Multispecific antigen-binding protein specifically binding to PDL1 and vegfr2, and protein-drug conjugate containing same

Abstract

A multispecific antigen-binding protein specifically binding to PDL1 and VEGFR2, a protein-drug conjugate containing same, and the use of the multispecific antigen-binding protein or protein-drug conjugate in the prevention or treatment of tumors.

Description

Multispecific antigen-binding proteins that specifically bind to PDL1 and VEGFR2, and protein-drug conjugates containing them.

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese patent applications No. 202510338561.0, filed on March 21, 2025, and No. 202511734930.4, filed on November 24, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to a multispecific antigen-binding protein that specifically binds to PDL1 and VEGFR2, and protein-drug conjugates comprising the same. This disclosure also relates to the use of the multispecific binding protein or the protein-drug conjugate for the prevention or treatment of tumors. Background Technology

[0004] Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein with a molecular weight of approximately 45 kDa. It is a signaling protein produced by various cells that stimulates angiogenesis. The VEGF protein family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIG. VEGF receptors (VEGFRs) include VEGFR1, VEGFR2, and VEGFR3. VEGF has a more significant activating effect on VEGFR-2. Therefore, VEGFR-2 is the main mediator of the VEGF-driven response in endothelial cells, promoting angiogenesis, vascular permeability, endothelial cell survival, proliferation, and migration.

[0005] VEGFR2 plays a crucial role in various physiological and pathological processes, regulating angiogenesis and lymphangiogenesis, and maintaining the proliferation and survival of vascular endothelial cells. Simultaneously, in the tumor microenvironment, VEGFR2 supports tumor growth and metastasis by promoting angiogenesis. Furthermore, VEGFR2 is involved in inflammatory responses and immune regulation.

[0006] As an important target for anti-angiogenic therapy, several antibodies or small molecule inhibitors targeting VEGFR2 have been developed, such as fruquintinib, sunitinib, lenvatinib, or ramucirumab (trade name: Cyramza). These inhibitors specifically bind to vascular endothelial growth factor receptor 2 (VEGFR-2), blocking its binding to ligands (such as VEGF-A, VEGF-C, and VEGF-D), thereby inhibiting tumor angiogenesis and controlling tumor growth and metastasis.

[0007] In recent years, the combined use of VEGFR2 inhibitors and immunotherapy has become a research hotspot. For example, the combined use of VEGFR2 inhibitors and PD-1 inhibitors can significantly improve anti-tumor efficacy. Meanwhile, the development of multi-target inhibitors that simultaneously target VEGFR2 and other targets is also a hot topic in anti-tumor drug research.

[0008] Programmed death receptor-1 (PD-1) is a member of the CD28 receptor family, which includes CD28, CTLA-4, ICOS, PD-1, and BTLA. The original members of this family, CD28 and ICOS, were discovered by enhancing T cell proliferation upon the addition of monoclonal antibodies. Two cell surface glycoprotein ligands of PD-1, PD-L1 and PD-L2, have been identified and shown to downregulate T cell activation and cytokine secretion upon binding to PD-1.

[0009] Existing results show that PD-L1, highly expressed in tumor cells, plays a crucial role in tumor immune escape by increasing T cell apoptosis. Researchers have found that the P815 tumor cell line transfected with the PD-L1 gene can resist the lysis of specific CTLs in vitro, and exhibits stronger tumorigenicity and invasiveness after inoculation into mice. These biological characteristics can all be reversed by blocking PD-L1. Recently, immunotherapy using antibodies to block the PD1 / PD-L1 interaction has shown remarkable clinical efficacy, demonstrating sustained tumor suppression and improved patient survival. Furthermore, clinical results indicate that immunotherapy blocking PD-1 / PD-L1 also has excellent efficacy against other immunosuppression-related diseases.

[0010] Antibody-drug conjugates (ADCs), which link antibodies to other bioactive substances (such as toxin molecules) via linkers, can simultaneously exert the high targeting specificity of antibodies and the therapeutic effects of bioactive substances. As a representative example, more than a dozen ADCs have been approved for marketing in the treatment of cancer, with many more conjugate molecules in clinical or preclinical research stages.

[0011] Therefore, there is a need in the field to develop multispecific antigen-binding proteins that specifically bind to VEGFR2 and PDL1, as well as protein-drug conjugates constructed based on them. Summary of the Invention

[0012] This disclosure therefore provides a multispecific antigen-binding protein, such as a multispecific antibody, particularly a bispecific antibody, that specifically binds to VEGFR2 and PDL1.

[0013] In some embodiments, the multispecific antigen-binding protein comprises a PDL1-binding region that specifically binds to PDL1 and a VEGFR2-binding region that specifically binds to VEGFR2, wherein the PDL1-binding region is an immunoglobulin single variable domain such as VHH, and the VEGFR2-binding region that specifically binds to VEGFR2 is Fab. Optionally, the multispecific antigen-binding protein further comprises an Fc region, which is preferably linked to Fab (e.g., linked to the C-terminus of the Fab heavy chain). Optionally, the PDL1-binding region is linked to the VEGFR2-binding region of the Fab heavy chain, for example, to the N-terminus or C-terminus, preferably the N-terminus, preferably via a linker.

[0014] In some embodiments, this disclosure provides a protein-drug conjugate comprising the multispecific antigen-binding protein and a bioactive molecule, having the structure of Formula I: P-(L1-sp1-L2-sp2-D)n (I),

[0015] Wherein, protein P comprises the multispecific antigen-binding protein described in this disclosure, D is a bioactive molecule, L1 is a linker for connecting with P, sp1 is a first spacer unit, L2 is a cleavable linker, sp2 is a second spacer unit connected to D, and n is an integer from 1 to 20, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0016] This disclosed multispecific antigen-binding protein can simultaneously target two key targets, VEGF and PD-L1, inhibiting tumor growth and immune escape through a dual mechanism. In some embodiments, the multispecific antigen-binding protein can not only specifically target PDL1-positive tumors but also block the binding of VEGF to its receptor VEGFR, thereby inhibiting tumor angiogenesis. Simultaneously, it can block the PDL1 / PD1 signaling pathway, reactivating T cells and enhancing the immune system's ability to attack tumors. In some embodiments, the multispecific binding protein can simultaneously block the VEGF and PDL1 pathways, producing a synergistic effect and more comprehensively inhibiting tumor growth and immune escape.

[0017] After constructing the multispecific antigen-binding protein into a protein-drug conjugate, the protein-drug conjugate can fully utilize the tumor-killing ability of the conjugated drug to more effectively kill tumor cells, based on its specific targeting of PDL1-positive tumors and / or VEGFR2-mediated signaling pathways. Attached Figure Description

[0018] Figure 1 shows the binding activity of ADC molecules to NCI-H441 tumor cells;

[0019] Figure 2 shows the binding activity of ADC molecules to HCC4006 tumor cells;

[0020] Figure 3 shows the binding activity of ADC molecules to Karpas299 tumor cells;

[0021] Figure 4A shows a schematic diagram of the structure of a multispecific antigen-binding protein; Figure 4B shows a schematic diagram of a protein-drug conjugate linked to a bioactive molecule.

[0022] Figure 5 shows the binding activity of the ADC and antibody to (a) human colorectal cancer cell line RKO, (b) non-small cell lung cancer cell line HCC827, (c) human lung squamous cell carcinoma cell line EBC-1, and (d) recombinant cell line 293T-KDR overexpressing VEGFR2.

[0023] Figure 6 shows the endocytosis of antibodies in (a) RKO and (b) 293T-KDR cells.

[0024] Figure 7 shows the in vitro killing effect of ADC on (a) PD-L1 overexpressing cell lines 293T-PD-L1, (b) 293T-KDR, (c) RKO, (d) HCC827 and (e) EBC-1.

[0025] Figures 8(a) and (b) show the in vivo efficacy results of ADC in a human non-small cell lung cancer EBC-1 cell line subcutaneous xenograft model.

[0026] Figures 9(a) and (b) show the in vivo efficacy results of ADC in a human colorectal cancer RKO cell line subcutaneous xenograft model.

[0027] Figures 10(a) and (b) show the in vivo efficacy results of the ADC in the B-hVEGFA / hPD-L1plus MC38 colon cancer animal model based on B-hPD-1 / hPD-L1 / hVEGFR2 humanized mice.

[0028] Invention Details

[0029] I. Definition

[0030] Unless otherwise indicated or defined, all terms used herein have their ordinary meaning as will be understood by those skilled in the art. References include, for example, standard manuals such as Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd edition), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); Lewin, “Genes IV”, Oxford University Press, New York (1990); and Roitt et al., “Immunology” (2nd edition), Gower Medical Publishing, London, New York (1989), and general prior art cited herein; furthermore, unless otherwise stated, all methods, steps, techniques, and operations not specifically detailed herein can and have been performed in a manner known per se as will be understood by those skilled in the art. Also refer to, for example, standard manuals, the aforementioned general prior art, and other references cited therein.

[0031] The terms "complete antibody," "full-length antibody," or "all antibody" are used interchangeably herein and generally refer to an immunoglobulin molecule composed of two identical pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions can be linked by a "J" region of approximately 12 or more amino acids, and the heavy chain may also contain a "D" region of approximately 3 or more amino acids. The heavy chain can consist of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region can consist of three domains (CH1, CH2, and CH3). The light chain can consist of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region can consist of a single domain, CL. The constant regions of an antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). For example, VH and VL may each contain or consist of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antibody binding sites.

[0032] The term "antibody fragment" or "antigen-binding fragment" refers to a molecule distinct from a full-length antibody that comprises a portion of the full-length antibody and retains the ability to specifically bind an antigen (i.e., bind the same antigen to a full-length antibody from which the portion or fragment is derived). Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies; bivalent antibodies or fragments thereof; and camelid antibodies (heavy chain antibodies). The term also covers fragments of bispecific or multispecific antibodies, and / or bispecific or multispecific antibodies formed from antibody fragments.

[0033] The term "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains containing an immunoglobulin heavy chain variable domain (VH), a heavy chain constant domain (CH1), a light chain variable domain (VL), and a light chain constant domain (CL). One polypeptide chain contains VH and a constant region selected from CH1 and CL from its N-terminus to its C-terminus, while the other polypeptide chain contains VL and another constant region selected from CL and CH1 from its N-terminus to its C-terminus. The VH and VL domains pair to form an antigen-binding site. In this document, the Fab polypeptide chain containing the heavy chain constant region CH1 is also referred to as the "Fab heavy chain," and correspondingly, the Fab polypeptide chain containing the light chain constant region CL is also referred to as the "Fab light chain."

[0034] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (www.imgt.cines.fr) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)).

[0035] In this disclosure, unless otherwise specified, the Kabat assignment system is used to determine the CDR region:

[0036] Unless otherwise stated, in this disclosure, the terms “CDR” or “CDR sequence” cover a CDR sequence determined in any of the foregoing manner.

[0037] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0038] In this disclosure, the term "PD-L1" generally refers to programmed cell death protein 1 ligand 1, also known as CD274 or B7H1. In this disclosure, the PD-L1 can be a mammalian-derived PD-L1, for example, it can be derived from primates such as humans, or from rodents (e.g., mice or rats). Human PD-L1 can possess the amino acid sequence shown in NCBI accession number NP_054862.1. The PD-L1 can comprise natural PD-L1, or it can comprise variants, isotypes, species homologs, and analogs that share at least one common epitope with PD-L1.

[0039] In this disclosure, the term "VEGFR2" generally refers to vascular endothelial growth factor receptor 2. In this disclosure, the VEGFR2 can be a mammalian-derived VEGFR2, for example, it can be derived from primates such as humans, or from rodents (e.g., mice or rats). Human VEGFR2 can possess the amino acid sequence shown in UniProt accession number P35968. The VEGFR2 can comprise natural VEGFR2, or it can comprise variants, isotypes, species homologs, and analogs that share at least one common epitope with VEGFR2.

[0040] As used herein, the terms “antigen-binding protein,” “binding protein,” or “binding molecule” include a molecule containing at least one antigen-binding site, wherein said site specifically binds to a target molecule. An antigen-binding protein may be an antibody, such as a full-length antibody, or an antigen-binding fragment of an antibody, or a chimeric antigen receptor (CAR), or any other polypeptide with a higher-than-normal affinity, such as a scaffold protein, a cyclic peptide, a soluble receptor, a receptor-antibody (Rab) protein, or fragments of these polypeptides. The target molecule may be any molecule, but in particular herein it may be PDL1, VEGFR2, and especially conformational epitopes of PDL1 and VEGFR2.

[0041] As used herein, the term "multispecific antigen-binding protein" is used in the broadest sense to refer to a binding protein capable of binding (preferably specifically binding) two or more antigens. Without being bound by theory, in general, a multispecific antigen-binding protein comprises a plurality of relatively independent structures, each providing binding specificity against said plurality of antigens. For example, said plurality of structures independently contain one or more binding sites against their respective target antigens; for example, at least one of said plurality of structures is an antibody or antibody fragment. In some aspects, at least one binding specificity is provided by an antibody. In some aspects, at least one binding specificity is provided by a full-length antibody. In some aspects, at least one binding specificity is provided by a VHH domain. In some aspects, a multispecific binding protein is a multispecific antibody, such as a bispecific antibody.

[0042] In this disclosure, the term "bispecific antibody" generally refers to an antibody capable of binding to two antigens or antigenic epitopes, respectively. In some embodiments of this disclosure, the bispecific antibody may include light and heavy chains of an antibody capable of specifically binding to a first antigen or antigenic epitope, and light and heavy chains of an antibody capable of specifically binding to a second antigen or antigenic epitope. In some embodiments of this disclosure, the bispecific antibody may include a VHH domain capable of specifically binding to a first antigen or antigenic epitope, and light and heavy chains of an antibody capable of specifically binding to a second antigen or antigenic epitope.

[0043] The term "epitope" or "antigenic epitope" generally refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also known in the art as an "antigenic determinant." An epitope or antigenic determinant typically consists of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and usually has specific three-dimensional structural features and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation, which can be "linear" or "conformal." See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, points of interaction are separated by protein amino acid residues.

[0044] The term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein can bind. Specificity can be determined based on the affinity and / or cohesion of the antigen-binding protein. Affinity, expressed as the dissociation equilibrium constant (KD) between the antigen and the antigen-binding protein, is a measure of the strength of binding between the epitope and the antigen-binding site on the antigen-binding protein: the smaller the KD value, the stronger the binding between the epitope and the antigen-binding protein (or, affinity can also be expressed as the association constant (KA), which is 1 / KD). As those skilled in the art will understand, affinity can be determined in known ways depending on the specific antigen of interest. Affinity is a measure of the strength of binding between an antigen-binding protein (e.g., an immunoglobulin, antibody, a single variable domain of an immunoglobulin, or a polypeptide containing such a domain) and the associated antigen. Affinity relates to both the affinity between the antigen and the antigen-binding site on the antigen-binding protein and the number of associated binding sites present on the antigen-binding protein.

[0045] As used in this disclosure, the term "domain" (of a polypeptide or protein) refers to a folded protein structure that is able to maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of a protein and in many cases can be added to, removed from, or transferred to other proteins without loss of the function of the rest of the protein and / or the domain itself.

[0046] As used herein, the term "immunoglobulin domain" refers to a globular region of an antibody chain (e.g., a chain of a conventional four-chain antibody or a chain of a heavy-chain antibody), or to a polypeptide that is substantially composed of such globular regions. An immunoglobulin domain is characterized by its ability to maintain the immunoglobulin folding characteristics of the antibody molecule.

[0047] As used herein, the term "immunoglobulin variable domain" refers to an immunoglobulin domain substantially composed of four "frame regions" referred to in the art and hereinafter as "frame region 1" or "FR1", "frame region 2" or "FR2", "frame region 3" or "FR3", and "frame region 4" or "FR4", respectively, and three "complementarity-determining regions" or "CDRs" spaced apart from the frame regions, referred to in the art and hereinafter as "complementarity-determining region 1" or "CDR1", "complementarity-determining region 2" or "CDR2", and "complementarity-determining region 3" or "CDR3", respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain confers the specificity of antibodies to antigens by possessing antigen-binding sites. The variable domains of the heavy and light chains of natural antibodies typically have similar structures.

[0048] As used in this disclosure, the term "immunoglobulin single variable domain" refers to an immunoglobulin variable domain capable of specifically binding to an antigenic epitope without pairing with other immunoglobulin variable domains. A single heavy chain variable (VH) or light chain variable (VL) domain may be sufficient to provide antigen-binding specificity. An example of an immunoglobulin single variable domain as understood in this disclosure is a "domain antibody," such as an immunoglobulin single variable domain VH or VL (VH domain or VL domain). Another example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below, sometimes also referred to as a "single-domain antibody" or "nanobody."

[0049] The “VHH domain,” also known as a single-domain antibody, VHH, VHH domain, VHH antibody fragment, and VHH antibody, is a variable domain of an antigen-binding immunoglobulin called a “heavy chain antibody” (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)). The term “VHH domain” is used to distinguish the variable domain from the heavy chain variable domain (referred to herein as the “VH domain”) present in conventional 4-chain antibodies. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains (unlike the VH or VL domains in conventional 4-chain antibodies, in which case the epitope is recognized by the VL domain along with the VH domain). The VHH domain is a small, stable, and efficient antigen recognition unit formed by a single immunoglobulin domain. VHHs are derived, for example, from camels, such as alpacas, or from their humanized or sequence-optimized forms (e.g., affinity-matured forms to increase binding affinity). In some embodiments, the VHH is a monovalent, monospecific polypeptide molecule consisting of, or substantially consisting of, a single heavy chain variable region (e.g., the heavy chain variable region of a heavy chain antibody). Preferably, in one embodiment, this disclosure provides an immunoglobulin single variable domain comprising a humanized VHH. Preferably, in one embodiment, this disclosure provides an antibody comprising a humanized VHH. Preferably, in one embodiment, this disclosure provides a multispecific antigen-binding protein (e.g., a multispecific antibody) comprising a humanized VHH.

[0050] In the context of this disclosure, the terms “VHH domain”, “VHH”, “VHH domain”, “VHH antibody fragment”, and “VHH antibody” are used interchangeably. For example, as shown in Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999) (see Figure 2), the amino acid residues used in the VHH domain can be numbered according to the general numbering system for VH domains given by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0051] Alternative methods for numbering the amino acid residues of the VH domain are known in the art, and these alternatives can be similarly applied to the VHH domain. For example, the Chothia CDR refers to the position of the structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917(1987)). The AbM CDR represents a compromise between the Kabat hypervariable region and the Chothia structural loop, and is used in Oxford Molecular's AbM antibody modeling software. The "Contact" CDR is based on the analysis of the available crystal structure of the complex.

[0052] Single-domain antibodies or VHHs can also be contained within larger peptides / proteins. Examples of peptides / proteins containing VHHs include, but are not limited to, heavy chain antibodies (HcAbs). The term "heavy chain antibody" as used in this disclosure refers to an antibody that does not have a light chain, for example, which may contain VH-Fc or VH-CH2-CH3 or VH-hinge region-CH2-CH3 from its N-segment to C-segment, or may contain VH-CH1-CH2-CH3. The heavy chain antibodies of this disclosure may also cover homodimers, such as heavy chain dimer antibodies that do not have a light chain. Heavy chain antibodies may contain VHs derived from standard antibodies or VHs derived from single-domain antibodies. For example, the VH in a heavy chain antibody may be a VHH. For example, a heavy chain antibody may be a heavy chain antibody having a framework region and / or a heavy chain constant region derived from camels (lambs, camels, especially alpacas), in its humanized form or its sequence-optimized form (affinity-matured form), or a fragment thereof (e.g., a fragment containing at least a portion of the constant region). Heavy chain antibodies can also encompass antibodies formed by fusing the variable region or VHH of the heavy chain with an Fc region (e.g., the human IgG Fc region, such as the human IgG1 or IgG4 Fc region).

[0053] The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or 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. In this document, the term "Fc region" excludes the heavy chain variable region VH and light chain variable region VL of immunoglobulins, as well as the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. For example, an Fc region containing a partial N-terminal hinge region corresponds to Asp221 of the IgG1 constant region extending to the C-terminus of the heavy chain, or extending from Cys226 to the C-terminus of the heavy chain; or an Fc region without a hinge region corresponds to Asp231 of the IgG1 constant region extending to the C-terminus of the heavy chain. In this article, unless otherwise specified, the Fc region refers to the Fc region containing the portion of the hinge region corresponding to the Asp221 extension of the IgG1 constant region to the carboxyl terminus of the heavy chain.

[0054] "Conservative substitution" refers to the replacement of a polypeptide sequence without substantially altering its intended functional activity. For example, conservative substitution often results in an amino acid being replaced by a chemically similar amino acid. Eight exemplary groups of amino acids containing mutually conserved substitutions are listed below: 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).

[0055] In this article, when a domain is followed by an antigen name in subscript form, it indicates that the domain is a domain of an antibody or binding region that specifically binds to that antigen, such as VHH. PDL1 It is VHH that specifically binds to PDL1, VH VEGFR2 With VL VEGFR2 These refer to antibodies that specifically bind to VEGFR2 or the VH and VL regions of the binding domain, and Fab VEGFR2 It refers to Fab that specifically binds to VEGFR2.

[0056] The term "protein-drug conjugate" generally refers to a binding protein (e.g., an antigen-binding protein such as an antibody) linked to one or more bioactive molecules, such as antibody-drug conjugates (ADCs). The chemical drug can be any therapeutic agent and / or cytotoxic agent. The antibody-drug conjugate can have any number of drugs conjugated to the antibody from 1 to 16, for example, it can include 2, 4, 6, or 8 drug-loaded species. In this disclosure, the drug can include mitotic inhibitors, antitumor antibiotics, immunomodulators, vectors for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing agents, chemotherapeutic agents, hormones, anti-hormonal agents, corticosteroids, photosensitizing agents, oligonucleotides, radioactive isotopes, topoisomerase inhibitors, tyrosine kinase inhibitors, and / or radiosensitizers, etc.

[0057] The term "bioactive molecule" refers to a therapeutic compound intended to treat or prevent a disease or disorder in a subject. The term "bioactive molecule" is not intended to be particularly limited and can be any beneficial therapeutic compound that targets a specific target, receptor, gene, cell, tissue, or organism. Non-limiting examples of bioactive molecules in this disclosure include small molecule drugs, radioisotopes and their chelates, nucleic acids, peptides and antibodies or their antigen-binding fragments, and combinations thereof.

