Multispecific antibody targeting trop2 and PD-1 and use thereof

Abstract

The present invention belongs to the field of antibody drugs, and specifically relates to a monoclonal antibody targeting Trop2, a multispecific antibody targeting Trop2 and PD-1, a pharmaceutical composition comprising the monoclonal antibody or the multispecific antibody, and the use thereof in terms of treating tumors.

Description

Multispecific antibodies targeting Trop2 and PD-1 and uses thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202410032366.0 filed on January 9, 2024, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] 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 comprising the monoclonal antibody or the multispecific antibody, and their use in treating tumors. Background Art

[0004] Trophoblast cell surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), is a cell surface glycoprotein associated with the development, invasion, and metastasis of malignant tumors. TROP2 is overexpressed in a variety of malignancies, including breast cancer, pancreatic cancer, gallbladder cancer, colon cancer, gastric cancer, non-small cell lung cancer, prostate cancer, uterine cancer, and oral squamous cell carcinoma, while rarely or not expressed in normal adult tissues. Therefore, TROP2 is expected to become a target for tumor therapy.

[0005] IMMU 132, an antibody-drug conjugate targeting TROP2, has been approved by the FDA for third-line treatment of triple-negative breast cancer. Clinical studies are also underway in breast cancer, non-small cell lung cancer, ovarian cancer, urothelial carcinoma, endometrial cancer, and head and neck cancer. IMMU 132, consisting of the TROP2-targeting antibody hRS7 conjugated to the active metabolite of irinotecan, SN-38, is indicated for the treatment of various epithelial malignancies, including breast cancer (triple-negative breast cancer), ovarian cancer, and small cell lung cancer, demonstrating its feasibility as a therapeutic target. While immunotherapies targeting TROP2 have been reported, new drugs targeting TROP2 remain unresolved. Summary of the Invention

[0006] The present invention provides monoclonal antibodies targeting Trop2, multispecific antibodies targeting Trop2 and PD-1, and methods for treating cancer using such monoclonal or multispecific antibodies. The multispecific antibodies targeting Trop2 and PD-1 of the present invention can specifically bind to effector cells and tumor cells, exhibiting efficient cell bridging efficiency, thereby directing effector cells to tumor cells and selectively enhancing anti-tumor activity at the tumor site. This provides the following aspects.

[0007] Trop2 Antibody

[0008] 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 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.

[0009] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises: heavy chain CDR1, CDR2 and CDR3 whose sequences are shown in SEQ ID NOs: 1, 2 and 3, respectively, and light chain CDR1, CDR2 and CDR3 whose sequences are shown in SEQ ID NOs: 4, 5 and 6, respectively.

[0010] 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:

[0011] The VH comprises: a sequence as 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 that differs therefrom in only one or several (e.g., 1, 2 or 3) amino acid substitutions, deletions or additions; and / or,

[0012] The VL comprises: a sequence as set forth 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 that differs therefrom in only one or a few (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions. Preferably, the substitutions are conservative substitutions.

[0013] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises: a VH sequence as shown in SEQ ID NO:7 and a VL sequence as shown in SEQ ID NO:8.

[0014] In certain embodiments, the monoclonal antibody or its antigen-binding fragment further comprises a constant region. The constant region sequence can be derived from a mammalian (e.g., human) immunoglobulin. In certain embodiments, the heavy chain of the monoclonal antibody or its antigen-binding fragment comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG, such as IgG1, IgG2, IgG3, or IgG4), and the light chain of the monoclonal antibody or its antigen-binding fragment comprises a light chain constant region derived from a human immunoglobulin (e.g., κ or λ).

[0015] In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises the heavy chain constant region set forth in SEQ ID NO: 25 and / or the light chain constant region set forth in SEQ ID NO: 26.

[0016] 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 invention: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function. Functional changes can be produced by replacing at least one amino acid residue in the antibody constant region with a different residue or chemically modifying it, for example, by altering the affinity of the antibody for an effector ligand (such as FcR or complement C1q), thereby altering effector function (e.g., reducing or enhancing it). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC, and the like.

[0017] In certain embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, disulfide-linked Fv, scFv, diabody, and single domain antibody (sdAb).

[0018] In certain embodiments, the monoclonal antibody is a murine antibody, a chimeric antibody, a humanized antibody, a bispecific antibody, or a multispecific antibody.

[0019] Another aspect of the present invention provides a multispecific antibody comprising a monoclonal antibody or antigen-binding fragment thereof that specifically binds to Trop2. 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 tetraspecific antibody.

