Multispecific antibodies and uses thereof

Abstract

The present invention relates to multispecific antibodies targeting programmed death receptor-1 (PD-1) and vascular endothelial growth factor (VEGF), pharmaceutical compositions comprising said multispecific antibodies, nucleic acid molecules and vectors encoding said multispecific antibodies, host cells, and their use for treating tumors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS 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.

[0002] The present invention relates to the field of immunology. In particular, the present invention relates to multispecific antibodies targeting programmed death receptor-1 (PD-1) and vascular endothelial growth factor (VEGF), pharmaceutical compositions comprising said multispecific antibodies, nucleic acid molecules, vectors and host cells encoding said multispecific antibodies, and their use in the treatment of tumors. [Background technology]

[0003] Programmed death receptor-1 (PD-1) is a type I transmembrane glycoprotein receptor belonging to the CD28 immunoglobulin superfamily. It is widely expressed on immune cells such as T cells and NK cells. PD-1 has two natural ligands, PD-L1 and PD-L2, both of which belong to the B7 superfamily and are constitutively or inducibly expressed on the surface of various cells, including non-hematopoietic cells, immune cells, and tumor cells. Interaction between PD-1 and its ligands inhibits the activation and proliferation of lymphocytes (e.g., T cells and NK cells), the secretion of cytokines (e.g., IL-2, IFN-γ), and the killing of tumor cells.

[0004] Vascular endothelial growth factor (VEGF) is a highly specific growth factor that promotes endothelial cell proliferation, enhances the formation of new blood vessels, and increases vascular permeability. The VEGF family includes VEGFA, VEGFB, VEGFC, VEGFD, and PIGF. VEGF 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 present on the surface of vascular endothelial cells, while VEGFR3 is primarily present on lymphatic endothelial cells. Neovascularization provides the nutrients and oxygen necessary for tumor growth, progression, and metastasis and is an important target for inhibiting tumor growth. Bevacizumab, a humanized monoclonal antibody targeting VEGF-A, is approved for the treatment of various cancers, including non-small cell lung cancer, renal cell carcinoma, cervical cancer, and metastatic colorectal cancer. Ranibizumab, an affinity-enhanced monoclonal antibody Fab fragment specific for VEGF-A, is approved for the treatment of "wet" age-related macular degeneration, diabetic retinopathy, and macular edema due to branch retinal vein occlusion or central retinal vein occlusion.

[0005] Anti-PD-1 / PD-L1 monoclonal antibodies combined with VEGF pathway inhibitors (such as bevacizumab or tyrosine kinase inhibitors like lenvatinib and sorafenib) have shown breakthrough efficacy in a variety of tumor types, including liver, lung, gastric, renal, and some gynecological cancers. However, the side effects of combination therapy are significantly higher than those observed with monotherapy.

[0006] Therefore, it is necessary to develop multispecific antibodies that target both the PD-1 / PD-L1 and VEGF pathways to provide synergistic antitumor effects while reducing side effects, which also makes clinical treatment more convenient. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides a multispecific antibody that targets both PD-1 and VEGF and is capable of specifically blocking the PD-1 and VEGF signaling pathways, thereby exerting a synergistic anti-tumor effect. The present application provides the following aspects. [Means for solving the problem]

[0008] multispecific antibodies In one aspect, the present invention provides a multispecific antibody comprising a first antigen-binding domain targeting programmed death receptor-1 (PD-1) and a second antigen-binding domain targeting vascular endothelial growth factor (VEGF), wherein said first antigen-binding domain comprises a VHH domain, and said VHH domain comprises CDR1 set forth in SEQ ID NO: 2 or a variant thereof, CDR2 set forth in SEQ ID NO: 3 or a variant thereof, and CDR3 set forth in SEQ ID NO: 4 or a variant thereof, wherein said 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.

[0009] In certain embodiments, the VHH domain comprises a CDR1 set forth in SEQ ID NO:2, a CDR2 set forth in SEQ ID NO:3, and a CDR3 set forth in SEQ ID NO:4.

[0010] In one embodiment, the VHH domain comprises CDR1, CDR2 and CDR3 contained in the VHH domain set forth in SEQ ID NO: 1, preferably wherein the CDRs are determined according to the Kabat, IMGT or Chothia numbering system.

[0011] In one embodiment, the VHH domain typically consists 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 may be truncated at the N- or C-terminus to include only a portion of FR1 and / or FR4, or may lack one or two of these framework regions, as long as it substantially retains its antigen-binding specificity.

[0012] In one embodiment, the VHH domain may comprise framework region sequences derived from a camelid heavy chain antibody.

[0013] In some embodiments, the VHH domain may be humanized, i.e., one or more of the framework regions have been replaced with human framework regions. In some embodiments, the VHH domain comprises heavy chain framework regions derived from a human immunoglobulin (e.g., heavy chain framework regions comprised in an amino acid sequence encoded by a human germline antibody gene), and the heavy chain framework regions optionally comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) backmutations from human residues to camelid residues.

