Antibody and drug conjugate thereof, and use thereof

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.

Description

Antibodies and their drug conjugates and their uses

[0001] Cross-references to related applications

[0002] This application was filed on January 8, 2026 as a PCT international patent application, claiming priority to PCT international patent application No. PCT / CN2025 / 071762, filed on January 10, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] 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 Technology

[0004] Non-Hodgkin's lymphoma (NHL) accounts for approximately 4% of all cancers. Despite improvements in existing therapies, treatment options for relapsed / refractory (R / R, sometimes referred to as r / r) NHL patients remain limited and have poor prognoses. For example, adoptive immunotherapy with T cells expressing chimeric antigen receptors (CARs) has shown promising efficacy in treating CD19-positive B-cell malignancies, yet 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.

[0005] 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 various B-cell lymphomas, and its expression is correlated with cancer cell viability in most DLBCL tumor models. Therefore, the likelihood of resistance to CD79b-targeting drugs developing through antigen loss is relatively low, making CD79b an attractive target for developing novel immunotherapies.

[0006] Studies have shown that 90% of diffuse large B-cell lymphomas (DLBCL), 97% of follicular lymphomas (FL), 23% of chronic lymphocytic leukemia (CLL), 95% of marginal-zone lymphomas (MZL), 100% of hairy cell leukemia (HCL), and 95% of mantle cell lymphomas (MCL) express sufficient amounts of CD79b on their cell surface to elicit a response to CD79b-targeted drugs (CD79b-vc-MMAE) (Blood. 2009; 114:2721-2729).

[0007] Polatuzumab vedotin (POLIVY) TM Polatuzumab vedotin (PV) is an antibody-drug conjugate (ADC) molecule targeting CD79b developed by Roche Pharmaceuticals. It was approved by the FDA in 2019 for the treatment of r / r DLBCL. After binding to CD79b, PV is internalized by cells and releases monomethyl guanylate E (MMAE). MMAE then binds to microtubules and kills dividing cells by inhibiting cell division and inducing apoptosis. In combination with standard therapy (bendamustine and rituximab), PV treatment increases the complete response (CR) rate and duration of response (DOR) in patients with DLBCL. In combination with R-CHP (rituximab, cyclophosphamide, doxorubicin, and prednisone), PV significantly improves the 2-year progression-free survival (PFS) in patients with untreated DLBCL (N Engl J Med. 2022 Jan 27; 386(4):351-363.).

[0008] Although the approval of polatuzumab vedotin (PV) has established a more effective treatment option 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 revealed that the reasons for their insensitivity include: upregulation of the anti-apoptotic gene Bcl-xL; low CD79b expression level; and upregulation of the MMAE efflux pump (MDR-1) (Br J Haematol. 2022 Oct; 199(2):245-255.).

[0009] Nanobodies are small proteins composed of single-chain antibody molecules. They possess high specificity and affinity, and compared to traditional antibodies, they exhibit 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 therapeutic modalities such as photodynamic therapy and immunotherapy. Therefore, the application of nanobodies in cancer treatment is receiving widespread attention and is expected to become one of the important tools for future cancer therapy.

[0010] Given the unmet needs in the treatment of NHL cancer, there remains a need in the field to develop new drug molecules, especially those with improved antitumor resistance and / or improved tumor specificity and selectivity, to meet the treatment needs of different NHL cancer patients, particularly those with relapsed and refractory NHL cancer.

[0011] Invention Overview

[0012] 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-cell and B-cell malignancies, but their expression density shows tumor heterogeneity (see http: / / hematologyoutlines.com / atlas_topics / 69.html); in addition, their co-expression rate is extremely low in normal non-B-cell tissues.

[0013] Given the expression patterns of the two targets mentioned above, the inventors proposed and designed antibody molecules that simultaneously target CD79b and CD20 to reduce drug resistance and improve efficacy. Building upon 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 this invention is easier to prepare than conventional mAb-based bispecific antibodies and avoids light chain mismatch issues; it also facilitates more specific enrichment of the antibody molecule in tumor tissues, and correspondingly enhances efficacy. Based on these designs and findings, the inventors established the multispecific antibody of this invention, its drug conjugate, and its uses, particularly in the treatment of cancer and autoimmune diseases.

[0014] Therefore, in a first aspect, this disclosure provides multispecific antibodies against CD79b and CD20. In some embodiments of the multispecific antibody according to the invention, 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), preferably the VH comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO:16 and the VL comprises LCDR1, LCDR2, and LCDR3 of SEQ ID NO:15. In other embodiments of the multispecific antibody according to the invention, the antigen-binding domain specifically binding to CD20 comprises or is composed of a VHH domain, preferably the VHH domain comprises CDR1, CDR2, and CDR3 of one of SEQ ID NO:1 and 5-13.

[0015] In a second aspect, this disclosure provides antigen-binding molecules comprising the antibodies of the present invention.

[0016] In a third aspect, this disclosure provides a polynucleotide encoding an antibody or antigen-binding molecule according to the first and second aspects of this disclosure, a vector comprising the polynucleotide, and a host cell. This disclosure also provides methods for preparing antibody or antigen-binding molecules according to the first and second aspects of this disclosure.

[0017] In a fourth aspect, this disclosure provides immune conjugates and antibody-drug conjugates (ADCs) comprising antibodies or antigen-binding molecules as described in the first and second aspects of this disclosure, particularly anti-CD79b / CD20 multispecific antibody-drug conjugates.

[0018] In a fifth aspect, this disclosure provides pharmaceutical compositions and 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 selected from inhibitors of oncolytic agents, 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.

[0019] In a sixth aspect, this disclosure provides the use of antibodies or antigen-binding molecules according to the first and second aspects of this disclosure, or the immunoconjugates or ADCs of the fourth aspect of this disclosure, as medicaments or for the preparation of medicaments, wherein the medicaments are for the treatment of cancer and B-cell-related autoimmune diseases, for example, the cancers being selected from CD79b-positive and / or CD20-positive tumors, more preferably B-cell-related lymphomas and leukemias; the 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 regard, this disclosure also provides a method for treating cancer and B-cell-related autoimmune diseases, the 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 a pharmaceutical composition according to the fifth aspect of this disclosure, 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-related lymphoma and leukemia; and the B-cell-related autoimmune disease is selected from, for example, rheumatoid arthritis, NMDAR encephalitis, idiopathic thrombocytopenic purpura, multiple sclerosis, pemphigus vulgaris, systemic sclerosis, and systemic lupus erythematosus.

[0020] In some embodiments, the antibodies and ADCs 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.

[0021] In other embodiments, the antibody and ADC drugs of the present invention can simultaneously address drug specificity and tumor heterogeneity by dually targeting CD79b and CD20, thereby 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.

[0022] In some further embodiments, compared to treatment regimens combining CD79b ADCs with CD20 monoclonal antibodies, the present invention’s multi(bi)specific ADCs, which simultaneously target CD79b and CD20, can achieve comparable or improved efficacy while reducing the number of drug administrations.

[0023] In some further embodiments, the present invention’s multi-(bi)specific ADC can overcome MMAE-related drug low responsiveness and drug resistance by using novel payloads.

[0024] In some further embodiments, the ADC drugs of the present invention also have one or more advantages compared to CD79b ADC monotherapy (e.g., polatuzumab vedotin) or combination therapy of CD79b ADC with CD20 monoclonal antibody (e.g., PV / Rituximab):

[0025] - It exhibits superior killing activity in cells with low CD79b expression and / or low CD20 expression;

[0026] - It has a more significant killing effect on CD79b-targeted drug-resistant cell lines, wherein the resistance is preferably selected from one or more of the following: upregulation of the anti-apoptotic gene Bcl-xL; low expression level of CD79b; and upregulation of MMAE efflux pump (MDR-1);

[0027] - It has a wider range of applicability to patients;

[0028] - It can be used not only for previously untreated patients, but also for patients who have previously received CD79b ADC and / or anti-CD20 (e.g., PV and / or Rituximab treatment) but are intolerant to the treatment or have relapsed;

[0029] - Can be used in patients who are insensitive to or resistant to CD79b-targeted drugs (e.g., CD79b ADCs); and

[0030] - Can be used for patients with Richter conversion. Attached Figure Description

[0031] 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 presently 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.

[0032] Figures 1A and 1B show the binding of anti-CD20 antibody to CD20 on Raji cells (Figure 1A) and Daudi cells (Figure 1B) as determined by FACS, respectively.

[0033] Figures 2A and 2B show the binding of anti-CD20 antibody to HEK293-cynoCD20 cells expressing monkey CD20 as determined by FACS (Figure 2A) and to HEK293 cells not expressing CD20 (Figure 2B), respectively.

[0034] Figure 3 shows the binding of humanized anti-CD20 antibody and its parent antibody to CD20 on Daudi cells as determined by FACS.

[0035] 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.

[0036] Figures 5A and 5B show the endocytosis of anti-CD20 antibodies as measured on Ramos cells (Figure 5A) and Daudi cells (Figure 5B), respectively.

[0037] Figure 6 shows the endocytosis based on rProtein G-vc-MMAE kill.

[0038] Figure 7 shows a schematic diagram of the structure of a multispecific antibody.

[0039] 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.

[0040] Figures 9A, 9B, and 9C show the binding of multispecific antibodies and their parent antibodies to Ramos (Figure 9A), the binding of multispecific antibodies and their parent antibodies to JEKO-1 (Figure 9B), and the binding of multispecific antibodies and their parent antibodies to HEK293 (Figure 9C), respectively.

[0041] Figures 10A and 10B show the binding of multispecific antibodies and their parent antibodies to human CD20 (Figure 10A) and the binding of multispecific antibodies and their parent antibodies to monkey CD20 (Figure 10B), respectively.

[0042] Figure 11 shows that the multispecific antibody V-F2 binds to both CD20 and CD79b simultaneously.

[0043] 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.

[0044] Figures 13A and 13B show Ramos in vitro killing effect after 5 days (Figure 13A) and 6 days (Figure 13B), respectively.

[0045] Figures 14A, 14B, and 14C show the in vitro killing effects 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.

[0046] 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 changes in body weight of the Ramos-CDX model mice (Figure 15C), respectively.

[0047] 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 changes in body weight of mice in the WSU-DLCL2-CDX model (Figure 16C), respectively.

[0048] Figures 17A, 17B, and 17C show the antitumor efficacy of the Ramos-CDX model (Figure 17A), the tumor volume of Day 14 mice in the Ramos-CDX model (Figure 17B), and the changes in body weight of the Ramos-CDX model mice (Figure 17C), respectively.

[0049] Figures 18A, 18B, and 18C show the antitumor efficacy of the WSU-DLCL2-CDX model (Figure 18A), the tumor volume of WSU-DLCL2-CDX model mice on Day 40 (Figure 18B), and the changes in body weight of WSU-DLCL2-CDX model mice (Figure 18C), respectively.

[0050] Figures 19A, 19B, and 19C show the antitumor efficacy of the SU-DHL-8-CDX model (Figure 19A), the tumor volume of Day 16 mice in the SU-DHL-8-CDX model (Figure 19B), and the changes in body weight of mice in the SU-DHL-8-CDX model (Figure 19C), respectively.

[0051] Figures 20A, 20B, and 20C show the antitumor efficacy of the SU-DHL-2-CDX model (Figure 20A), the tumor volume of Day 18 mice in the SU-DHL-2-CDX model (Figure 20B), and the changes in body weight of mice in the SU-DHL-2-CDX model (Figure 20C), respectively.

[0052] Figures 21A, 21B, and 21C show the antitumor efficacy of the RT-PDX model (Figure 21A), the tumor size of Day 24 mice in the RT-PDX model (Figure 21B), and the changes in body weight of mice in the RT-PDX model (Figure 21C), respectively.

[0053] Figures 22A and 22B show the in vitro killing effect of ADC drugs on T cells and NK cells in PBMCs from normal healthy individuals. 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).

[0054] Invention Details

[0055] 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 become apparent from this specification and the accompanying drawings, and from the appended claims.

[0056] definition

[0057] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, 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 restrictive.

[0058] The term "about" when used in conjunction with a numerical value means to encompass a range of numerical values ​​having a lower limit that is 5%, 4%, 3%, 2%, or 1% smaller than the specified numerical value and an upper limit that is 5%, 4%, 3%, 2%, or 1% larger than the specified numerical value. When used in conjunction with a numerical range, it means to encompass a range consisting of a numerical range whose upper limit is 5%, 4%, 3%, 2%, or 1% smaller than the specified numerical value and a numerical range whose upper limit is 5%, 4%, 3%, 2%, or 1% larger than the specified numerical value, and a numerical range consisting of a numerical range whose lower limit is 5%, 4%, 3%, 2%, or 1% smaller than the specified numerical value and a numerical range whose lower limit is 5%, 4%, 3%, 2%, or 1% larger than the specified numerical value.

[0059] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0060] In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations where the variable region consists of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region that “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0061] When used with antigens, the terms "binding molecule" and "antigen-binding molecule" are used interchangeably, referring to a protein or polypeptide molecule that can specifically bind to an antigen or an epitope on an antigen. A binding molecule has "affinity" and / or "specificity" towards the antigen. Therefore, a CD79b binding molecule refers to a protein or polypeptide that specifically binds to CD79b, a CD20 binding molecule refers to a protein or polypeptide that specifically binds to CD20, and a CD79b and CD20 binding molecule refers to a protein or polypeptide that specifically binds to both CD79b and CD20. Examples of binding molecules include antibodies, antibody fragments, fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.

[0062] The domain in an antigen-binding molecule that actually binds to the antigen is referred to in this paper as the "antigen-binding site" or "antigen-binding domain". A "domain," as a folded structure in a protein or polypeptide, generally governs a single function of the protein or polypeptide. For example, conventional antibodies and immunoglobulins typically form an antigen-binding domain on the surface of a 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 between the antibody and the antigen. However, in some cases, a single immunoglobulin variable domain (e.g., the heavy chain variable domain (VH) or light chain variable domain (VL), and the heavy chain variable domain (VHH) derived from camel heavy chain antibodies, can confer antigen binding. That is, this single variable domain does not need to interact with another variable domain and can independently function as an "antigen-binding domain" for recognizing and binding the target antigen. 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 other domains of the protein or peptide. Therefore, various proteins and peptides constructed using antibody-containing antigen-binding sites are a class of antigen-binding molecules particularly considered in this disclosure.

[0063] The terms “binding” and “specific binding” are used interchangeably in this disclosure, meaning that the binding is selective for the antigen and can be distinguished from unwanted or nonspecific 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 K is detected by the SPR technique described in the examples. D .

[0064] The term "antibody" is used in the broadest sense herein to refer to a protein containing an antigen-binding site of an immunoglobulin, encompassing a wide range of natural and artificial antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies, monoepitope and polyepitope 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.

[0065] The terms "antibody fragment" or "antigen-binding fragment" of an antibody are used interchangeably to refer to a molecule distinct from the 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, 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 contain 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.

[0066] In this text, "monospecific" refers to the ability to bind to only one epitope. In contrast, the term "multispecific" refers to the ability to bind to multiple different epitopes. Accordingly, "bispecific" refers to the ability to bind to two different epitopes. These different epitopes can be different epitopes on different antigens or different epitopes on the same antigen.

[0067] In this article, 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 with 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.