[0020] Multispecific antibodies

[0021] In one aspect, the present invention provides a multispecific antibody comprising a first antigen-binding domain specific for Trop2 (eg, human Trop2) and a second antigen-binding domain specific for PD-1 (eg, human PD-1).

[0022] 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 (e.g., T cells) and Trop2 on tumor cells, thereby inducing tumor-specific cell killing activity.

[0023] In certain embodiments, the first antigen-binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), and the first light chain variable region (VL) and the first heavy chain variable region (VH) together form a domain that can specifically bind to Trop2; the second antigen-binding domain comprises a second light chain variable region (VL) and a second heavy chain variable region (VH), and the second light chain variable region (VL) and the second heavy chain variable region (VH) together form a domain that can specifically bind to PD-1.

[0024] I. Trop2 binding domain

[0025] 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.

[0026] In certain embodiments, the first antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0027] The VH comprises: a sequence as 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 that differs therefrom in only one or several (e.g., 1, 2 or 3) amino acid substitutions, deletions or additions; and / or,

[0028] The VL comprises: a sequence as set forth 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 that differs therefrom in only one or a few (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions. Preferably, the substitutions are conservative substitutions.

[0029] In certain embodiments, the first antigen binding domain comprises: a VH sequence as shown in SEQ ID NO:7 and a VL sequence as shown in SEQ ID NO:8.

[0030] In certain embodiments, the first antigen binding domain is a murine antibody, a chimeric antibody, or a humanized antibody.

[0031] In certain embodiments, the first antigen-binding domain is selected from a full-length antibody (e.g., an IgG antibody), an antigen-binding fragment (e.g., scFv, Fab, scFab), or any combination thereof. In certain embodiments, the first antigen-binding domain is selected from an IgG antibody, scFv, Fab, or scFab.

[0032] II. PD-1 binding domain

[0033] 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.

[0034] In certain embodiments, the second antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein

[0035] The VH comprises: a sequence as shown in SEQ ID NO: 17, 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 that differs therefrom in only one or several (e.g., 1, 2 or 3) amino acid substitutions, deletions or additions; and / or,

[0036] The VL comprises: a sequence as set forth in SEQ ID NO: 18, 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 that differs therefrom in only one or a few (e.g., 1, 2, or 3) amino acid substitutions, deletions, or additions. Preferably, the substitutions are conservative substitutions.

[0037] In certain embodiments, the second antigen binding domain comprises: a VH sequence as shown in SEQ ID NO: 17 and a VL sequence as shown in SEQ ID NO: 18.

[0038] In certain embodiments, the second antigen binding domain is a murine antibody, a chimeric antibody, or a humanized antibody.

[0039] In certain embodiments, the second antigen-binding domain is selected from a full-length antibody (e.g., an IgG antibody), an antigen-binding fragment (e.g., scFv, Fab, scFab), or any combination thereof. In certain embodiments, the second antigen-binding domain is selected from an IgG antibody, scFv, Fab, or scFab.

[0040] III. Structure

[0041] Those skilled in the art will appreciate that all multispecific antibody structures known in the art can be used in the present invention.

[0042] In certain embodiments, the various domains (e.g., antigen-binding domains, Fc domains, etc.) contained in the multispecific antibody are optionally connected by a peptide linker (e.g., a flexible peptide). In certain embodiments, the peptide linker is selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, the peptide linker is a flexible peptide comprising (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3, or 4. In certain embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO: 23.

[0043] 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) a single chain antibody specific for two different epitopes, for example via two scFvs connected in series via an additional peptide linker; (iii) a dual variable domain antibody (DVD-Ig TM ), in which each light and heavy chain contains two variable domains connected in series by a short peptide link; (iv) chemically linked bispecific (Fab')2 fragments; (v) 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) flexibodies, which are a combination of scFv and diabodies, resulting in multivalent molecules; (vii) so-called "dock and lock" molecules, based on the "dimerization and docking domain" in protein kinase A, which, when applied to Fab, can obtain a trivalent bispecific binding protein consisting of two identical Fab fragments linked to different Fab fragments; (viii) so-called scorpion molecules, which comprise, for example, two scFvs fused to the two ends of a human Fab arm; or (ix) diabodies.

[0044] 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., scFv, Fab, or 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., scFv, Fab, or scFab). In other embodiments, the first antigen-binding domain is an antigen-binding fragment (e.g., scFv, Fab, or scFab), and the second antigen-binding domain is a full-length antibody (e.g., an IgG antibody).

