Antibody and drug conjugate thereof, and use thereof

Abstract

The present disclosure relates to antibodies and antibody-drug conjugates (ADCs) targeting CD79b and / or CD20 and compositions containing the antibodies or ADCs and therapeutic applications thereof.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610020609.8 (22) Application Date 2026.01.08 (66) Domestic Priority Data PCT / CN2025 / 071762 2025.01.10 CN (71) Applicant Shanghai Chengfan Pharmaceutical Co., Ltd. Address Building 1, No. 215, Fute South Road, Waigaoqiao Free Trade Zone, Pudong New Area, Shanghai 200131 (72) Inventors Xu Man, Wang Wei, Li Jing, Bi Yanxia, ​​Yin Qun (74) Patent Agency Beijing Zhongzi Law Firm 11247 Patent Attorney Zhang Li, Huang Gesheng (51) Int.Cl. C07K 16 / 46 (2006.01) A61K 47 / 68 (2017.01) A61K 31 / 4745(2006.01) A61K 38 / 07(2006.01) A61P 35 / 00(2006.01) (54) Title of Invention Antibody and Drug Conjugate Thereof and Uses (57) Abstract This disclosure relates to antibodies and antibody-drug conjugates (ADCs) targeting CD79b and / or CD20, as well as compositions containing said antibodies or ADCs and their therapeutic uses. Claims 8 pages, Description 58 pages, Sequence Listing (Electronic Publication), Drawings 32 pages, CN 122356297 A 2026.07.10 CN 1 22 35 62 97 A 1. A multispecific antibody comprising at least one antigen-binding domain specifically binding to CD79b and at least one antigen-binding domain specifically binding to CD20, preferably wherein: the antigen-binding domain specifically binding to CD79b comprises or is composed of a heavy chain variable region (VH) and a light chain variable region (VL); and the antigen-binding domain specifically binding to CD20 comprises or is composed of a VHH domain; and / or wherein: (i) the CD79b binding domain binds to CD79b-expressing cells at an EC50 value of approximately 0.1–30 nM, for example, 1–10 nM EC50 value, as determined by flow cytometry (FACS); (ii) As determined by flow cytometry (FACS), the CD20 binding domain binds to CD20-expressing cells with an EC50 value of approximately 0.1–50 nM, for example, 1–30 nM EC50. 2. The multispecific antibody according to claim 1, wherein the CD20 binding domain comprises or is composed of a VHH domain, wherein: the VHH domain comprises the CDR1, CDR2, and CDR3 sequences of one of SEQ ID NOs: 1 and 5–13; preferably, the CDR1, CDR2, and CDR3 sequences (i) respectively comprise SEQ ID NOs: 2,The amino acid sequences of SEQ ID NOs:14, 3, and 4, or composed thereof; or (ii) the amino acid sequences of SEQ ID NOs:14, 3, and 4, or composed thereof; more preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NOs:1 and 5-13, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or composed thereof; more preferably, the VHH domain comprises an amino acid sequence of SEQ ID NO:6, or composed thereof. 3. The multispecific antibody according to claim 1 or 2, wherein the CD79b binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the heavy chain variable region comprises HCDR1-3 contained in the VH sequence of SEQ ID NO: 16; and the light chain variable region comprises LCDR1-3 contained in the VL sequence of SEQ ID NO: 15; preferably, wherein the HCDR1-3 comprises or consists of the amino acid sequences of SEQ ID NOs: 20, 21, and 22, respectively; and the LCDR1-3 comprises or consists of the amino acid sequences of SEQ ID NOs: 17, 18, and 19, respectively; more preferably, (a) the heavy chain variable region comprises SEQ ID NO: The amino acid sequence of SEQ ID NO: 16, or having at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or consisting thereof; and / or (b) the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15, or having at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or consisting thereof; more preferably, the heavy chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises or consists of the amino acid sequence of SEQ ID NO: 15. 4. The multispecific antibody according to any one of claims 1-3, wherein the antibody further comprises an immunoglobulin Fc region, and optionally wherein: (i) the Fc region comprises a mutation that reduces or eliminates the binding of the Fc region to FcγR, for example, an L234A or L235A mutation; (ii) the Fc region is IgG type, for example, IgG1 or IgG4 isotype; and / or (iii) the Fc region comprises the amino acid sequence of SEQ ID NO: 31 or 32, or at least 95%, 96%, 98% or more thereof.Claims 1 / 8 page 2 CN 122356297 A 99% identical amino acid sequence. 5. A multispecific antibody according to any one of claims 1-4, wherein the multispecific antibody comprises: (a) an anti-CD79b Fab domain and; (b) an immunoglobulin Fc region attached to the C-terminus of the anti-CD79b Fab domain; and (c) optionally, at least one (preferably one) CD20 binding domain attached to the N-terminus of the Fab domain or the C-terminus of the Fc region via a peptide linker, preferably, the peptide linker comprises the amino acid sequence of SEQ ID NO: 26 or 27, and more preferably, the CD20 binding domain is attached to the N-terminus of the heavy chain of the Fab domain. 6. A multispecific antibody according to any one of claims 1-5, wherein the valence ratio of the CD79b binding domain to the CD20 binding domain is 1:1. 7. The multispecific antibody according to any one of claims 1-6, wherein the multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus, the first polypeptide chain comprises: a VHCD79b-CH1 domain-immunoglobulin Fc region; the second polypeptide chain comprises: a VLCD79b-CL domain; wherein VHCD79b and VLCD79b represent the heavy chain variable region and the light chain variable region that bind to CD79b, respectively; wherein the first polypeptide chain optionally connects to the anti-CD20 VHH domain at the N-terminus or C-terminus via a peptide linker. 8. A multispecific antibody according to any one of claims 1-7, wherein the multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein: - the first and second polypeptide chains respectively comprise the amino acid sequences of SEQ ID NOs: 23 and 24, or have at least 85%, 90%, 95%, or 99% identity with them, or have one or more (preferably 1-10, more preferably 1-5) amino acid sequences with addition, deletion, and / or substitution, or are composed thereof; or - the first and second polypeptide chains respectively comprise the amino acid sequences of SEQ ID NOs: 25 and 24, or have at least 85%, 90%, 95%, or 99% identity with them, or have one or more (preferably 1-10, more preferably 1-5) amino acid sequences with addition, deletion, and / or substitution, or are composed thereof; preferably, the first and second polypeptide chains respectively comprise the amino acid sequences of SEQ ID NOs: 25 and 24, or are composed thereof. 9. An antigen-binding molecule comprising the antibody according to any one of claims 1-8. 10. A polynucleotide encoding an antibody according to any one of claims 1-8 or an antigen-binding molecule according to claim 9. 11. A vector, preferably an expression vector, comprising the polynucleotide of claim 10.12. A host cell comprising the polynucleotide of claim 10 or the vector of claim 11, optionally said host cell being a mammalian cell. 13. A method for producing an antibody according to any one of claims 1-8, said method comprising: culturing a host cell comprising a polynucleotide encoding said polypeptide chain under conditions suitable for producing said antibody or a polypeptide chain thereof. 14. An immunoconjugate comprising an antibody of any one of claims 1-8 or an antigen-binding molecule of claim 9. 15. An antibody-drug conjugate having formula (I) or a pharmaceutically acceptable salt or solvate thereof: Ab-(L-D)p (I) Wherein: Ab is the antibody according to any one of claims 1-8 or the antigen-binding molecule according to claim 9; L is a linker; AD is a drug, such as an antitumor compound; p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, 3-5, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. 16. The antibody-drug conjugate according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the drug is a cytotoxic agent, such as a camptothecin or aurestatin. 17. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15 or 16, wherein D has the structure shown in formula (D-1a) or formula (D-1b): or formula (D-1a) Formula (D-1b) wherein the wavy line indicates that the valence bond is connected to L; R1a is selected from H and C1-C6 alkyl; R2a is selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR5a and -SR5a; R3a is selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR5a; and R4a and R5a are independently selected from H and C1-C4 alkyl; R1b, R2b, R3b, R4b, R5b and R8b are each independently selected from C1-8 alkyl; preferably C1-4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; R6b and R7b are each independently selected from C1-8 alkoxy groups, such as methoxy, ethoxy, or propoxy; R9b is selected from C1-8 alkyl groups and COOH; preferably C1-4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and R10b is selected from OH and H. 18. The antibody-drug conjugate according to claim 17, or a pharmaceutically acceptable salt or solvation thereof, wherein D has the structure shown in formula (D-2a) or formula (D-2b): or formula (D-2a) formula (D-2b)R1a, R2a, R3a and R4a are defined as in claim 3 pairs of formulas (D-1a); R1b, R2b, R3b, R4b, R5b, R6b, R7b, R8b, R9b and R10b are defined as in claim 3 pairs of formulas (D-1b). 19. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15-18, wherein D has the structure of formula (D-1a) or (D-2a), and wherein R1a is H; R2a is C1-C4 alkyl, preferably methyl; R3a is halogen, preferably -F; R4a is C1-C4 alkyl, preferably ethyl; or D has the structure of formula (D-1b) or (D-2b), and wherein R1b, R4b and R8b are each independently selected from C1-2 alkyl; preferably methyl; R2b, R3b and R5b are each independently selected from C3-4 alkyl; R6b and R7b are each independently selected from C1-2 alkoxy; and R9b is selected from C1-4 alkyl and R10b is OH; or R9b is COOH and R10b is H. 20. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15-17, wherein D has a structure represented by formula (D-3a) or (D-3b): (D-3a) or (D-3b); preferably, D has a structure represented by formula (D-4a) or (D-4b): (D-4a) or (D-4b). 21. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15-19, wherein the drug is Exatecan, Dxd, SN-38, monomethylaurestatin E (MMAE), or MMAF. 22. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15-21, wherein -L- has the following structure: -Z-L1-L2-L3- where Z is selected from , , , , , and , and m is an integer selected from 1-10, for example 1, 2, 3, 4, 5, 6, 7 or 8, for example an integer from 1-5; L1 is selected from absent, , , , and , where n1 and m1 are each independently an integer selected from 0-20, for example an integer selected from 0-12, for example 1, 2, 3, 4, 5, 6, 7 or 8; L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and L3 is selected from: , , , , And, where X is selected from -NH-, -O-, and -S-; R1c is each independently selected from C1-8 alkyl, C1-8 haloalkyl, C1-8 alkoxy, halogen, nitro, and cyano; Su is each independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; andWherein, Z is connected to atoms on Ab, preferably S atoms, and L3 is connected to D. 23. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 22, wherein AZ is selected from and , where m is 1, 2, 3, 4, 5, 6, 7 or 8, for example, an integer from 1 to 5; preferably, Z is selected from, and . 24. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 22 or 23, wherein L1 is selected from non-existent, and , where n1 is independently an integer selected from 0 to 12, for example, 1, 2, 3, 4, 5, 6, 7 or 8; preferably, L1 is selected from non-existent, and . 25. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 22-24, wherein L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids; preferably, wherein each amino acid residue or amino acid is independently selected from valine (Val), alanine (Ala), glycine (Gly), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine ​​(Cys), histidine (His) and threonine (Thr); More preferably, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit), phenylalanine (Phe), lysine (Lys), glutamic acid (Glu), and glutamine (Gln); even more preferably, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit), and glutamic acid (Glu). 26. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 22-24, wherein L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-; preferably, L2 is selected from -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, and -Gly-Gly-Phe-Gly-; more preferably, L2 is selected from -Gly- and -Val-Cit-. 27. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 22-26.The antibody-drug conjugate according to any one of claims 22-27, wherein L3 is selected from: , and , wherein R1c is each independently selected from C1-8 alkyl, C1-8 haloalkyl, C1-8 alkoxy, halogen, nitro and cyano; Su is each independently selected from, , and ; n2 is 0, 1, 2, 3 or 4; and n5 is 0, 1, 2 or 3. 28. The antibody-drug conjugate according to any one of claims 22-28 or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: and . 29. The antibody-drug conjugate according to any one of claims 22-28 or a pharmaceutically acceptable salt or solvate thereof, wherein Su is each independently: ; preferably, Su is each independently , and further preferably, Su is each independently . 30. The antibody-drug conjugate according to any one of claims 22-29 or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: and . 31. The antibody-drug conjugate according to claim 22, or a pharmaceutically acceptable salt or solvate thereof, wherein -Z-L1-L2-L3- are each independently selected from the following structures: , and , wherein m are each independently an integer selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7 or 8; wherein the left side of the group is connected to an atom on Ab, preferably an S atom, and the right side is connected to D. 32. The antibody-drug conjugate according to claim 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is an antibody-drug conjugate having a structure selected from: (Claims 6 / 8, page 7, CN 122356297 A) where Ab is the antibody according to any one of claims 1-8 or the antigen-binding molecule according to claim 9, preferably the antibody according to claim 8; and p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. 33. The antibody-drug conjugate according to any one of claims 15-32, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, or 2-4, or 2-6. 34. A pharmaceutical composition comprising an antibody according to any one of claims 1-8, an antigen-binding molecule according to claim 9, an immunoconjugate according to claim 14, or an antibody-drug conjugate according to any one of claims 15-33, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, and optionally further comprising one or more other pharmaceutically active peptides and / or compounds, for example, other therapeutic agents selected from inhibitors of oncolytic drugs, cytotoxic agents, cytokines, and immune checkpoint molecules.35. Use of the antibody of any one of claims 1-8, the antigen-binding molecule of claim 9, the immunoconjugate of claim 14, or the antibody-drug conjugate of any one of claims 15-33, or a pharmaceutically acceptable salt or solvation thereof, as a medicine or for the preparation of a medicine, wherein preferably the medicine is used to treat cancer or B-cell-related autoimmune diseases. 36. A method of treating or preventing cancer or B-cell-related autoimmune diseases, comprising administering to an individual in need an effective amount of the antibody of any one of claims 1-8, the antigen-binding molecule of claim 9, the immunoconjugate of claim 14, the antibody-drug conjugate of any one of claims 15-33, or a pharmaceutically acceptable salt or solvation thereof, or the pharmaceutical composition of claim 34. 37. The use of claim 35 or the method of claim 36, wherein the cancer is a solid tumor or hematologic malignancy, preferably a CD79b-positive and / or CD20-positive tumor, more preferably a B-cell-associated lymphoid tumor and leukemia, for example selected from: non-Hodgkin's lymphoma (NHL), large B-cell lymphoma, DLBCL, RT (Richter's transformation / syndrome), BL (Burkitt lymphoma), FL (follicular lymphoma), MZL (marginal zone lymphoma), MCL (mantle cell lymphoma), acute lymphoblastic leukemia (ALL), and chronic lymphocyticle leukemia (CLL), optionally the cancer is a relapsed or refractory large B-cell lymphoma. Claims 7 / 8, page 8, CN 122356297 A 38. Use or method of claim 37, wherein the cancer is a CD79b-insensitive or resistant tumor, optionally wherein the tumor cells have one or more of the following characteristics: (a) the tumor cells have CD79b expression at 50%, 40%, 30%, 20%, 10%, 5%, or 2% lower than the CD79b expression level on Daudi cells; (b) the tumor cells have upregulated expression and / or activity of the anti-apoptotic gene Bcl-xL compared to Daudi cells or Ramos cells; (c) the tumor cells have upregulated expression and / or activity of the MMAE efflux pump (MDR-1) compared to Daudi cells or Ramos cells; and (d) the tumor cells have CD20 expression at 1%, 10%, 50%, 100%, 150%, 200%, 300%, or 400% higher than the CD20 expression level on Daudi cells.39. The use of claim 35 or the method of claim 36, wherein the B-cell-related autoimmune disease is selected from rheumatoid arthritis (RA); lupus; NMDAR encephalitis; multiple sclerosis; systemic sclerosis; immune thrombocytopenic purpura; simple erythrocytic aplasia; autoimmune anemia; cold agglutinin disease; severe insulin resistance type B syndrome; mixed cryoglobulinemia; myasthenia gravis; Wegener's granulomatosis; refractory pemphigus vulgaris; dermatomyositis; Sjogren's syndrome; active type II mixed cryoglobulinemia; pemphigus vulgaris; autoimmune nephropathy; neoplastic visual oculoclonus-myoclonus syndrome; and relapsing-remitting multiple sclerosis (RRMS). Claims 8 / 8 Page 9 CN 122356297 A Antibodies and Drug Conjugates Thereof and Their Uses Technical Field

[0001] This invention relates to antibodies and antibody-drug conjugates, and more particularly to antibodies and antibody-drug conjugates (ADCs) targeting CD79b and / or CD20, as well as compositions containing said antibodies or ADCs and their therapeutic applications. Background Art

[0002] Non-Hodgkin's lymphoma (NHL) accounts for approximately 4% of all cancers. Despite improvements in existing therapies, treatment options for patients with relapsed / refractory (R / R, sometimes referred to as r / r) NHL remain limited and have poor prognoses. For example, adoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) has shown good efficacy in treating CD19-positive B-cell malignancies, but only 40% of patients achieve long-term complete remission. Clinical data indicate that CD19 antigen loss occurs in patients with acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL), leading to disease relapse. Therefore, there is an unmet need for NHL treatment in this field.

[0003] B cells, or B lymphocytes, are a core component of adaptive immunity. B cells express B cell receptors (BCRs) on their surface to recognize antigens or pathogens. The B cell receptor is a multi-component receptor composed of transmembrane immunoglobulin molecules (mIg) and disulfide-linked CD79a and CD79b heterodimers. CD79b (Cluster of Differentiation 79b, also called B29, IGB, Igβ) is highly expressed in a variety of B-cell lymphomas, and its expression is associated with cancer cell viability in most DLBCL tumor models. Therefore, the likelihood of developing resistance to CD79b-targeted drugs through antigen loss is relatively small, making CD79b an attractive target for developing novel immunotherapies.

[0004] Studies have shown that 90% of diffuse large B-cell lymphomas (DLBCL) and 97% of follicular lymphomas (FL) are highly resistant to CD79b.23% of chronic lymphoblastic leukemia (CLL), 95% of marginal-zone lymphomas (MZL), 100% of hairy cell leukemia (HCL), and 95% of mantle cell lymphomas (MCL) express CD79b on the cell surface in sufficient quantities to elicit a response to CD79b-targeted drugs (CD79b-vc-MMAE) (Blood. 2009; 114:2721-2729).

[0005] Polatuzumab vedotin (POLIVY™) is an antibody-drug conjugate (ADC) molecule targeting CD79b developed by Roche Pharmaceuticals, which was approved by the FDA in 2019 for the treatment of r / r DLBCL. Polatuzumab vedotin (PV) binds to CD79b, is internalized by cells, and releases monomethylolpropionate E (MMAE). MMAE then binds to microtubules, killing dividing cells by inhibiting cell division and inducing apoptosis. In combination with standard therapy (bendamustine and rituximab), polatuzumab vedotin treatment increases the complete response (CR) rate and duration of response (DOR) in patients with DLBCL. Polatuzumab vedotin in combination with R-CHP (rituximab, cyclophosphamide, doxorubicin, and prednisone) significantly improves 2-year progression-free survival (PFS) in treatment-naïve DLBCL patients (N Engl J Med. 2022 Jan 27; 386(4):351-363.).

[0006] Although the approval of polatuzumab vedotin (PV) has established a more effective treatment for r / r DLBCL patients, there are still patients who are PV-insensitive / unresponsive, and with the approval of PV as a first-line therapy, treatment-related resistance is expected to emerge. Analysis of PV-insensitive cell lines showed that the reasons for their insensitivity included: upregulation of the anti-apoptotic gene Bcl-xL; low expression level of CD79b; and upregulation of the MMAE efflux pump (MDR-1) (Br J Haematol. 2022 Oct;199(2):245-255.). Specification 1 / 58 pages 10 CN 122356297 A

[0007] Nanobodies are small proteins composed of single-chain antibody molecules with high specificity and affinity.Compared with traditional antibodies, nanobodies have smaller size, higher stability, and deeper tissue penetration. Nanobodies can be designed to deliver drugs or radioisotopes to tumor cells to kill them. Furthermore, they can be used in various other treatment modalities such as photodynamic therapy and immunotherapy. Therefore, the application of nanobodies in the field of tumor treatment is receiving widespread attention and is expected to become one of the important means of future tumor treatment.

[0008] Given the unmet needs in the treatment of NHL cancer, there is still a need in the art to develop new drug molecules, especially new drug molecules with improved antitumor drug resistance and / or improved tumor specificity and selectivity, to meet the treatment needs of different NHL cancer patients, especially those with relapsed and refractory NHL cancer. Summary of the Invention

[0010] CD20 is a B cell-specific marker and a clinically validated therapeutic target for B cell malignancies and autoimmune diseases (see MAbs. 2013 Jan-Feb;5(1):22-33. doi: 10.4161 / mabs.22771. and Anderson et al. (1984) Blood 63(6):1424-1433). Furthermore, studies have shown that CD79b and CD20 proteins exhibit co-expression patterns in various B cells and B cell malignancies, but their expression density exhibits tumor heterogeneity (see http: / / hematologyoutlines.com / atlas_topics / 69.html); in addition, their co-expression ratio in non-B cell normal tissues is extremely low.

[0011] In view of the above-mentioned expression patterns of the two targets, the inventors proposed and designed antibody molecules that simultaneously target CD79b and CD20 to reduce drug resistance and improve efficacy. Based on this, the inventors further proposed an innovative antibody model based on nanobodies. By adding a CD20 VHH component to the full-length CD79b antibody, the antibody molecule of the present invention is easier to prepare than conventional mAb-based bispecific antibodies, and there is no light chain mismatch problem; and it is beneficial to more specific enrichment of antibody molecules in tumor tissues, and correspondingly further improvement in efficacy. Based on these designs and findings, the inventors have established the multispecific antibody of the present invention and its drug conjugate molecules and uses, especially in the treatment of cancer and autoimmune diseases.

[0012] Therefore, in a first aspect, the present disclosure provides anti-CD79b and CD20 multispecific antibodies. In some embodiments of the multispecific antibody according to the present invention, the antigen-binding domain that specifically binds to CD79b comprises or is composed of a heavy chain variable region (VH) and a light chain variable region (VL), preferably the VH comprises HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 16 and the VL comprises SEQ ID NO:LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 15. In some other embodiments of the multispecific antibody according to the present invention, the antigen-binding domain that specifically binds to CD20 comprises or is composed of a VHH domain, preferably, the VHH domain comprises CDR1, CDR2, and CDR3 in one of SEQ ID NO: 1 and 5-13.

[0013] In a second aspect, the present disclosure provides an antigen-binding molecule comprising the antibody of the present invention.

[0014] In a third aspect, the present disclosure provides a polynucleotide encoding an antibody or antigen-binding molecule according to the first and second aspects of the present disclosure, a carrier comprising the thereof, and a host cell. The present disclosure also provides a method for preparing an antibody or antigen-binding molecule according to the first and second aspects of the present disclosure.

[0015] In a fourth aspect, the present disclosure provides an immunoconjugate and antibody-drug conjugate (ADC) comprising an antibody or antigen-binding molecule according to the first and second aspects of the present disclosure, particularly an anti-CD79b / CD20 multispecific antibody-drug conjugate.

[0016] In a fifth aspect, this disclosure provides pharmaceutical compositions and pharmaceutical formulations comprising an antibody or antigen-binding molecule according to the first and second aspects of this disclosure, or an immunoconjugate or ADC according to the fourth aspect of this disclosure, and a pharmaceutically acceptable carrier, and optionally further comprising one or more other pharmaceutically active peptides and / or compounds, such as other therapeutic agents that also contain inhibitors of autolytic oncolytic drugs, cytotoxic agents, cytokines, and immune checkpoint molecules. In this aspect, this disclosure also provides combination products or kits comprising an antibody or antigen-binding molecule according to the first and second aspects of this disclosure, or an immunoconjugate or ADC according to the fourth aspect of this disclosure.

[0017] In a sixth aspect, this disclosure provides the use of an antibody or antigen-binding molecule according to the first and second aspects of this disclosure, or an immunoconjugate or ADC according to the fourth aspect of this disclosure, as a medicament or for the preparation of a medicament, wherein the medicament is for the treatment of cancer and B-cell-related autoimmune diseases, for example, said cancer being selected from CD79b-positive and / or CD20-positive tumors, more preferably B-cell-related lymphoma and leukemia; said B-cell-related autoimmune diseases being selected from, for example, rheumatoid arthritis, NMDAR encephalitis, idiopathic thrombocytopenic purpura, multiple sclerosis, pemphigus vulgaris, systemic sclerosis, and systemic lupus erythematosus. In this aspect, this disclosure also provides a method of treating cancer and B-cell-related autoimmune diseases, said method comprising administering to a subject in need an effective amount of an antibody or antigen-binding molecule according to the first and second aspects of this disclosure, an immunoconjugate or ADC according to the fourth aspect of this disclosure, or the fourth aspect of this disclosure.The pharmaceutical composition comprises five aspects, wherein the subject is a mammal; preferably, the subject is a human; wherein the cancer is, for example, a CD79b-positive and / or CD20-positive tumor, more preferably B-cell-associated lymphoid tissue and leukemia; wherein the B-cell-associated autoimmune disease is selected from, for example, rheumatoid arthritis, NMDAR encephalitis, idiopathic thrombocytopenic purpura, multiple sclerosis, pemphigus vulgaris, systemic sclerosis, and systemic lupus erythematosus.

[0018] In some embodiments, the antibody and ADC drug of the present invention can effectively kill malignant B cells by targeting more than one lymphoma tumor antigen, thereby minimizing or making minimal residual disease (MRD) negative.

[0019] In other embodiments, the antibody and ADC drug of the present invention can simultaneously take into account drug specificity and tumor heterogeneity by dually targeting CD79b and CD20, thereby achieving the effects of preventing tumor antigen escape, effectively targeting clonal populations (including, for example, capturing tumor cells that do not express sufficient CD79b or CD20), and improving tumor efficacy through affinity effects.