[0058] The term "drug:antibody ratio" or "DAR" refers to the ratio of a bioactive molecule coupled to the protein described herein to the protein itself. In some embodiments described herein, the DAR may be determined by n 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, or 12. 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 coupled to the ligand portion in the product to the ligand portion as determined by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA, electrophoresis, and / or HPLC), this DAR is referred to herein as the average DAR or the measured DAR. In some embodiments, the average DAR value of the conjugates disclosed herein is 1 to 16, for example 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 3-6, 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,

[0059] 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, with two of these values ​​as endpoints. It should be understood that when referring to the average DAR value, the couplings of this disclosure refer to a population or mixture of coupling molecules containing coupling molecules having the same and / or different DARs.

[0060] The term "linker" refers to a structural segment that connects a drug (e.g., a small molecule drug) to an antibody. It should be understood that a linker has functional groups that can form bonds with functional groups of the antibody or its antigen-binding fragment before it is linked to the antibody or its antigen-binding fragment.

[0061] The term "N-glycan" refers to a glycan chain attached to asparagine (Asn) of a protein's Asn-X-Ser / Thr sequence (Ser is serine, Thr is threonine, and X is any amino acid except proline). The term "O-glycan" refers to a glycan chain covalently linked to the -OH group of a serine or threonine residue in a protein via a terminal monosaccharide residue. O-glycosylation sites do not have conserved sequences, and glycans do not have a fixed core structure; they can be composed of a monosaccharide or a polysaccharide.

[0062] The term "G0 glycoform" refers to a glycosemystique in which the N-glycan molecule does not contain terminal galactose residues or a core fucose residue, such as... in It is N-acetylglucosamine. It is mannitol.

[0063] The term "GOF sugar type" refers to sugar molecules, such as N-glycan molecules, that do not contain terminal galactose residues but contain fucose linked to the core N-acetylglucosamine.

[0064] As used herein, the term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms.

[0065] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 10 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclohepttrienyl, cyclooctyl, etc.; polycyclic cycloalkyl groups include spirocyclic, fused-ring, and bridged-ring cycloalkyl groups.

[0066] The term "cycloalkylene" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group, which is a residue derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms.

[0067] The term "heterocyclic alkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon.

[0068] The term "heterocyclic alkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon group containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m(where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS- or -SS-, the remaining ring atoms are carbon, and residues derived from the same carbon atom or two different carbon atoms of the parent alkane by removing two hydrogen atoms.

[0069] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), wherein the definitions of alkyl or cycloalkyl are as described above.

[0070] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 10-membered, such as phenyl and naphthyl, with phenyl being more preferred. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring. The aryl group may be substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, and heterocyclic alkylthio.

[0071] The term "arylene" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic ring with a conjugated π-electron system, and is derived from residues derived by removing two hydrogen atoms from two different carbon atoms of the parent aromatic ring.

[0072] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5- or 6-membered, such as furanyl, thiophene, pyridyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally substituted or unsubstituted; when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic alkyl, aryl, heteroaryl, cycloalkoxy, heterocyclic alkoxy, cycloalkylthio, and heterocyclic alkylthio.

[0073] The term "hybrid aryl" refers to a heteroaromatic polycyclic aromatic compound containing 1 to 4 heteroatoms and 5 to 14 ring atoms, and is derived from residues derived by removing two hydrogen atoms from two different carbon atoms of the parent aromatic ring.

[0074] 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.

[0075] The term "glycosidic bond" refers to the chemical bond that connects the sugar and the other hydroxyl-containing compound (such as an alcohol, phenol, or another sugar) to form an acetal derivative through dehydration condensation of the hydroxyl group on the hemiacetal structure of a sugar.

[0076] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effects and properties of the conjugates of this disclosure, and that such salt is not biologically or otherwise undesirable. The conjugates of this disclosure may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In this disclosure, a pharmaceutically acceptable non-toxic acid addition salt refers to a salt formed by the conjugate of this disclosure 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 couplings in this disclosure 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 the couplings with N-containing organic bases.

[0077] Where there is no contradiction in the context, "pharmaceutical acceptable" and "medicinal" are used interchangeably in this article.

[0078] As used herein, the term “and / or” refers to any one of the options or two or more of the options.

[0079] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps.

[0080] The term "administration" generally refers to a method of giving a dose of a compound or pharmaceutical composition to a subject (e.g., a patient). Administerment can be made by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration (if necessary for local treatment). Parenteral infusion includes, for example, intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0081] In this disclosure, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Unless otherwise specified, all values ​​mentioned in this disclosure are considered to be modified by "about". In case of doubt, or where the range of error for a particular value or parameter is not generally understood in the art, "about" means ±5% of that value or parameter.

[0082] The term "effective amount" refers to such an amount or dose of the antibody, fragment, composition, or combination thereof as disclosed, 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 "prophylactic effective amount".

[0083] "Therapeutic effective dose" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. A therapeutic effective dose is also a dose in which any toxic or harmful effects of the antibody, antibody fragment, composition, or combination are less than the beneficial therapeutic effect. "Prophylactic effective dose" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired preventative outcome. Typically, because prophylactic doses are administered in subjects before or at an earlier stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0084] "Individual" or "subject" includes mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0085] The terms "cancer" and "cancerous" refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth. Cancer can be in its early, middle, or late stages, or it can be metastatic.

[0086] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. “Tumor” encompasses solid tumors and hematologic malignancies as well as metastatic lesions. The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusive when used in this article.

[0087] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0088] 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.

[0089] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0090] II. Multispecific antigen-binding proteins

[0091] In one aspect of this disclosure, the disclosure relates to a multispecific antigen-binding protein comprising a structural portion that specifically binds to PDL1 (PDL1 binding region) and a structural portion that specifically binds to VEGFR2 (VEGFR2 binding region). Optionally, the multispecific antigen-binding protein further comprises one or more additional binding regions that provide different antigen-binding specificities, i.e., specific binding to other antigens or other epitopes.

[0092] In some embodiments, the multispecific antigen-binding protein is a multispecific antibody, such as a bispecific antibody.

[0093] In some embodiments, the multispecific antigen-binding protein, such as a multispecific antibody like a bispecific antibody, comprises one or more PDL1 binding regions and one or more VEGFR2 binding regions. In some embodiments, the multispecific antigen-binding protein, such as a multispecific antibody like a bispecific antibody, comprises two PDL1 binding regions and two VEGFR2 binding regions.

[0094] In some embodiments, the PDL1 binding region is a single variable immunoglobulin domain capable of binding programmed cell death ligand 1 (PDL1).

[0095] In some embodiments, the immunoglobulin single variable domain that specifically binds to PDL1 is a single-domain antibody VHH (VHH PDL1 In some embodiments, the single-domain antibody of this disclosure is a VHH antibody comprising or composed of a heavy chain variable region, the heavy chain variable region typically having the following structure: FR1-VHH CDR1-FR2-VHH CDR2-FR3-VHH CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4; and VHH CDR1 to VHH CDR3 refer to complementarity-determining regions 1 to 3.

[0096] In some implementations, the PDL1 binding region is, for example, VHH. PDL1Includes VHH CDR1, VHH CDR2, and VHH CDR3, among which

[0097] (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:6;

[0098] (ii) VHH CDR1 contains the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 contains the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 contains the amino acid sequence shown in SEQ ID NO:6; or

[0099] (iii) VHH CDR1 consists of the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 consists of the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 consists of the amino acid sequence shown in SEQ ID NO:6.

[0100] In some implementations, the PDL1 binding region is, for example, VHH. PDL1 Include

[0101] (i) The amino acid sequence shown in SEQ ID NO: 3.

[0102] (ii) An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; for example, an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; or

[0103] (iii) Compared with the amino acid sequence shown in SEQ ID NO:3, it contains one or more amino acid substitutions, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.

[0104] In some implementations, the PDL1 binding region is, for example, VHH. PDL1 It contains or consists of the amino acid sequence shown in SEQ ID NO:3.

[0105] In some implementations, the PDL1 binding region is a humanized VHH.

[0106] In some implementations, the PDL1 binding region is, for example, VHH. PDL1 The CDR1, CDR2, and CDR3 contain envafolimab (also known as KN035), or a variable region structure containing envafolimab (also known as KN035). In some embodiments, the PDL1 binding region is, for example, VHH. PDL1 This refers to the variable region structure of envorimab.

[0107] Unless otherwise specified, the location of the variable region in a single variable domain (single-domain antibody, VHH) of an immunoglobulin is described using the Kabat numbering system.

[0108] Therefore, in some embodiments, the PDL1 binding region, for example, VHH PDL1 The heavy chain variable region of the VHH shown in SEQ ID NO: 3 contains three complementary determination regions (CDRs), such as CDR1, CDR2, and CDR3, which can be defined according to any CDR assignment system. For example, CDR1, CDR2, and CDR3 can be defined according to the Kabat, Chothia, AbM, or IMGT assignment system or any combination of assignment systems. For example, CDR1, CDR2, and CDR3 are defined according to the Kabat assignment system.

[0109] In some embodiments, the VEGFR2 binding region is derived from an anti-VEGFR2 antibody, such as ramucirumab. In some embodiments, the VEGFR2 binding region is an antigen-binding fragment of an anti-VEGFR2 antibody, such as a Fab fragment (also referred to herein as Fab). VEGFR2 For example, it is a Fab fragment of ramucirumab.

[0110] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 It contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or contains a heavy chain variable region (also referred to as VH in this paper) containing HCDR1, HCDR2, and HCDR3. VEGFR2 ) and light chain variable regions containing LCDR1, LCDR2, and LCDR3 (also referred to as VL in this paper) VEGFR2 );in

[0111] (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:9, HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, and HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:11, LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:12, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:14.

[0112] (ii) HCDR1 contains the amino acid sequence shown in SEQ ID NO:9, HCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains the amino acid sequence shown in SEQ ID NO:12, LCDR2 contains the amino acid sequence shown in SEQ ID NO:13, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:14; or

[0113] (iii) HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:9, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:10, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:11. LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:12, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:13, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:14.

[0114] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 It contains HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or contains a heavy chain variable region (also referred to as VH in this paper) containing HCDR1, HCDR2, and HCDR3. VEGFR2 ) and light chain variable regions containing LCDR1, LCDR2, and LCDR3 (also referred to as VL in this paper) VEGFR2 );in

[0115] (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:35, HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:36, and HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:37, LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:38, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:39, and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:40.

[0116] (ii) HCDR1 contains the amino acid sequence shown in SEQ ID NO:35, HCDR2 contains the amino acid sequence shown in SEQ ID NO:36, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:37; LCDR1 contains the amino acid sequence shown in SEQ ID NO:38, LCDR2 contains the amino acid sequence shown in SEQ ID NO:39, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:40; or

[0117] (iii) HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:35, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:36, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:37. LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:38, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:39, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:40.

[0118] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:7, or is composed of said amino acid sequence; and / or the light chain variable region comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:8, or is composed of said amino acid sequence.

[0119] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.

[0120] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:8.

[0121] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:33, or is composed of said amino acid sequence; and / or the light chain variable region comprises an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:34, or is composed of said amino acid sequence.

[0122] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34.

[0123] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 It comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34.

[0124] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 Include

[0125] The three complementary determination regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 7, for example, HCDR1, HCDR2, and HCDR3 can be defined according to any CDR assignment system, such as the Kabat, Chothia, AbM, or IMGT assignment system or any combination of assignment systems, for example, HCDR1, HCDR2, and HCDR3 are defined according to the Kabat assignment system; and

[0126] The three complementary determination regions (CDRs) LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 8 can be defined according to any CDR assignment system, such as the Kabat, Chothia, AbM, or IMGT assignment system or any combination of assignment systems. For example, LCDR1, LCDR2, and LCDR3 can be defined according to the Kabat assignment system.

[0127] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 Include

[0128] The three complementary determination regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 33 can be defined according to any CDR assignment system, such as the Kabat, Chothia, AbM, or IMGT assignment systems or any combination of assignment systems. For example, HCDR1, HCDR2, and HCDR3 can be defined according to the Kabat assignment system; and

[0129] The three complementary determination regions (CDRs) LCDR1, LCDR2, and LCDR3 contained in the light chain variable region shown in SEQ ID NO: 34 can be defined according to any CDR assignment system, such as the Kabat, Chothia, AbM, or IMGT assignment system or any combination of assignment systems. For example, LCDR1, LCDR2, and LCDR3 can be defined according to the Kabat assignment system.

[0130] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 Include

[0131] The heavy chain variable region of ramucirumab contains three complementary determinant regions (CDRs) HCDR1, HCDR2, and HCDR3. For example, HCDR1, HCDR2, and HCDR3 can be defined according to any CDR assignment system, such as the Kabat, Chothia, AbM, or IMGT assignment systems, or any combination of assignment systems. For instance, HCDR1, HCDR2, and HCDR3 are defined according to the Kabat assignment system.

[0132] The three complementary determination regions (CDRs) LCDR1, LCDR2, and LCDR3 contained in the light chain variable region of ramucirumab can be defined according to any CDR assignment system, such as the Kabat, Chothia, AbM, or IMGT assignment system or any combination of assignment systems. For example, LCDR1, LCDR2, and LCDR3 can be defined according to the Kabat assignment system.

[0133] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 Include

[0134] The heavy chain variable region of antibody DC101 contains three complementarity-determining regions (CDRs) HCDR1, HCDR2, and HCDR3. For example, HCDR1, HCDR2, and HCDR3 can be defined according to any CDR assignment system, such as the Kabat, Chothia, AbM, or IMGT assignment system, or any combination of assignment systems. For instance, HCDR1, HCDR2, and HCDR3 are defined according to the Kabat assignment system.

[0135] The three complementarity-determining regions (CDRs) LCDR1, LCDR2, and LCDR3 contained in the light chain variable region of antibody DC101 can be defined according to any CDR assignment system, such as the Kabat, Chothia, AbM, or IMGT assignment system or any combination of assignment systems. For example, LCDR1, LCDR2, and LCDR3 can be defined according to the Kabat assignment system.

[0136] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 It contains both heavy chain and light chain variable regions of ramucirumab.

[0137] In some implementations, the VEGFR2 binding region is, for example, Fab VEGFR2 It contains the heavy chain variable region and the light chain variable region of antibody DC101.

[0138] In some embodiments, the PDL1 binding region of the multispecific antigen-binding protein, such as a multispecific antibody like a bispecific antibody, is directly or via a linker to the VEGFR2 binding region.

[0139] In some embodiments, the multispecific antigen-binding protein, such as a multispecific antibody like a bispecific antibody, further comprises an immunoglobulin Fc region, for example, the Fc region being bound to a VEGFR2 binding region (e.g., Fab). VEGFR2 Heavy chain (LCL) connection, for example, the N-terminal connection (e.g., direct connection) of the Fc region to the Fab. VEGFR2 The C-terminus of the heavy chain.

[0140] In some embodiments, the multispecific antigen-binding protein, such as a multispecific antibody like a bispecific antibody, comprises VHH. PDL1 Fab VEGFR2 and the Fc region, where the N-terminus of the Fc region is connected to the Fab region. VEGFR2 The C-ends of the Fab heavy chain are connected, for example, directly; and

[0141] (i)VHH PDL1 C-terminus and Fab VEGFR2 The N-terminal connection of the Fab heavy chain, or

[0142] (ii)VHH PDL1 C-terminus and Fab VEGFR2 The N-terminal connection of the Fab light chain, or

[0143] (iii)VHH PDL1 The N-terminus of Fab VEGFR2 The C-end connection of the Fab light chain; or

[0144] (iv)VHH PDL1 The N-terminus connects to the C-terminus of the Fc region;

[0145] For example, in (i)-(iv) above, the connection is a direct connection or a connection via a connector, preferably a connection via a connector.

[0146] In some embodiments, a schematic diagram of the structure of the multispecific antigen-binding protein is shown in Figure 4A.

[0147] In some embodiments, the multispecific antigen-binding protein, such as a multispecific antibody like a bispecific antibody, comprises a heavy chain and a light chain, wherein

[0148] Heavy chain: From N-terminus to C-terminus, it contains or consists of the following: VHH PDL1 -VH VEGFR2 -CH1-Fc;

[0149] Light chain: From the N-terminus to the C-terminus, it contains or consists of the following: VL VEGFR2- CL;

[0150] Among them, VHH PDL1 It is the PDL1 binding region as defined in this article.

[0151] VH VEGFR2 -CH1 and VL VEGFR2- CL is constructed as the VEGFR2 binding region Fab as defined in this paper. VEGFR2 ,

[0152] CH1 is the CH1 domain of the heavy chain constant region, CL is the light chain constant region, and Fc is the Fc region of immunoglobulin.

[0153] Preferably, the VHH PDL1 -VH VEGFR2 They are connected via a connector.

[0154] In some embodiments, the multispecific antigen-binding protein, such as a multispecific antibody like a bispecific antibody, comprises two heavy chains and two light chains, and the two heavy chains polymerize through an Fc region to form a dimer.

[0155] In some embodiments, the multispecific antigen-binding protein, such as a multispecific antibody like a bispecific antibody, comprises two identical heavy chains and two identical light chains, or is composed of two identical heavy chains and two identical light chains.

[0156] In some embodiments, the structure of the multispecific antigen-binding protein may also be characterized as a full-length antibody that specifically binds to VEGFR2, and a VHH that specifically binds to PDL1 attached to the N-terminus of the full-length antibody (e.g., via a linker). For example, the C-terminus of the VHH is directly or via a linker attached to the N-terminus of the full-length VEGFR2 antibody (e.g., the N-terminus of the full-length antibody heavy chain or the N-terminus of the full-length antibody light chain), wherein the VH, VL, and Fc regions of the full-length VEGFR2 antibody are as defined herein, the VHH that specifically binds to PDL1 is as defined herein, and the linker is as defined herein.

[0157] In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region, preferably the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence described in SEQ ID NO:18, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity with it, or is composed of said amino acid sequence. For example, the immunoglobulin Fc region comprises or is an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:18.

[0158] In some specific embodiments, the amino acid sequence of the Fc region of the immunoglobulin is shown in SEQ ID NO:18.

[0159] In some embodiments, the immunoglobulin Fc region is a variant of the human immunoglobulin Fc region, such as a variant of the human IgG1 Fc region.

[0160] In some embodiments, the immunoglobulin Fc region variant may contain mutations that reduce or eliminate effector function. The effector function may be antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), etc. Specifically, the mutations that reduce or eliminate effector function weaken or eliminate the binding between the immunoglobulin and FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and / or C1q.

[0161] The mutation that weakens or eliminates the effector function can be any known mutation or combination of mutations in the prior art, for example, Esohe EI, J Immunol 2000; 164:4178-4184; Hutchins, JT, Proc. Natl. Acad. Sci. USA 1995, 92, 11980–11984; Xu D., Cell Immunol. 2000, 200, 16–26; Hezareh, M., J. Virol. 2001, 75, 12161–12168; Schlothauer, T., Protein Eng. Des. Sel. 2016, 29, 457–466; Chu, SY, Mol. Immunol. 2008, 45, 3926–3933; Sazinsky, SL, Proc. Natl. Acad. Sci. USA 2008, 105, 20167–20172; Oganesyan, V., Acta Crystallogr.Sect.D Biol.Crystallogr.2008, 64Pt 6, 700–704; An, Z., MAbs 2009, 1, 572–579; Moore, GL, Methods 2019, 154, 38–50; Schlothauer, T., Protein Eng.Des.Sel.2016, 29, 457–466; Strohl, W., US20150337053; Engelberts, PJ, EBioMedicine 2020, 52, 102625, etc.

[0162] In some embodiments, the immunoglobulin Fc region variant may further comprise mutations selected from the following that reduce or eliminate effector function: D265A, D270A, N297A, N297Q, N297G, N297D, K322A, P329A, P331G, D265A / P331G, L235A / G237A / E318A, L234A / L235A, S228P / L235E, G236R / L328R, S298G / T29 9A, L234F / L235E / P331S, L234F / L235E / D265A, H268Q / V309L / A330S / P331S, E233P / L234V / L235A / G236d el / S267K, L234A / L235A / P329G, L234F / L235E / D265A or V234A / G237A / P238S / H268A / V309L / A330S / P331S. Preferably, the immunoglobulin Fc variant may further comprise mutations selected from the following that reduce or eliminate effector function: D265A, P331G, D265A / P331G (abbreviated as AG), L234F / L235E / P331S (abbreviated as FES), L234F / L235E / D265A (abbreviated as FEA), or L234A / L235A (abbreviated as AA).

[0163] In some embodiments, the immunoglobulin Fc variant may contain mutations selected from the following that reduce or eliminate effector function: D265A, P331G, D265A / P331G (abbreviated as AG).

[0164] In some implementations, the immunoglobulin Fc region variant may comprise D265A / P331G.

[0165] In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19, or comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:19 and comprising D265A / P331G.

[0166] In some embodiments, the Fc region contains or is composed of the amino acid shown in SEQ ID NO: 18 or 19.

[0167] In some implementations, a connector suitable for connecting the PDL1 mating region and the VEGFR2 mating region, such as VHH, is preferred. PDL1 -VH VEGFR2The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length, without secondary or higher structures. In some embodiments, the linker is a flexible linker, such as a linker composed of one or more glycine, serine, or alanine, for example, one or more of (GGG)n, (GGGS)n, (GGGA)n, (GGGAA)n, (GGGGS)n, and (GGGGGS)n, where n is an integer selected from 1 to 30, an integer selected from 1 to 20, or an integer selected from 1 to 10. In some embodiments, the linker may comprise an N-terminal fragment of the antibody constant region CH1 domain, for example, 1-20 amino acids starting from the N-terminus, such as 10 amino acids starting from the N-terminus, 11 amino acids starting from the N-terminus, or 12 amino acids starting from the N-terminus. In some embodiments, the linker may comprise the N-terminal fragment of the antibody constant region CH1 domain, and a GAP attached to the C-terminus of the fragment. In some embodiments, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO:20.

[0168] In some embodiments, CH1 is a CH1 domain of the antibody heavy chain constant region, for example, derived from the CH1 domain of the IgG heavy chain constant region, such as the CH1 domain of IgG1, IgG2, IgG3, or IgG4. In some embodiments, CH1 is a CH1 domain of the heavy chain constant region of human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, CH1 comprises the amino acid sequence shown in SEQ ID NO:17, or comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:17, or is composed of said amino acid sequence. In some embodiments, CH1 comprises or is composed of the amino acid shown in SEQ ID NO:17.

[0169] In some embodiments, the CL is an antibody light chain constant region, such as derived from the Kappa light chain constant region or the Lambda light chain constant region, for example, the human Kappa light chain constant region or the human Lambda light chain constant region. In some embodiments, the CL comprises the amino acid sequence shown in SEQ ID NO:23, or comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:23, or is composed of said amino acid sequence. In some embodiments, the CL comprises or is composed of the amino acid shown in SEQ ID NO:23.

