatives and salts thereof.

[0073] The term "immunoconjugate" is an antibody bound to one or more other substances, including, but not limited to, a cytotoxic agent or a label.

[0074] The term "individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, goats, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, an individual or subject is a human.

[0075] The term "isolated" antibody means an antibody that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), and capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr., B848:79-87 (2007).

[0076] Calculation of sequence identity between sequences is performed as follows: To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison). In a preferred embodiment, the length of the reference sequence aligned for comparison is at least 30%, at least 40%, at least 50% or 60% of the length of the reference sequence, and even more preferably at least 70%, 80%, 90% or 100%. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at this position.

[0077] A mathematical algorithm can be used to compare two sequences and calculate the percent identity between the sequences. In one preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch algorithm ((1970) J. Mol. Biol., 48:444-453; available at http: / / www.gcg.com) incorporated in the GAP program of the GCG software package, using a Blossum62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percent identity between two nucleotide acid sequences is determined using the GAP program of the GCG software package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and that which should be used unless otherwise specified) is a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4 and a frameshift gap penalty of 5.

[0078] The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0) using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0079] Additionally or alternatively, the nucleic acid and protein sequences described herein can further be used as "query sequences" to conduct searches against public databases, e.g., to identify other family member or related sequences.

[0080] The term "pharmaceutical auxiliary material" refers to diluents, adjuvants (eg, Freund's adjuvant (complete and incomplete)), excipients, carriers, stabilizers, and the like, administered with an active substance.

[0081] The term "pharmaceutical composition" refers to a composition that is present in a form that allows for effective biological activity of the active ingredients contained therein, and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0082] The term "combination therapy" or "combined therapy" means that two or more therapeutic agents are administered to treat cancer or an infectious disease as described herein. Such administration includes co-administration of these therapeutic agents substantially simultaneously, for example, in a single capsule containing a fixed ratio of active ingredients. Alternatively, such administration includes co-administration of the active ingredients in various or separate containers, such as tablets, capsules, powders, and liquids. The powders and / or liquids can be reconstituted or diluted to the desired dose before administration. Furthermore, such administration also includes the use of each type of therapeutic agent at about the same time or sequentially at different times. In either case, the therapeutic regimen will provide the beneficial effect of the pharmaceutical combination in treating the disorder or condition as described herein.

[0083] As used herein, "treatment" (or "treat" or "treating") refers to slowing, hindering, arresting, ameliorating, halting, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease.

[0084] As used herein, "prevention" (or "prevent" or "preventing") includes preventing the onset or progression of a disease or disorder or symptoms of a particular disease or disorder. In some embodiments, subjects with a family history of cancer are candidates for a preventative regimen. In general, in the context of cancer, the term "prevention" (or "prevent" or "preventing") refers to the administration of a drug before signs or symptoms of cancer appear, particularly in subjects at risk of cancer.

[0085] The term "vector" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that function as self-replicating nucleic acid structures and vectors that integrate into the genome of a host cell into which the vector is introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0086] II. Antibodies As used herein, the terms "CD79b", "target CD79b" and "human CD79b" refer to CD79b recombinant protein. The extracellular domain of CD79b consists of amino acids 29-159 according to UniProt. As used herein, "antibody against CD79b" and "anti-CD79b antibody" refer to an antibody that specifically binds to CD79b. In one embodiment, the antibody that binds to CD79b is -9 M or less, preferably 10 -9 M~10 -13 It has a dissociation constant (Kd) of M. In one embodiment, the anti-CD79b antibody binds to an epitope of CD79b that is conserved in CD79b from different species, preferably humans.

[0087] In one embodiment, an "anti-CD79b antibody" described herein comprises a heavy chain variable region comprising the CDRs of SEQ ID NO: 4, 14, or 24, and a light chain variable region comprising the CDRs of SEQ ID NO: 9, 19, or 29.

[0088] T cells or T lymphocytes are a class of lymphocytes that play a central role in cell-mediated immunity. The specificity of T cell responses is mediated by the recognition of antigens by the TCR, which are presented in the context of the major histocompatibility complex (MHC). As part of the TCR, the CD3 receptor complex is a protein complex that includes the CD3γ (gamma) chain, the CD3δ (delta) chain, and two CD3ε (epsilon) chains present on the cell surface and is involved in the activation of cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). Clustering of CD3 on T cells, such as by immobilized anti-CD3 antibodies, leads to the activation of the T cell, which resembles the engagement of the T cell receptor but is independent of the typical specificity of its clone.

[0089] CD3 has been found to bind to the membrane of all mature T cells. This high specificity and the presence of CD3 at various stages of T cell development make it a useful immunohistochemical marker of T cells in tissue sections. It is generally and advantageously contemplated that the antibody construct according to the present invention shows little non-specific T cell activation, which is not required for specific immunotherapy. This means that the risk of side effects is reduced.

[0090] As used herein, an "anti-CD3 antibody" refers to an antibody that binds to CD3. In one embodiment, an "anti-CD3 antibody" as used herein comprises a heavy chain variable region comprising the CDRs of SEQ ID NO: 34 or 44 and a light chain variable region comprising the CDRs of SEQ ID NO: 39 or 49.

[0091] As described herein, the terms "bispecific antibodies against CD79b and CD3", "bispecific antibodies specifically binding to CD79b and CD3", "anti-CD79b x CD3 bispecific antibodies", "CD79b / CD3 bispecific antibodies" and the like refer to bispecific antibodies capable of binding to targets CD79b and CD3 with sufficient affinity, which can recruit T cells and allow redirected lysis of the target cells. The bound T cells are capable of continuously lysing the target cells and are not subject to immune evasion mechanisms that prevent peptide antigen processing and presentation or clonal T cell differentiation. In one embodiment, the "bispecific antibodies against CD79b and CD3" described herein comprise a heavy chain variable region comprising the CDRs of SEQ ID NO: 4, 14, or 24 targeting CD79b and a light chain variable region comprising the CDRs of SEQ ID NO: 9, 19, or 29 targeting CD3, and a heavy chain variable region comprising the CDRs of SEQ ID NO: 34 or 44 targeting CD3 and a light chain variable region comprising the CDRs of SEQ ID NO: 39 or 49. The antibodies may be used as diagnostic and / or therapeutic agents to target cancers that express CD79b.

[0092] The anti-CD79b x CD3 bispecific antibody provided herein has the following advantages: In some embodiments, the anti-CD79b x CD3 bispecific antibody of the present invention formed by combining different anti-CD79b antibodies with anti-CD3 antibodies having different affinities can facilitate comprehensive evaluation of the efficacy and safety of CD79b / CD3 bispecific antibodies at an early stage (in vitro screening stage) by detecting the degree of T cell activation and the release levels of several cytokines in tumor cell killing experiments by CD8+T induced by anti-CD79b x CD3 bispecific antibodies. Different molecules with similar maximum killing levels and low cytokine release levels in the in vitro screening stage can be selected for in vivo experiments and toxicity experiments, thus reducing the risk of cytokine storm when such bispecific antibodies are used in clinical settings.

[0093] In some embodiments, the CD79b / CD3 bispecific antibodies of the invention simultaneously bind to CD79b on the surface of B cell non-Hodgkin's lymphoma cells and CD3 on the surface of primary T cells and mediate T cell killing of CD79b positive tumor cells. In some embodiments, the bispecific antibodies of the invention are capable of dose-dependently inducing CD8+ T killing of B cell non-Hodgkin's lymphoma cells expressing different levels of CD79b.

[0094] In some embodiments, the anti-CD79bxCD3 antibodies of the invention mediate killing of human B-cell non-Hodgkin's lymphoma cells and activate CD8+ and CD4+ T cells isolated from PBMCs in a dose-dependent manner. In some embodiments, the antibodies of the invention enhance the proliferative capacity of human CD8+ and CD4+ T cells.

[0095] In some embodiments, the antibodies of the invention are effective in inhibiting tumor growth, where tumor growth inhibition may be 67% or even 100% compared to controls.

[0096] In one embodiment of the invention, anti-CD79bxCD3 bispecific antibodies having amino acid changes are encompassed herein, which include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0097] In preferred embodiments, the amino acid changes described herein occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid changes described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0098] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the replacement of an amino acid with another amino acid of the same class, such as replacing an acidic amino acid with another acidic amino acid, a basic amino acid with another basic amino acid, or a neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the table below. [Table 2]

[0099] In certain embodiments, the substitutions occur in the CDRs of the antibody. Generally, the resulting variant has modified (e.g., improved) certain biological properties (e.g., increased affinity) compared to the parent antibody and / or substantially retains certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody.

[0100] In certain embodiments, the antibodies provided herein are modified to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites of an antibody can be conveniently accomplished by modifying the amino acid sequence to create or remove one or more glycosylation sites. If the antibody contains an Fc region, the carbohydrate attached to it can be modified. In some applications, it may be useful to remove undesired modifications to glycosylation sites, for example, removing the fucose module to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) (see Shield et al., (2002) JBC, 277:26733). In other applications, galactosidyl modifications can be made to modify complement-dependent cytotoxicity (CDC).