[0020] In some further embodiments, compared to a treatment regimen combining a CD79b ADC with a CD20 monoclonal antibody, the multi(bi)specific ADC of the present invention, which simultaneously targets CD79b and CD20, can achieve comparable or improved efficacy while reducing the number of drug administrations.

[0021] In some further embodiments, the multi(bi)specific ADC of the present invention can overcome drug inresponsiveness and resistance associated with MMAEs by using novel payloads.

[0022] In some further embodiments, compared with CD79b ADC monotherapy (e.g., polatuzumab vedotin) or CD79b ADC in combination with CD20 monoclonal antibodies (e.g., PV / Rituximab), the ADC drugs of the present invention also have one or more of the following advantages: - Superior killing effect in cells with low CD79b expression and / or low CD20 expression; - More significant killing effect in CD79b-targeted drug-resistant cell lines, wherein the resistance cause is preferably selected from one or more of the following: upregulation of the anti-apoptotic gene Bcl-xL; low CD79b expression level; and upregulation of the MMAE efflux pump (MDR-1); - Wider patient applicability; - Can be used not only in previously untreated patients, but also in patients who have previously received CD79b ADC and / or anti-CD20 (e.g., PV and / or Rituximab treatment) but are intolerant or have relapsed; - Can be used for CD79b-targeted drugs (e.g., CD79b For patients who are insensitive to or resistant to ADCs; and – for patients with Richter conversion. Instructions for use 3 / 58 pages 12 CN 122356297 A Figure Description

[0023] The preferred embodiments of the invention described in the following detailed description will be better understood when read in conjunction with the accompanying drawings. The drawings show currently preferred embodiments for illustrative purposes. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0024] Figures 1A and 1B show the binding of anti-CD20 antibodies to CD20 on Raji cells (Figure 1A) and Daudi cells (Figure 1B) as measured by FACS, respectively.

[0025] Figures 2A and 2B show the binding of anti-CD20 antibodies to HEK293-cynoCD20 cells expressing monkey CD20 as measured by FACS (Figure 2A) and to HEK293 cells not expressing CD20 as measured by FACS (Figure 2B).

[0026] Figure 3 shows the binding of humanized anti-CD20 antibodies and their parent antibodies to CD20 on Daudi cells as measured by FACS.

[0027] Figures 4A and 4B show the binding of humanized anti-CD20 antibody and its parent antibody to HEK293-cynoCD20 cells as determined by FACS (Figure 4A) and to HEK293 cells (Figure 4B), respectively.

[0028] Figures 5A and 5B show the endocytosis of anti-CD20 antibody as determined on Ramos cells (Figure 5A) and Daudi cells (Figure 5B), respectively.

[0029] Figure 6 shows endocytosis based on rProtein G-vc-MMAE killing.

[0030] Figure 7 shows a schematic diagram of the multispecific antibody structure.

[0031] Figures 8A and 8B show the expression of CD20 on the surface of B cell-derived cell lines (Figure 8A) and the expression of CD79b on the surface of B cell-derived cell lines (Figure 8B), respectively.

[0032] Figures 9A, 9B, and 9C show the binding of the multispecific antibody and its parent antibody to Ramos cells (Figure 9A), the binding of the multispecific antibody and its parent antibody to JEKO-1 cells (Figure 9B), and the binding of the multispecific antibody and its parent antibody to HEK293 cells (Figure 9C), respectively.

[0033] Figures 10A and 10B show the binding of the multispecific antibody and its parent antibody to human CD20 cells (Figure 10A) and the binding of the multispecific antibody and its parent antibody to monkey CD20 cells (Figure 10B), respectively.

[0034] Figure 11 shows the multispecific antibody V-F2 binding to both CD20 and CD79b.

[0035] Figures 12A and 12B show the endocytosis of the multispecific antibody V-F2 and its parent antibody on Ramos cells (Figure 12A) and the endocytosis of the multispecific antibody V-F2 and its parent antibody on WSU-DLCL2 cells (Figure 12B), respectively.

[0036] Figures 13A and 13B show the effects of Ramos in vitro killing for 5 days (Figure 13A) and 6 days (Figure 13B), respectively.

[0037] Figures 14A, 14B, and 14C show the in vitro killing effect of SU-DHL-8 for 5 days (Figure 14A), SU-DHL-2 for 4 days (Figure 14B), and RC-K8 for 6 days (Figure 14C), respectively.

[0038] Figures 15A, 15B, and 15C show the antitumor efficacy of the Ramos-CDX model (Figure 15A), the tumor volume of Day 14 mice in the Ramos-CDX model (Figure 15B), and the change in body weight of the Ramos-CDX model mice (Figure 15C), respectively.

[0039] Figures 16A, 16B, and 16C show the antitumor efficacy of the WSU-DLCL2-CDX model (Figure 16A), the tumor volume of Day 19 mice in the WSU-DLCL2-CDX model (Figure 16B), and the change in body weight of the WSU-DLCL2-CDX model mice (Figure 16C), respectively.

[0040] Figures 17A, 17B, and 17C show the antitumor efficacy of the Ramos-CDX model (Figure 17A), the tumor volume of the Ramos-CDX model mice on Day 14 (Figure 17B), and the change in body weight of the Ramos-CDX model mice (Figure 17C), respectively.

[0041] Figures 18A, 18B, and 18C show the antitumor efficacy of the WSU-DLCL2-CDX model (Figure 18A), the tumor volume of the WSU-DLCL2-CDX model mice on Day 40 (Figure 18B), and the change in body weight of the WSU-DLCL2-CDX model mice (Figure 18C), respectively.

[0042] Figures 19A, 19B, and 19C show the antitumor efficacy of the SU-DHL-8-CDX model (Figure 19A), the tumor volume of the SU-DHL-8-CDX model mice on Day 16 (Figure 19B), and the change in body weight of the SU-DHL-8-CDX model mice (Figure 19C), respectively.

[0043] Figures 20A, 20B, and 20C show the antitumor efficacy of the SU-DHL-2-CDX model (Figure 20A), the tumor volume of mice in the SU-DHL-2-CDX model on Day 18 (Figure 20B), and the change in body weight of mice in the SU-DHL-2-CDX model (Figure 20C), respectively.

[0044] Figures 21A, 21B, and 21C show the antitumor efficacy of the RT-PDX model (Figure 21A), the tumor size of mice in the RT-PDX model on Day 24 (Figure 21B), and the change in body weight of mice in the RT-PDX model (Figure 21C), respectively.

[0045] Figures 22A and 22B show the in vitro killing effect of ADC drugs on T cells and NK cells in normal healthy human PBMCs, respectively. The figures show the viability of CD3-positive T cells (Figure 22A) and CD16-positive NK cells in PBMCs 7 days after the addition of ADC drugs (Figure 22B). Detailed Description of the Invention

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. Furthermore, the materials, methods and examples described herein are illustrative only and are not intended to be limiting. Other features, objects and advantages of the invention will be apparent from this specification and the accompanying drawings and from the appended claims.

[0048] Definitions

[0049] The following definitions will be used to interpret this specification, and terms used in the singular may also include plural forms, and vice versa, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0050] The term “about” when used in conjunction with a numerical value means to encompass a range of numerical values ​​having a lower limit of 5%, 4%, 3%, 2%, or 1% less than the specified numerical value and an upper limit of 5%, 4%, 3%, 2%, or 1% greater than the specified numerical value, and when used in conjunction with a numerical range, means to encompass a range consisting of values ​​whose upper limit is 5%, 4%, 3%, 2%, or 1% less than the specified value and values ​​whose upper limit is 5%, 4%, 3%, 2%, or 1% greater than the specified value, and values ​​whose lower limit is 5%, 4%, 3%, 2%, or 1% less than the specified value and values ​​whose lower limit is 5%, 4%, 3%, 2%, or 1% greater than the specified value.

[0051] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0052] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover the situation consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “containing” a specific sequence, it is also intended to encompass the antibody variable region composed of that specific sequence.

[0053] When used with antigens, the terms “binding molecule” and “antigen-binding molecule” are used interchangeably and refer to a protein or polypeptide molecule that can specifically bind to an antigen, an epitope on an antigen. A binding molecule has “affinity” and / or “specificity” to an antigen. Thus, a CD79b binding molecule refers to a protein or polypeptide that can specifically bind to CD79b, a CD20 binding molecule refers to a protein or polypeptide that can specifically bind to CD20, and a CD79b and CD20 binding molecule refers to a protein or polypeptide that can specifically bind to both CD79b and CD20. Some examples of binding molecules include antibodies, antibody fragments, fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.

[0054] The structural domain in an antigen-binding molecule that actually binds to the antigen is referred to herein as an “antigen-binding site” or “antigen-binding domain”. A “domain” is a folded structure in a protein or polypeptide, generally responsible for the protein or polypeptideIndividual functions of peptides. For example, conventional antibodies and immunoglobulins typically form antigen-binding domains on the surface of the VH-VL dimer via three complementarity-determining regions (HCDR1-3) in their heavy chain variable region (VH) and three complementarity-determining regions (LCDR1-3) in their light chain variable region (VL), where six CDRs confer specific binding of the antibody to the antigen. However, in some cases, a single immunoglobulin variable domain (e.g., the heavy chain variable domain (VH) or the light chain variable domain (VL), and the heavy chain variable domain (VHH) derived from camel heavy chain antibodies, can confer antigen binding independently. That is, this single variable domain does not need to interact with another variable domain as described on page 5 / 58 of the specification (CN 122356297 A) and can function independently as an "antigen-binding domain" for recognizing and binding target antigens. Typically, through engineering modifications, the "antigen-binding domains" of antibodies, including the aforementioned monoimmunoglobulin variable domains and the variable domain pairs of conventional antibodies, can be added, removed, or transferred to other proteins or peptides while still maintaining their antigen-binding function without losing the function of the remaining portions and / or remaining domains of the protein or peptide. Therefore, various proteins and peptides constructed by incorporating antigen-binding sites of antibodies are a class of antigen-binding molecules particularly considered in this disclosure.

[0055] The terms "binding" and "specific binding" are used interchangeably in this disclosure, meaning that the binding is selective to the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding site to bind to a specific antigen can be determined by conventional binding assays known in the art. For example, the binding ability of an antibody to an antigen is detected by the ELISA assay described in the examples, or the binding ability of an antibody to cells expressing an antigen on their surface is detected by the FACS assay described in the examples, or the affinity constant KD is detected by the SPR technique described in the examples.

[0056] The term “antibody” is used herein in the broadest sense to refer to a protein containing an antigen-binding site of an immunoglobulin, encompassing a variety of natural and artificial antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies, monoepitope and multiepitope antibodies (e.g., biepitope antibodies), monospecific and multispecific antibodies (e.g., bispecific antibodies), single-chain and multi-chain antibodies, nanobodies, single-domain antibodies, heavy-chain antibodies, chimeric antibodies, humanized antibodies, intact antibodies, and antibody fragments. In some embodiments, preferably, the antibodies of the present invention are bispecific or multispecific antibodies.

[0057] The terms “antibody fragment” or “antigen-binding fragment” of an antibody are used interchangeably to refer to a molecule distinct from an intact antibody that contains a portion of the intact antibody and is capable of binding the antigen bound by the intact antibody. Examples of antibody fragments include, but are not limited to, those described above.This includes, but is not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; single-chain antibody fragments (e.g., scFv, scFab); single immunoglobulin domains; variable domain fragments of camel heavy chain antibodies; and various monospecific, bispecific, or multispecific antibody structures formed from antibody fragments, such as linear antibody fragments, diabody fragments, etc. In this disclosure, unless otherwise stated or explicitly contradicted by the context, reference to the term "antibody" is equivalent to reference to "antibody and antibody fragments thereof." In some embodiments of the invention, antibodies and antibody fragments thereof comprise amino acid residues for coupling chemistry. Such amino acid residues include, but are not limited to, native cysteine ​​residues that form disulfide bonds between antibody polypeptide chains, and site-directed mutant cysteine ​​residues.

[0058] Hereinafter, "monospecific" refers to the ability to bind only one epitope. In contrast, the term "multispecific" refers to the ability to bind multiple different epitopes. Accordingly, "bispecific" refers to the ability to bind two different epitopes. The different epitopes may be different epitopes on different antigens or different epitopes on the same antigen.

[0059] In this document, the antibody-related terms “valence” or “valence number” refer to the total number of antigen-binding sites in an antibody molecule, or the number of antigen-binding sites having the same antigen-binding specificity. For example, a tetravalent antibody means that the antibody molecule contains a total of 4 antigen-binding sites; the antibody molecule can be a “2+2” type bispecific antibody, that is, the antibody has two different antigen-binding specificities, with 2 antigen-binding sites for one antigen-binding specificity and 2 antigen-binding sites for the other antigen-binding specificity.

[0060] In this document, the terms “first,” “second,” and “third,” etc., when used in conjunction with elements such as Fc regions, peptide linkers, or polypeptide chains, are intended to conveniently distinguish two or more elements belonging to the same category. However, it should be noted that unless explicitly stated otherwise, the use of these terms is not intended to assign a specific order, orientation, or position to the elements.

[0061] In this document, the term “CD20” refers to the B lymphocyte surface antigen CD20 (also known as B lymphocyte restriction differentiation antigen, membrane spanning 4-domains A1, or MS4A1). The human CD20 molecule is a hydrophobic transmembrane protein present on the surface of over 90% of peripheral blood or lymphoid organ B cells, expressed from the onset of pre-B cell development until differentiation into plasmablasts. This antigen is overexpressed in over 90% of B-cell non-Hodgkin lymphomas (NHL) (Anderson et al. (1984) Blood 63(6):1424-1433), and has therefore been proposed as a B-cell surface antigen.Candidates for targeted therapy of lymphocyte tumors. In this document, unless otherwise stated, the term CD20 includes any variant of human CD20, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and covers its species homologs. In some cases, the term specifically refers to the CD20 antigen expressed on the surface of tumor cells. An example of CD20 is the human CD20 protein containing the amino acid sequence under UniProtKB-P11836. In this disclosure, unless specifically indicated, the term "antigen-binding specificity against CD20," that is, "antigen-binding domain that specifically binds to CD20," refers to binding specificity against human CD20.

[0062] In this document, the term "CD79b" refers to the B lymphocyte surface antigen CD79b (also known as the B cell antigen receptor complex-associated protein β chain, or B29, IGB, Igβ). The human CD79b molecule is a transmembrane protein of 229 amino acid residues in length, containing an immunoglobulin-like domain. On the B cell membrane, CD79b forms a complex with CD79a and the B cell antigen receptor (BCR), mediating signal transduction and endocytosis. In this document, unless otherwise stated, the term CD79b includes any variant of human CD79b, including sequence variants, especially naturally occurring variants, allelic variants, and post-translational modification variants and conformational variants, and encompasses its species homologs. In some cases, the term specifically refers to the CD79b antigen expressed on the surface of tumor cells. An example of CD79b is the human CD79b protein containing the amino acid sequence under UniProtKB-P40259. In this disclosure, unless otherwise specified, the term "antigen-binding specificity against CD79b," i.e., "antigen-binding domain specifically binding to CD79b," refers to binding specificity against human CD79b.

[0063] In this document, the term "CD20-positive" cell refers to a cell that is positive for CD20 cell surface expression. The term "CD79b-positive" cell refers to a cell that is positive for CD79b cell surface expression. The term "CD20 and CD79b positive" cells refer to cells that are double-positive for both CD20 and CD79b expression on their cell surface. The expression levels of CD20, CD79b, or both, on the cell surface can be determined by any conventional method known in the art for determining cell surface antigen expression levels, such as FACS detection methods, immunohistochemical staining, or immunofluorescence staining methods. In some aspects of this disclosure, the positive cells refer to positive tumor cells expressing CD20, CD79b, or both.

[0064] In this document, the term "affinity" or "binding affinity" refers to the affinity of a molecule (e.g., an antibody) for...The strength of the sum of all non-covalent interactions between a single binding site and its binding partner (e.g., an antigenic epitope). Hereinafter, “binding affinity” reflects the intrinsic binding affinity of a 1:1 interaction between members of a binding pair. Binding affinity is typically expressed as a binding dissociation equilibrium constant (KD) and can be measured using methods known in the art, such as surface plasmon resonance (SPR) techniques.

[0065] Hereinafter, the term “avidity” or “binding affinity” refers to the combined strength of the interactions of multiple binding sites of a molecule (e.g., an antibody) with the same target. Therefore, a necessary condition for affinity is the multivalent nature of the molecule (e.g., an antibody) to a target.

[0066] Hereinafter, the term “immunoglobulin” refers to a protein having the structure of naturally occurring antibodies. For example, IgG immunoglobulins are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two light chains and two heavy chains linked by disulfide bonds. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, followed by a heavy chain constant region consisting of three heavy chain constant domains (CH1, CH2, and CH3). From the N-terminus to the C-terminus, each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, followed by a light chain constant region consisting of one light chain constant domain (CL). Immunoglobulin heavy chains can be classified into one of five categories based on the type of their constant domains, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further subdivided into subclasses, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). Immunoglobulin light chains can also be classified into one of two types based on the amino acid sequence of their constant domains, called κ and λ. In some embodiments of the antibody having a constant region according to page 7 / 58 of this specification, CN 122356297 A, the constant region refers to a constant region of an immunoglobulin or a sequence variant thereof.

[0067] In this document, the term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. Typically, the heavy chain variable region and the light chain variable region are identical, each containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs), arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. One or more residues in the variable region of the antibody may be modified, for example, by modifying one or more CDR regions and / or by modifying one or more framework regions, especially by substituting conserved residues, to obtain at least one biological property (e.g., antigen-binding ability) that still substantially retains the parent antibody.Antibody variants. Furthermore, antibody variable regions can be modified via CDR transplantation. In such antibody variants, a CDR sequence from a known antibody is transplanted onto the framework region of a different antibody with different properties, and one to several residues can be mutated as needed, such as reverting mutations, to refine the desired properties of the antibody. In some cases, for the therapeutic application of antibodies or their derivatives, it is desirable to reduce their immunogenicity and improve their drugability. For this purpose, the variable domains of antibodies can be engineered to construct humanized, deimmunogenic, and / or PTM (post-translational modification) de-mutated variants. The properties of the mutated and / or modified antibodies, such as target antigen binding properties or other desired functional properties, such as T cell activation activity and / or tumor cell killing activity, can be determined and screened in vitro or in vivo using methods known in the art and described herein. It should be understood that such functional variants of any variable regions (e.g., VH and / or VL regions, VHH regions) given herein are within the scope of this invention.

[0068] In this document, the terms "complementarity-determining region" and "CDR region," "CDR," and "hypervariant region" are used interchangeably, referring to regions within the variable domain of an antibody that are highly variable in sequence and form structurally defined loops ("hypervariant loops") and / or contain antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. In the VH and VL domains, CDRs are sequentially numbered starting from the N-terminus and are typically referred to as HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively. In the VHH domain, CDRs are sequentially numbered starting from the N-terminus and are typically referred to as CDR1, CDR2, and CDR3, respectively. The CDR sequence within a defined variable region can be determined using schemes known in the art, such as the Kabat, AbM, Chothia, Contact, and IMGT schemes or any combination thereof. Kabat complementarity-determining regions (CDRs) are determined based on sequence variability and are the most commonly used scheme (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The Chothia scheme refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196: 901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chothia structural loops, developed by Oxford Molecular's AbM HVR.Antibody modeling software is used. The “Contact” HVR is based on the analysis of the available crystal structure of the complex.

[0069] The following are examples of CDR region ranges defined using the Kabat, AbM, IMGT and Contact schemes. Specification 8 / 58 pages 17 CN 122356297 A

[0070]

[0071] Unless otherwise stated, in this invention, the term “CDR” or “CDR sequence” covers a CDR sequence determined in any of the above methods and combinations thereof. In addition, a CDR may also be determined based on having the same Kabat number position as a reference CDR sequence (e.g., the exemplary CDR of this invention). Furthermore, it is known in the art that although CDRs are different between antibodies, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM and Contact methods, a minimum overlapping region can be determined, thereby providing a “minimum binding unit” for antigen binding. Such a minimum binding unit may be a sub-part of a CDR. The remaining residues of the CDR sequence, as will be apparent to those skilled in the art, can be determined by the structure of the antibody and protein folding. Therefore, the present invention also considers any variants of the CDR given herein. For example, in a variant of a CDR, the amino acid residue of the smallest binding unit may remain unchanged, while the remaining CDR residues may be substituted.

[0072] In this document, unless otherwise stated, references to the positions of antibody variable regions and residues in the CDR refer to the positions according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0073] In this document, the terms “VHH” and “VHH domain” are used interchangeably to refer to a heavy chain variable domain derived from a heavy chain antibody lacking a light chain. VHHs can be derived from antibodies produced from camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids can also produce naturally occurring heavy-chain antibodies lacking the light chain, and these VHHs are also within the scope of this invention. Structurally, a VHH is a single-chain antibody fragment containing FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1 from the C-terminus to the N-terminus. Unlike the variable domains from conventional four-chain antibodies, VHHs do not require pairing with additional immunoglobulin variable domains to specifically recognize and bind to the target antigen. Therefore, the VHH domain (alone, or as a subset of the VHH domain) can be used.Using a larger peptide (part of a larger peptide) to recognize and bind to target antigens offers several significant advantages over conventional VH and VL domains, scFv, or conventional antibody fragments (e.g., Fab or F(ab')2 fragments): - Only a single domain is required to bind antigens with high affinity and selectivity, thus eliminating the need for two separate domains or ensuring that the two domains are in appropriate spatial conformation and configuration (e.g., scFv generally requires specially designed linkers); - The VHH domain can be easily modified into multivalent and multispecific formats; - The VHH domain is highly soluble and has no tendency to aggregate; - The VHH domain is highly stable to heat, pH, proteins or peptidases, and other denaturing agents or conditions; - The VHH domain is easy to prepare and relatively inexpensive; - The VHH domain is relatively small compared to conventional tetrapeptide antibodies and their antigen-binding fragments, thus exhibiting higher tissue permeability and allowing for relatively high doses. - The VHH domain can exhibit so-called cavity binding properties (compared to conventional VH domains, VHH has an extended CDR3 loop, thereby reaching target epitopes that are inaccessible to conventional tetrapeptide antibodies and their antigen-binding fragments).

[0074] VHHs include humanized VHHs, camel-derived VHs, or VHHs obtained through affinity maturation. VHH sequences may comprise fully human sequences, humanized sequences, chimeric sequences, or sequences optimized in other ways. Further description of VHHs can be found in WO 94 / 04678, WO 95 / 04079, and WO 96 / 34103. In some embodiments, the CD20 antigen-binding site of the antigen-binding molecule and antibody according to the invention is preferably provided by the VHH domain. This VHH domain is also referred to herein as anti-CD20 VHH or VHHCD20.

[0075] As used herein, the terms “Fab” and “Fab domain” are used interchangeably to refer to a structure similar to that in a conventional four-chain IgG antibody, formed by pairing a heavy chain variable region VH and a heavy chain constant region CH1 (VH-CH1) with complementary light chain variable regions VL and light chain constant regions CL (VL-CL). The term also covers structures in which CH1 and CL are exchanged, i.e., structures formed by pairing VH-CL with VL-CH1. In some embodiments of the antibody according to this disclosure, the Fab domain is fused to the Fc region of an immunoglobulin, including but not limited to, by fusing a fragment containing VH to the N-terminus of the Fc region of an immunoglobulin; or by fusing a fragment containing VL to the N-terminus of the Fc region of an immunoglobulin. In this case, the polypeptide chain covalently linked to the Fc region in the Fab domain is also referred to herein as the heavy chain of the Fab domain; correspondingly, the polypeptide chain not covalently linked to the Fc region is also referred to as the Fab domain.The light chain. In some embodiments, the CD79b antigen-binding site of the antigen-binding molecule and antibody according to the invention is preferably provided by a Fab domain. This Fab domain is also referred to herein as anti-CD79b Fab or FabCD79b.

[0076] As used herein, the terms “scFv” and “scFv domain” are used interchangeably to refer to a single-chain polypeptide comprising a VH domain and a VL domain linked together by a flexible linker, wherein the VH domain and the VL domain located on the polypeptide chain pair to form an antigen-binding domain responsible for antigen binding. In some embodiments of the antigen-binding molecule and antibody of the invention comprising anti-CD79b Fab, the antigen-binding molecule and antibody, as an alternative to the Fab domain, comprise an anti-CD79b scFv domain.

[0077] The term “immunoglobulin Fc region” is used interchangeably herein with “Fc region” and “Fc domain” to define a C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of the heavy chain constant region. The term "Fc region" or "Fc domain" excludes the heavy chain variable region VH and light chain variable region VL, as well as the heavy chain constant region CH1 and light chain constant region CL of immunoglobulins; however, it may include all or part of the immunoglobulin hinge region. In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or Pro230 to the C-terminus of the heavy chain. In other embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from E216 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (Gly446Lys447) residues in the Fc region may or may not be present. The sequence constituting the Fc region can be a native sequence or a variant sequence. Therefore, the term "Fc region" encompasses both native Fc regions and variant Fc regions.

[0078] In this document, the term “natural sequence Fc region” encompasses the Fc region sequences of various naturally occurring immunoglobulins, such as the Fc region sequences of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520.). Specification 10 / 58 pages 19 CN 122356297 A

[0079] In this document, the term “variant sequence Fc region” refers to a polypeptide containing a modified Fc region relative to the natural sequence Fc region polypeptide. The modification may be the addition, deletion, and / or substitution of amino acid residues. Substitution may include naturally occurring amino acids.Amino acid substitution and non-naturally occurring amino acid substitution. The purpose of modification includes, but is not limited to, altering the binding of the Fc region to its receptor and the resulting effector function, preventing undesirable heavy chain mismatches, or site-directed introduction of amino acid mutations that can be used to alter interchain disulfide bond formation.