[0170] In some specific embodiments, the multispecific antigen-binding protein comprises a heavy chain and a light chain, or consists of two heavy chains and two light chains, wherein

[0171] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and

[0172] The light chain comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0173] In some specific embodiments, the multispecific antigen-binding protein comprises a heavy chain and a light chain, or consists of two heavy chains and two light chains, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:1, and the light chain comprises the amino acid sequence shown in SEQ ID NO:2.

[0174] In some specific embodiments, the multispecific antigen-binding protein comprises a heavy chain and a light chain, or consists of two heavy chains and two light chains, wherein the heavy chain consists of the amino acid sequence shown in SEQ ID NO:1, and the light chain consists of the amino acid sequence shown in SEQ ID NO:2.

[0175] In some specific embodiments, the multispecific antigen-binding protein comprises a heavy chain and a light chain, or consists of two heavy chains and two light chains, wherein

[0176] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and

[0177] The light chain comprises the amino acid sequence shown in SEQ ID NO:30, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0178] In some specific embodiments, the multispecific antigen-binding protein comprises a heavy chain and a light chain, or consists of two heavy chains and two light chains, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:29, and the light chain comprises the amino acid sequence shown in SEQ ID NO:30.

[0179] In some specific embodiments, the multispecific antigen-binding protein comprises a heavy chain and a light chain, or consists of two heavy chains and two light chains, wherein the heavy chain consists of the amino acid sequence shown in SEQ ID NO:29, and the light chain consists of the amino acid sequence shown in SEQ ID NO:30.

[0180] In some specific implementations, the multispecific antigen-binding protein is a bispecific antibody.

[0181] In some specific implementations, this disclosure relates to the following implementations:

[0182] 1. A bispecific antibody comprising a heavy chain and a light chain, wherein...

[0183] Heavy chain: From N-terminus to C-terminus, it contains or consists of the following: VHH PDL1 -VH VEGFR2 -CH1-Fc;

[0184] Light chain: From the N-terminus to the C-terminus, it contains or consists of the following: VL VEGFR2- CL;

[0185] Among them, VHH PDL1 It is a VHH that specifically binds to PDL1.

[0186] VH VEGFR2 -CH1 and VL VEGFR2- CL constitutes a Fab that specifically binds to VEGFR2. VEGFR2 ,

[0187] CH1 is the CH1 domain of the heavy chain constant region, CL is the light chain constant region, and Fc is the Fc region of immunoglobulin.

[0188] Preferably, the VHH PDL1 -VH VEGFR2 They are connected via a connector.

[0189] 2. The bispecific antibody of implementation scheme 1, wherein the VHH PDL1 Includes VHH CDR1, VHH CDR2, and VHH CDR3, and

[0190] (i) VHH CDR1 contains the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 contains the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 contains the amino acid sequence shown in SEQ ID NO:6; or

[0191] (ii) VHH CDR1 consists of the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 consists of the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 consists of the amino acid sequence shown in SEQ ID NO:6.

[0192] 3. The bispecific antibody according to implementation scheme 1 or 2, wherein the VH VEGFR2 Includes HCDR1, HCDR2, and HCDR3, the VL VEGFR2 It includes LCDR1, LCDR2 and LCDR3, among which

[0193] (i) HCDR1 contains the amino acid sequence shown in SEQ ID NO:9, HCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains the amino acid sequence shown in SEQ ID NO:12, LCDR2 contains the amino acid sequence shown in SEQ ID NO:13, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:14; or

[0194] (ii) HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:9, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:10, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:11. LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:12, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:13, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:14.

[0195] 4. The bispecific antibody according to implementation scheme 1 or 2, wherein the VH VEGFR2 Includes HCDR1, HCDR2, and HCDR3, the VL VEGFR2 It includes LCDR1, LCDR2 and LCDR3, among which

[0196] (i) HCDR1 contains the amino acid sequence shown in SEQ ID NO:35, HCDR2 contains the amino acid sequence shown in SEQ ID NO:36, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:37; LCDR1 contains the amino acid sequence shown in SEQ ID NO:38, LCDR2 contains the amino acid sequence shown in SEQ ID NO:39, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:40; or

[0197] (ii) HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:35, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:36, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:37. LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:38, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:39, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:40.

[0198] 5. The bispecific antibody according to any one of embodiments 1-4, wherein the VHH PDL1 Include

[0199] (i) The amino acid sequence shown in SEQ ID NO: 3, or

[0200] (ii) An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; for example, an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3.

[0201] 6. The bispecific antibody according to any one of embodiments 1-3 and 5, wherein the VH VEGFR2 Contains or consists of an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:7; and / or the VL VEGFR2 Contains or consists of an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:8;

[0202] Preferably, the VHVEGFR2 Contains the amino acid sequence shown in SEQ ID NO:7, and the VL VEGFR2 Contains the amino acid sequence shown in SEQ ID NO:8; or the VH VEGFR2 Composed of the amino acid sequence shown in SEQ ID NO:7, and the VL VEGFR2 It consists of the amino acid sequence shown in SEQ ID NO:8.

[0203] 7. The bispecific antibody according to any one of embodiments 1, 2 and 4-5, wherein the VH VEGFR2 Contains or consists of an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:33; and / or the VL VEGFR2 Contains or consists of an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:34;

[0204] Preferably, the VH VEGFR2 Contains the amino acid sequence shown in SEQ ID NO:33, and the VL VEGFR2 Contains the amino acid sequence shown in SEQ ID NO:34; or the VH VEGFR2 Composed of the amino acid sequence shown in SEQ ID NO:33, and the VL VEGFR2 It consists of the amino acid sequence shown in SEQ ID NO:34.

[0205] 8. The bispecific antibody according to any one of embodiments 1 to 7, wherein the Fc region is the Fc region of human immunoglobulin, preferably the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4.

[0206] 9. The bispecific antibody according to any one of embodiments 1 to 8, wherein the Fc region further comprises a mutation that reduces or eliminates effector function, such as D265A / P331G.

[0207] 10. The bispecific antibody according to any one of embodiments 1 to 9, wherein the Fc region

[0208] (i) Contains the amino acid sequence shown in SEQ ID NO:18;

[0209] (ii) Consists of the amino acid sequence shown in SEQ ID NO:18;

[0210] (iii) Contains the amino acid sequence shown in SEQ ID NO:18, or contains an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:18.

[0211] 11. The bispecific antibody according to any one of embodiments 1 to 10, wherein the Fc region

[0212] (i) Contains the amino acid sequence shown in SEQ ID NO:19;

[0213] (ii) Consists of the amino acid sequence shown in SEQ ID NO:19;

[0214] (iii) Contains the amino acid sequence shown in SEQ ID NO:19, or contains an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:19 and containing D265A / P331G.

[0215] 12. The bispecific antibody according to any one of embodiments 1 to 11, wherein the linker comprises an N-terminal fragment of the antibody constant region CH1 domain and a GAP attached to its C-terminus, for example, the N-terminal fragment being 1-20 amino acids starting from the N-terminus, such as 10 amino acids starting from the N-terminus, 11 amino acids starting from the N-terminus, or 12 amino acids starting from the N-terminus.

[0216] Preferably, the connector comprises or consists of the amino acid sequence shown in SEQ ID NO:20.

[0217] 13. The bispecific antibody according to any one of embodiments 1 to 12, wherein CH1 is derived from the CH1 domain of the heavy chain constant region of IgG, such as the CH1 domain of the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the CH1 domain of the heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4.

[0218] For example, CH1 comprises the amino acid sequence shown in SEQ ID NO:17, or comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:17, or is composed of said amino acid sequence.

[0219] 14. The bispecific antibody according to any one of embodiments 1 to 13, wherein the CL is a light chain constant region from the Kappa light chain constant region or the Lambda light chain constant region, for example, the human Kappa light chain constant region or the human Lambda light chain constant region;

[0220] For example, the CL comprises the amino acid sequence shown in SEQ ID NO:23, or comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:23, or is composed of said amino acid sequence.

[0221] 15. The bispecific antibody according to any one of embodiments 1-14, comprising two heavy chains and two light chains, wherein the Fc regions of the two heavy chains are polymerized to form a dimer.

[0222] 16. The bispecific antibody according to any one of implementation schemes 1-15, wherein

[0223] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and

[0224] The light chain comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0225] Preferably, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:1, and the light chain comprises the amino acid sequence shown in SEQ ID NO:2; or the heavy chain is composed of the amino acid sequence shown in SEQ ID NO:1, and the light chain is composed of the amino acid sequence shown in SEQ ID NO:2.

[0226] 17. The bispecific antibody according to any one of implementation schemes 1-15, wherein

[0227] The heavy chain comprises the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and

[0228] The light chain comprises the amino acid sequence shown in SEQ ID NO:30, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence;

[0229] Preferably, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:29, and the light chain comprises the amino acid sequence shown in SEQ ID NO:30; or the heavy chain is composed of the amino acid sequence shown in SEQ ID NO:29, and the light chain is composed of the amino acid sequence shown in SEQ ID NO:30.

[0230] III. Protein-Drug Conjugates

[0231] On the other hand, this disclosure also relates to protein-drug conjugates having the structure of Formula I: P-(L1-sp1-L2-sp2-D)n (I),

[0232] Wherein, protein P comprises the multispecific antigen-binding protein described in this disclosure, D is a bioactive molecule, L1 is a linker for connecting with P, sp1 is a first spacer unit, L2 is a cleavable linker, sp2 is a second spacer unit connected to D, and n is an integer from 1 to 20, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0233] In some implementations, L1 is selected from: (The side connected to the protein is labeled P, and the side connected to the first spacer unit is labeled sp1), where Ar represents C. 6-10 aryl groups, which are optionally converted by halogens, C 1-6 Alkyl substitution; R1 is selected from hydrogen, halogen, and C. 1-6 Alkyl group; Z is selected from straight bond, C 2-6 Ethyne group, C 2-6 imidene group, C 6-10 Aryl, 5-10 heteroaryl, amide, sulfonamide, imino and CF2.

[0234] In some implementations, L1 is selected from

[0235] In some implementations, the structure of the first spacer unit sp1 is shown in Equation II:

[0236] (The side connected to L1 is labeled L1, and the side connected to L2 is labeled L2), where a1 = 0 or 1, a2 = an integer from 0 to 6, b1 = 0 or 1, b2 = an integer from 0 to 16, b3 = an integer from 0 to 16, c = an integer from 0 to 6, and at least one of b2 and b3 is 0.

[0237] In some implementations, a1 = 1 in the structure of sp1; in other implementations, a1 = 0.

[0238] In some implementations, a2 = 0, 1, 2, 3, 4, 5, or 6.

[0239] In some implementations, b1 = 0 or 1.

[0240] In some implementations, b2 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0241] In some implementations, b3 = 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0242] In some implementations, c = 0, 1, 2, 3, 4, 5, or 6.

[0243] The above options a1, a2, b1, b2, b3 and c can be combined in any way, provided that at least one of b2 and b3 is 0.

[0244] In some specific implementations, the structure of sp1 is selected from the following group:

[0245] (1) a1 = 0, a2 = 2, 3, 4, 5 or 6, b1 = 0, b2 = 0, b3 = 0, c = 0;

[0246] (2) a1 = 0, a2 = 0, b1 = 0, b2 = 0, b3 = 0, c = 2, 3, 4, 5 or 6;

[0247] (3) a1 = 1, a2 = 2, 3, 4, 5 or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0;

[0248] (4) a1 = 0, a2 = 2, 3, 4, 5 or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0; and

[0249] (5) a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 2, 3, 4, 5, 6, 7 or 8, c = 2, 3, 4, 5 or 6;

[0250] In some implementations, the structure of sp1 is specifically selected from the following group:

[0251] (1.1) a1=0, a2=2, b1=0, b2=0, b3=0, c=0;

[0252] (1.2) a1=0, a2=3, b1=0, b2=0, b3=0, c=0;

[0253] (1.3) a1=0, a2=4, b1=0, b2=0, b3=0, c=0;

[0254] (1.4) a1=0, a2=5, b1=0, b2=0, b3=0, c=0;

[0255] (1.5) a1=0, a2=6, b1=0, b2=0, b3=0, c=0;

[0256] (2.1) a1=0, a2=0, b1=0, b2=0, b3=0, c=2;

[0257] (2.2) a1=0, a2=0, b1=0, b2=0, b3=0, c=3;

[0258] (2.3) a1=0, a2=0, b1=0, b2=0, b3=0, c=4;

[0259] (2.4) a1=0, a2=0, b1=0, b2=0, b3=0, c=5;

[0260] (2.5) a1=0, a2=0, b1=0, b2=0, b3=0, c=6;

[0261] (3.1) a1=1, a2=2, b1=1, b2=2, b3=0, c=0;

[0262] (3.2) a1=1, a2=2, b1=1, b2=3, b3=0, c=0;

[0263] (3.3) a1=1, a2=2, b1=1, b2=4, b3=0, c=0;

[0264] (3.4)a1=1, a2=2, b1=1, b2=5, b3=0, c=0;

[0265] (3.5)a1=1, a2=2, b1=1, b2=6, b3=0, c=0;

[0266] (3.6)a1=1, a2=2, b1=1, b2=7, b3=0, c=0;

[0267] (3.7)a1=1, a2=2, b1=1, b2=8, b3=0, c=0;

[0268] (3.8)a1=1, a2=3, b1=1, b2=2, b3=0, c=0;

[0269] (3.9)a1=1, a2=3, b1=1, b2=3, b3=0, c=0;

[0270] (3.10)a1=1, a2=3, b1=1, b2=4, b3=0, c=0;

[0271] (3.11)a1=1, a2=3, b1=1, b2=5, b3=0, c=0;

[0272] (3.12)a1=1, a2=3, b1=1, b2=6, b3=0, c=0;

[0273] (3.13)a1=1, a2=3, b1=1, b2=7, b3=0, c=0;

[0274] (3.14)a1=1, a2=3, b1=1, b2=8, b3=0, c=0;

[0275] (3.15)a1=1, a2=4, b1=1, b2=2, b3=0, c=0;

[0276] (3.16)a1=1, a2=4, b1=1, b2=3, b3=0, c=0;

[0277] (3.17)a1=1, a2=4, b1=1, b2=4, b3=0, c=0;

[0278] (3.18)a1=1, a2=4, b1=1, b2=5, b3=0, c=0;

[0279] (3.19)a1=1, a2=4, b1=1, b2=6, b3=0, c=0;

[0280] (3.20)a1=1, a2=4, b1=1, b2=7, b3=0, c=0;

[0281] (3.21)a1=1, a2=4, b1=1, b2=8, b3=0, c=0;

[0282] (4.1)a1=0, a2=2, b1=1, b2=2, b3=0, c=0;

[0283] (4.2)a1=0, a2=2, b1=1, b2=3, b3=0, c=0;

[0284] (4.3)a1=0, a2=2, b1=1, b2=4, b3=0, c=0;

[0285] (4.4)a1=0, a2=2, b1=1, b2=5, b3=0, c=0;

[0286] (4.5)a1=0, a2=2, b1=1, b2=6, b3=0, c=0;

[0287] (4.6)a1=0, a2=2, b1=1, b2=7, b3=0, c=0;

[0288] (4.7)a1=0, a2=2, b1=1, b2=8, b3=0, c=0;

[0289] (4.8)a1=0, a2=3, b1=1, b2=2, b3=0, c=0;

[0290] (4.9)a1=0, a2=3, b1=1, b2=3, b3=0, c=0;

[0291] (4.10)a1=0, a2=3, b1=1, b2=4, b3=0, c=0;

[0292] (4.11)a1=0, a2=3, b1=1, b2=5, b3=0, c=0;

[0293] (4.12)a1=0, a2=3, b1=1, b2=6, b3=0, c=0;

[0294] (4.13)a1=0, a2=3, b1=1, b2=7, b3=0, c=0;

[0295] (4.14)a1=0, a2=3, b1=1, b2=8, b3=0, c=0;

[0296] (4.15) a1=0, a2=4, b1=1, b2=2, b3=0, c=0;

[0297] (4.16) a1=0, a2=4, b1=1, b2=3, b3=0, c=0;

[0298] (4.17) a1=0, a2=4, b1=1, b2=4, b3=0, c=0;

[0299] (4.18) a1=0, a2=4, b1=1, b2=5, b3=0, c=0;

[0300] (4.19) a1=0, a2=4, b1=1, b2=6, b3=0, c=0;

[0301] (4.20) a1=0, a2=4, b1=1, b2=7, b3=0, c=0;

[0302] (4.21) a1=0, a2=4, b1=1, b2=8, b3=0, c=0;

[0303] (5.1) a1=1, a2=0, b1=0, b2=0, b3=2, c=2;

[0304] (5.2) a1=1, a2=0, b1=0, b2=0, b3=3, c=2;

[0305] (5.3) a1=1, a2=0, b1=0, b2=0, b3=4, c=2;

[0306] (5.4) a1=1, a2=0, b1=0, b2=0, b3=5, c=2;

[0307] (5.5) a1=1, a2=0, b1=0, b2=0, b3=6, c=2;

[0308] (5.6) a1=1, a2=0, b1=0, b2=0, b3=7, c=2; and

[0309] (5.7) a1=1, a2=0, b1=0, b2=0, b3=8, c=2.

[0310] In some implementations, the cleavable linker L2 is a dipeptide, tripeptide, or tetrapeptide residue.

[0311] In some embodiments, L2 is selected from the following dipeptide residues: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Val-Cit-, -Ala-Lys-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, -Trp-Cit-, -Gly-Gly-, -Ala-Ala-, -Gly-Val-, and -Gly-Glu-; the left side of the dipeptide residue is linked to sp1, and the right side is linked to sp2. Preferably, L2 is selected from -Val-Ala-, -Val-Lys-, and -Val-Cit-.

[0312] In some embodiments, L2 is selected from the following tripeptide residues: -Glu-Val-Ala-, -Glu-Val-Cit-, -αGlu-Val-Ala-, -αGlu-Val-Cit-, -Val-Lys-Gly, and -Val-Cit-Gly-; the left side of the tripeptide residue is linked to sp1 and the right side is linked to sp2.

[0313] In some embodiments, L2 is selected from the following tetrapeptide residues: -Gly-Gly-Phe-Gly- and -Gly-Phe-Gly-Gly-; the left side of the tetrapeptide residue is linked to sp1 and the right side is linked to sp2.

[0314] In some implementations, the second spacer unit sp2 is absent, or sp2 is selected from:

[0315] (The side connected to L2 is labeled L2, and the side connected to the bioactive molecule D is labeled D), where,

[0316] R2 is independently selected from hydrogen, C 1-6 Alkyl, hydroxyl, amino, halogen, nitro, cyano d is an integer from 1 to 20, and e is an integer from 1 to 20; R3 and R4 are each independently selected from hydrogen and C. 1-6 alkyl;

[0317] The alkyl group may optionally be substituted with hydroxyl, amino, halogen, nitro, and cyano groups.

[0318] In some implementations, sp2 is

[0319] In some implementations, sp2 is

[0320] In some implementations, sp2 is selected from: Where d is an integer from 1 to 10, and e is an integer from 1 to 10.

[0321] In some implementations, -L1-sp1-L2-sp2- is selected from the following structures:

[0322] (The side linked to protein P is labeled P, and the side linked to bioactive molecule D is labeled D); where k is an integer from 1 to 20.

[0323] In some implementations, k is an integer from 2 to 16, for example, k = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0324] In some implementations, -L1-sp1-L2-sp2- is further selected from the following structures:

[0325] The definition of k is as described above.

[0326] In some embodiments, the bioactive molecule D may be selected from cytotoxins, protein kinase inhibitors, immune agonists, glucocorticoids, oligonucleotides, radioisotopes, peptides, and any combination thereof.

[0327] In some implementations, D is a cytotoxic agent selected from: DNA alkylating agents, DNA destructive agents, topoisomerase I inhibitors, topoisomerase II inhibitors, microtubule inhibitors, ribosome inhibitors, and any combination thereof.

[0328] In some specific implementation schemes, D is selected from: auristatin derivatives, maidansine derivatives, eribulin derivatives, tubulysin derivatives, pyrrolobenzodiazepine (PDB) derivatives, ducarmycin derivatives, calicacin derivatives, PNU-159682 and its derivatives, camptothecin derivatives, amatoxin derivatives, and any combination thereof.

[0329] In some embodiments, D is selected from camptothecin derivatives, which refer to compounds having the same 5-membered fused core structure as naturally derived camptothecin and having substitution modifications at positions 7, 9, 10, and 11, and having equal or stronger topoisomerase I inhibitory activity as natural camptothecin.

[0330] The optional camptothecin derivatives may be derived from the prior art as a whole, such as patent applications WO2014057687, WO2020063676, CN111689980A, WO2022068878, WO2022068878, WO2020259258, WO2020219287, WO2022121981, WO2021173773, WO2019195665, WO2021067861, WO2022170971, WO2020200880, WO2021148501, WO2023109965 and WO2022015656. The disclosures of the above patents are incorporated into this disclosure as a whole.

[0331] In some implementations, D has the structure shown in Formula III:

[0332] Wherein, X is selected from CH2, NH, O, S or SO2;

[0333] Y does not exist, or Y has The structure shown in (IV-b);

[0334] W1 and W3 are each independently selected from O, S, and NH, while W2 is selected from C, CH, and N.

[0335] R a R b Each is independently selected from hydrogen, deuterium, halogens, and carbon. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocyclic alkyl, hydroxyl, amino, cyano, and nitro; or, R a and R b Together with the carbon atom it is attached to, it forms a 3-6 membered cycloalkyl, a 3-6 membered heterocycloalkyl, or a carbonyl group; or, R a The N atom of the amide moiety is attached to form a 3-6 membered heterocyclic alkyl group and R b It is hydrogen;

[0336] Ring A is selected from the group consisting of 4-10-membered cycloalkylene, 4-10-membered heteroalkylene, 6-10-membered arylene and 5-10-membered heteroarylene;

[0337] The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, cycloalkylene, heterocycloalkylene, aryl and heteroaryl groups are each optionally and independently further substituted with groups selected from deuterium, halogen, hydroxyl, amino, cyano, carbonyl and nitro groups;

[0338] d and e are each independently selected from integers from 0 to 5.

