Multispecific antibodies and uses thereof

Abstract

Relates to a multispecific antibody targeting a programmed cell death receptor-1 (PD-1) and a vascular endothelial growth factor (VEGF), a pharmaceutical composition containing the multispecific antibody, a nucleic acid molecule encoding the multispecific antibody, a carrier and a host cell, and uses thereof in tumor treatment.

Description

Multispecific antibodies and their uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202210852642.9 filed on July 20, 2022, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to the field of immunology, and in particular, to multispecific antibodies targeting programmed cell death receptor-1 (PD-1) and vascular endothelial growth factor (VEGF), pharmaceutical compositions comprising the multispecific antibodies, nucleic acid molecules, vectors and host cells encoding the multispecific antibodies, and their use in treating tumors. Background Art

[0004] Programmed cell death protein (PD-1) is a type I transmembrane glycoprotein surface receptor belonging to the CD28 immunoglobulin superfamily and is ubiquitously expressed on immune cells such as T cells and NK cells. PD-1 has two natural ligands, PD-L1 and PD-L2. Both PD-L1 and PD-L2 belong to the B7 superfamily and are constitutively or inducibly expressed on the surface of various cell membranes, including non-hematopoietic cells, immune cells, and tumor cells. The interaction between PD-1 and its ligands can inhibit the activation and proliferation of lymphocytes, such as T cells and NK cells, the secretion of cytokines (such as IL-2 and IFN-γ), and the killing of tumor cells.

[0005] Vascular endothelial growth factor (VEGF) is a highly specific vascular endothelial growth factor that promotes endothelial cell division and proliferation, angiogenesis, and increased vascular permeability. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, and PIGF. Vascular endothelial growth factor receptors (VEGFRs) include VEGFR1 (also known as Flt1), VEGFR2 (also known as KDR or Flk1), VEGFR3 (also known as Flt4), and Neuropilin-1 (NRP-1). VEGFR1 and VEGFR2 are primarily located on the surface of vascular endothelial cells, while VEGFR3 is primarily located on the surface of lymphatic endothelial cells. The formation of new blood vessels provides essential nutrients and oxygen for tumor growth and metastasis, making it an important target for inhibiting tumor growth. Bevacizumab is a humanized monoclonal antibody targeting VEGF A and has been approved for the treatment of various tumors, including non-small cell lung cancer, renal cell carcinoma, cervical cancer, and metastatic colorectal cancer. Ranibizumab, a Fab fragment of a monoclonal antibody with enhanced affinity for VEGF A, has been approved for the treatment of neovascular (wet) age-related macular degeneration and diabetic macular edema.

[0006] The combination of PD-1 / PD-L1 monoclonal antibodies and VEGF pathway inhibitors (bevacizumab or tyrosine kinase inhibitors such as lenvatinib and sorafenib) has achieved breakthroughs in the treatment of many tumors, including liver cancer, lung cancer, gastric cancer, kidney cancer, and various gynecological tumors. However, the incidence of adverse reactions caused by combination therapy is much higher than that of monotherapy.

[0007] Therefore, there is a need in this field to develop multispecific antibodies targeting the PD-1 / PD-L1 and VEGF pathways to provide synergistic anti-tumor efficacy while reducing the incidence of adverse reactions and making clinical use more convenient.

[0008] Summary of the Invention

[0009] The inventors of the present application have developed a multispecific antibody against PD-1 and VEGF, which can specifically block the PD-1 and VEGF signaling pathways, thereby producing a synergistic anti-tumor effect, thereby providing the following aspects.

[0010] Multispecific antibodies

[0011] In one aspect, the present invention provides a multispecific antibody comprising a first antigen-binding domain targeting programmed cell death receptor-1 (PD-1) and a second antigen-binding domain targeting vascular endothelial growth factor (VEGF), wherein the first antigen-binding domain comprises a VHH domain, wherein the VHH domain comprises: a CDR1 as shown in SEQ ID NO: 2 or a variant thereof, a CDR2 as shown in SEQ ID NO: 3 or a variant thereof, and a CDR3 as shown in SEQ ID NO: 4 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.

[0012] In certain embodiments, the VHH domain comprises: a CDR1 as set forth in SEQ ID NO:2, a CDR2 as set forth in SEQ ID NO:3, and a CDR3 as set forth in SEQ ID NO:4.

[0013] In certain embodiments, the VHH domain comprises: CDR1, CDR2 and CDR3 contained in the VHH as shown in SEQ ID NO: 1; preferably, the CDRs are determined by the Kabat, IMGT or Chothia numbering systems.

[0014] In certain embodiments, the VHH domain is typically composed of four framework regions (FRs) and three complementarity determining regions (CDRs), referred to as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The VHH domain can be truncated at the N-terminus or C-terminus to include only part of FR1 and / or FR4, or lack one or two of those framework regions, as long as it substantially retains antigen binding and specificity.

