Multi-specific antibodies targeting Trop2 and PD-1 and uses thereof

Abstract

The invention belongs to the field of antibody drugs, and particularly relates to a Trop2-targeting monoclonal antibody, a Trop2 and PD-1-targeting multispecific antibody, a pharmaceutical composition containing the monoclonal antibody or the multispecific antibody, and application of the monoclonal antibody and the multispecific antibody in tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of antibody drugs, and specifically relates to a monoclonal antibody targeting Trop2 and a multispecific antibody targeting Trop2 and PD-1, a pharmaceutical composition containing the monoclonal antibody or the multispecific antibody, and their use for treating tumors. Background Art

[0002] Trophoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a cell surface glycoprotein that is associated with the occurrence, invasion, and metastasis of malignant tumors. TROP2 is overexpressed in a variety of malignant tumors, including, for example, breast cancer, pancreatic cancer, gallbladder cancer, colon cancer, gastric cancer, non-small cell lung cancer, prostate cancer, uterine cancer, and oral squamous cell carcinoma, etc., and is rarely expressed or not expressed in normal adult tissues. Therefore, TROP2 is expected to become a target for tumor treatment.

[0003] The antibody-drug conjugate IMMU-132 of TROP2 has been approved by the FDA for marketing and is used for the third-line treatment of triple-negative breast cancer, and clinical studies have been conducted in breast cancer, non-small cell lung cancer, ovarian cancer, urothelial cancer, endometrial cancer, and head and neck cancer. IMMU-132 is composed of the antibody hRS7 targeting TROP2 conjugated with the active metabolite SN-38 of irinotecan, and can be used to treat a variety of epithelial malignancies such as breast cancer (triple-negative breast cancer), ovarian cancer, small cell lung cancer, etc., which confirms the feasibility as a tumor treatment target. Although immunotherapies targeting TROP2 have been reported, there is still a need for new drugs targeting TROP2. Summary of the Invention

[0004] The present invention provides a monoclonal antibody targeting Trop2, a multispecific antibody targeting Trop2 and PD-1, and a method for treating cancer using such a monoclonal antibody or multispecific antibody. The multispecific antibody targeting Trop2 and PD-1 of the present invention can specifically bind to effector cells and tumor cells, showing high cell bridging efficiency, thereby directing effector cells to tumor cells and selectively enhancing the anti-tumor activity at the tumor site. The following aspects are thus provided.

[0005] Trop2 antibody

[0006] In one aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that can specifically bind to Trop2, wherein the monoclonal antibody or the antigen-binding fragment thereof comprises: heavy chain CDR1, CDR2, and CDR3 respectively comprising SEQ ID NOs: 1, 2, and 3, and light chain CDR1, CDR2, and CDR3 respectively comprising SEQ ID NOs: 4, 5, and 6.

[0007] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises: heavy chain CDR1, CDR2, and CDR3 having sequences as set forth in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 having sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively.

[0008] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein,

[0009] the VH comprises: the sequence shown in SEQ ID NO: 7, a sequence having at least 80% (e.g., 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%) identity thereto, or a sequence differing therefrom by only one or several (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions; and / or,

[0010] the VL comprises: the sequence shown in SEQ ID NO: 8, a sequence having at least 80% (e.g., 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%) identity thereto, or a sequence differing therefrom by only one or several (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions. Preferably, the substitution is a conservative substitution.

[0011] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a VH having the sequence shown in SEQ ID NO: 7 and a VL having the sequence shown in SEQ ID NO: 8.

[0012] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof further comprises a constant region. The constant region sequence may be derived from mammalian (e.g., human) immunoglobulin. In certain embodiments, the heavy chain of the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3, or IgG4), and the light chain of the monoclonal antibody or antigen-binding fragment thereof comprises a light chain constant region derived from human immunoglobulin (e.g., κ or λ).

[0013] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region shown in SEQ ID NO: 25 and / or a light chain constant region shown in SEQ ID NO: 26.

[0014] In certain embodiments, the heavy chain constant region may comprise one or more amino acid mutations or chemical modifications to alter one or more of the following properties of the antibodies of the present invention: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function, etc. Functional alterations can be generated by replacing at least one amino acid residue in the antibody constant region with a different residue or by chemical modification. For example, altering the affinity of the antibody for effector ligands (such as FcR or complement C1q) can change effector function (e.g., reduce or enhance). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.

[0015] In certain embodiments, the antigen-binding fragment is selected from Fab, Fab’, (Fab’)2, Fv, disulfide-linked Fv, scFv, diabody, and single-domain antibody (sdAb).

[0016] In certain embodiments, the monoclonal antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0017] On the other hand, the present invention also provides a multispecific antibody comprising a monoclonal antibody specifically binding to Trop2 of the present invention or an antigen-binding fragment thereof. In certain embodiments, the multispecific antibody specifically binds to Trop2 and additionally specifically binds to one or more other targets. In certain embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetra-specific antibody.

[0018] Multispecific antibody

[0019] In one aspect, the present invention provides a multispecific antibody comprising a first antigen-binding domain specific for Trop2 (such as human Trop2) and a second antigen-binding domain specific for PD-1 (such as human PD-1).

[0020] In certain embodiments, the multispecific antibody promotes the targeting and recruitment of effector cells to tumor cells by binding to PD-1 present on effector cells (such as T cells) and Trop2 on tumor cells, inducing tumor-specific cell killing activity.

[0021] In certain embodiments, the first antigen-binding domain comprises a first variable light chain (VL) and a first variable heavy chain (VH), and the first variable light chain (VL) and the first variable heavy chain (VH) together form a domain capable of specifically binding to Trop2; the second antigen-binding domain comprises a second variable light chain (VL) and a second variable heavy chain (VH), and the second variable light chain (VL) and the second variable heavy chain (VH) together form a domain capable of specifically binding to PD-1.

[0022] I. Trop2 binding domain

[0023] In certain embodiments, the first antigen-binding domain comprises: heavy chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 4, 5, and 6, respectively. In certain embodiments, the first antigen-binding domain comprises: heavy chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 4, 5, and 6, respectively.

[0024] In certain embodiments, the first antigen-binding domain comprises a variable heavy chain (VH) and a variable light chain (VL), wherein,

[0025] the VH comprises: the sequence shown in SEQ ID NO: 7, a sequence having at least 80% (such as 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%) identity thereto, or a sequence differing therefrom only by substitution, deletion, or addition of one or several (such as 1, 2, or 3) amino acids; and / or,

[0026] the VL comprises: the sequence shown in SEQ ID NO: 8, a sequence having at least 80% (such as 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%) identity thereto, or a sequence differing therefrom only by substitution, deletion, or addition of one or several (such as 1, 2, or 3) amino acids. Preferably, the substitution is a conservative substitution.

[0027] In certain embodiments, the first antigen-binding domain comprises: a VH having a sequence as shown in SEQ ID NO: 7 and a VL having a sequence as shown in SEQ ID NO: 8.

[0028] In certain embodiments, the first antigen-binding domain is a murine antibody, a chimeric antibody, or a humanized antibody.

[0029] In certain embodiments, the first antigen-binding domain is selected from full-length antibodies (such as IgG antibodies), antigen-binding fragments (such as scFv, Fab, scFab), or any combination thereof. In certain embodiments, the first antigen-binding domain is selected from IgG antibodies, scFv, Fab, or scFab.

[0030] II. PD-1 binding domain

[0031] In certain embodiments, the second antigen-binding domain comprises: heavy chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 11, 12, and 13, respectively, and light chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 14, 15, and 16, respectively. In certain embodiments, the first antigen-binding domain comprises: heavy chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 11, 12, and 13, respectively, and light chain CDR1, CDR2, and CDR3 having sequences as shown in SEQ ID NOs: 14, 15, and 16, respectively.