[0080] In this document, unless otherwise stated, the amino acid residues in the Fc region and the heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., SEQuences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0081] In this document, the term "effector function" refers to those biological activities attributable to the Fc region of immunoglobulins that vary with immunoglobulin isotype. Examples of immunoglobulin effector functions include: Fc receptor binding, C1q binding and complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated cytotoxicity (ADCC). Depending on the intended use of the antibody molecule, the Fc region of the antibody may be selected and / or modified to give it effector functions appropriate to the intended use, such as weakened, reduced, or eliminated Fcγ receptor binding, ADCC activity, and / or CDC activity compared to the wild-type IgG1 Fc region.

[0082] In this document, the terms “flexible linker” or “peptide linker” are used interchangeably and refer to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or from the hinge region of an immunoglobulin.

[0083] In this document, the “identity percentage (%)” of an amino acid sequence refers to the percentage of positions in the candidate sequence that have the same amino acid residues at the corresponding positions in the alignment with the specific amino acid sequence shown in this disclosure, without taking into account any conserved substitutions as part of sequence identity, after comparing the candidate sequence with the specific amino acid sequence shown in this disclosure and, if necessary, introducing vacancies to achieve the maximum sequence identity percentage. In some embodiments, this disclosure contemplates variants of the antibody sequences of the present invention that have a considerable degree of identity with respect to the antibody sequences specifically disclosed herein within a comparison window, for example, an identity of at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or higher. Where no comparison window is specified herein (i.e., the antibody region of interest to be compared), the alignment is performed over the full length of the reference antibody sequence. In some embodiments, the variants may contain conserved modifications.

[0084] In this document, for a polypeptide sequence, “conserved modification” includes substitutions, deletions, or additions to the polypeptide sequence that do not significantly affect or alter the desired properties of the polypeptide containing the modification (e.g., binding characteristics and / or T cell activation characteristics). Tables of conserved substitutions for functionally similar amino acids are well known in the art. The following eight groups contain amino acids that are conservedly substituted for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)).

[0085] In this document, the term "chimeric antibody" refers to an antibody in which a portion (e.g., the variable region sequence) is derived from one species and another portion (e.g., the constant region sequence) is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0086] In this document, a "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) of a humanized antibody correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of human antibodies. The antibody may optionally contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized. In this document, in some embodiments, the humanized antibody of the present invention has a framework region sequence "derived" from a specific human lineage sequence. Here, "derived from" means that the amino acid sequence of the antibody framework region has at least 85% and 90% identity with the corresponding amino acid sequence of the framework region encoded by the human immunoglobulin gene, and that the antibody retains antigen-binding activity.

[0087] In this document, if the antibody amino acid sequence is specific to two different antigens or antigenic determinants (e.g., CD20 from different mammalian species, such as human and cynomolgus monkey CD20), it is said to be "cross-reactive" to these two different antigens or antigenic determinants. Antibodies possessing human-monkey species cross-reactivity, especially having similar human-monkey antigen-binding affinity, is advantageous, as this property can facilitate preclinical drug development of the antibody, such as toxicological assays of antigen-binding molecules composed of antibodies. In some embodiments, the antibodies of the present invention preferably possess human-monkey species cross-reactivity.

[0088] In this document, "isolated" antibody or antibody fragment refers to artificial antibody or antibody fragment, recombinant antibody or antibody fragment, and antibody or antibody fragment that has been at least partially isolated from components in the natural environment from which it was produced. In some embodiments, the antibody or antibody fragment according to the invention is "isolated". In some embodiments, the isolated antibody or antibody fragment is purified to a purity of more than 90%, 95%, or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0089] In this document, the term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny of such cells. Host cells include "transformers" and "transformed cells", which include primary transformed cells and progeny derived therefrom. Host cells are any type of cell system that can be used to produce the antibody molecules of the invention, including eukaryotic cells, such as mammalian cells, insect cells, yeast cells; and prokaryotic cells, such as Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0090] In this document, the term "expression vector" refers to a vector capable of directing the expression of a nucleotide sequence operatively linked thereto. Expression vectors typically contain cis-acting elements for the expression of said nucleotide sequence; while other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include, for example, but not limited to, granules, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0091] In this document, the terms "endocytosis" and "internalization" are used interchangeably, referring to the process by which a ligand / receptor complex is internalized and delivered into the cytosol or transferred to a suitable intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface. In some embodiments, the antibodies of the present invention initiate endocytosis mediated by CD79b and / or CD20 receptors upon binding to CD79b and / or CD20 expressed on the cell surface. In this document, the absolute endocytosis and endocytosis rate can be determined by methods described, for example, in the embodiments, to characterize the endocytic activity of the antibody. In some embodiments, the antibody of the present invention having endocytic activity can be used as a tool for delivering antitumor drugs into cancer cells in the ADC of the present invention.

[0092] The terms “conjugate” or “coupler” herein refer to a molecule formed by conjugating one or more immunoglobulin-related molecules or fragments thereof with one or more other molecules. Conjugates typically contain at least one non-protein chemical structural moiety, such as a chemical linker for achieving the conjugation. In some cases, the other molecules mayThese are all immunoglobulin-related molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-related molecules or fragments thereof. The one or more additional molecules may be the same or different from each other. For example, the other molecules may be target-binding elements and / or effector elements, such as chemotherapeutic agents, toxins, drugs (such as immunotherapeutic agents), radioactive elements, probes, or signaling molecules, etc.

[0093] "Antibody-drug conjugate (ADC)" refers to a compound obtained by linking an antibody or antigen-binding molecule to a drug (small molecule) via a linker. In this document, the term "antibody-drug conjugate" or "ADC" includes its pharmaceutically acceptable salts and solvent compounds, as well as other equivalent forms. The drug compound portion in the ADC may be referred to herein as "payload" or "toxin".

[0094] The term "linker-payload" refers to a compound formed by linking a payload to a linker. In some cases, the linker-payload is used as an intermediate in the synthesis of the ADC.

[0095] The term "linker" refers to a structural segment that covalently links a drug (e.g., a small molecule drug) to a portion of an antibody or antigen-binding molecule. It should be understood that the linker has functional groups that can form bonds with functional groups of the antibody or antigen-binding molecule before being linked to it. In some cases, the linker may also have a degradable portion and optionally a hydrophilicity modulating module such as a PEG segment. In some embodiments of the ADC according to the invention, the linker is preferably "degradable," thereby being able to break and release the payload after the ADC is delivered to the target region (e.g., the target tumor tissue site). Such "degradable linkers" available include, for example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers.

[0096] The term "therapeutic agent" covers any substance effective in preventing or treating diseases such as cancer, including chemotherapeutic agents, cytotoxic agents, immunomodulators (e.g., immunosuppressants), other antibodies, small molecule drugs, angiogenesis inhibitors, or cytokines.

[0097] The term "drug" refers to a compound capable of regulating biological processes, particularly altering or preventing pathological processes. In this document, "drug" preferably refers to an antitumor compound.

[0098] The term "small molecule drug" refers to a drug with a low molecular weight capable of regulating biological processes, particularly altering or preventing pathological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa, more preferably less than 500 kDa. Small molecule drugs include, but are not limited to, organic molecules having the molecular weights defined above, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can...It can penetrate cells more easily than macromolecules, is less susceptible to degradation, and is less likely to trigger an immune response.

[0099] "Antitumor compound" is a pharmaceutically active compound that has an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773, US5658920, camptothecin compounds such as eczemaconazole and Dxd (Exatecan derivatives), and auristatin compounds such as monomethyl auristatin E (MMAE) and MMAF.

[0100] The term "cytotoxic agent" is used in this invention to mean a substance that inhibits or prevents cell function and / or causes cell death or destruction.

[0101] "Chemotherapy agent" includes chemical compounds useful in the treatment of cancer or immune system diseases.

[0102] The term "alkyl" as used herein refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 1 to 16 carbon atoms, such as 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc.

[0103] The terms “halogen” or “halogenated” refer to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0104] The term “halogenated alkyl” refers to an alkyl group as defined herein that is substituted with one or more halogen groups as defined herein. Halogenated alkyl groups may preferably be monohalogenated alkyl groups, dihalogenated alkyl groups, or polyhalogenated alkyl groups (including perhalogenated alkyl groups). Monohaloalkyl groups may contain one iodine, bromine, chlorine, or fluorine atom in the alkyl group. Dihaloalkyl and polyhaloalkyl groups may contain two or more identical halogen atoms or combinations of different halogen groups in the alkyl group. Preferably, polyhaloalkyl groups contain up to 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of halogen alkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhaloalkyl refers to an alkyl group in which all hydrogen atoms are replaced by halogen atoms.

[0105] The terms “alkoxy” and “alkyl-O-” are used interchangeably to refer to alkyl groups as defined above that are linked by an oxygen atom. Preferably, the alkoxy group has 1-8 carbon atoms (C1-8 alkoxy), 1-6 carbon atoms (C1-6 alkoxy), 1-4 carbon atoms (C1-4 alkoxy), or 1-3 carbon atoms (C1-3 alkoxy). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, and propionic acid.Oxygen groups (including n-propoxy and isopropoxy), butoxy groups (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy groups (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexoxy, heptoxy, octoxy, etc.

[0106] The term "amino acid" refers to naturally occurring and synthetic amino acids, amino acid analogs, and their artificially modified forms. Amino acids can be L or D isomers. In this disclosure, 20 naturally occurring amino acids are represented by single-letter and three-letter abbreviations known in the art, such as: phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn; N), isoleucine (Ile; I), arginine (Arg; R), proline (Pro; P), and glutamine (Gln; Q). The remaining amino acids are represented by their full names or multi-letter abbreviations known in the art; for example, citrulline can be represented by Cit; cyclobutane-1,1-dicarboxamide-citrulline is represented by cBu-Cit. Unless otherwise specified, the amino acids of this invention refer to L-amino acids.

[0107] The terms “pentose” or “hexose” refer to polyhydroxy aldehydes or ketones with 5 or 6 carbon atoms, respectively. Pentoses and hexoses applicable to the present invention include, but are not limited to, ribose, rhamnose, arabinose, xylose, glucose, lythose, mannose, and galactose.

[0108] The term “penturonic acid” refers to a compound formed by oxidizing the primary hydroxyl group of a pentose as defined above to a carboxyl group. Examples of penturonic acids include, but are not limited to, xyuronic acid and arabinuronic acid.

[0109] The term “hexuronic acid” refers to a compound formed by oxidizing the primary hydroxyl group of a hexose as defined above to a carboxyl group. Examples of hexuronic acids include, but are not limited to, glucuronic acid, galacturonic acid, and mannuronic acid.

[0110] The terms “optional” or “optionally” mean that the event or condition described below occurs or does not occur, and the description includes instances where the event or condition occurs and instances where the event or condition does not occur. For example, when a group or structure is “optionally substituted,” the group or structure may be substituted or not substituted.

[0111] In this document, “pharmaceutically acceptable” means that it can be administered to an individual or subject without producing biologically or otherwise undesirable side effects, such as serious, intolerable side effects. Where there is no conflict in the context, “pharmaceutically acceptable” and “pharmaceutical-grade” are used interchangeably herein.

[0112] The term “pharmaceutically acceptable salt” refers to a salt that retains the biological effects and properties of the ADC conjugate of the present invention.Salts, and such salts are not biologically or otherwise undesirable. The ADC conjugates of the present invention can exist in their pharmaceutically acceptable salt forms, including acid addition salts and base addition salts. In the present invention, pharmaceutically acceptable non-toxic acid addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic acids, including but not limited to hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. Pharmaceutically acceptable non-toxic base addition salts refer to salts formed by the ADC conjugates of the present invention with organic or inorganic bases, including but not limited to alkali metal salts, such as lithium, sodium, or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; and organic base salts, such as ammonium salts formed by reacting with an organic base containing an N-group. Instructions for Use, Page 14 / 58, 23 CN 122356297 A

[0113] The term “solvent” refers to an association formed by one or more solvent molecules with the ADC antibody-drug conjugate of the present invention. Solvents forming solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0114] The term “drug:antibody ratio” or “DAR” refers to the ratio of the drug portion (D) to the Ab portion conjugated herein. In some embodiments described herein, the DAR may be determined by p in Formula I, for example, the DAR may be 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. DAR can also be calculated as the average DAR of the molecular population in the product, which is the overall ratio of the drug fraction (D) coupled to the Ab fraction described herein to the Ab fraction in the product as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assay, electrophoresis and / or HPLC). This DAR is referred to herein as the average DAR. In some embodiments, the average DAR value of the conjugates of the present invention is 1 to 16, for example 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 4-6, 6-10, for example 1.0-8.0, 2.0-6.0, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, a range with two of these values ​​as endpoints. It should be understood that when referring to the average DAR value, the ADC of the present invention refers to a population or mixture of ADC molecules containing ADC molecules having the same and / or different DARs.

[0115] The term “half-maximum effective concentration (EC50)” refers to the concentration of a drug, antibody, ADC, or toxin that induces a 50% response between baseline and maximum after a specific exposure time. In the context of this application, EC50 is expressed in “nM”.

[0116] The term “pharmaceutical composition” refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0117] The term “pharmaceutical excipient” refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), carrier, or stabilizer, etc., administered with the active substance.

[0118] The terms “drug combination,” “combination product,” “drug conjugate,” or “combination product” refer to non-fixed combination products or fixed combination products, including but not limited to pillboxes and pharmaceutical compositions. The term “non-fixed combination” means that the active ingredients (e.g., (i) the antibody or antigen-binding molecule of the present invention or the ADC molecule of the present invention, including pharmaceutically acceptable salts thereof, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of two or more active agents. In some embodiments, the antibody, antigen-binding molecule, or ADC molecule of the present invention and other therapeutic agents used in the drug combination are administered at levels not exceeding those obtained when used alone. The term “fixed combination” means that two or more active agents are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active agents are selected so that the combined use of the portions can produce an effect greater than that obtained by using any one component alone in treating a disease or condition. The components may each be in separate formulations, and their formulations may be the same or different.

[0119] The terms “individual” or “subject” are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual or subject is a human.

[0120] The terms “tumor” and “cancer” are used interchangeably herein and refer to a physiological disorder in mammals in which cell growth is not regulated, encompassing both solid and liquid tumors, and including both malignant and benign tumors, and all precancerous and cancerous cells and tissues.

[0121] In this document, the term “treatment” refers to a clinical intervention intended to alter the natural course of a disease in an individual receiving treatment. Desired therapeutic effects include, but are not limited to, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In cases involving tumor or cancer treatment, “treatment” encompasses antitumor biological effects that can be induced by human intervention (e.g., through the administration of a drug), including but not limited to, for example, reduction in tumor volume, reduction in the number of tumor cells, reduction in tumor cell proliferation, or reduction in tumor cell survival.

[0122] When used herein, “prevention” includes the suppression of the occurrence or development of a disease or condition or symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for preventative protocols. Generally, in the context of cancer, the term “prevention” refers to the administration of a drug prior to the occurrence of signs or symptoms of cancer, particularly prior to occurrence in subjects at risk of cancer.

[0123] The term “effective amount” refers to such an amount or dose of the antibody, antigen-binding molecule, or ADC molecule or composition or combination of the present invention, which, after being administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention. Depending on the intended effect, it may include “therapeutic effective amount” and “preventative effective amount”.

[0124] The term “therapeutic effective amount” refers to the amount at the required dose and sustained for the required period of time that effectively achieves the desired therapeutic outcome. The therapeutically effective dose of an antibody or ADC can vary depending on a variety of factors such as disease state, individual age, sex, weight, and the ability of the antibody or ADC to elicit the desired response in the individual. A therapeutically effective dose is also a dose in which any toxic or harmful effects of the antibody or ADC are less than the beneficial therapeutic effect. Relative to untreated subjects, a "therapeuticly effective dose" preferably inhibits measurable parameters (e.g., tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%. This can be used to predict efficacy in human tumors.The ability of compounds to inhibit measurable parameters (e.g., cancer) is evaluated in animal model systems.

[0125] "Prophylactic effective amount" refers to the amount that effectively achieves the desired preventive outcome at the required dose and for the required duration. Typically, because the prophylactic dose is used in subjects before or at an earlier stage of the disease, the prophylactic effective amount is less than the therapeutic effective amount.

[0126] The term "antitumor effect" refers to a biological effect that can be demonstrated by a variety of means, including but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0127] The present invention is described in detail below. It will be understood by those skilled in the art that, unless the context clearly indicates otherwise, any technical feature described in any of the following sections, subsections, or embodiments may be combined with any technical feature described in any other section, subsection, or embodiment, and such combinations are all within the scope of consideration of the present invention.

[0128] I. First aspect of the present disclosure: Multispecific antibody of the present invention

[0129] In a first aspect, the present disclosure provides a multispecific antibody that specifically binds to B cell-associated antigens CD79b and CD20. The multispecific antibody of the present invention can specifically target CD79b single-positive, CD20 single-positive, and CD79b and CD20 double-positive tumor cells, achieving effective tumor killing; and can deplete B cells, improving B cell-related autoimmune diseases. In some embodiments, the multispecific antibody of the present invention comprises at least one antigen-binding domain specifically binding to CD79b and at least one antigen-binding domain specifically binding to CD20. In some embodiments, the multispecific antibody of the present invention further comprises an immunoglobulin Fc region. The multispecific antibody of the present invention can take any suitable form, wherein the domains located on the same chain can be linked by peptide linkers or directly linked as needed.

[0130] The components of the multispecific antibody of the present invention are described in detail below. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features of these components is within the scope of the present invention. Furthermore, those skilled in the art will understand that, unless the context clearly indicates otherwise, the antibody of the present invention (including any form of antibody) can contain any such combination of features.

[0131] Antigen-binding domain

[0132] In their research, the inventors found that, in some cases, it is advantageous to administer a multispecific antibody that can simultaneously target CD79b and CD20 for the treatment of B-cell-associated lymphoid tumors and leukemia. Such a multispecific antibody can take into account the heterogeneity of CD79b and CD20 expression on different malignant B cells, thus expanding the patient applicability of the antibody. Furthermore, it enables the antibody to…One specific target for CD20 can promote the specific binding of antibodies and antibody-based ADCs to B tumor cells; while another specific target for the antibody on the CD79b surface receptor can promote the endocytosis and degradation of antibodies and antibody-based ADCs. Simultaneously, the two specificities of the antibody can provide a mechanism against tumor drug resistance. Furthermore, the inventors have also found that using the VHH domain, which has high CD20 cell binding affinity, as the CD20 specificity source of the antibody can significantly enhance endocytosis mediated by the antibody's CD79b binding arm, especially on tumor cells with low CD79b expression.

[0133] In some embodiments, the present disclosure thereby provides a multispecific antibody comprising at least one antigen-binding domain that specifically binds to CD79b and at least one antigen-binding domain that specifically binds to CD20. In some embodiments, the CD79b binding domain comprises a VH and VL domain pair, or comprises or is composed of a Fab domain; the CD20 binding domain comprises or is composed of a VHH domain.

[0134] In some embodiments, the multispecific antibody according to the present invention is a multispecific antibody against CD79b and CD20. In some embodiments, the antibody is a 3-5 valent antibody (i.e., the total number of antigen-binding domains is 3-5). In some embodiments, the valence ratio of the CD79b binding domain to the CD20 binding domain is 1:1. In some embodiments, the CD79b binding domain is 2 valent, and the CD20 binding domain is 2 valent. In some embodiments, the antibody is a bispecific antibody against CD79b and CD20.

[0135] In some embodiments, the multispecific antibody according to the invention has antigen-binding activity selected from one or more of the following: (i) the CD79b binding domain binds to CD79b-expressing cells at an EC50 value of about 0.1-30 nM, for example, 1-10 nM EC50 value, as determined by flow cytometry (FACS); (ii) the CD20 binding domain binds to CD20-expressing cells at an EC50 value of about 0.1-50 nM, for example, 1-30 nM EC50 value, as determined by flow cytometry (FACS).

[0136] In some embodiments, the multispecific antibody according to the invention has excellent antitumor activity and safety properties. In some embodiments, the multispecific antibody according to the invention also has excellent drug-likeness.

[0137] Anti-CD20 antigen-binding domain

[0138] In some embodiments of the multispecific antibody according to the invention, the CD20 binding domain is provided by a VHH domain. Some exemplary VHH sequences applicable to the CD20 binding domain of the present invention include, but are not limited to, the VHH sequences shown in SEQ ID NOs: 1 and 5-13.

[0139] In some embodiments, the CD20 binding domain according to the present invention comprises the CDR1, CDR2, and CDR3 sequences contained in any VHH sequence shown in SEQ ID NOs: 1 and 5-13.

[0140] The CDR sequence in the CD20 binding domain according to the present invention may be a CDR sequence defined according to AbM, Chothia, Kabat, IMGT, or any combination thereof. Preferably, the CDR is defined according to Kabat or AbM, or a combination thereof, and more preferably, the CDR is defined according to AbM. However, it should be understood that the CDR may also be defined in any other manner known in the art. Specification 17 / 58 pages 26 CN 122356297 A

[0141] In some embodiments, the CD20 binding domain according to the present invention comprises CDR1, CDR2, and CDR3. In some embodiments, the CDR1, CDR2, and CDR3: (i) comprise or consist of the amino acid sequences of SEQ ID NOs:2, 3, and 4, respectively; or (ii) comprise or consist of the amino acid sequences of SEQ ID NOs:14, 3, and 4, respectively. In some embodiments, the CD20 binding domain according to the invention as defined in (i) is preferred.

[0142] Based on the above-described CDR regions of the invention, different framework regions can be selected to construct a variety of CD20-binding anti-CD20 binding domains of the invention with equivalent effectiveness in binding CD20. Therefore, the CD20 binding domains applicable to the invention include not only anti-CD20 binding domains having the VHH sequence listed in one of SEQ ID NOs:1 and 5-13; but also anti-CD20 binding domains having the same or similar CDR sequence (preferably the same CDR sequence) but different framework region sequences as the VHH sequence.

[0143] In some embodiments, the CD20 binding domain according to the invention comprises a VHH domain. In some embodiments, the VHH domain comprises an amino acid sequence selected from SEQ ID NO: 1 and 5-13, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions, and / or substitutions, or is composed of therewith.

[0144] In some preferred embodiments, the CD20 binding domain according to the invention comprises a VHH domain, wherein the VHH domain comprises an amino acid sequence of one of SEQ ID NOs: 1 and 5-13, or is composed of therewith.

[0145] In some more preferred embodiments, the CD20 binding domain according to the invention comprises a VHH domain, wherein the VHH domain comprises an amino acid sequence of SEQ ID NO: 6, or is composed of therewith.

[0146] Anti-CD79b antigen-binding domain

[0147] In some embodiments of the multispecific antibody according to the present invention, the CD79b binding domain is provided by a VH and VL domain pair. In other embodiments, the CD79b binding domain comprises or is composed of a Fab domain.

[0148] Some exemplary VH and VL sequence combinations suitable for the present invention include, but are not limited to, combinations of SEQ ID NO: 16 and 15.

[0149] In some embodiments, the CD79b binding domain according to the present invention comprises the HCDR1, HCDR2 and HCDR3 sequences contained in the VH sequence of SEQ ID NO: 16 and the LCDR1, LCDR2 and LCDR3 sequences contained in the VL sequence of SEQ ID NO: 15.

[0150] The CDR sequence in the CD79b binding domain according to the present invention may be a CDR sequence defined according to AbM, Chothia, Kabat, IMGT or any combination thereof. Preferably, the CDR is defined according to Kabat or AbM or a combination thereof, more preferably, the CDR is defined according to AbM. However, it should be understood that the CDR can also be defined in any other manner known in the art.

[0151] In some embodiments, the CD79b binding domain according to the present invention comprises HCDR1-3 and LCDR1-3, wherein HCDR1-3 and LCDR1-3 respectively comprise or consist of the amino acid sequences of SEQ ID NOs: 20-22 and SEQ ID NOs: 17-19.

[0152] Since the antigen-binding properties of the antibody are mainly responsible for the CDR sequence, different framework regions can be selected based on the above-mentioned CDR region of the present invention to construct a variety of anti-CD79b binding domains of the present invention that are equally effective in binding CD79b. Therefore, the CD79b binding domain applicable to the present invention includes not only anti-CD79b binding domains having the VH and VL sequence combinations listed above (SEQ ID NOs 16 / 15); but also anti-CD79b binding domains having the same or similar CDR sequences (preferably the same CDR sequences) but different frame region sequences as the VH and VL sequence combinations.

[0153] In some embodiments, the CD79b binding domain according to the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has one or more (preferably 1-10, more preferably 1-(a) an amino acid sequence consisting of the addition, deletion, and / or substitution of 5 amino acids, or composed thereof; and / or (b) the light chain variable region comprises an amino acid sequence selected from SEQ ID NO: 15, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence consisting of the addition, deletion, and / or substitution of one or more (preferably 1-10, more preferably 1-5) amino acids, or composed thereof.

[0154] In some preferred embodiments, the CD79b binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: (a) the heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 16; and / or (b) the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0155] In some preferred embodiments, the CD79b binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 15.