[0014] In certain embodiments, the first antigen-binding domain comprises a VHH sequence as set forth in SEQ ID NO: 1 or a variant thereof, wherein said 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), preferably wherein said substitutions are conservative substitutions.

[0015] In one exemplary embodiment, the first antigen-binding domain comprises a VHH set forth in SEQ ID NO:1.

[0016] 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.

[0017] In certain embodiments, the antibody that specifically binds to VEGF is selected from bevacizumab or a variant thereof, and ranibizumab or a variant thereof.

[0018] In one embodiment, 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: (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, and 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; (ii) the VH comprises CDR-H1, CDR-H2, and CDR-H3 contained in the VH set forth in SEQ ID NO: 5, and the VL comprises CDR-L1, CDR-L2, and CDR-L3 contained in the VL set forth in SEQ ID NO: 6, preferably wherein the CDRs are determined according to the Kabat, IMGT, or Chothia numbering system;

[0019] In some embodiments, the VH and / or VL further comprise a framework region derived from a mammalian (e.g., human or mouse) immunoglobulin, hi some embodiments, the VH and / or VL further comprise a framework region derived from a human immunoglobulin.

[0020] In one embodiment, the VH comprises the sequence set forth in SEQ ID NO: 5 or a variant thereof, and / or the VL comprises the sequence set forth in SEQ ID NO: 6 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, or 5 amino acid substitutions, deletions, or additions), preferably wherein the substitutions are conservative substitutions.

[0021] In one exemplary embodiment, the second antigen-binding domain comprises a VH set forth in SEQ ID NO:5 and a VL set forth in SEQ ID NO:6.

[0022] In one embodiment, 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: (i) the VH comprises CDR-H1 set forth in SEQ ID NO: 15, CDR-H2 set forth in SEQ ID NO: 16, and CDR-H3 set forth in SEQ ID NO: 17, and the VL comprises CDR-L1 set forth in SEQ ID NO: 18, CDR-L2 set forth in SEQ ID NO: 19, and CDR-L3 set forth in SEQ ID NO: 20; or (ii) the VH comprises CDR-H1, CDR-H2, and CDR-H3 contained in the VH set forth in SEQ ID NO: 13, and the VL comprises CDR-L1, CDR-L2, and CDR-L3 contained in the VL set forth in SEQ ID NO: 14, preferably, the CDRs are determined according to the Kabat, IMGT, or Chothia numbering system.

[0023] In certain embodiments, the VH and / or VL further comprise a framework region derived from a mammalian (eg, human or murine) immunoglobulin.

[0024] In one embodiment, the VH comprises the sequence set forth in SEQ ID NO: 13 or a variant thereof, and / or the VL comprises the sequence set forth in SEQ ID NO: 14 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, or 5 amino acid substitutions, deletions, or additions), preferably wherein the substitutions are conservative substitutions.

[0025] In one exemplary embodiment, the second antigen-binding domain comprises the VH set forth in SEQ ID NO:13 and the VL set forth in SEQ ID NO:14.

[0026] In certain embodiments, unless otherwise specified, the CDRs contained in the first antigen-binding domain and the second antigen-binding domain are determined by the Kabat numbering system.

[0027] In one embodiment, the first antigen-binding domain and the second antigen-binding domain are optionally linked via a linker.

[0028] In certain embodiments, the linker is a peptide linker, eg, a flexible peptide linker.

[0029] In some embodiments, the linker is a peptide linker comprising one or more glycines and / or one or more serines. In some embodiments, the linker is a flexible peptide linker comprising (G4S)n (wherein n is 1, 2, 3, or 4), for example, the linker comprises the sequence set forth in SEQ ID NO:35.

[0030] In certain embodiments, the second antigen-binding domain is a full-length antibody (eg, an IgG antibody), an Fv fragment, a Fab fragment, an F(ab')2 fragment, or an scFv.

[0031] In one embodiment, the second antigen-binding domain comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprises a light chain variable region (VL) and a light chain constant region (CL). In one embodiment, the first antigen-binding domain is optionally linked to the N-terminus and / or C-terminus of the heavy chain and / or the N-terminus and / or C-terminus of the light chain via a linker.

[0032] In one embodiment, the heavy chain variable region (VH) and light chain variable region (VL) are defined as described in any of the previous embodiments.

[0033] In one embodiment, the heavy chain constant region (CH) is an IgG, e.g., IgG1, IgG2, IgG3, or IgG4. In one exemplary embodiment, the heavy chain constant region (CH) comprises the sequence set forth in SEQ ID NO:21.

[0034] In one embodiment, the Fc domain contained in the heavy chain constant region (CH) is a native Fc region, comprising an amino acid sequence identical to that of an Fc region found in nature, which may exhibit effector functions such as binding to Fc receptors, C1q binding and complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCC), antibody-dependent cellular phagocytosis (ADCP), down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0035] In some embodiments, the Fc domain contained in the heavy chain constant region (CH) may be a modified Fc region that contains amino acid mutations or chemical modifications compared to the native Fc region to alter its effector function, hi some embodiments, the modified Fc region may contain modifications that promote dimerization.