[0032] In certain embodiments, the second antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein,

[0033] the VH comprises: the sequence shown in SEQ ID NO: 17, a sequence having at least 80% (such as 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%) identity thereto, or a sequence differing therefrom only by substitution, deletion, or addition of one or several (such as 1, 2, or 3) amino acids; and / or,

[0034] the VL comprises: the sequence shown in SEQ ID NO: 18, a sequence having at least 80% (such as 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%) identity thereto, or a sequence differing therefrom only by substitution, deletion, or addition of one or several (such as 1, 2, or 3) amino acids. Preferably, the substitution is a conservative substitution.

[0035] In certain embodiments, the second antigen-binding domain comprises: a VH having a sequence as set forth in SEQ ID NO:17 and a VL having a sequence as set forth in SEQ ID NO:18.

[0036] In certain embodiments, the second antigen-binding domain is a murine antibody, a chimeric antibody, or a humanized antibody.

[0037] In certain embodiments, the second antigen-binding domain is selected from full-length antibodies (such as IgG antibodies), antigen-binding fragments (such as scFv, Fab, scFab), or any combination thereof. In certain embodiments, the second antigen-binding domain is selected from IgG antibodies, scFv, Fab, or scFab.

[0038] III. Structure

[0039] Those skilled in the art will understand that all forms of multispecific antibody structures known in the art can be used in the present invention.

[0040] In certain embodiments, the various domains (such as antigen-binding domains, Fc domains, etc.) comprised by the multispecific antibody are optionally linked by a peptide linker (such as a flexible peptide). In certain embodiments, the peptide linker is selected from peptide linkers comprising one or more glycines (G) and / or serines (S). In certain embodiments, the peptide linker is a flexible peptide comprising (G4S)n, where n is an integer not less than 0, such as 1, 2, 3, or 4. In certain embodiments, the peptide linker comprises an amino acid sequence as set forth in SEQ ID NO:23.

[0041] In certain embodiments, the multispecific antibody is a bispecific antibody. The bispecific antibodies of the present invention can be: (i) a single antibody having two arms comprising different antigen-binding regions, (ii) for example, a single-chain antibody specific for two different epitopes, such as two scFvs tandemly linked via an additional peptide linker; (iii) a dual variable domain antibody (DVD-Ig TM), wherein each light chain and heavy chain contains two variable domains connected in series by a short peptide; (iv) a chemically linked bispecific (Fab’)2 fragment; (v) a TandAb, which is a fusion of two single-chain diabodies, resulting in a tetravalent bispecific antibody with two binding sites for each target antigen; (vi) a flexibody, which is a combination of an scFv and a diabody, resulting in a multivalent molecule; (vii) a so-called “dock and lock” molecule, based on the “dimerization and docking domain” in protein kinase A, which can obtain a trivalent bispecific binding protein composed of two identical Fab fragments linked to different Fab fragments when applied to Fab; (viii) a so-called Scorpion molecule, which contains, for example, two scFvs fused to the two ends of a human Fab arm; or (ix) a diabody.

[0042] In certain embodiments, one of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the other is an antigen-binding fragment (e.g., an scFv, a Fab, or an scFab). In some embodiments, the first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the second antigen-binding domain is an antigen-binding fragment (e.g., an scFv, a Fab, or an scFab). In other embodiments, the first antigen-binding domain is an antigen-binding fragment (e.g., an scFv, a Fab, or an scFab), and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody).

[0043] In certain embodiments, one of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the other is an scFv. In some embodiments, the first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the second antigen-binding domain is an scFv. In other embodiments, the first antigen-binding domain is an scFv, and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody).

[0044] In certain embodiments, the antigen-binding fragment (e.g., an scFv, a Fab, or an scFab) is optionally linked to the N-terminus and / or C-terminus of the heavy chain and / or light chain of the full-length antibody (e.g., an IgG antibody) via a peptide linker. In certain embodiments, the antigen-binding fragment is linked to the C-terminus of the heavy chain and / or light chain. In certain embodiments, the antigen-binding fragment is linked to the C-terminus of the heavy chain.

[0045] In certain embodiments, the peptide linker is a flexible peptide linker. In certain embodiments, the peptide linker is selected from peptide linkers comprising one or more glycines (G) and / or serines (S). In certain embodiments, the peptide linker is a flexible peptide comprising (G4S)n, where n is an integer not less than 0, such as 1, 2, 3, or 4. In certain embodiments, the peptide linker comprises the amino acid sequence as set forth in SEQ ID NO:23.

[0046] In certain embodiments, the multispecific antibody described herein comprises an Fc domain, and the Fc domain comprises first and second Fc domain monomers.

[0047] In some embodiments, the first and second Fc domain monomers are native Fc region sequences.

[0048] In other embodiments, the first and second Fc domain monomers each independently comprise one or more amino acid modifications that can alter effector function or promote heterodimerization of the first and second Fc domain monomers, such as knob-into-hole modifications.

[0049] In certain embodiments, one of the N-terminus and C-terminus of the first Fc domain monomer is optionally linked to one of the first antigen-binding domains through a linker, and the other of them is optionally linked to one of the second antigen-binding domains through a linker.

[0050] In certain embodiments, one of the N-terminus and C-terminus of the second monomer is optionally linked to one of the first antigen-binding domains through a linker, and the other of them is optionally linked to one of the second antigen-binding domains through a linker.

[0051] In some embodiments, one of the first antigen-binding domains is respectively linked to the N-termini of the first monomer and the second monomer; one of the second antigen-binding domains is respectively linked to the C-termini of the first monomer and the second monomer.

[0052] In other embodiments, one of the second antigen-binding domains is respectively linked to the N-termini of the first monomer and the second monomer; one of the first antigen-binding domains is respectively linked to the C-termini of the first monomer and the second monomer.

[0053] IgG-scFv

[0054] By way of example, the multispecific antibody of the present invention can have an IgG-scFv structure.

[0055] In some embodiments, the first antigen-binding domain is a full-length antibody (e.g., an IgG antibody), and the second antigen-binding domain is a scFv. In certain embodiments, the multispecific antibody comprises:

[0056] (i) A first peptide chain that comprises (e.g., from the N-terminus to the C-terminus) the VL and the light chain constant region (CL) of the first antigen-binding domain;

[0057] (ii) A second peptide chain that comprises (e.g., from the N-terminus to the C-terminus) the VH of the first antigen-binding domain, the CH1 region of the heavy chain, a monomer of the Fc domain, and the second antigen-binding domain.

[0058] In other embodiments, the first antigen-binding domain is a scFv and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody). In certain embodiments, the multispecific antibody comprises:

[0059] (i) A first peptide chain that comprises (e.g., from the N-terminus to the C-terminus) the VL and the light chain constant region (CL) of the second antigen-binding domain;

[0060] (ii) A second peptide chain that comprises (e.g., from the N-terminus to the C-terminus) the VH of the second antigen-binding domain, the CH1 region of the heavy chain, a monomer of the Fc domain, and the first antigen-binding domain.

[0061] In certain embodiments, the CL is a kappa light chain constant region, such as a human kappa light chain constant region. In certain embodiments, the CL comprises the sequence shown in SEQ ID NO:26.

[0062] In certain embodiments, the monomer of the Fc domain is the Fc domain monomer of IgG, such as the Fc domain monomer of IgG1 or IgG4. In certain embodiments, the monomer of the Fc domain is the Fc domain monomer of human IgG, such as the Fc domain monomer of human IgG1 or IgG4.

[0063] In certain embodiments, the monomer of the Fc domain comprises CH2 and CH3. In certain embodiments, the monomer of the Fc domain comprises the hinge region, CH2, and CH3.

[0064] In certain embodiments, the heavy chain constant region (CL) comprising the CH1 region of the heavy chain and the monomer of the Fc domain comprises the sequence shown in SEQ ID NO:24 or 25.