[0156] In some embodiments, the CD79b binding domain according to the invention comprises or is composed of a Fab domain. In some embodiments, the Fab domain comprises (1) a VH-CH1 chain (i.e., a chain composed of the heavy chain variable region and the immunoglobulin CH1 domain) and (2) a VL-CL chain (i.e., a chain composed of the light chain variable region and the immunoglobulin light chain constant domain CL). In other embodiments, the Fab domain comprises (1) a VH-CL chain (i.e., a chain composed of the heavy chain variable region and the CL domain) and (2) a VL-CH1 chain (i.e., a chain composed of the light chain variable region and the CH1 constant domain). In some embodiments, the CL domain is a κ or λ light chain constant region, such as the human κ light chain constant region. In some embodiments, the CH1 domain is a CH1 domain derived from IgG immunoglobulins, such as from IgG1, IgG2, IgG3, or IgG4, or a subtype thereof. Preferably, the CH1 domain comprises a CH1 domain sequence derived from humans.

[0157] In some embodiments, the CD79b binding domain according to the invention comprises or is composed of a Fab domain, wherein the Fab domain comprises a CH1 domain and a CL domain, wherein: (a) the CH1 domain comprises the amino acid sequence of SEQ ID NO: 30, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has or is composed of an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids; and / or (b)The CL domain comprises the amino acid sequence of SEQ ID NO: 29, or has at least 85%, 90%, 95%, or 99% identity with respect to the amino acid sequence, or has an amino acid sequence comprising one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids, or is composed thereof.

[0158] Immunoglobulin Fc Region

[0159] In some embodiments, in addition to the aforementioned antigen-binding domain, the multispecific antibody according to the invention may also comprise an immunoglobulin Fc region. In other embodiments, as an alternative, a half-life extension domain, such as serum albumin or serum albumin-binding peptide, may also be included to adjust the circulating half-life of the antibody after administration to an animal. In some embodiments, preferably, the antibody of the invention comprises an immunoglobulin Fc region.

[0160] The immunoglobulin Fc region used in the multispecific antibody of the invention may be an Fc region from any immunoglobulin. In some embodiments, the immunoglobulin Fc region comprises at least an immunoglobulin CH2 domain and a CH3 domain. In some embodiments, the immunoglobulin Fc region further includes a hinge region or a partial hinge region. In some embodiments, the immunoglobulin Fc region includes, from the N-terminus to the C-terminus, an immunoglobulin hinge region or a partial hinge region, a CH2 domain and a CH3 domain, or is composed of the latter. In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2 or IgG4, or a subtype thereof. Preferably, the immunoglobulin Fc region includes an Fc region sequence derived from humans. Specification 19 / 58 pages 28 CN 122356297 A

[0161] The immunoglobulin Fc region can be fused to the C or N-terminus of the antigen-binding domain according to the invention. In some cases, the immunoglobulin Fc can also optionally be fused to other domains via a peptide linker, or directly fused to other domains. Fusion at the N-terminus of the immunoglobulin Fc region is preferably performed via an immunoglobulin hinge region. Fusion at the C-terminus of the immunoglobulin Fc region is preferably performed via a flexible peptide linker.

[0162] In some cases, it is advantageous for the immunoglobulin Fc region to include a hinge region sequence, which can, for example, facilitate the dimerization of the antibody polypeptide chain and / or provide cysteine ​​residues for coupling with other active molecules. Such a hinge sequence may substantially or partially correspond to the hinge region of IgG1, IgG2, IgG3, or IgG4. For example, the hinge region sequence may include all or part of the core hinge region and, optionally, all or part of the upper hinge region. The core hinge region has the amino acid sequence CPPC in IgG1, IgG2, and IgG3, and the CPSC sequence in IgG4. In some embodiments, the hinge region sequence comprises the hinge region sequence E216 to T225 from IgG1.(According to EU number), or a corresponding hinge region sequence from other immunoglobulin isotypes.

[0163] The immunoglobulin Fc region used in the multispecific antibody of the present invention can be a natural Fc region sequence. Alternatively, the Fc region can contain mutations relative to the natural Fc sequence. Mutations include substitutions, insertions, and / or deletions. Mutations can be introduced into the immunoglobulin Fc region for the purpose of introducing desired therapeutic properties. As an example, the Fc region can contain mutations that alter effector function, depending on the specific application of the antibody of the present invention or an antigen-binding molecule based on the antibody. In some embodiments, the mutations include mutations that reduce or eliminate effector function, such as the LALA mutation where leucine (L) at positions 234 and 235 of the Fc region is replaced with alanine (A). Additionally or alternatively, the Fc region of the antibody of the present invention may also contain other mutations, including but not limited to: mutations for increasing binding to FcRn and / or removing protease sites; introducing amino acid modifications that can be used to couple active molecules; and removing or replacing amino acids that may undergo post-translational modifications (e.g., glycosylation) to provide improved drugability and developability of the therapeutic antibody.

[0164] In some embodiments of the multispecific antibody according to the invention, the antibody comprises an immunoglobulin Fc region, wherein the immunoglobulin Fc region has one or more of the following characteristics:

[0165] (i) the Fc region contains a mutation that reduces or eliminates the binding of the Fc region to FcγR, for example, L234A or L235A mutations; (ii) the Fc region is IgG type, for example, IgG1 or IgG4 isotype; and / or (iii) the Fc region comprises the amino acid sequence of SEQ ID NO: 31 or 32, or an amino acid sequence that is at least 95%, 96%, 98% or 99% identical thereto.

[0166] Peptide Linker

[0167] In the multispecific antibody according to the invention, the antibody components (i.e., the antigen-binding domain and optionally the immunoglobulin Fc or half-life-binding domain) may be linked by a peptide linker. There are no particular limitations on the peptide linkers that can be used in the antibodies of the invention. Peptide linkers are generally flexible. It can be primarily composed of amino acids with large side chains, such as glycine, alanine, and serine, that do not have the potential to limit flexibility. Alternatively, it can be composed of sequences derived from the hinge region of immunoglobulins. Depending on the linkage location and the component to be linked, those skilled in the art can readily determine the available peptide linker sequence or optimal length. Suitable peptide linker lengths can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids long, or longer. In some cases, the peptide...The length of the linker sequence can be relatively short, for example less than about 20 or 15 amino acids, such as 2-15 amino acids or 5-10 amino acids. Suitable linker sequences include, but are not limited to, G4S (SEQ ID NO: 40); (G4S)2 (SEQ ID NO: 41); (G4S)3 (SEQ ID NO: 27); GGGSG (SEQ ID NO: 42); GGSGG (SEQ ID NO: 43); GSGGG (SEQ ID NO: 44); GSGGGP (SEQ ID NO: 45); GGEPS (SEQ ID NO: 46); GGEGGGP (SEQ ID NO: 47) and GGEGGGSEGGGS (SEQ ID NO: 48); and (G4S)n (SEQ ID NO: 26), where n is an integer equal to or greater than 1; TS(G4S)n (SEQ ID NO: 49), where n is an integer equal to or greater than 1; G(G4S)n (SEQ ID NO: 49) (SEQ ID NO: 40); G(G4S)n (SEQ ID NO: 40) (SEQ ID NO: 41); GGGSG (SEQ ID NO: 42); GGSGG (SEQ ID NO: 43); GGSGG (SEQ ID NO: 44); GGSGGP (SEQ ID NO: 45); GGEPS (SEQ ID NO: 46); GGEGGGP (SEQ ID NO: 47) and GGEGGGSEGGGS (SEQ ID NO: 48); and (G4S)n (SEQ ID NO: 49) (SEQ ID NO: 40); GGEGGGP (SEQ ID NO: 41); GGEGGGP (SEQ ID NO: 45); GGEPS (SEQ ID NO: 46); GGEGGGP (SEQ ID NO: 47); GGEGGGP (SEQ ID NO: 48); GGEGGGP (SEQ ID NO: 49); GGEGGGP (SEQ ID NO: 48); GGEGGP (SEQ ID NO: 49); GGEGGP (SEQ ID NO: 49); GGEGGP (SEQ ID NO: 50), where n is an integer equal to or greater than 1; (G4)n (SEQ ID NO: 51), where n is an integer equal to or greater than 1; (GRPGS)n (SEQ ID NO: 52), where n is an integer equal to or greater than 1. Suitable flexible linker peptides can be rationally designed using computer programs to simulate the three-dimensional structures of proteins and peptides, or by phage display methods. In some embodiments, the peptide linker used in the antibody of the present invention is a flexible linker peptide of 5-50 amino acids, preferably comprising a linker peptide containing glycine (G) and / or serine (S) and / or threonine residues (T). In one embodiment, the peptide linker has a length of 5-50 amino acids, for example, 5, 10, 15, 20, 25, or 30 amino acids, or has an amino acid length falling between any two integers. In some embodiments, the peptide linker comprises the amino acid sequence (G4S)n (SEQ ID NO: 26), where n is an integer equal to or greater than 1, for example, n is an integer from 1 to 7, such as n = 1, 2, 3, 4, 5, 6, or 7.

[0168] In some embodiments of the multispecific antibody according to the invention, the anti-CD20 domain according to the invention can be linked to the C-terminus of the Fc region of an immunoglobulin via a peptide linker. In some embodiments, the peptide linker can be 5 to 20 amino acid lengths, for example, about 10 or 15 amino acid lengths. In some embodiments, the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 26 or 27.

[0169] In some embodiments of the multispecific antibody according to the present invention, the anti-CD20 domain according to the present invention can be linked to the VH or VL domain of the anti-CD79b Fab domain according to the present invention via a peptide linker. In some embodiments, the peptide linker can be 5-15 amino acid lengths, for example, about 5, 10, or 15 amino acid lengths. In some embodiments, the peptide linker comprises or is composed of the amino acid sequence of SEQ ID NO: 26 or 27.

[0170] Structural Forms of Multispecific Antibodies

[0171] Multispecific antibodies (e.g., bispecific antibodies) can be classified into many types according to different components and construction methods. For example, according to the substantially left-right symmetry of the multispecific antibody structure, they can be classified into symmetrical structures and asymmetrical structures; according to the presence or absence of the Fc region of IgG, they can be classified into antibody patterns with Fc regions and antibody patterns without Fc regions; according to the number of antigen-binding sites in the multispecific antibody, they can be classified into bivalent, trivalent, tetravalent, or more valent antibodies, etc.; according to the number of polypeptide chains constituting the multispecific antibody, they can be classified into single-chain or multi-chain forms. See, for example, Brinkmann U. and Kontermann RE, The making of bispecific antibodies, Mabs, 2017, 9(2): 182-212. These multispecific antibody structures known in the art are all within the scope of consideration of this invention.

[0172] In some embodiments, this disclosure provides a multispecific antibody comprising at least one anti-CD20 VHH domain, at least one anti-CD79b binding domain, and at least one half-life extension domain, wherein preferably the half-life extension domain is an immunoglobulin Fc region.

[0173] In some embodiments, the multispecific antibody according to the invention comprises: (a) an anti-CD79b binding domain and; (b) an immunoglobulin Fc region attached to the C-terminus of (a) domain; and (c) at least one (preferably one) CD20 binding domain, optionally attached to the N-terminus of (a) domain or the C-terminus of (b) domain via a peptide linker. In some embodiments, the anti-CD79b binding domain comprises a VH and VL domain pair. In some embodiments, the anti-CD79b binding domain comprises a Fab domain or an scFv domain, preferably a Fab domain, or is composed of thereof. In some embodiments, the CD20 binding domain comprises a VHH domain or is composed of thereof. Preferably, the peptide linker comprises the amino acid sequence of SEQ ID NO: 26 or 27. Preferably, the multispecific antibody has a symmetrical structure formed by dimerization of the Fc region. Specification 21 / 58 pages 30 CN 122356297 A

[0174] In some embodiments, the multispecific antibody according to the present invention comprises: (a) an anti-CD79b Fab domain; (b) an immunoglobulin Fc region attached to the C-terminus of the (a) domain; and (c) an anti-CD20 VHH domain attached to the N-terminus of the (a) domain or the C-terminus of the (b) domain via a peptide linker.

[0175] In some embodiments, preferably, the anti-CD20 VHH domain is attached to the N-terminus of the Fab domain heavy chain. In some embodiments, the Fab domain heavy chain comprises a VH domain and a CH1 domain from the N-terminus to the C-terminus, and the Fab domain light chain comprises a VL domain and a CL domain from the N-terminus to the C-terminus; or, in other embodiments, the Fab domain heavy chain comprises a VH domain and a CL domain from the N-terminus to the C-terminus, and the Fab domain light chain comprises a VL domain and a CH1 domain from the N-terminus to the C-terminus. Preferably, the Fab domain heavy chain comprises a VH domain and a CH1 domain from the N-terminus to the C-terminus; the Fab domain light chain comprises a VL domain and a CL domain from the N-terminus to the C-terminus. Preferably, the peptide linker comprises the amino acid sequence of SEQ ID NO: 26 or 27.

[0176] In some further embodiments, the multispecific antibody according to the present invention comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus, the first polypeptide chain comprises: a VHCD79b-CH1 domain-immunoglobulin Fc region; the second polypeptide chain comprises: a VLCD79b-CL domain; wherein VHCD79b and VLCD79b represent the heavy chain variable region and the light chain variable region that bind to CD79b, respectively; wherein the first polypeptide chain is optionally connected to the anti-CD20 VHH domain at the N-terminus or C-terminus via a peptide linker.

[0177] In some further embodiments, the multispecific antibody according to the present invention comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus, the first polypeptide chain comprises: a VHCD79b-CH1 domain-immunoglobulin Fc region; the second polypeptide chain comprises: a VLCD79b-CL domain; wherein VHCD79b and VLCD79b represent the heavy chain variable region and light chain variable region that bind to CD79b, respectively; wherein the first polypeptide chain is connected to the anti-CD20 VHH domain at the N-terminus via a peptide linker.

[0178] In some preferred embodiments, the multispecific antibody according to the present invention has a symmetrical structure formed by dimerization of the Fc region.

[0179] In some preferred embodiments, the multispecific antibody according to the present invention has the structure shown in FIG. 7A. In some more preferred embodiments, the multispecific antibody according to the present invention has the structure shown in FIG. 7B.

[0180] In some embodiments of the multispecific antibody of the present invention having the above-described structural form, preferably, the...The CD79b bonding domain is the CD79b bonding domain according to the present invention. More preferably, the CD20 bonding domain is the CD20 bonding domain according to the present invention.

[0181] Exemplary Multispecific Antibody

[0182] In some embodiments, the present invention provides a multispecific antibody, wherein the antibody comprises a first and a second polypeptide chain, wherein: - the first and second polypeptide chains respectively comprise the amino acid sequences of SEQ ID NOs: 23 and 24, or have at least 85%, 90%, 95% or 99% identity with them, or have one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or are composed thereof; or - the first and second polypeptide chains respectively comprise the amino acid sequences of SEQ ID NOs: 25 and 24, or have at least 85%, 90%, 95% or 99% identity with them, or have one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or are composed thereof.

[0183] Preferably, the first and second polypeptide chains respectively comprise, or consist of, the amino acid sequences of SEQ ID NOs: 25 and 24.

[0184] Preferably, the multispecific antibody has a symmetrical structure formed by dimerization of the Fc region.

[0185] Properties of the Multispecific Antibody of the Invention

[0186] In some embodiments, the multispecific antibody of the present invention has one or more of the following properties: (i) binding with high affinity to tumor cells expressing CD20 and / or CD79b; (ii) species cross-reactivity with cynomolgus monkey CD20; (iii) exhibiting endocytic activity on tumor cells expressing human CD20 or CD79b and on tumor cells co-expressing human CD20 and CD79b.

[0187] In some aspects, the multispecific antibody of the present invention has high binding affinity to tumor cells expressing CD20. The EC50 value and / or maximum binding amount of the antibody of the present invention to CD20-positive tumor cells can be determined by FACS or ELISA (e.g., the determination described in the examples) and optionally compared with a reference antibody to reflect the cell binding affinity of the antibody.

[0188] In some aspects, the multispecific antibody of the present invention exhibits species cross-reactivity with human and monkey CD20. In some embodiments, the EC50 value of the CD20 binding domain of the antibody of the present invention binding to human CD20 is approximately equivalent to the EC50 value of the binding domain binding to monkey CD20, for example, the ratio between the two is between 1 and 10, for example between 1 and 5, more preferably between approximately 1 and 3.

[0189] In some embodiments, the antibody according to the invention also has one or more properties selected from the following: (iv) better tumor cell targeting compared to single-target antibodies targeting CD20 or CD79b; (v) higher endocytosis efficiency on tumor cells; (vi) higher binding affinity on tumor cells.

[0190] In still some embodiments, the multispecific antibody of the invention also has one or more of the following characteristics: (1) good developability; (2) good stability; and (3) favorable pharmacokinetic properties.

[0191] II. Second aspect of the disclosure: Antigen-binding molecule of the invention

[0192] In a second aspect, the invention provides an antigen-binding molecule comprising an antibody according to the first aspect of the disclosure. In some embodiments, the antigen-binding molecule is a fusion produced by fusing the antibody of the invention to a heterologous protein, polypeptide, or peptide.

[0193] In one embodiment of the fusion according to the invention, the antibody (or its antigen-binding fragment) of the invention is linked to a heterologous peptide or polypeptide molecule directly or via an amino acid linker. Heterogeneous peptides or polypeptides that may be mentioned include, but are not limited to, proteins or polypeptides that impart another functional activity to the fusion, or tag peptides that facilitate the purification or detection of the immunofusion. For example, a chimeric antigen receptor (CAR) comprising an antibody of the present invention or an antigen-binding fragment thereof is an example of an immunofusion according to the present invention.

[0194] III. Third aspect of the disclosure: nucleic acid, vector, host, and production method

[0195] In a third aspect, the present disclosure provides encoding nucleic acids, vectors, host cells, and production methods for antibody and antigen-binding molecules according to the first and second aspects of the present disclosure.

[0196] In one embodiment, the present disclosure provides a method for preparing the antibody and antigen-binding molecule of the present invention, wherein the method comprises: culturing host cells comprising the nucleic acid or an expression vector comprising the nucleic acid under conditions suitable for expressing a nucleic acid encoding the antibody and antigen-binding molecule, and optionally isolating the antibody and antigen-binding molecule. In one embodiment, the method further comprises recovering the antibody and antigen-binding molecule from the host cell (or host cell culture medium).

[0197] To recombinantly generate the antibody and antigen-binding molecules of the present invention, the nucleic acid encoding the antibody and antigen-binding molecule can first be isolated and said nucleic acid inserted into a vector for further cloning and / or expression in host cells. Such nucleic acids are easily isolated and sequenced using conventional procedures, for example, by using oligonucleotide probes capable of specifically binding to the nucleic acid encoding the antibody and antigen-binding molecule of the present invention.

[0198] The antibody and antigen-binding molecules of the present invention prepared as described herein can be obtained using known prior art such as high-performance liquid crystals (HPLC).Purification methods include high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibody and antigen-binding molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc.

[0199] IV. Fourth aspect of the present disclosure: Conjugates and antibody-drug conjugates (ADCs)

[0200] In a fourth aspect, the present disclosure provides conjugates and antibody-drug conjugates comprising antibody and antigen-binding molecules according to the first and second aspects of the present disclosure.

[0201] Conjugates

[0202] In one embodiment, the present disclosure provides conjugates generated by conjugating antibody and antigen-binding molecules according to the first and second aspects of the present disclosure to a heterologous molecule. In one embodiment, the antigen-binding molecule of the present invention (such as an antibody) is conjugated with a therapeutic agent, diagnostic agent, or detectable agent. In the conjugates, chemical connectors can be used to covalently link different entities of the conjugate. In some cases, it is advantageous that the chemical linker is a “cleavable linker” that facilitates the release of the antigen-binding polypeptide upon delivery to the target site. For example, acid-instable linkers, peptidase-sensitive linkers, photostable linkers, dimethyl linkers, or disulfide-containing linkers can be used.

[0203] In embodiments conjugated with therapeutic agents, suitable therapeutic agents for the conjugate include, but are not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), drugs, or radioisotopes.

[0204] In embodiments conjugated with diagnostic or detectable agents, such conjugates can be used as part of a clinical testing method (e.g., to determine the efficacy of a particular therapy) to monitor or predict the onset, development, progression, and / or severity of a disease or condition. Such diagnostics and detections can be achieved by conjugating antibodies with detectable agents, including but not limited to a variety of enzymes, such as horseradish peroxidase; prosthetic groups, such as streptavidin / biotin and avidin / biotin; fluorescent substances; luminescent substances; radioactive substances; and positron-emitting metal and non-radioactive paramagnetic metal ions used in various positron emission tomography (PET) imaging techniques.

[0205] In some embodiments, therapeutic agents suitable for the conjugate include, but are not limited to, pharmaceuticals (e.g., antitumor drugs); in other embodiments, diagnostic agents suitable for the conjugate include, but are not limited to, radiodiagnostic agents, fluorescent substances, or luminescent substances.

[0206] Antibody-Drug Conjugates (ADCs)

[0207] In some preferred embodiments, this disclosure provides antibody-drug conjugates (ADCs).

[0208] In some embodiments, this disclosure provides an antibody-drug conjugate (ADC) having formula (I) or a pharmaceutically acceptable salt or solvate thereof: Ab-(L-D)p(I) Wherein: 24 / 58 pages 33 CN 122356297 A Ab is the antibody or antigen-binding molecule of the present invention; L is a linker; D is a drug, such as an antitumor compound; and p is an integer selected from 1 to 16, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0209] In some embodiments, Ab is a multispecific antibody against CD79b and CD20 according to the first aspect of this disclosure, especially a bispecific antibody. In some particularly preferred embodiments, the multispecific antibody comprises first and second polypeptide chains, wherein: (i) the first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 23; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 24; (ii) the first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 25; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 24; preferably, the first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 25; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 24.

[0210] It is understood that the -L-D portion can be covalently linked to the Ab in any manner known in the art. In some embodiments, the -L-D portion is covalently linked to the Ab via a sulfur (S) atom from the Ab, i.e., the -L-D portion and the Ab are linked via -S-. In some embodiments, the sulfur atom originates from the opening of interchain disulfide bonds of the Ab. In some embodiments, the sulfur atom is derived from (engineered or natural) cysteine ​​in Ab.

[0211] It is understood that p refers to the number of -L-D atoms linked to Ab in the antibody-drug conjugate molecule of formula (I), also known as DAR.

[0212] In some embodiments, D in formula (I) of the present invention can be any antitumor compound, as long as it has an antitumor effect and has a structural portion that can be linked to the linker, without particular limitation. The antitumor compound can be a pharmaceutically active compound that has an effect on tumors. For antitumor compounds, part or all of the linker is preferably cleaved within tumor cells, releasing the antitumor compound portion, thereby exhibiting an antitumor effect.

[0213] In some embodiments, the antitumor compound can be, for example, a cytotoxic agent, such as camptothecin compounds or auratestatin compounds.

[0214] In some embodiments, D has the structure shown in formula (D-1a) or formula (D-1b): Formula (D-1a) wherein R1a is selected from H and -C1-C6 alkyl;R2a is selected from H, halogen, -C1-C6 alkyl, -C1-C6 haloalkyl, -OR5a, and -SR5a; R3a is selected from H, halogen, CN, -C1-C6 alkyl, -C1-C6 haloalkyl, and -OR5a; and R4a and R5a are independently selected from H and -C1-C4 alkyl.

[0215] In some embodiments, R1a is H.

[0216] In some embodiments, R2a is -C1-C6 alkyl, preferably -C1-C4 alkyl, more preferably methyl. Specification 25 / 58 pages 34 CN 122356297 A

[0217] In some embodiments, R3a is halogen, preferably -F.

[0218] In some embodiments, R4a is -C1-C4 alkyl, preferably ethyl.

[0219] In some embodiments, R1a is H; R2a is -C1-C4 alkyl, preferably methyl; R3a is halogen, preferably -F; R4a is -C1-C4 alkyl, preferably ethyl.

[0220] In some embodiments, D has the structure shown in formula (D-1b), wherein R1b, R2b, R3b, R4b, R5b and R8b are each independently selected from -C1-8 alkyl; preferably -C1-C4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; R6b and R7b are each independently selected from -C1-C8 alkoxy, such as methoxy, ethoxy or propoxy; R9b is selected from -C1-C8 alkyl and -COOH; preferably -C1-C4 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or sec-butyl; and R10b is selected from -OH and H.

[0221] In some embodiments, R1b, R4b and R8b are each independently selected from -C1-C2 alkyl; preferably methyl.

[0222] In some embodiments, R2b and R3b are each independently selected from -C3-C4 alkyl; preferably isopropyl.

[0223] In some embodiments, R5b is -C3-C4 alkyl; preferably sec-butyl.

[0224] In some embodiments, R6b and R7b are each independently selected from -C1-C2 alkoxy; preferably methoxy.

[0225] In some embodiments, R9b is selected from -C1-C4 alkyl and R10b is OH; or R9b is -COOH and R10b is H.

[0226] In some embodiments, R1b, R4b, and R8b are each independently selected from -C1-C2 alkyl; preferably methyl; R2b, R3b, and R5b are each independently selected from -C3-C4 alkyl; R6b and R7b are each independently selected from -C1-C2 alkoxy; and R9b is selected from -C1-C4 alkyl and R10b is -OH; or R9b is -COOH and R10b is H.

[0227] It is understood that the wavy line in the structural formula indicates that the valence bond is connected to the rest of the molecule. For example, the wavy line in the D structural formula indicates that the valence bond is connected to L.

[0228] In some embodiments, D has the structure shown in formula (D-2a) or formula (D-2b): Formula (D-2a), or formula (D-2b) where R1a, R2a, R3a and R4a are as defined above; where R1b, R2b, R3b, R4b, R5b, R6b, R7b, R8b, R9b and R10b are as defined above.

[0229] In some embodiments, D has the structure shown in formula (D-3a) or (D-3b): (D-3a) or (D-3b).

[0230] In some embodiments, D has the structure shown in formula (D-4a) or (D-4b): Specification 26 / 58 pages 35 CN 122356297 A (D-4a) or (D-4b).

[0231] In some embodiments, in the ADC of this disclosure, the drug is Exatecan, Dxd, SN-38, monomethylaurestatin E (MMAE) or MMAF. The structural formulas are shown below: , , , .