[0036] In one embodiment, the light chain constant region (CL) is a kappa or lambda light chain constant region. In one exemplary embodiment, the light chain constant region (CL) comprises the sequence set forth in SEQ ID NO:22.

[0037] In one embodiment, 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.

[0038] In one embodiment, the second antigen-binding domain comprises two identical heavy chains and two identical light chains, and the first antigen-binding domain is optionally linked to the N- or C-terminus of the two heavy chains or to the N-terminus of the two light chains via a linker.

[0039] In one embodiment, the multispecific antibody comprises a first peptide chain and a second peptide chain, wherein: (1) the first peptide chain comprises the following structure: [VHH]-[L]-[VH]-[CH], and the second peptide chain comprises the following structure: [VL]-[CL]; (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 (3) the first peptide chain comprises the following structure: [VH]-[CH]-[L]-[VHH], and the second peptide chain comprises the following structure: [VL]-[CL]; Here, [L] is a peptide linker.

[0040] In an exemplary embodiment, the multispecific antibody comprises: (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. Includes:

[0041] In some embodiments, the multispecific antibody is a bispecific antibody.

[0042] In some embodiments, the multispecific antibody further comprises at least one additional antigen-binding domain that targets an antigen different from those bound by the first and second antigen-binding domains, allowing binding to at least three or more different binding sites and / or target molecules. In some embodiments, the multispecific antibody is a trispecific or tetraspecific antibody.

[0043] Antibody preparation Multispecific antibodies of the present invention can be prepared by various methods known in the art, such as genetic engineering techniques and recombinant technologies. For example, multispecific antibodies of the present invention can be produced by coexpressing multiple polynucleotides encoding the various polypeptide chains of the multispecific antibody. The polypeptide chains produced by coexpression can be linked, for example, via disulfide bonds or other means, to form a functional multispecific antibody.

[0044] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a multispecific antibody of the invention or at least one polypeptide chain thereof.

[0045] In some embodiments, the isolated nucleic acid molecules comprise a nucleotide sequence encoding each of the polypeptide chains of a multispecific antibody of the invention, wherein the nucleotide sequences are present on the same isolated nucleic acid molecule or on different isolated nucleic acid molecules.

[0046] In another aspect, the present invention provides a vector (eg, a cloning vector or an expression vector) comprising an isolated nucleic acid molecule as described above.

[0047] In some embodiments, the vectors comprise nucleotide sequences encoding each of the polypeptide chains of the multispecific antibody of the invention, and the nucleotide sequences are present on the same or different vectors. For example, the vectors of the invention may comprise a first vector comprising a nucleotide sequence encoding the first polypeptide chain and a second vector comprising a nucleotide sequence encoding the second polypeptide chain.

[0048] In another aspect, the present invention provides a host cell comprising the nucleic acid molecule or vector described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells, human cells, etc.).

[0049] In another aspect, the present invention provides a method for preparing a multispecific antibody of the present invention, comprising culturing the host cells described above under conditions that allow expression of the protein, and recovering said multispecific antibody from the culture of said cultured host cells.

[0050] Pharmaceutical Composition In another aspect, the present invention provides a pharmaceutical composition comprising said multispecific antibody of the invention, said isolated nucleic acid molecule of the invention, said vector of the invention or said host cell of the invention, and a pharmaceutically acceptable carrier and / or excipient.

[0051] In some embodiments, the pharmaceutical composition comprises a multispecific antibody of the invention.

[0052] In some embodiments, the pharmaceutical composition may further comprise an additional pharmaceutically active agent.

[0053] In some embodiments, the additional pharmaceutically active agent is an anti-tumor agent.

[0054] In some embodiments, in the pharmaceutical composition, the multispecific antibody, isolated nucleic acid molecule, vector or host cell of the invention, and the additional pharmaceutically active agent may be provided as separate or mixed components. Thus, the multispecific antibody, isolated nucleic acid molecule, vector or host cell of the invention, and the additional pharmaceutically active agent may be administered simultaneously, separately or sequentially.

[0055] The multispecific antibodies, isolated nucleic acid molecules, vectors or host cells of the invention, or the pharmaceutical compositions of the invention can be formulated into any dosage form known in the medical arts, such as tablets, pills, suspensions, emulsions, liquids, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and injectable concentrated solutions), inhalers, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic application.

[0056] A preferred dosage form is an injectable preparation. Such an injectable preparation may be a sterile injectable solution. For example, a sterile injectable solution can be prepared by mixing the required dose of a multispecific antibody of the present invention in an appropriate solvent, optionally with other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Furthermore, a sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or lyophilization) for storage and use. Such a sterile lyophilized powder can be reconstituted before use in a suitable carrier, such as water for injection (WFI), bacteriostatic water for injection (BWFI), saline (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, or any combination thereof.

[0057] In some exemplary embodiments, the pharmaceutical composition of the present invention comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, the sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), saline (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, or any combination thereof.