[0068] 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.

[0069] In this paper, 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 B cells in peripheral blood or lymphoid organs, expressing itself 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 candidate for targeted therapy of B-lymphocyte tumors. In this paper, 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 and conformational variants, and encompasses its species homologs. In some contexts, 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 UniProtKB-P11836. In this disclosure, unless otherwise specified, the term "antigen-binding specificity against CD20," i.e., "antigen-binding domain that specifically binds to CD20," refers to binding specificity against human CD20.

[0070] 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 full-length 229-amino acid transmembrane protein 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, as well as 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 UniProtKB-P40259. In this disclosure, unless otherwise specified, the term "antigen binding specificity against CD79b," that is, "antigen binding domain that specifically binds to CD79b," refers to the binding specificity against human CD79b.

[0071] In this document, the term "CD20-positive" cell refers to a cell that is positive for CD20 expression on its cell surface. The term "CD79b-positive" cell refers to a cell that is positive for CD79b expression on its cell surface. The term "CD20 and CD79b-positive" cell refers to a cell that is positive for both CD20 and CD79b expression on its 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 cell refers to a positive tumor cell expressing CD20, CD79b, or both.

[0072] In this paper, the term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigenic epitope). In this context, "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 commonly known in the art, such as surface plasmon resonance (SPR) techniques.

[0073] In this paper, the term "affinity" or "binding affinity" refers to the combined strength of interactions between multiple binding sites of a molecule (e.g., an antibody) and the same target. Therefore, a necessary condition for affinity is the multivalent nature of the molecule (e.g., an antibody) to a target.

[0074] In this article, the term "immunoglobulin" refers to a protein with a structure that contains 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. Each immunoglobulin heavy chain has a heavy chain variable region (VH), also called a heavy chain variable domain, from the N-terminus to the C-terminus, followed by a heavy chain constant region consisting of three heavy chain constant domains (CH1, CH2, and CH3). Each immunoglobulin light chain has a light chain variable region (VL), also called a light chain variable domain, from the N-terminus to the C-terminus, followed by a light chain constant region consisting of a light chain constant domain (CL). The heavy chains of immunoglobulins can be classified into one of five categories based on the type of their constant regions, referred to as 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). The light chains of immunoglobulins can also be classified into one of two types based on the amino acid sequence of their constant domains, referred to as κ and λ. In some embodiments of the antibodies according to the invention having antibody constant regions, the constant regions refer to the constant regions of immunoglobulins or sequence variants thereof.

[0075] In this paper, the term "variable region" or "variable domain" refers to the 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 sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. One or more residues in the variable region of an antibody can be modified, for example, by modifying one or more CDR regions and / or one or more framework regions, particularly by substituting conserved residues, to obtain antibody variants that still substantially retain at least one biological property (e.g., antigen-binding ability) of the parent antibody. Furthermore, the antibody variable region can be modified by 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 residue mutations, such as reversion mutations, can be made as needed 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 druggability. To this end, the variable domains of antibodies can be engineered to construct humanized, deimmunogenic, and / or PTM (post-translational modification) removed 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 any such functional variants of any variable regions (e.g., VH and / or VL regions, VHH regions) presented herein are within the scope of this invention.

[0076] In this paper, the terms "complementarity-determining region" and "CDR region," "CDR," and "hypervariant region" are used interchangeably. They refer 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 specific 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, to define the extent of the CDR. 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 the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chothia structural loops, used by the AbM antibody modeling software from Oxford Molecular. “Contact” HVR is based on the analysis of the available crystal structure of the complex.

[0077] The following are examples of CDR region ranges defined using the Kabat, AbM, IMGT, and Contact schemes.

[0078] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the above-described methods and combinations thereof. Furthermore, a CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., an exemplary CDR of this invention). Moreover, it is understood in the art that although CDRs differ between antibodies, only a limited number of amino acid positions within a CDR directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, and Contact methods, a minimal overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. Such a minimum binding unit can 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 antibody's structure and protein folding. Therefore, the invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues may be substituted.

[0079] In this article, unless otherwise stated, references to the positions of residues in the antibody variable region and CDR refer to the positions numbered 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)).

[0080] 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 in camelid species (e.g., camels, alpacas, dromedaries, llamas, and guanacos). Other species besides camelids may also produce naturally occurring heavy chain antibodies lacking a light chain, and such VHHs are also within the scope of this invention. Structurally, a VHH is a single-chain antibody fragment comprising FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1 from the C-terminus to the N-terminus. Unlike variable domains derived 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, using the VHH domain (alone, or as part of a larger polypeptide) to recognize and bind to the target antigen offers many significant advantages over using conventional VH and VL domains, scFv, or conventional antibody fragments (e.g., Fab or F(ab')2 fragments):

[0081] - Only a single domain is needed to bind to the antigen with high affinity and high selectivity, so that there is no need for two separate domains, nor is it necessary to ensure that the two domains exist in the appropriate spatial conformation and configuration (for example, scFv generally requires the use of specially designed adapters).

[0082] -VHH domains can be easily modified into multivalent and multispecific formats;

[0083] - The VHH domain is highly soluble and has no tendency to aggregate;

[0084] The -VHH domain is highly stable to heat, pH, protein or peptide enzymes, and other denaturing agents or conditions.

[0085] -VHH domains are easy to prepare and relatively inexpensive;

[0086] The -VHH domain is relatively small compared to conventional tetrapeptide chain antibodies and their antigen-binding fragments, thus exhibiting higher tissue penetration and allowing for relatively high doses of administration.

[0087] - The VHH domain can exhibit so-called cavity binding properties (compared to the conventional VH domain, the VHH has an extended CDR3 loop, thus reaching target epitopes that are inaccessible to conventional tetrapeptide antibodies and their antigen-binding fragments).

[0088] VHHs include humanized VHHs, camel-derived VHHs, or VHHs obtained through affinity maturation. VHH sequences may comprise fully human sequences, humanized sequences, chimeric sequences, or sequences optimized in other ways. Further descriptions 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 a VHH domain. This VHH domain is also referred to herein as anti-CD20 VHH or VHH. CD20 .

[0089] 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 the pairing of 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 the pairing of VH-CL and 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 herein as the light chain of the Fab domain. In some embodiments, the CD79b antigen-binding site of the antigen-binding molecule and antibody according to the invention is preferably provided by the Fab domain. This Fab domain is also referred to in this disclosure as the CD79b-resistant Fab or Fab. CD79b .

[0090] 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 molecules and antibodies of the present invention comprising anti-CD79b Fab, the antigen-binding molecules and antibodies comprise an anti-CD79b scFv domain as an alternative to the Fab domain.

[0091] The term "immunoglobulin Fc region," used interchangeably with "Fc region" and "Fc domain" herein, defines the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the heavy chain constant region. The terms "Fc region" or "Fc domain" do not include the heavy chain variable region VH and light chain variable region VL, or the heavy chain constant region CH1 and light chain constant region CL; however, they 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 from 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) of the Fc region may or may not be present. The sequence constituting the Fc region may be a native or variant sequence. Therefore, the term "Fc region" encompasses both the natural sequence Fc region and the variant Fc region.

[0092] In this paper, 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 subclasses 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.).

[0093] In this document, the term "variant sequence Fc region" refers to a polypeptide containing a modified Fc region relative to the native Fc region sequence. The modification can be the addition, deletion, and / or substitution of amino acid residues. Substitution can include naturally occurring amino acid substitutions and non-naturally occurring amino acid substitutions. The purpose of the 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.

[0094] In this paper, 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.

[0095] In this document, the term "effective 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 can be selected and / or modified to give it effector functions appropriate to said use, such as attenuated, reduced, or eliminated Fcγ receptor binding, ADCC activity, and / or CDC activity compared to the wild-type IgG1 Fc region.

[0096] 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.

[0097] In this document, the “percentage of identity (%)” for 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 aligned specific amino acid sequence shown in this disclosure, after aligning the candidate sequence with the specific amino acid sequence shown in this disclosure and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. In some embodiments, this disclosure contemplates variants of the antibody sequences of the 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. In this document, if no comparison window (i.e., the antibody region of interest to be compared) is specified, the alignment is performed over the full length of the reference antibody sequence. In some embodiments, the variants may contain conserved modifications.

[0098] 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)).

[0099] In this paper, the term "chimeric antibody" refers to an antibody in which one part (e.g., the variable region sequence) comes from one species and another part (e.g., the constant region sequence) comes from another species, such as an antibody in which the variable region sequence comes from a mouse antibody and the constant region sequence comes from a human antibody.

[0100] In this document, a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from nonhuman CDRs and amino acid residues from human FRs. In some embodiments, all or substantially all of the CDRs (e.g., CDRs) in a humanized antibody correspond to those in nonhuman antibodies, and all or substantially all of the FRs correspond to those in human antibodies. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. The “humanized form” of an antibody (e.g., a nonhuman antibody) refers to an antibody that has been humanized. In some embodiments, the humanized antibody of the present invention has a framework region sequence “derived” from a specific human lineage sequence. Here, “derived” means that the amino acid sequence of the antibody framework region has at least 85% or 90% identity with the corresponding framework region amino acid sequence encoded by the human lineage immunoglobulin gene, and that the antibody retains antigen-binding activity.

[0101] In this document, an antibody is described as "cross-reactive" to two different antigens or antigenic determinants if its 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). Antibodies exhibiting human-monkey species cross-reactivity, particularly having similar human-monkey antigen-binding affinities, 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 exhibit human-monkey species cross-reactivity.

[0102] 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 separated from components in the natural environment in 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).

[0103] In this document, the term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including progeny cells of this type. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny. Host cells can be any type of cell system that can be used to produce the antibody molecules of this invention, including eukaryotic cells, such as mammalian cells, insect cells, and yeast cells; and prokaryotic cells, such as *E. coli* cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0104] 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 a cis-acting element for the expression of said nucleotide sequence; other elements for expression may be provided by a host cell or by an in vitro expression system. Expression vectors include, for example, but not limited to, entrapments, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0105] 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 translocated to a suitable intracellular compartment, triggered by the binding of a ligand to a corresponding receptor on the cell surface. In some embodiments, the antibody of the present invention initiates 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, for example, by the methods described in the examples, 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.

[0106] The term "conjugate" or "coupler" herein refers to a molecule formed by conjugating one or more immunoglobulin-associated molecules or fragments thereof with one or more other molecules. A conjugate typically contains at least one non-protein chemical structural part, such as a chemical linker for achieving the conjugation. In some cases, the other molecules may be the same as immunoglobulin-associated molecules or fragments thereof. In some cases, the other molecules may be different from immunoglobulin-associated molecules or fragments thereof. The one or more additional molecules may be the same as 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 (e.g., immunotherapeutic agents), radioactive elements, probes, or signaling molecules.

[0107] "Antibody-drug conjugate (ADC)" refers to a compound obtained by linking an antibody or antigen-binding molecule to a (small molecule) drug 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 equivalents. The drug compound portion of an ADC may be referred to herein as the "payload" or "toxin."

[0108] The term "linker-payload" refers to a compound formed by the connection of a payload and a linker. In some cases, linker-payloads are used as intermediates in ADC synthesis.

[0109] 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. 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 enabling it to break and release the payload after the ADC is delivered to the target region (e.g., a 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.

[0110] The term "therapeutic agent" encompasses any substance that is effective in preventing or treating diseases such as cancer, including chemotherapeutic agents, cytotoxic agents, immunomodulators (such as immunosuppressants), other antibodies, small molecule drugs, angiogenesis inhibitors, or cytokines.

[0111] The term "drug" refers to a compound that can regulate biological processes, particularly altering or preventing pathological processes. In this article, "drug" preferably refers to antitumor compounds.

[0112] The term "small molecule drug" refers to a low molecular weight drug that can regulate 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 penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0113] "Antitumor compounds" are pharmaceutically active compounds that have an effect on tumors, including but not limited to cytotoxic agents or chemotherapeutic agents, such as the cytotoxic agents disclosed in WO2021 / 173773 and US5658920, camptothecin compounds such as eczetidine and Dxd (Exatecan derivatives), and auristatin compounds such as monomethyl auristatin E (MMAE) and MMAF.

[0114] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0115] "Chemotherapy agents" include chemical compounds that are useful in treating cancer or immune system diseases.

[0116] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. Alkyl groups preferably contain 1-16 carbon atoms, for example, 1-12 carbon atoms, 1-10 carbon atoms, 1-6 carbon atoms, or 1-4 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.

[0117] The term “halogen” or “halogenated” refers to fluorine (-F), chlorine (-Cl), bromine (-Br), or iodine (-I).

[0118] The term "haloalkyl" refers to an alkyl group as defined herein, which is substituted with one or more halogen groups. Haloalkyl groups may preferably be monohaloalkyl, dihaloalkyl, or polyhaloalkyl (including perhaloalkyl). Monohaloalkyl groups may contain one iodine, bromine, chlorine, or fluorine group 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 at most 12, 10, 8, 6, 4, 3, or 2 halogen groups. Non-limiting examples of haloalkyl 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.

[0119] The terms "alkoxy" and "alkyl-O-" are used interchangeably to refer to an alkyl group as defined above, linked by an oxygen atom. Preferably, the alkoxy group has 1-8 carbon atoms (C... 1-8 alkoxy group), 1-6 carbon atoms (C 1-6 alkoxy group), 1-4 carbon atoms (C 1-4 alkoxy group or 1-3 carbon atoms (C 1-3 Alkoxy groups. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), butoxy (including n-butoxy, sec-butoxy, isobutoxy, tert-butoxy, etc.), pentoxy (including n-pentoxy, isopentoxy, neopentoxy, etc.), hexoxy, heptoxy, octoxy, etc.

[0120] 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.

[0121] The terms "pentose" or "hexose" refer to polyhydroxy aldehydes or ketones with 5 or 6 carbon atoms, respectively. Pentoses and hexoses applicable to this invention include, but are not limited to, ribose, rhamnose, arabinose, xylose, glucose, lythose, mannose, and galactose.

[0122] The term "penturonic acid" refers to compounds formed by oxidizing the primary hydroxyl group of a pentose sugar as defined above to a carboxyl group. Examples of penturonic acids include, but are not limited to, xyuronic acid and arabinuronic acid.

[0123] The term "hexuronic acid" refers to compounds 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.

[0124] The term "optional" or "optionally" means that the event or condition described below either occurs or does not occur, and the description includes instances where the event or condition occurs as well as instances where the event or condition does not occur. For example, when a group or structure is "optionally substituted," the group or structure may or may not be substituted.

[0125] In this article, "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 contradiction in the context, "pharmaceutically acceptable" and "medicinal" are used interchangeably.

[0126] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the ADC conjugates of the present invention, and that such salt is not biologically or otherwise undesirable. The ADC conjugates of the present invention can exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the ADC conjugates of the present invention with an organic or inorganic acid, 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.

[0127] The term "solvent" refers to an association formed by one or more solvent molecules with the ADC antibody-drug conjugate of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N,N-dimethylformamide, dimethyl sulfoxide, etc.

[0128] The term "drug:antibody ratio" or "DAR" refers to the ratio of the drug portion (D) coupled to the Ab portion described herein to the Ab portion. 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. The DAR may also be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug portion (D) coupled to the Ab portion described herein to the Ab portion 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 that contains ADC molecules having the same and / or different DAR values.