[0065] In certain embodiments, the scFv has the structure shown as VH-Linker-VL. In certain embodiments, the scFv has the structure shown as VL-Linker-VH. The Linker is a peptide linker, preferably a flexible peptide. In certain embodiments, the Linker is selected from peptide linkers comprising one or more glycines (G) and / or serines (S). In certain embodiments, the peptide linker is a flexible peptide comprising (G4S)n, where n is an integer not less than 0, such as 1, 2, 3, or 4. In certain embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO:23.

[0066] In certain embodiments, the first antigen-binding domain or the second antigen-binding domain is linked to the C-terminus of the Fc domain monomer via a peptide linker. In certain embodiments, the peptide linker is a flexible peptide linker. In certain embodiments, the peptide linker is selected from peptide linkers comprising one or more glycines (G) and / or serines (S). In certain embodiments, the peptide linker is a flexible peptide comprising (G4S)n, where n is an integer not less than 0, such as 1, 2, 3, or 4. In certain embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO:23.

[0067] In certain embodiments, the multispecific antibody comprises: a first peptide chain comprising the sequence shown in SEQ ID NO:10, and a second peptide chain comprising the sequence shown in SEQ ID NO:21.

[0068] Preparation of antibody

[0069] The monoclonal antibodies and multispecific antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant techniques. For example, monoclonal antibodies can be produced as follows: DNA molecules encoding the heavy and light chain genes of the antibodies of the present application are obtained by chemical synthesis or PCR amplification; the obtained DNA molecules are inserted into an expression vector and then transfected into a host cell; then, the transfected host cell is cultured under specific conditions to express the antibodies of the present application. For example, multispecific antibodies can be produced by co-expressing multiple polynucleotides encoding the respective polypeptide chains of the multispecific antibody. The polypeptide chains produced by co-expression can be joined via, for example, disulfide bonds or other means to form a functional multispecific antibody. For example, the light chain portion of a Fab fragment can be encoded by a separate polynucleotide from the portion of the multispecific antibody that contains the heavy chain portion of the Fab fragment (which portion can further contain an Fc domain monomer and optionally other antigen-binding domains). When co-expressed, the polypeptide containing the heavy chain portion of the Fab fragment will join with the polypeptide containing the light chain portion of the Fab fragment to form a Fab fragment. Again, for example, the portion of the multispecific antibody provided herein that contains one of the two Fc domain monomers (which portion can further contain an antigen-binding domain) can be encoded by a separate polynucleotide from the portion that contains the other of the two Fc domain monomers (which portion can further contain an antigen-binding domain). When co-expressed, the two Fc domain monomers will join to form an Fc domain.

[0070] In another aspect, the present invention provides an isolated nucleic acid molecule comprising (i) a nucleotide sequence encoding a multispecific antibody of the present invention or at least one of its peptide chains or (ii) a nucleotide sequence encoding a monoclonal antibody of the present invention or an antigen-binding fragment thereof or its heavy chain variable region and / or light chain variable region.

[0071] In certain embodiments, the isolated nucleic acid molecule comprises nucleotide sequences encoding the respective peptide chains of the multispecific antibody of the present invention, and the nucleotide sequences encoding the respective peptide chains are present on the same or different isolated nucleic acid molecules.

[0072] In certain embodiments, the isolated nucleic acid molecule comprises nucleotide sequences encoding the heavy chain (or heavy chain variable region) and light chain (or light chain variable region) of a monoclonal antibody of the present invention or an antigen-binding fragment thereof, and the nucleotide sequences encoding the heavy chain (or heavy chain variable region) and light chain (or light chain variable region) are present on the same or different isolated nucleic acid molecules.

[0073] In another aspect, the present invention provides a vector (such as an expression vector) comprising the above-described isolated nucleic acid molecule.

[0074] In certain embodiments, the vector comprises nucleotide sequences encoding each of the peptide chains of the multispecific antibody of the present invention, and the nucleotide sequences encoding each of the peptide chains are 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.

[0075] In certain embodiments, the vector comprises nucleotide sequences encoding the heavy chain (or heavy chain variable region) and the light chain (or light chain variable region) of the monoclonal antibody of the present invention or an antigen-binding fragment thereof, and the nucleotide sequences encoding the heavy chain (or heavy chain variable region) and the light chain (or light chain variable region) are present on the same or different vectors.

[0076] In another aspect, the present invention provides a host cell comprising the nucleic acid molecule or vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., Escherichia 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.).

[0077] In another aspect, the present invention provides a method for preparing the multispecific antibody of the present invention or the monoclonal antibody of the present invention or an antigen-binding fragment thereof, which comprises culturing the host cell as described above under conditions permitting protein expression, and recovering the multispecific antibody or the monoclonal antibody or an antigen-binding fragment thereof from the cultured host cell culture.

[0078] Pharmaceutical composition

[0079] In another aspect, the present invention provides a pharmaceutical composition comprising the multispecific antibody of the present invention, the monoclonal antibody of the present invention or an antigen-binding fragment thereof, an isolated nucleic acid molecule, a vector, or a host cell, and a pharmaceutically acceptable carrier and / or excipient.

[0080] In certain embodiments, the pharmaceutical composition comprises an effective amount of the multispecific antibody.

[0081] In certain embodiments, the pharmaceutical composition comprises an effective amount of the monoclonal antibody or an antigen-binding fragment thereof.

[0082] The pharmaceutical composition of the present invention can be formulated into a dosage form compatible with its intended route of administration. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by incorporating the required dose of the multispecific antibody described herein in a suitable solvent, and optionally, other desired components (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof) at the same time, followed by filtration sterilization. In addition, the sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for ease of storage and use.

[0083] The pharmaceutical composition of the present invention can be administered by any suitable method known in the art. Preferred routes of administration include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration. Parenteral administration refers to a mode of administration that is usually by injection rather than enteral and topical administration, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, it can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration, e.g., intranasal, oral, vaginal, rectal, sublingual or topical.

[0084] Therapeutic use

[0085] In another aspect, the present invention provides a method for treating a tumor, which comprises administering to a subject in need thereof the multispecific antibody, monoclonal antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition of the present invention. The present invention also relates to the use of the multispecific antibody, monoclonal antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, or pharmaceutical composition for treating a tumor, or the use in the preparation of a drug for treating a tumor.

[0086] In certain embodiments, the tumor is Trop2 positive. Trop2 positivity can be determined at the nucleic acid level or the protein level. Methods for determining expression at the nucleic acid level include but are not limited to RT-PCR or real-time RT-PCR. Methods for determining expression at the protein level include but are not limited to immunological assays, such as enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance, etc.

[0087] In certain embodiments, the tumor is a solid tumor.

[0088] In certain embodiments, the tumor is selected from breast cancer, pancreatic cancer, colorectal cancer, cervical cancer, ovarian cancer, prostate cancer, thyroid cancer, gastric cancer, brain cancer, esophageal cancer, bladder cancer, head and neck cancer, endometrial cancer, lung cancer, oral cancer, or any combination thereof.

[0089] The multispecific antibody, monoclonal antibody, or antigen-binding fragment thereof, or pharmaceutical composition 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. The multispecific antibody, monoclonal antibody, or antigen-binding fragment thereof, or pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injection solution can be prepared by incorporating the required dose of the active ingredient in a suitable solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH regulators, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, a sterile injection 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), solution containing a surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0090] The multispecific antibody, monoclonal antibody, or antigen-binding fragment thereof, or pharmaceutical composition of the present 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, intracisternal, inguinal, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art should understand that the route of administration and / or mode will vary according to the intended purpose. In certain embodiments, the multispecific antibody, monoclonal antibody, or antigen-binding fragment thereof, or pharmaceutical composition of the present invention is administered by intravenous injection or bolus injection.