[0015] In certain embodiments, the VHH domain may comprise a framework region sequence derived from a camelid heavy chain antibody.

[0016] In certain embodiments, the VHH domain can also be humanized, i.e., wherein one or more framework regions have been substantially replaced by human framework regions. In certain embodiments, the VHH domain comprises a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region contained in the amino acid sequence encoded by a human heavy chain germline antibody gene), optionally comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) back mutations from human residues to camelid residues.

[0017] In certain embodiments, the first antigen binding domain comprises: a VHH comprising the sequence shown in SEQ ID NO: 1 or a variant thereof, wherein the variant has a sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions.

[0018] In certain exemplary embodiments, the first antigen binding domain comprises a VHH as shown in SEQ ID NO:1.

[0019] In certain embodiments, the second antigen binding domain comprises an antibody or antigen binding fragment thereof (eg, a heavy chain variable region and / or a light chain variable region thereof) that specifically binds to VEGF.

[0020] In certain embodiments, the antibody that specifically binds to VEGF is selected from bevacizumab or a variant thereof, ranibizumab or a variant thereof.

[0021] In certain embodiments, the second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) derived from bevacizumab or a variant thereof; wherein,

[0022] (i) the VH comprises CDR-H1 set forth in SEQ ID NO: 7, CDR-H2 set forth in SEQ ID NO: 8, and CDR-H3 set forth in SEQ ID NO: 9; the VL comprises CDR-L1 set forth in SEQ ID NO: 10, CDR-L2 set forth in SEQ ID NO: 11, and CDR-L3 set forth in SEQ ID NO: 12;

[0023] (ii) the VH comprises: CDR-H1, CDR-H2 and CDR-H3 contained in the VH as shown in SEQ ID NO: 5; the VL comprises: CDR-L1, CDR-L2 and CDR-L3 contained in the VL as shown in SEQ ID NO: 6; preferably, the CDRs are determined by the Kabat, IMGT or Chothia numbering systems.

[0024] In certain embodiments, the VH and / or VL further comprises a framework region derived from a mammalian (eg, human or mouse) immunoglobulin. In certain embodiments, the VH and / or VL further comprises a framework region derived from a human immunoglobulin.

[0025] In certain embodiments, the VH comprises the sequence shown in SEQ ID NO: 5 or a variant thereof, and / or the VL comprises the sequence shown in SEQ ID NO: 6 or a variant thereof; the variant has a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions.

[0026] In certain exemplary embodiments, the second antigen binding domain comprises a VH as set forth in SEQ ID NO:5 and a VL as set forth in SEQ ID NO:6.

[0027] In certain embodiments, the second antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) derived from ranibizumab or a variant thereof; wherein,

[0028] (i) the VH comprises CDR-H1 of SEQ ID NO: 15, CDR-H2 of SEQ ID NO: 16, and CDR-H3 of SEQ ID NO: 17; the VL comprises CDR-L1 of SEQ ID NO: 18, CDR-L2 of SEQ ID NO: 19, and CDR-L3 of SEQ ID NO: 20; or,

[0029] (ii) the VH comprises: CDR-H1, CDR-H2 and CDR-H3 contained in the VH as shown in SEQ ID NO: 13; the VL comprises: CDR-L1, CDR-L2 and CDR-L3 contained in the VL as shown in SEQ ID NO: 14; preferably, the CDRs are determined by the Kabat, IMGT or Chothia numbering systems.

[0030] In certain embodiments, the VH and / or VL further comprises a framework region derived from a mammalian (eg, human or murine) immunoglobulin.

[0031] In certain embodiments, the VH comprises the sequence shown in SEQ ID NO: 13 or a variant thereof, and / or the VL comprises the sequence shown in SEQ ID NO: 14 or a variant thereof; the variant has a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions.

[0032] In certain exemplary embodiments, the second antigen binding domain comprises a VH as set forth in SEQ ID NO:13 and a VL as set forth in SEQ ID NO:14.

[0033] In certain embodiments, unless otherwise indicated, the CDRs contained in the first antigen-binding domain and the second antigen-binding domain are identified by the Kabat numbering system.

[0034] In certain embodiments, the first antigen binding domain and the second antigen binding domain are optionally connected by a linker.

[0035] In certain embodiments, the linker is a peptide linker, such as a flexible peptide linker.

[0036] In certain embodiments, the linker is a peptide linker comprising one or more glycine and / or one or more serine. In certain embodiments, the linker is a flexible peptide comprising (G4S)n, where n is 1, 2, 3 or 4, such as the sequence shown in SEQ ID NO:35.

[0037] In certain embodiments, the second antigen binding domain is a full-length antibody (eg, an IgG antibody), an Fv fragment, a Fab fragment, a F(ab')2 fragment, or a scFv.