[0129] The term "half-maximum effective concentration (EC50)" 50 "EC" refers to the concentration of a drug, antibody, ADC, or toxicant that induces a 50% response between baseline and maximum after a specific exposure time. In the context of this application, EC 50 The unit is “nM”.

[0130] 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 which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0131] The term "pharmaceutical excipient" refers to diluents, adjuvants (such as Freund's adjuvants (complete and incomplete)), carriers, or stabilizers that are applied together with the active substance.

[0132] 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 antibodies, antigen-binding molecules, or ADC molecules of the present invention used in the drug combination are administered at levels not exceeding those achieved 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 components produces an effect greater than that achieved 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.

[0133] 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 being.

[0134] The terms “tumor” and “cancer” are used interchangeably in this document to refer to a physiological disorder in mammals in which cell growth is not regulated, encompassing both solid and liquid tumors, as well as malignant and benign tumors, and all precancerous and cancerous cells and tissues.

[0135] In this article, 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, symptom relief, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or mitigation of the disease state, and relief or improvement of prognosis. In cases involving tumor or cancer treatment, "treatment" encompasses antitumor biological effects that can be induced by artificial intervention (e.g., through the administration of drugs), including but not limited to, reductions in tumor volume, number of tumor cells, proliferation, or survival.

[0136] 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 implementations, 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 onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0137] 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, when 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 "preventive effective amount".

[0138] The term "therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. Therapeutic effective doses of antibodies or ADCs can vary depending on various 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 therapeutic 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 "therapeutic 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%. The ability of a compound to inhibit measurable parameters (e.g., cancer) can be evaluated in animal model systems that predict efficacy in human tumors.

[0139] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered in subjects before or at an early stage of the disease, the prophylactic effective dose is less than the therapeutic effective dose.

[0140] The term "antitumor effect" refers to biological effects that can be demonstrated through a variety of means, 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.

[0141] The present invention will now be described in detail. Those skilled in the art will understand 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 this invention.

[0142] I. First aspect of this disclosure: The multispecific antibody of the present invention

[0143] In a first aspect, this disclosure provides a multispecific antibody that specifically binds to B cell-associated antigens CD79b and CD20. The multispecific antibody of this invention can specifically target CD79b-positive, CD20-positive, and CD79b-and CD20-positive tumor cells, achieving effective tumor killing; and can deplete B cells, improving B cell-related autoimmune diseases. In some embodiments, the multispecific antibody according to the 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 invention further comprises an immunoglobulin Fc region. The multispecific antibody of the 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.

[0144] 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 this 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) may contain any such combination of features.

[0145] Antigen-binding domain

[0146] In their research, the inventors discovered that, in some cases, it is advantageous to administer multispecific antibodies that simultaneously target CD79b and CD20 for the treatment of B-cell-associated lymphoid tumors and leukemia. Such multispecific antibodies can accommodate the heterogeneity of CD79b and CD20 expression on different malignant B cells, expanding the patient applicability of the antibody. Furthermore, targeting one specificity of the antibody to CD20 can promote the specific binding of the antibody and antibody-based ADCs to B tumor cells; while targeting the other specificity of the antibody to the CD79b surface receptor can promote the endocytosis and degradation of the antibody and antibody-based ADCs. Simultaneously, the dual specificity of the antibody can provide a mechanism against tumor resistance. In addition, the inventors also found that using the VHH domain, which has high CD20 cell-binding affinity, as the source of antibody CD20 specificity can significantly enhance endocytosis mediated by the antibody's CD79b binding arm, especially on tumor cells with low CD79b expression.

[0147] In some embodiments, this disclosure 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.

[0148] 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 trivalent-pentavalent 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 bivalent, and the CD20 binding domain is bivalent. In some embodiments, the antibody is a bispecific antibody against CD79b and CD20.

[0149] In some embodiments, the multispecific antibody according to the present invention has antigen-binding activity selected from one or more of the following:

[0150] (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;

[0151] (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. 50The value binds to CD20-expressing cells.

[0152] In some embodiments, the multispecific antibody according to the present invention exhibits excellent antitumor activity and safety properties. In some embodiments, the multispecific antibody according to the present invention also exhibits excellent drug-like properties.

[0153] Anti-CD20 antigen binding domain

[0154] In some embodiments of the multispecific antibody according to the present invention, the CD20 binding domain is provided by a VHH domain. Some exemplary VHH sequences suitable for 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.

[0155] In some embodiments, the CD20 binding domain according to the invention comprises the CDR1, CDR2 and CDR3 sequences contained in any of the VHH sequences shown in SEQ ID NOs: 1 and 5-13.

[0156] The CDR sequence in the CD20 binding domain according to the present invention can 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 can also be defined in any other manner known in the art.

[0157] In some embodiments, the CD20 binding domain according to the invention comprises CDR1, CDR2, and CDR3. In some embodiments, CDR1, CDR2, and CDR3: (i) comprise or consist of the amino acid sequences of SEQ ID NOs:2,3, and4, respectively; or (ii) comprise or consist of the amino acid sequences of SEQ ID NOs:14,3, and4, respectively. In some embodiments, the CD20 binding domain according to the invention as defined in (i) is preferred.

[0158] Based on the CDR region described above in this invention, different framework regions can be selected to construct various anti-CD20 binding domains of this invention that are equally effective at binding CD20. Therefore, the CD20 binding domains applicable to this invention include not only anti-CD20 binding domains having the VHH sequence listed in 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.

[0159] 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) added, deleted and / or substituted amino acids, or is composed of the above.

[0160] In some preferred embodiments, the CD20 binding domain according to the invention comprises a VHH domain, wherein the VHH domain comprises, or is composed of, one of the amino acid sequences of SEQ ID NOs:1 and 5-13.

[0161] In some preferred embodiments, the CD20 binding domain according to the invention comprises a VHH domain, wherein the VHH domain comprises or is composed of the amino acid sequence of SEQ ID NO:6.

[0162] Anti-CD79b antigen-binding domain

[0163] In some embodiments of the multispecific antibody according to the 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.

[0164] Some exemplary combinations of VH and VL sequences applicable to the anti-CD79b binding domain of the present invention include, but are not limited to, combinations of SEQ ID NO: 16 and 15.

[0165] In some embodiments, the CD79b binding domain according to the present invention includes 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.

[0166] The CDR sequence in the CD79b binding domain according to the present invention can 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.

[0167] In some embodiments, the CD79b binding domain according to the present invention comprises HCDR1-3 and LCDR1-3, wherein HCDR1-3 and LCDR1-3 comprise or are composed of the amino acid sequences of SEQ ID NOs:20-22 and SEQ ID NOs:17-19, respectively.

[0168] Since the antigen-binding properties of antibodies are primarily governed by the CDR sequence, different framework regions can be selected based on the CDR region described above to construct various anti-CD79b binding domains of this invention that are equally effective at binding CD79b. Therefore, the CD79b binding domains applicable to this invention include 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 framework region sequences as the VH and VL sequence combinations.

[0169] In some embodiments, the CD79b binding domain according to the invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0170] (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 comprising, or consisting of, one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids; and / or

[0171] (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 having one or more (preferably 1-10, more preferably 1-5) amino acid additions, deletions and / or substitutions, or is composed of the amino acid sequence.

[0172] 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:

[0173] (a) The heavy chain variable region comprises or is composed of the amino acid sequence of SEQ ID NO:16; and / or

[0174] (b) The light chain variable region contains or is composed of the amino acid sequence of SEQ ID NO:15.

[0175] 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.

[0176] 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 consisting of a heavy chain variable region and an immunoglobulin CH1 domain) and (2) a VL-CL chain (i.e., a chain consisting of a light chain variable region and an immunoglobulin light chain constant domain CL). In other embodiments, the Fab domain comprises (1) a VH-CL chain (i.e., a chain consisting of a heavy chain variable region and a CL domain) and (2) a VL-CH1 chain (i.e., a chain consisting of a light chain variable region and a 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 human CH1 domain sequence.

[0177] 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:

[0178] (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 an amino acid sequence comprising, or consisting of, one or more (preferably 1-10, more preferably 1-5) added, deleted, and / or substituted amino acids; and / or

[0179] (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 having one or more (preferably 1-10, more preferably 1-5) added, deleted and / or substituted amino acids, or is composed of the amino acid sequence.

[0180] Immunoglobulin Fc region

[0181] In some embodiments, the multispecific antibody according to the present invention may further comprise an immunoglobulin Fc region in addition to the aforementioned antigen-binding domain. In other embodiments, alternatively, it may comprise a half-life extension domain, such as serum albumin or a serum albumin-binding peptide, to adjust the circulating half-life of the antibody after administration to animals. In some embodiments, preferably, the antibody of the present invention comprises an immunoglobulin Fc region.

[0182] The immunoglobulin Fc region used in the multispecific antibody of the present invention can be an Fc region derived 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 comprises a hinge region or a partial hinge region. In some embodiments, the immunoglobulin Fc region comprises, or consists of, an immunoglobulin hinge region or a partial hinge region, a CH2 domain, and a CH3 domain from the N-terminus to the C-terminus. In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, or IgG4, or a subtype thereof. Preferably, the immunoglobulin Fc region comprises an Fc region sequence derived from humans.

[0183] 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 peptide linkers, or directly to other domains. Fusion at the N-terminus of the immunoglobulin Fc region is preferably carried out via the immunoglobulin hinge region. Fusion at the C-terminus of the immunoglobulin Fc region is preferably carried out via a flexible peptide linker.

[0184] In some cases, it is advantageous for the Fc region of an immunoglobulin to include a hinge region sequence, which can, for example, facilitate the dimerization of antibody polypeptide chains 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 a core hinge region and, optionally, all or part of an 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 sequences E216 to T225 from IgG1 (according to EU designations), or corresponding hinge region sequences from other immunoglobulin isotypes.

[0185] The immunoglobulin Fc region of the multispecific antibody of the present invention can be the native Fc region sequence. Alternatively, the Fc region can contain mutations relative to the native 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, in the specific application of the antibody of the present invention or an antigen-binding molecule based on said antibody, the Fc region can contain mutations that alter effector function. In some embodiments, said 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 for coupling with 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.

[0186] In some embodiments of the multispecific antibody according to the present invention, the antibody comprises an immunoglobulin Fc region, wherein the immunoglobulin Fc region has one or more of the following characteristics:

[0187] (i) The Fc region contains mutations that reduce or eliminate the binding of the Fc region to FcγR, for example, L234A or L235A mutations.

[0188] (ii) The Fc region is of the IgG type, such as the IgG1 or IgG4 isotype; and / or

[0189] (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.

[0190] peptide linkers

[0191] In the multispecific antibody according to the invention, antibody components (i.e., the antigen-binding domain and optionally the immunoglobulin Fc or half-life-binding domain) can be linked using peptide linkers. There are no specific limitations on the peptide linkers that can be used in the antibodies of the invention. Peptide linkers are generally flexible. They can consist primarily of amino acids with large side chains that do not limit flexibility, such as glycine, alanine, and serine. Alternatively, they can consist of sequences from the hinge region of immunoglobulins. Depending on the linking location and the components 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 length of the peptide 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 connective 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:50), where n is an integer equal to or greater than 1; (G4)n (SEQ ID NO:49); G(G4S)n (SEQ ID NO:50), where n is an integer equal to or greater than 1; (G4)n (SEQ ID NO:49); G4S)n (SEQ ID NO:40 ... 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 linker peptides 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 an 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.

[0192] 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 5-20 amino acids in length, for example, about 10 or 15 amino acids in length. In some embodiments, the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 26 or 27.

[0193] In some embodiments of the multispecific antibody according to the invention, the anti-CD20 domain according to the invention can be linked via a peptide linker to the VH or VL domain of the anti-CD79b Fab domain according to the invention. In some embodiments, the peptide linker can be 5-15 amino acids long, for example, about 5, 10, or 15 amino acids long. In some embodiments, the peptide linker comprises or consists of the amino acid sequence of SEQ ID NO: 26 or 27.

[0194] Structural forms of multispecific antibodies

[0195] Multispecific antibodies (e.g., bispecific antibodies) can be classified into many types based on their different components and construction methods. For example, based on the substantially symmetrical structure of the multispecific antibody, they can be divided into symmetrical and asymmetrical structures; based on the presence or absence of the Fc region of IgG, they can be divided into antibody patterns with and without the Fc region; based on the number of antigen-binding sites in the multispecific antibody, they can be divided into bivalent, trivalent, tetravalent, or more valent antibodies; based on the number of polypeptide chains constituting the multispecific antibody, they can be divided 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 known multispecific antibody structures are all within the scope of consideration of this invention.

[0196] 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.

[0197] In some embodiments, the multispecific antibody according to the present invention comprises:

[0198] (a) Anti-CD79b binding domain and;

[0199] (b) The immunoglobulin Fc region attached to the C-terminus of domain (a); and

[0200] (c) Optionally, at least one (preferably one) CD20 binding domain is attached to the N-terminus of domain (a) or the C-terminus of domain (b) 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.

[0201] In some embodiments, the multispecific antibody according to the present invention comprises:

[0202] (a) Anti-CD79b Fab domain and;

[0203] (b) The immunoglobulin Fc region attached to the C-terminus of domain (a); and

[0204] (c) An anti-CD20 VHH domain is attached to the N-terminus of (a) or the C-terminus of (b) via a peptide linker.

[0205] 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; and 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.

[0206] In some further embodiments, the multispecific antibody according to the invention comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus,

[0207] The first polypeptide chain comprises: VHCD79b -CH1 domain-immunoglobulin Fc region;

[0208] The second polypeptide chain comprises: VL CD79b -CL structural domain;

[0209] VH CD79b and VL CD79b These represent the heavy chain variable region and the light chain variable region that bind to CD79b, respectively.

[0210] The first polypeptide chain is optionally connected to the anti-CD20 VHH domain at the N-terminus or C-terminus via a peptide linker.

[0211] In some further embodiments, the multispecific antibody according to the invention comprises a first polypeptide chain and a second polypeptide chain, wherein, from the N-terminus to the C-terminus,

[0212] The first polypeptide chain comprises: VH CD79b -CH1 domain-immunoglobulin Fc region;

[0213] The second polypeptide chain comprises: VL CD79b -CL structural domain;

[0214] VH CD79b and VL CD79b These represent the heavy chain variable region and the light chain variable region that bind to CD79b, respectively.

[0215] The first polypeptide chain is connected to the anti-CD20 VHH domain at its N-terminus via a peptide linker.

[0216] In some preferred embodiments, the multispecific antibody according to the invention has a symmetrical structure formed by dimerization of the Fc region.

[0217] 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.

[0218] In some embodiments of the multispecific antibody of the present invention having the above-described structural form, preferably, the CD79b binding domain is the CD79b binding domain according to the present invention. More preferably, the CD20 binding domain is the CD20 binding domain according to the present invention.

[0219] Exemplary multispecific antibodies

[0220] In some embodiments, the present invention provides a multispecific antibody, wherein the antibody comprises first and second polypeptide chains, wherein:

[0221] - 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

[0222] - 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.

[0223] Preferably, the first and second polypeptide chains comprise, or consist of, the amino acid sequences of SEQ ID NOs:25 and 24, respectively.