[0232] In some embodiments, -L- has the following structure: -Z-L1-L2-L3-

[0233] Wherein

[0234] Z is selected from , , , , , and , where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; L1 is selected from non-existent, , , , and , where n1 and m1 are each independently an integer selected from 0 to 20, for example, an integer selected from 0 to 12, for example, 1, 2, 3, 4, 5, 6, 7 or 8; L2 is an amino acid residue or a peptide residue consisting of 2 to 8 amino acids; and L3 is selected from: , , , , and , Where X is selected from -NH-, -O-, and -S-; R1c is each independently selected from -C1-8 alkyl, -C1-8 haloalkyl-, -C1-8 alkoxy, halogen, nitro, and cyano; Su is each independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and Z is connected to an atom on Ab, for example, an S atom, and L3 is connected to D.

[0235] In some embodiments, -L- has the following structure: -Z-L1-L2-L3-

[0236] Wherein

[0237] Z is selected from , , where m is an integer selected from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7 or 8, for example an integer from 1 to 5; Specification 27 / 58 page 36 CN 122356297 A L1 is selected from non-existent, and , where n1 is independently an integer selected from 0 to 12, for example 1, 2, 3, 4, 5, 6, 7 or 8;L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and L3 is selected from: , and , wherein R1c is selected from: -C1-C6 alkyl; n2 is 0, 1, 2, 3 or 4; and n5 is 0, 1, 2 or 3.

[0238] In some embodiments, -L- has the following structure: -Z-L1-L2-L3-

[0239] wherein

[0240] Z is selected from, wherein m is an integer selected from 1-10, for example 1, 2, 3, 4, 5, 6, 7 or 8, for example an integer from 1-5; L1 is selected as not present; L2 is an amino acid residue or a peptide residue consisting of 2-4 amino acids; and L3 is selected from: , and , wherein R1c is selected from: C1-C6 alkyl; n2 is 0, 1, 2, 3 or 4; and n5 is 0, 1, 2 or 3.

[0241] It should be understood that in the above -Z-L1-L2-L3-, Z is connected to Ab, for example, connected to S on Ab, and L3 is connected to D.

[0242] In some embodiments, Z is selected from m, where m is 1, 2, 3, 4, 5, 6, 7 or 8, for example, an integer from 1 to 5.

[0243] In some embodiments, Z is selected from n1, n2, n3, n4, n5, n6, n7 or 8.

[0244] In some embodiments, L1 is selected from n1, n2, n3, n4, n5, n6, n7 or 8, where n1 is independently an integer selected from 0 to 12, for example, 1, 2, 3, 4, 5, 6, 7 or 8.

[0245] In some embodiments, L1 is selected from n1, n2, n3, n4, n5, n6, n7 or 8.

[0246] In some embodiments, L1 is selected from n1, n3, n4, n5, n6 or 7.

[0247] In some embodiments, L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids. In some embodiments, L2 is an amino acid residue. In some embodiments, L2 is a peptide residue consisting of 2, 3, 4, 5, 6, or 7 amino acids. In some embodiments, L2 is a peptide residue consisting of 2, 3, or 4 amino acids.

[0248] In some embodiments, the amino acid residue or amino acid is preferably an L-amino acid. Moreover, in addition to α-amino acids, the amino acid residue or amino acid can be an amino acid residue or amino acid with the structure of β-alanine, ε-aminohexanoic acid, γ-aminobutyric acid, etc., and can also be a non-natural amino acid, such as an N-methylated amino acid.

[0249] In some embodiments, the amino acid residue or amino acid is each independently selected from valine (Val), alanine (Ala), glycine (Gly), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine, etc.The amino acid residues are selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit), phenylalanine (Phe), lysine (Lys), glutamic acid (Glu), and glutamine (Gln). In some embodiments, the amino acid residues are selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit), and glutamic acid (Glu).

[0250] In some embodiments, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly- (SEQ ID NO: 53).

[0251] In some embodiments, L2 is selected from -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, and -Gly-Gly-Phe-Gly- (SEQ ID NO: 53). In some embodiments, L2 is -Gly-. In some embodiments, L2 is selected from -Val-Ala-, -Gly-Gly-Phe-Gly- (SEQ ID NO: 53), -Val-Cit-, and -Glu-Val-Cit-. In some embodiments, L2 is selected from -Gly- and -Val-Cit-.

[0252] It should be understood that L2 is linked to L1 or Z via the amino group of the left-hand amino acid and to L3 via the carbonyl group of the right-hand amino acid, consistent with the explanation below.

[0253] In some embodiments, L3 is selected from: , and , for example selected from and , wherein R1c is each independently selected from C1-8 alkyl, C1-8 haloalkyl-, C1-8 alkoxy, halogen, nitro and cyano; Su is each independently selected from, , and ; n2 is 0, 1, 2, 3 or 4; and n5 is 0, 1, 2 or 3.

[0254] In some embodiments, L3 is selected from: , , wherein each variable is as defined herein.

[0255] In some embodiments, Su is selected from xylose, arabinose, xyuronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid and mannuronic acid.

[0256] In some embodiments, Su is selected from.

[0257] In some embodiments, Su is each independently: (See specification 29 / 58 pages 38 CN 122356297 A).

[0258] In some embodiments, Su is independent of each other.

[0259] In some embodiments, Su is independently selected.

[0260] In some embodiments, L3 is selected from and.

[0261] It should be understood that L3 is connected to L2 via an amino group on the left and to D via a carbonyl group on the right, consistent with the explanation below.

[0262] In some embodiments, -Z-L1-L2-L3- is independently selected from the following structures: , , where m is independently selected from an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; and where the left side of the group is connected to an atom on Ab, for example S, and the right side is connected to D.

[0263] In some embodiments, -Z-L1-L2-L3- is independently selected from the following structures: Specification 30 / 58 pages 39 CN 122356297 A.

[0264] It should be understood that, unless otherwise stated and in accordance with the context, for the ADCs of the present invention, the left-hand bond of the divalent group shown herein is connected to an Ab or a group near the Ab end, and the right-hand bond of the divalent group is connected to a D or a group near the D end. For example, when L2 is , the left-hand amino group is connected to L1, and the right-hand carbonyl group is connected to L3; In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, or 2-4 or 2-6.

[0265] In some embodiments, the antibody-drug conjugate is selected from:

[0266] wherein Ab is an antibody or antigen-binding molecule of the present invention; p is as defined above, for example, p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. Preferably, the antibody-drug conjugate has an average DAR of, for example, 2-10, 6-10, 4-8, 7-9 or 2-4 or 2-6. In some embodiments, the multispecific antibody is a multispecific antibody according to the first aspect of this disclosure. In some particularly preferred embodiments, the multispecific antibody comprises first and second polypeptide chains, wherein: the first polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 25; and the second polypeptide chain comprises or is composed of the amino acid sequence of SEQ ID NO: 24. Specification page 31 / 58 40 CN 122356297 A

[0267] It should be understood that the S atom linked to Ab in the above ADC comes from the antibody Ab. Ab opens the disulfide bond (e.g., interchain disulfide bond) under the action of a reducing agent such as TCEP to generate a thiol-SH, which is then linked to the terminal functional group of the linker, such as the maleimide portion. In some embodiments, the S atom linked to Ab comes from the cysteine ​​of Ab.

[0268] It should be noted that the above and other technical solutions and one or more features of this disclosure can be arbitrarily combined to constitute technical solutions not directly described herein, and these undescribed technical solutions are also covered within the scope of this application.

[0269] In some embodiments, the antibody-drug conjugates according to the present invention have the following advantages: (1) they bind to tumor cells expressing human CD79b or CD20 and tumor cells co-expressing human CD79b and CD20; (2) they exhibit endocytic activity on tumor cells expressing human CD79b or CD20 and tumor cells co-expressing human CD79b and CD20; (3) they have a side-by-side killing effect; (4) they have a broad spectrum of antitumor activity and exhibit significant killing activity against tumors with different CD79b and CD20 expression densities.

[0270] In some embodiments, the antibody-drug conjugates according to the present invention also have one or more advantages selected from the following: (5) better tumor targeting compared to CD79b or CD20 single-target ADC drugs; (6) higher endocytosis efficiency on tumor cells; (7) higher affinity on tumor cells; (8) stronger killing effect on tumor cells; (9) stronger inhibitory effect on tumor growth; and (10) fairly good in vivo safety.

[0271] In some embodiments, the antibody-drug conjugates according to the present invention also have one or more advantages selected from the following: (11) good product homogeneity; (12) good product stability; and (13) good drugability.

[0272] Preparation of ADC molecules of the present invention

[0273] Another aspect of the present invention provides a method for preparing an ADC using the antibody of the present invention. In the present invention, “ADC” is defined as an antibody conjugated to an active substance D having biological and pharmaceutical activity via a linker L. The method includes conjugating the antibody (Ab) of the present invention to one or more active substances D via one or more adapters L defined in the present invention. Preferably, the adapter-active substance is conjugated to the antibody in a site-specific manner.

[0274] In some embodiments, the method includes preparing an Ab for an ADC comprising culturing a host cell containing a nucleic acid encoding the Ab (e.g., any one polypeptide chain and / or multiple polypeptide chains) or an expression vector containing the nucleic acid, as provided above, under conditions suitable for expression of the Ab or its chain, and optionally recovering the Ab from the host cell (or host cell culture medium).

[0275] In some embodiments, the method includes the steps of: (a) adding the antibody Ab to a buffer solution, adding a reducing agent, and then incubating until the reaction is complete; (b) removing the reducing agent by ultrafiltration; (c) adding the adapter-loador to the reaction solution in step (b) for conjugation to obtain a crude product; Specification 32 / 58Page 41 CN 122356297 A (d) Optionally, the crude product is purified to obtain the antibody-drug conjugate of the present invention; wherein Ab is as defined above.

[0276] It should be understood that the reaction of the linker-load with Ab provides the -L-D portion of the compound of formula I, and the structure of the linker-load can be determined according to the prior art when -L-D is clearly defined.

[0277] In some embodiments, the buffer solution of step a) is PBS buffer, preferably with a pH of 5.0-9.0, for example 6.0-8.0.

[0278] In some embodiments, the reducing agent of step a) is TCEP.

[0279] In some embodiments, the steps are performed under the specific reaction conditions disclosed in the examples.

[0280] It should be noted that embodiments obtained by varying the range or specific values ​​of the specific reaction conditions disclosed in the examples by 100%, 80%, 60%, 40%, 20% or 10% are also considered in the present invention.

[0281] V. Fifth aspect of this disclosure: Pharmaceutical compositions and pharmaceutical formulations, as well as combination products and kits

[0282] In some embodiments, this disclosure provides compositions comprising antibodies, antigen-binding molecules, or ADCs described herein, preferably pharmaceutical compositions or pharmaceutical formulations. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition comprises an antibody, antigen-binding molecule, or ADC of the present invention, and a combination of one or more other therapeutic agents (e.g., chemotherapeutic agents, tumor vaccines, antibodies that bind to other specific antigens on tumor cells, other antibodies that deplete tumor cells).

[0283] In some embodiments, the compositions of the present invention are pharmaceutical compositions or pharmaceutical formulations comprising suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, as known in the art. As used herein, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents, etc. Pharmaceutical carriers suitable for the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.

[0284] The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. In the treatment of cancer, such active ingredients include, but are not limited to, anticancer agents and chemotherapeutic agents; in the treatment of infectious diseases, such active ingredients include, but are not limited to, antiviral agents and antibiotics. The active ingredients are suitably combined in amounts effective for the intended use.

[0285] In some embodiments, this disclosure also provides a combination product comprising at least one antibody, antiviral agent, or anti-inflammatory agent of the present invention.The original binding molecule or ADC, or may also contain one or more other antitumor agents.

[0286] In some embodiments, two or more components of the combination product may be administered to the subject sequentially, separately or simultaneously.

[0287] In some embodiments, this disclosure also provides a kit comprising the antibody, antigen-binding molecule, ADC, pharmaceutical composition or combination product of the present invention, and optionally a package insert for instructions of administration.

[0288] In some embodiments, this disclosure also provides a pharmaceutical article comprising the antibody, antigen-binding molecule, ADC, pharmaceutical composition or combination product of the present invention, optionally further comprising a package insert for instructions of administration.

[0289] VI. Sixth aspect of the disclosure: Uses and methods

[0290] In a sixth aspect, this disclosure provides the use and methods of antibodies or antigen-binding molecules according to the first and second aspects of the present disclosure, conjugates or antibody-drug conjugates according to the fourth aspect of the present disclosure, or pharmaceutical compositions or combinations according to the fifth aspect of the present disclosure in the treatment and prevention of CD79b-related diseases and / or cancers and / or CD20-related diseases and / or cancers.

[0291] Tumor-associated antigens such as CD79b and CD20 are overexpressed on the surface of cancer cells in B-cell lymphoma and leukemia, making them suitable targets for cancer immunotherapy. In one embodiment, therefore, this disclosure provides the use of the multispecific antibody, ADC, or combination thereof of the present invention for the prevention and / or treatment of CD79b and / or CD20-positive tumors in a subject. In said use, the antibody, ADC, or combination thereof of the present invention may be administered to the subject as the sole active agent or may be administered to the subject in combination with other therapies or therapeutic agents. The other therapies and therapeutic agents include, for example, drugs that target antigens on the surface of tumor cells to eliminate tumors by binding to and / or blocking these molecules; and drugs that activate the subject's immune system to induce spontaneous elimination of tumors. In another aspect, the present invention also provides a method for the prevention or treatment of CD79b and / or CD20-positive tumors in a subject, comprising administering the antibody, ADC, or combination of drugs of the present invention to a subject in need.

[0292] The tumors suitable for the methods and uses of the present invention can be selected from various solid tumors or hematologic malignancies. The tumors can be early, intermediate, or late-stage cancers, or metastatic cancers. Furthermore, the tumors suitable for the methods and uses of the present invention can be tumors that have previously received treatment and have experienced immune escape. In some embodiments, the tumor is a hematologic malignancy, preferably a CD79b and / or CD20 positive tumor.

[0293] Since the expression of CD79b and CD20 covers almost all B-cell-related tumors, this disclosure considers that the present invention...This invention relates to the application of a CD79b / CD20 multispecific (bispecific) ADC in the treatment of various B-cell-related lymphomas and leukemias. The B-cell-related lymphomas and leukemias include, but are not limited to, non-Hodgkin lymphoma (NHL), large B-cell lymphoma, DLBCL, RT (Richter's transformation / syndrome), BL (Burkitt lymphoma), FL (follicular lymphoma), MZL (marginal zone lymphoma), MCL (mantle cell lymphoma), hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and chronic lymphocytoid leukemia (CLL). In some embodiments, the cancer described is relapsed or refractory non-Hodgkin lymphoma (RRNHL). In some embodiments, the cancer patient is insensitive to first-line NHL treatment or has relapsed after treatment; in other embodiments, the cancer patient is insensitive to CAR-T cell therapy or has relapsed after treatment. In some embodiments, the cancer patient is a newly diagnosed cancer patient with B-cell-related tumors.

[0294] In some embodiments, this disclosure provides the use of the present invention's multi(bi)specific ADC targeting CD79b and CD20 in the treatment of B-cell malignancies, such as B-cell non-Hodgkin lymphoma and its subtypes. In some embodiments, the cancer is a CD79b-insensitive or resistant tumor. In some embodiments, the tumor has one or more of the following characteristics: (a) compared to Daudi cells, the tumor cells have a low CD79b expression level, for example, less than 50%, 40%, 30%, 20%, 10%, 5%, or 2% of the CD79b expression level on Daudi cells; (b) compared to Daudi cells or Ramos cells, the tumor cells have upregulated expression and / or activity of the anti-apoptotic gene Bcl-xL; (c) compared to Daudi cells or Ramos cells, the tumor cells have upregulated expression and / or activity of the MMAE efflux pump (MDR-1); and (d) compared to the CD20 expression level on Daudi cells, the tumor cells have CD20 expression that is 1%, 10%, 50%, 100%, 150%, 200%, 300%, or 400% higher than that expression level.

[0295] In autoimmune diseases, B cells coordinate antigen presentation, cytokine production, and autoantibody production, playing a key role in the pathogenesis of autoimmune diseases. It has been proposed to use antibodies against B cell surface antigens (e.g.)Anti-CD20 antibodies are therapeutic strategies that deplete B cells to improve or even cure autoimmune diseases. See, for example, pages 34 / 58 of the specification, CN 122356297 A, CN102281902A, and the review “B-cell depletion in autoimmune diseases”. Therefore, in one embodiment, this disclosure provides the use of the multispecific antibodies, ADCs, or combinations thereof of the present invention for the prevention and / or treatment of B-cell-related autoimmune diseases in a subject. In said use, the antibodies, ADCs, or combinations thereof of the present invention may be administered to the subject as the sole active agent or may be administered to the subject in combination with other therapies or therapeutic agents. These other therapies and therapeutic agents include, for example, corticosteroids, immunosuppressive drugs, and biologics. In another aspect, the present invention also provides a method for the prevention or treatment of B-cell-related autoimmune diseases in a subject, comprising administering the antibodies, ADCs, or drug combinations of the present invention to a subject in need.

[0296] B-cell-related autoimmune diseases suitable for the methods and uses of the present invention include autoimmune diseases in which B cells are involved or associated, and preferably will benefit from B-cell depletion therapy. Examples of such autoimmune diseases include, but are not limited to, rheumatoid arthritis (RA); lupus; NMDAR encephalitis; multiple sclerosis; systemic sclerosis; immune thrombocytopenic purpura; simple erythrocytic aplasia; autoimmune anemia; cold agglutinin disease; severe insulin resistance type B syndrome; mixed cryoglobulinemia; myasthenia gravis; Wegener's granulomatosis; refractory pemphigus vulgaris; dermatomyositis; Sjogren's syndrome; active type II mixed cryoglobulinemia; pemphigus vulgaris; autoimmune nephropathy; tumor prodromal visual oculoclonus-myoclonus syndrome; and relapsing-remitting multiple sclerosis (RRMS). In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, NMDAR encephalitis, idiopathic thrombocytopenic purpura, multiple sclerosis, pemphigus vulgaris, systemic sclerosis, and systemic lupus erythematosus.

[0297] In any of the above embodiments of the method of the present invention, the administration of the antibody, antigen-binding molecule, or ADC according to the present invention, or the pharmaceutical composition or combination according to the present invention, may include 1) a therapeutic measure that cures, slows, alleviates, reduces the symptoms of a diagnosed pathological condition or disease, and / or stops the progression of the diagnosed pathological condition or disease; or 2) a preventive or preventative measure that prevents and / or slows the development of a pathological condition or disease. Therefore, in the method of the present invention, the subject may be an individual who has already contracted the disease, an individual who is susceptible to the disease, or an individual who wishes to prevent the disease. The individual will benefit from the therapeutic or preventative measures and, compared to an individual who has not received the treatment, showThe invention relates to the reduction or improvement of the occurrence, recurrence, or development of a disease, condition, symptom, and / or symptoms. In some embodiments, the invention relates to the treatment of a disease or condition; in other embodiments, the invention relates to the prevention of a disease or condition.

[0298] Antibodies, antigen-binding molecules, or ADCs according to the invention, as well as pharmaceutical compositions or combinations thereof according to the invention, can be administered by any suitable method, including parenteral administration, intratumoral administration, and intranasal administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Various dosing schedules are covered herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulsatile infusion.

[0299] To prevent or treat a disease, the antibody, antigen-binding molecule, or ADC according to the invention, or the pharmaceutical composition or combination of drugs according to the invention, is administered at a suitable dose, which means that, when used alone or in combination with one or more other therapeutic agents, will depend on the type of disease to be treated, the specific type of drug used, the severity and course of the disease, whether the drug is administered for preventive or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0300] In some embodiments, this disclosure also provides for the use of the antibody, antigen-binding molecule, or ADC according to the invention, or the pharmaceutical composition or combination of drugs according to the invention, as a drug or for the preparation of a drug. In some embodiments, the drug is a drug for use in the foregoing treatment and prevention methods.

[0301] Any or all of the features described above and throughout this application may be combined in various embodiments of the invention. The following examples further illustrate the invention; however, it should be understood that the examples are for illustrative purposes only and should not be construed as constituting any limitation. Instructions for Use, Pages 35 / 58, 44, CN 122356297 A

[0302] Examples

[0303] Example Section I. Antibody Preparation and Characterization

[0304] Materials and Methods

[0305] Reference Antibody and its Preparation

[0306] In this example, CD79b reference antibody Polatuzumab and CD20 reference antibodies Rituximab, Ofatumumab, and Obinutuzumab were used.

[0307] The “reference antibody Polatuzumab” (heavy chain sequence: SEQ ID NOs: 38, light chain sequence: SEQ ID NO: 39) is the antibody portion of the approved CD79b ADC “Polatuzumab vedotin”. Its sequence was retrieved from the IMGT database as: IMGT / 3Dstructure-DB card, IMGT / 2Dstructure-DB card for INN: 9714.

[0308] “Reference antibody Ofatumumab” (heavy chain sequence: SEQ ID NOs: 34, light chain sequence: SEQ ID NO: 35) is an approved CD20 monoclonal antibody. Its sequence was retrieved from the IMGT database as: IMGT / 3Dstructure-DB card, IMGT / 2Dstructure-DB card for INN: 8606.

[0309] “Reference antibody Obinutuzumab” (heavy chain sequence: SEQ ID NOs: 36, light chain sequence: SEQ ID NO: 37) is an approved CD20 monoclonal antibody. Its sequence was retrieved from the IMGT database as: IMGT / 3Dstructure-DB card, IMGT / 2Dstructure-DB card for INN: 9043.

[0310] The above amino acid sequences were optimized according to human codon preference, and the gene was synthesized and subcloned into the pcDNA3.4 vector; after verification by Sanger sequencing, plasmid was extracted for later use. The constructed eukaryotic expression vectors were used to transiently transfect Expi293F cells; protein expression supernatant was collected from the transfected cell culture, and the target protein was purified by protein A affinity chromatography; the purity of the protein was detected by SDS-PAGE and SEC-HPLC experiments, and the purity was >95%. The protein purification process was carried out under aseptic conditions and the endotoxin level was controlled to <5 EU / mg.

[0311] The “reference antibody Rituximab” is a monoclonal antibody targeting CD20, which binds to CD20 molecules expressed on the surface of B lymphocytes and kills tumor B cells through antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The reference antibody was purchased from MedChemExpress, catalog number: CAT#HY-P9913.

[0312] Antigen and its preparation

[0313] Considering that CD20 is a transmembrane protein, its antigen may differ from the natural structural conformation of the cell membrane surface, so CD20 antigen was not prepared. The extracellular sequence information (Ala 29 - Asp 159) of human CD79b (UniProt_P40259-1) was retrieved from the database. His tags were added to the C-terminus of each sequence, and after optimization according to human codon preferences, the gene was synthesized and subcloned into the pcDNA3.4 vector. After verification by Sanger sequencing, plasmid extraction was performed for later use. HEK293 cells were transiently transfected with these constructed eukaryotic expression vectors. The protein expression supernatant was collected from the transfected cell culture, and the target protein was purified using a nickel column. SDS-PAGE was performed to determine protein purity, which was >90%. The aforementioned reference anti-CD79b antibody was used.(Polatuzumab), the activity of the prepared human huCD79b-His recombinant antigen protein was confirmed by ELISA.

[0314] Recombinant antigen-expressing engineered cell lines and their preparation

[0315] HEK293 engineered cell lines expressing human and monkey CD20, HEK293-hCD20 and HEK293-cynoCD20, were prepared as follows. The full-length genes of human and monkey CD20 (full-length sequence information of human CD20 (UniProt_P11836-1) and cynomolgus monkey CD20 (UniProt_M4ZHZ6-1) (Met 1-Pro 297)) were inserted into the lentiviral expression vector pLVX-puro, respectively. Lentiviral virus was packaged in 293T cells and infected HEK293 cells. The cells were cultured in a medium containing 5ug / ml puromycin selection pressure to obtain polyclonal cell lines. Using the reference antibody Rituximab for FACS detection, the antigen expression positivity rate of the stable cell line was >90%. Instructions for use, pages 36 / 58, CN 122356297 A

[0316] ELISA binding assay

[0317] The antigen was diluted with PBS to 1.0 µg / ml, 100 µl / well was coated onto the plate, and incubated overnight at 2-8℃ or for 2 hours at 37℃. The plate was washed 3 times with 0.05% PBST, and 200 µl / well blocking buffer (1% BSA or 8% skim milk powder in PBS) was added, and the plate was incubated at room temperature for at least 1 hour. The sample to be tested was diluted to a suitable concentration with blocking buffer. The plate was washed 3 times with 0.05% PBST, and 100 µl / well of the diluted sample was added, and the plate was incubated at room temperature for about 1 hour. Wash the plate 3 times with 0.05% PBST using a plate washer, add 100 µl / well of secondary antibody-HRP diluted to the target dilution factor, and let stand at room temperature for about 0.5 or 1 hour. Wash the plate 3 times with 0.05% PBST using a plate washer, add 100 µl / well of TMB, observe the color change, and add 100 µl / well of stop solution immediately when the color is appropriate. Read OD450-OD650.