[0038] In certain embodiments, the second antigen-binding domain comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprising a light chain variable region (VL) and a light chain constant region (CL). In certain embodiments, the first antigen-binding domain is optionally connected to the N-terminus and / or C-terminus of the heavy chain and / or to the N-terminus and / or C-terminus of the light chain via a linker.

[0039] In certain embodiments, the heavy chain variable region (VH) and light chain variable region (VL) are as defined in any of the embodiments above.

[0040] In certain embodiments, the heavy chain constant region (CH) is IgG, such as IgG1, IgG2, IgG3 or IgG4. In certain exemplary embodiments, the heavy chain constant region (CH) comprises the sequence shown in SEQ ID NO: 21.

[0041] In certain embodiments, the Fc domain contained in the heavy chain constant region (CH) is a native Fc region, which comprises an amino acid sequence consistent with the amino acid sequence of the Fc region found in nature. The native Fc region may have effector functions, such as binding to Fc receptors; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); antibody-dependent cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0042] In certain embodiments, the Fc domain included in the heavy chain constant region (CH) may also be a variant Fc region, which may include amino acid mutations or chemical modifications compared to a native Fc region to alter its effector function. In certain embodiments, the variant Fc region may have modifications that promote dimerization.

[0043] In certain embodiments, the light chain constant region (CL) is κ or λ. In certain exemplary embodiments, the light chain constant region (CL) comprises the sequence shown in SEQ ID NO: 22.

[0044] In certain embodiments, the second antigen binding domain comprises: at least one heavy chain and at least one light chain, and the first antigen binding domain is optionally linked to the N-terminus or C-terminus of the heavy chain, or to the N-terminus of the light chain via a linker.

[0045] In certain embodiments, the second antigen-binding domain comprises: two identical heavy chains and two identical light chains, and the first antigen-binding domain is optionally connected to the N-terminus or C-terminus of the two heavy chains, or to the N-terminus of the two light chains via a linker.

[0046] In certain embodiments, the multispecific antibody comprises a first peptide chain and a second peptide chain, wherein:

[0047] (1) The first peptide chain comprises the following structure: [VHH]-[L]-[VH]-[CH], and the second peptide chain comprises the following structure: [VL]-[CL];

[0048] (2) the first peptide chain comprises the following structure: [VH]-[CH], and the second peptide chain comprises the following structure: [VHH]-[L]-[VL]-[CL]; or,

[0049] (3) The first peptide chain comprises the following structure: [VH]-[CH]-[L]-[VHH], and the second peptide chain comprises the following structure: [VL]-[CL];

[0050] Wherein, [L] is a peptide linker.

[0051] In certain exemplary embodiments, the multispecific antibody comprises:

[0052] (1) a first peptide chain comprising the sequence shown in SEQ ID NO: 23, and a second peptide chain comprising the sequence shown in SEQ ID NO: 24;

[0053] (2) a first peptide chain comprising the sequence shown in SEQ ID NO: 25, and a second peptide chain comprising the sequence shown in SEQ ID NO: 26;

[0054] (3) a first peptide chain comprising the sequence shown in SEQ ID NO: 27, and a second peptide chain comprising the sequence shown in SEQ ID NO: 28;

[0055] (4) a first peptide chain comprising the sequence shown in SEQ ID NO: 29, and a second peptide chain comprising the sequence shown in SEQ ID NO: 30;

[0056] (5) a first peptide chain comprising the sequence shown in SEQ ID NO: 31, and a second peptide chain comprising the sequence shown in SEQ ID NO: 32; or

[0057] (6) A first peptide chain comprising the sequence shown in SEQ ID NO: 33, and a second peptide chain comprising the sequence shown in SEQ ID NO: 34.

[0058] In certain embodiments, the multispecific antibody is a bispecific antibody.

[0059] In certain embodiments, the multispecific antibody further comprises at least one additional antigen-binding domain that targets an antigen other than the first and second antigen-binding domains, thereby being able to bind to at least three or more different binding sites and / or target molecules. In certain embodiments, the multispecific antibody is a trispecific antibody or a tetraspecific antibody.

[0060] Antibody preparation

[0061] The multispecific antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant techniques. For example, the multispecific antibodies of the present invention can be produced by co-expressing multiple polynucleotides encoding the individual polypeptide chains of the multispecific antibody. The polypeptide chains produced by co-expression can be combined, for example, via disulfide bonds or other means, to form a functional multispecific antibody.

[0062] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a multispecific antibody of the present invention or at least one peptide chain thereof.

[0063] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of the present invention, and the nucleotide sequence encoding each peptide chain is present on the same or different isolated nucleic acid molecules.

[0064] In another aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising the isolated nucleic acid molecule described above.