[0224] Preferably, the multispecific antibody has a symmetrical structure formed by dimerization of the Fc region.

[0225] Properties of the multispecific antibodies of this invention

[0226] In some embodiments, the multispecific antibody of the present invention has one or more of the following properties:

[0227] (i) It binds with high affinity to tumor cells expressing CD20 and / or CD79b;

[0228] (ii) It exhibits species cross-reactivity with CD20 of cynomolgus monkeys;

[0229] (iii) Endocytotic activity was observed in tumor cells expressing human CD20 or CD79b and in tumor cells co-expressing human CD20 and CD79b.

[0230] In some respects, the multispecific antibodies of the present invention exhibit high binding affinity to CD20-expressing tumor cells. The cell-binding affinity of the antibodies to CD20-positive tumor cells can be reflected by FACS or ELISA assays (e.g., the assays described in the examples) to determine the EC50 value and / or maximum binding amount, and optionally by comparison with a reference antibody.

[0231] In some aspects, the multispecific antibodies of the present invention exhibit 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 with human CD20 is approximately equivalent to the EC50 value of the binding domain binding with 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.

[0232] In some embodiments, the antibodies according to the invention further have one or more properties selected from the following: compared to single-target antibodies targeting CD20 or CD79b,

[0233] (iv) It has better tumor cell targeting;

[0234] (v) It has higher endocytosis efficiency on tumor cells;

[0235] (vi) It has a higher binding affinity on tumor cells.

[0236] In some further embodiments, the multispecific antibody of the present invention also has one or more of the following characteristics: (1) good developability; (2) good stability; and (3) favorable pharmacokinetic properties.

[0237] II. Second aspect of this disclosure: The antigen-binding molecule of the present invention

[0238] In a second aspect, the present invention provides an antigen-binding molecule comprising an antibody according to a first aspect of this disclosure. In some embodiments, the antigen-binding molecule is a fusion product produced by fusing the antibody of the present invention to a heterologous protein, polypeptide, or peptide.

[0239] In one embodiment of the fusion compound 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. Heterologous 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 compound. For example, a chimeric antigen receptor (CAR) comprising the antibody of the invention or its antigen-binding fragment is an example of an immunofusion compound according to the invention.

[0240] III. The third aspect of this disclosure: nucleic acids, vectors, hosts, and production methods

[0241] In a third aspect, this disclosure provides encoded nucleic acids, vectors, host cells, and methods of production of antibodies and antigen-binding molecules according to the first and second aspects of this disclosure.

[0242] In one embodiment, this disclosure provides a method for preparing the antibody and antigen-binding molecule of the present invention, wherein the method includes: culturing a host cell containing the nucleic acid or an expression vector containing 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 includes recovering the antibody and antigen-binding molecule from the host cell (or host cell culture medium).

[0243] To recombinantly generate the antibody and antigen-binding molecules of the present invention, nucleic acids encoding the antibody and antigen-binding molecules can first be isolated and 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 acids encoding the antibody and antigen-binding molecules of the present invention.

[0244] The antibody and antigen-binding molecules of the present invention, prepared as described herein, can be purified using known prior art techniques such as high-performance liquid chromatography (HPLC), ion-exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography. 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, and HPLC.

[0245] IV. Fourth aspect of this disclosure: Conjugates and antibody-drug conjugates (ADCs)

[0246] In a fourth aspect, this disclosure provides conjugates and antibody-drug conjugates comprising antibodies and antigen-binding molecules according to the first and second aspects of this disclosure.

[0247] Conjugate

[0248] In one embodiment, this disclosure provides conjugates generated by conjugating an antibody and an antigen-binding molecule according to the first and second aspects of this disclosure to a heterologous molecule. In one embodiment, the antigen-binding molecule of the present invention (such as an antibody) is conjugated to a therapeutic agent, diagnostic agent, or detectable agent. In said conjugates, chemical linkers 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 molecule 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.

[0249] In embodiments where a therapeutic agent is conjugated, the therapeutic agent suitable for the conjugation includes, but is not limited to, cytotoxins (e.g., cell growth inhibitors or cell killers), drugs, or radioisotopes.

[0250] In embodiments conjugated with diagnostic or detectable agents, such conjugates can be used as part of clinical testing methods (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 to 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.

[0251] In some embodiments, therapeutic agents suitable for the conjugate include, but are not limited to, drugs (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.

[0252] Antibody-drug conjugates (ADCs)

[0253] In some preferred embodiments, this disclosure provides antibody-drug conjugates (ADCs).

[0254] In some embodiments, this disclosure provides antibody-drug conjugates (ADCs) having formula (I) or pharmaceutically acceptable salts or solvates thereof:

[0255] Ab-(LD) p (I)

[0256] in:

[0257] Ab is the antibody or antigen-binding molecule of the present invention;

[0258] L is the connector;

[0259] D represents a drug, such as an anti-tumor compound; and

[0260] 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.

[0261] In some embodiments, Ab is a multispecific antibody against CD79b and CD20 according to the first aspect of this disclosure, particularly a bispecific antibody. In some particularly preferred embodiments, the multispecific antibody comprises first and second polypeptide chains, wherein:

[0262] (i) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:23; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:24;

[0263] (ii) The first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:25; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:24;

[0264] Preferably, the first polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:25; and the second polypeptide chain contains or is composed of the amino acid sequence of SEQ ID NO:24.

[0265] It is understood that the -LD portion can be covalently linked to the Ab in any manner known in the art. In some embodiments, the -LD portion is covalently linked to the Ab via a sulfur (S) atom from the Ab, i.e., the -LD portion and the Ab are linked via -S-. In some embodiments, the sulfur atom originates from the opening of interchain disulfide bonds in the Ab. In some embodiments, the sulfur atom originates from (engineered or natural) cysteine ​​residues in the Ab.

[0266] It can be understood that p refers to the number of -LDs linked to Ab in the antibody-drug conjugate molecule of formula (I), which can also be called DAR.

[0267] In some embodiments, D in formula (I) of the present invention can be any antitumor compound, as long as it has antitumor effects and has a structural portion that can be attached to the linker, without particular limitation. The antitumor compound can be a pharmaceutically active compound that acts on tumors. For antitumor compounds, it is preferable that part or all of the linker can be cleaved within tumor cells to release the antitumor compound portion, thereby exhibiting an antitumor effect.

[0268] In some implementations, the antitumor compound may be, for example, a cytotoxic agent, such as camptothecin or aurestatin.

[0269] In some implementations, D has the structure shown in formula (D-1a) or formula (D-1b):

[0270] Where R 1a Selected from H and -C1-C6 alkyl groups;

[0271] R 2a Selected from H, halogen, -C1-C6 alkyl, -C1-C6 haloalkyl, -OR 5a and -SR5a ;

[0272] R 3a Selected from H, halogen, CN, -C1-C6 alkyl, -C1-C6 haloalkyl and -OR 5a ;and

[0273] R 4a and R 5a Independently selected from H and -C1-C4 alkyl groups.

[0274] In some implementation schemes, R 1a For H.

[0275] In some implementation schemes, R 2a It is a -C1-C6 alkyl group, preferably a -C1-C4 alkyl group, and more preferably a methyl group.

[0276] In some implementation schemes, R 3a It is a halogen, preferably -F.

[0277] In some implementation schemes, R 4a It is a -C1-C4 alkyl group, preferably ethyl.

[0278] In some implementation schemes, 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.

[0279] In some implementations, D has the structure shown in formula (D-1b).

[0280] Where R 1b R 2b R 3b R 4b R 5b and R 8b Each is independently selected from -C 1-8 Alkyl group; preferably -C 1- C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl;

[0281] R 6b and R 7b Each is independently selected from -C 1- C8 alkoxy groups, such as methoxy, ethoxy, or propoxy groups;

[0282] R 9b Selected from -C 1- C8 alkyl group and -COOH; preferably -C 1-C4 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or sec-butyl; and

[0283] R 10b Selected from -OH and H.

[0284] In some implementation schemes, R 1b R 4b and R 8b Each is independently selected from -C 1- C2 alkyl; preferably methyl.

[0285] In some implementation schemes, R 2b and R 3b Each is independently selected from -C 3- C4 alkyl; preferably isopropyl.

[0286] In some implementation schemes, R 5b -C 3- C4 alkyl; preferably sec-butyl.

[0287] In some implementation schemes, R 6b and R 7b Each is independently selected from -C 1- C2 alkoxy; preferably methoxy.

[0288] In some implementation schemes, R 9b Selected from -C 1- C4 alkyl and R 10b For OH; or R 9b It is -COOH and R 10b For H.

[0289] In some implementation schemes,

[0290] R 1b R 4b and R 8b Each is independently selected from -C 1- C2 alkyl; preferably methyl;

[0291] R 2b R 3b and R 5b Each is independently selected from -C 3- C4 alkyl;

[0292] R 6b and R 7b Each is independently selected from -C 1- C2 alkoxy group; and

[0293] R 9b Selected from -C 1- C4 alkyl and R 10b -OH; or R9b It is -COOH and R 10b For H.

[0294] It can be 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.

[0295] In some implementations, D has the structure shown in formula (D-2a) or formula (D-2b):

[0296] Where R 1a R 2a R 3a and R 4a As defined above;

[0297] Where R 1b R 2b R 3b R 4b R 5b R 6b R 7b R 8b R 9b and R 10b As defined above.

[0298] In some implementations, D has the structure shown in formula (D-3a) or (D-3b):

[0299] In some implementations, D has the structure shown in formula (D-4a) or (D-4b):

[0300] In some embodiments, in the ADC of this disclosure, the drug is Exatecan, Dxd, SN-38, monomethylauratestatin E (MMAE), or MMAF. The structural formula is shown below:

[0301] In some implementations, -L- has the following structure: -Z-L1-L2-L3-

[0302] in

[0303] 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;

[0304] L1 is selected from non-existent, Where n1 and m1 are each 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;

[0305] L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and

[0306] L3 is selected from: Where X is selected from -NH-, -O-, and -S-; R 1c Each is 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

[0307] In this configuration, Z is connected to atoms on Ab, such as S atoms, and L3 is connected to D.

[0308] In some implementations, -L- has the following structure: -Z-L1-L2-L3-

[0309] in

[0310] 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;

[0311] L1 is selected from non-existent, Where n1 is an integer independently selected from 0-12, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0312] L2 is an amino acid residue or a peptide residue consisting of 2-8 amino acids; and

[0313] L3 is selected from: Where R 1c Selected from: -C1-C6 alkyl; n2 is 0, 1, 2, 3 or 4; and n5 is 0, 1, 2 or 3.

[0314] In some implementations, -L- has the following structure: -Z-L1-L2-L3-

[0315] in

[0316] 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;

[0317] L1 is selected from non-existent;

[0318] L2 is an amino acid residue or a peptide residue consisting of 2-4 amino acids; and

[0319] L3 is selected from: Where R 1c Selected from: C1-C6 alkyl; n2 is 0, 1, 2, 3 or 4; and n5 is 0, 1, 2 or 3.

[0320] 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.

[0321] In some implementation schemes, Z is selected from Where m is 1, 2, 3, 4, 5, 6, 7 or 8, for example, an integer from 1 to 5.

[0322] In some implementation schemes, Z is selected from

[0323] In some implementations, L1 is selected from non-existent, Where n1 is an integer independently selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0324] In some implementations, L1 is selected from non-existent,

[0325] In some implementations, L1 is selected as not existing.

[0326] 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.

[0327] 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 structures such as 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.

[0328] In some embodiments, the amino acid residues or amino acids are 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 (Ile), arginine (Arg), proline (Pro), methionine (Met), tryptophan (Trp), cysteine ​​(Cys), histidine (His), and threonine (Thr). In some embodiments, 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). In some embodiments, the amino acid residues or amino acids are each independently selected from glycine (Gly), valine (Val), alanine (Ala), citrulline (Cit), and glutamic acid (Glu).

[0329] In some implementations, L2 is selected from -Ala-, -Val-, -Gly-, -Val-Ala-, -Val-Cit-, -Glu-Val-Cit-, -Gly-Gly-Phe-Gly- (SEQ ID NO:53).

[0330] 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-.

[0331] It should be understood that L2 is connected to L1 or Z through the amino group of the amino acid on the left, and to L3 through the carbonyl group of the amino acid on the right, which is consistent with the explanation below.

[0332] In some implementations, L3 is selected from:

[0333] For example, selected from

[0334] Where R 1c Each is independently selected from C 1-8 Alkyl, C1-8 Haloalkyl-, C 1-8 Alkyl, halogen, nitro, and cyano groups; Su is selected independently from each of the following groups: n2 is 0, 1, 2, 3 or 4; and n5 is 0, 1, 2 or 3.

[0335] In some implementations, L3 is selected from:

[0336] The variables are as defined in this paper.

[0337] In some embodiments, Su is selected from xylose, arabinose, xyuronic acid, arabinuronic acid, glucose, galactose, mannose, glucuronic acid, galacturonic acid, and mannuronic acid.

[0338] In some implementation schemes, Su is selected from

[0339] In some implementations, Su is independently:

[0340] In some implementation schemes, Su is independently

[0341] In some implementation schemes, Su is independently

[0342] In some implementations, L3 is selected from

[0343] It should be understood that L3 is connected to L2 via the amino group on the left and to D via the carbonyl group on the right, which is consistent with the explanation below.

[0344] In some implementations, -Z-L1-L2-L3- are each independently selected from the following structures:

[0345] Where each m is an independent integer selected from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, or 8; and

[0346] The group is connected to an atom on Ab, such as S, on the left side and to D on the right side.

[0347] In some implementations, -Z-L1-L2-L3- are each independently selected from the following structures:

[0348] It should be understood that, unless otherwise specified and without contradiction in the context, for the ADCs of this invention, the left-hand bond of the divalent group shown herein is connected to an Ab group or a group near the Ab end, and the right-hand bond of the divalent group is connected to a D group or a group near the D end. For example, when L2 is In this case, the amino group on the left is connected to L1, and the carbonyl group on the right is connected to L3;

[0349] In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, 2-4, or 2-6.

[0350] In some embodiments, the antibody-drug conjugate is selected from:

[0351] Wherein Ab is the 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, for example 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, 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.

[0352] It should be understood that the S atom linked to the Ab in the above ADC originates from the antibody Ab. The Ab breaks its disulfide bond (e.g., an interchain disulfide bond) under the action of a reducing agent such as TCEP, generating a thiol group (-SH), which then links to the terminal functional group of the linker, such as the maleimide moiety. In some embodiments, the S atom linked to the Ab originates from the cysteine ​​residue of the Ab.

[0353] 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.

[0354] In some embodiments, the antibody-drug conjugate according to the present invention has the following advantages:

[0355] (1) Combined with tumor cells expressing human CD79b or CD20 and tumor cells co-expressing human CD79b and CD20;

[0356] (2) Endocytotic activity was observed in tumor cells expressing human CD79b or CD20 and in tumor cells co-expressing human CD79b and CD20;

[0357] (3) It has a lateral killing effect;

[0358] (4) It has a broad spectrum of antitumor activity and exhibits significant killing activity against various tumors with different CD79b and CD20 expression densities.