[0091] The multispecific antibodies, monoclonal antibodies, antigen-binding fragments thereof, or pharmaceutical compositions of the present invention can be formulated in dosage unit forms for easy administration. A dosage unit form refers to physically discrete units suitable as a single dose for a subject to be treated; each unit contains a predetermined quantity of the active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0092] The multispecific antibodies, monoclonal antibodies, antigen-binding fragments thereof, or pharmaceutical compositions of the present invention can be administered alone or in combination with other pharmaceutically active agents (such as anti-tumor agents) or other therapies (such as anti-tumor therapies). In certain embodiments, the anti-tumor agent can be selected from, for example, alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, antibodies specifically targeting tumor cells, or immune checkpoint inhibitors.

[0093] The subjects described herein can be mammals, such as humans. In certain embodiments, the subject has a tumor, such as a Trop2-positive tumor.

[0094] Definition of terms

[0095] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the virological, biochemical, and immunological laboratory procedures used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, for a better understanding of the present invention, the definitions and explanations of relevant terms are provided below.

[0096] As used herein, the term "trophoblast cell surface antigen 2 (TROP2)" refers to a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, also known as tumor-associated calcium signal transducer 2 (TACSTD2). TROP2 has been found to be overexpressed in a variety of malignant tumors and is an oncogene associated with the occurrence, invasion, and metastasis of malignant tumors. TROP2 is preferably human-derived and can also be a homologous gene from other species (such as non-human mammals, fish, reptiles, or birds, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, primates, zebrafish, etc.). The sequence of TROP2 is well-known to those skilled in the art and can be found in various public databases, such as NCBI: Gene ID: 4070.

[0097] As used herein, the term "programmed death molecule 1 (PD-1)", also known as CD279, is an immunosuppressive molecule in the CD28 family. PD-1 is preferably of human origin, and can also be a homologous gene from other species (e.g., non-human mammals, fish, reptiles or birds, such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, primates, zebrafish, etc.). The sequence of PD-1 is well known to those skilled in the art and can be found in various public databases, such as NCBI: Gene ID: 5133.

[0098] As used herein, the term "antibody" in its broadest sense refers to a molecule that specifically binds to an epitope and can include various antibody structures as long as they exhibit the required antigen-binding activity. Typically, an antibody can be an immunoglobulin molecule composed 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 the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined 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 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 domains do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin 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 further divided into regions with high variability (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each V H and V LComposed of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form the antigen-binding site respectively. The amino acid assignment in each region or domain may follow the definition of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883.

[0099] 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). Multispecific antibodies contain multiple antigen-binding domains that have binding specificity for different antigens (or epitopes), thereby being able to bind at least two different binding sites and / or target molecules. Each antigen-binding domain contained in the multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., Fv, Fab, scFab, or scFv). In some cases, each antigen-binding domain is linked by a peptide linker. In certain embodiments, the multispecific antibody can be a bispecific antibody, and the term "bispecific antibody" refers to an antibody that has binding specificity for two different antigens (or epitopes).

[0100] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, 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 of ordinary skill in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to a person of ordinary skill in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably determined by the IMGT numbering system.

[0101] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0102] The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0103] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Herein, a "full-length heavy chain" refers to a polypeptide chain that, in the N-terminal to C-terminal direction, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is of the IgE isotype, optionally further includes a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. A full-length antibody contains two antigen-binding sites formed by pairs of VH and VL, respectively, which two antigen-binding sites specifically recognize / bind the same antigen.

[0104] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind 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 which is also referred to as an "antigen-binding portion". Antigen-binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of a whole antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab’, F(ab’)2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (technology from Domantis), domain antibodies (technology from Ablynx), probodies, and such polypeptides that contain at least a portion of an antibody sufficient to confer upon the polypeptide the ability to specifically bind an antigen.

[0105] As used herein, the term "Fab fragment" refers to an antibody fragment consisting of a light chain containing VL and CL and a heavy chain fragment containing VH and CH1, which typically consists of one peptide chain containing VL and CL and another peptide chain containing VH and CH1. However, those skilled in the art understand that the Fab domain can be arranged according to the above natural orientation, but can also contain domain substitutions or exchanges that promote correct VH and VL pairing (such as domain exchange in the Crossmab form); the term "scFab" refers to a single polypeptide chain containing VL, VH, CL, and CH1 domains, where each adjacent domain is optionally connected by a linker. In the typical structure of scFab, the single polypeptide chain contained in scFab contains, from the N-terminus to the C-terminus: VL, CL, VH, and CH1, where CL and VH are usually connected by a peptide linker, or VH, CH1, VL, and CL, where CH1 and VL are usually connected by a peptide linker.

[0106] As used herein, the term "scFv" refers to a single polypeptide chain containing VL and VH domains, where the VL and VH are connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repetitive GGGGS amino acid sequence or variants thereof. In some cases, a disulfide bond can also exist between the VH and VL of scFv.

[0107] As used herein, the term "Fc domain" or "Fc region" or "Fc domain" has the meaning commonly understood by those skilled in the art and can be used interchangeably, which refers to a part of the heavy chain constant region containing CH2 and CH3. The Fc region of an antibody has a variety of different functions but does not participate in antigen binding. "Effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptors); and B cell activation, etc. In some embodiments, the Fc region contains a hinge, CH2, and CH3. When the Fc region contains a hinge, the hinge regulates the dimerization between two Fc-containing polypeptides. The Fc region can be any heavy chain constant region isotype of an antibody, such as IgG1, IgG2, IgG3, or IgG4.

[0108] The Fc domain may include either a native Fc region or a variant Fc region. The native Fc region contains an amino acid sequence that is identical to the amino acid sequence of the Fc region found in nature. For example, the native sequence human Fc region includes the native sequence human IgG1 Fc region (non-A and A allotypes); the native sequence human IgG2 Fc region; the native sequence human IgG3 Fc region; and the native sequence human IgG4 Fc region, as well as its naturally occurring variants. The variant Fc region contains an amino acid sequence that is different from the amino acid sequence of the native sequence Fc region due to at least one amino acid modification. In some embodiments, the variant Fc region may have altered effector functions compared to the native Fc region (e.g., Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function). In some embodiments, the variant Fc region may have a modification that promotes dimerization. As used herein, a "monomer" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide that contains the C-terminal constant region of an immunoglobulin heavy chain capable of stabilizing its own association.

[0109] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first amino acid sequence or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). Then the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecule is identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions / total number of positions × 100%). The optimal alignment of sequences for comparison can be performed by a computerized implementation of known algorithms or by visual inspection. Existing sequence alignment and multiple sequence alignment algorithms include BLAST, ClustalW / ClustalW2 / Clustal Omega, etc.

[0110] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The strength or affinity of a specific binding interaction can be represented by the equilibrium dissociation constant (K D ) of the interaction. In the present invention, the term "K D"refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. 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 property between two molecules can be determined using methods well known in the art. One method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation. The ratio of kdis / kon is equal to the dissociation constant K D K can be measured by any effective method D , kon and kdis values. For example, surface plasmon resonance (SPR) can be used in Biacore to measure the dissociation constant, and bioluminescence interferometry or Kinexa can also be used to measure the dissociation constant.

[0111] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection, such that the genetic elements carried by the vector are 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, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (such as SV40). A vector can contain multiple elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can also contain an origin of replication.

[0112] 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 fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0113] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing an 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 an amino acid residue with another amino acid residue having a similar side chain, e.g., substitutions with residues that are physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having 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 substitute the corresponding amino acid residue with another amino acid residue from the same side chain family. In addition, amino acid residues can also be grouped into classes defined by alternative physical and functional characteristics. For example, alcohol-containing residues (S and T), aliphatic residues (I, L, V, and M), cycloalkenyl-related residues (F, H, W, and Y), hydrophobic residues (A, C, F, G, H, I, L, M, R, T, V, W, and Y), negatively charged residues (D and E), polar residues (C, D, E, H, K, N, Q, R, S, and T), positively charged residues (H, K, and R), small residues (A, C, D, G, N, P, S, T, and V), very small residues (A, G, and S), residues involved in turn formation (A, C, D, E, G, H, K, N, Q, R, S, P, and T), flexible residues (Q, T, K, S, G, P, D, E, and R). Methods for identifying conservative amino acid substitutions are well known in the art.