[0339] In some implementations, D has the structure shown in Formula III-a:

[0340] Where W1 is O or NH;

[0341] R a R b Each is independently selected from hydrogen, deuterium, halogens, and carbon. 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Cycloalkyl, 3-6 membered heterocyclic alkyl, hydroxyl, amino, cyano, and nitro; or, R a and R b Together with the carbon atom it is attached to, it forms a 3-6 membered cycloalkyl, a 3-6 membered heterocycloalkyl, or a carbonyl group; or, R a The N atom of the amide moiety is attached to form a 3-6 membered heterocyclic alkyl group and R b It is hydrogen;

[0342] Each of the alkyl, alkoxy, cycloalkyl and heterocycloalkyl groups is independently and optionally further substituted with a group selected from deuterium, halogen, hydroxyl, amino, cyano and nitro groups;

[0343] d is an integer from 0 to 5.

[0344] In some implementations, W1 is 0.

[0345] In some implementations, d is 0, 1, or 2.

[0346] In some implementations, R a Selected from hydrogen, deuterium, halogens, and C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocyclic alkyl, hydroxyl and amino, R b The alkyl, alkoxy, cycloalkyl, and heterocycloalkyl groups are each optionally further substituted with groups selected from deuterium, halogen, hydroxyl, amino, cyano, and nitro groups.

[0347] In some implementations, R a and R bTogether with the carbon atom attached thereto, they form a 3-6 membered cycloalkyl or a 3-6 membered heterocycloalkyl. Each of the cycloalkyl and heterocycloalkyl groups may be further substituted independently and optionally with a group selected from deuterium, halogen, hydroxyl, amino, cyano, and nitro groups.

[0348] In some implementations, D may be selected from the following group:

[0349] In other embodiments, D has the structure shown in Formula III-b:

[0350] Where W3 is O or NH, and W2 is C, CH or N.

[0351] Ring A is selected from the group consisting of 4-10-membered cycloalkylene, 4-10-membered heteroalkylene, 6-10-membered arylene and 5-10-membered heteroarylene;

[0352] Each of the cycloalkylene, heteroalkylene, arylene, and heteroarylene groups is optionally and independently further substituted with a group selected from deuterium, halogen, hydroxyl, amino, cyano, carbonyl, and nitro groups;

[0353] e is an integer from 0 to 5.

[0354] In some implementations, W3 is O; in other implementations, W2 is CH.

[0355] In some implementations, e is 0, 1, or 2.

[0356] In some embodiments, ring A is a 4-10 membered cycloalkylene group. The cycloalkylene group may be further substituted with a group selected from deuterium, halogen, hydroxyl, amino, cyano, carbonyl, and nitro groups.

[0357] In some implementations, D may be selected from the following group:

[0358] In some implementations, D may also have the structure shown in Formula III-c:

[0359] Among them, R5 is selected from hydrogen, C 1-6 Alkyl, hydroxyl, amino, and halogen compounds, where p is an integer from 0 to 5. In some embodiments, D may be selected from the following structures:

[0360] In some embodiments, D, as a camptothecin derivative, may also be selected from the following structures:

[0361] (i.e., Exatecan) (i.e., SN-38), and (i.e., DXd, ixotecan derivative).

[0362] In some specific implementations, -L1-sp1-L2-sp2-D is selected from the following structures:

[0363] Where k is an integer from 1 to 20.

[0364] In some implementations, k is an integer from 2 to 16, for example, k = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0365] In some implementations, n = 1 to 15, for example n = 1 to 10, n = 1 to 8, n = 2 to 6, n = 2 to 5, n = 2.5 to 3.5 or n = 3.5 to 4.0.

[0366] In some embodiments, in Formula I, the linker unit L1 is linked to protein P via a thiol group; preferably, the thiol group is derived from the multispecific binding protein; more preferably, the thiol group is obtained by reducing the disulfide bonds between heavy chains and / or between heavy chains and light chains.

[0367] In other embodiments, the linker unit L1 is connected to protein P via an oligosaccharide; preferably, the oligosaccharide is derived from the natural glycan chain of the multispecific binding protein. In some embodiments, the glycan chain can be an N-glycan chain or an O-glycan chain, preferably an N-glycan chain.

[0368] In some embodiments, the oligosaccharide is derived from the N-glycan chain of the multispecific binding protein.

[0369] In some embodiments, the oligosaccharide is composed of 2 to 15 monosaccharides; preferably, the oligosaccharide is composed of 2 to 10 monosaccharides.

[0370] In some embodiments, the oligosaccharide has the structure shown in formula Va or formula Vb:

[0371] Wherein, P* is a multispecific binding protein that specifically binds PDL1 and VEGFR2 as described in this disclosure (e.g., a multispecific binding protein as described in Part I, or more specifically, a bispecific antibody as described in any one of Embodiments 1-17), GlcNAc is N-acetylglucosamine, Fuc is fuc, Man is mannose, f is 0 or 1, and j is an integer from 1 to 20, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0372] Gal* is a modified galactose selected from the following structures:

[0373] The oligosaccharide is connected to P* via a core GlcNAc.

[0374] In some embodiments, the oligosaccharide has the structure shown in formula V-a' or formula V-b' (the side connected to P* is labeled P*, and the side connected to L1 is labeled L1):

[0375] Wherein, P* is a multispecific binding protein that specifically binds to PDL1 and VEGFR2 as described in this disclosure (e.g., a multispecific binding protein as described in Part I, or more specifically, a bispecific antibody as described in any one of Embodiments 1-17), GlcNAc is N-acetylglucosamine, Fuc is fuc, Man is mannose, f is 0 or 1, Gal* is modified galactose, and the structure is selected from the following:

[0376] In some implementations, the modified galactose is linked to GlcNAc via a β-1,4-glycosidic bond.

[0377] In some embodiments, the oligosaccharide is linked to the Fc fragment of P*; preferably to the CH2 domain of the Fc fragment; more preferably to the Asn297 of the Fc fragment (according to the EU index number of Kabat).

[0378] In some specific embodiments, the protein-drug conjugates described herein have the structure shown in Formula VI:

[0379] Wherein, P* is a multispecific binding protein that specifically binds PDL1 and VEGFR2 as described in this disclosure (e.g., a multispecific binding protein as described in Part I, or more specifically, a bispecific antibody as described in any one of Embodiments 1-17), GlcNAc is N-acetylglucosamine, Fuc is fuc, Man is mannose, f is 0 or 1, and j is an integer from 1 to 20, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0380] Gal* is a modified galactose selected from the following structures:

[0381] LP is selected from one of the following structures (a) to (g):

[0382] (a) and / or

[0383] (b) and / or

[0384] (c) and / or

[0385] (d) and / or

[0386] (e) and / or

[0387] (f) and / or as well as,

[0388] (g) and / or k is an integer from 1 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10;

[0389] The core GlcNAc is directly connected to P*.

[0390] In some embodiments, the average DAR value of the coupling is 1 to 8. In some embodiments, the average DAR value of the coupling is about 2.0 to 16.0, preferably about 3.0 to 10.0, more preferably about 3.0 to 5.0, for example about 3.5, about 3.6, about 3.7, about 3.8, 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4 or about 4.5.

[0391] In some implementation schemes, j = 1 to 10, for example j = 1 to 8, j = 1.2 to 6, j = 1.5 to 3, j = 1.5 to 2.5.

[0392] In some embodiments, the antigen-binding protein comprises the heavy chain shown in SEQ ID NO:1 and the light chain shown in SEQ ID NO:2.

[0393] In some embodiments, the antigen-binding protein comprises the heavy chain shown in SEQ ID NO:29 and the light chain shown in SEQ ID NO:30.

[0394] In some embodiments, the antigen-binding protein is a bispecific antibody as described in any one of embodiments 1 to 17.

[0395] In some embodiments, the oligosaccharide in the protein-drug conjugate is linked to the Fc fragment of P*; preferably to the CH2 domain of the Fc fragment; more preferably to the Asn297 (according to the Kabat EU index number) of the Fc fragment.

[0396] As mentioned above, the oligosaccharide can be derived from the natural sugar chain of the multispecific antigen-binding protein. Specifically, the preparation method of the oligosaccharide precursor (or referred to herein as a protein derivative) includes the following steps: reacting an antibody with an N-glycosylation mainly of G0F with UDP-GalNAz or its salt, or with other UDP-GalNAc azide derivatives, in the presence of a catalyst to obtain the aforementioned oligosaccharide precursor.

[0397] The UDP-GalNAz has the following structure:

[0398] Methods for obtaining G0F glycoform antibodies (in this article, G0F-type multispecific antigen-binding proteins) are well known in the art. For example, antibodies expressed in eukaryotic cells can be post-translational modified to convert the glycan into the G0F form by galactosidase treatment, which removes any terminal galactose residues and leaves terminal N-acetylglucosamine residues.

[0399] In other embodiments, the starting glycan of the G0F glycoform described in this disclosure can also be obtained by expression and purification using a cell line with the B4GALT1 gene knocked out. One example of knocking out the B4GALT1 gene in a cell line is through homologous recombination technology. Other examples of knocking out the B4GALT1 gene include the use of zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), as described in Nature Biotechnology, volume 33, pages 842-844 (2015). Int. J. Mol. Sci. 2015, 16(10), 23849-23866, etc.

[0400] In some embodiments, the aforementioned catalyst is a galactosyltransferase or a functional variant or fragment thereof.

[0401] In some embodiments, the catalyst is β-1,4-galactosyltransferase or a functional variant or fragment thereof.

[0402] In some embodiments, the catalyst is bovine β-1,4-galactosyltransferase, human β-1,4-galactosyltransferase, or a functional variant or fragment thereof.

[0403] In some embodiments, the catalyst is human β-(1,4)-GalT1 with the Y285L mutation, for example, containing or consisting of the amino acid sequence shown in SEQ ID NO:26, 27 or 41.

[0404] In some embodiments, the catalyst is bovine β-(1,4)-GalT1 with the Y289L mutation, for example, containing or consisting of the amino acid sequence shown in SEQ ID NO:28.

[0405] In some embodiments, the catalyst is β-1,4-acetylgalactosyltransferase disclosed in patent application WO2016170186.

[0406] In some embodiments, the catalyst comprises or consists of any of the sequences shown in SEQ ID NO: 26, 27, 28 or 41.

[0407] protein derivatives

[0408] On the other hand, this disclosure also relates to a protein derivative having the structure shown in Formula VII:

[0409] Wherein, P* is a multispecific binding protein that specifically binds PDL1 and VEGFR2 as described in this disclosure (e.g., a multispecific binding protein as described in Part I, or more specifically, a bispecific antibody as described in any one of Embodiments 1-17), GlcNAc is N-acetylglucosamine, Fuc is fuc, Man is mannose, f is 0 or 1, and j is an integer from 1 to 20, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0410] Gal** is a modified galactose selected from the following structures:

[0411] The oligosaccharide is connected to P* via a core GlcNAc.

[0412] The protein derivative can be obtained by reacting an antigen-binding protein with an N-glycosylation mainly of G0F with UDP-GalNAz or its salt, or with other UDP-GalNAc azide derivatives, in the presence of a catalyst.

[0413] IV. VEGFR2 binding protein-drug conjugates

[0414] In another aspect, this disclosure provides a VEGFR2 binding protein-drug conjugate having the structure shown in Formula VIII: P VEGFR2 -(L1-sp1-L2-sp2-D)n (VIII),

[0415] Among them, P VEGFR2 It contains VEGFR2 binding protein, with D, L1, sp1, L2, sp2 and n as defined in Part III, “Protein-Drug Conjugates”.

[0416] In some implementations, the P VEGFR2 The antibody comprises an anti-VEGFR2 antibody or its antigen-binding fragment thereof, said VEGFR2 antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3. VEGFR2 ) and light chain variable regions (VL) containing LCDR1, LCDR2, and LCDR3 VEGFR2 ),in

[0417] (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:9, HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:10, and HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:11, LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:12, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:13, and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:14.

[0418] (ii) HCDR1 contains the amino acid sequence shown in SEQ ID NO:9, HCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains the amino acid sequence shown in SEQ ID NO:12, LCDR2 contains the amino acid sequence shown in SEQ ID NO:13, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:14; or

[0419] (iii) HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:9, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:10, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:11. LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:12, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:13, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:14.

[0420] In some implementations, the P VEGFR2 The antibody comprises an anti-VEGFR2 antibody or its antigen-binding fragment thereof, said VEGFR2 antibody or its antigen-binding fragment comprising a heavy chain variable region (VH) containing HCDR1, HCDR2 and HCDR3. VEGFR2 ) and light chain variable regions (VL) containing LCDR1, LCDR2, and LCDR3 VEGFR2 ),in

[0421] (i) HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:35, HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:36, and HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:37, LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:38, LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:39, and LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:40.

[0422] (ii) HCDR1 contains the amino acid sequence shown in SEQ ID NO:35, HCDR2 contains the amino acid sequence shown in SEQ ID NO:36, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:37; LCDR1 contains the amino acid sequence shown in SEQ ID NO:38, LCDR2 contains the amino acid sequence shown in SEQ ID NO:39, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:40; or

[0423] (iii) HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:35, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:36, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:37. LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:38, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:39, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:40.

[0424] In some implementations, the P VEGFR2 The device comprises an anti-VEGFR2 antibody or its antigen-binding fragment thereof, wherein the VEGFR2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:7, or is composed of said amino acid sequence; and / or the light chain variable region comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:8, or is composed of said amino acid sequence.

[0425] In some implementations, the P VEGFR2The substance comprises an anti-VEGFR2 antibody or an antigen-binding fragment thereof, wherein the VEGFR2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:8.

[0426] In some implementations, the P VEGFR2 The substance comprises an anti-VEGFR2 antibody or an antigen-binding fragment thereof, wherein the VEGFR2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:7 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:8.

[0427] In some implementations, the P VEGFR2 The molecule comprises an anti-VEGFR2 antibody or an antigen-binding fragment thereof, wherein the VEGFR2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:33, or is composed of said amino acid sequence; and / or the light chain variable region comprises an amino acid sequence identical to or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:34, or is composed of said amino acid sequence.

[0428] In some implementations, the P VEGFR2 The substance comprises an anti-VEGFR2 antibody or an antigen-binding fragment thereof, wherein the VEGFR2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:34.

[0429] In some implementations, the P VEGFR2 The substance comprises an anti-VEGFR2 antibody or an antigen-binding fragment thereof, wherein the VEGFR2 antibody or the antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:33 and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:34.

[0430] In some implementations, the P VEGFR2It comprises an anti-VEGFR2 antibody or its antigen-binding fragment, said anti-VEGFR2 antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region of ramucirumab. In some embodiments, the P VEGFR2 It contains an anti-VEGFR2 antibody or its antigen-binding fragment, wherein the anti-VEGFR2 antibody or its antigen-binding fragment contains a heavy chain variable region and a light chain variable region of DC101.

[0431] In some implementations, the P VEGFR2 It also contains the Fc region of immunoglobulins.

[0432] In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region, preferably the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence described in SEQ ID NO:18, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity with it, or is composed of said amino acid sequence. For example, the immunoglobulin Fc region comprises or is an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:18.

[0433] In some specific embodiments, the amino acid sequence of the Fc region of the immunoglobulin is shown in SEQ ID NO:18.

[0434] In some embodiments, the immunoglobulin Fc region is a variant of the human immunoglobulin Fc region, such as a variant of the human IgG1 Fc region.

[0435] In some embodiments, the immunoglobulin Fc region variant may contain mutations that reduce or eliminate effector function. The effector function may be antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), etc. Specifically, the mutations that reduce or eliminate effector function weaken or eliminate the binding between the immunoglobulin and FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and / or C1q.

[0436] In some embodiments, the immunoglobulin Fc variant may contain mutations selected from the following that reduce or eliminate effector function: D265A, P331G, D265A / P331G (abbreviated as AG).

[0437] In some implementations, the immunoglobulin Fc region variant may comprise D265A / P331G.

[0438] In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19, or comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:19 and comprising D265A / P331G.

[0439] In some embodiments, the Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO: 18 or 19.

[0440] In some implementations, the P VEGFR2 The product contains an anti-VEGFR2 antibody, which comprises, or is composed of, a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 2.

[0441] In some implementations, the P VEGFR2 The anti-VEGFR2 antibody comprises, or is composed of, a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 2.

[0442] In some implementations, the P VEGFR2 The product contains an anti-VEGFR2 antibody, which comprises, or is composed of, a heavy chain as shown in SEQ ID NO: 31 and a light chain as shown in SEQ ID NO: 30.

[0443] In some implementations, the P VEGFR2 The anti-VEGFR2 antibody comprises, or is composed of, the heavy chain as shown in SEQ ID NO: 31 and the light chain as shown in SEQ ID NO: 30.

[0444] In some implementations, the connection unit L1 and P VEGFR2 The connection method is as defined in Part III, “Protein-Drug Conjugates”.

[0445] In some embodiments, this disclosure provides a coupling having a structure as shown in Formula IX:

[0446] Among them, P* VEGFR2Anti-VEGFR2 antibody: (1) comprising, or consisting of, a heavy chain as shown in SEQ ID NO: 15 and a light chain as shown in SEQ ID NO: 2; or (2) comprising, or consisting of, a heavy chain as shown in SEQ ID NO: 31 and a light chain as shown in SEQ ID NO: 30;

[0447] GlcNAc is N-acetylglucosamine, Fuc is fuc, Man is mannose, f is 0 or 1, and j is an integer from 1 to 20, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0448] Gal* is a modified galactose selected from the following structures:

[0449] LP is selected from the following structures:

[0450] and / or In some implementations, the core GlcNAc in the aforementioned formula IX is related to P* VEGFR2 Direct connection.

[0451] V.PDL1 binding protein-drug conjugate

[0452] In another aspect, this disclosure provides a PDL1-binding protein-drug conjugate having the structure shown in Formula X: P PDL1 -(L1-sp1-L2-sp2-D)n (X),

[0453] Among them, P PDL1 It contains PDL1 binding protein, with D, L1, sp1, L2, sp2 and n as defined in Part III, “Protein-Drug Conjugates”.

[0454] In some implementation schemes, P PDL1 Immunoglobulins containing a single variable domain that specifically binds to PDL1, such as the single-domain antibody VHH (VHH) that specifically binds to PDL1. PDL1 ).

[0455] In some implementations, the P PDL1 VHH containing PDL1-specific binding PDL1 The VHH PDL1 Includes VHH CDR1, VHH CDR2, and VHH CDR3, among which

[0456] (i) VHH CDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:6;

[0457] (ii) VHH CDR1 contains the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 contains the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 contains the amino acid sequence shown in SEQ ID NO:6; or

[0458] (iii) VHH CDR1 consists of the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 consists of the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 consists of the amino acid sequence shown in SEQ ID NO:6.

[0459] In some implementations, the VHH PDL1 Include:

[0460] (i) The amino acid sequence shown in SEQ ID NO: 3.

[0461] (ii) An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; for example, an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; or

[0462] (iii) Compared with the amino acid sequence shown in SEQ ID NO:3, it contains one or more amino acid substitutions, preferably conservative amino acid substitutions, for example, an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.

[0463] In some implementations, the VHH PDL1 It contains or consists of the amino acid sequence shown in SEQ ID NO:3.

[0464] In some implementations, the VHH PDL1 It is a humanized VHH.

[0465] In some implementations, the VHH PDL1The VHH contains CDR1, CDR2, and CDR3 of envafolimab (also known as KN035), or a variable region structure containing envafolimab (also known as KN035). In some embodiments, the VHH... PDL1 This refers to the variable region structure of envorimab.

[0466] In some implementations, the P PDL1 It also contains the Fc region of immunoglobulins.

[0467] In some embodiments, the immunoglobulin Fc region is a human immunoglobulin Fc region, preferably the Fc region of human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the immunoglobulin Fc region comprises the amino acid sequence described in SEQ ID NO:18, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity with it, or is composed of said amino acid sequence. For example, the immunoglobulin Fc region comprises or is an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:18.

[0468] In some specific embodiments, the amino acid sequence of the Fc region of the immunoglobulin is shown in SEQ ID NO:18.

[0469] In some embodiments, the immunoglobulin Fc region is a variant of the human immunoglobulin Fc region, such as a variant of the human IgG1 Fc region.

[0470] In some embodiments, the immunoglobulin Fc region variant may contain mutations that reduce or eliminate effector function. The effector function may be antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), etc. Specifically, the mutations that reduce or eliminate effector function weaken or eliminate the binding between the immunoglobulin and FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, and / or C1q.

[0471] In some embodiments, the immunoglobulin Fc variant may contain mutations selected from the following that reduce or eliminate effector function: D265A, P331G, D265A / P331G (abbreviated as AG).

[0472] In some implementations, the immunoglobulin Fc region variant may comprise D265A / P331G.

[0473] In some embodiments, the Fc region comprises the amino acid sequence shown in SEQ ID NO:19, or comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:19 and comprising D265A / P331G.

[0474] In some embodiments, the Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO: 18 or 19.

[0475] In some implementations, the P PDL1 It contains or consists of an amino acid sequence as shown in SEQ ID NO: 16.

[0476] In some embodiments, this disclosure provides a coupling having a structure as shown in Formula IX:

[0477] Among them, P* PDL1 It is a PDL1 binding protein that contains or is composed of the amino acid sequence shown in SEQ ID NO: 16;

[0478] GlcNAc is N-acetylglucosamine, Fuc is fuc, Man is mannose, f is 0 or 1, and j is an integer from 1 to 20, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12;

[0479] Gal* is a modified galactose selected from the following structures:

[0480] LP is selected from the following structures:

[0481] and / or In some implementations, the core GlcNAc in the aforementioned formula XI is related to P* VEGFR2 Direct connection.

[0482] VI. The nucleic acids disclosed herein, as well as the vectors and host cells containing them.

[0483] In one aspect, this disclosure provides nucleic acids encoding any of the above-described multispecific antigen-binding proteins or any fragment thereof (e.g., antigen / target binding fragments). This disclosure also covers nucleic acids that hybridize with the above-described nucleic acids under stringent conditions, nucleic acids having one or more substitutions (e.g., conserved substitutions), deletions, or insertions compared to the above-described nucleic acids, or nucleic acid sequences having at least 80%, at least 85%, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity compared to the above-described nucleic acids.

[0484] For example, the nucleic acid disclosed herein comprises a nucleic acid encoding an amino acid sequence selected from SEQ ID NO: 1 or 2, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from SEQ ID NO: 1 or 2.

[0485] For example, the nucleic acid disclosed herein comprises a nucleic acid encoding an amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 29 or 30, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from the amino acid sequence shown in SEQ ID NO: 29 or 30.

[0486] As will be apparent to those skilled in the art, due to codon degeneracy, the amino acid sequence of each binding protein, single variable domain, VHH, fusion protein, antibody, multispecific antigen-binding protein, or any fragment thereof can be encoded by multiple nucleic acid sequences. The nucleic acid sequence encoding the molecules of this disclosure can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or by PCR amplification.