[0045] In certain embodiments, one of the first and second antigen-binding domains 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).

[0046] In certain embodiments, the antigen binding fragment (e.g., scFv, Fab, or scFab) is optionally connected to the N-terminus and / or C-terminus of the heavy chain and / or light chain of the full-length antibody (e.g., IgG antibody) via a peptide linker. In certain embodiments, the antigen binding fragment is connected to the C-terminus of the heavy chain and / or light chain. In certain embodiments, the antigen binding fragment is connected to the C-terminus of the heavy chain.

[0047] In certain embodiments, the peptide linker is a flexible peptide linker. In certain embodiments, the peptide linker is selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, the peptide linker is a flexible peptide comprising (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3 or 4. In certain embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO:23.

[0048] In certain embodiments, the multispecific antibodies described herein comprise an Fc domain comprising first and second Fc domain monomers.

[0049] In some embodiments, the first and second Fc domain monomers are native Fc region sequences.

[0050] In other embodiments, the first and second Fc domain monomers each independently comprise one or more amino acid modifications that alter effector function or promote heterodimerization of the first and second Fc domain monomers, such as knob-into-hole modifications.

[0051] In certain embodiments, one of the N-terminus and the C-terminus of the first Fc domain monomer is optionally connected to a first antigen-binding domain via a linker, and the other of the N-terminus and the C-terminus is optionally connected to a second antigen-binding domain via a linker.

[0052] In certain embodiments, one of the N-terminus and the C-terminus of the second monomer is optionally linked to a first antigen-binding domain via a linker, and the other of the N-terminus and the C-terminus is optionally linked to a second antigen-binding domain via a linker.

[0053] In some embodiments, the N-termini of the first monomer and the second monomer are respectively connected to one of the first antigen-binding domains; and the C-termini of the first monomer and the second monomer are respectively connected to one of the second antigen-binding domains.

[0054] In other embodiments, the N-termini of the first monomer and the second monomer are respectively connected to one of the second antigen-binding domains; and the C-termini of the first monomer and the second monomer are respectively connected to one of the first antigen-binding domains.

[0055] IgG-scFv

[0056] As an example, the multispecific antibody of the present invention may have an IgG-scFv structure.

[0057] 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:

[0058] (i) a first peptide chain comprising (e.g., from N-terminus to C-terminus) the VL and light chain constant region (CL) of the first antigen-binding domain;

[0059] (ii) a second peptide chain comprising (eg, from N-terminus to C-terminus) the VH, heavy chain CH1 region, Fc domain monomer of the first antigen-binding domain and the second antigen-binding domain.

[0060] 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:

[0061] (i) a first peptide chain comprising (eg, from N-terminus to C-terminus) the VL and light chain constant region (CL) of the second antigen-binding domain;

[0062] (ii) a second peptide chain comprising (eg, including from N-terminus to C-terminus) the VH, heavy chain CH1 region, Fc domain monomer of the second antigen-binding domain and the first antigen-binding domain.

[0063] 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.

[0064] In certain embodiments, the Fc domain monomer is an Fc domain monomer of IgG, such as an Fc domain monomer of IgG1 or IgG4. In certain embodiments, the Fc domain monomer is an Fc domain monomer of human IgG, such as an Fc domain monomer of human IgG1 or IgG4.

[0065] In certain embodiments, the Fc domain monomer comprises CH2 and CH3. In certain embodiments, the Fc domain monomer comprises a hinge region, CH2 and CH3.

[0066] In certain embodiments, the heavy chain constant region (CL) comprising the heavy chain CH1 region and Fc domain monomer comprises the sequence shown in SEQ ID NO: 24 or 25.

[0067] In certain embodiments, the scFv has the structure shown in VH-Linker-VL. In certain embodiments, the scFv has the structure shown in VL-Linker-VH. The linker is a peptide linker, preferably a flexible peptide. In certain embodiments, the linker is selected from a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, the peptide linker is a flexible peptide comprising (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3 or 4. In certain embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO:23.

[0068] In certain embodiments, the first antigen-binding domain or the second antigen-binding domain is connected 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 a peptide linker comprising one or more glycine (G) and / or serine (S). In certain embodiments, the peptide linker is a flexible peptide comprising (G4S)n, where n is an integer not less than 0, for example, 1, 2, 3 or 4. In certain embodiments, the peptide linker comprises the amino acid sequence shown in SEQ ID NO:23.

[0069] 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.