[0058] The multispecific antibodies, isolated nucleic acid molecules, vectors, or host cells of the invention, or the pharmaceutical compositions of the invention may 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., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral (e.g., intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). One skilled in the art will appreciate that the route and / or method of administration will vary depending on the intended purpose. In some embodiments, the multispecific antibodies, isolated nucleic acid molecules, vectors, or host cells of the invention, or the pharmaceutical compositions of the invention are administered by intravenous injection or bolus injection.

[0059] The pharmaceutical compositions of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the multispecific antibody, isolated nucleic acid molecule, vector, or host cell of the present invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, suppress, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially suppress a disease, along with 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 being treated, the general condition of the patient's immune system, and the patient's general condition, such as age, weight, and sex, the method of administration, and other concomitant treatments.

[0060] therapeutic use In one aspect, the present invention provides a method for preventing and / or treating tumors, comprising administering to a subject in need thereof said multispecific antibody, isolated nucleic acid molecule, vector, host cell or pharmaceutical composition of the invention. The present invention also provides use of said multispecific antibody, isolated nucleic acid molecule, vector, host cell or pharmaceutical composition of the invention for preventing and / or treating tumors, or for the manufacture of a medicament for preventing and / or treating tumors.

[0061] In certain embodiments, the tumor is selected from a solid tumor.

[0062] In some embodiments, the tumor is selected from lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), liver cancer (e.g., hepatocellular carcinoma), kidney cancer (e.g., renal cell carcinoma), gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, breast cancer (e.g., triple-negative breast cancer), colon cancer, and brain cancer (e.g., glioblastoma).

[0063] In certain embodiments, the agent provided by the present invention 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, hormonal therapy, gene therapy, or palliative care), selected from said multispecific antibody, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition of the present invention. Said second therapeutic agent or treatment may be applied before, simultaneously with, or after the administration of the aforementioned agent of the present invention.

[0064] Definition of Terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.In addition, the experimental procedures of virology, biochemistry and immunology described herein refer to the conventional methods that are widely used in their respective technical fields.In order to further clarify the present invention, the definitions and explanations of relevant terms are provided below.

[0065] As used herein, the term "antibody" refers to a molecule that specifically binds to an epitope and can encompass a variety of antibody structures as long as it exhibits the desired antigen-binding activity. Typically, antibodies are immunoglobulin molecules composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as κ (kappa) or λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, which define the antibody isotype as IgM, IgD, IgG, IgA, or IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and heavy chains also contain 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 consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domain is not directly involved in antibody binding to antigens but exhibits various effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including binding to the first component (C1q) of the classical complement system in various cells of the immune system (e.g., effector cells). The VH and VL regions can be further subdivided into hypervariable regions known as complementarity-determining regions (CDRs), interspersed with more conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions (VH and VL) of each heavy / light chain pair form an antigen-binding site.

[0066] 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 regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The exact 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, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989), or the IMGT numbering system (Lefranc et al., Dev. Comp. Immunol. 27:55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined by the respective numbering systems. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (Lefranc et al., Dev. Comp. Immunol. 27:55-77, 2003).

[0067] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof can be determined according to various numbering systems known in the art. In one embodiment, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined according to the IMGT, Kabat, or Chothia numbering system. In one embodiment, unless otherwise specified, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined according to the Kabat numbering system.

[0068] As used herein, the term "framework region" or "FR" refers to amino acid residues in the variable region of an antibody other than the residues of the CDRs as defined above.

[0069] The term "antibody" is not limited by any particular method of antibody production. Examples include recombinant antibodies, monoclonal antibodies, polyclonal antibodies, etc. The antibody may be of a different isotype, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0070] As used herein, the term "multispecific antibody" refers to an antibody that can specifically bind to at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody contains multiple antigen-binding domains that specifically bind to different antigens (or epitopes), allowing them to interact with at least two different binding sites and / or target molecules. Each antigen-binding domain of 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 (Fab')2 fragment, or an scFv). In some cases, the antigen-binding domains are 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 can be a trispecific or tetraspecific antibody.

[0071] 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 composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region). These antibodies are typically derived from the variable regions of heavy chain antibodies (such as camelids and sharks). A typical sdAb is composed of four framework regions and three CDRs arranged in the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The sdAb may be truncated at the N- or C-terminus to include only a portion of FR1 and / or FR4, or one or two of these framework regions may be absent, provided that antigen-binding activity and specificity are substantially maintained. Single domain antibodies are also called nanobodies, and these terms can be used interchangeably. The variable regions of single domain antibodies or nanobodies are called VHH domains.

[0072] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, also referred to as an "antigen-binding portion," that retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen. 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 antibodies can be obtained by recombinant DNA techniques or enzymatic or chemical cleavage of full-length antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, dis-scFv, (scFv)2, and other such polypeptides that contain at least a portion of an antibody sufficient to confer specificity to the polypeptide with antigen-binding ability.