[0359] In some embodiments, the antibody-drug conjugate according to the invention also has one or more advantages selected from the following: compared to CD79b or CD20 single-target ADC drugs,

[0360] (5) Better tumor targeting;

[0361] (6) It has higher endocytosis efficiency on tumor cells;

[0362] (7) It has a higher affinity for tumor cells;

[0363] (8) It has a stronger killing effect on tumor cells;

[0364] (9) It has a stronger inhibitory effect on tumor growth; and

[0365] (10) Quite good in vivo safety.

[0366] In some embodiments, the antibody-drug conjugate according to the present invention also has one or more advantages selected from the following:

[0367] (11) It has good product uniformity;

[0368] (12) It has good product stability; and

[0369] (13) It has good drug-like properties.

[0370] Preparation of ADC molecules of the present invention

[0371] Another aspect of the present invention provides a method for preparing an antibody-adjuvant antibody (ADC) using the antibody of the present invention. In this invention, "ADC" is defined as an antibody coupled to an active substance D having biological and pharmaceutical activity via a linker L. The method comprises coupling an antibody (Ab) of the present invention to one or more active substances D via one or more linkers L defined in the present invention. Preferably, the linker-active substance is coupled to the antibody in a site-specific manner.

[0372] 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, as provided above, and optionally recovering the Ab from the host cell (or host cell culture medium).

[0373] In some implementations, the method includes the following steps:

[0374] (a) Add antibody Ab to buffer solution, add reducing agent, and then incubate until the reaction is complete;

[0375] (b) Removal of reducing agent by ultrafiltration;

[0376] (c) Add a connector-load to the reaction solution in step (b) for coupling to obtain the crude product;

[0377] (d) Optionally, the crude product is purified to obtain the antibody-drug conjugate of the present invention;

[0378] Ab is defined as above.

[0379] It should be understood that the connector-load and the reaction of Ab provide the -LD portion in the compound of formula I. Given that -LD is clearly defined, the structure of the connector-load can be determined according to the prior art.

[0380] In some implementations, the buffer solution in step a) is a PBS buffer, preferably with a pH of 5.0-9.0, for example 6.0-8.0.

[0381] In some implementations, the reducing agent in step a) is TCEP.

[0382] In some implementations, the steps are performed under the specific reaction conditions disclosed in the embodiments.

[0383] It should be noted that implementation schemes obtained by varying the range or specific values ​​of the specific reaction conditions disclosed in the embodiments by 100%, 80%, 60%, 40%, 20%, or 10% are also under consideration in this invention.

[0384] V. Fifth aspect of this disclosure: Pharmaceutical compositions and pharmaceutical formulations, as well as combination products and reagent kits

[0385] In some embodiments, this disclosure provides compositions comprising the 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 the 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).

[0386] In some embodiments, the compositions of the present invention are pharmaceutical compositions or pharmaceutical preparations comprising suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. As used herein, "pharmaceutical carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. 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.

[0387] The pharmaceutical compositions or formulations of the present invention may further comprise 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.

[0388] In some embodiments, this disclosure also provides combination products comprising at least one antibody, antigen-binding molecule or ADC of the present invention, or further comprising one or more other antitumor agents.

[0389] In some implementations, two or more components of the combined product may be administered to the subject sequentially, separately, or simultaneously.

[0390] In some embodiments, this disclosure also provides kits comprising the antibodies, antigen-binding molecules, ADCs, pharmaceutical compositions, or combination products of the present invention, as well as optional packaging inserts providing instructions for administration.

[0391] In some embodiments, this disclosure also provides pharmaceutical articles comprising antibodies, antigen-binding molecules, ADCs, pharmaceutical compositions, and combination products of the present invention, optionally including a packaging insert with instructions for administration.

[0392] VI. Sixth aspect of this disclosure: Use and method

[0393] 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 this disclosure, conjugates or antibody-drug conjugates according to the fourth aspect of this disclosure, or pharmaceutical compositions or combinations according to the fifth aspect of this disclosure in the treatment and prevention of CD79b-related diseases and / or cancers and / or CD20-related diseases and / or cancers.

[0394] 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, this disclosure therefore provides the use of the multispecific antibodies, ADCs, or combinations 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 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, 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 antibodies, ADCs, or drug combinations of the present invention to a subject in need.

[0395] 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.

[0396] Since CD79b and CD20 expression covers almost all B-cell-related tumors, this disclosure contemplates the use of the CD79b / CD20 multispecific (bispecific) ADC of the present invention 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 lymphocytic leukemia (CLL). In some embodiments, the cancer described is relapsed or refractory non-Hodgkin lymphoma (rr NHL). In some embodiments, the cancer patient is unresponsive to first-line NHL treatment or has relapsed after treatment; in other embodiments, the cancer patient is unresponsive to CAR-T cell therapy or has relapsed after treatment. In some embodiments, the cancer patient is a newly diagnosed B-cell-related tumor.

[0397] In some embodiments, this disclosure provides the use of the present invention's multi(bi)specific ADCs 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 CD79b-resistant tumor. In some embodiments, the tumor has one or more of the following characteristics:

[0398] (a) Compared with Daudi cells, tumor cells have low CD79b expression levels, for example, less than 50%, 40%, 30%, 20%, 10%, 5% or 2% of the CD79b expression level on Daudi cells;

[0399] (b) Compared with Daudi cells or Ramos cells, tumor cells have upregulated expression and / or activity of the anti-apoptotic gene Bcl-xL;

[0400] (c) Compared to Daudi cells or Ramos cells, tumor cells exhibited upregulated expression and / or activity of the MMAE efflux pump (MDR-1), and

[0401] (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.

[0402] In autoimmune diseases, B cells coordinate antigen presentation, cytokine production, and autoantibody generation, playing a crucial role in the pathogenesis of these diseases. Treatment strategies have been proposed to improve or even cure autoimmune diseases by depleting B cells using antibodies against B cell surface antigens (e.g., anti-CD20 antibodies). See, for example, 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 application, 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.

[0403] B-cell-related autoimmune diseases suitable for the methods and uses of this 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-predictive visual oculoclonus-myoclonus syndrome; and relapsing-remitting multiple sclerosis (RRMS). In some implementations, the autoimmune disease is selected from rheumatoid arthritis, NMDAR encephalitis, idiopathic thrombocytopenic purpura, multiple sclerosis, pemphigus vulgaris, systemic sclerosis, and systemic lupus erythematosus.

[0404] 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 or stops the progression of a 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 already has a disease, an individual susceptible to a disease, or an individual who wishes to prevent a disease. The individual will benefit from the therapeutic or preventative measures and, compared to an individual who has not received the treatment, exhibit a reduction or improvement in the occurrence, recurrence, or development of the disease, condition, symptom, and / or symptoms. In some embodiments, the present invention relates to the treatment of a disease or condition; in other embodiments, the present invention relates to the prevention of a disease or condition.

[0405] The antibodies, antigen-binding molecules, or ADCs according to the invention, as well as the 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, intra-arterial, intraperitoneal, or subcutaneous administration. Various dosing schedules are covered herein, including, but not limited to, single-dose or multiple-dose administration at multiple time points, bolus administration, and pulsatile infusion.

[0406] To prevent or treat a disease, the antibody, antigen-binding molecule, or ADC according to the invention, or the pharmaceutical composition or combination according to the invention, is administered at an appropriate dose, which, 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.

[0407] In some embodiments, this disclosure also provides for the use of the antibodies, antigen-binding molecules, or ADCs of the present invention, or the pharmaceutical compositions or combinations thereof, as medicines or for the preparation of medicines. In some embodiments, the medicine is a medicine for use in the aforementioned treatment and prevention methods.

[0408] 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. Example

[0409] Example Section I. Antibody Preparation and Characterization

[0410] Materials and methods

[0411] Reference antibody and its preparation

[0412] In this embodiment, CD79b reference antibody Polatuzumab and CD20 reference antibodies Rituximab, Ofatumumab, and Obinutuzumab are used.

[0413] 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.

[0414] The 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.

[0415] The 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.

[0416] The above amino acid sequences were optimized according to human codon preference, and the resulting gene was synthesized and subcloned into the pcDNA3.4 vector. After verification by Sanger sequencing, plasmid extraction was performed for later use. These constructed eukaryotic expression vectors were transiently transfected into Expi293F cells. The protein expression supernatant was collected from the transfected cell culture, and the target protein was purified using protein A affinity chromatography. SDS-PAGE and SEC-HPLC experiments were performed to determine protein purity, which was >95%. Aseptic techniques were used throughout the protein purification process, and endotoxin levels were controlled to <5 EU / mg.

[0417] The reference antibody, Rituximab, is a monoclonal antibody targeting CD20. It binds to the CD20 molecule expressed on the surface of B lymphocytes and kills tumor B cells through antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). This reference antibody was purchased from MedChemExpress under the catalog number CAT#HY-P9913.

[0418] Antigens and their preparation

[0419] Considering that CD20 is a transmembrane protein, its antigen may differ from the native structure on the cell membrane surface; therefore, CD20 antigen was not prepared. The extracellular sequence information (Ala 29-Asp 159) of human CD79b (UniProt_P40259-1) was retrieved from the database. A His tag was added to the C-terminus, and the gene was synthesized and subcloned into the pcDNA3.4 vector after optimization according to human codon preferences. After verification by Sanger sequencing, plasmid extraction was performed for later use. HEK293 cells were transiently transfected using the 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 activity of the prepared human huCD79b-His recombinant antigen protein was confirmed by ELISA using the aforementioned reference anti-CD79b antibody (Polatuzumab).

[0420] Recombinant engineered cell lines expressing antigens and their preparation

[0421] 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 (the 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 viruses were packaged in 293T cells and used to infect HEK293 cells. Cells were cultured in medium containing 5 μg / ml puromycin for selection pressure to obtain polyclonal cell lines. FACS analysis using the reference antibody Rituximab showed that the antigen expression positivity rate of stable transfected cell lines was >90%.

[0422] ELISA combination assay

[0423] Dilute the antigen to 1.0 μg / ml with PBS, apply 100 μl / well to each plate, and incubate overnight at 2-8°C or for 2 hours at 37°C. Wash the plate three times with 0.05% PBST, add 200 μl / well of blocking buffer (1% BSA or 8% skim milk powder in PBS), and incubate at room temperature for at least 1 hour. Dilute the sample to be tested to the appropriate concentration with blocking buffer. Wash the plate three times with 0.05% PBST, add 100 μl / well of the diluted sample, and incubate at room temperature for about 1 hour. Wash the plate three times with 0.05% PBST, add 100 μl / well of secondary antibody-HRP diluted to the target dilution, and incubate at room temperature for about 0.5 or 1 hour. Wash the plate three times with 0.05% PBST, add 100 μl / well of TMB, observe the color change, and immediately add 100 μl / well of stop buffer when the color reaches the appropriate level. Read OD450-OD650.

[0424] Direct labeling FACS for detecting tumor cell surface antigen expression abundance

[0425] Target cells were distributed at (1-2) × 10 per well. 5 Cells were seeded at a density of 100 g / well in 96-well plates and centrifuged at 300 g for 5 minutes at 4°C. The direct-labeled antibody was diluted with FACS buffer (1% BSA or 2% FBS in PBS) at the recommended concentration and incubated at 2-8°C for approximately 0.5 hours. After centrifugation at 4°C, the supernatant was removed, and the cells were washed twice with 200 μl / well of FACS buffer and centrifuged again at 4°C. Cells were resuspended in 100 μl / well of FACS buffer and analyzed using flow cytometry (Beckman Coulter). The MFI of cells was measured using a flow cytometer (Beckman Coulter). The ratio of CD20 or CD79b MFI of each target cell to that of the unstained wells was calculated, and the cells were normalized using Daudi cells (i.e., defining the expression levels of CD20 and CD79b on the surface of Daudi cells as 100%). The fold change in the ratio of other target cells to Daudi cells at the corresponding target sites was statistically analyzed.

[0426] FACS Combined Test

[0427] Target cells were distributed at (1-2) × 10 per well. 5 Seed cells at a density of 100 μL / well in 96-well plates and centrifuge at 300 g for 5 min at 4 °C. Add the test sample diluted to the appropriate concentration and incubate at 2-8 °C for approximately 1 h. Centrifuge at 4 °C, remove the supernatant, wash twice with 200 μL / well FACS buffer (1% BSA or 2% FBS in PBS), and centrifuge at 4 °C. Add 100 μL / well of flow cytometry secondary antibody diluted to the target dilution, resuspend the cells, and incubate at 2-8 °C in the dark for approximately 0.5 h or 1 h. Wash twice with 200 μL / well FACS buffer, resuspend the cells in 100 μL / well FACS buffer, and perform flow cytometry analysis. Measure the MFI of the cells using a Beckman Coulter flow cytometer.

[0428] FACS examination of target cross-reactivity in humans and monkeys

[0429] The immunoreactivity of anti-CD20 antibodies (including monospecific and bispecific antibodies) was detected using an engineered HEK293 cell line stably expressing human / monkey CD20 via FACS binding assays. The experimental conditions were as follows: target cells (1 × 10⁻⁶ cells / year)... 5 ( / well) + sample (200nM, 4× dilution, 4℃ 1h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5h). The monospecific and bispecific antibodies of this invention tested showed similar binding activity to human-monkey antigens, exhibiting cross-reactivity with monkeys.

[0430] FACS detection of bispecific antibodies combined with dual-target assay

[0431] CD20-overexpressing cells were used at a density of (1-2) × 10 cells per well. 5 Seed cells at a density of 100 μg / well in 96-well plates and centrifuge at 300 g for 5 min at 4 °C. Add the test sample diluted to the appropriate concentration (200 nM) and incubate at 2-8 °C for about 1 h. Centrifuge at 4 °C, remove the supernatant, wash twice with 200 μl / well FACS buffer (1% BSA or 2% FBS in PBS), and centrifuge at 4 °C. Resuspend cells in 100 μl / well biotinylated CD79b antigen (Acro, CAT#CDB-H82E3) at 5 μg / ml and incubate at 2-8 °C in the dark for about 0.5 h or 1 h. Centrifuge at 4 °C, remove the 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. After washing twice with 200 μl / well FACS buffer, cells were resuspended in 100 μl / well FACS buffer and analyzed by flow cytometry. The MFI of the cells was measured using a Beckman Coulter flow cytometer.

[0432] Internalization test - I (4℃ / 37℃)

[0433] Target cells were distributed at (2-4) × 10 per well. 5Seed cells onto plates, add the test sample diluted to an appropriate concentration, and incubate at 2-8°C for approximately 0.5 hours to allow the test sample to bind to the cells. Centrifuge at 300g for 4 minutes at 4°C, remove the supernatant, and wash the cells 2-3 times with pre-chilled 200 μl / well FACS buffer to remove excess unbound test sample. Resuspend the cells in pre-chilled 200 μl / well FACS buffer. Divide the cells into two groups, with 100 μl / well for each group, and incubate at 4°C and 37°C for 2-4 hours, respectively. Immediately after incubation, add FACS buffer on ice to terminate the endocytosis experiment. Centrifuge at 300g for 4 minutes at 4°C, and wash the cells twice with pre-chilled 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°C in the dark for approximately 30 minutes to 1 hour. Wash cells 2-3 times with 200 μL / well FACS buffer, then resuspend cells in 100 μL / well FACS buffer for flow cytometry analysis. Measure the MFI of cells using a Beckman Coulter flow cytometer. Calculate the internalization level of antibodies bound to the cell surface using the following formula: Absolute internalization = MFI of sample incubated at 4°C - MFI of sample incubated at 37°C. Internalization rate % = 100% - (MFI of sample incubated at 37°C / MFI of sample incubated at 4°C) × 100%.