[0318] Detection of tumor cell surface antigen expression abundance using direct label FACS

[0319] Seed target cells at a density of (1-2)×105 cells per well in a 96-well plate and centrifuge at 300g for 5 minutes at 4℃. Dilute the direct label antibody with FACS buffer (1% BSA or 2% FBS in PBS) according to the recommended concentration, and incubate at 2-8℃ for about 0.5 hours. Centrifuge at 4℃, remove the supernatant, wash twice with 200 µl / well of FACS buffer, and centrifuge at 4℃. Resuspend cells in 100 µl / well FACS buffer and perform flow cytometry analysis. Measure MFI of cells using a Beckman Coulter flow cytometer. Calculate CD20 or CD79b for each target cell.The ratio of MFI to blank unstained wells was normalized using Daudi cells (i.e., the expression levels of CD20 and CD79b on the surface of Daudi cells were defined as 100%), and the fold change of the ratio of other target cells to Daudi cells at the corresponding target sites was statistically analyzed.

[0320] FACS binding assay

[0321] Target cells were seeded into 96-well plates at a density of (1-2) × 10⁵ cells per well and centrifuged at 300g for 5 minutes at 4°C. The test sample diluted to an appropriate concentration was added and incubated at 2-8°C for about 1 hour. After centrifugation at 4°C, the supernatant was removed, and the cells were washed twice with 200 µl / well FACS buffer (1% BSA or 2% FBS in PBS) and centrifuged at 4°C. 100 µl / well of flow cytometry secondary antibody diluted to the target dilution was added, the cells were resuspended, and incubated at 2-8°C in the dark for about 0.5 h or 1 h. After washing twice with 200 µl / well FACS buffer, cells were resuspended in 100 µl / well FACS buffer and analyzed. The MFI of the cells was measured using a flow cytometer (Beckman Coulter).

[0322] FACS examination of target human-monkey cross-reactivity

[0323] The immunoreactivity of anti-CD20 antibodies (including monospecific and bispecific antibodies) was detected by FACS binding assay using engineered HEK293 cell lines stably expressing human / monkey CD20. The experimental conditions were as follows: target cells (1×10⁵ / well) + sample (200 nM, 4× dilution, 4℃ 1 h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5 h). The monospecific and bispecific antibodies of the present invention tested showed similar binding activity to human-monkey antigens, exhibiting immunoreactivity with monkeys.

[0324] FACS Detection of Bispecific Antibody Binding to Dual Targets

[0325] CD20 overexpressing cells were seeded into 96-well plates at a density of (1-2) × 10⁵ cells per well and centrifuged at 300g for 5 minutes at 4°C. The test sample (200 nM) diluted to an appropriate concentration was added and incubated at 2-8°C for about 1 hour. After centrifugation at 4°C, the supernatant was removed, and the cells were washed twice with 200 µl / well FACS buffer (1% BSA or 2% FBS in PBS) and centrifuged at 4°C. The cells were resuspended in 100 µl / well biotinylated CD79b antigen (Acro, CAT# CDB-H82E3) at 5 µg / ml and incubated at 2-8°C in the dark for about 0.5 h or 1 h. Centrifuge at 4°C, remove supernatant, add 100 µl / well of PE-coupled streptavidin (Invitrogen, CAT# 12-4317-87) diluted to the target dilution, resuspend cells, and incubate at 2-8°C in the dark for 0.5 h. Wash twice with 200 µl / well of FACS buffer, then add 100 µl / well of FACS buffer.Cells were resuspended in buffer and analyzed. The MFI of cells was measured using a flow cytometer (Beckman Coulter).

[0326] Endocytosis assay-I (4℃ / 37℃)

[0327] Target cells were seeded at (2-4)×10⁵ cells per well, and the test sample diluted to an appropriate concentration was added. The cells were incubated at 2-8℃ for about 0.5h to allow the test sample to bind to the cells. The cells were centrifuged at 300g for 4min at 4℃, the supernatant was removed, and the cells were washed 2-3 times with pre-cooled 200 µl / well FACS buffer to remove excess unbound test sample. The cells were resuspended in pre-cooled 200 µl / well FACS buffer. The cells were divided into two groups, 100 µl / well for each group, and incubated at 4℃ and 37℃ for 2-4h respectively. After incubation, FACS buffer in an ice bath was added immediately to terminate the endocytosis experiment. Centrifuge at 300g for 4 min at 4 ℃, and wash the cells twice with pre-cooled 200 µl / well FACS buffer. Immediately add 100 µl / well of flow cytometry secondary antibody diluted to the target dilution, resuspend the cells, and incubate at 2-8 ℃ in the dark for 30 min to 1 h. After washing the cells 2-3 times with 200 µL / well FACS buffer, add 100 µl / well FACS buffer to resuspend the cells and perform flow cytometry. The MFI of the cells was measured using a flow cytometer (Beckman Coulter). The internalization level of the antibody bound to the cell surface was calculated using the following formula: Absolute endocytosis = MFI of sample incubated at 4 ℃ - MFI of sample incubated at 37 ℃.

[0328] Endocytosis rate % = 100% - (MFI of sample incubated at 37 ℃ / MFI of sample incubated at 4 ℃) × 100%.

[0329] Endocytosis assay-II (rProtein G-vc-MMAE, cytotoxic endocytosis)

[0330] rProtein G-vc-MMAE was prepared internally (it is a protein-drug conjugate formed by linking Protein G and MMAE through the linker valine-citrulline (VC), wherein protein G can bind to the Fc region of mammalian immunoglobulins). The endocytosis assay was performed as follows: a) Cells were plated (15000 / 100µl / well); b) rProtein G-D4-vc-MMAE was diluted to 200 nM with complete culture medium (1640 / 10% FBS), and the test sample was also diluted to 200 nM with complete culture medium. The test sample and rProtein G-vc-MMAE were mixed at a volume ratio of 1:1 (molar concentration ratio of 1:1) and incubated at room temperature for about 1 hour; c) The test sample and rProtein G-vc-MMAE mixture was diluted 1:2.5 with complete culture medium, resulting in 9 concentration points (starting concentration of 50 μL).nM); d) Add the sequence-diluted sample to a 96-well plate and incubate for 3 days; e) Add 20 µl of CCK8 to each well and incubate at 37℃ for 2-4 h, then read OD450-OD650 using a microplate reader.

[0331] Endocytosis assay-III (DT3C, cytotoxic endocytosis)

[0332] 1) Take cells in the logarithmic growth phase. Count, centrifuge, and resuspend the cells in medium containing 20% ​​FBS to the required concentration (2x105 / ml).

[0333] 2) Inject the cell suspension into a 96-well plate at 2x104 / 100 µl / well.

[0334] 3) Dilute DT3C (Jiman Biotechnology, GM-046001) and antibody to a suitable concentration (2x) using serum-free medium, mix in proportion, and filter to remove bacteria. Incubate at 37℃ for 30 min. Serially dilute the mixture with serum-free medium. Add serially diluted mixtures, 100 µl / well, to the 96-well plate.

[0335] 4) Incubate in an incubator for 48–72 h. Discard 100 µl from each well. Add 100 µl of CellTiter-Lumi steady-state assay reagent (Beyotime Cat.C0069) to each well. Read the luciferase signal value after 10 min.

[0336] SEC-HPLC

[0337] Load an appropriate amount of protein sample onto a TSK-gel G3000SWxL column (Tosoh Corporation) or a Zenix-C SEC-300 column (Sepax Technologies) at ambient column temperature. Using an Agilent 1260 HPLC system, use a mobile phase consisting of 0.05 M sodium phosphate, 0.3 M sodium chloride, and pH 6.8 ± 0.1, and elute the sample isocratically at a flow rate of 0.8 mL / min for 20 min. The eluted proteins were detected using UV absorbance at 280 nm.

[0338] CEX-HPLC

[0339] An appropriate amount of protein sample was loaded onto a ProPac™ WCX-10 BioLC column (Thermo SCIENTIFIC) at ambient column temperature. Using an Agilent 1260 HPLC system, the sample was eluted at a gradient rate of 0.9 mL / min for 70 min using mobile phase A consisting of 2-methylpiperazine, imidazole, Tris, pH 5.0 and mobile phase B consisting of 100 mM sodium chloride, 2-methylpiperazine, imidazole, and Tris, pH 10.8±0.1. The eluted proteins were detected using UV absorbance at 280 nm.

[0340]

[0341] An appropriate amount of protein sample was loaded onto a MAbPac™ HIC-Butyl column (Thermo SCIENTIFIC) at ambient column temperature. Using an Agilent 1260 HPLC system, mobile phases A and B were used to gradient elute the sample for 35 minutes at a flow rate of 0.8 mL / min. Mobile phase A consisted of 1.5 M ammonium sulfate, 0.05 M sodium phosphate, and 5% isopropanol, pH 6.0 ± 0.1. Mobile phase B consisted of 0.05 M sodium phosphate and 5% isopropanol, pH 6.0 ± 0.1. The eluted protein was detected using UV absorbance at 280 nm.

[0342] Thermal stability was determined by DSF

[0343] Based on nano-differential scanning fluorescence (nanoDSF) technology, the temperature range was 25-95 ℃, the temperature change rate was 1 ℃ / min. The denaturation temperature (Tm and Tonset) of the antibody protein and the onset temperature (Tagg) of protein aggregation were accurately determined by the fluorescence spectrum of 280-450 nm and the intensity change of laser scattering light of 266 nm or 473 nm, and the thermal stability of the antibody protein was evaluated.

[0344] Example 1 Construction and characterization of bispecific antibodies

[0345] Example 1.1 VHH screening

[0346] Alpaca immunization and magnetic sorting technology were used to screen candidate VHH sequences that bind to CD20 through preliminary property characterization. In short, Raji and Daudi cells expressing human CD20 membrane protein were used to immunize alpacas alternately, with an immunization interval of 14 days. Starting with the second immunization, peripheral blood was collected seven days after each immunization, and the serum titer was monitored using FACS experiments. Once the serum titer reached the standard for blood bank construction, peripheral blood was collected from immunized alpacas, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted from PBMCs, and cDNA was prepared using the PrimeScript™ II 1st Strand cDNA Synthesis Kit (Takara) as a template for reverse transcription. Using the cDNA as a template, the first round of PCR amplification produced nucleic acid fragments of conventional IgG (containing VH) and pure heavy chain IgG lacking the CH1 domain (containing VHH). These two types of nucleic acids were separated on an agarose gel. The VHH-encoding nucleic acid was extracted, purified, and then subjected to a second round of PCR amplification. The VHH fragment was then separated, purified, and recovered using gel electrophoresis. The recovered VHH gene fragment was mixed with the linearized yeast display vector pDisplay and co-transfected into competent yeast cells by electroporation to generate a yeast display library displaying VHH antibodies on the yeast cell surface. Yeast cells bound to the target antigen were enriched from a constructed library by magnetic sorting using streptavidin magnetic beads that had been incubated with the target antigen beforehand and thus bound to the antigen.

[0347] The yeast culture obtained after sorting by magnetic beads was plated on SDCAA plates, and single-clone cells were picked and cultured. After 48 h of induction expression, the single-clone cell culture was incubated with biotin-antigen and PE-Streptavidin. After incubation, flow cytometry (FACS) was performed to identify positive single-clone yeast cells that bound the target antigen. Genomic DNA was extracted from the culture of the obtained positive yeast cell clones for PCR amplification of antibody sequences and sequencing.

[0348] Based on the sequencing results, candidate VHH sequences were selected and ligated into the expression vector pcDNA3.4 in the form of C-terminal fusion with human IgG1Fc sequence. After the vector was verified by sequencing, it was transiently transfected into HEK-293F cells (hereinafter referred to as "293F cells"). The culture supernatant was used to characterize the binding and endocytic properties of the expressed antibody, and finally the anti-CD20 antibody was obtained by screening: V-n6D11: QVQLQESGGGLVQAGGSLRLSCAASGRTFSSSNMGWFRQAPGKERDFVAVISWSGSSPYY IDSVRGRFTISRDNAKNAMYLQMNSLKPEDTAVYYCAAGMSYGSRWLADYWGQGTQVTVSS (SEQ ID NO: 1)

[0349] Example 1.2. In vitro characterization of candidate VHH

[0350] Candidate VHH-Fc antibody expression and purification instructions 39 / 58 pages 48 CN 122356297 A

[0351] The encoding gene of the above VHH antibody sequence was synthesized and inserted into the expression vector pcDNA3.4, so that it was fused with the hIgG1 Fc sequence (SEQ ID NO: 33) at the C-terminus. The constructed expression vector was transiently transfected into 293F cells. After 3 days of continuous culture of transfected cells, the culture supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a Protein A column. The purity of the antibody product was determined by SEC-HPLC.

[0352] FACS Antigen Binding Properties Detection

[0353] The binding of the anti-CD20 VHH-Fc candidate antibody molecule V-n6D11 to target cells was detected using the FACS binding assay. The assay was performed under the following conditions: Raji / Daudi target cells (2×10⁵ / well) + VHH-Fc or reference antibody (375 nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h). The FACS binding results are shown in Figures 1A and 1B. The candidate antibody showed good target cell binding properties on both cell lines.In FACS binding on Raji cells (Figure 1A), V-6D11 had an EC50 value similar to the reference antibody Ofatumumab, and was superior to the maximum binding (Bmax) of Ofatumumab and Obinutuzumab; in FACS binding on Daudi cells (Figure 1B), V-6D11 had an EC50 value and maximum binding (Bmax) similar to the reference antibody Ofatumumab, and was superior to the maximum binding (Bmax) of Obinutuzumab.

[0354] Detection of FACS cross-reactivity between humans and monkeys

[0355] The binding of the anti-CD20 VHH-Fc candidate antibody molecule V-n6D11 to cynoCD20 overexpressing cells was detected using the FACS binding assay. The experiment was conducted under the following conditions: HEK293-cynoCD20 target cells (2×10⁵ / well) + VHH-Fc or reference antibody (375 nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h). The FACS binding results are shown in Figures 2A and 2B. The candidate antibody exhibited good target cell binding properties on HEK293-cynoCD20 cells, showing superior maximum binding (Bmax) compared to the reference antibodies Ofatumumab and Obinutuzumab; and no significant binding signal was observed with blank negative cells HEK293, suggesting that the binding is antigen-specific.

[0356] As shown in the figure above, in the CD20 FACS binding assay, V-n6D11 showed good binding to human and monkey CD20 antigens, and the EC50 ratio of binding to monkey and human CD20 antigens was between 1 and 10, indicating that the affinity of the candidate to human and monkey CD20 antigens was close.

[0357] Example 1.3. Sequence optimization and characterization of VHH components

[0358] Sequence optimization of anti-CD20 VHH components

[0359] Based on the in vitro activity characterization results, PTM risk assessment, and physicochemical property analysis, the sequence optimization of the anti-CD20 VHH sequence V-n6D11 was carried out.

[0360] The original VHH sequence was humanized using the "best-matching method". The amino acid sequence of the VHH framework region was compared and analyzed using the human germline V gene database to select the best germline sequence. The best-matching human CDR sequence was replaced with the VHH CDR sequence to generate the humanized VHH sequence. Sequence analysis of V-n6D11 revealed that it did not require removal of post-translational modifications (PTMs). The humanized sequence was reverse-translated and synthesized by Genewiz (Shanghai, China). It was then constructed into the pcDNA 3.4 expression vector, with hIgG1 fused to the C-terminus.The Fc sequence (SEQ ID NO: 33) expresses humanized VHHs in the form of human IgG1, thereby obtaining VHH antibody protein.

[0361] The VHH sequence of the humanized V-n6D11 antibody is shown in Table 1 below.

[0362] Table 1. Anti-CD20 VHH sequence specification 40 / 58 pages 49 CN 122356297 A

[0363] The obtained antibodies V-zn6D11.m1 to m9 were subjected to FACS detection. The binding of the humanized antibody to the target cells Daudi was similar to that of the parent antibody (Figure 3 and Table 2). The FACS binding test conditions were as follows: target cells (2×105 / well) + anti-CD20 antibody (375 nM, 5x dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h).

[0364] Table 2. Binding activity of V-zn6D11 humanized antibody with Daudi

[0365] The obtained antibodies V-zn6D11.m1 to m9 were subjected to FACS to verify the human-monkey cross-reactivity (Figures 4A and 4B). The binding of the humanized antibody to target cells HEK293-cynoCD20 was similar to that of the maternal antibody (Figure 4A and Table 3), and none of them bound to negative cells HEK293 (Figure 4B). The FACS binding assay conditions were as follows: target cells (2×105 / well) + anti-CD20 antibody (375nM, 5x dilution, 4℃ 1h incubation) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h).

[0366] Table 3. Binding activity of V-zn6D11 humanized antibody with cynoCD20, specification 41 / 58 pages, 50 CN 122356297 A

[0367] In summary, all nine humanization modifications of V-n6D11 were successful.

[0368] Internalization assay

[0369] Method 1: The internalization ability of the CD20 antibody molecule of the present invention on different target cells, Ramos and Daudi cells, was detected using the 4℃ / 37℃ internalization assay-I. As shown in Figure 5, the tested V-zn6D11.m2 molecule has a certain internalization ability, but it is weaker than Rituximab.

[0370] Method 2: The internalization ability of the CD20 antibody molecule on Ramos cells was detected using the internalization assay-II based on rProtein G-vc-MMAE killing. The endocytic capacity of V-zn6D11.m2 of the present invention was tested using Ramos cell lines positive for the target antigen. As shown in Figure 6, V-zn6D11.m2 exhibited significant endocytic activity compared with the IgG1 isotype control.

[0371] In summary, V-zn6D11.m2 has a certain endocytic capacity and exhibits good endocytosis effect based on MMAE killing.

[0372] Example 1.4 Generation of multispecific anti-CD79b / CD20 antibody molecules

[0373] This example describes the structure of an exemplary anti-CD79b / CD20 bispecific antibody (BsAb) and the design and construction of its expression vector.

[0374] Multispecific antibody molecule design

[0375] The bispecific antibody molecule constructs shown in Table 4 were designed. The constructs consist of two parts: a full-length anti-CD79b antibody and an anti-CD20 VHH domain; the antigen-binding domains targeting both targets maintain bivalent properties.

[0376] Table 4. Symmetrical antibody forms against CD79b and CD20: "-" indicates peptide linker (G4S)n; HC indicates heavy chain; LC indicates light chain; VHH indicates VHH domain. Due to the dimerization of the Fc region of immunoglobulin, two antibody heavy chains can associate to form a homodimer, thereby producing a symmetrical bispecific binding molecule.

[0377] Construction of multispecific antibody molecules

[0378] Specifically, the exemplary multi-chain bispecific antibodies shown in Table 5 are constructed.

[0379] In Table 5, the following constituent elements are used: VHH CD20 is the anti-CD20 VHH domain, derived from V-zn6D11.m2, and its amino acid sequence is shown in SEQ ID NO: 6 on pages 42 / 58 of the specification, CN 122356297 A; HCCD79b is the anti-CD79b heavy chain, whose VH amino acid sequence is shown in SEQ ID NO: 16, and has the human IgG1 constant region with LALA mutation shown in SEQ ID NO: 28; LCCD79b is the anti-CD79b light chain, whose VL amino acid sequence is shown in SEQ ID NO: 15, and has the human Kappa light chain constant region shown in SEQ ID NO: 29; the symbol "-" indicates that the two domains are connected by a peptide linker (G4S)3.

[0380] Table 5. Multi-chain multispecific antibodies

[0381] In this disclosure, V-F1S1.1 uIgG1 LALA and V-F1S1.2 uIgG1 LALA are also referred to as V-F1 and V-F2, respectively, as shown in Figures 7A and 7B.

[0382] The heavy and light chains of the bispecific antibodies shown in Table 5 were constructed into the pcDNA 3.4 expression vector and transfected into HEK293F cells. The cells were cultured for 3 days, and the culture supernatant of the transfected cells was collected and loaded into a Protein A column (MabSelect PrismA, Cytiva) for purification. The antibodies were eluted with acetate-sodium acetate solution (pH 3.5) and then immediately neutralized with 2M Tris. NanoAntibody concentration was measured by drop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and then stored at -80°C.

[0383] Example 1.5 Characterization of multispecific anti-CD79b / CD20 antibody molecules

[0384] Analysis of target antigen expression levels on tumor cells

[0385] Considering that the BCR (B cell receptor) on the surface of B cell-derived tumor cells may bind directly to the conventionally used anti-human IgG Fc or anti-hIgG (H+L) secondary antibody, direct-labeled antibodies anti-CD20 PE or BV421 (Clone: ​​2H7, Invitrogen) and anti-CD79b PE or APC (Clone: ​​CB3-1, Invitrogen) were used. The relative expression of CD20 antigen and CD79b antigen on various target tumor cells was determined by using the direct-labeled FACS method to detect the abundance of tumor cell surface antigen expression.

[0386] The detection results are shown in Figures 8A and 8B. Among all B-cell-derived tumor cell lines tested, WSU-DLCL2 showed high CD20 expression, SU-DHL-8 showed very low CD20 expression, Raji showed low CD20 expression, and the remaining cell lines showed moderate CD20 expression. Among all B-cell-derived tumor cell lines tested, Ramos, JEKO-1, and Daudi showed high CD79b expression, WSU-DLCL2, SU-DHL-8, and SU-DHL-2 showed low CD79b expression, and RC-K8 showed almost no CD79b expression.

[0387] Tumor Cell Binding Activity

[0388] Based on the above analysis of antigen expression on tumor cells, cell lines JEKO-1 (CD79b+++, CD20+++) and Ramos (CD79b++++, CD20++) with different target antigen expression levels were selected. The binding ability of the bispecific antibody (referred to as "V BsAb") of the present invention on different target cells was tested by FACS binding assay. The BCR on the surface of JEKO-1 and Ramos cells, as determined internally, did not directly bind to the detection secondary antibody (anti-human IgG Fc-PE). The FACS binding assay conditions were as follows: target cells (2×10⁵ / well) + sample (100 nM, 5× dilution, 4℃ 1 h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5 h).

[0389] In Ramos cell lines with high CD79b expression (Fig. 9A), and in JEKO-1 cell lines with relatively high expression of both CD79b and CD20 (Fig. 9B), the Fc-containing V BsAb of the present invention showed binding to the cells, with V-F2 showing the binding on pages 43 / 58 of the cell manual (52 CN).122356297 A Superior to Polatuzumab and CD20 VHH parent V-zn6D11.m2 in combination with EC50, Bmax, or both. The V BsAb of this invention does not bind to control cells HEK293, which are negative for both CD79b and CD20 (Figure 9C).

[0390] Developability Assessment

[0391] The physicochemical properties of candidate molecules V-F1 and V-F2 were analyzed by SEC-HPLC, CEX-HPLC, HIC-HPLC, and DLS. The samples showed physicochemical properties indicating developability.

[0392] Table 6. Physicochemical properties of multispecific antibodies (DA data)

[0393] FACS examination of target human-monkey cross-reactivity

[0394] The immunoreactivity of the humanized V BsAb (V-F2) and the humanized CD20 monospecific antibody (V-zn6D11.m2) of the present invention was detected by FACS binding assay using an engineered HEK293 cell line stably expressing human / monkey CD20. The experimental conditions were as follows: target cells (1×105 / well) + sample (200 nM, 4× dilution, 4℃ 1 h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5 h). The tested monospecific and bispecific antibodies of the present invention showed similar binding activity to human-monkey antigens, exhibiting immunoreactivity with monkeys. The detection results of V-F2 antibody are shown in Figures 10A and 10B. The anti-HEL isotype in the figure is the anti-HEL-human IgG1 (LALA) isotype control (Baiying Biotechnology, CAT# B109802, also referred to as aHEL or anti-HEL in this disclosure).

[0395] FACS detection of dual antibody binding to dual targets

[0396] The ability of the humanized V BsAb (V-F2) of the present invention to bind to both CD20 and CD79b dual targets was detected by FACS binding assay using an engineered HEK293 cell line that stably expresses human CD20. The experimental conditions were as follows: target cells (1×105 / well) + sample (200 nM, 4℃ 1 h) + biotinylated CD79b protein (5 μg / ml) + SAV-PE secondary antibody (Invitrogen, 1:500, 4℃ 0.5 h). The bispecific antibody of the present invention tested could bind to CD79b after binding to CD20, that is, it has the activity of simultaneously binding to CD20 and CD79b. The detection results of the V-F2 antibody are shown in Figure 11.

[0397] Internalization Assay

[0398] The internalization ability of the bispecific antibody molecule of the present invention on different target cells, Ramos and WSU-DLCL2 cells, was detected using the endocytosis assay-III (DT3C, cytotoxic endocytosis).

[0399] The endocytic ability of the antibody V-F2 of the present invention was tested in the above cell lines with different target antigen expression. As shown in Figure 12, antibody V-F2 showed good endocytic activity in both cell lines; moreover, the endocytosis of antibody V-F2 was stronger than that of Polatuzumab and CD20 VHH parent V-zn6D11.m2, suggesting that the V BsAb of the present invention can achieve more effective killing through efficient endocytosis.

[0400] Example Section II. Preparation and Characterization of Antibody-Drug Conjugate (ADC)

[0401] Materials and Methods

[0402] General Method for ADC Synthesis

[0403] General Synthesis Method A

[0404] 2.3 mg / ml of the antibody of the present invention or 7 mg / ml of aHEL LALA (Baiying Biotechnology, CAT# B109802) isotype control antibody in 50 mM NaAc-Ac, pH5.5 buffer was placed in Eppendorf tubes. Add 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 10 mM concentration) to the antibody buffer (TCEP:antibody molar ratio = 6:1). Place the Eppendorf tube containing the reaction mixture on a shaker (x500, manual page 44 / 58, 53 CN 122356297 A, rpm) and react at 37°C for 3 hours. Then add another 6 molar equivalents of TCEP (10 mM, TCEP:antibody = 6:1) to the mixture and continue reacting for 3 hours under the same conditions. Then, ultrafilter (MWCO 30 kd, Millipore filter) 6 times, each time to half the solution volume, and replenish the reduced antibody solution with 10 mM His-hac buffer to the same volume to remove TECP. Add 10 molar equivalents of linker-payload (5 mg / ml DMA solution) dropwise to the completely reduced antibody until the linker-payload to antibody molar ratio reaches 10:1. Maintain the DMA concentration below 20% (v / v) during addition; otherwise, adjust with buffer. Incubate the reaction at room temperature (500 RPM) for 1 hour using a shaker. Detect the conversion using HIC-HLPC. Once conversion is complete, purify the mixture. Transfer the reaction mixture to an ultrafiltration tube (MWCO 30 kd) and centrifuge the sample at 10,000 rpm for 5 minutes to half the solution volume. Replenish the volume with Glutamate buffer (containing 10% DMA). Discard the flow-through. Repeat the washing step 10 times. Then, wash 10 times with 10 mM Glutamate solution (pH=5.0) for solution replacement. Finally, transfer the remaining solution and adjust to the appropriate concentration.