[0065] In certain embodiments, the vector comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of the present invention, and the nucleotide sequence encoding each peptide chain is present on the same or different vectors. For example, the vectors of the present invention comprise: a first vector comprising a nucleotide sequence encoding a first peptide chain, and a second vector comprising a nucleotide sequence encoding a second peptide chain.

[0066] On the other hand, the invention provides host cells comprising nucleic acid molecules or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as Escherichia coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0067] In another aspect, the present invention provides a method for preparing the multispecific antibody of the present invention, comprising culturing the host cell as described above under conditions allowing protein expression, and recovering the multispecific antibody from the cultured host cell.

[0068] Pharmaceutical composition

[0069] In another aspect, the present invention provides a pharmaceutical composition comprising the multispecific antibody, isolated nucleic acid molecule, vector, or host cell of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0070] In certain embodiments, the pharmaceutical composition comprises a multispecific antibody of the invention.

[0071] In certain embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0072] In certain embodiments, the additional pharmaceutically active agent is an anti-tumor agent.

[0073] In certain embodiments, the multispecific antibodies, isolated nucleic acid molecules, vectors, or host cells of the present invention and the additional pharmaceutically active agent can be provided as separate components or as mixed components in the pharmaceutical composition. Thus, the multispecific antibodies, isolated nucleic acid molecules, vectors, or host cells of the present invention and the additional pharmaceutically active agent can be administered simultaneously, separately, or sequentially.

[0074] The multispecific antibodies, isolated nucleic acid molecules, vectors or host cells of the present invention, or the pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use.

[0075] A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by incorporating the required dose of the multispecific antibody of the present invention into an appropriate solvent, and optionally, other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for ease of storage and use. Such a sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0076] In certain exemplary embodiments, the pharmaceutical composition of the present invention comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0077] The multispecific antibodies, isolated nucleic acid molecules, vectors or host cells of the invention, or pharmaceutical compositions of the invention can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic, inguinal, intravesical, topical (e.g., powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral (e.g., intravenous or bolus, subcutaneous, intraperitoneal, intramuscular). It will be understood by those skilled in the art that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the multispecific antibodies, isolated nucleic acid molecules, vectors or host cells of the invention, or pharmaceutical compositions of the invention are administered by intravenous or bolus injection.

[0078] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of the multispecific antibodies, isolated nucleic acid molecules, vectors, or host cells of the present invention. A "prophylactically effective amount" is an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered therapies.

[0079] Therapeutic uses

[0080] In another aspect, the present invention provides a method for preventing and / or treating tumors, comprising administering a multispecific antibody, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition of the present invention to a subject in need thereof. The present invention also provides the use of the multispecific antibody, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition of the present invention for preventing and / or treating tumors, or for preparing a medicament for preventing and / or treating tumors.

[0081] In certain embodiments, the tumor is selected from a solid tumor.

[0082] In certain embodiments, the tumor is selected from lung cancer (such as non-small cell lung cancer or small cell lung cancer), liver cancer (such as hepatocellular carcinoma), kidney cancer (such as renal cell carcinoma), gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, breast cancer (such as triple-negative breast cancer), colorectal cancer, brain tumor (such as glioblastoma).

[0083] In certain embodiments, an agent provided herein is administered in combination with a second therapeutic agent (e.g., an anti-tumor agent) or treatment (e.g., surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care) selected from the group consisting of the multispecific antibodies, isolated nucleic acid molecules, vectors, host cells, or pharmaceutical compositions described herein. The second therapeutic agent or treatment can be administered before, simultaneously with, or after administration of the agent provided herein.

[0084] Definition of terms

[0085] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0086] As used herein, the term "antibody" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant and can include various antibody structures as long as they exhibit the desired antigen-binding activity. Typically, an antibody can be an immunoglobulin molecule consisting of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of a domain CL. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as mediating 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 also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site.

[0087] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable region of each of the heavy and light chains contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0088] In the present invention, the CDRs contained in the antibodies or antigen-binding fragments thereof can be identified according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof involved in the present invention are preferably identified by the IMGT, Kabat, or Chothia numbering systems. In certain embodiments, unless otherwise specified, the CDRs contained in the antibodies or antigen-binding fragments thereof involved in the present invention are preferably identified by the Kabat numbering system.

[0089] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0090] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0091] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity for at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises a plurality of antigen-binding domains that have binding specificity for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules. Each antigen-binding domain comprised by a multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, a Fab fragment, a F(ab')2 fragment, or a scFv). In some cases, each antigen-binding domain is connected by a peptide linker. In certain embodiments, the multispecific antibody of the present invention can be a bispecific antibody. In certain embodiments, the multispecific antibody of the present invention can be a trispecific antibody or a tetraspecific antibody.