[0070] Antibody preparation

[0071] 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 technology. 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 resulting DNA molecules are inserted into expression vectors and then transfected into host cells; then, the transfected host cells are cultured under specific conditions and the antibodies of the present application are expressed. For example, multispecific antibodies can be produced by co-expressing multiple polynucleotides encoding the various polypeptide chains of the multispecific antibodies. The polypeptide chains produced by co-expression can be combined via, for example, disulfide bonds or other means to form functional multispecific antibodies. For example, the light chain portion of a Fab fragment can be encoded by a separate polynucleotide with a portion of the heavy chain portion of the Fab fragment in the multispecific antibody (the portion may further include an Fc domain monomer and optionally other antigen binding domains). When co-expressed, the polypeptide comprising the heavy chain portion of the Fab fragment is combined with the polypeptide comprising the light chain portion of the Fab fragment to form a Fab fragment. For another example, the portion comprising one of the two Fc domain monomers in the multispecific antibodies provided herein (which portion may further comprise an antigen-binding domain) and the portion comprising the other of the two Fc domain monomers (which portion may further comprise an antigen-binding domain) may be encoded by separate polynucleotides. When co-expressed, the two Fc domain monomers will associate to form an Fc domain.

[0072] 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 peptide chain thereof or (ii) a nucleotide sequence encoding a monoclonal antibody of the present invention or an antigen-binding fragment thereof or a heavy chain variable region and / or a light chain variable region thereof.

[0073] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of the present invention, and the nucleotide sequence encoding each peptide chain is present on the same or different isolated nucleic acid molecules.

[0074] In certain embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding the heavy chain (or heavy chain variable region) and the light chain (or light chain variable region) of the monoclonal antibody or antigen-binding fragment thereof of the present invention, 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 isolated nucleic acid molecules.

[0075] In another aspect, the present invention provides a vector (eg, an expression vector) comprising a nucleic acid molecule encoding the above-described isolation.

[0076] In certain embodiments, the vector comprises a nucleotide sequence encoding each peptide chain of the multispecific antibody of the present invention, and the nucleotide sequence encoding each peptide chain is present on the same or different vectors. For example, the vectors of the present invention comprise: a first vector comprising a nucleotide sequence encoding a first peptide chain and a second vector comprising a nucleotide sequence encoding a second peptide chain.

[0077] In certain embodiments, the vector comprises nucleotide sequences encoding the heavy chain (or heavy chain variable region) and light chain (or light chain variable region) of the monoclonal antibody or antigen-binding fragment thereof of the present invention, 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 vectors.

[0078] On the other hand, the invention provides host cells comprising nucleic acid molecules or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as Escherichia coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).

[0079] 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, comprising culturing the host cell as described above under conditions that allow protein expression, and recovering the multispecific antibody or the monoclonal antibody or an antigen-binding fragment thereof from the cultured host cell culture.

[0080] Pharmaceutical composition

[0081] 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.

[0082] In certain embodiments, the pharmaceutical composition comprises an effective amount of the multispecific antibody.

[0083] In certain embodiments, the pharmaceutical composition comprises an effective amount of the monoclonal antibody or antigen-binding fragment thereof.

[0084] 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 the following method: incorporating the necessary dose of the multispecific antibody described herein into an appropriate 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, the sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use.

[0085] Pharmaceutical composition of the present invention can be used by any suitable method known in the art.Preferred route of administration includes intravenous, intraperitoneal, intramuscular, subcutaneous, spinal column or other parenteral routes of administration. Parenteral administration refers to the administration mode 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, through trachea, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, spinal column, epidural and intrasternal injection and infusion.Or, can be used via non-parenteral route, such as local, epidermal or mucosal route of administration, for example intranasal, oral, vaginal, rectal, sublingual or local.

[0086] Therapeutic uses

[0087] In another aspect, the present invention provides a method for treating a tumor, comprising administering to a subject in need thereof a 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 in the preparation of a medicament for treating a tumor.

[0088] In certain embodiments, the tumor is TROP2 positive. TROP2 positivity can be measured at the nucleic acid level or the protein level. Methods for measuring expression at the nucleic acid level include, but are not limited to, RT-PCR or real-time RT-PCR. Methods for measuring expression at the protein level include, but are not limited to, immunological assays, such as enzyme immunoassay (EIA), chemiluminescent immunoassay (CLIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), Western blotting, immunoturbidimetry, surface plasmon resonance, and the like.

[0089] In certain embodiments, the tumor is a solid tumor.