[0073] As used herein, the term "Fab" refers to an antibody fragment composed of a light chain comprising VL and CL and a heavy chain fragment comprising VH and CH1; the term "(Fab')2" refers to an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region; and the term "Fab'" refers to a fragment obtained by reducing the disulfide bond linking two heavy chain fragments in an F(ab')2 fragment composed of an intact light chain and the Fd fragment of a heavy chain (composed of the VH and CH1 domains).

[0074] As used herein, the term "Fv" refers to an antibody fragment consisting of the variable regions of the light chain (VL) and heavy chain (VH) of a single arm of an antibody. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site.

[0075] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH region, wherein the VL and VH regions are connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers from the prior art are typically composed of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, although variants thereof can also be used. In some cases, a disulfide bond can exist between the VH and VL of the scFv. In some cases, scFvs can form disulfide-linked scFvs, which refer to antibodies formed by linking two or more individual scFvs in series. In some cases, scFvs can form (scFv)2, which refers to antibodies formed by linking two or more individual scFvs in parallel.

[0076] As used herein, the term "variant" in the context of a polypeptide (including a single polypeptide) refers to a polypeptide or peptide containing an amino acid sequence altered by the introduction of substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently attaching any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide can be modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cellular ligands, or other proteins. 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, and metabolic synthesis of tunicamycin. Furthermore, the variant possesses similar, identical, or improved function as the polypeptide or peptide from which it is derived.

[0077] As used herein, the term "specific binding" refers to a non-random interaction between two molecules, such as the interaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (KD) of the interaction. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The specific binding properties between two molecules can be measured using methods well known in the art, such as surface plasmon resonance (SPR) using Biacore, biolayer interferometry (BLI), or Kinexa, which measures dissociation constants.

[0078] As used herein, the term "vector" refers to a nucleic acid delivery tool into which a polynucleotide can be inserted. If the vector can express a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, allowing the genetic material elements carried by the vector 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, cosmid vectors, artificial chromosomes (e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs)), bacteriophages such as λ 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 viruses), poxviruses, baculoviruses, papilloma viruses, and polyoma viruses (such as SV40). Vectors can contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, vectors may contain an origin of replication.

[0079] 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, eukaryotic cells such as fungal cells (e.g., yeast or Aspergillus), insect cells such as S2 or Sf9 cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells, and immune cells (e.g., T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells). Host cells can include individual cells or populations of cells.

[0080] As used herein, the term "identity" refers to the degree of sequence identity between two polypeptides or two nucleic acids. If a position in the compared sequences is occupied by the same base or amino acid monomer subunit (for example, if a position in each of two DNA molecules is occupied by adenine, or if a position in each of two peptides is occupied by lysine), then each molecule is identical at that position. The "percent identity" between two sequences is the ratio of the number of identical positions to the total number of positions compared, expressed as a percentage. For example, if 6 out of 10 positions are identical, the sequences have 60% identity. The DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions are identical). Typically, sequences are compared by alignment to maximize identity. Such alignment can be achieved using methods such as the Needleman-Wunsch algorithm implemented in computer programs such as ALIGN (DNAstar, Inc.) (J. Mol. Biol. 1970 Mar;48(3):443-53.). Percent identity between amino acid sequences can also be calculated using the algorithm of E. Meyers and W. Miller, available in the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 (Comput. Appl. Biosci., 4:11-17 (1988)). Additionally, the Needleman and Wunsch (J Mol Biol. 48:44-543 (1970)) algorithm, integrated into the GAP program of the GCG software package (available at www.gcg.com), can determine the percentage identity between two amino acid sequences using a Blossum62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0081] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide containing 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 those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, for example, an amino acid residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties including the ability to form covalent 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a 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 (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. Sci. USA 94:412-417, 1997).

[0082] The 20 conventional amino acids referred to herein are written according to conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR 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 one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0083] 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 active ingredient (see, e.g., 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, osmotic pressure maintainers, absorption delaying agents, 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. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, etc. Absorption delaying agents include, but are not limited to, monostearate salts and gelatin. The diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, and polyols (e.g., glycerol). The preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, and sorbic acid. The stabilizer has the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the pharmaceutical, and includes, but is not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or their degradation products (e.g., lactalbumin hydrolysate), etc. In an exemplary embodiment, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution).In certain exemplary embodiments, such a sterile injectable fluid is selected from the group consisting of 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), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0084] As used herein, the term "prevention" refers to a method performed to prevent or delay the occurrence of a disease, disorder, or symptom in a subject. As used herein, the term "treatment" refers to a method performed to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include (but are not limited to) alleviation of symptoms, reduction of the extent of the disease, stabilization of the disease state (i.e., no longer worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and alleviation of symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonging survival compared to expected survival (if not receiving treatment).

[0085] As used herein, the term "subject" refers to a mammal, such as a primate, including a human. In some embodiments, the subject (e.g., a human) has a tumor.