[0114] The writing of the twenty common amino acids involved in this article follows the common usage. In the present invention, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. And in the present invention, amino acids are generally represented by the single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0115] 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 and includes, but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining reagents, absorption delaying reagents, preservatives. For example, pH regulators include, but are not limited to, phosphate buffer. 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, etc. Osmotic pressure maintaining reagents include, but are not limited to, sugars, NaCl and the like. Absorption delaying reagents 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), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art, and can stabilize the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, saccharides (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.

[0116] As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the 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 deteriorating) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or not. In addition, "treatment" can also refer to prolonging the survival period compared to the expected survival period (if not treated). For "anti-tumor effect", it includes, but is not limited to, for example, reduction in tumor volume, reduction in the number of cancer cells, reduction in the number of metastatic foci, increase in life expectancy, reduction in cancer cell proliferation, reduction in cancer cell survival, or improvement in various physiological symptoms associated with the cancer condition.

[0117] As used herein, the term "effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of a particular disorder. The effective amount herein can vary with factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit the desired response in an individual. The effective amount is also the amount at which the therapeutic benefit exceeds any toxic or detrimental effects of the treatment. For prophylactic use, beneficial or desired results include results such as eliminating or reducing risk, alleviating severity, or delaying the onset of a disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during disease formation. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms resulting from the disease, improving the quality of life of subjects suffering from the disease, reducing the dosage of other medications required to treat the disease, enhancing the effect of another medication (such as via targeting), delaying the progression of the disease, and / or prolonging survival. In the case of cancer or a tumor, the effective amount of a drug can have an effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slowing to some extent and desirably stopping) the infiltration of cancer cells into peripheral organs; inhibiting (i.e., slowing to some extent and desirably stopping) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating one or more symptoms associated with the disorder to some extent. The effective amount can be administered in one or more administrations. For the purposes of the present invention, the effective amount of a multispecific antibody or pharmaceutical composition is the amount sufficient to directly or indirectly effect a prophylactic or therapeutic treatment.

[0118] As used herein, the terms "cancer" and "tumor" are used interchangeably and refer to a large group 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 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 and also hematological malignancies such as lymphoma, leukemia, myeloma, or lymphoid malignancies, as well as cancers of the spleen and lymph nodes.

[0119] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (such as a human) has a tumor (such as a Trop2-positive tumor).

[0120] Advantages of the Invention

[0121] The present invention provides multispecific antibodies targeting Trop2 and PD-1, which can specifically bind to effector cells and tumor cells, exhibit significant cell bridging efficiency, thereby directing effector cells to tumor cells and selectively enhancing the anti-tumor activity at the tumor site. The multispecific antibodies of the present invention have good cytological activity and in vivo pharmacodynamic activity, and have important clinical value.

[0122] The embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0123] Figure 1 Shows the structure of the anti-PD-1 / anti-Trop2 bispecific antibody molecule.

[0124] Figure 2 Schematic diagram of the SDS-PAGE results of the bispecific antibody.

[0125] Figure 3 Schematic diagram of the HPLC results of the bispecific antibody.

[0126] Figure 4 Schematic diagram of the DSC results of the bispecific antibody and the corresponding monoclonal antibody. In the figure: A is h2T81, B is 17D5, C is the bispecific antibody h2T81-G4-aPD1s, and D is the Tm value comparison table.

[0127] Figure 5 Schematic diagram of the ELISA results of the bispecific antibody and the corresponding monoclonal antibody.

[0128] Figure 6 Schematic diagram of the affinity results of the bispecific antibody and the corresponding monoclonal antibody. A is the affinity of h2T81-G4-aPD1s for Human Trop2-His (NCBI Gene ID: 4070), B is the affinity of h2T81 for Human Trop2-His, C is the affinity of h2T81-G4-aPD1s for Human PD1-His (NCBI Gene ID: 5133), D is the affinity of 17D5 for Human PD1-His, E is the affinity of h2T81-G4-aPD1s for Cynomolgus Trop2-His (NCBI Gene ID: 716334), F is the affinity of h2T81-G4-aPD1s for Cynomolgus PD1-His (NCBI Gene ID: 100135775), and G is the affinity KD value comparison table of the bispecific antibody and the corresponding monoclonal antibody.

[0129] Figure 7 Cell binding results of bispecific antibody and corresponding monoclonal antibody.

[0130] Figure 8 Flow cytometry bridging results of bispecific antibody and corresponding monoclonal antibody.

[0131] Figure 9 In vitro killing results of bispecific antibody against different tumors for 24 hours.

[0132] Figure 10 Evaluation of in vivo tumor targeting of bispecific antibody and anti-PD-1 monoclonal antibody.

[0133] Figure 11 Evaluation of tumor inhibition experiment of bispecific antibody and monoclonal antibody. A shows the change in tumor volume, and B shows the change in mouse body weight.

[0134] Sequence information

[0135] The description of the sequences involved in this application is provided in the following table.

[0136] Table 1: Sequence information

[0137]

[0138]

[0139]

[0140]

[0141]

[0142] Examples

[0143] The present invention will now be described with reference to the following examples which are intended to illustrate the invention (but not to limit the invention).

[0144] Those skilled in the art will appreciate that the examples describe the invention by way of illustration and are not intended to limit the scope claimed in this application. The experimental methods in the examples are all conventional methods unless otherwise specified. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0145] The exemplary materials and reagents used in the following examples include:

[0146]

[0147]

[0148] Example 1: Preparation of Bispecific Antibody

[0149] 1. Preparation of Monoclonal Antibody Against Trop2

[0150] The antigen was obtained by recombinant expression of the extracellular domain of human Trop2 fused with mouse IgG2a Fc. Female Balb / c mice at about 6 weeks old were immunized with the antigen, and the antigen mixed with Freund's adjuvant was injected subcutaneously into multiple sites of the mice. After 4 times of antigen immunization, candidate hybridoma cell lines were obtained by screening through fusion with Sp2 / 0 cells. The hybridoma cell lines were preferably selected through evaluation of binding activity, affinity, immunofluorescence, flow cytometry and internalization level, and the antibody variable region genes of the candidate hybridoma cell lines were retrieved to obtain 2T81. According to the principle of humanization transformation, CDR transplantation and pairing were carried out to obtain the optimal combination h2T81 antibody molecule, whose heavy chain variable region (VH) and light chain variable region (VL) were SEQ ID NO:7 and SEQ ID NO:8 respectively, and the heavy chain CDR1, CDR2 and CDR3 of the IMGT number were SEQ ID NOs:1, 2 and 3 respectively, and the light chain CDR1, CDR2 and CDR3 were SEQ ID NOs:4, 5 and 6 respectively.

[0151] 2. Construction of Bispecific Antibody Expression Vector

[0152] 2-1. Structure of Bispecific Antibody

[0153] The structure of h2T81-G4-aPD1s bispecific antibody is as Figure 1 shown in and Table 2, and it contains h2T81-IgG4 (derived from antibody h2T81) that binds Trop2 and scFv (derived from antibody 17D5, which can be referred to in Chinese Patent Application CN201910654839.X) that binds PD-1.

[0154] PD-1-scFv is linked to the C-terminus of the heavy chain of h2T81-IgG4 through L1 (SEQ ID NO:23). The heavy chain variable region and light chain variable region (Trop2-VH, Trop2-VL) of h2T81-IgG4 are shown as SEQ ID NO:7 and SEQ ID NO:8 respectively. PD-1-scFv has the structure shown as VL2-L2-VH2, VH2 and VL2 are shown as SEQ ID NO:17 and SEQ ID NO:18 respectively, and L2 is shown as SEQ ID NO:23.