[0090] 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.

[0091] The multispecific antibodies, monoclonal antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as 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, and the like. The preferred dosage form depends on the intended route of administration and therapeutic use. The multispecific antibodies, monoclonal antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such an injection can be a sterile injectable solution. For example, a sterile injectable solution can be prepared by incorporating the necessary dose of the active ingredient into an appropriate solvent, and optionally, other desired ingredients (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0092] The multispecific antibodies, monoclonal antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions 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, intracytoplasmic, inguinal, intravesical, topical (e.g., powders, ointments, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by those skilled in the art that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the multispecific antibodies, monoclonal antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions of the present invention are administered by intravenous or bolus injection.

[0093] The multispecific antibodies, monoclonal antibodies, or antigen-binding fragments thereof, or pharmaceutical compositions of the present invention can be formulated in dosage unit form for ease of administration. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0094] The multispecific antibodies, monoclonal antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present invention can be administered alone or in combination with another pharmaceutically active agent (e.g., an anti-tumor agent) or another therapy (e.g., an anti-tumor therapy). 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.

[0095] The subject described herein can be a mammal, such as a human. In certain embodiments, the subject has a tumor, such as a Trop2-positive tumor.

[0096] Definition of terms

[0097] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the virology, biochemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the respective fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0098] As used herein, the term "trophoblast cell surface antigen 2 (TROP2)" refers to a cell surface glycoprotein expressed by the TACSTD2 gene encoding, 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, but may 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, cattle, 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.

[0099] As used herein, the term "programmed death 1 (PD-1)," also known as CD279, is an immunosuppressive molecule in the CD28 family. PD-1 is preferably human, but may 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, cattle, 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.

[0100] As used herein, the term "antibody" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant and can include various antibody structures as long as they exhibit the desired antigen-binding activity. Typically, an antibody can be an immunoglobulin molecule consisting of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The constant domain is not directly involved in antibody-antigen binding but exhibits various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions, known as complementarity-determining regions (CDRs), interspersed with more conserved regions known as framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding site. The distribution of amino acids among regions or domains can follow the definitions 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.

[0101] As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity to at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises a plurality of antigen-binding domains that have binding specificity to different antigens (or epitopes), thereby being able to bind to at least two different binding sites and / or target molecules. Each antigen-binding domain comprised by a multispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or an antigen-binding fragment thereof (e.g., Fv, Fab, scFab, or scFv). In some cases, each antigen-binding domain is connected 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 to two different antigens (or epitopes).

[0102] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable region of each of the heavy and light chains contains three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, a person skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art (e.g., see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). In certain embodiments, the CDRs contained in the antibodies or antigen-binding fragments thereof of the present invention are preferably identified by the IMGT numbering system.

[0103] As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0104] The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0105] As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that, in the direction from N-terminus to C-terminus, 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 an IgE isotype, optionally further comprises 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 direction from N-terminus to C-terminus. 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 direction from N-terminus to C-terminus. The two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. A full-length antibody contains two antigen-binding sites formed by a VH and VL pair, respectively, and these two antigen-binding sites specifically recognize / bind to the same antigen.

[0106] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion thereof". Antigen-binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementary 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 polypeptides comprising at least a portion of an antibody sufficient to impart specific antigen-binding ability to a polypeptide.

[0107] As used herein, the term "Fab fragment" means an antibody fragment consisting of a light chain comprising VL and CL and a heavy chain fragment comprising VH and CH1, which is generally composed of one peptide chain comprising VL and CL and another peptide chain comprising VH and CH1. However, those skilled in the art understand that the Fab domains can be arranged according to the above-mentioned natural orientation, but can also contain domain substitutions or exchanges that promote correct VH and VL pairing (such as domain exchanges in the form of Crossmab); the term "scFab" refers to a single polypeptide chain comprising VL, VH, CL and CH1 domains, wherein adjacent domains are optionally connected by linkers. In the typical structure of scFab, the single polypeptide chain contained in scFab comprises, from N-terminus to C-terminus: VL, CL, VH and CH1, wherein CL and VH are generally connected by a peptide linker, or VH, CH1, VL and CL, wherein CH1 and VL are generally connected by a peptide linker.

[0108] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. In some cases, a disulfide bond may also exist between the VH and VL of the scFv.