[0434] Internalization Test-II (rProtein G-vc-MMAE, lethality-based internalization)

[0435] Internal preparation of rProtein G-vc-MMAE (a protein-drug conjugate formed by linking Protein G and MMAE via a valine-citrulline (VC) linker, where protein G can bind to the Fc region of mammalian immunoglobulins). The endocytosis assay method is as follows:

[0436] a) Cell plating (15000 / 100μl / well);

[0437] b) Dilute rProtein G-D4-vc-MMAE to 200 nM with complete culture medium (1640 / 10% FBS), and also dilute the test sample to 200 nM with complete culture medium. Mix the test sample and rProtein G-vc-MMAE at a volume ratio of 1:1 (molar concentration ratio of 1:1) and incubate at room temperature for about 1 hour.

[0438] c) The test sample and rProtein G-vc-MMAE mixture were diluted 1:2.5 with complete culture medium to obtain a total of 9 concentration points (starting concentration of 50 nM);

[0439] d) Add the sequence-diluted sample to a 96-well plate and incubate for 3 days;

[0440] e) Add 20 μl of CCK8 to each well and incubate at 37°C for 2-4 hours. Read OD450-OD650 using a microplate reader.

[0441] Endocytosis Test-III (DT3C, lethality-based endocytosis)

[0442] 1) Collect cells in the logarithmic growth phase. Count, centrifuge, and resuspend the cells in medium containing 20% ​​FBS to the desired concentration (2 x 10⁻⁶). 5 / ml).

[0443] 2) 96-well plate, 2x10 4 Inject 100 μl / well of cell suspension.

[0444] 3) Dilute DT3C (Jiman Biotechnology, GM-046001) and antibody to an appropriate concentration (2x) using serum-free medium, mix according to the ratio, and filter to sterilize. Incubate at 37℃ for 30 min. Serially dilute the mixture with serum-free medium. Add the serially diluted mixture to 96-well plates, 100 μl / well.

[0445] 4) Incubate in an incubator for 48-72 hours. 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 minutes.

[0446] SEC-HPLC

[0447] At ambient column temperature, an appropriate amount of protein sample was loaded onto a TSK-gel G3000SWxL column (Tosoh Corporation) or a Zenix-C SEC-300 column (Sepax Technologies). Using an Agilent 1260 HPLC system, the sample was eluted isocratically for 20 minutes at a flow rate of 0.8 mL / min using a mobile phase consisting of 0.05 M sodium phosphate, 0.3 M sodium chloride, and pH 6.8 ± 0.1. The eluted protein was detected using UV absorbance at 280 nm.

[0448] CEX-HPLC

[0449] 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, mobile phase A (composed of 2-methylpiperazine, imidazole, and Tris, pH 5.0 ± 0.1) and mobile phase B (composed of 100 mM sodium chloride, 2-methylpiperazine, imidazole, and Tris, pH 10.8 ± 0.1) were used for gradient elution at a flow rate of 0.9 mL / min for 70 min. The eluted proteins were detected using UV absorbance at 280 nm.

[0450] HIC-HPLC

[0451] 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, the sample was eluted at a gradient flow rate of 0.8 mL / min for 35 min using mobile phases A and B. Mobile phase A consisted of 1.5 M ammonium sulfate, 0.05 M sodium phosphate, and 5% isopropanol, pH 6.0 ± 0.1, while 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.

[0452] Thermal stability was determined by DSF.

[0453] Based on nano-differential scanning fluorescence (nanoDSF) technology, with a temperature range of 25-95℃ and a temperature change rate of 1℃ / min, the denaturation temperature (Tm and Tonset) of antibody proteins and the onset temperature (Tagg) of protein aggregation are accurately determined by using fluorescence spectra of 280-450nm and changes in the intensity of scattered light from 266nm or 473nm lasers, thus assessing the thermal stability of antibody proteins.

[0454] Example 1: Construction and Characterization of Bispecific Antibodies

[0455] Example 1.1 VHH Screening

[0456] Alpaca immunization and magnetic sorting techniques were used to screen for candidate VHH sequences binding to CD20 through preliminary characterization. In short, alpaca were alternately immunized using Raji and Daudi cells expressing human CD20 membrane protein, with an interval of 14 days. Peripheral blood was collected seven days after each immunization, starting from the second immunization, and serum titers were monitored using FACS experiments. Once the serum titers reached the criteria for blood bank construction, peripheral blood was collected from immunized alpaca, and peripheral blood mononuclear cells (PBMCs) were isolated. Total RNA was extracted from PBMCs, and PrimeScript was used as a template for RNA extraction. TM II. Reverse transcription was performed using the 1st Strand cDNA Synthesis Kit (Takara) to prepare cDNA. Using the cDNA as a template, a first-round 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 and purified, followed by a second-round PCR amplification. The VHH fragment was then isolated and purified by gel electrophoresis. The recovered VHH gene fragment was mixed with the linearized yeast display vector pDisplay and co-transformed 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 the constructed library by magnetic sorting using streptavidin beads that had been pre-incubated with and thus bound to the target antigen.

[0457] Yeast culture obtained after magnetic bead sorting was plated on SDCAA plates, and single-clonal cells were picked and cultured. After 48 hours of induction, the single-clonal cell cultures were incubated sequentially with biotin-antigen and PE-Streptavidin. After incubation, flow cytometry (FACS) was performed to identify positive single-clonal yeast cells that bound the target antigen. Genomic DNA was extracted from the cultures of the obtained positive yeast cell clones for PCR amplification of antibody sequences and sequencing.

[0458] 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 a human IgG1Fc sequence. After the vectors were verified by sequencing, they were 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 antibodies, and finally, anti-CD20 antibodies were obtained through screening.

[0459] V-n6D11:

[0460] Example 1.2. In vitro characterization of candidate VHH

[0461] Candidate VHH-Fc antibody expression and purification

[0462] The encoding gene for the VHH antibody sequence described above was synthesized and inserted into the expression vector pcDNA3.4, fusing the hIgG1 Fc sequence (SEQ ID NO:33) at its C-terminus. The constructed expression vector was transiently transfected into 293F cells. After continuous culturing of transfected cells for 3 days, the culture supernatant was collected and filtered through a 0.45 μm filter membrane. The filtrate was transferred to sterile centrifuge tubes, and the antibody was purified using a Protein A column. The purity of the antibody product was determined by SEC-HPLC.

[0463] FACS antigen binding property detection

[0464] The binding of the anti-CD20 VHH-Fc candidate antibody molecule V-n6D11 to target cells was detected using a FACS binding assay. The assay was performed under the following conditions: Raji / Daudi target cells (2 × 10⁶ cells / year). 5 / well) + VHH-Fc or reference antibody (375nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h). FACS binding results are shown in Figures 1A and 1B. The candidate antibodies showed good target cell binding properties on both cell lines. In Raji cells (Figure 1A), V-6D11 had an EC50 value similar to the reference antibody Ofatumumab, and a better maximum binding (Bmax) than Ofatumumab and Obinutuzumab. In Daudi cells (Figure 1B), V-6D11 had an EC50 value and a maximum binding (Bmax) similar to the reference antibody Ofatumumab, and a better maximum binding (Bmax) than Obinutuzumab.

[0465] FACS Human-Monkey Cross-Reactivity Detection

[0466] The binding of the anti-CD20 VHH-Fc candidate antibody molecule V-n6D11 to cynoCD20-overexpressing cells was detected using a FACS binding assay. The assay was performed under the following conditions: HEK293-cynoCD20 target cells (2 × 10⁻⁶ cells). 5 / well) + VHH-Fc or reference antibody (375nM, 5× dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h). FACS binding results are shown in Figures 2A and 2B. The candidate antibody showed good target cell binding properties on HEK293-cynoCD20 cells, with a better maximum binding (Bmax) than the reference antibodies Ofatumumab and Obinutuzumab; and there was no obvious binding signal with blank negative cells HEK293, suggesting that the binding is antigen-specific.

[0467] As shown in the figure above, in the CD20 FACS binding assay, V-n6D11 showed good binding with human and monkey CD20 antigens, and the EC50 ratio of binding with monkey and human CD20 antigens was between 1 and 10, indicating that the affinity of the candidate to human and monkey CD20 antigens is close.

[0468] Example 1.3. Sequence optimization and characterization of VHH components

[0469] Sequence optimization of anti-CD20 VHH components

[0470] Based on in vitro activity characterization results, PTM risk assessment, and physicochemical property analysis, the anti-CD20 VHH sequence V-n6D11 was optimized.

[0471] The original VHH sequence was humanized using the "best-matching method." Amino acid sequences of the VHH framework region were compared and analyzed using a human germline V gene database to select the optimal germline sequence. The best-matching human CDR sequence was replaced with the VHH CDR sequence to generate the humanized VHH sequence. Analysis of the V-n6D11 sequence revealed that it did not require removal of post-translational modification (PTM). The humanized sequence was reverse-translated and synthesized by Genewiz (Shanghai, China). It was then constructed into the pcDNA 3.4 expression vector, with the C-terminus fused with the hIgG1 Fc sequence (SEQ ID NO:33) to generate human IgG1 and express humanized VHHs, thereby obtaining the VHH antibody protein.

[0472] Table 1 below shows the VHH sequence of the V-n6D11 humanized antibody.

[0473] Table 1. Anti-CD20 VHH sequences

[0474] The obtained antibodies V-zn6D11.m1 to m9 were subjected to FACS detection. The binding of the humanized antibodies to target cells Daudi was similar to that of the maternal antibodies (Figure 3 and Table 2). The FACS binding assay conditions were as follows: target cells (2 × 10⁻⁶ cells / year) 5 ( / well) + anti-CD20 antibody (375nM, 5x dilution, 4℃ 1h) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ 0.5h).

[0475] Table 2. Binding activity of V-zn6D11 humanized antibody with Daudi

[0476] The obtained antibodies V-zn6D11.m1 to m9 were subjected to FACS verification to confirm their human-monkey cross-reactivity (Figures 4A and 4B). The binding of the humanized antibodies to target cells HEK293-cynoCD20 was similar to that of the maternal antibodies (Figure 4A and Table 3), and none of them bound to HEK293 negative cells (Figure 4B). The FACS binding assay conditions were as follows: target cells (2 × 10⁻⁶ cells / year) 5 ( / well) + anti-CD20 antibody (375nM, 5x dilution, 4℃ for 1h incubation) + anti-hIgG Fc-PE secondary antibody (eBioscience / 12-4998-82, 1:500, 4℃ for 0.5h).

[0477] Table 3. Binding activity of V-zn6D11 humanized antibody to cynoCD20

[0478] In conclusion, all nine humanization modifications of V-n6D11 were successful.

[0479] Internalization assay

[0480] Method 1: The internalization ability of the CD20 antibody molecule of this 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 showed some internalization, but weaker than Rituximab.

[0481] Method 2: The internalization capacity of CD20 antibody molecules on Ramos cells was detected using an endocytosis assay-II based on rProtein G-vc-MMAE killing. The endocytic capacity of V-zn6D11.m2 of this invention was tested in Ramos cell lines positive for the target antigen. As shown in Figure 6, V-zn6D11.m2 exhibited significant endocytic activity compared to the IgG1 isotype control.

[0482] In conclusion, V-zn6D11.m2 has a certain internalization capability, and its internalization effect based on MMAE lethality is good.

[0483] Example 1.4 Generation of multispecific anti-CD79b / CD20 antibody molecules

[0484] This embodiment describes the structure of an exemplary anti-CD79b / CD20 bispecific antibody (BsAb) and the design and construction of its expression vector.

[0485] Multispecific antibody molecule design

[0486] 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.

[0487] Table 4. Forms of anti-CD79b and CD20 symmetric antibodies* *: "-" indicates peptide linker (G4S)n; HC indicates heavy chain; LC indicates light chain; VHH indicates VHH domain.

[0488] Due to the dimerization of the Fc region of immunoglobulins, two antibody heavy chains can associate to form a homodimer, thereby producing a symmetrical bispecific binding molecule.

[0489] Construction of multispecific antibody molecules

[0490] Specifically, bispecific antibodies in exemplary multi-chain forms are constructed as shown in Table 5 below.

[0491] In Table 5, the following components are used: VHH CD20 The anti-CD20 VHH domain is derived from V-zn6D11.m2, and its amino acid sequence is shown in SEQ ID NO:6; HC CD79b It is an anti-CD79b heavy chain, with its VH amino acid sequence as shown in SEQ ID NO:16, and possesses the human IgG1 constant region with the LALA mutation shown in SEQ ID NO:28; LC CD79b It is an anti-CD79b light chain, its VL amino acid sequence is shown in SEQ ID NO:15, and it 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.

[0492] Table 5. Multi-chain multispecific antibodies

[0493] 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.

[0494] 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. Cells were cultured for 3 days, and the culture supernatant 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. Antibody concentration was measured using Nano Drop. Protein purity was determined by SDS-PAGE and analytical HPLC-SEC, and the proteins were then stored at -80°C.

[0495] Example 1.5 Characterization of multispecific anti-CD79b / CD20 antibody molecules

[0496] Analysis of target antigen expression levels on tumor cells

[0497] Considering that the BCR (B cell receptor) on the surface of B cell-derived tumor cells may directly bind to conventionally used anti-human IgG Fc or anti-hIgG (H+L) secondary antibodies, direct-labeled antibodies anti-CD20 PE or BV421 (Clone: ​​2H7, Invitrogen) and anti-CD79b PE or APC (Clone: ​​CB3-1, Invitrogen) were used. The method for detecting the abundance of tumor cell surface antigen expression using direct-labeled FACS was applied to determine the relative expression of CD20 and CD79b antigens on various target tumor cells.

[0498] 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 levels, WSU-DLCL2, SU-DHL-8, and SU-DHL-2 showed low CD79b expression, and RC-K8 showed almost no CD79b expression.

[0499] Tumor cell binding activity

[0500] Based on the above analysis of antigen expression on tumor cells, the cell line JEKO-1 (CD79b) with different levels of target antigen expression was selected. +++ CD20+++ ) and Ramos (CD79b ++++ CD20 ++ The binding ability of the bispecific antibody (VBsAb) of this invention on different target cells was tested by FACS binding assay. Internal assays showed that neither JEKO-1 nor Ramos surface BCR directly bound to the detection secondary antibody (anti-human IgG Fc-PE). The FACS binding assay conditions were as follows: target cells (2 × 10⁻⁶ cells / year). 5 ( / well) + sample (100nM, 5× dilution, 4℃ 1h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5h).

[0501] In the Ramos cell line with high CD79b expression (Fig. 9A), and in the JEKO-1 cell line with relatively high expression of both CD79b and CD20 (Fig. 9B), the Fc-containing V BsAbs of this invention showed binding to cells. Among them, V-F2 was superior to Polatuzumab and CD20 VHH parent V-zn6D11.m2 in binding to EC50, Bmax, or both. The V BsAbs of this invention did not bind to the control cells HEK293, which were negative for both CD79b and CD20 (Fig. 9C).