[0155] Table 2: Structure of h2T81-G4-aPD1s

[0156]

[0157] 2-2. Construction of expression vectors

[0158] The nucleic acid sequences encoding the light chain (i.e., h2T81-K, SEQ ID NO:31) and heavy chain (i.e., h2T81-G4-aPD1s-H, SEQ ID NO:29) of the above bispecific antibody were obtained, and were respectively ligated into the PTT5 vector with PmeI / BamHI to obtain the plasmids PTT5-h2T81-K and PTT5-h2T81-G4-aPD1s-H. The gene synthesis was completed by General Biosystems (Anhui) Co., Ltd. In addition, based on the nucleic acid sequences encoding the light chain (i.e., h2T81-K, SEQ ID NO:31) and heavy chain (i.e., h2T81-H, SEQ ID NO:28) of the Trop2 parental monoclonal antibody h2T81, the plasmids PTT5-h2T81-K and PTT5-h2T81-H were respectively obtained; based on the nucleic acid sequences encoding the light chain (i.e., 17D5-K, SEQ ID NO:30) and heavy chain (i.e., 17D5-H, SEQ ID NO:27) of the PD-1 parental monoclonal antibody 17D5, the plasmids PTT5-17D5-K and PTT5-17D5-H were respectively obtained.

[0159] 3. Protein expression and purification:

[0160] Adjust ExpiCHO cells to 5 - 6×10 6 / mL, and the concentration of the plasmid (i.e., antibody DNA) is 0.8 μg / mL (the ratio of light chain to heavy chain is 1:1). Dilute DNA and the transient transfection reagent ExpiFectamine TM with pre-cooled OptiPROTM medium to the working concentration; add the diluted ExpiFectamine TM to the diluted DNA, immediately gently invert and mix well, and place at room temperature for 1 - 5 minutes. Add the DNA and ExpiFectamine TM mixture to the cells, and gently shake the flask during the addition process. Culture in a shaker, conditions: 37°C, 5% CO2, 110 rpm. After 18 - 22 hours, supplement ExpiCHO TM Feed, and adjust the culture temperature to 32°C. Centrifuge to harvest the supernatant after 10 - 12 days. Purify using an AKTA pure Protein A column to obtain the final antibody product h2T81-G4-aPD1s.

[0161] 4. SDS-PAGE and HPLC detection results

[0162] 4-1. SDS-PAGE

[0163] (1) Take the purified protein sample with a loading amount of 3 μg, and make the loading volume of each protein 15 μL (if the volume is less than 15 μL, make it up with PBS);

[0164] (2) Add 3 μL of reducing and non-reducing loading buffer to the protein sample respectively (add 1 μL of loading buffer to every 5 μL of protein sample). The reducing sample needs to be heated in a metal bath at 100 °C for 10 minutes;

[0165] (3) Perform SDS-PAGE (ExpressPlus PAGE Gel, 10*8, 4-12%) under reducing and non-reducing conditions. The electrophoresis conditions are: 80 V, 90 min. Use a rapid coomassie staining instrument for color development and photographing. The results are as Figure 2 shown.

[0166] 4-2. SEC-HPLC

[0167] Determine the antibody purity by high performance liquid chromatography. First, insert the pipelines into PBS and ultrapure water respectively, connect G3000 and equilibrate for 100 min, then start sample injection (the protein sample concentration is above 0.5 μg / mL, add 150 μL of sample to each sample cup), 30 min / sample. After sample injection, equilibrate again and then remove the sample injection column. The HPLC peak map is exported by the instrument, as Figure 3 shown.

[0168] 5. DSC detection

[0169] Use a differential scanning calorimeter (VP-Capillary DSC) to measure the Tm value of the antibody. Add 300 μL of 1 μg / mL sample to be measured to the sample well, and add an equal volume of PBS to the control well. After removing the bubbles, tighten the metal lid to a pressure above 40, and run the program to increase the temperature from 10 °C to 90 °C. Calculate the Tm values of the monoclonal antibody and bispecific antibody samples respectively based on PBS as the baseline and export the running curve. As Figure 4 shown, the results show that the thermal stability of the bispecific antibody h2T81-G4-aPD1s is basically the same as that of its corresponding monoclonal antibodies h2T81 and 17D5, and it has stable physical properties.

[0170] Example 2: Determination of the binding ability of the bispecific antibody

[0171] ELISA assay

[0172] Evaluate the antibody-antigen binding activity by ELISA

[0173] (1) Dilute Trop2-his and PD1 proteins in PBS to a final concentration of 1 μg / mL, and incubate at 37 °C for 1 hour in an enzyme-linked immunosorbent assay plate at 100 μL / well;

[0174] (2) Discard the liquid in the plate, add PBST washing solution to each well, wash once, and centrifuge dry using a plate centrifuge;

[0175] (3) Add 200 μL / well of NRA blocking solution and incubate at 37 °C for 2 hours;

[0176] (4) Discard the liquid in the plate and centrifuge dry using a plate centrifuge;

[0177] (5) Dilute the antibody in PBS. The highest concentration of the bispecific antibody is 10 μg / mL, and the highest concentration of the parental monoclonal antibody is calculated to be 7.5 μg / mL according to the molecular weight. Perform 2-fold dilution on a shaker, set a total of 12 concentration gradients, and add the diluted antibody correspondingly, 100 μL / well, and incubate at 37 °C for 1 hour;

[0178] (6) Discard the liquid in the plate, add PBST washing solution to each well, wash 5 times, and centrifuge dry using a plate centrifuge;

[0179] (7) Add Goat Anti-Human IgG / HRP Ab (diluted 1:5000 in NRAED enzyme dilution solution), 100 μL / well, and incubate at 37 °C for 30 minutes;

[0180] (8) Discard the liquid in the plate, add PBST washing solution to each well, wash 5 times, and centrifuge dry using a plate centrifuge;

[0181] (9) Add AB mixed chromogenic solution, 100 μL / well, and develop color at 37 °C for 10 minutes;

[0182] (10) Add 50 μL / well of stop solution to terminate color development, and immediately read OD450 using an ELISA reader.

[0183] The results are as Figure 5 shown. The in vitro binding activity of the bispecific antibody is basically consistent with that of the parental antibody.

[0184] Biacore assay

[0185] Use a BIAcore 8k instrument to measure the affinity of the antibody against the antigen. Reagents used: buffer PBS-P (commercial); 0.5 mM NiSO4 (6.57125 mg nickel sulfate hexahydrate added to 50 mL of ultrapure water); 350 mM EDTA (ethylenediaminetetraacetic acid); 3 mM EDTA.

[0186] ① Trop2 or PD-1 protein is captured onto the NTA sensor chip through Ni ions,

[0187] ②The chip was adjusted by reacting with 30 μL of 350 mM EDTA for 1 min, and after washing with buffer, the chip was activated.

[0188] ③The chip was regenerated with 10 μL of 0.5 mM NiSO4 and washed for the first time with 3 mM EDTA.

[0189] ④The capture protein was added and passed through for 180 s at a flow rate of 8 μL / min, and then through the buffer for 120 s.

[0190] ⑤The chip was regenerated with 350 mM EDTA passed through at a flow rate of 30 μL / min for 60 s, and then washed for the second time with buffer.

[0191] ⑥The analyte was added for the experiment. After protein capture, the analyte was bound at a flow rate of 25 μL / min for 120 s and dissociated for 1200 s.

[0192] ⑦The KD value was obtained by calculation with the instrument, and the image data processing and plotting were from Graphpad Prism 8.