[0086] As used herein, the terms "cancer" and "tumor" are used interchangeably to refer to a broad range of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or infiltrating cells in surrounding tissues, which can 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 both solid tumors and hematologic malignancies. [Effects of the Invention]

[0087] The present invention provides a multispecific antibody targeting PD-1 and VEGF, which can specifically block the PD-1 and VEGF signaling pathways, thereby producing a synergistic anti-tumor effect while simultaneously reducing the incidence of side effects and making clinical administration more convenient. The multispecific antibody of the present invention has significant clinical value.

[0088]

[0023] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are used only to illustrate the present invention and are not intended to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the drawings and the following detailed description of the preferred embodiments. [Brief explanation of the drawings]

[0089] [Figure 1] FIG. 1 shows the results of PD1-blocking activity in cell-based experiments. [Figure 2] FIG. 2 shows the results of VEGF blocking activity in cell-based experiments. [Figure 3] FIG. 3 shows the change in tumor volume in an in vivo antitumor experiment. [Figure 4] FIG. 4 shows changes in mouse body weight in an in vivo antitumor experiment. DETAILED DESCRIPTION OF THE INVENTION

[0090] Sequence information The sequence information involved in the present invention is provided in the table below.

[0091] [Table 1] TIFF2025527160000003.tif247164TIFF2025527160000004.tif251164TIFF2025527160000005.tif247164 [Example]

[0092] The invention will now be illustrated by the following non-limiting examples.

[0093] Those skilled in the art will understand that the examples illustrate the present invention by way of example and are not intended to limit the scope of protection sought by this application. The experimental methods in the examples are conventional methods unless otherwise specified. If specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. If the manufacturers of reagents or equipment used are not specified, they were all commercially available conventional products. The present invention will now be described with reference to the following examples, which are intended to illustrate, but not limit, the present invention.

[0094] Example 1. Design and expression of bispecific antibodies

[0095] 1. Preparation of Anti-PD-1 Single Domain Antibodies Healthy adult female alpacas were immunized with recombinant human PD-1 protein. After immunization, peripheral blood was collected from the alpacas, lymphocytes were isolated, total RNA was extracted, and cDNA was obtained for establishing an anti-PD-1 single domain antibody phage display library. VHH single domain antibodies were screened for strong binding activity to human PD-1, blocking the binding of human PD-L1 to human recombinant PD-1, and competing with pembrolizumab for binding to the same or overlapping PD-1 epitope. The detailed screening process is described in International Patent Application PCT / CN2022 / 074550. Finally, a VHH single domain antibody was screened and obtained. Its VHH sequence is shown in SEQ ID NO: 1, and its CDR1-CDR3 are shown in SEQ ID NOs: 2-4, respectively, according to the Kabat numbering system.

[0096] 2. Design and Expression of Bispecific Antibodies Bispecific antibodies were constructed based on the above anti-PD-1 VHH antibodies and anti-VEGF antibodies. The anti-VEGF antibodies used were bevacizumab (variant sequence from U.S. Patent No. 7,575,893) and ranibizumab sequences. The VH and VL of bevacizumab (variant) are shown in SEQ ID NOS: 5-6, respectively. According to the Kabat numbering system, its CDR-H1 to CDR-H3 are shown in SEQ ID NOS: 7-9, respectively, and its 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, its CDR-H1 to CDR-H3 are shown in SEQ ID NOS: 15-17, respectively, and its CDR-L1 to CDR-L3 are shown in SEQ ID NOS: 18-20, respectively.

[0097] Six bispecific antibodies were constructed, and their structures are shown in Table 2. For 162-17 and 162-20, their heavy chains had the structure shown in [VHH]-[L]-[VH]-[CH], and their light chains had the structure shown in [VL]-[CL]. For 162-18 and 162-21, their heavy chains had the structure shown in [VH]-[CH], and their light chains had the structure shown in [VHH]-[L]-[VL]-[CL]. For 162-19 and 162-22, their heavy chains had the structure shown in [VH]-[CH]-[L]-[VHH], and their light chains had the structure shown in [VL]-[CL]. [L] above represents the linker shown in SEQ ID NO: 35.

[0098] [Table 2]

[0099] Nucleic acid molecules encoding each polypeptide chain of the bispecific antibody were constructed into expression vectors and transiently expressed in HEK cells. After expression, the antibody was purified with Protein A. The expression yield and purity after purification are shown in Table 3.

[0100] [Table 3]

[0101] Example 2. Experiments on PD1 blocking activity

[0102] The activity of bispecific antibodies to block the PD-1 / PD-L1 signaling pathway was measured in this example.