[0487] In one aspect, this disclosure provides nucleic acids encoding any of the above-mentioned multispecific antigen-binding proteins or any peptide chains / fragments thereof. When expressed from a suitable expression vector, the polypeptides encoded by said nucleic acids are capable of exhibiting human PDL1 and / or VEGFR2 binding ability.

[0488] In one embodiment, the nucleic acids encoding the strands of the multispecific antigen-binding protein of this disclosure may be in the same vector or in different vectors. In yet another embodiment, the nucleic acids encoding the strands of the multispecific antigen-binding protein of this disclosure may be introduced into the same or different host cells for expression. Therefore, in some embodiments, a method for producing the multispecific antigen-binding protein of this disclosure includes the steps of: culturing host cells containing nucleic acids encoding the strands of the molecule under conditions suitable for expression of the strands of the molecule to produce the multispecific antigen-binding protein of this disclosure.

[0489] On the other hand, this disclosure provides a vector comprising the aforementioned nucleic acid. In a preferred embodiment, the vector is an expression vector. Those skilled in the art will readily understand that vectors commonly used in the technical field to which this disclosure pertains can be applied.

[0490] In one embodiment, this disclosure provides a host cell comprising the nucleic acid or the vector.

[0491] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in terms of nucleic acid content and may contain mutations. Mutant progeny with the same function or biological activity screened or selected from the initially transformed cells are included herein. Host cells are any type of cell system that can be used to produce the antibody molecules disclosed herein, including eukaryotic cells, such as mammalian cells (e.g., CHO cells or HEK293 cells), insect cells, 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.

[0492] VII. Composition

[0493] On the other hand, this disclosure provides a composition, for example, preferably, a pharmaceutical composition, which contains one or a combination of the multispecific antigen-binding protein or protein-drug conjugate of this disclosure formulated together with a pharmaceutically acceptable carrier.

[0494] As used herein, "pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, buffers, stabilizers, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., the antibody molecule, may be encapsulated in a material to protect it from acids and other natural conditions that could inactivate it.

[0495] The amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation varies depending on the target population and the specific route of administration. Generally, the amount of active ingredient that can be combined with a carrier material to prepare a single-dose formulation is the amount of the composition that produces the therapeutic effect. Typically, this amount, expressed as 100%, ranges from about 0.01% to about 99% of the active ingredient, for example, from about 0.1% to about 70%, or from about 1% to about 30% of the active ingredient, combined with a pharmaceutically acceptable carrier.

[0496] The actual dose level of the active ingredient in the pharmaceutical compositions disclosed herein may be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response in a particular patient, composition, and route of administration, without toxicity to the patient. The selected dose level depends on a variety of pharmacokinetic factors, including the activity of the specific composition of this disclosure or its esters, salts, or amides, the route of administration, the time of administration, the excretion rate of the specific compound applied, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition applied, the age, sex, weight, condition, general health status, and medical history of the patient receiving treatment, and similar factors known in the medical field.

[0497] The compositions of this disclosure can be administered via one or more routes of administration using one or more methods known in the art. Those skilled in the art will understand that the route and / or manner of administration varies depending on the desired outcome. Preferred routes of administration for the multispecific antigen-binding proteins or protein-drug conjugates of this disclosure include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes, such as injection or infusion. As used herein, the phrase "parenteral administration" refers to a mode of administration other than enteral and local administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intra-bursal, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, and intrasternal injections and infusions.

[0498] In some embodiments, this disclosure provides a composition comprising the protein-drug conjugate described herein, and optionally a pharmaceutically acceptable carrier. Preferably, the DAR value of the composition, for example, an average DAR value of 1 to 16, such as 2-16, 4-16, 5-12, 6-10, 2-8, 3-8, 2-6, 3-6, 6-10, such as 1.0-8.0, 2.0-6.0, 3.0-5.0, 3.5-4.0, such as 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.

[0499] VIII. Drug Combinations

[0500] The terms "drug combination" or "combination product" refer to non-fixed or fixed combinations, including but not limited to pillboxes. The term "non-fixed combination" means that the active ingredients (e.g., (i) the multispecific antigen-binding protein of this disclosure or (iii) the protein-drug conjugate of this disclosure) 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. The term "fixed combination" means that two or more active ingredients 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 ingredients are selected so that the combined use of the components produces an effect greater than that achievable by using any one ingredient alone in treating a disease or condition. The components may be in separate formulations, and their formulations may be the same or different.

[0501] Therefore, in another aspect, this disclosure also provides a pharmaceutical combination or combination product comprising the multispecific antigen-binding protein and protein-drug conjugate of this disclosure, as well as one or more other therapeutic agents.

[0502] This disclosure also provides a complete set of medicine boxes containing the aforementioned drug combination, for example, the complete set of medicine boxes containing, within the same package:

[0503] -A first container containing the multispecific antigen-binding protein or protein-drug conjugate disclosed herein;

[0504] - Optionally, it also includes a second container containing a pharmaceutical composition comprising one or more other therapeutic agents (in some embodiments, the two or more other therapeutic agents are in the same container or in separate containers).

[0505] In some embodiments, other therapeutic agents encompass any therapeutic agent used to treat the disease described in this disclosure. For example, if the molecules of this disclosure are used to treat tumors, the therapeutic agents include, for example, various therapeutic agents used to treat tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, or small molecule drugs.

[0506] IX. Treatment Methods and Uses

[0507] In another aspect, this disclosure also provides a method for treating and / or preventing tumors, comprising administering to a patient in need one or more of the multispecific antigen-binding protein, protein-drug conjugate, nucleic acid, carrier, pharmaceutical composition or combination of drugs disclosed herein.

[0508] In some embodiments, this disclosure provides the use of the multispecific antigen-binding protein, protein-drug conjugate, nucleic acid, carrier, pharmaceutical composition, or combination of drugs of this disclosure for the treatment and / or prevention of tumors.

[0509] In some embodiments, this disclosure provides the use of the multispecific antigen-binding protein, protein-drug conjugate, nucleic acid, carrier, pharmaceutical composition, or combination of drugs of this disclosure for the preparation of medicaments for the treatment and / or prevention of tumors.

[0510] This disclosure provides multispecific antigen-binding proteins, protein-drug conjugates, nucleic acids, carriers, pharmaceutical compositions, or combinations thereof for use in therapies, such as for the treatment and / or prevention of tumors.

[0511] In some embodiments, the tumor described in this disclosure is a PDL1-positive tumor or a tumor with high PDL1 expression. In some embodiments, the tumor is an angiogenesis-dependent tumor or a tumor that benefits from angiogenesis. In some embodiments, the tumor is a tumor that can benefit from treatment by inhibiting the PDL1 / PD1 signaling pathway, and / or a tumor that can benefit from treatment by inhibiting the VEGF / VEGFR signaling pathway, and / or a tumor that can benefit from treatment by inhibiting angiogenesis.

[0512] In some embodiments, the tumor is a solid tumor, a non-solid tumor, or a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in an early, intermediate, or late stage, or it can be metastatic. In some embodiments, the tumor exhibits tumor immune evasion.

[0513] In some embodiments, the PD-L1-positive tumor or cancer refers to abnormal expression or activity of PD-L1 in a subject suffering from said tumor or cancer. In some embodiments, the subject (particularly an adult subject) has PD-L1 expression (e.g., low, intermediate, or high expression). In some embodiments, the subject has PD-L1 (e.g., elevated levels, such as nucleic acid or protein levels or activity) (e.g., compared to healthy subjects). In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue) has PD-L1 (e.g., elevated levels, such as nucleic acid or protein levels or activity) (e.g., compared to biological samples from healthy subjects (e.g., corresponding tissues or cells in healthy subjects), or compared to PD-L1 in adjacent healthy tissues or cells of the subject).

[0514] In some embodiments, a PD-L1 positive tumor refers to a tumor cell that abnormally expresses PD-L1. In some embodiments, abnormal PD-L1 expression refers to the expression of PD-L1 on the cell membrane of the tumor cells. In some embodiments, abnormal PD-L1 expression refers to higher PD-L1 expression on tumor cells compared to PD-L1 expression in control cells (e.g., healthy cells from a corresponding tissue of a healthy individual, or healthy cells adjacent to tumor cells). In some embodiments, abnormal PD-L1 activity refers to abnormal activation of the PD-L1 / PD-1 signaling pathway.

[0515] In some embodiments, the tumor or cancer refers to a PD-L1-positive tumor or cancer. In some embodiments, the tumor or cancer refers to abnormal PD-L1 expression or activity in a subject suffering from said tumor or cancer. In some embodiments, the subject (particularly an adult subject) has PD-L1 expression (e.g., low, intermediate, or high expression). In some embodiments, the subject has (e.g., elevated levels, such as nucleic acid or protein levels or activity) of PD-L1 (e.g., compared to healthy subjects).

[0516] In some embodiments, the tumor or cancer refers to a tumor or cancer with angiogenesis, such as a tumor or cancer with VEGFR2-mediated angiogenesis. In some embodiments, the tumor or cancer refers to a subject with said tumor or cancer who has angiogenesis, such as VEGFR2-mediated angiogenesis. In some embodiments, the subject (particularly adult subjects) has angiogenesis, such as VEGFR2-mediated angiogenesis, particularly increased angiogenesis and / or VEGFR2-mediated angiogenesis compared to healthy subjects.

[0517] In some implementations, the cancer is selected from lymphomas such as large cell lymphoma or lung cancers such as lung adenocarcinoma, pancreatic cancer, ovarian cancer, colon cancer, rectal cancer, melanoma, kidney cancer, bladder cancer, breast cancer, liver cancer, lymphoma, hematologic malignancies, head and neck cancer, glioma, gastric cancer, nasopharyngeal carcinoma, laryngeal cancer, cervical cancer, endometrial cancer, and osteosarcoma.

[0518] Depending on their therapeutic use, the multispecific antigen-binding proteins, protein-drug conjugates, nucleic acids, carriers, or pharmaceutical compositions of this disclosure may also be combined or administered in combination with one or more other therapies, such as modes of treatment and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the tumors described herein.

[0519] In some embodiments, the therapeutic agents combined or concomitant with the multispecific antigen-binding proteins, protein-drug conjugates, nucleic acids, carriers, or pharmaceutical compositions of this disclosure include, for example, various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, or small molecule drugs. In some embodiments, the treatment methods include surgery; radiotherapy, local irradiation, or focused irradiation, etc.

[0520] In this disclosure, references to combinations or regimens, such as "administration of a combination of drugs" or "in combination with...", refer to the administration of two or more therapeutic agents or modes of treatment to treat the disease described herein. Such administration includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed proportion of active ingredients. Alternatively, such administration includes co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Such administration also includes separate administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids), such as sequential administration. Example

[0521] Instruments and equipment

[0522] Reagents and Consumables

[0523] Example 1: Preparation of linker-toxin

[0524] 1.1 Synthesis of LP1

[0525] Linker-toxin LP1 can be prepared according to the following method (A) or (B):

[0526] (A) The following linker-toxin LP1 is prepared according to the process described in patent application CN113264983A (the contents of which are incorporated herein by reference):

[0527] The linker-toxin was tested and found to have an MS m / z (ESI) of 1375.77.

[0528] (B) The following linker-toxin LP1 was prepared according to the process described in patent application PCT / CN2025 / 116919 (the contents of which are incorporated herein by reference):

[0529] Step 1

[0530] Compound Fmoc-PEG4-GGFG-CH2-O-CH2-Cbz (668.6 g, 0.68 mol, CAS No.: 2914227-56-6) and THF (5.98 kg) were added to the reaction vessel and stirred. 10% palladium on carbon (67.1 g) was then added. After the addition was complete, the mixture was purged with nitrogen three times and hydrogen three times, and pressurized to 0.2-0.3 MPa. The reaction was carried out at an internal temperature of 20-25℃ for 12±1 h, and monitored by HPLC.

[0531] After the reaction was completed, the pressure was released, and the mixture was filtered through diatomaceous earth. The filter cake was washed twice with THF (738.1 g * 2). The filtrate was concentrated to 1-2V at 15-25℃, and 1V of THF was added. MTBE (9.98 kg) and F-2 seed crystals (6.0 g) were placed in a crystallizer and stirred for 15-25 min at an external temperature of 10-20℃. A THF solution containing the product was added dropwise (completed over 3-4 h), and the mixture was stirred for 3-4 h at an external temperature of 10-20℃. The mixture was then filtered, washed once with MTBE (1.21 kg), and the solid was transferred to a vacuum drying oven and dried at 15-25℃ for 16 ± 1 h. After drying, intermediate IntA (503.3 g, 0.56 mol) was obtained with a yield of 82.4% and an HPLC purity of 98.0%. [M + Na] + (Measured value m / z = 915.3)

[0532] 1H NMR (400MHz, DMSO-d6) δ8.58(t,J=6.8Hz,1H),8.31(t,J=6.0Hz,1H),8.19(t,J=5.6Hz,1H),8.15(d,J=8.0Hz,1H),8.04(t,J=5.6Hz,1 H),7.89(d,J=7.6Hz,2H),7.68(ov,2H),7.41(t,J=7.6Hz,2H),7.33(ov,3H),7.24(ov,4H),7.21~7.15(m,1H),4.62(d,J=6.4Hz,2H), 4.55~4.45(m,1H),4.29(d,J=6.8Hz,2H),4.21(t,J=6.8Hz,1H),3.98(s,2H),3.83~3.67(ov,5H),3.64~3.56(ov,3H),3.48(t,J=6.0H z,12H),3.40(t,J=5.6Hz,2H),3.13(dd,J=11.6,6.0Hz,2H),3.10~3.02(ov,2H),2.81(dd,J=14.0,9.6Hz,1H),2.39(t,J=6.4Hz,2H).

[0533] Step Two

[0534] Add IntA (493.1g, 0.55mol) and DMAc (3.20kg) to the reaction vessel, stir, adjust the external temperature to -10~0℃, and control the internal temperature to -10~0℃ to add DBU (124.7g) dropwise. After the addition is complete, maintain the internal temperature at -10~0℃ for 4.5±0.5h. Take a sample (take 1-2ml of the reaction solution, adjust the pH to 6-7 with acetic acid, and send it for analysis) to HPLC control.

[0535] After the reaction was complete, HOBT (183.8 g) was added in batches at an internal temperature of -10 to 0℃. After the addition was complete, the internal temperature was adjusted to 15-25℃ and the mixture was stirred for 1-2 hours. THF (11.02 kg) and MTBE (9.08 kg) were added to the crystallization vessel, and the internal temperature was adjusted to 0-10℃ and stirred for 20±5 min. The solution in the reaction vessel was then added dropwise while maintaining the internal temperature at 0-10℃. After the addition was complete, the mixture was stirred at 0-10℃ for 2-3 hours. The mixture was filtered, and the filter cake was washed once with THF (2.20 kg). The solid was transferred to a vacuum drying oven and dried at 15-25℃ for 11±1 h to obtain intermediate IntB (347.2 g, 0.52 mol), with a yield of 94.5% and an HPLC purity of 94.7%. [M+H] + (Measured value m / z = 671.3).

[0536] 1H NMR (400MHz, DMSO-d6) δ8.70(t,J=6.0Hz,2H),8.63(t,J=5.6Hz,2H),8.53(ov,3H),8.41(t ,J=5.6Hz,2H),7.30~7.14(ov,8H),4.70~4.52(ov,4H),4.45~4.34(m,2H),3.85(d,J=6.8Hz ,1H),3.81(d,J=6.4Hz,1H),3.76(ov,2H),3.67(ov,2H),3.64~3.56(ov,7H),3.08(dd,J=1 3.6, 4.8Hz, 2H), 2.93 (t, J = 5.2Hz, 3H), 2.85 (dd, J = 14.0, 9.6Hz, 2H), 2.42 (t, J = 6.4Hz, 3H).

[0537] Step 3

[0538] Compound IntB (340.8 g, 0.51 mol) and DCM (4.50 kg) were added to the reaction vessel. The mixture was stirred, and under nitrogen protection, the reaction temperature was adjusted to 10–20 °C. DBCO-NHS ester (214.3 g, 0.53 mol, CAS No.: 1353016-71-3) was added dropwise while maintaining the temperature at 0–10 °C. The reaction was carried out for 2–3 hours under HPLC control. After the reaction was complete, column chromatography was performed, and the crude product was concentrated.

[0539] MTBE (12.62 kg) was added to a crystallization container, stirring was started, and the temperature was controlled at 15 ± 5 °C. The crude product's DCM solution (dissolved by diluting 4.50 kg of DCM) was slowly added dropwise. After the addition was complete, the mixture was stirred at 15 ± 5 °C for 2 ± 0.5 h to allow crystallization. The mixture was filtered under nitrogen protection, and the filter cake was washed with MTBE (1.74 kg). The mixture was then vacuum dried at 20 ± 5 °C for 10 ± 2 h. After drying, intermediate IntC (356.0 g, 0.37 mol) was obtained, with a yield of 72.5% and an HPLC purity of 98.2%. [M+Na] + (Measured value m / z = 980.3).

[0540] 1H NMR (400MHz, DMSO-d6) δ8.10(ov,3H),8.00(ov,2H),7.61~7.41(m,2H),7.35~7.27(ov,3H),7.2 7~7.05(ov,10H),6.66(d,J=19.2Hz,1H),5.23(s,1H),5.05(dd,J=14.0,7.6Hz,1H),4.63(ov,2H ),4.53(m,1H),3.90(s,2H),3.60(ov,2H),3.52~3.41(ov,12H),3.36(ov,3H),3.24(m,3H),2.96 (m,2H),2.73(m,1H),2.37(m,3H),2.09(m,1H),1.85(m,1H),1.62(m,1H),1.30(d,J=7.2Hz,2H).

[0541] Step Four

[0542] Compound IntC (333.0 g, 0.35 mol) and DMF (2.48 kg) were added to the reaction flask, stirred and dissolved, and then purged with nitrogen for protection. EDCI (85.9 g, 0.45 mol) and HOBt (61.2 g, 0.45 mol) were added at room temperature, and the mixture was stirred for about 15 min. The temperature was lowered to below 10 °C, and eczema mesylate (178.5 g, 0.34 mol) (eczema was previously suspended in DMF) was added. DIPEA (89.3 g, 0.69 mol) was added dropwise while maintaining an internal temperature ≤10 °C. After the addition was complete, the mixture was stirred vigorously at below 10 °C for 16-20 h.

[0543] After the reaction was complete, water was added dropwise to quench the reaction, and the internal temperature was kept below 20°C. The quenched reaction solution was transferred to a separatory funnel, diluted with DCM, stirred, and allowed to stand for separation. The organic phase was collected. DCM was added to the aqueous phase for extraction (5.95 kg * 2), the organic phases were combined, and water was added for washing (7.27 kg * 3). The organic phase was collected and dried with anhydrous sodium sulfate. After filtration, the solution was transferred to a rotary evaporator and concentrated under reduced pressure at 25–35°C until no obvious fraction was observed. The concentrated solution was purified by reversed-phase preparative chromatography (10 μm C18 silica gel reversed-phase column), and the target component was collected. The solution was lyophilized to obtain 201.51 g of a white solid compound LP1, with a yield of 42.2% and an HPLC purity of 99.5%. [M+H]+ (measured value m / z = 1375.5).

[0544] 1H NMR (400MHz, DMSO-d6) δ8.63(t,J=6.4Hz,1H),8.49(d,J=8.8Hz,1H),8.30(t,J= 5.2Hz,1H),8.16(t,J=5.2Hz,1H),8.11(d,J=8.0Hz,1H),8.00(t,J=5.6Hz,1H),7 .75(ov,1H),7.74(ov,1H),7.67(d,J=7.2Hz,1H),7.60(d,J=7.2Hz,1H),7.51~7. 40(ov,3H),7.40~7.11(ov,9H),6.51(s,1H),5.58(m,1H),5.41(s,2H),5.14(dd, J=32.0,19.2Hz,2H),5.01(d,J=14.0Hz,1H),4.64(s,2H),4.47(m,1H),4.03(s,2 H),3.81~3.65(ov,5H),3.65~3.52(ov,4H),3.50~3.40(ov,12H),3.29(t,J=5.2H z,2H),3.25~2.97(ov,5H),2.78(dd,J=13.6,10.0Hz,1H),2.57(m,1H),2.38(ov, 5H),2.20(ov,3H),2.00(m,1H),1.85(m,2H),1,75(m,1H),0.87(t,J=7.2Hz,3H).

[0545] 1.2 Synthesis of LP2

[0546] 1.2.1 Synthesis of CPT2

[0547] 3 g (7.65 mmol, 1.0 eq) of 10,11-methylenedioxycamptothecin (CAS: 135415-73-5) was dissolved in methanol (80 mL) and water (70 mL). The reaction solution was cooled to 0 °C, and 60 mL of 75% H₂SO₄ was added, followed by 6.38 g (22.95 mmol, 3.0 eq) of FeSO₄·7H₂O. 15 mL of 30% H₂O₂ was slowly added dropwise at 0 °C, and the reaction solution was stirred at room temperature for 16 h. LC-MS analysis showed that the reaction was complete, and the reaction was stopped. The reaction solution was poured directly into ice water, filtered, and the filter cake was dried to obtain 3.1 g of crude product, a yellowish-brown solid. The crude product was slurried with DMF to obtain 1.2 g of yellow solid compound CPT₂, with a yield of 37%. ESI-MS (+) m / z = 423.1 [M+H] + .

[0548] 1.2.2 Synthesis of Int1

[0549] CPT2 (500 mg, 1.184 mmol, 1.0 eq) and (5S,8S)-1-(9H-fluorene-9-yl)-5-isopropyl-8-methyl-3,6,9-trioxo-2-oxa-4,7,10-triazaundecane-11-ylacetate (CAS: 2505045-86-1) (683 mg, 1.421 mmol, 1.2 eq) were dissolved in 10 mL DMSO, and BF3Et2O (500 mg, 3.552 mmol, 3.0 eq) was added. The reaction mixture was allowed to react overnight at room temperature. The reaction solution was quenched in an ice-cold aqueous solution of NaHCO3, resulting in the formation of a large amount of solid. The mixture was filtered, the filter cake was washed with water, and the filter cake was lyophilized to obtain 500 mg of crude yellow solid compound Int1. The crude product was used directly in the next reaction. ESI-MS (+) m / z = 844.2 [M+H] + .

[0550] 1.2.3 Synthesis of Int2

[0551] Compound Int1 (3.0 g, 3.55 mmol, 1.0 eq) was dissolved in DMF (6 mL), and triethylenediamine (3.99 g, 35.55 mmol, 10 eq) was added. After addition, the mixture was stirred at room temperature for 3 h. LC-MS showed that the reaction was complete. The reaction solution was directly and rapidly separated by RP flash chromatography (ACN in H2O 30%, 0.1% TFA). Lyophilization yielded 220 mg of a yellow solid, Int2, with an overall yield of 29%. ESI-MS(+) m / z = 622.2 [M+H] + .