[0109] As used herein, the terms "Fc domain" or "Fc region" or "Fc domain" have the meanings commonly understood by those skilled in the art and are used interchangeably, meaning a portion of the heavy chain constant region comprising CH2 and CH3. The Fc region of an antibody has a variety of different functions, but is not involved in antigen binding. The "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 (e.g., B cell receptors); and B cell activation, among others. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. When the Fc region comprises a hinge, the hinge regulates the dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype, such as IgG1, IgG2, IgG3, or IgG4.

[0110] The Fc domain may include both a native Fc region and a variant Fc region. A native Fc region comprises an amino acid sequence that is consistent with the amino acid sequence of an Fc region found in nature, for example, a native sequence human Fc region includes a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may have effector functions (e.g., Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function) that are altered compared to a native Fc region. In some embodiments, a variant Fc region may have modifications that promote 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 comprising a C-terminal constant region of an immunoglobulin heavy chain that is capable of stabilizing its own association.

[0111] As used herein, the term "identity" refers to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are 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%). Optimal alignment of sequences for comparison can be performed by computerized implementations of known algorithms or by visual inspection. Existing sequence alignment and multiple sequence alignment algorithms include BLAST, ClustalW / ClustalW2 / Clustal Omega, etc.

[0112] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as an antibody and its antigen. The strength or affinity of a specific binding interaction can be measured by the equilibrium dissociation constant (K) of the interaction. D ) indicates. 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 the antibody and the 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 properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. 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, the dissociation constant can be measured using surface plasmon resonance (SPR) in Biacore, bioluminescence interferometry or Kinexa.

[0113] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0114] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0115] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Furthermore, amino acid residues can be further divided into categories defined by alternative physical and functional properties. 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.

[0116] The twenty conventional amino acids referred to herein are designated according to conventional usage. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0117] 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 adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). 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 meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein), or degradation products thereof (such as lactalbumin hydrolysate), etc.

[0118] As used herein, the term "treatment" refers to a method implemented in order to obtain a beneficial or desired clinical outcome. For purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the development of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partially or completely), whether detectable or undetectable. In addition, "treatment" can also refer to, compared to the expected survival period (if not receiving treatment), extending the survival period. For "anti-tumor effect", including but not limited to, for example, tumor volume reduction, cancer cell number reduction, metastatic lesion number reduction, life expectancy increase, cancer cell proliferation reduction, cancer cell survival reduction, or improvement of the various physiological symptoms associated with cancer conditions.

[0119] As used herein, the term "effective amount" is at least the minimum concentration required to achieve a measurable improvement or prevention of a particular condition. The effective amount herein can vary with factors such as the patient's disease state, age, sex, and weight, and the ability of the antibody to elicit a desired response in an individual. An effective amount is also the amount at which the therapeutic benefit exceeds any toxic or adverse effect of the treatment. For preventive use, the beneficial or desired result includes the following results, such as eliminating or reducing risk, alleviating severity, or delaying the onset of the disease, including the biochemistry of the disease, histology and / or behavioral symptoms, the intermediate pathological phenotype presented during its complication and disease formation. For therapeutic use, the beneficial or desired result includes clinical results, such as reducing one or more symptoms from the disease, improving the quality of life of those subjects suffering from the disease, reducing the dosage of other drugs needed for treating the disease, enhancing the effect of another drug (such as via targeting), delaying the progression of the disease, and / or prolonging survival. In the case of cancer or tumors, an effective amount of the drug can have an effect in reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., slowing down or desirably stopping) cancer cell infiltration into peripheral organs; inhibiting (i.e., slowing down and desirably stopping) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating to some extent one or more symptoms associated with the condition. An effective amount can be administered in one or more administrations. For the purposes of the present invention, an effective amount of a multispecific antibody or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment.

[0120] As used herein, the terms "cancer" and "tumor" are used interchangeably to refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors, or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer includes both benign and malignant cancers, as well as dormant tumors or micrometastases. Cancer includes solid tumors, as well as hematologic malignancies such as lymphomas, leukemias, myelomas, or lymphoid malignancies, as well as spleen cancer and lymph node tumors.

[0121] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (eg, a human) has a tumor (eg, a Trop2-positive tumor).

[0122] Advantageous Effects of the Invention

[0123] The present invention provides multispecific antibodies targeting Trop2 and PD-1. These antibodies can specifically bind to both effector cells and tumor cells, exhibiting significant cell-bridging efficiency. This directs effector cells to tumor cells, selectively enhancing anti-tumor activity at the tumor site. These multispecific antibodies exhibit excellent cytological activity and in vivo pharmacodynamic activity, and possess significant clinical value.