[0103] PDL1-CHO cells (GenScript, Cat. No. RD00703) were plated at 3.5 × 10 in a 96-well white plate (Thermo Cat. No. 136101). 4 Cells were seeded at 4 × 10 cells / well, where cells were seeded in the center 6 × 10 wells of a 96-well plate, and 200 μl of culture medium was added to the surrounding wells. After overnight incubation at 37°C and 5% CO for 16–20 h, the next day, the cells were seeded at 4 × 10 cells / well using complete medium (RPMI 1640 + 10% FBS). 5 PD1-Jurkat cell solution (GenScript, Catalog No. RD00702) at 1000 cells / ml and the antibody to be measured (initial concentration 300 nM, 4-fold diluted) were prepared, and the two were added to a 96-well plate from which the culture medium had been discarded. The plate was then incubated at 37°C and 5% CO2 for 6 hours, and a Fire-LumiNova was used to detect the chemical signal. TM Luciferase detection reagent (GenScript, Cat. No. L00877C) was added. A four-parameter curve was drawn using Graphpad.

[0104] The results are shown in Figure 1 and Table 4. The bispecific antibodies of the present application had significant activity in blocking PD1 / PD-L1.

[0105] [Table 4]

[0106] Example 3. Experiments on VEGF blocking activity

[0107] The activity of the bispecific antibodies to block the VEGF signaling pathway was measured in this example.

[0108] HEK293 cells overexpressing VEGFR2 (GenScript, catalog no. RD00704) were seeded at 5,000 cells per well in a 96-well plate and cultured overnight for 16-20 hours. 2x VEGF165 (Sino Biological, catalog no. 11066-HNAH) and the test antibody were prepared in complete medium (DMEM + 10% FBS). The recommended concentration of 2x VEGF165 was 30 ng / ml, and the concentration of the test antibody was 20 μg / ml. Five 3-fold dilutions and five 10-fold dilutions were made. 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, mixed, and incubated at 37°C, 5% CO2. 100 μl of complete medium was added to cell-free wells to obtain background absorbance values. 6 hours later, Fire-Lumi TM The reagent was added at 100 μl / well, incubated for 5 to 60 minutes, and chemical signals were detected.

[0109] The results are shown in Figure 2 and Table 5. The bispecific antibodies of the present application had significant activity in blocking the VEGF signaling pathway.

[0110] [Table 5]

[0111] Example 4. In vivo antitumor activity experiment

[0112] The in vivo antitumor efficacy of bispecific antibodies in animal tumor models was determined in this example.

[0113] Human breast cancer cells MDA-MB-231 (purchased from the Chinese Academy of Sciences) were cultured and expanded in vitro. 5 × 10 6The cells were resuspended in PBS and subcutaneously implanted into 6- to 8-week-old female NSG mice (obtained from Shanghai Southern Model Organism Technology Co., Ltd.) to establish tumor-bearing models. PBMCs (obtained from Miaoshun (Shanghai) Biotechnology Co., Ltd.) were added at 4 × 10 cells / mL at 7 days after tumor bearing. 6 The mice were then injected into the tail vein with 100 cells / mouse. After 14 days, the mice were then resuspended in approximately 100 mm 3 The mice were divided into groups according to tumor volume and intraperitoneally injected (twice weekly). Body weight and tumor volume were recorded twice weekly. A comparative analysis of the tumor volume and body weight of the mice after 39 days is shown in Figures 3 and 4, respectively. The results showed that the bispecific antibody of the present application could significantly inhibit tumor growth, and was significantly better than nivolumab. The bispecific antibody of the present invention also had good in vivo safety.

[0114] Although specific embodiments of the present invention have been described in detail, it will be understood that those skilled in the art can make various modifications and changes to the details based on all the teachings disclosed, and these modifications are within the scope of protection of the present invention. All of the present invention is provided by the appended claims and any equivalents thereof.

Claims

1. A multispecific antibody comprising a first antigen-binding domain targeting programmed 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, and the VHH domain comprises a CDR1 set forth in SEQ ID NO: 2 or a variant thereof, a CDR2 set forth in SEQ ID NO: 3 or a variant thereof, and a CDR3 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 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; Preferably, the VHH domain comprises CDR1, CDR2 and CDR3 contained in the VHH shown in SEQ ID NO: 1, and preferably the CDRs are determined by the Kabat, IMGT or Chothia numbering system.

2. 2. The multispecific antibody of claim 1, wherein the first antigen-binding domain comprises a VHH comprising the sequence set forth in 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), wherein the substitutions are preferably conservative substitutions; Preferably, the first antigen-binding domain comprises the VHH shown in SEQ ID NO:

1.

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

4. 4. The multispecific antibody according to claim 1, wherein the second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL); (i) the VH comprises CDR-H1 as set forth in SEQ ID NO: 7, CDR-H2 as set forth in SEQ ID NO: 8, and CDR-H3 as set forth in SEQ ID NO: 9, and the VL comprises CDR-L1 as set forth in SEQ ID NO: 10, CDR-L2 as set forth in SEQ ID NO: 11, and CDR-L3 as set forth in SEQ ID NO: 12; or (ii) the VH comprises CDR-H1, CDR-H2, and CDR-H3 contained in the VH set forth in SEQ ID NO: 5, and the VL comprises CDR-L1, CDR-L2, and CDR-L3 contained in the VL set forth in SEQ ID NO: 6, preferably wherein the CDRs are determined by the Kabat, IMGT, or Chothia numbering system; Preferably, said VH and / or VL further comprise a framework region derived from a mammalian (e.g., human or murine) 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, wherein said 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, or 5 amino acid substitutions, deletions, or additions), preferably wherein said substitutions are conservative substitutions; Preferably, the second antigen-binding domain comprises the VH set forth in SEQ ID NO:5 and the VL set forth in SEQ ID NO:

6.