[0552] 1.2.4 Synthesis of LP2

[0553] Compound Int2 (100 mg, 94.15 μmol, 1.0 eq) was dissolved in DMF (6 mL). DBCO-PEG4-COOH (CAS: 1537170-85-6) (35 mg, 0.053 mmol, 1.2 eq), DIEA (61 mg, 0.47 mmol, 5 eq), and HATU (47 mg, 0.122 mmol, 1.3 eq) were added under ice bath conditions. The reaction was allowed to proceed at room temperature for 1 h. LC-MS showed the reaction was complete. The reaction solution was filtered and purified by high-performance liquid chromatography (Ultimate XB-C18, 50*250 mm, 10 μm, 0.1% FA, ACN in H2O 35%–65%). The prepared solution was lyophilized to obtain a yellow solid compound LP2 (55 mg), yield 50%, purity 98%. ESI-MS (+) m / z = 1156.4 [M+H] + .

[0554] 1 H NMR (400MHz, DMSO): δ8.83(t,J=6.3Hz,1H),8.13(d,J=6.5Hz,1H),7.80(d,J=8.9Hz,1H),7.74(t,J=5.9Hz,1H),7.69–7.64(m,1H),7.60(d,J=6.7Hz,1 H),7.52(s,1H),7.48(d,J=6.2Hz,2H),7.46–7.43(m,1H),7.39–7.30(m,2H ),7.30–7.26(m,1H),7.25(s,1H),6.48(s,1H),6.29(s,2H),5.42(s,2H),5 .29(d,J=5.2Hz,2H),5.10–4.97(m,3H),4.82–4.72(m,2H),4.24–4.15(m,2 H),3.61–3.54(m,3H),3.50–3.40(m,12H),3.27(d,J=6.2Hz,1H),3.12–2.9 7(m,2H),2.62–2.52(m,2H),2.46–2.34(m,2H),2.27–2.17(m,1H),2.08–1. 59(m,5H),1.18(d,J=7.2Hz,3H),0.90–0.82(m,6H),0.79(d,J=6.8Hz,3H).

[0555] Example 2: Preparation and Characterization of Antibodies and ADCs

[0556] 2.1 Construction of eukaryotic expression vectors

[0557] A bispecific antibody against PDL1×VEGFR2, hu56-Ramu-hIgG1ag, was constructed. The anti-PDL1 antibody was derived from a camel single-domain antibody obtained by phage display, and the sequence hu56 (SEQ ID NO: 3) was obtained through humanization. The VH (SEQ ID NO: 7) and VL (SEQ ID NO: 8) of the anti-VEGFR2 antibody were derived from ramucirumab (WHO Drug Information, Vol. 23, No. 3, 2009; Recommended INN list R62 (2009)). The VH of hu56 and ramucirumab was synthesized by tandem gene synthesis via a linker (SEQ ID NO: 20) and fused with the human IgG1 constant region (D265A / P331G mutation to remove ADCC effect, named IgG1ag) to obtain the heavy chain of the hu56-Ramu-hIgG1ag antibody (SEQ ID NO: 1). The VL region of ramucirumab was fused with the Kappa region of the light chain to obtain the light chain of the hu56-Ramu-hIgG1ag antibody (SEQ ID NO:2). A schematic diagram of the bispecific antibody molecule is shown in Figure 4A.

[0558] Genes were synthesized based on the heavy chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 2) sequences of the bispecific antibody hu56-Ramu-hIgG1ag, respectively. PCR amplification was performed using primers, and the results were cloned into the eukaryotic expression vector pCDNA3.4 to obtain eukaryotic expression vectors pCDNA3.4-hu56-ramu-hIgG1ag and pCDNA3.4-ramu-kappacl, respectively, expressing the heavy chain and light chain.

[0559] The heavy chain variable region (anti-HEL) of the negative control antibody Iso-IgG1ag, the single-target control hu56, and the heavy chain variable region of Ramucirumab were fused with the constant region of human IgG1ag to obtain the negative control heavy chain (SEQ ID NO:24), the single-target control hu56-Fcag (SEQ ID NO:16), and the Ramucirumab-ag heavy chain (SEQ ID NO:15). The light chain variable region of the negative control antibody and the light chain variable region of Ramucirumab were fused with the light chain constant region Kappa to obtain the negative control light chain (SEQ ID NO:25) and the Ramucirumab-ag light chain (SEQ ID NO:2).

[0560] The heavy and light chain sequences of the negative control antibody were synthesized, amplified by PCR using primers, and cloned into the eukaryotic expression vector pCDNA3.4 to obtain eukaryotic expression vectors pCDNA3.4-IsoVH-hIgG1agCH and pCDNA3.4-IsoVL-kappacl, which express the heavy and light chains, respectively.

[0561] The single-target control hu56-Fcag (SEQ ID NO:16) was synthesized, amplified by PCR using primers, and cloned into the eukaryotic expression vector pCDNA3.4 to obtain the vector pCDNA3.4-hu56-Fcag expressing hu56-Fcag.

[0562] The heavy and light chain sequences of Ramucirumab were synthesized, amplified by PCR using primers, and cloned into the eukaryotic expression vector pCDNA3.4 to obtain the eukaryotic expression vectors pCDNA3.4-RamuVH-hIgG1CHag and pCDNA3.4-RamuLC, which express the heavy and light chains, respectively.

[0563] Based on similar methods, eukaryotic expression vectors for murine bispecific antibody hu56-DC101-hIgG1ag, murine monoclonal antibody DC101-hIgG1ag, and hu56-Ramu-hIgG1 without AG mutation were constructed.

[0564] The amino acid sequences of the antibodies involved in the examples are summarized below:

[0565] 2.2 Antibody Expression and Purification

[0566] For a 100ml transient transduction system, the CHO cell density was first adjusted to 6×10⁶ cells / mL using expression culture medium. 6 Cells / mL were prepared for use. PEI and plasmid (150ug) were mixed at a mass ratio of 6:1 and allowed to react at room temperature for 5 minutes to form a complex. This complex was then added to the prepared cell suspension and incubated at 36.5±0.5℃, 7%±3% CO2, and 100±10rpm for 4 hours. The incubation speed was then adjusted to 130±10rpm and continued for 24 hours. After that, the temperature was lowered to 32℃ and incubated for another 10 days, with inhibitors and feed added as needed.

[0567] After transient cell culture, the cells were centrifuged at 4000 rpm for 10 min at 4℃, and the cell supernatant was collected. Supernatant particles were removed using a 0.22 μm filter membrane. The treated supernatant was then loaded onto a Protein A affinity column (Mabselect SuRe) equilibrated with 10 mM PB (pH 6.0). TM Unadsorbed impurities were removed by rinsing with the same buffer solution, and weakly adsorbed impurities were washed off with a high-salt buffer (25 mM PB, 500 mM NaCl, pH 7.0). The target protein was eluted with elution buffer (20 mM citrate buffer, pH 3.6), and the pH of the eluted protein was adjusted to 7.0 with neutralization buffer (2 M Tris-HCl, pH 9.5). The purified fractions were then analyzed by electrophoresis.

[0568] 2.3 Preparation and Characterization of ADC

[0569] Bispecific antibody hu56-ramu-hIgG1ag, negative control antibody Iso-IgG1ag, single-target control antibody hu56-Fcag, single-target control antibody Ramucirumab-ag, murine bispecific antibody hu56-DC101-IgG1ag, murine monoclonal antibody DC101-IgG1ag, and hu56-Ramu-hIgG1 without AG mutation were mixed with substrate UDP-GalNAz (Qingdao Tangzhi Pharmaceutical Technology Co., Ltd.), GalT1, and MnCl2 in Tris-HCl buffer. The pH was adjusted to 7.30±0.1, and the reaction was carried out overnight at 25-30℃ and 400 rpm. The mixture was then concentrated into DPBS by ultrafiltration to obtain the intermediate product antibody-(N3)4.

[0570] Then, LP1 (16 μL, 50 mM), DMSO (25 μL), and the previously obtained antibody-(N3)4 (10 mg / ml, 800 μL) were added sequentially to a 1.5 mL centrifuge tube. The pH was adjusted to 5.1 ± 0.2, and the reaction was carried out overnight at 25-30 °C and 400 rpm. After the reaction was complete, the solution was desalted into DPBS to obtain antibody-DXD, and its molecular weight was detected by mass spectrometry.

[0571] Using a similar method, antibody-CPT2 molecules linked to LP2 were obtained. The purity of the obtained ADC molecules was determined by DAR and SEC assays, and the results are shown in Table 1.

[0572] A schematic diagram of the obtained ADC molecule is shown in Figure 4B.

[0573] SEC testing: After diluting the samples with phosphate solution, isocratic separation was performed using a TOSOH G3000 SWxl column at a wavelength of 280 nm. Sample purity was calculated using the peak area normalization method. The purity of each ADC sample was above 95%.

[0574] Average DAR value detection: After dilution with ultrapure water, the samples were separated using a Waters / ACQUITY UPLC Protein BEH C4 column. At a wavelength of 280 nm, molecular weight was determined by retention time and peak area to calculate the number of couplings. The percentage content of each component was calculated using the response formula, and the DAR value was then calculated based on the component content and the number of couplings. As shown in Table 1, the DAR values ​​of all ADC samples were above 3.7.

[0575] Table 1. Structural composition and average DAR value of ADC molecules

[0576] Example 3: Detection of the binding ability of antibodies and ADCs to receptors

[0577] 3.1 ELISA method for detecting the binding ability of antibodies and ADCs to VEGFR family proteins.

[0578] Preparation of coating solution: Weigh 1.59g Na2CO3 and 2.93g NaHCO3 and dissolve them in 900mL of deionized water. Adjust the pH to 9.6, add water to make up to 1L, and mix well.

[0579] Coating antigen: Prepare human VEGFR1, VEGFR2, and VEGFR3 His solutions using coating buffer to a final concentration of 1 μg / mL. Add 100 μL of the above solution to each well of a 96-well ELISA plate, seal the plate with sealing film, and incubate overnight at 2-8°C.

[0580] Detection: Discard the liquid in the plate, add 300 μL of washing buffer (1×PBST) to each well, wait 2 min, discard the liquid in the plate, and wash 3 times. Add 300 μL of blocking buffer (3% BSA-PBST) to each well of the ELISA plate, seal the plate with sealing film, and incubate at 25±2℃ for 2 h. Before the end of incubation, prepare the sample as follows (using one sample as an example): Prepare 500 μL of sample solution (30 nM) in a centrifuge tube using diluent (1% BSA-PBST). Pipette 140 μL of the diluent into the sample dilution plate, from column 2 to column 12. Pipette 250 μL of the prepared sample solution into the dilution plate, from column 1 to column 11, using a multichannel pipette. Perform serial dilutions from column 1 to column 11, with a dilution factor of 3. After washing the plate, use a multichannel pipette to transfer 100 μL of the sample solution from the dilution plate to the microplate, one well at a time. Seal the microplate and incubate at 25 ± 2 °C for 2 h. After washing the plate again, add 100 μL of Goat Anti-Human IgG (Fc specific) Secondary Antibody to each well. Seal the plate with sealing film and incubate at 25 ± 2 °C for 2 h. After washing the plate again, add 100 μL of TMB solution to each well and incubate at room temperature in the dark for 5 min. Add 100 μL of stop solution (1 M H2SO4) to each well to stop the reaction. Read the absorbance at 450 nm using a microplate reader with 650 nm as the reference wavelength. Analyze the data using a 4-parameter curve fitting method. The results showed that hu56-Ramu-hIgG1ag-DXd, hu56-Ramu-hIgG1ag, and Ramucirumab-ag could all bind to human VEGFR2 protein, and the binding EC10 was 100%. 50 The values ​​were 0.180 nM, 0.204 nM, and 0.193 nM, respectively, indicating comparable affinity; however, they did not bind to human VEGFR1 and VEGFR3 proteins.

[0581] 3.2 ELISA method was used to detect the binding ability of antibodies and ADCs to VEGFR2 proteins of different species.

[0582] Following the method described in 3.1, the sample to be tested was added to an ELISA plate pre-coated with antigen (VEGFR2 protein from different species) and incubated. Then, the detection antibody (human IgG Fc tag specific) was added, forming a solid-phase antigen-sample-ELISA antibody complex. The complex was then analyzed using EC... 50The binding affinity of the samples to VEGFR2 proteins from different species was assessed. Results showed that hu56-Ramu-hIgG1ag-DXd, hu56-Ramu-hIgG1ag, and Ramucirumab-ag did not bind to VEGFR2 proteins from rats or mice. The EC50 values ​​of hu56-Ramu-hIgG1ag-DXd for binding to human and cynomolgus monkey VEGFR2 proteins were 0.238 nM and 0.320 nM, respectively; the EC50 values ​​of hu56-Ramu-hIgG1ag for binding to human and cynomolgus monkey VEGFR2 proteins were 0.214 nM and 0.304 nM, respectively; and the EC50 values ​​of Ramucirumab-ag for binding to human and cynomolgus monkey VEGFR2 proteins were 0.184 nM and 0.219 nM, respectively.

[0583] Data showed that ADC hu56-Ramu-hIgG1ag-DXd significantly bound to VEGFR2 protein in both humans and cynomolgus monkeys, but did not bind to VEGFR2 protein in rats or mice.

[0584] 3.3 ELISA method for detecting the binding ability of antibodies and ADCs to PD-L1 in different species

[0585] Following the method described in 3.1, the binding affinity of the samples to PD-L1 from different species was assessed. The results showed that hu56-Ramu-hIgG1ag-DXd, hu56-Ramu-hIgG1ag, and hu56-Fcag did not bind to PD-L1 protein in rats and mice. The EC50 values ​​of hu56-Ramu-hIgG1ag-DXd with human and cynomolgus monkey PD-L1 proteins were 0.287 nM and 0.136 nM, respectively; the EC50 values ​​of hu56-Ramu-hIgG1ag with human and cynomolgus monkey PD-L1 proteins were 0.318 nM and 0.147 nM, respectively; the EC50 values ​​of hu56-Fcag-C9 with human and cynomolgus monkey PD-L1 proteins were 0.394 nM and 0.152 nM, respectively; and the affinity of hu56-Ramu-hIgG1ag-DXD, hu56-Ramu-hIgG1ag, and hu56-Fcag with human and cynomolgus monkey PD-L1 proteins were comparable.

[0586] 3.4 Detection of the affinity of antibodies and ADCs for human and cynomolgus monkey VEGFR2 proteins using BLI technology

[0587] The test sample solutions were prepared using PBST, with a final concentration of 10 μg / mL. A VEGFR2 gradient solution was prepared using PBST, starting at 250 nM and diluted 2-fold, for a total of 8 spots. The last well was set as a blank well (without protein). Equilibration buffer (PBST), test sample solutions, VEGFR2 gradient solutions, regeneration buffer (10 mM Glycine-HCl), and neutralization buffer (PBST) were added to the blackboard. Detection was performed using the proA probe, and the data were analyzed using Data Analysis HT 12.0 software. Background subtraction was performed using a reference sensor, and a 1:1 model was used for fitting to obtain the affinity results.

[0588] The results showed that, regarding binding to human VEGFR2 protein, the KD values ​​of hu56-Ramu-hIgG1ag-DXd were 0.647 nM, hu56-Ramu-hIgG1ag was 0.336 nM, and Ramucirumab-ag was 0.725 nM, indicating similar affinity. Regarding binding to cynomolgus monkey VEGFR2 protein, the KD values ​​of hu56-Ramu-hIgG1ag-DXd were 3.47 nM, hu56-Ramu-hIgG1ag was 2.03 nM, and Ramucirumab-ag was 2.62 nM, also indicating similar affinity.

[0589] 3.5 Detection of the affinity of antibodies and ADCs for human and cynomolgus monkey PD-L1 using BLI technology

[0590] Following the method described in section 3.4, the affinity of the antibody and ADC for human and cynomolgus monkey PD-L1 was determined. The results showed that for human PD-L1 protein, the KD values ​​of hu56-Ramu-hIgG1ag-DXd were 0.447 nM, hu56-Ramu-hIgG1ag was 0.448 nM, and hu56-Fcag was 0.591 nM, indicating similar affinity. For binding to cynomolgus monkey PD-L1 protein, the KD values ​​of hu56-Ramu-hIgG1ag-DXd were 1.24 nM, hu56-Ramu-hIgG1ag was 1.06 nM, and hu56-Fcag was 1.31 nM, also indicating similar affinity.

[0591] 3.6 ELISA method for detecting the simultaneous binding activity of antibody and ADC to PD-L1 and VEGFR2.

[0592] Coating antigen: Prepare human VEGFR2 Fc solution using coating buffer (preparation method as in 3.1) to a final concentration of 2 μg / mL. Add 100 μL of the above human VEGFR2 Fc solution to each well of a 96-well ELISA plate, seal the plate with sealing film, and incubate overnight at 2-8°C.

[0593] Detection: Discard the liquid in the plate, add 300 μL of washing buffer (1×PBST) to each well, wait 2 min, discard the liquid in the plate, and wash 3 times. Add 300 μL of blocking buffer (3% BSA-PBST) to each well of the ELISA plate, seal the plate with sealing film, and incubate at 25±2℃ for 2 h. Before the end of incubation, prepare the sample as follows (using one sample as an example): Prepare 500 μL of sample solution (30 nM) in a centrifuge tube using diluent (1% BSA-PBST). Pipette 140 μL of the diluent into the sample dilution plate, from column 2 to column 12. Pipette 250 μL of the prepared sample solution into the dilution plate, from column 1 to column 11, using a multichannel pipette. Perform serial dilutions from column 1 to column 11, with a dilution factor of 3. Wash the plate, then use a multichannel pipette to transfer 100 μL of the sample solution from the dilution plate to the microplate, 100 μL per well. Seal the microplate and incubate at 25 ± 2 °C for 2 h. After washing the plate again, prepare the PDL1-His solution to a final concentration of 300 ng / mL using dilution buffer. Add 100 μL of the PDL1-His solution to each well of the microplate, seal the plate with sealing film, and incubate at 25 ± 2 °C for 1 h. Add 100 μL of the Anti-6X His tag antibody (HRP) detection antibody to each well of the microplate, seal the plate with sealing film, and incubate at 25 ± 2 °C for 2 h. After washing the plate again, add 100 μL of TMB solution to each well of the microplate and incubate at room temperature in the dark for 5 min. Add 100 μL of stop solution (1 M H2SO4) to each well of the microplate to stop the reaction. On the microplate reader, absorbance values ​​were read at 450 nm with 650 nm as the reference wavelength, and data analysis was performed using a 4-parameter fitting curve analysis method.

[0594] The results showed that hu56-Ramu-hIgG1ag-DXd simultaneously binds to both VEGFR2 and PDL1 proteins in EC. 50 The value was 0.106 nM, indicating that hu56-Ramu-hIgG1ag simultaneously binds to VEGFR2 and PDL1 proteins in EC. 50 The value was 0.090 nM; neither Ramucirumab-ag nor hu56-Fcag could bind to VEGFR2 and PDL1 proteins simultaneously.

[0595] Example 4: Detection of the ability of antibodies and ADCs to block ligand-receptor binding

[0596] 4.1 ELISA method for detecting the ability of antibodies and ADCs to block the binding of PD-L1 and PD1

[0597] Coating antigen: Prepare PDL1-Fc solution using coating buffer (preparation method as in 3.1) to a final concentration of 5 μg / mL. Add 100 μL of the above PDL1-Fc solution to each well of a 96-well ELISA plate, seal the plate with sealing film, and incubate overnight at 2-8℃.

[0598] Detection: Discard the liquid in the plate, add 300 μL of washing buffer (1×PBST) to each well, wait 2 min, discard the liquid in the plate, and wash 3 times. Add 300 μL of blocking buffer (3% BSA-PBST) to each well of the ELISA plate, seal the plate with sealing film, and incubate at 25±2℃ for 2 h. Before the end of incubation, prepare the sample as follows, taking one sample as an example: In a centrifuge tube, prepare 500 μL of sample solution with a concentration of 50 nM using diluent (1% BSA-PBST) containing 10 μg / mL PD1-muFc. Pipe the diluent into the sample dilution plate, 140 μL per well from columns 2 to 12. Use a pipette to pipette the prepared sample solution into the dilution plate, 250 μL per well from column 1. Use a multichannel pipette to perform serial dilutions from columns 1 to 11, with a dilution factor of 3. After washing the plate, use a multichannel pipette to transfer 100 μL of the sample solution from the dilution plate to the ELISA plate in each well. Seal the ELISA plate and incubate at 25 ± 2 °C for 2 h. After washing the plate again, add 100 μL of Goat anti-mouse IgG1 HRP detection antibody to each well of the ELISA plate, seal the plate with sealing film, and incubate at 25 ± 2 °C for 2 h. After washing the plate again, add 100 μL of TMB solution to each well of the ELISA plate and incubate at room temperature in the dark for 5 min. Add 100 μL of stop solution (1 M H2SO4) to each well to stop the reaction. Read the absorbance at 450 nm using a microplate reader with 650 nm as the reference wavelength. Analyze the data using a 4-parameter curve fitting method.

[0599] The results showed that hu56-Ramu-hIgG1ag-DXd blocks the IC50 of PD-L1 and PD-1 protein binding. 50 The value was 1.718 nM; hu56-Ramu-hIgG1ag had an IC50 value of 1.718 nM for blocking the binding of PD-L1 and PD-1 proteins. 50 The value was 1.647 nM; the IC50 value of hu56-Fcag in blocking the binding of PD-L1 and PD-1 proteins was 1.647 nM. 50 The value is 1.547 nM. The bispecific antibody and its ADC disclosed herein can block the binding of PD-L1 and PD-1 proteins, and the blocking ability is similar to that of the single-target parent antibody hu56-Fcag.

[0600] 4.2 ELISA method for detecting the ability of antibodies and ADCs to block the binding of VEGF-A to VEGFR2.