[0124] The embodiments of the present invention will be described in detail below with reference to 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 rather than to limit the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] FIG1 shows the structure of an anti-PD-1 / anti-Trop2 bispecific antibody molecule.

[0126] FIG2 is a schematic diagram of the SDS-PAGE results of the bispecific antibody.

[0127] FIG3 is a schematic diagram of the HPLC results of the bispecific antibody.

[0128] Figure 4 is a schematic diagram of the DSC results of bispecific antibodies and corresponding monoclonal antibodies. In the figure: A is h2T81, B is 17D5, C is the bispecific antibody h2T81-G4-aPD1s, and D is a Tm value comparison table.

[0129] FIG5 is a schematic diagram of the ELISA results of the bispecific antibody and the corresponding monoclonal antibody.

[0130] FIG6 is a schematic diagram showing the affinity results of bispecific antibodies and corresponding monoclonal antibodies. 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 a comparison table of affinity KD values ​​for bispecific antibodies and corresponding monoclonal antibodies.

[0131] FIG7 is a graph showing the cell binding results of the bispecific antibody and the corresponding monoclonal antibody.

[0132] FIG8 is a graph showing the bridging flow cytometry results of the bispecific antibody and the corresponding monoclonal antibody.

[0133] Figure 9 shows the results of the bispecific antibody's 24-hour in vitro killing of different tumors.

[0134] FIG10 is an evaluation of the in vivo tumor targeting ability of bispecific antibodies and PD-1 monoclonal antibodies.

[0135] Figure 11 shows the tumor inhibition evaluation of bispecific antibodies and monoclonal antibodies. A shows the change in tumor volume, and B shows the change in mouse body weight.

[0136] Sequence information

[0137] A description of the sequences involved in this application is provided in the table below.

[0138] Table 1: Sequence information Example

[0139] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0140] Those skilled in the art will appreciate that the examples are provided to illustrate the present invention by way of example and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

[0141] Exemplary materials and reagents used in the following examples include:

[0142] Example 1: Preparation of bispecific antibodies

[0143] 1. Preparation of Trop2 monoclonal antibody

[0144] The antigen was recombinantly expressed by fusing the extracellular domain of human Trop2 to mouse IgG2a Fc. Female Balb / c mice approximately six weeks old were immunized with the antigen and injected subcutaneously at multiple sites with Freund's adjuvant. After four immunizations, candidate hybridoma cell lines were screened by fusion with Sp2 / 0 cells. Hybridoma cell lines were selected based on binding activity, affinity, immunofluorescence, flow cytometry, and internalization evaluation. Antibody variable region genes were then extracted from these candidate hybridoma cell lines to generate 2T81. CDR grafting and pairing were performed according to humanization principles to generate the optimally matched h2T81 antibody molecule. The heavy chain variable region (VH) and light chain variable region (VL) are SEQ ID NOs: 7 and 8, respectively. The IMGT numbering for the heavy chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 1, 2, and 3, respectively, and the light chain CDR1, CDR2, and CDR3 are SEQ ID NOs: 4, 5, and 6, respectively.

[0145] 2. Construction of bispecific antibody expression vector

[0146] 2-1. Bispecific Antibody Structure

[0147] The structure of the h2T81-G4-aPD1s bispecific antibody is shown in Figure 1 and Table 2, and comprises h2T81-IgG4 (derived from antibody h2T81) that binds to Trop2 and scFv (derived from antibody 17D5, which can be found in Chinese patent application CN201910654839.X) that binds to PD-1.

[0148] The PD-1-scFv is linked to the C-terminus of the h2T81-IgG4 heavy chain via L1 (SEQ ID NO: 23). The heavy and light chain variable regions (Trop2-VH and Trop2-VL) of h2T81-IgG4 are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. The PD-1-scFv has the structure shown in VL2-L2-VH2, with VH2 and VL2 shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively, and L2 shown in SEQ ID NO: 23.