[0193] As Figure 6 shown in A - G, the affinities of h2T81 - G4 - aPD1s and h2T81 for hTrop2 were 6.51e - 11 M and 5.15e - 11 M respectively, the affinities of h2T81 - G4 - aPD1s and 17D5 for hPD - 1 were 9.00e - 10 M and 6.52e - 11 M respectively, and the affinities of h2T81 - G4 - aPD1s for cynomolgus monkey Trop2 and PD - 1 were 1.60e - 10 M and 6.93e - 9 M respectively. The results showed that h2T81 - G4 - aPD1 retained the respective binding activities of the parental monoclonal antibodies.

[0194] Cell binding assay

[0195] Flow cytometry was used to evaluate the antibody - cell binding activity.

[0196] (1) Cell treatment: Digest CHO - Trop2 and CHO - PD1 cells, count them, and make each sample contain 1×10 6 cells. Remove the cell supernatant under the condition of centrifuging at 1500 rpm for 5 minutes, resuspend the cell pellet with PBS, and wash the cells twice.

[0197] (2) Antibody dilution: Dilute the antibody using RPMI 1640 medium containing 2% serum. The initial antibody concentration in the first well is 10 μg / mL, and perform serial dilutions on the dilution plate. Resuspend the cells with the diluted antibody at a volume of 100 μL and incubate at 37 °C for 1 hour. After one hour, remove the antibody by centrifugation at 1500 rpm for 5 minutes, resuspend and wash twice with PBS. Resuspend the cells with the diluted fluorescent secondary antibody (Goat anti-Human IgG(H+L)Cross-Adsorbed Secondary Antibody, Alexa Fluor 488) at a volume of 100 μL and incubate at 37 °C in the dark for 30 minutes.

[0198] (3) Analyze the stained cells using a flow cytometer. Analyze the fluorescence intensity values under different antibody concentration incubations using FlowJo software, and evaluate the binding of the antibody to the cells based on the fluorescence values at different concentrations.

[0199] The results are as Figure 7 shown. The binding activity of the bispecific antibody to the cells is consistent with that of the parental antibody.

[0200] Example 3: Detection of cell bridging of bispecific antibody

[0201] The cell bridging experiment was performed by detecting the connection of the antibody to the CHO cell model expressing Trop2 or PD1 after modification. CHO-Trop2 and CHO-PD1 cells were infected with lentivirus to express Trop2 or PD1 and cultured using RPMI 1640 medium containing 10% FBS. Reagents used: CellTrace TM CFSE cell Proliferation Kit; CellTrace TM Far red cell Proliferation Kit.

[0202] ① According to the kit instructions, stain CHO-PD1 cells with CellTrace Far red and CHO-Trop2 cells with CFSE.

[0203] ② Incubate 200 μL of the antibody (20 μg / mL) with 5×10 5 CHO-Trop2 for 1 h.

[0204] ③ Wash once with PBS, add 5×10 5 CHO-PD1 and incubate for 1 h.

[0205] ④ Wash twice with PBS and resuspend in an appropriate volume of PBS.

[0206] ⑤ Use LSRFortessaX-20 to analyze FITC and APC channels after screening, and calculate the percentage of cells that are positive in both FITC and APC channels.

[0207] like Figure 8 As shown, the positive rate of the bispecific antibody h2T81-G4-aPD1s group was 29.2%, the positive rates of the monoclonal antibody h2T81 and 17D5 groups were 2.05% and 2.22% respectively, and the positive rate of the monoclonal antibody h2T81 and 17D5 combination group was 2.37%. This shows that bispecific antibodies can efficiently bridge effector cells and tumor cells, showing a cell bridging efficiency that is better than that of monoclonal antibodies and monoclonal antibody combinations.

[0208] Example 4: In vitro killing results of bispecific antibodies

[0209] The cell killing assay is performed by measuring the killing level of human PBMC or T cells against tumor cells that highly express TROP2. Cell culture: healthy human PBMC; tumor cells MDA-MB-468 (human breast cancer cells), BxPc-3 (human pancreatic cancer cells), HCT116 (human colon cancer cells), HCT-8 (human colorectal adenocarcinoma cells). Reagents used: Gibco Dynabeads TM HumanT-Activator CD3 / CD28; CellTrace TM CFSE cell proliferation kit.

[0210] ① PBMC separation: Taking 10mL whole blood separation as an example, first, evenly mix 10mL fresh human whole blood with an equal volume of serum-free 1640, slowly adhere to the wall and add to the upper layer of 14mL Ficoll-Plaque premium1.084, centrifuge at 500g for 20min, and then reduce the speed to "1". Carefully aspirate the PBMC in the middle layer of the culture medium and density gradient separation solution after centrifugation into a new centrifuge tube with a pipette, add serum-free 1640 to 45mL, centrifuge at 1500rpm for 5 minutes, remove the supernatant, add 3-5mL red blood cell lysis solution (Solarbio), wait for 3 minutes, add five times the volume of culture medium to terminate the reaction, centrifuge at 1500rpm for 5 minutes, remove the supernatant and use.

[0211] ② According to the kit instructions, each 1×10 6 Each cell was activated in vitro with 25 μL CD3 / CD28 Dynabeads to induce PD1 for about three days.

[0212] ③ According to the kit instructions, use CellTrace to stain PBMC cells with Far red and tumor cells with CFSE.

[0213] ④ Add 5000 tumor cells per well to a 96-well plate. After they adhere to the wall, add PBMC with a cell amount 10 times that of the tumor cells and incubate with 50 μg / mL of antibody for 24 h. Take pictures every 6 h using a high-content cell screening imaging analysis system (Opera Phenix).

[0214] ⑤ Use the Columbus website to calculate the fluorescence value of each well. Finally, calculate the Cell lysis (%) of the whole well through the formula = (fluorescence value of the control group - fluorescence value of the experimental group) / fluorescence value of the control group × 100. The data processing and graphing are from Graphpad Prism 8.

[0215] As Figure 9 shown, the bispecific antibody h2T81-G4-aPD1s has significant killing effects on various human tumor cell lines such as triple-negative breast cancer, colon cancer, and pancreatic cancer.

[0216] Example 5: In vivo tumor suppression experiment of bispecific antibody

[0217] In vivo targeting experiment

[0218] (1) Dye labeling: The molecular weights of the bispecific antibody and the monoclonal antibody are 200 kDa and 150 kDa respectively, and the Cy5.5 labeling dye is 767.6 Da. Modulate the mass-volume concentration of the antibody to more than 2 mg, and then mix the Cy5.5 dye with the antibody in a molar ratio of 10 times and keep it in the dark at 4 °C. Mix it with a rotator for 12 h, then transfer the liquid labeling solution to a dialysis bag and dialyze for 12 h, changing the liquid every 4 h. Collect the labeling solution and store it in the dark at 4 °C for standby.

[0219] (2) Small animal in vivo imaging: Use the IVIS Lumina II imaging system of Caliper company for in vivo imaging. Modulate the excitation wavelength to 640 nm, the exposure time to 20 s, and the imaging mode: fluorescence. Inject 200 μg / needle / animal of the bispecific antibody labeled with Cy5.5 dye and 150 μg / needle / animal of the monoclonal antibody into the tail vein of mice respectively, and perform in vivo imaging at 24 h and 48 h.

[0220] The results are as Figure 10 shown, the bispecific antibody can efficiently target the tumor site.

[0221] Tumor suppression experiment

[0222] In the tumor suppression experiment, inoculate the corresponding amount of tumor cells into mice. After the tumor volume grows to a certain size, inject the antibody drug into the abdominal cavity of the mice for treatment, and monitor the growth change of the tumor volume after treatment.