[0405] Purity was determined by SEC-HPLC. The average DAR value and residual free linker-load content were determined by HIC and RP-HPLC.

[0406] General Synthesis Method B

[0407] 2.3 mg / ml of the antibody V-F2 of the present invention or 7 mg / ml of aHEL LALA (Baiying Biotechnology, CAT# B109802) isotype antibody in 50 mM NaAc-Ac, pH 5.5 buffer were placed in an Eppendorf tube. 2.0 molar equivalent (V-F2) or 2.5 molar equivalent (aHEL LALA) of TCEP (Tris(2-carboxyethyl) phosphine hydrochloride, 10 mM concentration) was added to the antibody buffer (TCEP: antibody molar ratio = 3.6 / 2.5:1). Place the Eppendorf tube containing the reaction mixture on a shaker (x500 rpm) and react at 37°C for 4 hours. Then, ultrafilter (MWCO 30 kd, Millipore filter) 6 times, replenishing the reduced antibody solution to the same volume with 10 mM His-hac buffer each time to remove TECP. Add 10 molar equivalents of linker-payload (5 mg / ml DMA solution) dropwise to the completely reduced antibody until the linker-payload to antibody molar ratio reaches 10:1. Maintain the DMA concentration below 20% (v / v) during addition; otherwise, adjust with buffer. Incubate the reaction at room temperature (500 RPM) for 1 hour. Detect the conversion using HIC-HLPC; once conversion is complete, add 10 molar equivalents of cysteine ​​(10 mM concentration). The reaction was kept at room temperature on a shaker for 1 hour (cysteine:antibody = 10:1) to deplete excess linker-load. After the reaction was completed, the reaction mixture was transferred to an ultrafiltration tube (MWCO 30 kd) and centrifuged at 10,000 rpm for 5 minutes to half the solution volume. The volume was then replenished with 10 mM His-hac (10% DMA). The flow-through was discarded. The washing step was repeated 10 times. The solution was then replaced by washing 10 times with 10 mM His-hac. Finally, the remaining solution was transferred and adjusted to the appropriate concentration.

[0408] Purity was determined by SEC-HPLC. The average DAR value and residual free linker-load content were determined by HIC and RP-HPLC methods.

[0409] General methods and / or parameters for determining or detecting ADC

[0410] Size exclusion chromatography (SEC) method (for ADC purity determination)

[0411] SEC-HPLC method parameters are shown in the table below: Specification 45 / 58Page 54 CN 122356297 A

[0412] Reversed-phase HPLC (RP HPLC) method (for free drug detection)

[0413] RP HPLC parameters are shown in the table below:

[0414] Elution is performed according to the table below

[0415] Post run: 10 mins

[0416] HIC-HPLC method (for free antibody and DAR distribution detection)

[0417] HIC-HPLC conditions are shown in the table below: Instruction manual 46 / 58 Page 55 CN 122356297 A

[0418] Elution is performed according to the table below

[0419] ADC activity characterization method

[0420] In vitro killing test of ADC

[0421] Target cells are plated at (0.3-2)×104 cells / 100 µl per well, and 2x ADC sample to be tested is added with culture medium diluted to an appropriate concentration. The cells are incubated in a carbon dioxide incubator for 96-144 hours. Add 20 µL of CCK8 reagent to each well of a 96-well plate, incubate in an incubator for 4-6 hours, and read OD450-OD650 using a microplate reader.

[0422] Cell viability = (OD value of sample well - OD value of blank culture medium) / (OD value of control well without sample - OD value of blank culture medium) × 100%.

[0423] In vitro killing test of ADC on normal human PBMCs

[0424] After revival, normal healthy human PBMCs were seeded at 1×10⁵ cells / 100 µL per well, and 2x ADC sample to be tested was added with culture medium diluted to an appropriate concentration. The plates were incubated in a carbon dioxide incubator for 168 hours. On the day of testing, centrifuge and remove the supernatant. The cell pellet is used for flow cytometry. The specific staining steps are as follows: 1. Add 200 µl of FACS buffer to each well, centrifuge and remove the supernatant, then wash the cells once; 2. Add 1 µl / well of Fc receptor blocking agent (Biolegend, 422302), incubate at room temperature for 15 min with a 50 µl / well incubation system; 3. Add 150 µl of FACS buffer to each well, centrifuge and remove the supernatant, then wash the cells again; 4. Prepare the flow cytometry staining mixture: anti-CD19 PE (Invitrogen, 12-0199-42, 1:100) + anti-huCD16 FITC (Biolegend, 302006, 1:400) + anti-CD3 APC (Biolegend, 300412, 1:50) (Instructions for use, pages 47 / 58, 56, CN 122356297 A) 5. 50 µl 6. Incubate the cells in the / well incubation system at 4°C for 30 min; 7. Add 150-200 µl of FACS buffer to each well, centrifuge to remove the supernatant, and wash the cells twice; 8. Add 100 µl of FACS buffer to each well.Resuspend cells in FACS buffer containing DAPI (Biolegend, 422801, 1:5000); 8. Perform detection by flow cytometry.

[0425] Assay for anti-tumor effect of ADC drug in mouse CDX subcutaneous xenograft model

[0426] Model establishment: Female severe combined immunodeficiency (SCID) mice (CB17-SCID, Beijing Vital River Laboratory Animal Technology Co., Ltd.) or NSG mice (NSG, from Shanghai Model Organisms Center, Inc.) or nude mice (nude mouse, Beijing Vital River Laboratory Animal Technology Co., Ltd.) aged 7-8 weeks were used. After one week of acclimation, cell suspension resuspended in PBS (3×106 or 1×107 cells per animal) was injected into the right scapular region. When the tumor volume grew to 100-200 mm3, the mice were randomly grouped according to the average tumor volume (n=4 or 5). The day of grouping was defined as D0, and the test article was administered intravenously or intraperitoneally on D0 as a single dose.

[0427] Administration volume: adjusted according to mouse body weight (administration volume for mice = 10 μL / g × mouse body weight (g))

[0428] Data collection: After the start of administration, the body weight of mice was weighed twice a week, the tumor volume was measured twice a week, and the animals were observed twice a day.

[0429] Test endpoint: The endpoint was determined based on the tumor volume (1500-2000 mm3) or the animal status. At the endpoint, all surviving animals were euthanized and tumors were collected. The tumors were photographed and weighed for subsequent processing.

[0430] Endpoint analysis: At the end of the test, the following indicators were analyzed: tumor volume change, percentage change in tumor volume relative to baseline (TGITV) and body weight change.

[0431] Calculation formula of TGITV: TGITV = {1‑[(Vt‑V0) / (Ct‑C0)]} × 100% : Average tumor volume of the test article administration group on day t; : Average tumor volume of the test article administration group on day 0; : Average tumor volume of the vehicle group on day t; : Average tumor volume of the vehicle group on day 0.

[0432] Assay for anti-tumor effect of ADC drug in mouse PDX subcutaneous xenograft model

[0433] Experimental animals:

[0434] NU / NU mice, female, aged 6‑8 weeks, weighing about 18‑22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK (Jing) 2021‑0006). The laboratory animal use license number is SYXK (Shan) 2023‑008. Feeding environment: SPF grade.

[0435] Establishment and in vivo passage of patient-derived tumor xenografts:

[0436] Human tumor tissue was inoculated subcutaneously into immunodeficient mice and observed until tumor formation and a tumor volume of approximately 500-800 mm3 were observed. The tumor tissue was then peeled off and evenly cut into small tissue pieces of approximately 3 mm × 3 mm × 3 mm. The small tumor tissue pieces were then inoculated subcutaneously into mice, and tumor growth was observed. When the average tumor volume reached approximately 100-200 mm3 (average 120 ± 30 mm3), the tumor-bearing mice were grouped and administered drugs for observation.

[0437] Grouping design: Before grouping, the tumor volume and weight of all tumor-bearing mice were measured. The tumor-bearing mice were randomly grouped according to the measured tumor volume. The randomized block design principle was adopted. First, the mice were divided into blocks according to the tumor volume. Then, the mice in each block were randomly assigned to each treatment group.

[0438] Experimental observation: Throughout the experiment, the use and observation of experimental animals were carried out in accordance with the regulations of AAALAC. After the experimental animals were inoculated with tumor tissue, their morbidity and mortality were recorded daily. During the routine experiment, all experimental animals were monitored for behavior, food intake, water intake, weight changes, hair luster, and other abnormalities.

[0439] Evaluation indicators: Tumor volume measurement: Measured twice a week using vernier calipers. The tumor volume calculation formula is V = 0.5a × b2, where a and b represent the long and short diameters of the tumor, respectively; Tumor growth inhibition rate TGI (%) = [1 - (Ti - T0) / (Vi - V0)] × 100, where Ti - T0 > 0; TGI (%) = [1 - (Ti - T0) / T0] × 100, where Ti - T0 < 0.

[0440] Ti is the average tumor volume after the start of administration of the compound group, T0 is the average tumor volume at the first administration of the compound group, V0 is the average tumor volume at the first administration of the solvent control group, and Vi is the average tumor volume after the start of administration of the solvent control group.

[0441] The weight of all tumor-bearing mice was measured twice a week. The weight change rate after drug administration was calculated as follows: RCBW (%) = (BWi – BW0) / BW0 × 100, where BWi is the average weight after drug administration and BW0 is the average weight at the time of the first drug administration.

[0442] After the experiment, the tumors were removed, weighed, and photographed.

[0443] Experiment termination: When individual tumor-bearing mice became extremely emaciated and near death, or when the tumor volume reached 3000 mm3 (including the average tumor volume of 2000 mm3 in the treatment group), the tumor-bearing mice were euthanized prematurely.

[0444] Safety evaluation of the test drug: During the experiment, when the weight of the treated mice continued to decrease and the RCBW reached more than 15%, the treatment was considered safe.Mice were observed after drug withdrawal until their condition recovered and RCBW < 10%, at which point communication was conducted to confirm whether to resume drug administration. When RCBW reached 20% or higher, euthanasia of the treated mice was considered.

[0445] Statistical Analysis

[0446] Analysis and Reporting. Quantitative indicators were described using mean ± standard error (Mean ± SEM / SD). Quantitative indicators were analyzed using one-way ANOVA or two-way ANOVA. For inter-group comparisons, a t-test was used; p < 0.05 was considered statistically significant. Both statistical and biological significance were considered in the results analysis.

[0447] Example 2. In vitro killing activity detection of CD79bxCD20 bispecific antibody ADC

[0448] Example 2.1 Preparation and characterization of ADC molecules

[0449] Preparation of ADC molecules conjugated with Mal-Gly-Exatecan-D-glucuronic acid:

[0450] Ab is the antibody VBsAb (V-F2) prepared in this application or the control antibody aHEL LALA, with p mainly at 8. Instructions for Use, pages 49 / 58, CN 122356297 A

[0451] According to the above synthesis method A, V-F2-D8-GLUC-Exd, MW 181.13, average Dar 8, yield 40%, purity 96.80% was prepared by using antibody V-F2 or aHEL LALA and linker-effective load Mal-Gly-Exatecan-D-glucuronic acid (CAS No.: 2763252-25-9; MedChemExpress, HY-153179); and aHEL-D8-GLUC-Exd, MW 151.88, average Dar 8, yield 62%, purity 98.00%.

[0452] Preparation of ADC molecules conjugated with MC-VC-PAB-MMAE:

[0453] Ab is the antibody V BsAb (V-F2) prepared in this application or the control antibody aHEL LALA, with p mainly at 3.3~3.4.

[0454] According to the above synthesis method B, V-F2-D4-VC-MMAE was prepared using antibody V-F2 or aHEL LALA and linker-loaded MC-VC-PAB-MMAE (CAS No.: 646502-53-6; MedChemExpress, HY-15575), with MW 178.51, average Dar 3.4, yield 50%, and purity 94.69%; and aHEL-D4-VC-MMAE, with MW 149.26, average Dar 3.3, yield 60%, and purity 93.4%.

[0455] The characterization data of the exemplary ADC obtained are as follows:

[0456] Example 2.2Polatuzumab Vedotin Sensitive Cell Line In Vitro Killing

[0457] Polatuzumab Vedotin analogue (abbreviated as PV, MedChemExpress, CAT# HY-132253) was used as the BMK ADC control. PV-sensitive Ramos cells expressing CD79b and CD20 antigens were selected as target cells. The in vitro killing assay of ADC was used to detect the in vitro killing activity of candidate CD79bxCD20 bispecific antibodies V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD ADC.

[0458] As shown in Figure 13A, the test drug was incubated with 10000 / well Ramos in vitro for 5 days. The PV BMK control group showed obvious cell killing. The in vitro killing activity of V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD was better than that of the PV BMK control group. V-F2-D4-VC-MMAE had the most advantage in the 5-day killing system.

[0459] As shown in Figure 13B, the test drug was incubated with 7500 / well Ramos in vitro for 6 days. The PV BMK control group showed obvious cell killing. The in vitro killing activity of V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD was significantly better than that of the PV BMK control group. The in vitro killing advantage of V-F2-D8-Gluc-EXD increased with the extension of culture time.

[0460] Example 2.3 In vitro killing of Polatuzumab Vedotin insensitive cell lines, instruction manual, pages 50 / 58, 59 CN 122356297 A

[0461] Polatuzumab Vedotin analogues were used as BMK ADC controls. Three PV-insensitive cell lines were selected: SU-DHL-8 (CD79b+CD20+, PV insensitivity mainly due to upregulation of the anti-apoptotic gene Bcl-xL), SU-DHL-2 (CD79b+CD20+++, PV insensitivity due to low expression of CD79b), and RC-K8 (CD79b-CD20++, PV insensitivity due to upregulation of MDR-1 (MMAE efflux pump), upregulation of the anti-apoptotic gene Bcl-xL, and low expression of CD79b) as target cells. The in vitro killing activity of the candidate CD79bxCD20 bispecific antibodies V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD ADC was detected using an in vitro ADC killing assay.

[0462] As shown in Figure 14A, the test drug was incubated with 20000 / well SU-DHL-8 cells in vitro for 5 days. The PV BMK control group showed better killing effect on the cells than the isotype ADC.(aHEL-D4-VC-MMAE) showed no significant advantage, while V-F2-D4-VC-MMAE was slightly better at killing cells than the PV BMK control group. The in vitro killing activity of V-F2-D8-Gluc-EXD was significantly better than that of the PV BMK control group and the isotype ADC control group (aHEL-D8-Gluc-EXD and aHEL-D4-VC-MMAE).

[0463] As shown in Figure 14B, the test drugs were incubated with 10000 / well SU-DHL-2 in vitro for 4 days. The PV BMK control group had very weak killing effect on the cells, similar to that of the isotype ADC (aHEL-D4-VC-MMAE). The in vitro killing activity of V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD was significantly better than that of the PVBMK control group and the isotype ADC control group (aHEL-D8-Gluc-EXD and aHEL-D4-VC-MMAE), and V-F2-D4-VC-MMAE was more advantageous in the 4-day in vitro killing system.

[0464] As shown in Figure 14C, the test drugs were incubated with 10000 / well RC-K8 in vitro for 6 days. The PVBMK control group had very weak killing effect on the cells, similar to that of the isotype ADC (aHEL-D4-VC-MMAE). The in vitro killing activity of V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD was significantly better than that of the PV BMK control group and the isotype ADC control group. Furthermore, due to the high expression of MDR-1 (MMAE efflux pump) in these cells, the in vitro killing advantage of V-F2-D4-VC-MMAE was weakened, and V-F2-D8-Gluc-EXD showed superior killing power compared to V-F2-D4-VC-MMAE at high concentrations.

[0465] The above results suggest that the CD79bxCD20 bispecific antibody ADC of the present invention can overcome PV resistance caused by the above three reasons: upregulation of MDR-1 (MMAE efflux pump), upregulation of the anti-apoptotic gene Bcl-xL, and low expression of CD79b. Moreover, compared to the BMK CD79b single-target ADC drug, the addition of CD20 can overcome single-target resistance (cell killing effect: V-F2-D4-VC-MMAE > PV). Therefore, the CD79bxCD20 bispecific antibody ADC of the present invention has advantages in both PV-sensitive and PV-insensitive cell lines.

[0466] Example 3. In vivo efficacy experiment of CD79bxCD20 bispecific antibody ADC

[0467] Example 3.1 Antitumor efficacy of Ramos-CDX model

[0468] Based on the in vitro killing results of the aforementioned CD79bxCD20 bispecific antibody ADC, the ADC drug to be tested was selected and subjected to Ramos-CDX model in mice.The CDX model was used to evaluate the efficacy of the drug. The antitumor effect of the ADC drug in a mouse CDX subcutaneous transplantation model was tested. The in vivo antitumor efficacy of the candidate V-F2 ADC drug and the PV BMK control group was detected using the NSG mouse Ramos-CDX model. Specifically, when the tumor volume reached 100-200 mm3, tumor-bearing mice were randomly divided into 3 groups (n=5) based on the average tumor volume. The day of grouping was defined as D0, and the test drug was administered intravenously on D0. The dose of the test V-F2-D8-Gluc-EXD ADC drug was 5 mg / kg, a single dose, and the dose of the reference drug, the Polatuzumab Vedotin analog (PV), was 1.5 mg / kg, a single dose. Tumor volume and mouse weight were measured periodically after administration.

[0469] As shown in Figure 15A, the efficacy at the experimental endpoint is as follows: The antitumor efficacy of the V-F2-D8-Gluc-EXD ADC drug (5 mg / kg) of the present invention is significantly better than that of the reference drug Polatuzumab Vedotin analog (1.5 mg / kg), and the results show a significant statistical difference (Figure 15B). As shown in Figure 15C, at the experimental endpoint, the weight changes of mice in all treatment groups were within the normal range, indicating that all tested drugs are safe in this model.

[0470] Example 3.2 Antitumor efficacy of WSU-DLCL2-CDX model

[0471] Considering the support effect of the CD20 end, the WSU-DLCL2 (CD20 ++++CD79b+) CDX model of diffuse large B lymphoma with high CD20 expression was selected for efficacy evaluation. The antitumor efficacy of ADC drugs in a mouse CDX subcutaneous transplantation model was tested using an NSG mouse WSU-DLCL2-CDX model. Specifically, tumor-bearing mice were randomly divided into three groups (n=5) based on the average tumor volume when the tumor volume reached 100-200 mm³. The day of grouping was defined as D0, and the test drug was administered intravenously on D0. The dose of the test drug, V-F2-D8-Gluc-EXD, was 5 mg / kg, administered as a single dose. The dose of the reference drug, Polatuzumab Vedotin analog (PV), was 2 mg / kg, administered as a single dose. Tumor volume and mouse weight were measured periodically after administration. (Instructions for use: Pages 51 / 58, 60 CN 122356297 A)

[0472] As shown in Figure 16A, the efficacy at the experimental endpoint is as follows: The antitumor efficacy of the V-F2-D8-Gluc-EXD ADC drug (5 mg / kg) of this invention is significantly better than that of the reference drug Polatuzumab Vedotin analog (2 mg / kg), and the results show significant efficacy.Significant statistical differences were observed (Figure 16B). As shown in Figure 16C, at the experimental endpoint, the weight changes of mice in all treatment groups were within the normal range, indicating that all tested drugs had good safety in this model.

[0473] Example 3.3 Antitumor efficacy of the Ramos-CDX model

[0474] Based on the aforementioned in vitro killing and in vivo efficacy results of CD79bxCD20 bispecific antibody ADCs, the drug dosage was reduced, and the ADC drugs to be tested were selected for efficacy evaluation using the mouse Ramos-CDX model. The antitumor effect of ADC drugs in the mouse CDX subcutaneous transplantation model was tested, and the in vivo antitumor efficacy of the candidate V-F2 ADC drug and the PVBMK control group was detected using the SCID mouse Ramos-CDX model. Considering that polatuzumab vedotin is used clinically in combination with rituximab for the treatment of DLBCL, this embodiment also compares the in vivo efficacy of the candidate ADC drug V-F2-D8-Gluc-EXD of the present invention with that of PV+Rituximab (MedChemExpress, CAT#HY-P9913). Specifically, tumor-bearing mice were randomly divided into 8 groups (n=4) based on the average tumor volume when the tumor volume reached 100-200 mm3. The day of grouping was defined as D0. On D0, the test drug V-F2-D8-Gluc-EXD ADC, the BMK control group PV, and the isotype ADC were administered intravenously, and Rituximab was administered intraperitoneally. The dosage of the test V-F2-D8-Gluc-EXD ADC drug was 3 mg / kg and 5 mg / kg, administered as a single dose. Considering the maximum limiting dose (MTD) of MMAE-type ADCs in mouse models, the dosage of the reference drug Polatuzumab Vedotin analog was 1 mg / kg and 3 mg / kg, and the dosage of the Rituximab analog was 30 mg / kg, all as a single dose. Tumor volume and body weight of mice were measured periodically after administration.

[0475] As shown in Figures 17A to C, the efficacy at the experimental endpoint is as follows: 1. The antitumor efficacy of the low-dose group (3 mg / kg) of the V-F2-D8-Gluc-EXD ADC drug of the present invention is significantly better than that of the low-dose group (1 mg / kg) of the reference drug Polatuzumab Vedotin analog (Figure 17A).

[0476] 2. The high-dose group (5 mg / kg) of the V-F2-D8-Gluc-EXD ADC drug, the high-dose group (3 mg / kg) of the Polatuzumab Vedotin analogue, and the PV+Rituximab combination group of the present invention all achieved long-term (Day 40 after group dosing) complete remission (CR) (Figures 17A and 17B).

[0477] 3. As shown in Figure 17C, at the experimental endpoint, the weight changes of all mice in the drug administration groups were within the normal range, indicating that all tested drugs had good safety in this model.

[0478] Example 3.4 Antitumor efficacy of WSU-DLCL2-CDX model

[0479] Based on the aforementioned in vitro killing and in vivo efficacy results of CD79bxCD20 bispecific ADC, the drug dosage was reduced, and the ADC drug to be tested was selected for efficacy evaluation through the mouse WSU-DLCL2-CDX model. The antitumor effect detection experiment of ADC drug in mouse CDX subcutaneous transplantation model was applied, and the in vivo antitumor efficacy of candidate V-F2 ADC drug and PV BMK control group was detected using SCID mouse WSU-DLCL2-CDX model. Considering that Polatuzumab Vedotin is used in combination with Rituximab in clinical practice to treat DLBCL, this example also compares the in vivo efficacy of the candidate ADC drug V-F2-D8-Gluc-EXD of the present invention in combination with PV+ Rituximab. Specifically, tumor-bearing mice were randomly divided into 7 groups (n=5) based on the average tumor volume when the tumor volume reached 100-200 mm3. The day of grouping was defined as D0. On D0, the test drug V-F2-D8-Gluc-EXD ADC and PV were administered intravenously, and Rituximab was administered intraperitoneally. The dosage of the test drug V-F2-D8-Gluc-EXD ADC was 3 mg / kg and 5 mg / kg, a single dose. The dosage of the reference drugs, Polatuzumab Vedotin analogues, was 1 mg / kg and 3 mg / kg, and the dosage of Rituximab analogues was 30 mg / kg, a single dose. Tumor volume and mouse weight were measured periodically after administration.

[0480] As shown in Figure 18, the efficacy at the experimental endpoint is as follows: 1. The antitumor efficacy of the low-dose group (3 mg / kg) of the V-F2-D8-Gluc-EXD ADC drug of the present invention is significantly better than that of the low-dose group (1 mg / kg) and high-dose group (3 mg / kg) of the reference drug Polatuzumab Vedotin analog (Figure 18A, Figure 18B).

[0481] 2. The antitumor efficacy of the low-dose group (3 mg / kg) of the V-F2-D8-Gluc-EXD ADC drug of the present invention is significantly better than that of the low-dose group (1 mg / kg PV + 30 mg / kg Rituximab) of the reference drug PV + Rituximab combination (Figure 18A, Figure 18B).

[0482] 3. The high- and low-dose groups (3 mg / kg and 5 mg / kg) of the V-F2-D8-Gluc-EXD ADC drug of the present inventionThe combination of PV and Rituximab (3 mg / kg PV + 30 mg / kg Rituximab) and the high-dose group (3 mg / kg PV + 30 mg / kg Rituximab) both achieved long-term (Day 40 after group administration) complete remission (CR) (Figure 18A, Figure 18B).

[0483] 4. As shown in Figure 18C, at the experimental endpoint, the weight changes of mice in all treatment groups were within the normal range, indicating that all tested drugs were safe in this model.