[0101] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (such as a human IgG1, IgG2, IgG3, or IgG4 Fc region) that comprises an amino acid modification (such as a substitution) at one or more amino acid positions. For examples of Fc variants, see U.S. Patent No. 7,332,581, U.S. Patent No. 6,737,056, U.S. Patent No. 6,737,056; WO 2004 / 056312 and Shields et al., J. Biol. Chem., 9(2):6591-6604 (2001), U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol., 164:4178-4184 (2000), U.S. Patent No. 7,371,826, Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0102] In certain embodiments, it may be necessary to generate antibodies modified by cysteine ​​engineering, such as "sulfoMAbs," in which one or more residues of an antibody are replaced by a cysteine ​​residue. Cysteine ​​modified antibodies can be generated, for example, as described in U.S. Patent No. 7,521,541.

[0103] In certain embodiments, the antibodies provided herein can be further modified to include other non-proteinaceous moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, glucan, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and glucan or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof.

[0104] III. Nucleic Acids of the Invention and Vectors and Host Cells Containing Them In one aspect, the invention provides nucleic acids encoding any of the above-mentioned anti-CD79b antibodies, anti-CD3 antibodies, and anti-CD79bxCD3 antibodies, or antigen-binding fragments thereof. The invention also encompasses nucleic acids that hybridize to the above-mentioned nucleic acids under stringent conditions, nucleic acids that have one or more substitutions (e.g., conservative substitutions), deletions, or insertions compared to the above-mentioned nucleic acids, or nucleic acid sequences that have at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to the above-mentioned nucleic acids.

[0105] In another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid. In a preferred embodiment, the vector is an expression vector. It is well understood by those skilled in the art that vectors commonly used in the technical field to which the present invention belongs may be applied to the present invention.

[0106] In one embodiment, the invention provides a host cell comprising the nucleic acid or vector. Suitable host cells for cloning or expressing the antibody-encoding vector include prokaryotic or eukaryotic cells as described herein. In one embodiment, the host cell is a eukaryotic cell. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells), and other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0107] IV. Pharmaceutical Compositions In some embodiments, the invention provides a composition comprising any of the anti-CD79bxCD3 antibodies or antigen-binding fragments thereof described herein, preferably the composition is a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical auxiliary material. In one embodiment, the composition (e.g., a pharmaceutical composition) comprises a combination of an anti-CD79bxCD3 antibody or antigen-binding fragment thereof of the invention and one or more other therapeutic agents (e.g., a chemotherapeutic agent, a cytotoxic agent, a vaccine, another antibody, an anti-infective active agent, a small molecule drug, or an immunomodulatory agent).

[0108] The pharmaceutical composition of the present invention may further comprise one or more other active ingredients as required for the specific indication to be treated, preferably active ingredients that do not adversely affect each other's activity.For example, it may be desirable to provide other anti-cancer active ingredients, such as chemotherapeutic agents, cytotoxic agents, vaccines, other antibodies, anti-infectious disease active agents, small molecule drugs, or immunomodulators.The active ingredients are suitably combined in an amount effective for the intended purpose. EXAMPLES

[0109] The following examples are described to help understand the present invention. The examples are not intended to limit the scope of protection of the present invention in any way, and should not be interpreted as limiting, and a person skilled in the art can make various modifications according to the description of the specification of this application.

[0110] Unless otherwise indicated, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology known in the art are used in the practice of the present invention. Descriptions of such methods can be found, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual (3 rd Ed.,2001);Sambrook et al.,Molecular Cloning:A Laboratory Manual(2 ndEd.,1989);Maniatis et al.,Molecular Cloning:A Laboratory Manual(1982);Ausubel et al.,Current Protocols in Molecular Biology(John Wiley and Sons,updated in July 2008);Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience;Glover,DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford,1985);Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Transcription and Translation(B.Hames&S.Higgins,Eds.,1984);Perbal,A Practical Guide to Molecular Cloning(1984);Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Current Protocols in Immunology (QE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991); Annual Review of Immunology, and Advances in Immunolog.

[0111] Molecular structure design of the antibody of the present invention In one aspect, the present invention designs monospecific antibodies targeting CD79b that can bind to CD79b on the surface of B-cell non-Hodgkin's lymphoma cells. Humanization is performed based on five anti-CD79b antibodies (34F1, 11G10, 38D9, 54H6, and 60D9) to obtain five humanized anti-CD79b antibodies (hz34F1.14, hz11G10.9.p1, hz38D9B3.11, hz54H6A3.13, and hz60D9H6.2).

[0112] In another embodiment, the present invention designs T cell engager bispecific antibodies that simultaneously target CD79b and CD3, which can simultaneously bind to CD79b on the surface of B cell non-Hodgkin's lymphoma cells and CD3 on the surface of T cells. Based on three anti-CD79b antibodies (38D9B3.11, 11G10.9.p1, and 34F1.14) and two anti-CD3 antibodies with different affinities (sp34.24 and sp34.87), bispecific antibodies targeting both CD79b and CD3 were designed.

[0113] Six bispecific antibody molecules with 1+1 format (38D9B3.11 / sp34.87, 38D9B3.11 / sp34.24, 11G10.9.p1 / sp34.87, 11G10.9.p1 / sp34.24, 34F1.14 / sp34.87, and 34F1.14 / sp34.24) are designed. The mispairing of the heavy chains of the bispecific antibodies is resolved using the Fc "knobs-in-holes" technology, where Fc is the heavy chain constant region of IgG1, and the amino acid mutations L234A and L235A (according to Kabat's "EU" numbering) that attenuate effector function are introduced.

[0114] Example 1. Preparation of hybridoma cells immunization CD79b protein (sino, catalog number 29750-H08H) was emulsified with Freund's complete adjuvant (Sigma, catalog number F5881) and used to immunize Balb / c mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.), and two weeks later, CD79b protein was emulsified with Freund's incomplete adjuvant (Sigma, catalog number F5506) and used to immunize the mice three times by intramuscular injection (50 μg protein per mouse) once every two weeks.

[0115] cell fusion When the serum titer met the requirement, the mouse spleen was harvested to prepare a B lymphocyte suspension. The B lymphocyte suspension and SP2 / 0 myeloma cells (ATCC) were mixed in a ratio of 1:2 to 1:1 and subjected to electric fusion. The fused cells were transferred from the electrode dish to a 50 mL centrifuge tube and diluted with HAT-containing medium to 1–2 × 10 4 The cells were diluted to 100 cells / mL, and the cell suspension was added to a 96-well plate at 100 μL / mL. The screening medium was replaced on the 7th day after fusion, and after 10 days of culture (or longer depending on the cell growth state), the resulting products were detected by flow cytometry (FACS) to select positive clones.

[0116] Screening for positive clones of hybridoma cells (FACS) Hybridoma cells specifically expressing anti-CD79b antibodies were selected using a flow cytometer (FACS). The cells to be detected (Ramos) were counted and 1 × 10 6The cells were diluted to 100 cells / mL and added to a U-bottom 96-well plate at 100 μL / well. The cells were centrifuged at 500g for 5 min to remove the cell culture medium. The hybridoma culture supernatant in the 96-well plate was added to the U-bottom plate at 100 μL / well, the cells were resuspended, and left on ice for 30 min. The suspension was centrifuged at 500g for 5 min to remove the supernatant, and the cells were washed once with PBS. The PBS was removed by centrifugation at 500g for 5 min, and FITC-labeled anti-mouse Fab secondary antibody (Jackson Immunoresearch, Cat. No. 115-545-006, diluted 1:500 in PBS) was added at 100 μL / well, and 100 μL of PE-labeled anti-human Fc secondary antibody (Biolegend, Cat. No. 409304) was added as a positive control antibody. The resulting mixture was incubated on ice in the dark for 30 min. The mixture was centrifuged at 500g for 5 min to remove the supernatant, and the cells were washed once with PBS. The cells were resuspended in 50 μL of 1×PBS, and the suspension was loaded for FACS assay. Positive clones were subcloned and monoclonal cells were selected.

[0117] Subcloning of positive hybridoma cells Subcloning procedure: Culture medium was added to 96-well plates at 200 μL / well. Compared with the screening medium, this medium contains the same components, except that HAT was replaced with HT (Gibco, Cat. No. 11067-030). The cells in the positive wells selected by fusion were prepared into a cell suspension, then the cell suspension was added to the first column at 100 μL / well, then, after mixing well, the cell suspension of the first column was added to the second column at 100 μL / well, then, after mixing well, the cell suspension of the second column was added to the next column at 100 μL / well. The above procedure was repeated. The 96-well plate was left for 30 minutes, and the cells were observed and counted under a microscope. The corresponding volume containing about 100 cells was added to 20 mL of culture medium, mixed well, and plated at 200 μL / well. After one week, the cells were observed under a microscope, and the monoclonal wells were identified and marked. When the cell confluence in each well reached 50% or more, the cells were detected by the same method as the FACS screening method described above to select the target positive wells, and the affinity of the resulting hybridoma cell supernatant to the antigen was detected by using biolayer interference method (ForteBio). All detection results are shown in Table 1. [Table 3]

[0118] Example 2. Preparation of recombinant mouse antibodies The gene sequences of the light and heavy chains of the antibody were extracted from the hybridoma candidate clones obtained in Example 1. 6Freshly cultured cells were harvested for RNA extraction (Macherey-Nagel, Cat. No. 740984.250). Reverse transcription was performed using PrimeScript II 1st Strand cDNA Synthesis Kit (Takara) to obtain cDNA. The upstream primer was designed using the base sequence located in the 5'-end FR1 region, and the downstream primer was designed using the base located in the antibody constant region or FR4 region, and then amplification was performed to obtain gene fragments of the antibody light chain variable region and heavy chain variable region. The gene fragments were ligated into T vectors (Mighty TA-cloning Kit, Takara), monoclones were selected for sequencing, and the sequencing results were analyzed and compared using MEGA7 software. Finally, recombinant mouse antibodies were constructed according to the specificity of the antibody light chain variable region sequence and heavy chain variable region sequence and the affinity of the antibody.