5. 4. The multispecific antibody according to claim 1, wherein the second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL); (i) the VH comprises CDR-H1 as set forth in SEQ ID NO: 15, CDR-H2 as set forth in SEQ ID NO: 16, and CDR-H3 as set forth in SEQ ID NO: 17, and the VL comprises CDR-L1 as set forth in SEQ ID NO: 18, CDR-L2 as set forth in SEQ ID NO: 19, and CDR-L3 as set forth in SEQ ID NO: 20; or (ii) the VH comprises CDR-H1, CDR-H2, and CDR-H3 contained in the VH set forth in SEQ ID NO: 13, and the VL comprises CDR-L1, CDR-L2, and CDR-L3 contained in the VL set forth in SEQ ID NO: 14, preferably wherein the CDRs are determined by the Kabat, IMGT, or Chothia numbering system; Preferably, said VH and / or VL further comprise a framework region derived from a mammalian (e.g., human or murine) immunoglobulin; Preferably, the VH comprises the sequence set forth in SEQ ID NO: 13 or a variant thereof, and / or the VL comprises the sequence set forth in SEQ ID NO: 14 or a variant thereof, wherein said 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, or 5 amino acid substitutions, deletions, or additions), preferably wherein said substitutions are conservative substitutions; Preferably, the second antigen-binding domain comprises the VH set forth in SEQ ID NO: 13 and the VL set forth in SEQ ID NO:

14.

6. 6. The multispecific antibody of claim 1, wherein the first antigen-binding domain and the second antigen-binding domain are optionally linked via a linker; Preferably, the linker is a peptide linker, e.g., a flexible peptide linker; Preferably, the linker is a peptide linker containing one or more glycines and / or one or more serines, such as (G ４ S) A multispecific antibody, wherein n is 1, 2, 3, or 4.

7. 7. The multispecific antibody of claim 1, wherein the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), an Fv fragment, a Fab fragment, an F(ab') fragment, or a Fab fragment. ２ A multispecific antibody that is a scFv, a fragment thereof, or an scFv.

8. 8. The multispecific antibody of claim 7, wherein the second antigen-binding domain comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, and the light chain comprising 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, e.g., 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 a kappa or lambda light chain constant region; Preferably, the multispecific antibody wherein the light chain constant region comprises the sequence shown in SEQ ID NO:

22.

9. 9. The multispecific antibody of claim 8, wherein the first antigen-binding domain is optionally linked to the N-terminus and / or C-terminus of the heavy chain and / or 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 linked to the N-terminus or C-terminus of the heavy chain or to the N-terminus of the light chain via a linker; Preferably, said second antigen-binding domain comprises two identical heavy chains and two identical light chains, and said first antigen-binding domain is linked via a linker optionally to the N-terminus or C-terminus of both heavy chains or to the N-terminus of both light chains.

10. The multispecific antibody according to any one of claims 1 to 9, (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. A multispecific antibody comprising:

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

12. 11. The multispecific antibody of claim 1, further comprising at least one additional antigen-binding domain that targets an antigen different from the antigen bound by the first and second antigen-binding domains, Preferably, the multispecific antibody is a trispecific or tetraspecific antibody.

13. 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, said isolated nucleic acid molecules comprise a nucleotide sequence encoding each of the polypeptide chains of said multispecific antibody, wherein said nucleotide sequences are present on the same isolated nucleic acid molecule or on different isolated nucleic acid molecules.

14. A vector comprising the nucleic acid molecule of claim 13, Preferably, said vector comprises a nucleotide sequence encoding each of the polypeptide chains of the multispecific antibody according to any one of claims 1 to 12, said nucleotide sequences being present on the same vector or on different vectors.

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

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

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, said pharmaceutical composition further comprises an additional pharmaceutically active agent, such as an anti-tumor agent.

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

19. 19. A method for preventing and / or treating tumors, comprising administering to a subject in need thereof an effective amount of the multispecific antibody of any one of claims 1 to 12, the isolated nucleic acid molecule of claim 13, the vector of claim 14, the host cell of claim 15, or the pharmaceutical composition of claim 17, Preferably, the tumor is selected from solid tumors, Preferably, the tumor is selected from lung cancer (e.g., non-small cell lung cancer or small cell lung cancer), liver cancer (e.g., hepatocellular carcinoma), kidney cancer (e.g., renal cell carcinoma), gastric cancer, ovarian cancer, cervical cancer, endometrial cancer, breast cancer (e.g., triple-negative breast cancer), colon cancer and brain cancer (e.g., glioblastoma); Preferably, the subject is a mammal, e.g., a human; Preferably, the method further comprises administering 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 therapy) to the subject.

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