[0484] Example 3.5 Antitumor efficacy of SU-DHL-8-CDX model

[0485] Based on the in vitro killing and in vivo efficacy results of the aforementioned CD79bxCD20 bispecific antibody ADC, the drug dosage was reduced, and the ADC drug to be tested was selected for efficacy evaluation through the mouse SU-DHL-8-CDX model. The antitumor effects of ADC drugs in a mouse CDX subcutaneous transplantation model were investigated. The in vivo antitumor efficacy of the candidate V-F2 ADC drug and the PV BMK control group in PV-resistant cell lines was tested using the SCID mouse SU-DHL-8-CDX model. Considering that polatuzumab vedotin is used clinically in combination with rituximab for the treatment of DLBCL, this embodiment also compared the in vivo efficacy of the candidate ADC drug V-F2-D8-Gluc-EXD with the combination of PV and rituximab (MedChemExpress, CAT#HY-P9913). Specifically, when the tumor volume of tumor-bearing mice reached 100-200 mm3, they were randomly divided into 7 groups (n=5) based on the average tumor volume. The day of grouping was defined as D0. On D0, the test drug V-F2-D8-Gluc-EXD ADC and PV were administered intravenously, and rituximab was administered intraperitoneally. The V-F2-D8-Gluc-EXD ADC drug to be tested was administered at doses of 3 mg / kg and 5 mg / kg, as a single dose. The reference drug, the Polatuzumab Vedotin analog, was administered at doses of 1 mg / kg and 3 mg / kg, and the Rituximab analog was administered at doses of 30 mg / kg, as a single dose. Tumor volume and body weight were measured periodically after administration.

[0486] As shown in Figure 19, the efficacy at the experimental endpoint was as follows: 1. The antitumor efficacy of the low-dose group (3 mg / kg) of the V-F2-D8-Gluc-EXD ADC drug of this invention was significantly better than that of the low-dose group (1 mg / kg) and high-dose group (3 mg / kg) of the reference drug, the Polatuzumab Vedotin analog (Figure 19A, Figure 19B).

[0487] 2. The antitumor efficacy of the low-dose group (3 mg / kg) of the V-F2-D8-Gluc-EXD ADC drug of this invention was significantly better than that of the reference drug, the Polatuzumab Vedotin analog (Figure 19A, Figure 19B).The reference drug PV + Rituximab combination low-dose group (1 mg / kg PV + 30 mg / kg Rituximab) (Figure 19A, Figure 19B).

[0488] 3. The high-dose group of the V-F2-D8-Gluc-EXD ADC drug of the present invention (5 mg / kg) and the high-dose group of PV + Rituximab combination (3 mg / kg PV + 30 mg / kg Rituximab) can achieve complete remission (CR) for a long time (Day 40 after group administration) (Figure 19A).

[0489] 4. As shown in Figure 19C, at the experimental endpoint, the weight changes of mice in all drug administration groups were within the normal range, indicating that all tested drugs have good safety in this model. Instructions for Use, Pages 53 / 58, 62 CN 122356297 A

[0490] Example 3.6 Antitumor Efficacy of SU-DHL-2-CDX Model

[0491] Based on the aforementioned in vitro killing and in vivo efficacy results of CD79bxCD20 bispecific ADC, different drug doses were set, and the ADC drugs to be tested were selected for efficacy evaluation using the mouse SU-DHL-2-CDX model. The antitumor effect of ADC drugs in the mouse CDX subcutaneous transplantation model was tested, and the in vivo antitumor efficacy of candidate V-F2 ADC drugs and PV BMK control group in PV-resistant cell lines was detected using the nude mouse SU-DHL-2-CDX model. Specifically, when the tumor volume of tumor-bearing mice grew to 100-200 mm3, they were randomly divided into 7 groups (n=5) according to the average tumor volume. The day of grouping was defined as D0, and the test products V-F2-D8-Gluc-EXD ADC and PV were administered intravenously on D0. The V-F2-D8-Gluc-EXD ADC drug to be tested was administered at doses of 0.5 mg / kg, 2 mg / kg, and 5 mg / kg, as a single dose. The dosage of the reference drug, Polatuzumab Vedotin analog, was also administered at doses of 0.5 mg / kg, 2 mg / kg, and 5 mg / kg, as a single dose. Tumor volume and body weight of mice were measured periodically after administration.

[0492] As shown in Figure 20, the efficacy at the experimental endpoint is as follows: 1. The antitumor efficacy of the V-F2-D8-Gluc-EXD ADC drug of the present invention in the three dosage groups (0.5 mg / kg, 2 mg / kg, and 5 mg / kg) was significantly better than that of the reference drug, Polatuzumab Vedotin analog (Figure 20A, Figure 20B).

[0493] 2. As shown in Figure 20C, at the experimental endpoint, the body weight changes of mice in all administration groups were within the normal range, indicating that all tested drugs had good safety in this model.

[0494] Example 3.7 Antitumor efficacy of RT-PDX model

[0495] Richter syndrome (RS / RT) is an aggressive histological transformation of chronic lymphocytic leukemia (CLL), most commonly into diffuse large B-cell lymphoma (DLBCL). Treatment outcomes for Richter syndrome are generally poor, with a complete remission (CR) rate of only about 20%, and long-term survival with chemoimmunotherapy is less than 20%, indicating a strong need for treatment in these patients. Considering the high heterogeneity of RT, it is hypothesized that dual anti-ADCs may have an advantage in efficacy. This study investigated the antitumor effects of ADC drugs in a mouse PDX subcutaneous transplantation model. The in vivo antitumor efficacy of the candidate V-F2 ADC drug and the PV BMK control group in the RT model was tested using a NU / NU mouse RT-PDX model. Specifically, tumor-bearing mice were randomly divided into 3 groups (n=5) based on the average tumor volume when the tumor volume reached 100-200 mm3. The day of grouping was defined as D0, and the test drugs V-F2-D8-Gluc-EXD ADC and PV were administered intravenously on D0. The dosage of the tested V-F2-D8-Gluc-EXD ADC drug and the reference drug Polatuzumab Vedotin analog was 2 mg / kg, administered as a single dose. Tumor volume and body weight were measured periodically after administration.

[0496] As shown in Figure 21, the efficacy at the experimental endpoint was as follows: 1. At the same dosage, the antitumor efficacy of the V-F2-D8-Gluc-EXD ADC drug of this invention was significantly better than that of the reference drug Polatuzumab Vedotin analog group (Figure 21A, Figure 21B).

[0497] 2. As shown in Figure 21C, at the experimental endpoint, the body weight changes of all administered mice were within the normal range, indicating that all tested drugs had good safety in this model.

[0498] The above in vitro and in vivo pharmacodynamic models suggest that the V-F2 ADC of the present invention can effectively kill both PV-sensitive and PV-resistant cell lines, and can effectively inhibit the growth of RT. This suggests that the V-F2 ADC of the present invention can be used to supplement clinical PV-insensitive patients and can also be used for RT patients, covering a larger patient population and benefiting more patients.

[0499] Example 4. Killing of irrelevant cells in normal healthy human PBMCs by CD79bxCD20 bispecific antibody ADC

[0500] Considering that ADCs in the blood may have blood toxicity, an experiment was designed to detect the effect of V-F2 ADC on irrelevant cells in peripheral PBMCs of normal healthy humans. According to the aforementioned in vitro killing test of ADCs on normal human PBMCs, the V-F2 of the present invention killed CD3-positive T cells in peripheral PBMCs slightly less than PV (Figure 22A), and the V-F2 of the present invention killed CD16 cells in peripheral PBMCs less than PV.The killing effect of positive NK cells was significantly lower than that of PV (Figure 22B). The results suggest that the ADC of this invention has a safety profile, especially in terms of blood toxicity, which is significantly superior to PV. Specification page 54 / 58, page 63, CN 122356297 A

[0501] Sequence List Description Specification page 55 / 58, page 64, CN 122356297 A Specification page 56 / 58, page 65, CN 122356297 A Specification page 57 / 58, page 66, CN 122356297 A Specification page 58 / 58, page 67, CN 122356297 A, Figure 1A Specification Figure 1 / 32, page 68, CN 122356297 A, Figure 1B Specification Figure 2 / 32, page 69, CN 122356297 A, Figure 2A Specification Figure 3 / 32, page 70, CN 122356297 A, Figure 2B Specification Figure 4 / 32, page 71, CN 122356297 A, Figure 3 Specification Figure 5 / 32, page 72, CN 122356297 A, Figure 4A Figure 4B, Appendix to the Instruction Manual, Page 6 / 32, 73 CN 122356297 A; Figure 5A, Figure 5B, Appendix to the Instruction Manual, Page 7 / 32, 74 CN 122356297 A; Figure 6, Figure 7, Appendix to the Instruction Manual, Page 8 / 32, 75 CN 122356297 A; Figure 8A, Figure 8B, Appendix to the Instruction Manual, Page 9 / 32, 76 CN 122356297 A; Figure 9A, Figure 9B, Appendix to the Instruction Manual, Page 10 / 32, 77 CN 122356297 A; Figure 9C, Figure 10A, Appendix to the Instruction Manual, Page 11 / 32, 78 CN 122356297 A; Figure 10B, Figure 11, Appendix to the Instruction Manual, Page 12 / 32, 79 CN 122356297 A; Figure 12A, Figure 12B, Appendix to the Instruction Manual, Page 13 / 32, 80 CN 122356297 A; Figure 13A, Appendix to the Instruction Manual, Page 14 / 32, 81 CN 122356297 A Figure 13B Figure 14A Instruction Manual Drawings 15 / 32 Page 82 CN 122356297 A Figure 14B Figure 14C Instruction Manual Drawings 16 / 32 Page 83 CN 122356297 A Figure 15A Figure 15B Instruction Manual Drawings 17 / 32 Page 84 CN 122356297 A Figure 15C Figure 16A Instruction Manual DrawingsFigure 18 / 32, page 85, CN 122356297 A; Figure 16B, Figure 16C; Instruction Manual Drawings; Figure 19 / 32, page 86, CN 122356297 A; Figure 17A; Instruction Manual Drawings; Figure 20 / 32, page 87, CN 122356297 A; Figure 17B, Figure 17C; Instruction Manual Drawings; Figure 21 / 32, page 88, CN 122356297 A; Figure 18A; Instruction Manual Drawings; Figure 22 / 32, page 89, CN 122356297 A; Figure 18B; Instruction Manual Drawings; Figure 23 / 32, page 90, CN 122356297 A; Figure 18C, Figure 19A; Instruction Manual Drawings; Figure 24 / 32, page 91, CN 122356297 A; Figure 19B; Instruction Manual Drawings; Figure 25 / 32, page 92, CN 122356297 A; Figure 19C, Figure 20A; Instruction Manual Drawings; Figure 26 / 32, page 93, CN 122356297 A Figure 20B Appendix to the Instruction Manual, Page 27 / 32, 94 CN 122356297 A Figure 20C Appendix to the Instruction Manual, Page 28 / 32, 95 CN 122356297 A Figure 21A Appendix to the Instruction Manual, Page 29 / 32, 96 CN 122356297 A Figure 21B Appendix to the Instruction Manual, Page 30 / 32, 97 CN 122356297 A Figure 21C Figure 22A Appendix to the Instruction Manual, Page 31 / 32, 98 CN 122356297 A Figure 22B Appendix to the Instruction Manual, Page 32 / 32, 99 CN 122356297 A Abstract Provided are an antibody targeting CD79b and / or CD20, an antibody-drug conjugate (ADC), a composition containing the antibody or ADC, and the therapeutic use thereof.

Claims

1. A multispecific antibody comprising at least one antigen-binding domain specifically binding to CD79b and at least one antigen-binding domain specifically binding to CD20, preferably in: The antigen-binding domain that specifically binds to CD79b comprises or is composed of a heavy chain variable region (VH) and a light chain variable region (VL); and the antigen-binding domain that specifically binds to CD20 comprises or is composed of a VHH domain; and / or in: (i) The CD79b binding domain was determined by flow cytometry (FACS) to have an EC50 value of approximately 0.1–30 nM, for example, 1–10 nM EC50. 50 Values ​​bind to CD79b-expressing cells; (ii) The CD20 binding domain was determined by flow cytometry (FACS) to have an EC50 value of approximately 0.1–50 nM, for example, 1–30 nM EC50. 50 The value binds to CD20-expressing cells.

2. The multispecific antibody according to claim 1, wherein the CD20 binding domain comprises or is composed of a VHH domain, wherein: The VHH domain contains the CDR1, CDR2, and CDR3 sequences of one of the amino acid sequences in SEQ ID NO: 1 and 5-13; Preferably, the CDR1, CDR2 and CDR3 sequences (i) comprise or consist of the amino acid sequences of SEQ ID NOs:2, 3 and 4, respectively; or (ii) comprise or consist of the amino acid sequences of SEQ ID NOs:14, 3 and 4, respectively. More preferably, the VHH domain comprises an amino acid sequence selected from SEQ ID NO: 1 and 5-13, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed of such amino acids. More preferably, the VHH domain comprises or is composed of the amino acid sequence of SEQ ID NO:

6.

3. The multispecific antibody according to claim 1 or 2, wherein the CD79b binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: The heavy chain variable region contains HCDR1-3 contained in the VH sequence of SEQ ID NO: 16; and the light chain variable region contains LCDR1-3 contained in the VL sequence of SEQ ID NO: 15; Preferably, the HCDR1-3 respectively comprises or consists of the amino acid sequences of SEQ ID NOs: 20, 21 and 22; and the LCDR1-3 respectively comprises or consists of the amino acid sequences of SEQ ID NOs: 17, 18 and 19; More preferably, (a) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 16, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof; and / or (b) the light chain variable region comprises the amino acid sequence of SEQ ID NO: 15, or has at least 85%, 90%, 95% or 99% identity with respect to the amino acid sequence, or has an amino acid sequence having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed thereof; More preferably, the heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO: 16, and the light chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:

15.

4. The multispecific antibody according to any one of claims 1-3, wherein the antibody further comprises an immunoglobulin Fc region, and optionally wherein: (i) The Fc region contains mutations that reduce or eliminate the binding of the Fc region to FcγR, for example, L234A or L235A mutations. (ii) The Fc region is of the IgG type, such as the IgG1 or IgG4 isotype; and / or (iii) The Fc region contains the amino acid sequence of SEQ ID NO: 31 or 32, or an amino acid sequence that is at least 95%, 96%, 98% or 99% identical to it.

5. The multispecific antibody according to any one of claims 1-4, wherein, The multispecific antibody comprises: (a) Anti-CD79b Fab domain and; (b) The immunoglobulin Fc region connected to the C-terminus of the anti-CD79b Fab domain; and (c) Optionally, at least one (preferably one) CD20 binding domain is attached to the N-terminus of the Fab domain or the C-terminus of the Fc region via a peptide linker. Preferably, the peptide linker comprises the amino acid sequence of SEQ ID NO:26 or 27. More preferably, the CD20 binding domain is connected to the N end of the heavy chain of the Fab domain.

6. The multispecific antibody according to any one of claims 1-5, wherein: The valence ratio of the CD79b binding domain to the CD20 binding domain is 1:

1.

7. The multispecific antibody according to any one of claims 1-6, wherein the multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, From N-end to C-end, The first polypeptide chain comprises: VH CD79b -CH1 domain-immunoglobulin Fc region; The second polypeptide chain comprises: VL CD79b - CL structure domain; VH CD79b and VL CD79b These represent the heavy chain variable region and the light chain variable region that bind to CD79b, respectively. The first polypeptide chain is optionally connected to the anti-CD20 VHH domain at the N-terminus or C-terminus via a peptide linker.

8. The multispecific antibody according to any one of claims 1-7, wherein the multispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein, - The first and second polypeptide chains respectively comprise the amino acid sequences of SEQ ID NOs: 23 and 24, or have at least 85%, 90%, 95% or 99% identity with them, or have one or more (preferably 1-10, more preferably 1-5) amino acid sequences with additions, deletions and / or substitutions, or consist of, or The first and second polypeptide chains respectively comprise the amino acid sequences of SEQ ID NOs: 25 and 24, or have at least 85%, 90%, 95%, or 99% identity with them, or have one or more (preferably 1-10, more preferably 1-5) amino acid sequences with additions, deletions, and / or substitutions, or are composed of them. Preferably, the first and second polypeptide chains comprise, or consist of, the amino acid sequences of SEQ ID NOs: 25 and 24, respectively.

9. An antigen-binding molecule comprising the antibody according to any one of claims 1-8.

10. A polynucleotide encoding the antibody of any one of claims 1-8 or the antigen-binding molecule of claim 9.

11. A vector, preferably an expression vector, comprising the polynucleotide of claim 10.

12. A host cell comprising the polynucleotide of claim 10 or the vector of claim 11, wherein the host cell is optionally a mammalian cell.

13. A method for producing the antibody according to any one of claims 1-8, the method comprising: Host cells containing polynucleotides encoding the polypeptide chain are cultured under conditions suitable for producing the antibody or its polypeptide chain.

14. An immunoconjugate comprising the antibody of any one of claims 1-8 or the antigen-binding molecule of claim 9.

15. An antibody-drug conjugate having formula (I) or a pharmaceutically acceptable salt or solvation thereof: Ab-(L-D) p (I) in: Ab is the antibody according to any one of claims 1-8 or the antigen-binding molecule according to claim 9; L is the connector; D represents a drug, such as an anti-tumor compound; p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, 3-5, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

16. The antibody-drug conjugate of claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the drug is a cytotoxic agent, such as a camptothecin or auratestatin.

17. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15 or 16, wherein D has the structure shown in formula (D-1a) or formula (D-1b): or Equation (D-1a) Equation (D-1b) in, The wavy line indicates that the price bond is connected to L; R 1a Selected from H and C1-C6 alkyl groups; R 2a Selected from H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR 5a and -SR 5a ; R 3a Selected from H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl and -OR 5a ;and R 4a and R 5a Independently selected from H and C1-C4 alkyl groups; R 1b R 2b R 3b R 4b R 5b and R 8b Each was independently selected from C 1-8 Alkyl; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; R 6b and R 7b Each was independently selected from C 1-8 Alkyl groups, such as methoxy, ethoxy, or propoxy; R 9b Selected from C 1-8 Alkyl groups and COOH; preferably C 1-4 Alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and R 10b Selected from OH and H.

18. The antibody-drug conjugate of claim 17 or a pharmaceutically acceptable salt or solvation thereof, wherein D has the structure shown in formula (D-2a) or formula (D-2b): or Equation (D-2a) Equation (D-2b) Where R 1a R 2a R 3a and R 4a As defined in claim 3 of equation (D-1a); R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined in formula (D-1b) as claimed in claim 3.

19. The antibody-drug conjugate according to any one of claims 15-18, or a pharmaceutically acceptable salt or solvate thereof, wherein... D has the structure of formula (D-1a) or (D-2a), and R 1a For H; R 2a It is a C1-C4 alkyl group, preferably methyl; R 3a It is a halogen, preferably -F; R 4a It is a C1-C4 alkyl group, preferably ethyl; or D has the structure of formula (D-1b) or (D-2b), and in which R 1b R 4b and R 8b Each was independently selected from C 1-2 Alkyl; preferably methyl; R 2b R 3b and R 5b Each was independently selected from C 3-4 alkyl; R 6b and R 7b Each was independently selected from C 1-2 alkoxy groups; and R 9b Selected from C 1-4 Alkyl and R 10b For OH; or R 9b It is COOH and R 10b For H.

20. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15-17, wherein D has the structure shown in formula (D-3a) or (D-3b): (D-3a) or (D-3b); Preferably, D has the structure shown in formula (D-4a) or (D-4b): (D-4a) or (D-4b).

21. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15-19, wherein the drug is Exatecan, Dxd, SN-38, monomethylaurestatin E (MMAE), or MMAF.

22. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15-21, wherein -L- has the following structure: -Z-L1-L2-L3- in Z is selected from , , , , and , where m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8, for example, an integer from 1 to 5; L1 is selected from non-existent, , , and , where n1 and m1 are each independently an integer selected from 0 to 20, for example, an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8; L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and L3 is selected from: , , , and Where X is selected from -NH-, -O-, and -S-; R 1c Each was independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is independently selected from pentose, penturonic acid, hexose, and hexuronic acid; n2 is 0, 1, 2, 3, or 4; n5 is 0, 1, 2, or 3; and in, Z is connected to atoms on Ab, preferably S atoms, and L3 is connected to D.

23. The antibody-drug conjugate according to claim 22, or a pharmaceutically acceptable salt or solvate thereof, wherein... Z is selected from and , where m is 1, 2, 3, 4, 5, 6, 7 or 8, for example, an integer from 1 to 5; Preferably, Z is selected from , and .

24. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to claim 22 or 23, wherein L1 is selected from those that do not exist. and , where n1 is an integer independently selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8; Preferably, L1 is selected from non-existent, and .

25. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 22-24, wherein L2 is an amino acid residue or a peptide residue consisting of 2, 3, 4, 5, 6 or 7 amino acids; preferably, wherein each amino acid residue or amino acid is independently selected from valine (Val), alanine (Ala), glycine (Gly), lysine (Lys), citrulline (Cit), glutamine (Gln), glutamic acid (Glu), phenylalanine (Phe), leucine (Leu), tyrosine (Tyr), serine (Ser), aspartic acid (Asp), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine ​​(Cys), histidine (His) and threonine (Thr); More preferably, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit), phenylalanine (Phe), lysine (Lys), glutamic acid (Glu), and glutamine (Gln); More preferably, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit) and glutamic acid (Glu).

26. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 22-24, wherein L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly-; Preferably, L2 is selected from -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, and -Gly-Gly-Phe-Gly-; More preferably, L2 is selected from -Gly- and -Val-Cit-.

27. The antibody-drug conjugate according to any one of claims 22-26, or a pharmaceutically acceptable salt or solvate thereof, wherein L3 is selected from: , and , Where R 1c Each was independently selected from C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is selected independently from each of the following groups: , , and n2 is 0, 1, 2, 3 or 4; and n5 is 0, 1, 2 or 3.

28. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 22-27, wherein L3 is selected from: and .

29. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 22-28, wherein each of Su is independently: ； Preferably, Su is independently , Alternatively, preferably, each Su is independently... .

30. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 22-29, wherein L3 is selected from: and .

31. The antibody-drug conjugate according to claim 22, or a pharmaceutically acceptable salt or solvate thereof, wherein... -Z-L1-L2-L3- are each independently selected from the following structures: ,and ， in, Each m is an integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7 or 8; The group is connected to an atom on Ab, preferably an S atom, on the left side, and to D on the right side.

32. The antibody-drug conjugate of claim 22 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is an antibody-drug conjugate having a structure selected from: Wherein Ab is the antibody according to any one of claims 1-8 or the antigen-binding molecule according to claim 9, preferably the antibody according to claim 8; and p is an integer selected from 1 to 16, such as an integer selected from 1-10, 1-9, 2-8, 4-10, 6-8, 3-7, 4-6, 2-6, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

33. The antibody-drug conjugate or its pharmaceutically acceptable salt or solvate according to any one of claims 15-32, wherein the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, 2-4, or 2-6.

34. A pharmaceutical composition comprising an antibody according to any one of claims 1-8, an antigen-binding molecule according to claim 9, an immunoconjugate according to claim 14, or an antibody-drug conjugate according to any one of claims 15-33, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, and optionally further comprising one or more other pharmaceutically active peptides and / or compounds, for example, other therapeutic agents selected from inhibitors of oncolytic drugs, cytotoxic agents, cytokines, and immune checkpoint molecules.

35. Use of the antibody of any one of claims 1-8, the antigen-binding molecule of claim 9, the immunoconjugate of claim 14, or the antibody-drug conjugate of any one of claims 15-33, or a pharmaceutically acceptable salt or solvate thereof, as a medicine or for the preparation of a medicine, wherein preferably the medicine is used to treat cancer or B-cell-related autoimmune diseases.

36. A method of treating or preventing cancer or B-cell-related autoimmune diseases, comprising administering to an individual in need an effective amount of an antibody of any one of claims 1-8, an antigen-binding molecule of claim 9, an immunoconjugate of claim 14, an antibody-drug conjugate of any one of claims 15-33 or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of claim 34.

37. The use of claim 35 or the method of claim 36, wherein the cancer is a solid tumor or hematologic malignancy, preferably a CD79b-positive and / or CD20-positive tumor, more preferably a B-cell-associated lymphoid tumor and leukemia, for example selected from: non-Hodgkin's lymphoma (NHL), large B-cell lymphoma, DLBCL, RT (Richter's transformation / syndrome), BL (Burkitt lymphoma), FL (follicular lymphoma), MZL (marginal zone lymphoma), MCL (mantle cell lymphoma), acute lymphoblastic leukemia (ALL), and chronic lymphocyticle leukemia (CLL), optionally the cancer is a relapsed or refractory large B-cell lymphoma.

38. The use or method of claim 37, wherein the cancer is a CD79b-insensitive or resistant tumor, optionally wherein the tumor cells have one or more of the following characteristics: (a) The tumor cells have CD79b expression levels that are 50%, 40%, 30%, 20%, 10%, 5% or 2% lower than the CD79b expression levels on Daudi cells. (b) Compared with Daudi cells or Ramos cells, the tumor cells have upregulated expression and / or activity of the anti-apoptotic gene Bcl-xL; (c) Compared to Daudi cells or Ramos cells, the tumor cells have upregulated expression and / or activity of the MMAE efflux pump (MDR-1), and (d) The tumor cells have CD20 expression levels that are 1%, 10%, 50%, 100%, 150%, 200%, 300%, or 400% higher than those on Daudi cells.

39. The use of claim 35 or the method of claim 36, wherein the B-cell-related autoimmune disease is selected from rheumatoid arthritis (RA); lupus; NMDAR encephalitis; multiple sclerosis; systemic sclerosis; immune thrombocytopenic purpura; simple erythrocytic aplasia; autoimmune anemia; cold agglutinin disease; severe insulin resistance type B syndrome; mixed cryoglobulinemia; myasthenia gravis; Wegener's granulomatosis; refractory pemphigus vulgaris; dermatomyositis; Sjogren's syndrome; active type II mixed cryoglobulinemia; pemphigus vulgaris; autoimmune nephropathy; neoplastic visual oculoclonus-myoclonus syndrome; and relapsing-remitting multiple sclerosis (RRMS).

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