[0223] (1) Tumor cell treatment: Digest the tumor cells to be inoculated with trypsin containing 0.25%. Collect the digested cell suspension into a 50 mL / 15 mL centrifuge tube. Use trypan blue solution for cell counting. Centrifuge at 1500 rpm for 5 minutes to remove the cell supernatant. Resuspend the cell pellet with serum-free cell culture medium. Centrifuge again at 1500 rpm for 5 minutes and repeat the operation once. The inoculation amount of tumor cells for each mouse is 5×10 6 cells / 100 μL, and resuspend the cells with the corresponding volume of serum-free cell culture medium according to the cell number.

[0224] (2) Tumor cell inoculation: Remove the hair at the tumor inoculation site of hPD1-BALB / c mice. Nude mice do not need to be shaved. Use a 1 mL syringe to inoculate the tumor cells at the right hind leg of the mouse.

[0225] (3) Tumor formation observation: Observe the tumor formation status of the mice according to the tumor formation speed of the inoculated tumor cells. Measure the tumor with a vernier caliper and monitor the body weight of the mice with an electronic scale. The tumor volume of the mice is calculated according to the formula:

[0226] tumor volume=(length × width × width) / 2 mm 3

[0227] Select tumors of appropriate size for grouping and drug administration treatment (intraperitoneal injection, 200 μL / mouse). Measure the tumor volume of the mice every two days to evaluate the inhibitory effect of the drug on the tumor growth of the mice.

[0228] As Figure 11 shown, the bispecific antibody shows stronger anti-tumor effects than the PD-1 monoclonal antibody or the Trop2 monoclonal antibody, and still maintains a good tumor regulation effect after stopping drug administration.

[0229] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A multispecific antibody comprising a first antigen-binding domain specific for Trop2 and a second antigen-binding domain specific for PD-1.

2. The multispecific antibody according to claim 1, wherein, The first antigen-binding domain comprises: heavy chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 1, 2, and 3 respectively, and light chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 4, 5, and 6 respectively; Preferably, the first antigen-binding domain comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 7 or a sequence having at least 80% identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 8 or a sequence having at least 80% identity thereto.

3. The multispecific antibody according to claim 1 or 2, wherein, The second antigen-binding domain comprises: heavy chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 11, 12, and 13 respectively, and light chain CDR1, CDR2, and CDR3 comprising SEQ ID NOs: 14, 15, and 16 respectively; Preferably, the second antigen-binding domain comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO: 17 or a sequence having at least 80% identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 18 or a sequence having at least 80% identity thereto.

4. The multispecific antibody according to any one of claims 1-3, wherein, The first antigen-binding domain and the second antigen-binding domain are each independently selected from full-length antibodies (such as IgG antibodies), antigen-binding fragments (such as scFv, Fab, scFab).

5. The multispecific antibody according to any one of claims 1-4, wherein, One of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (such as IgG antibody), and the other is an antigen-binding fragment (such as scFv, Fab, or scFab); Preferably, one of the first antigen-binding domain and the second antigen-binding domain is a full-length antibody (such as IgG antibody), and the other is scFv; Preferably, the antigen-binding fragment is optionally linked to the C-terminus of the heavy chain of the full-length antibody via a peptide linker.

6. The multispecific antibody according to claim 5, wherein, The first antigen-binding domain is a full-length antibody (such as IgG antibody), and the second antigen-binding domain is scFv; Preferably, the multispecific antibody comprises: (i) A first peptide chain comprising the VL of the first antigen-binding domain and a light chain constant region (CL); preferably, the CL is a kappa light chain constant region; (ii) A second peptide chain comprising the VH of the first antigen-binding domain, a heavy chain CH1 region, a monomeric Fc domain, and the second antigen-binding domain; preferably, the monomeric Fc domain is a monomeric Fc domain of IgG, such as a monomeric Fc domain of IgG1 or IgG4; preferably, the monomeric Fc domain comprises a hinge region, CH2, and CH3; preferably, the second antigen-binding domain is linked to the C-terminus of the monomeric Fc domain via a peptide linker.

7. The multispecific antibody according to any one of claims 1-6, wherein, The multispecific antibody comprises: a first peptide chain comprising the sequence shown in SEQ ID NO:10, and a second peptide chain comprising the sequence shown in SEQ ID NO:

21.

8. A monoclonal antibody or an antigen-binding fragment thereof that can specifically bind to Trop2, wherein, The monoclonal antibody or antigen-binding fragment thereof comprises: heavy chain CDR1, CDR2, and CDR3 respectively comprising SEQ ID NOs:1, 2, and 3, and light chain CDR1, CDR2, and CDR3 respectively comprising SEQ ID NOs:4, 5, and 6; Preferably, the monoclonal antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence shown in SEQ ID NO:7 or a sequence having at least 80% identity thereto, and a light chain variable region (VL) comprising the sequence shown in SEQ ID NO:8 or a sequence having at least 80% identity thereto.

9. The monoclonal antibody or antigen-binding fragment thereof according to claim 8, wherein, The antibody or antigen-binding fragment thereof further comprises a constant region; Preferably, the heavy chain of the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (such as IgG, e.g., IgG1, IgG2, IgG3, or IgG4), and the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (such as κ or λ).

10. The monoclonal antibody or antigen-binding fragment thereof according to claim 8 or 9, wherein, The antibody or antigen-binding fragment thereof is selected from Fab, Fab’, (Fab’)2, Fv, disulfide-linked Fv, scFv, diabody, single domain antibody (sdAb), murine antibody, chimeric antibody, humanized antibody, bispecific antibody, or multispecific antibody.

11. An isolated nucleic acid molecule comprising: (i) a nucleotide sequence encoding the multispecific antibody of any one of claims 1-7 or at least one peptide chain thereof, or (ii) a nucleotide sequence encoding the monoclonal antibody or antigen-binding fragment thereof or its heavy chain variable region and / or light chain variable region of any one of claims 8-10; Preferably, the isolated nucleic acid molecule comprises nucleotide sequences encoding each peptide chain of the multispecific antibody of any one of claims 1-7, and the nucleotide sequences encoding each peptide chain are present on the same or different isolated nucleic acid molecules.

12. A vector comprising the isolated nucleic acid molecule of claim 11; Preferably, the vector comprises nucleotide sequences encoding each peptide chain of the multispecific antibody of any one of claims 1-7, and the nucleotide sequences encoding each peptide chain are present on the same or different vectors.

13. A host cell comprising the isolated nucleic acid molecule of claim 11 or the vector of claim 12.

14. A method for preparing the multispecific antibody of any one of claims 1-7 or the monoclonal antibody or antigen-binding fragment thereof of any one of claims 8-10, comprising culturing the host cell of claim 13 under conditions that permit the expression of the multispecific antibody or the monoclonal antibody or antigen-binding fragment thereof, and recovering the multispecific antibody or the monoclonal antibody or antigen-binding fragment thereof from the cultured host cell culture.

15. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1-7, the monoclonal antibody or antigen-binding fragment thereof of any one of claims 8-10, the isolated nucleic acid molecule of claim 11, the vector of claim 12, or the host cell of claim 13, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is a drug having anti-tumor activity, such as an alkylating agent, a mitotic inhibitor, an anti-tumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, a radiosensitizer, an anti-angiogenic agent, a cytokine, a specific antibody targeting tumor cells or an immune checkpoint inhibitor.

16. Use of the multispecific antibody of any one of claims 1-7, the monoclonal antibody or antigen-binding fragment thereof of any one of claims 8-10, the isolated nucleic acid molecule of claim 11, the vector of claim 12, or the host cell of claim 13 or the pharmaceutical composition of claim 15 in the preparation of a drug for treating tumors; Preferably, the tumor is Trop2-positive; Preferably, the tumor is a solid tumor; Preferably, the tumor is selected from breast cancer, pancreatic cancer, colorectal cancer, cervical cancer, ovarian cancer, prostate cancer, thyroid cancer, gastric cancer, brain cancer, esophageal cancer, bladder cancer, head and neck cancer, endometrial cancer, lung cancer, oral cancer or any combination thereof.

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