[0119] The gene fragments of the antibody light chain variable region and heavy chain variable region were respectively ligated into the pcDNA3.1 vector (IgG1 subtype was selected as the constant region) using homologous recombinase from Nanjing Vazyme Biotech Co., Ltd. (Exnase® II, catalog number C112-01) to obtain expression plasmids for the antibody light chain and heavy chain.

[0120] Then, the light chain plasmid and the heavy chain plasmid of the same antibody were mixed at a molar ratio of 1:1 and transfected into 293F cells with polyethyleneimine (PEI) (Polysciences, catalog no. 23966). After 5–7 days of culture, when the cell viability was less than 60%, the cell culture supernatant was collected and purified by protein A affinity column to obtain the monoclonal antibody.

[0121] Example 3. Binding kinetics of chimeric antibodies of the present invention to antigens as determined by biolayer interferometry The equilibrium dissociation constant (KD) for the binding of the antibodies of the invention to CD79b was determined by biolayer interferometry (ForteBio). A conventional ForteBio affinity assay was performed (Estep, P., et al., "High throughput solution based measurement of antibody-antigen affinity and epitope binning", MAbs, 2013.5(2):270-8).

[0122] Briefly, AMQ (Pall, 1506091) sensors were equilibrated offline in assay buffer for 30 min, then online for 60 s to establish a baseline. Each of the purified antibodies obtained above was loaded online into an AHQ sensor (ForteBio) for ForteBio affinity assay. The antibody-loaded sensor was then exposed to the antigen CD79b, after which the sensor was transferred to assay buffer to measure the dissociation rate. The KD values ​​were analyzed using ForteBio analysis software. The results of the antibody affinity assay are shown in Table 2. [Table 4]

[0123] Example 4. Binding assays of chimeric antibodies and B-cell non-Hodgkin's lymphoma cell lines To verify whether the antibody of the present invention can bind to antigens expressed on the cell surface, in this study, flow cytometry was used to detect the binding of the antibody to Ramos cells and BJAB cells. The experimental process is as follows: Ramos (ATCC, Catalog No. CRL-1596) and BJAB (AddexBio, USA, Catalog No. C0003016) were passaged according to conventional procedures. After centrifugation and resuspension, the cells were counted and 4 × 10 6The cells were adjusted to a density of 1000 cells / mL, poured into a filling tank, and seeded into a 96-well plate at 50 μL / well using a multichannel pipettor. 50 μL of gradient-diluted antibody sample was added to 50 μL of cells in each well, and the mixture was then incubated at 37°C with 5% CO2 in an incubator for 30 minutes. The mixture was centrifuged at 400g for 5 minutes, then 200 μL of PBS was added, and the resulting mixture was centrifuged at 400g for 5 minutes. This procedure was repeated three times. Goat anti-human IgGPE (1:200) was added, and the mixture was incubated in the dark at room temperature for 30 minutes. The mixture was washed twice with 200 μL of PBS, centrifuged, and then resuspended in 100 μL of PBS, and the values ​​were read using a flow cytometer. The values ​​were fitted to obtain a curve, and the EC50 value was calculated.

[0124] In experiments performed using the assays described above, binding of the chimeric antibodies to CD79b on the surface of Ramos cells is shown in FIG. 1, and binding of the chimeric antibodies to CD79b on the surface of BJAB cells is shown in FIG.

[0125] Example 5. Humanization of chimeric antibodies The chimeric antibodies obtained in Example 2 were humanized according to conventional methods, and thus the humanized antibodies hz34F1.14, hz11G10.9.p1, hz38D9B3.11, hz54H6A3.13, and hz60D6H6.2, as well as their CDR sequences, light and heavy chain variable region sequences, and light and heavy chain amino acid sequences are shown in Example 9.

[0126] Example 6. Binding kinetics of humanized antibodies to antigens determined by ForteBio The equilibrium dissociation constants (KD) of the binding of the humanized antibodies of the present invention to human CD79b were determined by ForteBio. The assay was the same as in Example 3, and the data are shown in Table 3. [Table 5]

[0127] As can be seen from the affinities of the humanized antibodies to CD79b listed in Table 3, the humanized antibodies substantially maintained the binding of the previous chimeric antibodies to CD79b.

[0128] Example 7. Binding assays of humanized antibodies and B-cell non-Hodgkin's lymphoma cell lines Binding of humanized antibodies to Ramos and BJAB cells was detected by flow cytometry. The assay was the same as in Example 4. Binding of humanized antibodies to CD79b on the surface of Ramos cells is shown in Figure 3, and binding of humanized antibodies to CD79b on the surface of BJAB cells is shown in Figure 4.

[0129] Example 8. Effect of antibody-mediated ADCC effect In this example, the efficacy of the obtained antibodies in mediating the ADCC effect and thereby eliminating tumor cells is examined. In this study, Promega's Jurkat-ADCC NF-AT luciferase effector cell line (hereinafter referred to as ADCC effector cells) was used. The ADCC activity of the antibodies was detected by detecting the activation of the NF-AT signal. The specific experimental process is as follows:

[0130] 1) Preparation of cells The target cells 293T-hCD79b and ADCC effector cells were counted. The supernatant was removed by centrifugation. The cells were washed twice with PBS and resuspended in detection medium (1640 medium (Gibco) containing 5% low IgG serum). The concentration of ADCC effector cells was 2 × 10 7 Adjust the concentration of target cells to 2 x 10 cells / mL. 6 The concentration of cells was adjusted to 10:1 cells / mL. The two cell types were mixed at a 1:1 ratio, resulting in a final effector to target ratio of 10:1. 2) Plating: The mixed cells were plated at 50 μL / well. 3) Antibody dilution: Antibody samples were gradient diluted respectively. The final concentration in the first well was 300 nM, and 4-fold dilutions were performed to obtain a gradient of 10. 50 μL of antibody was added to 50 μL of cells in each well. 4) The above 96-well plate was incubated in an incubator at 37° C. for 7 hours. 5) After 7 hours, the 96-well plate was removed and thawed luciferase assay reagent was added at 100 μL / well. The cells were incubated at room temperature for 20 minutes and detected using a microplate reader. The concentration-dependent curve was fitted using GraphPad.

[0131] As shown in FIG. 5, the humanized antibodies can effectively activate the NF-AT signaling pathway downstream of ADCC.

[0132] Example 9. Expression and purification of bispecific antibodies of the invention A total of six bispecific antibodies, 38D9B3.11 / sp34.87, 38D9B3.11 / sp34.24, 11G10.9.p1 / sp34.87, 11G10.9.p1 / sp34.24, 34F1.14 / sp34.87, and 34F1.14 / sp34.24, were constructed separately by combining three different anti-human CD79b clones, 38D9B3.11, 11G10.9.p1, and 34F1.14 with two different anti-human CD3 clones, HzSP34.87 and HzSP34.24, according to the combinations shown in Table 4. Each bispecific antibody was transiently expressed in HEK293 cells. After 7 days of expression, the resulting cell fermentation broth was filtered and clarified, and then a protein A column (Hitrap Mabselect Sure, GE11-0034-95) was used for capture to obtain half antibodies. After detecting the concentration of half antibodies by the A280 method, the half antibodies were mixed in a 1:1 molar ratio. An appropriate amount of reducing agent GSH was added, and the mixture was reacted at room temperature overnight. The reducing agent was then removed by ultrafiltration to stop the reaction. Then, precision purification was performed using MonoS cation exchange chromatography. The eluate was ultrafiltered, and the buffer was exchanged into PBS (Gibco, 70011-044). The molecular weight was determined by mass spectrometry, and the purity was determined by SEC-HPLC. CD79b.A7v14b / 38E4v1 is a CD79b / CD3 bispecific antibody in patent US20180327492, and was expressed using the same method. The above seven bispecific antibodies obtained were used in the following examples. [Table 6]

[0133] The specific amino acid sequence listing (CDR regions defined by the Kabat rules) is as follows:

[0134] Amino acid sequences of the heavy and light chains of 38D9B3.11 The heavy chain CDR sequences are as follows: HCDR1 (SEQ ID NO: 1): GYTFTDYSMH; HCDR2 (SEQ ID NO:2): WINTETGEPSYADDFKG; HCDR3 (SEQ ID NO:3): GPY; Heavy chain variable region VH sequence (SEQ ID NO:4): QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLEWMGWINTETGEPSYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCYYGPYWGQGTLVTVSS; Full length heavy chain sequence (SEQ ID NO:5): QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLEWMGWINTETGEPSYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCYYGPYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. Light chain CDR sequences: LCDR1 (SEQ ID NO: 6): KASQSVDHDVDSYMD; LCDR2 (SEQ ID NO: 7): SASNLES; LCDR3 (SEQ ID NO: 8): QQINEYPYT; Light chain variable region VL sequence (SEQ ID NO:9): EIVLTQSPATLSLSPGERATLSCKASQSVDHDVDSYMDWYQQKPGQAPRLLIYSASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQINEYPYTFGGGTKVEIK; Full length light chain sequence (SEQ ID NO:10): EIVLTQSPATLSLSPGERATLSCKASQSVDHDVDSYMDWYQQKPGQAPRLLIYSASNLESGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQINEYPYTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0135] Amino acid sequences of the heavy and light chains of 11G10.9.p1 The heavy chain CDR sequences are as follows: HCDR1 (SEQ ID NO: 11): GYTFTDYWVN; HCDR2 (SEQ ID NO: 12): MIDPSDSETHYNQMFND; HCDR3 (SEQ ID NO: 13): SNYY; Heavy chain variable region VH sequence (SEQ ID NO:14): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWVNWVRQAPGQGLEWMGMIDPSDSETHYNQMFNDRVTMTVDTSTSTVYMELSSLRSEDTAVYYCTRSNYYWGQGTLVTVSS; Full length heavy chain sequence (SEQ ID NO:15): QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWVNWVRQAPGQGLEWMGMIDPSDSETHYNQMFNDRVTMTVDTSTSTVYMELSSLRSEDTAVYYCTRSNYYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The light chain CDR sequences are as follows: LCDR1 (SEQ ID NO: 16): KSSQSLLDSEGKTYLN; LCDR2 (SEQ ID NO: 17): LVSKLDS; LCDR3 (SEQ ID NO: 18): GTHFPLT; Light chain variable region VL sequence (SEQ ID NO: 19): DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSEGKTYLNWLLQKPGQSPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPLTFGGGTKVEIK; Full length light chain sequence (SEQ ID NO:20): DIVMTQTPLSLSVTPGQPASISCKSSQSLLDSEGKTYLNWLLQKPGQSPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPLTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0136] Amino acid sequences of the heavy and light chains of 34F1.14 The heavy chain CDR sequences are as follows: HCDR1 (SEQ ID NO:21): GYSFTTYWMN; HCDR2 (SEQ ID NO: 22): MIDPSDSETHYNHLFKD; HCDR3 (SEQ ID NO: 23): AIGY; Heavy chain variable region VH sequence (SEQ ID NO:24): QVQLVQSGAEVKKPGASVKVSCKASGYSFTTYWMNWVRQAPGQGLTWMGMIDPSDSETHYNHLFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCTRAIGYWGQGTLVTVSS; Full length heavy chain sequence (SEQ ID NO:25): QVQLVQSGAEVKKPGASVKVSCKASGYSFTTYWMNWVRQAPGQGLTWMGMIDPSDSETHYNHLFKDRVTMTVDKSTSTAYMELSSLRSEDTAVYYCTRAIGYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The light chain CDR sequences are as follows: LCDR1 (SEQ ID NO:26): KSSLSLLDSEGKTYLN; LCDR2 (SEQ ID NO:27): LVSKLDS; LCDR3 (SEQ ID NO:28): WQGTHFPLT; Light chain variable region VL sequence (SEQ ID NO:29): DVVMTQSPLSLPVTLGQPASISCKSSLSLLDSEGKTYLNWFQQRPGQSPRPLISLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPLTFGGGTKVEIK; Full length light chain sequence (SEQ ID NO:30): DVVMTQSPLSLPVTLGQPASISCKSSLSLLDSEGKTYLNWFQQRPGQSPRPLISLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPLTFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0137] Amino acid sequences of the heavy and light chains of HzSP34.87 The heavy chain CDR sequences are as follows: HCDR1 (SEQ ID NO:31): GFTFNTYAMN; HCDR2 (SEQ ID NO: 32): RIRSKYNNYATYYAD; HCDR3 (SEQ ID NO: 33): HYNFGQSYVSWFAY; Heavy chain variable region VH sequence (SEQ ID NO:34): EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKSTLYLQMNSLKTEDTAVYYCARHYNFGQSYVSWFAYWGQGTTVTVSS; Full length heavy chain sequence (SEQ ID NO:35): EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKSTLYLQMNSLKTEDTAVYYCARHYNFGQSYVSWFAY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The light chain CDR sequences are as follows: LCDR1 (SEQ ID NO:36): RSSTGAVTTSNYAN; LCDR2 (SEQ ID NO: 37): GTNKRAP; LCDR3 (SEQ ID NO:38): ALWYSNLWV; Light chain variable region VL sequence (SEQ ID NO:39): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGDKAALTLLGAQPEDEAEYYCALWYSNLWVFGQGTKLTVL; Full length light chain sequence (SEQ ID NO:40): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGDKAALTLLGAQPEDEAEYYCALWYSNLWVFGQGTKLTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0138] Amino acid sequences of the heavy and light chains of HzSP34.24 The heavy chain CDR sequences are as follows: HCDR1 (SEQ ID NO: 41): GFTFNTYAMN; HCDR2 (SEQ ID NO: 42): RIRSKYNNYATYYADSVKD; HCDR3 (SEQ ID NO: 43): HGNFGQSYVSWFAY; Heavy chain variable region VH sequence (SEQ ID NO:44): EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKSTLYLQMNSLKTEDTAVYYCARHGNFGQSYVSWFAYWGQGTTVTVSS; Full length heavy chain sequence (SEQ ID NO:45): EVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKSTLYLQMNSLKTEDTAVYYCARHGNFGQSYVSWFAY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The light chain CDR sequences are as follows: LCDR1 (SEQ ID NO: 46): RSSTGAVTTSNYAN; LCDR2 (SEQ ID NO: 47): GTNKRAP; LCDR3 (SEQ ID NO: 48): ALWYSNLWV; Light chain variable region VL sequence (SEQ ID NO:49): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGDKAALTLLGAQPEDEAEYYCALWYSNLWVFGQGTKLTVL; Full length light chain sequence (SEQ ID NO:50): QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPGVPARFSGSLLGDKAALTLLGAQPEDEAEYYCALWYSNLWVFGQGTKLTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS.

[0139] CD79b.A7v14b heavy and light chain amino acid sequences The heavy chain CDR sequences are as follows: HCDR1 (SEQ ID NO:51): GYTFTTYYMN; HCDR2 (SEQ ID NO:52): MIDPSDSETHYNQKFQG; HCDR3 (SEQ ID NO:53): SLAF; Heavy chain variable region VH sequence (SEQ ID NO:54): EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMNWVRQAPGQGLEWIGMIDPSDSETHYNQKFQGRATLTVDTSTSTAYLELSSLRSEDTAVYYCSRSLAFWGQGTLVTVSS; Full length heavy chain sequence (SEQ ID NO:55): EVQLVQSGAEVKKPGASVKVSCKASGYTFTTYYMNWVRQAPGQGLEWIGMIDPSDSETHYNQKFQGRATLTVDTSTSTAYLELSSLRSEDTAVYYCSRSLAFWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The light chain CDR sequences are as follows: LCDR1 (SEQ ID NO:56): KSSQSLLDSDGKTYLN; LCDR2 (SEQ ID NO:57): LVSKLDS; LCDR3 (SEQ ID NO:58): WQGTHFPQT; Light chain variable region VL sequence (SEQ ID NO:59): DIVMTQTPLSLPVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGQGTKVEIK; Full length light chain sequence (SEQ ID NO:60): DIVMTQTPLSLPVTPGQPASISCKSSQSLLDSDGKTYLNWLLQKPGQSPQSLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0140] Amino acid sequences of the heavy and light chains of 38E4v1 The heavy chain CDR sequences are as follows: HCDR1 (SEQ ID NO:61): GFTFTSYYIH; HCDR2 (SEQ ID NO:62):WIYPENDNTKYNEKFKD; HCDR3 (SEQ ID NO:63): DGYSRYYFDY; Heavy chain variable region VH sequence (SEQ ID NO:64): EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQGTLVTVSS; Full length heavy chain sequence (SEQ ID NO:65): EVQLVQSGAEVKKPGASVKVSCKASGFTFTSYYIHWVRQAPGQGLEWIGWIYPENDNTKYNEKFKDRVTITADTSTSTAYLELSSLRSEDTAVYYCARDGYSRYYFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. The light chain CDR sequences are as follows: LCDR1 (SEQ ID NO:66): KSSQSLLNSRTRKNYLA; LCDR2 (SEQ ID NO:67): WTSTRKS; LCDR3 (SEQ ID NO:68): KQSFILRT; Light chain variable region VL sequence (SEQ ID NO:69): DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSFILRTFGQGTKVEIK; Full length light chain sequence (SEQ ID NO:70): DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWTSTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSFILRTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0141] The amino acid sequences of the heavy and light chains of polmab are: Full length heavy chain sequence (SEQ ID NO:71): EVQLVESGGGLVQPGGSLRLSCAASGYTFSSYWIEWVRQAPGKGLEWIGEILPGGGDTNYNEIFKGRATFSADTSKNTAYLQMNSLRAEDTAVYYCTRRVPIRLDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Full length light chain sequence (SEQ ID NO:72): DIQLTQSPSSLSASVGDRVTITCKASQSVDYEGDSFLNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPLTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0142] Example 10. Affinity Assays of Antibodies of the Invention Surface plasmon resonance (BiacoreT200) was used to determine the binding kinetics of the exemplary anti-CD79b / CD3 antibodies of the present invention to human CD79b, human CD3, and cynomolgus monkey CD3. (1) Affinity assay of antibodies to human CD79b-his (Sino biological): 50 mM N-hydroxysuccinimide (NHS) and 200 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were freshly mixed and activated for 420 seconds at a flow rate of 10 μL / min. Human CD79b-His antigen was then diluted with 10 mM acetate (pH 5.0) to a concentration of 1 μg / mL before the antigen solution was subjected to coupling, with a value of approximately 50 RU. 1 M ethanolamine was then injected at a flow rate of 10 μL / min for 420 seconds to block the remaining activation sites. The buffer used in the experiment was HBS-EP+ solution at pH 7.4, and under high performance mode, the antibodies obtained after 2-fold gradient dilution (0-80nM) were injected into channels 1, 2, 3 and 4 of the chip at a flow rate of 30μL / min according to the ascending order based on concentration, respectively, one cycle was used for the measurement of one concentration, the binding time was 180 seconds, and the dissociation time was 600 seconds. (2) Affinity assay of antibodies against human CD3E and CD3G-biotin (Sino biological) and monkey CD3E and CD3G-biotin (Sino biological): 50mM NHS and 200mM EDC were freshly mixed and activated for 420 seconds at a flow rate of 10μL / min. Then, streptavidin (Thermo Fisher Scientific) protein was diluted with 10mM acetate (pH 4.5) to a concentration of 10μg / mL, and then the antigen solution was subjected to coupling, the value was about 3000RU. Then, 1M ethanolamine was injected at a flow rate of 10 μL / min for 420 seconds to block the remaining activation sites. Human CD3E and CD3G biotin proteins and monkey CD3E and CD3G biotin proteins were immobilized on the chip channels coupled with streptavidin protein, respectively. The antibodies obtained after two-fold gradient dilution (0-16 nM or 0-40 nM or 0-200 nM) were injected into channels 1, 2, 3, and 4, respectively, according to the ascending order based on concentration.The chip was injected at a flow rate of 30 μL / min, one cycle was used for the measurement of one concentration, the association time was 180 s, and the dissociation time was 300 s.