[0092] As used herein, the term "single-domain antibody (sdAb)" has the meaning commonly understood by those skilled in the art, and refers to an antibody fragment consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single domain antibody can be truncated at the N-terminus or C-terminus so that it contains only part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity. Single domain antibodies are also referred to as nanobodies, and the two are used interchangeably. The variable region of a single domain antibody or nanobody is called a VHH domain.

[0093] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of an antibody can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv)2, and polypeptides that comprise at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0094] As used herein, the term "Fab fragment" means an antibody fragment consisting of a light chain comprising VL and CL and a heavy chain fragment comprising VH and CH1; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab' fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0095] As used herein, the term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen binding site.

[0096] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 may be used, but variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv. In some cases, scFv may form a di-scFv, which refers to two or more single scFvs connected in series to form an antibody. In some cases, scFv may form a (scFv)2, which refers to two or more single scFvs connected in parallel to form an antibody.

[0097] As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived.

[0098] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates that the smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The specific binding properties between two molecules can be determined using methods well known in the art, for example, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore, or using bioluminescence interferometry or Kinexa.

[0099] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0100] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells, immune cells (such as T lymphocytes, NK cells, monocytes, macrophages or dendritic cells, etc.). Host cells can include single cells or cell populations.

[0101] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0102] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0103] The twenty conventional amino acids referred to herein are written in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0104] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Stabilizers have the meaning commonly understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w / v) NaCl), glucose solution (such as 5% glucose), a solution containing a surfactant (such as 0.01% polysorbate 20), a pH buffer solution (such as phosphate buffer solution), Ringer's solution, and any combination thereof.

[0105] As used herein, the term "prevention" refers to a method implemented in order to prevent or delay the occurrence of a disease or illness or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or entirely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival (if not receiving treatment), extending the survival period.

[0106] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, human) has a tumor.

[0107] As used herein, the terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer includes solid tumors as well as hematologic malignancies.

[0108] Advantageous Effects of the Invention

[0109] The present invention provides multispecific antibodies targeting PD-1 and VEGF, which can specifically block the PD-1 and VEGF signaling pathways, thereby producing a synergistic anti-tumor effect while reducing the incidence of adverse reactions and making clinical medication more convenient. The multispecific antibodies of the present invention have important clinical value.

[0110] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 shows the results of a cell-based PD1 blocking activity assay.

[0112] FIG2 shows the results of a cell-based VEGF blocking activity assay.

[0113] FIG3 shows the changes in tumor volume in the in vivo anti-tumor experiment.

[0114] FIG4 shows the changes in mouse body weight during the in vivo anti-tumor experiment.

[0115] Sequence information

[0116] A description of the sequences involved in this application is provided in the table below.

[0117] Table 1: Sequence information Example

[0118] The invention will now be described in the following non-limiting examples.

[0119] Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in this application. The experimental methods in the embodiments, unless otherwise specified, are conventional methods. Where specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. The present invention will now be described with reference to the following embodiments which are intended to illustrate the present invention (but not to limit the present invention).

[0120] Example 1. Design and expression of bispecific antibodies

[0121] 1. Preparation of PD-1 VHH single-domain antibody

[0122] Healthy adult female alpacas (Alpaca) were immunized with recombinant human PD-1 protein. After immunization, peripheral blood was collected from the alpacas, lymphocytes were isolated, and total RNA was extracted and reverse-transcribed to obtain cDNA to establish a PD-1 single-domain antibody phage display library. VHH single-domain antibodies with strong binding activity to human PD-1, the ability to block the binding of human PD-L1 to human recombinant PD-1, and the ability to compete with pembrolizumab for binding to the same or overlapping PD-1 epitopes were screened. The detailed screening process can be found in the international patent application PCT / CN2022 / 074550. Finally, a VHH single-domain antibody was screened, whose VHH sequence is shown in SEQ ID NO: 1. According to the Kabat numbering system, CDR1-CDR3 are shown in SEQ ID NO: 2-4, respectively.

[0123] 2. Design and expression of bispecific antibodies

[0124] Bispecific antibodies were further constructed based on the above-mentioned PD-1 VHH antibody and VEGF antibody. The VEGF antibody used Bevacizumab (mutant sequence derived from United States Patent 7575893) and Ranibizumab sequences. The VH and VL of Bevacizumab (mutant) are shown in SEQ ID NOs: 5-6, respectively. According to the Kabat numbering system, CDR-H1 to CDR-H3 are shown in SEQ ID NOs: 7-9, respectively, and CDR-L1 to CDR-L3 are shown in SEQ ID NOs: 10-12, respectively. The VH and VL of Ranibizumab are shown in SEQ ID NOs: 13-14, respectively. According to the Kabat numbering system, CDR-H1 to CDR-H3 are shown in SEQ ID NOs: 15-17, respectively, and CDR-L1 to CDR-L3 are shown in SEQ ID NOs: 18-20, respectively.