[0502] Developmentability assessment

[0503] 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 with potential for development.

[0504] Table 6. Physicochemical properties of multispecific antibodies (DA data)

[0505] FACS examination of target cross-reactivity in humans and monkeys

[0506] The immunoreactivity of the humanized VBsAb (V-F2) and the humanized CD20 monospecific antibody (V-zn6D11.m2) of this invention was detected using an engineered HEK293 cell line stably expressing human / monkey CD20 via FACS binding assay. The experimental conditions were as follows: target cells (1×10⁻⁶ cells / year) 5 / well) + sample (200nM, 4× dilution, 4℃ 1h) + anti-human IgG Fc-PE secondary antibody (Thermo, 1:500, 4℃ 0.5h). The monospecific and bispecific antibodies of this invention tested showed similar binding activity to human-monkey antigens, exhibiting cross-reactivity with monkeys. The detection results of V-F2 antibody are shown in Figures 10A and 10B. In the figures, anti-HEL-isotype is the anti-HEL-human IgG1 (LALA) isotype control (Baiying Biotechnology, CAT#B109802, also referred to as aHEL or anti-HEL in this disclosure).

[0507] FACS detection of bispecific antibodies combined with dual-target assay

[0508] The ability of the humanized V BsAb (V-F2) of this invention to simultaneously bind to both CD20 and CD79b targets was detected using an engineered HEK293 cell line stably expressing human CD20 via FACS binding assays. The experimental conditions were as follows: target cells (1×10⁻⁶ cells / year)... 5 / well) + sample (200nM, 4℃ 1h) + biotinylated CD79b protein (5μg / ml) + SAV-PE secondary antibody (Invitrogen, 1:500, 4℃ 0.5h). The bispecific antibody of this invention tested can still bind CD79b after binding CD20, that is, it has the activity of simultaneously binding CD20 and CD79b. The detection results of V-F2 antibody are shown in Figure 11.

[0509] Internalization assay

[0510] The internalization capacity of the bispecific antibody molecules of this invention on different target cells, Ramos and WSU-DLCL2 cells, was tested using the endocytosis assay-III (DT3C, kill-based endocytosis).

[0511] The endocytic ability of the antibody V-F2 of this invention was tested using the above-mentioned cell lines with different target antigen expression. As shown in Figure 12, antibody V-F2 exhibited 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 this invention can achieve more effective killing through efficient endocytosis.

[0512] Example Section II. Preparation and Characterization of Antibody-Drug Conjugates (ADCs)

[0513] Materials and methods

[0514] General methods for ADC synthesis

[0515] General Synthesis Method A

[0516] 2.3 mg / ml of the antibody of this invention or 7 mg / ml of aHEL LALA (Baiying Biotechnology, CAT#B109802) isotype antibody in 50 mM NaAc-Ac, pH 5.5 buffer was placed in an Eppendorf tube. 6 molar equivalents of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 10 mM) were added to the antibody buffer (TCEP:antibody molar ratio = 6:1). The Eppendorf tube containing the reaction mixture was placed on a shaker (x500 rpm) and reacted at 37°C for 3 hours. Then, another 6 molar equivalents of TCEP (10 mM, TCEP:antibody = 6:1) were added to the mixture, and the reaction was continued for another 3 hours under the same conditions. The mixture was then ultrafiltered (MWCO 30kd, Millipore filter) 6 times, each time to half the solution volume, and the reduced antibody solution was replenished 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 fully 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. Assess the conversion using HIC-HLPC. Once conversion is complete, purify the mixture. Transfer the reaction mixture to an ultrafiltration tube (MWCO 30 kDa) and centrifuge 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.

[0517] Purity was determined by SEC-HPLC. The average DAR value and residual free linker-load content were determined by HIC and RP-HPLC methods.

[0518] General Synthesis Method B

[0519] 2.3 mg / ml of the antibody V-F2 of this invention or 7 mg / ml of aHEL LALA (Baiying Biotechnology, CAT#B109802) isotype antibody in 50 mM NaAc-Ac, pH 5.5 buffer was placed in an Eppendorf tube. 2.0 molar equivalents (V-F2) or 2.5 molar equivalents (aHEL LALA) of TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, 10 mM) were added to the antibody buffer (TCEP:antibody molar ratio = 3.6 / 2.5:1). The Eppendorf tube containing the reaction mixture was placed on a shaker (x500 rpm) and reacted at 37°C for 4 hours. Afterwards, the mixture was ultrafiltered (MWCO 30kd, Millipore filter) 6 times, each time replenished 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 fully 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. Assess the conversion using HIC-HLPC. Once conversion is complete, add 10 molar equivalents of cysteine ​​(10 mM). Incubate the reaction at room temperature (cysteine:antibody = 10:1) for 1 hour to deplete excess linker-payload. After incubation, transfer the reaction mixture to an ultrafiltration tube (MWCO 30 kDa). Centrifuge the sample at 10,000 rpm for 5 minutes to half the solution volume. Replenish the volume with 10 mM His-hac (10% DMA). Discard the flow-through. Repeat the washing step 10 times. Then wash 10 times with 10mM His-hac to replace the solution, and finally transfer the remaining solution to adjust to the appropriate concentration.

[0520] Purity was determined by SEC-HPLC. The average DAR value and residual free linker-load content were determined by HIC and RP-HPLC methods.

[0521] General methods and / or parameters for determining or detecting ADCs

[0522] Size exclusion chromatography (SEC) method (for ADC purity determination)

[0523] The parameters for the SEC-HPLC method are shown in the table below:

[0524] Reversed-phase HPLC (RP HPLC) method (for the detection of free drugs)

[0525] The parameters for the RP-HPLC method are shown in the table below:

[0526] Perform elution according to the table below.

[0527] Post run: 10 mins

[0528] HIC-HPLC method (for the detection of free antibodies and DAR distribution)

[0529] The HCl-HPLC conditions are shown in the table below:

[0530] Perform elution according to the table below.

[0531] ADC activity characterization methods

[0532] In vitro killing test of ADC

[0533] Target cells were distributed at a rate of (0.3-2) × 10⁶ cells per well. 4 Seed each cell in 100 μL of medium to form a plate. Add 2x ADC sample diluted to the appropriate concentration with culture medium and incubate in a CO2 incubator for 96-144 hours. Add 20 μL of CCK8 reagent to each well of a 96-well plate and incubate for 4-6 hours. Measure OD450-OD650 using a microplate reader. Cell viability = (OD value of sample wells - OD value of blank medium) / (OD value of control wells without sample - OD value of blank medium) * 100%.

[0534] In vitro killing test of ADC on normal human PBMCs

[0535] After resuscitation of PBMCs in normal healthy individuals, administer 1×10⁻⁶ doses per well. 5 Seed cells at 100 μL / cell volume, add 2x ADC sample diluted to the appropriate concentration with culture medium, and incubate in a CO2 incubator for 168 hours. On the day of detection, centrifuge to remove supernatant; the cell pellet is used for flow cytometry analysis. Specific staining steps are as follows:

[0536] 1. Add 200 μl of FACS buffer to each well, centrifuge to remove the supernatant, and wash the cells once;

[0537] 2. 1 μl / well Fc receptor blocking agent (Biolegend, 422302), 50 μl / well incubation system, incubate at room temperature for 15 min;

[0538] 3. Add 150 μl of FACS buffer to each well, centrifuge to remove the supernatant, and wash the cells again;

[0539] 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)

[0540] 5. Incubate at 50 μl / well for 30 min at 4°C;

[0541] 6. Add 150-200 μl of FACS buffer to each well, centrifuge to remove the supernatant, and wash the cells twice;

[0542] 7. Resuspend cells in 100 μl of FACS buffer containing DAPI (Biolegend, 422801, 1:5000) in each well;

[0543] 8. Flow cytometry testing.

[0544] Antitumor effect assay of ADC drugs in mouse CDX subcutaneous transplantation model

[0545] Model construction: 7-8 week old female severe combined immunodeficiency (SCID) mice (CB17-SCID, Beijing Vital River Laboratory Animal Technology Co., Ltd.), NSG mice (NSG, from Shanghai Southern Model Biotechnology Co., Ltd.), or nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used. After acclimatization for 1 week, PBS resuspended cell solution (3×10⁻⁶ cells / mL) was injected into the right scapula. 6 Or 1×10 7 (Cells / animals), until the tumor volume grows to 100-200mm 3 Patients were randomly assigned to groups based on mean tumor volume (n = 4 or 5). The day of grouping was defined as D0, and the test drug was administered intravenously or intraperitoneally on D0 as a single dose.

[0546] Dosage volume: Adjusted according to mouse body weight (dosage volume for mice = 10 μL / g × mouse body weight (g))

[0547] Data collection: After the start of drug administration, the mice were weighed twice a week, the tumor volume was measured twice a week, and the animals were observed twice a day.

[0548] Trial endpoint: based on tumor volume (1500-2000 mmHg) 3) or the endpoint of animal status determination. At the endpoint, all surviving animals are euthanized and tumors are collected. After photographing and weighing the tumors, subsequent processing is performed.

[0549] Endpoint analysis:

[0550] At the end of the experiment, the following indicators are analyzed: tumor volume change, percentage change in tumor volume relative to the baseline (TGI TV ) and body weight change.

[0551] TGI TV Calculation formula: TGI TV ={1 - [(V t - V0) / (C t - C0)]} × 100%

[0552] V t : The average tumor volume of the mice in the test article administration group on the t-th day;

[0553] V0: The average tumor volume of the mice in the test article administration group on the 0-th day;

[0554] C t : The average tumor volume of the mice in the vehicle group on the t-th day;

[0555] C0: The average tumor volume of the mice in the vehicle group on the 0-th day.

[0556] Detection experiment of the anti-tumor effect of ADC drugs in a mouse PDX subcutaneous transplantation model

[0557] Experimental animals:

[0558] NU / NU mice, female, 6 - 8 weeks old, weighing about 18 - 22 g. Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK(Beijing)2021 - 0006). License number for the use of experimental animals: SYXK(Shaanxi)2023 - 008. Feeding environment: SPF level.

[0559] Establishment and in vivo passage of human-derived tumor xenografts:

[0560] Inoculate the established human tumor tissue subcutaneously in immunodeficient mice, and continuously observe until tumors form and the tumor volume reaches approximately 500 - 800 mm 3 . Then, dissect the tumor tissue and cut it evenly into tissue pieces of about 3 mm × 3 mm × 3 mm. After that, inoculate the tissue pieces subcutaneously in mice and observe the growth of the tumors. When the average tumor volume reaches approximately 100 - 200 mm 3 (average value 120 ± 30 mm 3 ), group the tumor-bearing mice and observe after administration.

[0561] Group design:

[0562] Before grouping, all tumor-bearing mice underwent tumor volume measurement and weight weighing. Based on the measured tumor volume, the mice were randomly assigned to different treatment groups. A randomized block design was employed: mice were first divided into blocks based on tumor volume, and then mice within each block were randomly assigned to their respective treatment groups.

[0563] Experimental observation:

[0564] Throughout the experiment, the use and observation of laboratory animals were conducted in accordance with AAALAC guidelines. After inoculation with tumor tissue, the animals were observed daily, and their morbidity and mortality were recorded. During routine experiments, all laboratory animals were monitored for behavior, food intake, water consumption, weight changes, coat luster, and other abnormalities.

[0565] Evaluation indicators:

[0566] • Tumor volume measurement: Measured twice weekly using vernier calipers. The tumor volume calculation formula is V = 0.5a × b 2 a and b represent the long and short diameters of the tumor, respectively;

[0567] 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.

[0568] Ti represents the average tumor volume after the start of administration in the compound group, T0 represents the average tumor volume at the first administration in the compound group, V0 represents the average tumor volume at the first administration in the solvent control group, and Vi represents the average tumor volume after the start of administration in the solvent control group.

[0569] • The body weight of all tumor-bearing mice was measured twice a week. The percentage change in body weight after administration was calculated as follows: RCBW(%) = (BWi – BW0) / BW0 × 100, where BWi is the average body weight after administration and BW0 is the average body weight at the time of the first administration.

[0570] • After the experiment, the tumor was removed, weighed, and photographed.

[0571] Experiment terminated:

[0572] In the experiment, some tumor-bearing mice became extremely emaciated and near death, or their tumor volume reached 3000 mmHg. 3 (Including patients in the treatment group with an average tumor volume of 2000 mm) 3 At this time, tumor-bearing mice will be euthanized in advance.

[0573] Safety evaluation of the investigational drug:

[0574] During the experiment, if the weight of the treated mice continued to decrease and the RCBW reached 15% or more, the treatment was discontinued for observation until the mice recovered and the RCBW was <10%. Then, communication was conducted to confirm whether to resume medication. If the RCBW reached 20% or more, euthanasia was considered for the treated mice.

[0575] Statistical analysis

[0576] Analysis, processing, and reporting. Quantitative indicators are described using mean ± standard error (Mean ± SEM / SD). One-way ANOVA or two-way ANOVA is used for analysis of quantitative indicators. For inter-group comparisons, a t-test is used, and p < 0.05 is considered statistically significant. Both statistical and biological significance are considered in the analysis of results.

[0577] Example 2. In vitro killing activity assay of CD79bxCD20 bispecific antibody ADC

[0578] Example 2.1 Preparation and characterization of ADC molecules

[0579] Preparation of ADC molecules coupled with Mal-Gly-Exatecan-D-glucuronic acid:

[0580] Ab is the antibody V BsAb (V-F2) prepared in this application or the control antibody aHEL LALA, with p mainly at 8.

[0581] Following the above-described synthesis method A, using antibody V-F2 or aHEL LALA and linker-loaded Mal-Gly-Exatecan-D-glucuronic acid (CAS No.: 2763252-25-9; MedChemExpress, HY-153179), V-F2-D8-GLUC-Exd (MW 181.13, average Dar 8, yield 40%, purity 96.80%) and aHEL-D8-GLUC-Exd (MW 151.88, average Dar 8, yield 62%, purity 98.00%) were prepared respectively.

[0582] Preparation of ADC molecules coupled with MC-VC-PAB-MMAE:

[0583] Ab refers to the antibody V BsAb (V-F2) or the control antibody aHEL LALA prepared in this application, with p mainly ranging from 3.3 to 3.4.

[0584] Following the above-described synthesis method B, using antibody V-F2 or aHEL LALA and linker-loaded MC-VC-PAB-MMAE (CAS No.: 646502-53-6; MedChemExpress, HY-15575), V-F2-D4-VC-MMAE was prepared with a MW of 178.51, an average Dar of 3.4, a yield of 50%, and a purity of 94.69%; and aHEL-D4-VC-MMAE with a MW of 149.26, an average Dar of 3.3, a yield of 60%, and a purity of 93.4%.

[0585] The characterization data of the exemplary ADC obtained from the fabrication are as follows:

[0586] Example 2.2 In vitro killing of Polatuzumab-Vedotin-sensitive cell lines

[0587] Polatuzumab Vedotin analogue (PV, MedChemExpress, CAT#HY-132253) was used as a BMK ADC control. PV-sensitive Ramos cells expressing both CD79b and CD20 antigens were selected as target cells. In vitro killing assays were performed to detect the in vitro killing activity of the candidate CD79bxCD20 bispecific antibodies V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD ADCs.