[0143] In experiments performed using the above assays, the affinity of 38D9B3.11 / sp34.87, 38D9B3.11 / sp34.24, 11G10.9.p1 / sp34.87, 11G10.9.p1 / sp34.24, 34F1.14 / sp34.87, 34F1.14 / sp34.24, and CD79b.A7v14b / 38E4v1 for human CD79b-His is shown in Table 5. The affinity of 38D9B3.11 / sp34.87, 38D9B3.11 / sp34.24, 11G10.9.p1 / sp34.87, 11G10.9.p1 / sp34.24, 34F1.14 / sp34.87, 34F1.14 / sp34.24, and CD79b.A7v14b / 38E4v1 for human CD3E and CD3G (Sino biological) and monkey CD3E and CD3GG is shown in Tables 6 and 7. [Table 7] [Table 8] [Table 9]

[0144] Example 11. Binding assays for anti-CD79b / CD3 antibodies of the invention and B-cell non-Hodgkin's lymphoma cell lines Flow cytometry was used to detect the binding ability of CD79b / CD3 bispecific antibodies to CD79b-positive B-cell non-Hodgkin's lymphoma cell lines. BJAB (AddexBio, USA, Cat. No. C0003016) and WSU-DLCL2 (Nanjing Kebai Biotechnology Co., Ltd., Cat. No. CBP60273) were cultured and passaged according to conventional procedures. After centrifugation and resuspension, cells were counted and 4 × 10 6The density was adjusted to 100 cells / mL, poured into a filling tank, and seeded into a 96-well plate at 50 μL / well using a multichannel pipettor. 50 μL of gradient diluted antibody sample was added to 50 μL of cells in each well, and the mixture was incubated for 30 min at 37 °C with 5% CO2 in an incubator. The mixture was centrifuged at 400 g for 5 min, then 200 μL of PBS was added, and the resulting mixture was centrifuged at 400 g for 5 min. This procedure was repeated three times. Goat anti-human IgGPE (1:200) was added, and the mixture was incubated in the dark at room temperature for 30 min. The mixture was washed twice with 200 μL of PBS, centrifuged, and then resuspended in 100 μL of PBS, and the values ​​were read using a flow cytometer. The values ​​were fitted to obtain a curve, and the EC50 value was calculated.

[0145] In experiments performed using the above assays, binding of exemplary antibodies to CD79b on the surface of BJAB cells, a CD79b high expressing cell line, is shown in FIG. 6, and binding of exemplary antibodies to CD79b on the surface of WSU-DLCL2 cells, a CD79b low expressing cell line, is shown in FIG. 7.

[0146] Example 12. Assay for activation of Jurkat-NFAT-Luc cells by anti-CD79b / CD3 antibodies of the present invention CD79b / CD3 bispecific antibodies simultaneously bind to CD79b on the surface of B-cell non-Hodgkin's lymphoma cells and CD3 on the surface of Jurkat-NFAT-Luc cells, and activate the downstream signaling pathway of NFAT-Luc through CD79b-dependent CD3 cross-linking. In this study, we used the luciferase reporter gene method to detect luciferase expression after overnight incubation of co-cultured Jurkat-NFAT-Luc cells and CD79b-positive cells with additional exemplary antibodies, demonstrating the activating ability of the antibodies.

[0147] Experimental Method Preparation of assay medium: 10% FBS + 90% RPMI medium 1640 Preparation of Bio-Glo Luciferase Assay System Detection Solution: Bio-Glo™ Buffer (Promega, G7940) was thawed and mixed well with Bio-Glo™ Substrate (Promega, G7940), and the mixture was stored in a -40°C refrigerator. 1. BJAB, WSU-DLCL2, NUGC4, and Jurkat-NFAT-Luc cells were each centrifuged at 300 g for 8 minutes, and the supernatant was discarded. BJAB cells and Jurkat-NFAT-Luc cells were each suspended in assay medium, and then the cell number was counted and the cell density was adjusted to 6.0 × 10 5 The tumor cells and Jurkat-NFAT-Luc cells were mixed in a 1:1 ratio for later use. 2. The cell suspension and gradient diluted test antibodies were added to 96-well white cell culture plates at 100 μL / well and the mixture was incubated overnight. 3. Remove the detection solution from the Bio-Glo Luciferase Assay System and thaw at room temperature. 4. Remove the samples incubated in step 2 and add 80 μL / well of Bio-Glo Luciferase Assay System Detection Solution. 5. The plate was read on a microplate reader (Molecular Device, SpectraMaxI3).

[0148] In experiments performed using the assays described above, the exemplary antibodies were able to activate NFAT signaling in a dose-dependent manner (see Figures 8A and 8B), and the exemplary antibodies showed no activation against non-target cells NUGC4 (see Figure 8C).

[0149] Example 13. Assay for killing of B-cell non-Hodgkin's lymphoma cell lines by anti-CD79b / CD3 antibodies of the invention The CD79b / CD3 bispecific antibody simultaneously binds to CD79b on the surface of B-cell non-Hodgkin's lymphoma cells and CD3 on the surface of primary T cells, activates T cells through CD79b-dependent CD3 crosslinking, and mediates the killing of CD79b-positive tumor cells by T cells. In this study, based on the propidium iodide (PI) staining method, the PI positivity rate of tumor cells 48 hours after the addition of an exemplary antibody to co-cultured human CD8+ T cells and CD79b-positive cells was detected, and the killing ability of human CD8+ T cells against CD79b-positive tumor cells was evaluated.

[0150] PBMCs were removed from the liquid nitrogen tank, quickly thawed at 37°C, and added dropwise to pre-warmed 1640 medium containing 10% FBS (containing 0.1% DNase) to obtain 10 mL of mixed solution. The mixed solution was centrifuged at 400g for 5 min, resuspended in 10 mL of 1640 medium containing 10% FBS, and 10 μL of DNase was added. The mixture was left at 37°C and 5% CO2 for 2 hours. Human CD8+ T cells were isolated using EasySep™ Human CD8+ T Cell Enrichment Kit, and the procedure was performed according to the manufacturer's instructions. The density of human CD8+ T cells was adjusted to 1 × 10 using 1640 medium containing 10% FBS. 6 The cells were adjusted to 10 cells / mL and used as effector cells. CD79b positive cells BJAB and WSU-DLCL2 were used as target cells, and NUGC4 was used as non-target cells. The cells were centrifuged at 400g for 5 min, resuspended in 1640 medium containing 10% FBS, and Cell Trace Far Red Cell staining solution (THERMO FISHER, C34564) was added. The mixture was incubated for 30 min, centrifuged at 400g for 5 min, and resuspended in 1640 medium containing 10% FBS, and the cell density was adjusted to 2 × 10 5The concentration was adjusted to 100 cells / mL. The tumor cell suspension and human CD8+ T cell suspension were mixed well in a ratio of 1:5, and the resulting cell suspension and gradient diluted test antibodies were added to a (standard) 96-well round-bottom culture plate (with cover) at 200 μL / well. The cells were placed in a carbon dioxide incubator and stimulated at 37°C for 48 hours. The cells were centrifuged at 400g for 5 minutes, the supernatant was removed, and PBS (containing a 1:1000 diluted propidium iodide solution) was added at 100 μL / well to resuspend the cells. The mixture was left at 4°C for 10 minutes, and the values ​​were read using a flow cytometer (BD, FACSCELESTA).