[0125] Six bispecific antibodies were constructed, and their structures are shown in Table 2. The heavy chains of 162-17 and 162-20 have the structure [VHH]-[L]-[VH]-[CH], and the light chains have the structure [VL]-[CL]. The heavy chains of 162-18 and 162-21 have the structure [VH]-[CH], and the light chains have the structure [VHH]-[L]-[VL]-[CL]. The heavy chains of 162-19 and 162-22 have the structure [VH]-[CH]-[L]-[VHH], and the light chains have the structure [VL]-[CL]. In all cases, [L] represents the linker shown in SEQ ID NO: 35.

[0126] Table 2: Bispecific antibody structures and sequences

[0127] Nucleic acid molecules encoding each polypeptide chain of the above-mentioned bispecific antibody were constructed into expression vectors and transiently expressed by HEK. After expression, the antibody was purified by Protein A. The expression level and purity after purification are shown in the table below.

[0128] Table 3: Expression level and purity of bispecific antibodies

[0129] Example 2. PD1 blocking activity experiment

[0130] This example investigates the blocking activity of bispecific antibodies on the PD1 / PD-L1 signaling pathway.

[0131] PDL1-CHO cells (GenScript, Cat#RD00703) were cultured at 3.5×104 Cells were seeded in a 96-well white plate (Thermo, Cat#136101). 6 × 10 cells were seeded in the center wells of the 96-well plate, and 200 μl of culture medium was added to the surrounding wells. Incubate overnight at 37°C, 5% CO₂ for 16–20 hours. The next day, 4 × 10 PD1-Jurkat cell suspension (GenScript, Cat#RD00702) was prepared using complete culture medium (RPMI 1640 + 10% FBS). 5 Cells / ml and the antibody to be tested (initial concentration of 300 nM, 4-fold dilution) were added to a 96-well plate with the culture medium discarded, incubated at 37°C, 5% CO2 for 6 hours, and Fire-Lumi TM Chemical signals were detected using luciferase assay reagent (GenScript, Cat# L00877C), and four-parameter curves were plotted using GraphPad.

[0132] The results are shown in Figure 1 and Table 4. The bispecific antibody of the present application has significant blocking activity against PD1 / PD-L1.

[0133] Table 4: PD1 blocking activity assay

[0134] Example 3. VEGF blocking activity experiment

[0135] This example investigates the blocking activity of bispecific antibodies on the VEGF signaling pathway.

[0136] HEK293 cells overexpressing VEGFR2 (GenScript, Cat#RD00704) were seeded in 96-well plates (Thermo, Cat#136101) at 5,000 cells per well and cultured overnight for 16-20 hours. 2× VEGF165 (Sino Biological, Cat#11066-HNAH) and the test antibody were prepared in complete medium (DMEM + 10% FBS). The recommended concentration of 2× VEGF165 was 30 ng / ml, and the 2× test antibody was 20 μg / ml. Five 3-fold dilutions and five 10-fold dilutions were prepared. After preparing the above solutions, the medium was discarded from the VEGFR2-HEK293 cells. 50 μl of VEGF165 and 50 μl of the test antibody were immediately added and mixed. The cells were incubated at 37°C, 5% CO2, and 100 μl of complete medium was added to the cell-free wells to measure the background absorbance. After 6 hours, add 100ul / well Fire-Lumi TM Reagent, incubate for 5-60 minutes, and detect chemical signals.

[0137] The results are shown in FIG2 and Table 5 , indicating that the bispecific antibody of the present application has significant blocking activity on the VEGF signaling pathway.

[0138] Table 5: VEGF blocking activity assay

[0139] Example 4. In vivo anti-tumor activity experiment

[0140] This example investigates the in vivo anti-tumor activity of bispecific antibodies in animal tumor models.

[0141] Human breast cancer cells MDA-MB-231 (obtained from the Chinese Academy of Sciences) were cultured and expanded in vitro. 5E6 cells were resuspended in PBS and subcutaneously implanted in female NSG mice aged 6-8 weeks (obtained from Shanghai Southern Model Biotechnology Co., Ltd.) to construct a tumor-bearing model. PBMC (obtained from Miaoshun (Shanghai) Biotechnology Co., Ltd.) were injected into the tail vein 7 days after tumor bearing, 4E6 / mouse. After 14 days, the mice were divided into groups according to the tumor volume of approximately 100mm^3 and intraperitoneally injected (twice a week). Body weight and tumor volume were recorded twice a week. After 39 days, the comparative analysis of mouse tumor volume and body weight is shown in Figures 3 and 4, respectively. The results showed that the bispecific antibody of the present application can significantly inhibit tumor growth and is significantly better than Nivolumab. In addition, the bispecific antibody of the present application also has good in vivo safety.