[0601] Using a method similar to 4.1, a mixture of hu56-Ramu-hIgG1ag-DXd and other samples co-incubated with VEGF-A was added to an ELISA plate pre-coated with antigen 1 (VEGFR2-Fc) and incubated. Horseradish peroxidase-labeled detection antibody (SA-HRP) was then added, forming a solid-phase antigen 1-sample-VEGF-A mixture-ELISA antibody complex. This complex was then analyzed using IC50. 50 The value was used to assess the ability of the test sample to block the binding of VEGF-A and VEGFR2. The results showed that hu56-Ramu-hIgG1ag-DXd had an IC50 value for blocking VEGF-A and VEGFR2 proteins. 50 The value was 0.872 nM; hu56-Ramu-hIgG1ag had an IC50 value of 0.872 nM for blocking VEGF-A and VEGFR2 proteins. 50 The value was 0.884 nM; the IC50 value of Ramucirumab-ag in blocking VEGF-A and VEGFR2 proteins was 0.884 nM. 50 The value is 0.941 nM. The bispecific antibody and its ADC disclosed herein can block the binding of VEGF-A to VEGFR2, and the blocking ability is similar to that of the single-target parent antibody Ramucirumab-ag.

[0602] Example 5: Binding of the ADC to be tested to tumor cells

[0603] 5.1 Binding of the ADC molecule to be tested to NCI-H441

[0604] NCI-H441 cells (human lung adenocarcinoma cells, purchased from Nanjing Kebai Biotechnology) were cultured in complete medium (RPMI-1640 + 10% FBS + 1% P / S). After the NCI-H441 cells (moderate PD-L1 expression, very low VEGFR2 expression) reached a sufficient cell volume, they were digested with 0.25% Trypsin-EDTA (1X) Solution (Gibco, catalog number 25200056) and harvested by centrifugation. The cell density was then adjusted to 1×10⁻⁶ cells / year. 6Cells / ml, 100 μl per well, was added to a 96-well flow cytometry plate and centrifuged. The ADC molecule was diluted with PBS (containing 3% BSA) to a maximum concentration of 100 nM, and serially diluted three-fold for a total of 9 spots. 100 μl of the diluted antibody sample was added to each well of the aforementioned 96-well flow cytometry plate containing cells, and incubated at 4°C for 30 min, followed by washing three times with PBS (containing 3% BSA). Then, 100 μl of mouse anti-human IgG-Fc (purchased from Biolegend, PE-labeled, catalog number 366904), diluted 100-fold with PBS, was added to each well, and incubated at 4°C for 30 min; after that, the cells were washed three times with PBS, and 100 μl of PBS was added to each well to resuspend the cells. Finally, the samples were detected using a CytoFlex flow cytometer (purchased from Bechman), and the corresponding median fluorescence intensity (Median-PE) was calculated. The binding of the ADC was evaluated based on the median fluorescence intensity.

[0605] The experimental results are shown in Figure 1. Both the test ADC molecule hu56-Ramu-hIgG1ag-DXd and the control hu56-Fcag-DXd could bind to NCI-H441 tumor cells. Ramucirumab-ag-DXd and the negative control ISOag-DXd did not bind to NCI-H441 tumor cells.

[0606] 5.2 Binding of the ADC molecule to be tested to HCC4006 cells

[0607] HCC4006 cells (human lung cancer adenocarcinoma cells, purchased from Beina Biotechnology) were cultured in complete medium (RPMI-1640 + 10% FBS + 1% P / S). After the HCC4006 cells (moderate PD-L1 expression, VEGFR2 negative) reached a sufficient cell volume, they were digested with 0.25% Trypsin-EDTA (1X) Solution (Gibco, catalog number 25200056) and harvested by centrifugation. The cell density was then adjusted to 1×10⁻⁶ cells / year. 6Cells / ml, 100 μl per well, was added to a 96-well flow cytometry plate and centrifuged. The ADC molecule was diluted with PBS (containing 3% BSA) to a maximum concentration of 200 nM, and serially diluted three-fold for a total of 9 spots. 100 μl of the diluted antibody sample was added to each well of the aforementioned 96-well flow cytometry plate containing cells, and incubated at 4°C for 30 min, followed by washing three times with PBS (containing 3% BSA). Then, 100 μl of mouse anti-human IgG-Fc (purchased from Biolegend, APC-labeled, catalog number 366906), diluted 100-fold with PBS, was added to each well, and incubated at 4°C for 30 min; after that, the cells were washed three times with PBS, and 100 μl of PBS was added to each well to resuspend the cells. Finally, the samples were detected using a CytoFlex flow cytometer (purchased from Bechman), and the corresponding median fluorescence intensity (Median-APC) was calculated. The binding of the ADC was evaluated based on the median fluorescence intensity.

[0608] The experimental results are shown in Figure 2. Both the test ADC molecule hu56-Ramu-hIgG1ag-DXd and the control hu56-Fcag-DXd can bind to tumor cells HCC4006.

[0609] 5.3 Binding of the ADC molecule to be tested to Karpas299 cells

[0610] Karpas299 cells (human large cell lymphoma cells, purchased from Nanjing Kebai Biotechnology) were cultured in complete medium (RPMI-1640 + 10% FBS + 1% P / S). After the Karpas299 cells (moderate PD-L1 expression, VEGFR2 negative) reached a sufficient cell volume, they were dispersed and harvested by centrifugation. The cell density was then adjusted to 1×10⁻⁶ cells / year. 6 Cells / ml, 100 μl per well, was added to a 96-well flow cytometry plate and centrifuged. Antibody was diluted with PBS (containing 3% BSA) to a maximum concentration of 200 nM, and serially diluted three-fold for a total of 9 spots. 100 μl of the diluted antibody sample was added to each well of the aforementioned 96-well flow cytometry plate containing cells, and incubated at 4°C for 30 min, followed by washing three times with PBS (containing 3% BSA). Then, 100 μl of mouse anti-human IgG-Fc (purchased from Biolegend, APC-labeled, catalog number 366906), diluted 100-fold with PBS, was added to each well, and incubated at 4°C for 30 min; after that, the cells were washed three times with PBS, and 100 μl of PBS was added to each well to resuspend the cells. Finally, the samples were analyzed using a CytoFlex (purchased from Bechman) flow cytometer to detect median fluorescence intensity (Median-APC), and the binding of the target ADC was evaluated based on the median fluorescence intensity.

[0611] The experimental results are shown in Figure 3. Both the test ADC molecule hu56-Ramu-hIgG1ag-DXd and the control hu56-Fcag-DXd can bind to the tumor cells Karpas299.

[0612] Following the methods described in 5.1-5.3, the binding affinity of ADC molecules to other tumor cell lines that express only PD-L1 but not VEGFR2 (KDR) was examined: human colorectal cancer cell line RKO (purchased from Nanjing Kebai Biotechnology, culture medium MEM + 10% FBS + 1% NEAA + 1mM NaP), non-small cell lung cancer cell line HCC827 (purchased from Nanjing Kebai Biotechnology, culture medium RPMI-1640 + 10% FBS), and human lung squamous cell carcinoma cell line EBC-1 (purchased from Nanjing Kebai Biotechnology, culture medium MEM + 10% FBS + 1% NEAA + 1mM NaP). The results, as shown in Figures 5a-5c, indicate that hu56-Ramu-hIgG1ag-DXd can effectively bind to PD-L1 positive cells. Furthermore, in HCC827 and EBC-1 cells, the binding affinity of hu56-Ramu-hIgG1ag-DXd is slightly higher than that of the corresponding bispecific antibody hu56-Ramu-hIgG1ag and the monoclonal antibody ADC hu56-Fcag-DXd.

[0613] Simultaneously, the binding ability of ADC molecules to 293T-KDR cells (a recombinant cell line overexpressing VEGFR2 but not PD-L1; construction method: constructing a VEGFR2 overexpression pCDNA3.4 plasmid, stably transfecting the plasmid into 293T cells using Lipo3000 transfection reagent, obtaining single clones through limiting dilution, and constructing a library after flow cytometry identification) was tested. The results are shown in Figure 5d. hu56-Ramu-hIgG1ag-DXd, hu56-Ramu-hIgG1ag, and Ramucirumab-ag-DXd effectively bound to 293T-KDR cells, while hu56-Fcag-DXd and Isoag-DXd did not show significant binding activity even at the highest detection concentration.

[0614] Example 6: Study on antibody endocytosis

[0615] 6.1 Internalization of antibodies in a human colorectal cancer RKO cell line model

[0616] Take RKO cells in the logarithmic growth phase from a T75 culture flask, discard the culture medium, wash twice with DPBS, and aspirate the liquid. Add 2 mL of 0.25% trypsin-EDTA to the T75 culture flask and incubate at 37°C for 2 min to dissociate the cells. Transfer the cell digestion solution to a 15 mL centrifuge tube, add 6 mL of growth medium to stop digestion, and prepare a single-cell suspension. Centrifuge at 1500 rpm for 5 min. Discard the supernatant, resuspend the cells in 2 mL of growth medium, mix 20 μL of the cell suspension with 20 μL of 0.2% trypan blue, and count the cells using a cell counter. Adjust the cell density to 4 × 10⁶ cells / mL with growth medium. 4 Cells / mL, then 100 μL / well of cell suspension was added to a 96-well plate and incubated overnight at 37°C.

[0617] Drug samples, including the control, were diluted to 30 nM with growth medium (working concentration on cells: 10 nM). A negative control group was also included, with growth medium added directly. Each sample was tested in triplicate. Incucyte human Fabfluor red fluorescent antibody labeling reagent was diluted to 90 nM with growth medium. The antibody-drug solution and antibody-labeled staining solution were mixed at a 1:1 volume ratio and incubated at room temperature for 15 min. The culture medium in the 96-well plate containing the cells was discarded, and 50 μL of fresh growth medium was added. Then, 100 μL of the labeling reagent and antibody mixture was added to the 96-well plate. After incubation at room temperature for 1 hour, the 96-well plate was placed in the Incucyte instrument, and the program was set to record changes in red fluorescence and bright-field signals over time to assess internalization.

[0618] As shown in Figure 6a, hu56-Ramu-hIgG1ag can be effectively endocytosed by RKO cells. In terms of endocytosis signal intensity, the endocytosis capacity of hu56-Ramu-hIgG1ag is higher than that of hu56-Fcag.

[0619] 6.2 Internalization of antibodies in the VEGFR2 overexpressing cell line 293T-KDR model

[0620] Following the method described in 6.1, the endocytosis of the antibody in the VEGFR2-overexpressing 293T-KDR cell line model was evaluated. The results are shown in Figure 6b. hu56-Ramu-hIgG1ag was endocytosed by 293T-KDR cells, and its endocytic capacity was comparable to that of Ramucirumab-ag.

[0621] Example 7: In vitro killing effect of ADC

[0622] 7.1 Killing effect of ADC on PD-L1 overexpressing cell line 293T-PD-L1 model

[0623] Take 293T-PD-L1 cells (a recombinant cell line overexpressing PD-L1, constructed by constructing a PD-L1 overexpressing pCDNA3.4 plasmid, stably transfecting 293T cells with Lipo3000 transfection reagent, obtaining single clones through limiting dilution, and constructing a library after flow cytometry identification) in the logarithmic growth phase from a T75 culture flask, discard the culture medium, wash twice with DPBS, and aspirate the liquid. Add 2 mL of 0.25% trypsin-EDTA to the T75 culture flask and incubate at 37°C for 1 min to dissociate the cells. Transfer the cell digestion solution to a 15 mL centrifuge tube, add 6 mL of growth medium to stop digestion, and prepare a single-cell suspension. Centrifuge at 1000 rpm for 5 min. Discard the supernatant and resuspend the cells in 5 mL of growth medium. Mix 20 μL of the cell suspension with 20 μL of 0.2% trypan blue and count the cells using a cell counter. Adjust the cell density to 1 × 10⁶ cells / mL with growth medium. 4 cells / mL. Then add 100 μL / well of cell suspension to a 96-well plate and incubate overnight.

[0624] The test samples were serially diluted 4-fold downwards from the highest concentration of 30 nM (corresponding to a working concentration of 10 nM) using growth medium, resulting in a total of 9 concentrations. A 96-well plate was removed from the incubator, and 50 μL / well of each diluted sample was added to the cells. A control group (NC) was also set up, with 50 μL / well of growth medium added. The 96-well plate was returned to a 37°C, 5% CO2 incubator for 120 h of incubation. After incubation, 50 μL of Cell Counting-Lite 2.0 Luminescent Cell Viability Assay reagent was added to each well, thoroughly mixed, and 100 μL / well of the mixture was transferred to a 96-well plate. The luminescence intensity was measured, and the cytotoxicity of different drugs to the cells was calculated.

[0625] As shown in Figure 7a, hu56-Ramu-hIgG1ag-DXd can effectively induce a decrease in the viability of 293T-PD-L1 cells, and its cell-killing ability is comparable to that of the parental single-target ADC hu56-Fcag-DXd.

[0626] 7.2 Killing effect of ADC on VEGFR2 overexpressing cell line 293T-KDR

[0627] The killing effects of hu56-Ramu-hIgG1ag-DXd and the control drug on VEGFR2 single-positive 293T-KDR cells were evaluated using the method described in section 7.1. The results, shown in Figure 7b, indicate that hu56-Ramu-hIgG1ag-DXd effectively induced a decrease in the viability of 293T-KDR cells, and its cell-killing ability was comparable to that of the parental single-target ADC Ramucirumab-ag-DXd.

[0628] 7.3 Killing effect of ADCs on tumor cell lines

[0629] Using the method described in 7.1, the in vitro killing activity of the ADC against human colorectal cancer cell line RKO, human non-small cell lung cancer cell line HCC827, and human lung squamous cell carcinoma cell line EBC-1 was tested. The results, shown in 7c-7e, indicate that hu56-Ramu-hIgG1ag-DXd exhibited superior tumor cell killing activity compared to the parental single-target ADCs hu56-Fcag-Dxd and Ramucirumab-ag-DXd.

[0630] Example 8: Detection of the affinity of antibodies and ADCs for Fcγ receptors, C1q, and FcRn using BLI technology

[0631] Taking the detection of the affinity between the antibody and ADC and CD64 as an example:

[0632] The test sample solution was prepared using PBST to a final concentration of 10 μg / mL. A CD64 gradient solution was prepared using PBST, starting at a concentration of 500 / 100 nM, diluted 2-fold, for a total of 8 spots. The last well was set as a blank well (without protein). Equilibration buffer (PBST), test sample solution, CD64 gradient solution, regeneration buffer (10 mM Glycine-HCl), and neutralization buffer (PBST) were added to the blackboard. Detection was performed using the proA probe, and the data were analyzed using Data Analysis HT 12.0 software. Background subtraction was performed using a reference sensor, and a 1:1 model was used for fitting to obtain the affinity results.

[0633] The results showed that, regarding binding to the CD64 protein, the affinity of hu56-Ramu-hIgG1ag-DXd containing a mutation reducing Fc function was significantly decreased by 664.1-fold compared to the unmutated hu56-Ramu-hIgG1-DXd. This indicates that the mutation in the Fc region of hu56-Ramu-hIgG1ag-DXd can significantly reduce its affinity for CD64. The binding abilities of hu56-Ramu-hIgG1ag-DXd and hu56-Ramu-hIgG1ag to the CD64 protein were comparable, indicating that coupling has no effect on the binding of CD64 to Fc.

[0634] Using a similar method, the affinity of the antibody and ADC for CD32a, CD16a, C1q, and FcRn was detected. The results showed that hu56-Ramu-hIgG1ag-DXd and hu56-Ramu-hIgG1ag, both containing Fc-reducing mutations, could not bind to CD32a, CD16a, and C1q. Compared to antibodies and ADCs containing wild-type Fc, the Fc mutations in hu56-Ramu-hIgG1ag-DXd and hu56-Ramu-hIgG1ag had no significant effect on their binding to FcRn. Furthermore, the affinity of hu56-Ramu-hIgG1ag-DXd for FcRn was similar to that of the antibody hu56-Ramu-hIgG1ag, indicating that conjugation had no significant effect on FcRn binding.

[0635] Example 9: In vivo pharmacodynamic study of ADC

[0636] 9.1 Pharmacodynamic study of ADC in a human non-small cell lung cancer EBC-1 cell line subcutaneous xenograft model

[0637] The anti-VEGFR2 moiety in hu56-Ramu-hIgG1ag-DXd recognizes human VEGFR2 protein but not mouse VEGFR2 protein. To evaluate the efficacy of the ADC in a mouse model, we replaced the anti-VEGFR2 moiety in the ADC with the sequence of the antibody DC101, which is derived from anti-mouse VEGFR2, to prepare the mouse ADC substitute hu56-DC101-IgG1ag-Dxd. hu56-DC101-IgG1ag-Dxd can bind to PD-L1 protein on human tumor cells and also bind to VEGFR2 protein on mouse-derived vascular endothelial cells, inhibiting angiogenesis in mice, thus making it suitable for efficacy evaluation in a CDX mouse model of human tumor cells.

[0638] EBC-1 cells (seeding quantity: 5 × 10⁶) were seeded. 6The tumor cells were resuspended in PBS and mixed 1:1 with matrix gel before being subcutaneously inoculated into BALB / c nude mice (purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.). The tumors were allowed to grow until the average tumor volume reached 176.32 mm². 3 Animals were randomly assigned to four groups based on tumor volume and body weight: a PBS control group, a hu56-DC101-IgG1ag-Dxd group (10 mpk), a hu56-DC101-IgG1ag-Dxd group (20 mpk), and a hu56-DC101-IgG1ag-Dxd group (40 mpk), with six animals in each group. Day 0 was defined as the day of grouping. Drug administration was performed via tail vein injection on days 0, 3, 7, 10, and 14. Tumor volume and body weight were measured 2-3 times per week during the experiment. Changes in tumor volume were used to assess antitumor efficacy; changes in body weight and survival were used to assess safety. The experiment ended on day 30, and the TGI TV on day 30 was calculated for efficacy evaluation. TGI TV Calculation of TGI (%): TGI (%) = (1 - average tumor volume at the end of treatment in a certain treatment group / average tumor volume at the end of treatment in the solvent control group) × 100%.

[0639] The results are shown in Figures 8a and 8b. During treatment, none of the test substances significantly affected the body weight of the mice, no mice died in any group, and the mice showed no obvious abnormalities or drug toxicity. The mice tolerated the drugs well. On day 30 after the start of treatment, the average tumor volume in the PBS control group reached 2085.97 mm. 3 Based on tumor volume data at this time point, the antitumor effect was analyzed. The hu56-DC101-IgG1ag-Dxd group (10 mpk) showed a tumor inhibition rate of 53.44%, significantly inhibiting tumor growth compared to the PBS group (p = 0.0002); the hu56-DC101-IgG1ag-Dxd group (20 mpk) showed a tumor inhibition rate of 82.97%, also demonstrating a significant antitumor effect (p = 0.0001); and the hu56-DC101-IgG1ag-Dxd group (40 mpk) showed a tumor inhibition rate of 95.90%, significantly inhibiting tumor growth (p = 0.0001). Therefore, the ADC disclosed in this study exhibits significant dose-dependent antitumor activity and good drug safety in a subcutaneous transplantation model of squamous non-small cell lung cancer EBC-1 cell line.

[0640] 9.2 Pharmacodynamic study of ADC in a human colorectal cancer RKO cell line subcutaneous xenograft model

[0641] This study systematically evaluated the antitumor activity and safety of the disclosed murine version of the ADC in a CDX model of human colorectal cancer RKO cell line expressing PD-L1. RKO cells (2 × 10⁻⁶) were used. 6Mixed with matrix gel at a 1:1 ratio, the mixture was subcutaneously injected into BALB / c nude mice. The tumor volume was increased until the average size reached approximately 104.35 mm². 3 Animals were randomly assigned to four groups based on tumor volume and body weight: a PBS control group, an Iso-hIgG1ag-DXd group (16 mpk), a hu56-DC101-IgG1ag group (19 mpk), a hu56-Fcag-DXd group (9 mpk), a DC101-hIgG1ag-DXd group (17 mpk), and a hu56-DC101-IgG1ag-DXd group (20 mpk). Dosage was calculated based on molecular weight using equimolar ratios. Six animals were assigned to each group. Day 0 was defined as the day of grouping. Drugs were administered via tail vein injection on days 0 and 7. In the PBS group, the average tumor volume exceeded 2000 mmHg on day 16. 3 Upon reaching the euthanasia endpoint, mice in the PBS group, Iso-hIgG1ag-Dxd group, and hu56-DC101-IgG1ag group were euthanized, while mice in other groups were preserved for continued observation of drug efficacy. Tumor volume and body weight were measured 2-3 times per week during the experiment; changes in tumor volume were used to assess antitumor efficacy; and safety was assessed based on changes in body weight and survival status.

[0642] The results are shown in Figures 9a and 9b. During treatment, none of the test substances significantly affected the body weight of the mice, no mice died in any group, and the mice showed no obvious abnormalities or drug toxicity. The mice tolerated the drugs well. On day 16 after the start of treatment, the average tumor volume in the PBS control group reached 2254.52 mm. 3 Based on tumor volume data at this time point, the anti-tumor effects were analyzed. Iso-hIgG1ag-DXd (16 mpk) showed a tumor inhibition rate of 18.44%, which was not significantly different from the PBS group (p = 0.0646); hu56-DC101-IgG1ag (19 mpk) showed a tumor inhibition rate of 29.53%, indicating a weak tumor-suppressing effect compared to the PBS group (p = 0.0012); the hu56-Fcag-DXd group (9 mpk) showed a tumor inhibition rate of 92.67%, significantly inhibiting tumor growth compared to the PBS group (p = 0.0001); the DC101-hIgG1ag-DXd (17 mpk) showed a tumor inhibition rate of 56.41%, significantly inhibiting tumor growth compared to the PBS group (p = 0.0001); and the hu56-DC101-IgG1ag-DXd (20 mpk) showed a tumor inhibition rate of 98.65%, significantly inhibiting tumor growth compared to the PBS group (p = 0.0001). It is evident that the ADC disclosed herein exhibits significant antitumor efficacy and good drug safety in a subcutaneous xenograft model of human colorectal cancer RKO cell line.

[0643] 9.3 Pharmacodynamic study of ADC in B-hVEGFA / hPD-L1 plus MC38 colorectal cancer animal model based on B-hPD-1 / hPD-L1 / hVEGFR2 humanized mice

[0644] Mouse colon cancer MC38 cells were purchased from Shunran Shanghai Biotechnology Co., Ltd. Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd. genetically modified the MC38 cells to express human VEGFA and PD-L1; these cells were named B-hVEGFA / hPD-L1 plus MC38 cells (clone number 1-B07). Cells were cultured at 37°C in a 5% CO2 incubator using Dulbecco's Modified Eagle's Medium containing 10% inactivated fetal bovine serum.