[0151] In experiments performed using the assays described above, the exemplary antibodies were able to induce killing of BJAB and WSU-DLCL2 target cells by human CD8+ T cells in a dose-dependent manner (see Figures 9A and 9B), and the exemplary antibodies showed no killing against the non-target cells NUGC4 (see Figure 9C).

[0152] Example 14. Levels of T cell activation and cytokine release upon killing of human B cell non-Hodgkin's lymphoma cell lines by anti-CD79b / CD3 antibodies of the invention In this study, gradient dilutions of exemplary antibodies were added to the co-incubation system of B-cell non-Hodgkin's lymphoma cells and human T cells, and the percentage of cells double positive for human T cells CD25 and CD69 was detected by flow cytometry after 18 hours to evaluate the degree of antibody-mediated T cell activation in the co-incubation system of human T cells and CD79b-positive tumor cells. The levels of various cytokines were simultaneously detected by using a multi-cytokine assay kit (human Th1 / Th2 / Th17, BD) to evaluate the antibody-mediated ability of T cells to release cytokines in the co-incubation system of human T cells and CD79b-positive tumor cells.

[0153] Experimental process Detection of human CD8+ T cell activation and cytokine release: PBMCs were removed from the liquid nitrogen tank, quickly thawed at 37°C, and added dropwise to pre-warmed 1640 medium containing 10% FBS (containing 0.1% DNase) to obtain 10 mL of mixed solution. The mixed solution was centrifuged at 400g for 5 min, resuspended in 10 mL of 1640 medium containing 10% FBS, and 10 μL of DNase was added. The mixture was left at 37°C and 5% CO2 for 2 hours. Human CD8+ T cells were isolated using EasySep™ Human CD8+ T Cell Enrichment Kit, and the procedure was performed according to the manufacturer's instructions. The density of human CD8+ T cells was adjusted to 1 × 10 using 1640 medium containing 10% FBS. 6 The tumor cells were resuspended in 1640 medium containing 10% FBS and the density was adjusted to 2 × 10 5 The tumor cell suspension and human CD8+ T cell suspension were mixed well in a 1:1 ratio, and the resulting cell suspension and gradient-diluted test antibodies (the highest concentration is 100 nM, 3-fold dilution, 12 gradients) were added to a (standard) 96-well round-bottom culture plate (with cover) at 200 μL / well. The cells were placed in a carbon dioxide incubator and stimulated at 37°C for 18 hours. The cells were centrifuged at 400g for 5 minutes, and PBS (containing 1:100 diluted APC anti-human CD8a, FITC anti-human CD69, and PE anti-human CD25) was added at 100 μL / well to resuspend the cells. The mixture was left at 4°C for 30 minutes, then washed and read using a flow cytometer (BD, FACSCELESTA). Meanwhile, the supernatant was collected, and the contents of cytokines TNFα and IFN-γ were detected according to the instructions of the assay kit (human Th1 / Th2 / Th17 kit, BD).

[0154] Detection of human CD4+ T cell activation: PBMCs were removed from the liquid nitrogen tank, quickly thawed at 37°C, and added dropwise to pre-warmed 1640 medium containing 10% FBS (containing 0.1% DNase) to obtain 10 mL of mixed solution. The mixed solution was centrifuged at 400g for 5 min, resuspended in 10 mL of 1640 medium containing 10% FBS, and 10 μL of DNase was added. The mixture was left at 37°C and 5% CO2 for 2 hours. Human CD4+ T cells were isolated using EasySep™ Human CD4+ T Cell Enrichment Kit, and the procedure was performed according to the manufacturer's instructions. The density of human CD4+ T cells was adjusted to 1 × 10 using 1640 medium containing 10% FBS. 6 The tumor cells were resuspended in 1640 medium containing 10% FBS and the density was adjusted to 2 × 10 5 The tumor cell suspension and human CD4+ T cell suspension were mixed well in a 1:1 ratio, and the resulting cell suspension and gradient diluted test antibodies were added to a (standard) 96-well round-bottom culture plate (with cover) at 200 μL / well. The cells were placed in a carbon dioxide incubator and stimulated at 37°C for 18 hours. The cells were centrifuged at 400g for 5 minutes, the supernatant was removed, and PBS (containing 1:100 diluted APC anti-human CD4, FITC anti-human CD69, and PE anti-human CD25) was added at 100 μL / well to resuspend the cells. The mixture was left at 4°C for 30 minutes, then washed and the values ​​were read using a flow cytometer (BD, FACSCELESTA).

[0155] result In experiments conducted using the above-described assays, the extent of CD8+ activation upon exemplary antibody-induced killing of BJAB cells by CD8+ T cells is shown in FIG. 10A, the extent of CD8+ activation upon exemplary antibody-induced killing of Ramos cells by CD8+ T cells is shown in FIG. 10B, the extent of CD8+ activation upon exemplary antibody-induced killing of WSU-DLCL2 cells by CD8+ T cells is shown in FIG. 10C, and the extent of CD8+ activation upon exemplary antibody-induced killing of NUGC4 cells by CD8+ T cells is shown in FIG. 10D. The level of IFN-γ release upon exemplary antibody-induced killing of various cells by CD8+ T cells is shown in FIG. 11, and the level of TNFα release upon exemplary antibody-induced killing of various cells by CD8+ T cells is shown in FIG. 12. The degree of CD4+ activation during co-incubation of CD4+ T cells and BJAB cells induced by the exemplary antibodies is shown in Figure 13A, the degree of CD4+ activation during co-incubation of CD4+ T cells and Ramos cells induced by the exemplary antibodies is shown in Figure 13B, the degree of CD4+ activation during co-incubation of CD4+ T cells and WSU-DLCL2 cells induced by the exemplary antibodies is shown in Figure 13C, and the degree of CD4+ activation during co-incubation of CD4+ T cells and NUGC4 cells induced by the exemplary antibodies is shown in Figure 13D. In the presence of CD79b-positive tumor cells, all of the exemplary antibodies can activate CD8+ and CD4+ T cells isolated from PBMCs in a dose-dependent manner, and the degree of T cell activation correlates to some extent with the affinity for CD3. The higher the affinity for CD3, the higher the ability to activate T cells.

[0156] Example 15. Assay for promotion of human CD8+ T cell proliferation capacity by exemplary antibodies CD8+ T cells isolated from PBMCs were labeled with CellTrace Far Red cell proliferation kit, and the labeled human CD8+ T cells were co-cultured with tumor cells, followed by the addition of gradient-diluted exemplary antibodies, and CD8+ T cell proliferation was detected after 96 hours using a flow cytometer.

[0157] Experimental procedure Isolation of human CD8+ T cells: PBMCs were removed from the liquid nitrogen tank, thawed quickly at 37°C, and added dropwise to 10mL 1640 medium (containing 0.1% DNase) containing 10% FBS at 37°C. The mixture was centrifuged at 300g for 8 minutes at 25°C, and the supernatant was removed. The cells were resuspended in 1640 medium (containing 0.1% DNase) containing 10% FBS at 37°C in a T75 culture flask, and the mixture was left in an incubator at 37°C, 5% CO2 for 3 hours. Human CD8+ T cells were isolated using EasySep™ Human CD8+ T Cell Enrichment Kit, and the procedure was performed according to the manufacturer's instructions. The density of human CD8+ T cells was adjusted to 5×10 using 1640 medium containing 10% FBS. 5 The tumor cells were resuspended in 1640 medium containing 10% FBS and the density was adjusted to 1 × 10 5 The concentration was adjusted to 100 cells / mL. The tumor cell suspension and human CD8+ T cell suspension were mixed well in a 1:1 ratio, and the resulting cell suspension and gradient diluted test antibodies were added to a (general) 96-well round-bottom culture plate (with cover) at 200 μL / well. The cells were placed in a carbon dioxide incubator and stimulated at 37°C for 72 hours. The cells were centrifuged at 400g for 5 minutes, the supernatant was removed, and PBS (containing PE anti-human CD8 diluted 1:100) was added at 100 μL / well to resuspend the cells. The cells were left at 4°C for 30 minutes, washed, and the values ​​were read using a flow cytometer (BD, FACSCELESTA).

[0158] result In experiments performed using the assays described above, the exemplary antibodies were able to effectively stimulate proliferation of CD8+ T cells in vitro in the presence of CD79b-positive cells (see Figures 14A, 14B, and 14C), but did not demonstrate CD79b-independent and non-specific proliferation of CD8+ T cells in the presence of CD79b-negative NUGC4 cells (see Figure 14D).

[0159] Example 16. Assay for promotion of human CD4+ T cell proliferation capacity by exemplary antibodies The exemplary antibodies were able to stimulate the proliferation of human CD4+ T cells in vitro in a dose-dependent manner in the presence of CD79b-positive tumor cells, whereas the exemplary antibodies did not show CD79b-independent and non-specific proliferation of CD4+ T cells in the presence of CD79b-negative NUGC4 cells.