[0588] As shown in Figure 13A, the test drugs were incubated with 10,000 / well Ramos in vitro for 5 days. The PV BMK control group showed significant cell killing. The in vitro killing activities of V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD were both better than those of the PV BMK control group. V-F2-D4-VC-MMAE showed the best killing effect in the 5-day killing system.

[0589] As shown in Figure 13B, after incubating the test drugs with 7500 / well Ramos for 6 days, significant cell killing was observed in the PV BMK control group. 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.

[0590] Example 2.3 In vitro killing of Polatuzumab-Vedotin-insensitive cell lines

[0591] Polatuzumab vedotin analogues were used as BMK ADC controls. The following three PV-insensitive cell lines were selected: SU-DHL-8 (CD79b...). + CD20 + The main reason for PV insensitivity is the upregulation of the anti-apoptotic gene Bcl-xL and SU-DHL-2 (CD79b). + CD20 +++ PV insensitivity is due to low CD79b expression and RC-K8 (CD79b). - CD20 ++ The reasons for PV insensitivity include upregulation of MDR-1 (MMAE efflux pump), upregulation of the anti-apoptotic gene Bcl-xL, and low expression of CD79b. Using these cells as target cells, the in vitro killing activity of candidate CD79bxCD20 bispecific antibodies V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD ADCs was detected using an in vitro killing assay.

[0592] As shown in Figure 14A, after incubating the test drug with 20,000 / well SU-DHL-8 cells in vitro for 5 days, the PV BMK control group showed no significant advantage in killing cells compared to the isotype ADC (aHEL-D4-VC-MMAE). The V-F2-D4-VC-MMAE showed slightly better killing effect 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).

[0593] As shown in Figure 14B, after incubation with 10,000 / well SU-DHL-2 cells for 4 days, the PV BMK control group showed weak cell killing activity, 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 superior to that of the PV BMK control group and the isotype ADC control group (aHEL-D8-Gluc-EXD and aHEL-D4-VC-MMAE), with V-F2-D4-VC-MMAE showing a greater advantage in the 4-day in vitro killing system.

[0594] As shown in Figure 14C, after incubation of the test drug with 10,000 / well RC-K8 cells for 6 days, the PVBMK control group showed weak killing effect on the cells, similar to that of the isotype ADC (aHEL-D4-VC-MMAE). The in vitro killing activities of V-F2-D4-VC-MMAE and V-F2-D8-Gluc-EXD were significantly superior to those of the PVBMK and isotype ADC control groups. 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 effect at high concentrations compared to V-F2-D4-VC-MMAE.

[0595] The above results suggest that the CD79bxCD20 bispecific antibody ADC of this invention can overcome PV resistance caused by the three reasons mentioned above: upregulation of MDR-1 (MMAE efflux pump), upregulation of the anti-apoptotic gene Bcl-xL, and low expression of CD79b. Furthermore, compared to BMK CD79b single-target ADC drugs, 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 this invention has advantages for both PV-sensitive and PV-insensitive cell lines.

[0596] Example 3. In vivo efficacy study of CD79bxCD20 bispecific antibody ADC

[0597] Example 3.1 Antitumor efficacy in the Ramos-CDX model

[0598] Based on the in vitro killing results of the aforementioned CD79bxCD20 bispecific antibody ADC, the ADC drug to be tested was evaluated for efficacy using a mouse Ramos-CDX model. The antitumor effect of the ADC drug in a mouse CDX subcutaneous transplantation model was investigated, and the in vivo antitumor efficacy of the candidate V-F2 ADC drug and the PVBMK control group was detected using an NSG mouse Ramos-CDX model. Specifically, in tumor-bearing mice, the tumor volume was increased to 100-200 mm. 3 Mice were randomly divided into 3 groups (n=5) based on mean tumor volume. The day of grouping was defined as D0, and the test drug was administered intravenously on D0. The test drug, V-F2-D8-Gluc-EXD ADC, was administered at a single dose of 5 mg / kg, while the reference drug, the Polatuzumab Vedotin analog (PV), was administered at a single dose of 1.5 mg / kg. Tumor volume and body weight were measured periodically after administration.

[0599] As shown in Figure 15A, the efficacy at the experimental endpoint was as follows: the antitumor efficacy of the V-F2-D8-Gluc-EXD ADC drug (5 mg / kg) of this invention was significantly better than that of the reference drug, the Polatuzumab Vedotin analog (1.5 mg / kg), with a statistically significant 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 had good safety in this model.

[0600] Example 3.2 Antitumor efficacy of the WSU-DLCL2-CDX model

[0601] Considering the reinforcing effect of the CD20 terminus, diffuse large B-cell lymphoma with high CD20 expression, WSU-DLCL2 (CD20-positive), was selected. ++++ CD79b + The efficacy of the drug was evaluated using a CDX model. 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 PVBMK control group was detected using an NSG mouse WSU-DLCL2-CDX model. Specifically, in tumor-bearing mice, the tumor volume was increased to 100-200 mm. 3 Mice were randomly divided into 3 groups (n=5) based on average tumor volume. The day of grouping was defined as D0, and the test drug was administered intravenously on D0. The test drug, V-F2-D8-Gluc-EXD ADC, was administered at a single dose of 5 mg / kg, while the reference drug, the Polatuzumab Vedotin analog (PV), was administered at a single dose of 2 mg / kg. Tumor volume and body weight were measured periodically after administration.

[0602] As shown in Figure 16A, the efficacy at the experimental endpoint was as follows: the antitumor efficacy of the V-F2-D8-Gluc-EXD ADC drug (5 mg / kg) of this invention was significantly better than that of the reference drug, the Polatuzumab Vedotin analog (2 mg / kg), with a statistically significant difference (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.

[0603] Example 3.3 Antitumor efficacy in the Ramos-CDX model

[0604] Based on the aforementioned in vitro cytotoxicity and in vivo efficacy results of the CD79bxCD20 bispecific antibody ADC, the drug dosage was reduced, and the ADC drug to be tested was evaluated for efficacy using a mouse Ramos-CDX model. An antitumor effect assay was performed using a mouse CDX subcutaneous transplantation model, and the in vivo antitumor efficacy of the candidate V-F2 ADC drug and the PV BMK control group was detected using a 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 this invention with that of PV+Rituximab (MedChemExpress, CAT#HY-P9913). Specifically, tumor-bearing mice were trained until the tumor volume reached 100-200 mm. 3 Based on mean tumor volume, mice were randomly divided into 8 groups (n=4). 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 test drug V-F2-D8-Gluc-EXD ADC was administered at doses of 3 mg / kg and 5 mg / kg, as a single dose. Considering the maximum limiting dose (MTD) of MMAE ADCs in mouse models, the reference drugs, Polatuzumab Vedotin analogs, were administered at doses of 1 mg / kg and 3 mg / kg, and the Rituximab analog was administered at a dose of 30 mg / kg, as a single dose. Tumor volume and mouse weight were measured periodically after administration.

[0605] As shown in Figures 17A to 17C, the efficacy at the experimental endpoint is as follows:

[0606] 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).

[0607] 2. The high-dose group (5 mg / kg) of V-F2-D8-Gluc-EXD ADC drug, the high-dose group (3 mg / kg) of Polatuzumab Vedotin analogue, and the PV+Rituximab combination group of the present invention can all achieve long-term (Day 40 after group dosing) complete remission (CR) (Figures 17A and 17B).

[0608] 3. As shown in Figure 17C, at the end of the experiment, the weight changes of mice in all drug-treated groups were within the normal range, indicating that all tested drugs were safe in this model.

[0609] Example 3.4 Antitumor efficacy of the WSU-DLCL2-CDX model

[0610] Based on the aforementioned in vitro killing and in vivo efficacy results of the CD79bxCD20 bispecific antibody ADC, the drug dosage was reduced, and the ADC drug to be tested was evaluated for efficacy using a mouse WSU-DLCL2-CDX model. The antitumor effect of the ADC drug in a mouse CDX subcutaneous transplantation model was tested, and the in vivo antitumor efficacy of the candidate V-F2 ADC drug and the PV BMK control group was detected using the SCID mouse WSU-DLCL2-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 this invention with the PV+Rituximab combination group. Specifically, tumor-bearing mice were trained until the tumor volume grew to 100-200 mm. 3 Mice were randomly divided into 7 groups (n=5) based on mean 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 test drug V-F2-D8-Gluc-EXD ADC was administered at a single dose of 3 mg / kg and 5 mg / kg. The reference drugs, polatuzumab vedotin analogs, were administered at 1 mg / kg and 3 mg / kg, and the Rituximab analog was administered at a single dose of 30 mg / kg. Tumor volume and body weight were measured periodically after administration.

[0611] As shown in Figure 18, the efficacy at the experimental endpoint is as follows:

[0612] 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).

[0613] 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 (Figure 18A, Figure 18B).

[0614] 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 invention, as well as 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 dosing) (Figure 18A, Figure 18B).

[0615] 4. As shown in Figure 18C, at the end of the experiment, the weight changes of mice in all drug-treated groups were within the normal range, indicating that all tested drugs were safe in this model.

[0616] Example 3.5 Antitumor efficacy of the SU-DHL-8-CDX model

[0617] Based on the aforementioned in vitro killing and in vivo efficacy results of the CD79bxCD20 bispecific antibody ADC, the drug dosage was reduced, and the ADC drug to be tested was evaluated for efficacy using a mouse SU-DHL-8-CDX model. The antitumor effect of the ADC drug in a mouse CDX subcutaneous transplantation model was tested, and the in vivo antitumor efficacy of the candidate V-F2 ADC drug and the PV BMK control group in PV-resistant cell lines was detected using a 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 compares the in vivo efficacy of the candidate ADC drug V-F2-D8-Gluc-EXD of this invention with the PV+Rituximab (MedChemExpress, CAT#HY-P9913) combination group. Specifically, tumor-bearing mice were trained until the tumor volume reached 100-200 mm. 3 Mice were randomly divided into 7 groups (n=5) based on mean 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 test drug V-F2-D8-Gluc-EXD ADC was administered at a single dose of 3 mg / kg and 5 mg / kg. The reference drugs, polatuzumab vedotin analogs, were administered at 1 mg / kg and 3 mg / kg, and the Rituximab analog was administered at a single dose of 30 mg / kg. Tumor volume and body weight were measured periodically after administration.

[0618] As shown in Figure 19, the efficacy at the experimental endpoint is as follows:

[0619] 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 19A, Figure 19B).

[0620] 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 (Figure 19A, Figure 19B).

[0621] 3. The high-dose group (5 mg / kg) of V-F2-D8-Gluc-EXD ADC drug and the high-dose group (3 mg / kg PV + 30 mg / kg Rituximab) of the present invention can achieve complete remission (CR) for a long time (Day 40 after group dosing) (Figure 19A).

[0622] 4. As shown in Figure 19C, at the end of the experiment, the weight changes of mice in all drug-treated groups were within the normal range, indicating that all tested drugs were safe in this model.

[0623] Example 3.6 Antitumor efficacy of the SU-DHL-2-CDX model

[0624] Based on the aforementioned in vitro cytotoxicity and in vivo efficacy results of the CD79bxCD20 bispecific antibody ADC, different drug dosages were set, and the ADC drugs to be tested were evaluated for efficacy using the mouse SU-DHL-2-CDX model. The antitumor effect of the ADC drugs in the mouse CDX subcutaneous transplantation model was detected, and 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 nude mouse SU-DHL-2-CDX model. Specifically, tumor-bearing mice were trained until the tumor volume reached 100-200 mm. 3 Mice were randomly divided into 7 groups (n=5) based on mean 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. The test drug V-F2-D8-Gluc-EXD ADC was administered at doses of 0.5 mg / kg, 2 mg / kg, and 5 mg / kg, as a single dose. The reference drug, the 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 were measured periodically after administration.

[0625] As shown in Figure 20, the efficacy at the experimental endpoint is as follows:

[0626] 1. The antitumor efficacy of the V-F2-D8-Gluc-EXD ADC drug of the present invention in 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 analogue (Figure 20A, Figure 20B).

[0627] 2. As shown in Figure 20C, at the end of the experiment, the weight changes of mice in all drug-treated groups were within the normal range, indicating that all tested drugs were safe in this model.

[0628] Example 3.7 Antitumor efficacy of RT-PDX model

[0629] 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, using a NU / NU mouse RT-PDX model to evaluate the in vivo antitumor efficacy of the candidate V-F2 ADC drug and the PVBMK control group in the RT model. Specifically, tumor-bearing mice were trained until the tumor volume reached 100-200 mm. 3 Mice were randomly divided into 3 groups (n=5) based on mean 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. The dose of the test drug V-F2-D8-Gluc-EXD ADC 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.

[0630] As shown in Figure 21, the efficacy at the experimental endpoint is as follows:

[0631] 1. At the same dosage, the antitumor efficacy of the V-F2-D8-Gluc-EXD ADC drug of the present invention is significantly better than that of the reference drug Polatuzumab Vedotin analog group (Figure 21A, Figure 21B).

[0632] 2. As shown in Figure 21C, at the end of the experiment, the weight changes of mice in all drug-treated groups were within the normal range, indicating that all tested drugs were safe in this model.

[0633] 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 RT patients, covering a larger patient population and benefiting more patients.

[0634] Example 4. Killing of irrelevant cells in normal healthy human PBMCs by CD79bxCD20 bispecific antibody ADC

[0635] Considering the potential hematologic toxicity of ADCs in the blood, an experiment was designed to detect the effect of V-F2 ADC on irrelevant cells in peripheral PBMCs of normal healthy individuals. Based on the aforementioned in vitro killing assay of ADCs on normal human PBMCs, the V-F2 of this invention showed slightly lower killing effect on CD3-positive T cells in peripheral PBMCs compared to PV (Figure 22A), and the V-F2 of this invention showed significantly lower killing effect on CD16-positive NK cells in peripheral PBMCs compared to PV (Figure 22B). These results suggest that the ADC of this invention has a safety profile, especially a significant advantage over PV in terms of hematologic toxicity.

[0636] Sequence List Description

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 in one of the amino acid sequences of SEQ ID NO:1 and 5-13; Preferably, the CDR1, CDR2 and CDR3 sequences (i) respectively contain or consist of the amino acid sequences of SEQ ID NOs:2,3 and 4; or (ii) respectively contain or consist of the amino acid sequences of SEQ ID NOs:14,3 and 4; 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) added, deleted, and / or substituted amino acids, or is composed of such an amino acid sequence. 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 is contained in HCDR1-3 of the VH sequence in SEQ ID NO:16; and the light chain variable region is contained in LCDR1-3 of the VL sequence in 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 structural 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): 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 is 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 is 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): 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 is independently selected from C 1-2 Alkyl; preferably methyl; R 2b R 3b and R 5b Each is independently selected from C 3-4 alkyl; R 6b and R 7b Each is 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): Preferably, D has the structure shown in formula (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 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, Where n1 and m1 are each 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: Where X is selected from -NH-, -O-, and -S-; R 1c Each is 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 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 24. The antibody-drug conjugate according to claim 22 or 23, or a pharmaceutically acceptable salt or solvate thereof, wherein L1 is selected from those that do not exist. 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, 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: Where R 1c Each is 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: 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:

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:

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: 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 those 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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