[0645] B-hVEGFA / hPD-L1 plus MC38 cells were loaded at 5×10⁻⁶. 5 0.1 mL / mouse was injected subcutaneously into the right back of 40 B-hPD-1 / hPD-L1 / hVEGFR2 humanized mice (provided by Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd.). The tumor volume was measured at 88 mm². 3 Twenty-four mice were randomly divided into four groups of six each, based on tumor volume and body weight: G1: PBS(-), G2: hu56-Ramu-hIgG1ag (10 mg / kg), G3: hu56-Ramu-hIgG1ag-DXd (10 mg / kg), and G4: hu56-Ramu-hIgG1ag-DXd (5 mg / kg). Administration was administered via tail vein for the first two injections and intraperitoneal injection for the third, once weekly for three consecutive weeks. The experiment ended on day 43 after grouping. Serum was collected from mice 72 hours after the last administration, at the humane endpoint, and at the experimental endpoint. Body weight and tumor volume were measured twice weekly during administration and observation, and the values ​​were recorded. At the end of the experiment, the animals were euthanized, the tumors were harvested, photographed, and the tumor growth inhibition rate was calculated.

[0646] The results are shown in Figures 10a and 10b. The animals maintained good activity and appetite during the administration period, and their body weight increased to some extent. Compared with the PBS control group, the test products hu56-Ramu-hIgG1ag and hu56-Ramu-hIgG1ag-DXd significantly inhibited the subcutaneous transplantation of B-hVEGFA / hPD-L1 plus MC38 tumor cells in B-hPD-1 / hPD-L1 / hVEGFR2 mice at the test dose. On Day 19, the mean tumor volume in the G1 control group was 1932 ± 387 mm.3 Based on the tumor volume data at this time point, the anti-tumor effect was analyzed. The average tumor volumes of groups G2-G4 were 406±43 mm3, 136±19 mm3, and 499±74 mm3, respectively, and the tumor growth inhibition rate (TGI) was [missing data]. TV The percentages were 79.0% (p<0.0001), 93.0% (p<0.0001), and 74.2% (p=0.0002), respectively.

[0647] Sequence information:

Claims

1. Bispecific antibodies, which consist of heavy and light chains, wherein... Heavy chain: From N-terminus to C-terminus, it contains or consists of the following: VHH PDL1 -VH VEGFR2 -CH1-Fc; Light chain: From the N-terminus to the C-terminus, it contains or consists of the following: VL VEGFR2- CL; in, VHH PDL1 It is a VHH that specifically binds to PDL1. VH VEGFR2 -CH1 and VL VEGFR2- CL constitutes a Fab that specifically binds to VEGFR2. VEGFR2 , CH1 is the CH1 domain of the heavy chain constant region, CL is the light chain constant region, and Fc is the Fc region of immunoglobulin. Preferably, the VHH PDL1 -VH VEGFR2 They are connected via a connector; Wherein, the VHH PDL1 Includes VHH CDR1, VHH CDR2, and VHH CDR3, and (i) VHH CDR1 contains the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 contains the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 contains the amino acid sequence shown in SEQ ID NO:6; or (ii) VHH CDR1 consists of the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 consists of the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 consists of the amino acid sequence shown in SEQ ID NO:6; and The VH VEGFR2 Includes HCDR1, HCDR2, and HCDR3, the VL VEGFR2 It includes LCDR1, LCDR2 and LCDR3, among which (i) HCDR1 contains the amino acid sequence shown in SEQ ID NO:9, HCDR2 contains the amino acid sequence shown in SEQ ID NO:10, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:11; LCDR1 contains the amino acid sequence shown in SEQ ID NO:12, LCDR2 contains the amino acid sequence shown in SEQ ID NO:13, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:14; or (ii) HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:9, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:10, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:

11. LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:12, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:13, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:

14.

2. Bispecific antibodies, which consist of heavy and light chains, wherein... Heavy chain: From N-terminus to C-terminus, it contains or consists of the following: VHH PDL1 -VH VEGFR2 -CH1-Fc; Light chain: From the N-terminus to the C-terminus, it contains or consists of the following: VL VEGFR2- CL; in, VHH PDL1 It is a VHH that specifically binds to PDL1. VH VEGFR2 -CH1 and VL VEGFR2- CL constitutes a Fab that specifically binds to VEGFR2. VEGFR2 , CH1 is the CH1 domain of the heavy chain constant region, CL is the light chain constant region, and Fc is the Fc region of immunoglobulin. Preferably, the VHH PDL1 -VH VEGFR2 They are connected via a connector; Wherein, the VHH PDL1 Includes VHH CDR1, VHH CDR2, and VHH CDR3, and (i) VHH CDR1 contains the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 contains the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 contains the amino acid sequence shown in SEQ ID NO:6; or (ii) VHH CDR1 consists of the amino acid sequence shown in SEQ ID NO:4; VHH CDR2 consists of the amino acid sequence shown in SEQ ID NO:5; and VHH CDR3 consists of the amino acid sequence shown in SEQ ID NO:6; and The VH VEGFR2 Includes HCDR1, HCDR2, and HCDR3, the VL VEGFR2 It includes LCDR1, LCDR2 and LCDR3, among which (i) HCDR1 contains the amino acid sequence shown in SEQ ID NO:35, HCDR2 contains the amino acid sequence shown in SEQ ID NO:36, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:37; LCDR1 contains the amino acid sequence shown in SEQ ID NO:38, LCDR2 contains the amino acid sequence shown in SEQ ID NO:39, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:40; or (ii) HCDR1 is composed of the amino acid sequence shown in SEQ ID NO:35, HCDR2 is composed of the amino acid sequence shown in SEQ ID NO:36, and HCDR3 is composed of the amino acid sequence shown in SEQ ID NO:

37. LCDR1 is composed of the amino acid sequence shown in SEQ ID NO:38, LCDR2 is composed of the amino acid sequence shown in SEQ ID NO:39, and LCDR3 is composed of the amino acid sequence shown in SEQ ID NO:

40.

3. The bispecific antibody according to claim 1 or 2, wherein the VHH PDL1 Include (i) The amino acid sequence shown in SEQ ID NO: 3, or (ii) An amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, or even more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; for example, an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

3.

4. The bispecific antibody according to claim 1 or 3, wherein the VH VEGFR2 Contains or consists of an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:7; and / or the VL VEGFR2 Contains or consists of an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:8; Preferably, the VH VEGFR2 Contains the amino acid sequence shown in SEQ ID NO:7, and the VL VEGFR2 Contains the amino acid sequence shown in SEQ ID NO:8; or the VH VEGFR2 Composed of the amino acid sequence shown in SEQ ID NO:7, and the VL VEGFR2 It consists of the amino acid sequence shown in SEQ ID NO:

8.

5. The bispecific antibody according to claim 2 or 3, wherein the VH VEGFR2 Contains or consists of an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:33; and / or the VL VEGFR2 Contains or consists of an amino acid sequence that is identical to or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:34; Preferably, the VH VEGFR2 Contains the amino acid sequence shown in SEQ ID NO:33, and the VL VEGFR2 Contains the amino acid sequence shown in SEQ ID NO:34; or the VH VEGFR2 Composed of the amino acid sequence shown in SEQ ID NO:33, and the VL VEGFR2 It consists of the amino acid sequence shown in SEQ ID NO:

34.

6. The bispecific antibody according to any one of claims 1 to 5, wherein the Fc region is a human immunoglobulin Fc region, preferably the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4. Optionally, the Fc region may also contain mutations that reduce or eliminate effector function, such as D265A / P331G; Preferably, the Fc region: (i) Contains the amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:19; (ii) Consists of the amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:19; (iii) Containing the amino acid sequence shown in SEQ ID NO:18, or containing an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:18; or (iv) Containing the amino acid sequence shown in SEQ ID NO:19, or containing an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:19 and containing D265A / P331G.

7. The bispecific antibody according to any one of claims 1 to 6, wherein the linker comprises an N-terminal fragment of the antibody constant region CH1 domain and a GAP attached to its C-terminus, for example, the N-terminal fragment being 1-20 amino acids starting from the N-terminus, such as 10 amino acids starting from the N-terminus, 11 amino acids starting from the N-terminus, or 12 amino acids starting from the N-terminus. Preferably, the connector comprises or consists of the amino acid sequence shown in SEQ ID NO:

20.

8. The bispecific antibody according to any one of claims 1 to 7, wherein the CH1 is derived from the CH1 domain of the heavy chain constant region of IgG, such as the CH1 domain of the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the CH1 domain of the heavy chain constant region of human IgG1, human IgG2, human IgG3 or human IgG4. For example, CH1 comprises the amino acid sequence shown in SEQ ID NO:17, or comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:17, or is composed of said amino acid sequence.

9. The bispecific antibody according to any one of claims 1 to 8, wherein the CL is a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region, for example, the human Kappa light chain constant region or the human Lambda light chain constant region; For example, the CL comprises the amino acid sequence shown in SEQ ID NO:23, or comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:23, or is composed of said amino acid sequence.

10. The bispecific antibody according to any one of claims 1-9, comprising two heavy chains and two light chains, wherein the two heavy chains are polymerized through an Fc region to form a dimer.

11. The bispecific antibody according to any one of claims 1-10, wherein The heavy chain comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and The light chain comprises the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; Preferably, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:1, and the light chain comprises the amino acid sequence shown in SEQ ID NO:2; or the heavy chain is composed of the amino acid sequence shown in SEQ ID NO:1, and the light chain is composed of the amino acid sequence shown in SEQ ID NO:

2.

12. The bispecific antibody according to any one of claims 1-10, wherein The heavy chain comprises the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and The light chain comprises the amino acid sequence shown in SEQ ID NO:30, or an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; Preferably, the heavy chain comprises the amino acid sequence shown in SEQ ID NO:29, and the light chain comprises the amino acid sequence shown in SEQ ID NO:30; or the heavy chain is composed of the amino acid sequence shown in SEQ ID NO:29, and the light chain is composed of the amino acid sequence shown in SEQ ID NO:

30.

13. Protein-drug conjugates having the structure of Formula I: P-(L1-sp1-L2-sp2-D)n (I), in, Protein P is a bispecific antibody as defined in any one of claims 1-12, D is a bioactive molecule, L1 is a linker for connecting with P, sp1 is a first spacer unit, L2 is a cleavable linker, sp2 is a second spacer unit connected to D, and n is an integer from 1 to 20, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

14. The protein-drug conjugate according to claim 13, wherein L1 is selected from: in, Ar represents C 6-10 aryl groups, which are optionally converted by halogens, C 1-6 Alkyl substitution; R1 is selected from hydrogen, halogen, and C. 1-6 Alkyl group; Z is selected from straight bond, C 2-6 Ethyne group, C 2-6 imidene group, C 6-10 Aryl, 5-10 heteroaryl, amide, sulfonamide, imino and CF2.

15. The protein-drug conjugate according to claim 13 or 14, wherein the structure of sp1 is as shown in Formula II: in, a1 = 0 or 1, a2 = integers from 0 to 6, b1 = 0 or 1, b2 = integers from 0 to 16, b3 = integers from 0 to 16, c = integers from 0 to 6, and at least one of b2 and b3 is 0.

16. The protein-drug conjugate according to claim 15, wherein: (1) a1 = 0, a2 = 2, 3, 4, 5 or 6, b1 = 0, b2 = 0, b3 = 0, c = 0; (2) a1 = 0, a2 = 0, b1 = 0, b2 = 0, b3 = 0, c = 2, 3, 4, 5 or 6; (3) a1 = 1, a2 = 2, 3, 4, 5 or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0; (4) a1 = 0, a2 = 2, 3, 4, 5 or 6, b1 = 1, b2 = 2, 3, 4, 5, 6, 7 or 8, b3 = 0, c = 0; or (5) a1 = 1, a2 = 0, b1 = 0, b2 = 0, b3 = 2, 3, 4, 5, 6, 7 or 8, c = 2, 3, 4, 5 or 6.

17. The protein-drug conjugate according to any one of claims 13-16, wherein, L2 is a dipeptide, tripeptide, or tetrapeptide amino acid residue.

18. The protein-drug conjugate according to claim 17, wherein L2 is selected from the following dipeptide amino acid residues: -Phe-Lys-, -Val-Ala-, -Val-Lys-, -Val-Cit-, -Ala-Lys-, -Phe-Cit-, -Leu-Cit-, -Ile-Cit-, -Phe-Arg-, -Trp-Cit-, -Gly-Gly-, -Ala-Ala-, -Gly-Val-, and -Gly-Glu-; wherein the left side of the dipeptide amino acid residue is connected to sp1 and the right side is connected to sp2.

19. The protein-drug conjugate according to claim 17, wherein L2 is selected from the following tripeptide amino acid residues: -Glu-Val-Ala-, -Glu-Val-Cit-, -αGlu-Val-Ala-, -αGlu-Val-Cit-, -Val-Lys-Gly, and -Val-Cit-Gly-; wherein the left side of the tripeptide amino acid residue is connected to sp1 and the right side is connected to sp2.

20. The protein-drug conjugate according to claim 17, wherein L2 is selected from the following tetrapeptide amino acid residues: -Gly-Gly-Phe-Gly- and -Gly-Phe-Gly-Gly-; the left side of the tetrapeptide amino acid residue is connected to sp1 and the right side is connected to sp2.

21. The protein-drug conjugate according to any one of claims 13-20, wherein sp2 is absent, or sp2 is selected from: R2 is independently selected from hydrogen, C 1-6 Alkyl, hydroxyl, amino, halogen, nitro, cyano d is an integer from 1 to 20, and e is an integer from 1 to 20; R3 and R4 are each independently selected from hydrogen and C. 1-6 alkyl; The alkyl group may optionally be substituted with hydroxyl, amino, halogen, nitro, and cyano groups.

22. The protein-drug conjugate according to any one of claims 13-21, wherein -L1-sp1-L2-sp2- is selected from the following structures: k is an integer from 1 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10.

23. The protein-drug conjugate according to any one of claims 13-22, wherein D is selected from cytotoxins, protein kinase inhibitors, immune agonists, glucocorticoids, oligonucleotides, radioisotopes, polypeptides, and any combination thereof.

24. The protein-drug conjugate according to claim 23, wherein D is a cytotoxic agent selected from: DNA alkylating agents, DNA destructive agents, topoisomerase I inhibitors, topoisomerase II inhibitors, microtubule inhibitors, ribosome inhibitors, and any combination thereof.

25. The protein-drug conjugate according to claim 24, wherein D is selected from: auristatin derivatives, maidansine derivatives, eribulin derivatives, tubulysin derivatives, pyrrolobenzodiazepine (PDB) derivatives, ducarmycin derivatives, calicacin derivatives, PNU-159682 and its derivatives, camptothecin derivatives, amatoxin derivatives, and any combination thereof.

26. The protein-drug conjugate according to claim 24, wherein D has the structure shown in Formula III: in, X is selected from CH2, NH, O, S or SO2; Y does not exist, or Y has The structure shown; W1 and W3 are each independently selected from O, S, and NH, while W2 is selected from CH and N. R a R b Each is independently selected from hydrogen, deuterium, halogens, and carbon. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocyclic alkyl, hydroxyl, amino, cyano, and nitro; or, R a and R b Together with the carbon atom it is attached to, it forms a 3-6 membered cycloalkyl, a 3-6 membered heterocycloalkyl, or a carbonyl group; or, R a The N atom of the amide moiety is attached to form a 3-6 membered heterocyclic alkyl group and R b It is hydrogen; Ring A is selected from the group consisting of 4-10-membered cycloalkylene, 4-10-membered heteroalkylene, 6-10-membered arylene and 5-10-membered heteroarylene; The alkyl, alkoxy, cycloalkyl, heterocycloalkyl, cycloalkylene, heterocycloalkylene, aryl and heteroaryl groups are each optionally and independently further substituted with groups selected from deuterium, halogen, hydroxyl, amino, cyano, carbonyl and nitro groups; d and e are each independently selected from integers from 0 to 5.

27. The protein-drug conjugate according to claim 26, wherein D has the structure shown in formula III-a: Where W1 is O or NH; R a R b Each is independently selected from hydrogen, deuterium, halogens, and carbon. 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocyclic alkyl, hydroxyl, amino, cyano, and nitro; or, R a and R b Together with the carbon atom it is attached to, it forms a 3-6 membered cycloalkyl, a 3-6 membered heterocycloalkyl, or a carbonyl group; or, R a The N atom of the amide moiety is attached to form a 3-6 membered heterocyclic alkyl group and R b It is hydrogen; Each of the alkyl, alkoxy, cycloalkyl and heterocycloalkyl groups is independently and optionally further substituted with a group selected from deuterium, halogen, hydroxyl, amino, cyano and nitro groups; d is an integer from 0 to 5.

28. The protein-drug conjugate according to claim 27, wherein, W1 is 0.

29. The protein-drug conjugate according to claim 27 or 28, wherein d is 0, 1 or 2.

30. The protein-drug conjugate according to any one of claims 27-29, wherein R a Selected from hydrogen, deuterium, halogens, and C 1-6 Alkyl, C 1-6 Alkoxy, C 3-6 Cycloalkyl, 3-6 membered heterocyclic alkyl, hydroxyl and amino, R b It is hydrogen.

31. The protein-drug conjugate according to any one of claims 27-29, wherein R a and R b Together with the carbon atoms attached thereto, they form 3-6 membered cycloalkyl or 3-6 membered heterocycloalkyl.

32. The protein-drug conjugate of claim 27, wherein D is selected from the group consisting of:

33. The protein-drug conjugate according to claim 27, wherein D has the structure shown in Formula III-b: Where W3 is O or NH, and W2 is CH and N. Ring A is selected from the group consisting of 4-10-membered cycloalkylene, 4-10-membered heteroalkylene, 6-10-membered arylene and 5-10-membered heteroarylene; Each of the cycloalkylene, heteroalkylene, arylene, and heteroarylene groups is optionally and independently further substituted with a group selected from deuterium, halogen, hydroxyl, amino, cyano, carbonyl, and nitro groups; e is an integer from 0 to 5.

34. The protein-drug conjugate according to claim 33, wherein, W3 is 0; and / or W2 is CH; and / or e is 0, 1, or 2; and / or Ring A is a 4-10 membered cycloalkylene group.

35. The protein-drug conjugate of claim 33, wherein D is selected from the group consisting of:

36. The protein-drug conjugate according to claim 25, wherein D has the structure shown in formula III-c: in, R5 is selected from hydrogen, C 1-6 Alkyl, hydroxyl, amino, and halogen, p is an integer from 0 to 5.

37. The protein-drug conjugate of claim 36, wherein D is selected from the following structures:

38. The protein-drug conjugate according to claim 25, wherein D is a camptothecin derivative; preferably from the following structures:

39. The protein-drug conjugate according to claim 13, wherein -L1-sp1-L2-sp2-D is selected from the following structures: k is an integer from 1 to 20.

40. The protein-drug conjugate according to any one of claims 13-39, wherein the L1 is linked to the multispecific antigen-binding protein P via a thiol group, the thiol group being obtained by reducing disulfide bonds between heavy chains and / or disulfide bonds between heavy chains and light chains.

41. The protein-drug conjugate according to any one of claims 13-40, wherein the L1 is linked to the multispecific antigen-binding protein P via an oligosaccharide.

42. The protein-drug conjugate according to claim 38, wherein the oligosaccharide has the structure shown in formula Va or formula Vb: in, P* is a bispecific antibody as defined in any one of claims 1-12, GlcNAc is N-acetylglucosamine, Fuc is fuc is fucose, Man is mannose, f is 0 or 1, and j is an integer from 1 to 20, for example, an integer selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Gal* is a modified galactose selected from the following structures: The oligosaccharide is connected to P* via a core GlcNAc.

43. The protein-drug conjugate according to claim 42, wherein the modified galactose is linked to GlcNAc via a β-1,4-glycosidic bond.

44. The protein-drug conjugate according to any one of claims 41-43, wherein the oligosaccharide is linked to the Fc fragment of P*; preferably linked to the CH2 domain of the Fc fragment; more preferably linked to the Asn297 (according to the EU index number of Kabat) of the Fc fragment.

45. The protein-drug conjugate according to any one of claims 13-44, wherein the protein-drug conjugate has the structure shown in Formula VI: in, P* is the bispecific antibody according to any one of claims 1-12, GlcNAc is N-acetylglucosamine, Fuc is fuc is fucose, Man is mannose, f is 0 or 1, and j is an integer from 1 to 20; Gal* is a modified galactose selected from the following structures: The oligosaccharide is connected to P* via a core GlcNAc; LP is selected from the following structures: (a)、 and / or (b) and / or (c) and / or (d) and / or (e) and / or (f) and / or as well as, (g) and / or k is an integer from 1 to 20, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10; The core GlcNAc is directly connected to P*.

46. ​​The protein-drug conjugate according to any one of claims 13-45, wherein the bispecific antibody comprises the heavy chain shown in SEQ ID NO:1 and the light chain shown in SEQ ID NO:

2.

47. The protein-drug conjugate according to any one of claims 13-45, wherein the bispecific antibody comprises the heavy chain shown in SEQ ID NO:29 and the light chain shown in SEQ ID NO:

30.

48. The protein-drug conjugate according to any one of claims 45-47, wherein the oligosaccharide is linked to the Fc fragment of P*; preferably linked to the CH2 domain of the Fc fragment; more preferably linked to the Asn297 (according to the EU index number of Kabat) of the Fc fragment.

49. The protein-drug conjugate according to any one of claims 13-48, wherein the average DAR of the protein-drug conjugate is about 2.0 to 16.0, preferably about 3.0 to 10.0, more preferably about 3.0 to 5.0, for example about 3.5, about 3.6, about 3.7, about 3.8, 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4 or about 4.

5.

50. A protein derivative having the structure shown in Formula VII: in, P* is the bispecific antibody according to any one of claims 1-12, GlcNAc is N-acetylglucosamine, Fuc is fuc, Man is mannose, f is 0 or 1, j is an integer from 1 to 20, for example selected from 1-10, 1-9, 2-8, 4-10, 3-7, 4-6 or 2-6, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; Gal** is a modified galactose selected from the following structures: The oligosaccharide is connected to P* via a core GlcNAc.

51. The protein derivative according to claim 50, wherein the core GlcNAc is linked to the Fc fragment of P*; preferably linked to the CH2 domain of the Fc fragment; more preferably linked to the Asn297 (according to the EU index number of Kabat) of the Fc fragment.

52. A composition comprising the protein-drug conjugate of any one of claims 13-49, and optionally a pharmaceutically acceptable carrier.

53. The composition according to claim 52, wherein the average DAR value of the composition is 1 to 16, preferably 2.0 to 8.0, more preferably 3.0 to 6.0, and even more preferably 3.5 to 4.

0.

54. Use of the bispecific antibody of any one of claims 1-12 or the protein-drug conjugate of any one of claims 13-49 in the preparation of a medicament for the treatment and / or prevention of tumors.

55. The use according to claim 54, wherein the tumor comprises a solid tumor and / or a non-solid tumor.

Images