[0160] Experimental procedure Isolation of human CD4+ T cells: PBMCs were removed from the liquid nitrogen tank, thawed quickly at 37°C, and added dropwise to 10mL 1640 medium (containing 0.1% DNase) containing 10% FBS at 37°C. The mixture was centrifuged at 300g for 8 minutes at 25°C, and the supernatant was removed. The cells were resuspended in 1640 medium (containing 0.1% DNase) containing 10% FBS at 37°C in a T75 culture flask, and the mixture was left in an incubator at 37°C and 5% CO2 for 3 hours. Human CD4+ T cells were isolated using EasySep™ Human CD4+ T Cell Enrichment Kit, and the procedure was performed according to the manufacturer's instructions. The density of human CD4+ T cells was adjusted to 5×10 using 1640 medium containing 10% FBS. 5 The tumor cells were resuspended in 1640 medium containing 10% FBS and the density was adjusted to 1 × 10 5 The concentration was adjusted to 100 cells / mL. The tumor cell suspension and human CD4+ T cell suspension were mixed well in a 1:1 ratio, and the resulting cell suspension and gradient diluted test antibodies were added to a (standard) 96-well round-bottom culture plate (with cover) at 200 μL / well. The cells were placed in a carbon dioxide incubator and stimulated at 37°C for 72 hours. The cells were centrifuged at 400g for 5 minutes, the supernatant was removed, and PBS (containing FITC anti-human CD4 diluted 1:100) was added at 100 μL / well to resuspend the cells. The cells were left at 4°C for 30 minutes, washed, and the values ​​were read using a flow cytometer (BD, FACSCELESTA).

[0161] result In experiments performed using the assays described above, the exemplary antibodies were able to effectively stimulate proliferation of CD4+ T cells in vitro in the presence of CD79b-positive cells (see Figures 15A, 15B, and 15C), but did not demonstrate CD79b-independent and non-specific proliferation of CD4+ T cells in the presence of CD79b-negative NUGC4 cells (see Figure 15D).

[0162] Example 17. Efficacy Assays of Anti-CD79b / CD3 Antibodies of the Invention in Animals In this study, the PBMC model of NOG mice was inoculated with human B-cell non-Hodgkin's lymphoma cell lines WSU-DLCl2 and Ramos cells to measure the anti-tumor efficacy of exemplary antibodies.

[0163] Anti-tumor Efficacy of Exemplary Antibodies in a WSU-DLCL2 Tumor-Bearing Humanized Mouse Model Experimental procedure Female NOG mice (14-17 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and were SPF grade. Upon arrival, the mice were acclimated and quarantined for 7 days before the start of the study. Mice were inoculated with 4 × 10 PBMC cells. 6 6 × 10 cells / mouse, 200 μL / mouse volume, were injected intravenously. WSU-DLCL2 cells were inoculated on day 3 after PBMC injection. WSU-DLCL2 cells were routinely passaged and harvested by centrifugation, and WSU-DLCL2 cells were dispersed in PBS. NOG mice were shaved on the right back and abdomen, and 6 × 10 WSU-DLCL2 cells were injected into the mice. 6 cells / mouse were inoculated in a volume of 200 μL / cell.

[0164] Administration: On the 8th day after inoculation with WSU-DLCL2 cells, the mice were divided into groups (8 mice per group) and administered drugs according to the tumor volume of the mice. The administration was performed once every 3-4 days, a total of 4 times. The tumor volume and body weight of the mice were monitored twice a week. On the 22nd day after inoculation, the relative tumor growth inhibition (TGI%) was calculated by the following formula: TGI%=100%*(tumor volume of hIgG control group-tumor volume of treatment group) / (tumor volume of hIgG control group-initial tumor volume of hIgG control group), and the initial tumor volume of the control group was approximately 90 mm 3 Tumor volume measurement: The maximum long axis (L) and the maximum short axis (W) of the tumor were measured with a caliper and calculated using the following formula: V = L × W 2 Tumor volumes were calculated by 1 / 2. Mice were weighed using an electronic balance. Throughout the study, mice were euthanized when the tumor reached the endpoint or when their body weight decreased by 20% or more. Tumor sizes were obtained and tumor growth inhibition (TGI%) was calculated.

[0165] result Tumor growth curves are shown in FIG. 16, and the exemplary antibodies were able to significantly inhibit the proliferation of WSU-DLCL2 cells. Tumor sizes were obtained on day 22, and tumor growth inhibition was calculated. Exemplary antibodies 38D9B3.11 / sp34.87, 38D9B3.11 / sp34.24, and 11G10.9.p1 / sp34.24 showed 78%, 93%, and 67% tumor growth inhibition, respectively, compared to hIgG. The TGI of CD79b.A7v14b / 38E4v1 was only 36%. Furthermore, no significant weight loss was observed in the treated mice groups.

[0166] Anti-tumor efficacy of exemplary antibodies in a Ramos tumor-bearing humanized mouse model Experimental procedure Female NOG mice (14-17 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and the mice were SPF grade. Upon arrival, the mice were acclimated and quarantined for 7 days before the study began. PBMC cells were thawed in prewarmed RPMI-1640 medium containing 0.1% DNase, then dispersed in PBS to a cell concentration of 20 × 10 6 The PBMC cell suspension was added to the mice at 0.2 mL / mouse, i.e., 4 × 10 6 The cells were injected intravenously at an inoculum size of 10 cells / mouse.

[0167] For subsequent in vivo experiments, Ramos cells were routinely passaged. Seven days after PBMC cell inoculation, Ramos cells were dispersed in PBS and Matrigel (1:1) and cultured at 7.5 × 10 6 A cell suspension with a cell concentration of 1.5 × 10 cells / mL was prepared. The right back of a NOG mouse was shaved, and the Ramos cell suspension was added to the mouse at 0.2 mL / mouse, i.e., 1.5 × 10 cells / mL. 6 The cells were injected subcutaneously at an inoculum size of 10 cells / mouse.

[0168] Six days after tumor cell inoculation, the mice were divided into groups (6 mice per group) and administered drugs according to the tumor volume of the mice, once every 3-4 days for 4 consecutive doses. The administration method was intraperitoneal injection, and the administration volume was 10mL / kg / dose. The tumor volume and body weight of the mice were monitored twice a week, and monitoring was terminated on the 20th day.

[0169] On the 20th day after inoculation, the relative tumor growth inhibition (TGI%) was calculated by the following formula: TGI%=100%×(tumor volume of hIgG control group−tumor volume of treatment group) / (tumor volume of hIgG control group−tumor volume of hIgG control group before administration). Tumor volume measurement: The maximum long diameter (L) and maximum short diameter (W) of the tumor were measured with a caliper, and the tumor volume was calculated by the following formula: V=L×W2 / 2. The body weight of the mice was measured using an electronic balance.

[0170] result Tumor growth curves are shown in FIG. 17, and the exemplary antibodies were able to significantly inhibit the proliferation of Ramos cells. Tumor sizes were obtained on day 20, and tumor growth inhibition was calculated. Exemplary antibodies 38D9B3.11 / sp34.87, 38D9B3.11 / sp34.24, and 11G10.9.p1 / sp34.24 showed 76%, 90%, and 100% tumor growth inhibition, respectively. The TGI of CD79b.A7v14b / 38E4v1 was 77%, which was similar to that of 38D9B3.11 / sp34.87. Furthermore, no significant weight loss was observed in the treated mice groups.

[0171] Example 18. PK assay of anti-CD79b / CD3 antibodies of the present invention in animals In this study, 38D9B3.11 / sp34.87, 38D9B3.11 / sp34.24, and 11G10.9.p1 / sp34.24 were administered at 10 mg / kg via tail vein injection to female Balb / C mice to study their pharmacokinetic properties in female Balb / C mice. Blood was collected from the eyeball at 0.083 hours, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 4 days, 7 days, 14 days, and 21 days, and centrifuged at 4°C and 3000 rpm for 10 minutes to collect serum. The half-life of 38D9B3.11 / sp34.87, 38D9B3.11 / sp34.24, and 11G10.9.p1 / sp34.24 in mice was calculated by measuring the antibody content in the serum by ELISA. The results are shown in Figure 18. The half-lives of 38D9B3.11 / sp34.87, 38D9B3.11 / sp34.24, and 11G10.9.p1 / sp34.24 in mice were 10.4 days, 7.3 days, and 7.0 days, respectively, and all three had PK similar to conventional monoclonal antibodies.

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), (i) the VH comprises three complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the VH set forth in SEQ ID NO: 4, and the VL comprises LCDR1, LCDR2, and LCDR3 contained in the VL set forth in SEQ ID NO: 9; or (ii) the VH comprises three complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the VH set forth in SEQ ID NO: 14, and the VL comprises LCDR1, LCDR2, and LCDR3 contained in the VL set forth in SEQ ID NO: 19; An antibody or antigen-binding fragment thereof.

2. 2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region VH and a light chain variable region VL, 1) the VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, the HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2, and the HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3; the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6, the LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7, and the LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 8; or 2) the VH comprises complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11, the HCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, and the HCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13; the VL comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, the LCDR2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17, and the LCDR3 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18; An antibody or antigen-binding fragment thereof.

3. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region VH and / or a light chain variable region VL, (a) the heavy chain variable region VH is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14; or (iii) comprising or consisting of an amino acid sequence having 1 to 10 amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in SEQ ID NO: 4 or SEQ ID NO: 14; and / or (b) the light chain variable region VL is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 19; or (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 19; or (iii) comprising or consisting of an amino acid sequence having 1 to 10 amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 19; An antibody or antigen-binding fragment thereof.

4. (1) A heavy chain variable region VH comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 4, and a light chain variable region VL comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 9; or (2) A heavy chain variable region VH comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 14, and a light chain variable region VL comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

19. The antibody or antigen-binding fragment thereof of claim 3, comprising:

5. (1) A heavy chain variable region VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4, and a light chain variable region VL comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9; or (2) A heavy chain variable region VH comprising or consisting of the amino acid sequence shown in SEQ ID NO: 14, and a light chain variable region VL comprising or consisting of the amino acid sequence shown in SEQ ID NO:

19. The antibody or antigen-binding fragment thereof of claim 4, comprising:

6. 2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain and / or a light chain: (a) the heavy chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:15; (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 15; or (iii) comprising or consisting of an amino acid sequence having 1 to 10 amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 15; and / or (b) the light chain is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 20; (ii) comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 20; or (iii) comprising or consisting of an amino acid sequence having 1 to 10 amino acid substitutions, insertions, or deletions compared to the amino acid sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 20; An antibody or antigen-binding fragment thereof.

7. (1) a heavy chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 5, and a light chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 10; or (2) A heavy chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 15, and a light chain comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO:

20. The antibody or antigen-binding fragment thereof of claim 6, comprising:

8. (1) A heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 10; or (2) A heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 15, and a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO:

20. The antibody or antigen-binding fragment thereof of claim 7, comprising:

9. The antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody is an IgG1 antibody.

10. The antibody is a monoclonal antibody molecule, a chimeric antibody molecule, a humanized antibody, or a human antibody molecule, or a bispecific or multispecific antibody molecule, and the antigen-binding fragment is Fab, Fab', Fab'-SH, Fv, a single chain antibody (e.g., scFv), or (Fab') ２ 2. The antibody or antigen-binding fragment thereof of claim 1, which is one of a single domain antibody, a diabody (dAb), a camelid antibody (heavy chain antibody), or a linear antibody.

11. 2. The antibody or antigen-binding fragment thereof of claim 1, which is a bispecific antibody comprising a first heavy chain, a first light chain, a second heavy chain, and a second light chain, wherein the first heavy chain and the first light chain comprise an antigen-binding site that binds to CD79b, and preferably the second heavy chain and the second light chain comprise an antigen-binding site that binds to CD3.

12. 1) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 6, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7, and the LCDR3 comprises or consists of SEQ ID NO: 8; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 31, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 32, and the HCDR3 comprises or consists of SEQ ID NO: 33; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

38. 2) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 6, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 7, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 8; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3; the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 41, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 42, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 43; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 46, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 47, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 48; 3) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 18; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

13. the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 31, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 32, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 33; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38; 4) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 18; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

13. the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 41, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 42, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 43; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 46, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 47, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 48; 5) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 28; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

23. the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 31, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 32, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 33; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 36, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 37, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 38; or 6) the first light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 26, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 27, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 28; the first heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 21, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 22, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:

23. the second heavy chain comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 41, the HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 42, and the HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 43; the second light chain comprises LCDR1, LCDR2, and LCDR3, wherein the LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 46, the LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 47, and the LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 48; The antibody or antigen-binding fragment thereof described in claim 11.

13. 1) the first light chain comprises a VL set forth in SEQ ID NO: 9, the first heavy chain comprises a VH set forth in SEQ ID NO: 4, the second heavy chain comprises a VH set forth in SEQ ID NO: 34, and the second light chain comprises a VL set forth in SEQ ID NO: 39; 2) the first light chain comprises a VL set forth in SEQ ID NO: 9, the first heavy chain comprises a VH set forth in SEQ ID NO: 4, the second heavy chain comprises a VH set forth in SEQ ID NO: 44, and the second light chain comprises a VL set forth in SEQ ID NO: 49; 3) the first light chain comprises a VL set forth in SEQ ID NO: 19, the first heavy chain comprises a VH set forth in SEQ ID NO: 14, the second heavy chain comprises a VH set forth in SEQ ID NO: 34, and the second light chain comprises a VL set forth in SEQ ID NO: 39; 4) the first light chain comprises a VL set forth in SEQ ID NO: 19, the first heavy chain comprises a VH set forth in SEQ ID NO: 14, the second heavy chain comprises a VH set forth in SEQ ID NO: 44, and the second light chain comprises a VL set forth in SEQ ID NO: 49; 5) the first light chain comprises a VL set forth in SEQ ID NO: 29, the first heavy chain comprises a VH set forth in SEQ ID NO: 24, the second heavy chain comprises a VH set forth in SEQ ID NO: 34, and the second light chain comprises a VL set forth in SEQ ID NO: 39; or 6) the first light chain comprises a VL set forth in SEQ ID NO: 29, the first heavy chain comprises a VH set forth in SEQ ID NO: 24, the second heavy chain comprises a VH set forth in SEQ ID NO: 44, and the second light chain comprises a VL set forth in SEQ ID NO: 49; The antibody or antigen-binding fragment thereof described in claim 12.

14. 1) the first light chain comprises or consists of SEQ ID NO: 10, the first heavy chain comprises or consists of SEQ ID NO: 5, the second heavy chain comprises or consists of SEQ ID NO: 35, and the second light chain comprises or consists of SEQ ID NO: 40; 2) the first light chain comprises or consists of SEQ ID NO: 10, the first heavy chain comprises or consists of SEQ ID NO: 5, the second heavy chain comprises or consists of SEQ ID NO: 45, and the second light chain comprises or consists of SEQ ID NO: 50; 3) the first light chain comprises or consists of the sequence of SEQ ID NO: 20, the first heavy chain comprises or consists of SEQ ID NO: 15, the second heavy chain comprises or consists of SEQ ID NO: 35, and the second light chain comprises or consists of SEQ ID NO: 40; 4) the first light chain comprises or consists of the sequence of SEQ ID NO: 20, the first heavy chain comprises or consists of SEQ ID NO: 15, the second heavy chain comprises or consists of SEQ ID NO: 45, and the second light chain comprises or consists of SEQ ID NO: 50; 5) the first light chain comprises or consists of the sequence of SEQ ID NO: 30, the first heavy chain comprises or consists of SEQ ID NO: 25, the second heavy chain comprises or consists of SEQ ID NO: 35, and the second light chain comprises or consists of SEQ ID NO: 40; or 6) the first light chain comprises or consists of the sequence of SEQ ID NO: 30, the first heavy chain comprises or consists of SEQ ID NO: 25, the second heavy chain comprises or consists of SEQ ID NO: 45, and the second light chain comprises or consists of SEQ ID NO: 50; The antibody or antigen-binding fragment thereof described in claim 13.

15. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 1.

16. A vector comprising the nucleic acid of claim 15, which is preferably an expression vector.

17. 16. A host cell comprising the nucleic acid of claim 15 or a vector comprising said nucleic acid.

18. 18. The host cell of claim 17, wherein the host cell is prokaryotic or eukaryotic, preferably the host cell is a yeast cell, a mammalian cell, or other cell suitable for preparing an antibody or antigen-binding fragment thereof, preferably the mammalian cell is a 293 cell or a CHO cell.

19. A method for preparing an antibody or antigen-binding fragment thereof, comprising culturing the host cell of claim 17 under conditions suitable for expressing nucleic acid encoding said antibody or antigen-binding fragment thereof, optionally comprising isolating said antibody or antigen-binding fragment thereof, and optionally further comprising recovering said antibody or antigen-binding fragment thereof from said host cell.

20. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of claim 1 and an additional substance.

21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or immunoconjugate thereof, according to claim 1, and optionally pharmaceutical auxiliary materials, and optionally one or more other therapeutic agents.

22. Use of the antibody or antigen-binding fragment thereof, or immunoconjugate thereof described in claim 1 in the preparation of a medicament for preventing and / or treating a disease or disorder associated with CD79b and / or CD3, such as a tumor.

23. 23. The use according to claim 22, wherein the antibody or antigen-binding fragment thereof, or immunoconjugate thereof can be administered in combination with one or more therapies.

24. 24. The use according to claim 23, wherein said treatment is a therapeutic modality and / or other therapeutic agent, preferably said therapeutic modality comprises surgical treatment and / or radiation therapy.

25. Use of the antibody or its antigen-binding fragment, or its immune complex described in claim 12, in the preparation of a medicament for preventing and / or treating a disease or disorder associated with CD79b and / or CD3, such as a tumor.

26. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof described in claim 1, or an immune complex thereof, for use in the prevention and / or treatment of a disease or disorder associated with CD79b and / or CD3.

27. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, or an immunoconjugate thereof, according to claim 12, for use in the prevention and / or treatment of a disease or disorder associated with CD79b and / or CD3.

28. 1. A method for detecting antigens CD79b and / or CD3 in a sample, comprising: (a) contacting the sample with the antibody or antigen-binding fragment thereof of claim 1; (b) detecting a complex formed by said antibody or antigen-binding fragment thereof and said antigen CD79b and / or CD3, wherein optionally said antibody is detectably labeled. A method comprising:

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