[0142] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A multispecific antibody comprising a first antigen-binding domain targeting programmed cell death receptor-1 (PD-1) and a second antigen-binding domain targeting vascular endothelial growth factor (VEGF), wherein: The first antigen-binding domain comprises a VHH domain, comprising: a CDR1 as set forth in SEQ ID NO: 2 or a variant thereof, a CDR2 as set forth in SEQ ID NO: 3 or a variant thereof, and a CDR3 as set forth in SEQ ID NO: 4 or a variant thereof; wherein the variant has one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived; Preferably, the VHH domain comprises: CDR1 as shown in SEQ ID NO: 2, CDR2 as shown in SEQ ID NO: 3, and CDR3 as shown in SEQ ID NO: 4; Preferably, the VHH domain comprises: CDR1, CDR2 and CDR3 contained in the VHH as shown in SEQ ID NO: 1; preferably, the CDRs are determined by the Kabat, IMGT or Chothia numbering system.

2. The multispecific antibody according to claim 1, wherein The first antigen-binding domain comprises: a VHH comprising the sequence of SEQ ID NO: 1 or a variant thereof, wherein the variant has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions; Preferably, the first antigen binding domain comprises the VHH shown in SEQ ID NO:

1.

3. The multispecific antibody according to claim 1 or 2, wherein The second antigen-binding domain comprises an antibody or antigen-binding fragment thereof that specifically binds to VEGF (e.g., a heavy chain variable region and / or a light chain variable region thereof); Preferably, the antibody that specifically binds to VEGF is selected from bevacizumab or its variants, ranibizumab or its variants.

4. The multispecific antibody according to any one of claims 1 to 3, wherein The second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein, (i) the VH comprises CDR-H1 shown in SEQ ID NO: 7, CDR-H2 shown in SEQ ID NO: 8, and CDR-H3 shown in SEQ ID NO: 9; the VL comprises CDR-L1 shown in SEQ ID NO: 10, CDR-L2 shown in SEQ ID NO: 11, and CDR-L3 shown in SEQ ID NO: 12; or, (ii) the VH comprises: CDR-H1, CDR-H2 and CDR-H3 contained in the VH as shown in SEQ ID NO: 5; the VL comprises: CDR-L1, CDR-L2 and CDR-L3 contained in the VL as shown in SEQ ID NO: 6; preferably, the CDRs are determined by the Kabat, IMGT or Chothia numbering systems; Preferably, the VH and / or VL further comprises a framework region derived from a mammalian (eg, human or mouse) immunoglobulin; Preferably, the VH comprises the sequence shown in SEQ ID NO: 5 or a variant thereof, and / or the VL comprises the sequence shown in SEQ ID NO: 6 or a variant thereof; the variant has a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions; Preferably, the second antigen binding domain comprises VH as shown in SEQ ID NO: 5 and VL as shown in SEQ ID NO:

6.

5. The multispecific antibody according to any one of claims 1 to 3, wherein The second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL); wherein, (i) the VH comprises CDR-H1 of SEQ ID NO: 15, CDR-H2 of SEQ ID NO: 16, and CDR-H3 of SEQ ID NO: 17; the VL comprises CDR-L1 of SEQ ID NO: 18, CDR-L2 of SEQ ID NO: 19, and CDR-L3 of SEQ ID NO: 20; or, (ii) the VH comprises: CDR-H1, CDR-H2 and CDR-H3 contained in the VH as shown in SEQ ID NO: 13; the VL comprises: CDR-L1, CDR-L2 and CDR-L3 contained in the VL as shown in SEQ ID NO: 14; preferably, the CDRs are identified by the Kabat, IMGT or Chothia numbering systems; Preferably, the VH and / or VL further comprises a framework region derived from a mammalian (eg, human or mouse) immunoglobulin; Preferably, the VH comprises the sequence shown in SEQ ID NO: 13 or a variant thereof, and / or the VL comprises the sequence shown in SEQ ID NO: 14 or a variant thereof; the variant has a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared thereto; preferably, the substitutions are conservative substitutions; Preferably, the second antigen binding domain comprises a VH as shown in SEQ ID NO: 13 and a VH as shown in SEQ ID NO: VL shown in NO:

14.

6. The multispecific antibody according to any one of claims 1 to 5, wherein The first antigen binding domain and the second antigen binding domain are optionally connected by a linker; Preferably, the linker is a peptide linker, such as a flexible peptide linker; Preferably, the linker is a peptide linker comprising one or more glycine and / or one or more serine, such as a flexible peptide comprising (G4S)n, where n is 1, 2, 3 or 4.

7. The multispecific antibody according to any one of claims 1 to 6, wherein The second antigen binding domain is a full-length antibody (eg, an IgG antibody), an Fv fragment, a Fab fragment, a F(ab')2 fragment, or a scFv.

8. The multispecific antibody according to claim 7, wherein The second antigen-binding domain comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region; Preferably, the heavy chain variable region and the light chain variable region are as defined in any one of claims 3 to 5; Preferably, the heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4; Preferably, the heavy chain constant region comprises the sequence shown in SEQ ID NO: 21; Preferably, the light chain constant region is κ or λ; Preferably, the light chain constant region comprises the sequence shown in SEQ ID NO:

22.

9. The multispecific antibody according to claim 8, wherein The first antigen binding domain is optionally connected to the N-terminus and / or C-terminus of the heavy chain and / or to the N-terminus and / or C-terminus of the light chain via a linker; Preferably, the second antigen-binding domain comprises: at least one heavy chain and at least one light chain, and the first antigen-binding domain is optionally connected to the N-terminus or C-terminus of the heavy chain, or to the N-terminus of the light chain via a linker; Preferably, the second antigen-binding domain comprises: two identical heavy chains and two identical light chains, and the first antigen-binding domain is optionally connected to the N-termini or C-termini of the two heavy chains, or to the N-termini of the two light chains via a linker.

10. The multispecific antibody according to any one of claims 1 to 9, comprising: (1) a first peptide chain comprising the sequence shown in SEQ ID NO: 23, and a second peptide chain comprising the sequence shown in SEQ ID NO: 24; (2) a first peptide chain comprising the sequence shown in SEQ ID NO: 25, and a second peptide chain comprising the sequence shown in SEQ ID NO: 26; (3) a first peptide chain comprising the sequence shown in SEQ ID NO: 27, and a second peptide chain comprising the sequence shown in SEQ ID NO: 28; (4) a first peptide chain comprising the sequence shown in SEQ ID NO: 29, and a second peptide chain comprising the sequence shown in SEQ ID NO: 30; (5) a first peptide chain comprising the sequence shown in SEQ ID NO: 31, and a second peptide chain comprising the sequence shown in SEQ ID NO: 32; or (6) A first peptide chain comprising the sequence shown in SEQ ID NO: 33, and a second peptide chain comprising the sequence shown in SEQ ID NO:

34.

11. The multispecific antibody according to any one of claims 1 to 10, wherein The multispecific antibody is a bispecific antibody.

12. The multispecific antibody according to any one of claims 1 to 10, wherein The multispecific antibody further comprises at least one additional antigen binding domain that targets an antigen other than the first and second antigen binding domains; Preferably, the multispecific antibody is a trispecific antibody or a tetraspecific antibody.

13. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the multispecific antibody of any one of claims 1 to 12 or at least one polypeptide chain thereof; Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody, and the nucleotide sequence encoding each peptide chain is present on the same or different isolated nucleic acid molecules.

14. A vector comprising the nucleic acid molecule according to claim 13; Preferably, the vector comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody according to any one of claims 1 to 12, and the nucleotide sequence encoding each peptide chain is present on the same or different vectors.

15. A host cell comprising the nucleic acid molecule of claim 13 or the vector of claim 14.

16. A method for preparing the multispecific antibody according to any one of claims 1 to 12, comprising culturing the host cell according to claim 15 under conditions that allow protein expression, and recovering the multispecific antibody from the cultured host cell.

17. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1 to 12, the isolated nucleic acid molecule of claim 13, the vector of claim 14, or the host cell of claim 15, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises another pharmaceutically active agent, such as an anti-tumor agent.

18. Use of the multispecific antibody according to any one of claims 1 to 12, the isolated nucleic acid molecule according to claim 13, the vector according to claim 14, the host cell according to claim 15, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for preventing and / or treating tumors; Preferably, the tumor is selected from solid tumors; Preferably, the tumor is selected from lung cancer (such as non-small cell lung cancer or small cell lung cancer), liver cancer (such as hepatocellular carcinoma), kidney cancer (such as renal cell carcinoma), gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, breast cancer (such as triple-negative breast cancer), colorectal cancer, brain tumor (such as glioblastoma); Preferably, the multispecific antibody, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition is administered in combination with another pharmaceutically active agent (eg, an anti-tumor agent).

19. A method for preventing and / or treating tumors, comprising administering to a subject in need thereof an effective amount of the multispecific antibody according to any one of claims 1 to 12, the isolated nucleic acid molecule according to claim 13, the vector according to claim 14, the host cell according to claim 15, or the pharmaceutical composition according to claim 17; Preferably, the tumor is selected from solid tumors; Preferably, the tumor is selected from lung cancer (such as non-small cell lung cancer or small cell lung cancer), liver cancer (such as hepatocellular carcinoma), kidney cancer (such as renal cell carcinoma), gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, breast cancer (such as triple-negative breast cancer), colorectal cancer, brain tumor (such as glioblastoma); Preferably, the subject is a mammal, such as a human; Preferably, the method further comprises administering to the subject a second therapeutic agent (such as an anti-tumor agent) or a treatment procedure (such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care).

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