[0149] Table 2: h2T81-G4-aPD1s structure

[0150] 2-2. Construction of expression vector

[0151] The light chain encoding nucleic acid sequence (i.e., h2T81-K, SEQ ID NO: 31) and the heavy chain encoding nucleic acid sequence (i.e., h2T81-G4-aPD1s-H, SEQ ID NO: 29) of the above-mentioned bispecific antibody were obtained and ligated into the PTT5 vector using PmeI / BamHI to obtain PTT5-h2T81-K and PTT5-h2T81-G4-aPD1s-H plasmids, respectively. Gene synthesis was completed by General Biotechnology (Anhui) Co., Ltd. In addition, based on the light chain encoding nucleic acid sequence (i.e., h2T81-K, SEQ ID NO: 31) and heavy chain encoding nucleic acid sequence (i.e., h2T81-H, SEQ ID NO: 28) of the Trop2 parental monoclonal antibody h2T81, PTT5-h2T81-K and PTT5-h2T81-H plasmids were obtained, respectively; based on the light chain encoding nucleic acid sequence (i.e., 17D5-K, SEQ ID NO: 30) and heavy chain encoding nucleic acid sequence (i.e., 17D5-H, SEQ ID NO: 27) of the PD-1 parental monoclonal antibody 17D5, PTT5-17D5-K and PTT5-17D5-H plasmids were obtained, respectively.

[0152] 3. Protein expression and purification:

[0153] Adjust the ExpiCHO cells to 5-6×10 6 / mL, the concentration of plasmid (i.e. antibody DNA) is 0.8μg / mL (the ratio of light chain to heavy chain is 1:1). Use pre-cooled OptiPROTM medium to dilute DNA and transient transfection reagent ExpiFectamine TM To the working concentration; dilute ExpiFectamine TM Add to the diluted DNA, gently invert and mix immediately, and let stand at room temperature for 15 minutes. TM Add the mixture to the cells and shake the flask gently during the addition. Incubate in a shaker at 37°C, 5% CO2, 110 rpm. After 18-22 hours, add ExpiCHO. TM Feed the cells and adjust the culture temperature to 32°C. After 10-12 days, the supernatant was harvested by centrifugation. The final antibody product, h2T81-G4-aPD1s, was purified using an AKTA pure Protein A column.

[0154] 4. SDS-PAGE and HPLC test results

[0155] 4-1. SDS-PAGE

[0156] (1) Take the purified protein sample and load it at a volume of 3 μg, so that the loading volume of each protein is 15 μL (if the volume is less than 15 μL, fill it up with PBS);

[0157] (2) Add 3 μL of reducing and non-reducing loading buffer to the protein sample (1 μL of loading buffer for every 5 μL of protein sample). The reduced sample needs to be heated in a metal bath at 100°C for 10 minutes.

[0158] (3) SDS-PAGE (ExpressPlus PAGE Gel, 10*8, 4-12%) was performed under reducing and non-reducing conditions. The electrophoresis conditions were: 80V, 90min. A rapid colorimeter was used for color development and photography was performed. The results are shown in FIG2 .

[0159] 4-2. SEC-HPLC

[0160] Antibody purity was determined using high-performance liquid chromatography. First, the tubing was inserted into PBS and ultrapure water, respectively. After equilibration for 100 minutes, the G3000 was connected. Sample injection (for protein samples with a concentration above 0.5 μg / mL, 150 μL of sample was added to each loading cup) was initiated at 30-minute intervals. After loading, the sample was equilibrated again and the loading column was removed. The HPLC peak profile was exported from the instrument, as shown in Figure 3.

[0161] 5.DSC detection

[0162] Antibody Tm values ​​were determined using a differential scanning calorimeter (VP-Capillary DSC). 300 μL of 1 μg / mL test sample was added to the sample well, and an equal volume of PBS was added to the control well. After removing air bubbles, the metal cap was tightened to a pressure of at least 40°C. The temperature was ramped from 10°C to 90°C. Tm values ​​for both the monoclonal antibody and bispecific antibody samples were calculated using PBS as a baseline, and a running curve was derived. As shown in Figure 4, the results demonstrate that the bispecific antibody h2T81-G4-aPD1s exhibits essentially the same thermal stability as its corresponding monoclonal antibodies h2T81 and 17D5, demonstrating stable physical properties.

[0163] Example 2: Determination of binding ability of bispecific antibodies

[0164] ELISA test

[0165] Evaluation of antibody-antigen binding activity using ELISA

[0166] (1) Dilute Trop2-his and PD1 proteins in PBS to a final concentration of 1 μg / mL, and incubate 100 μL / well in an ELISA plate at 37°C for 1 hour;

[0167] (2) Discard the liquid in the plate, add PBST solution to each well, wash once, and spin dry using a plate spinner;

[0168] (3) Add 200 μL / well of NRA blocking solution and incubate at 37°C for 2 hours;

[0169] (4) Discard the liquid in the plate and use a plate spinner to spin dry;

[0170] (5) Dilute the antibodies in PBS, with the highest concentration of the bispecific antibody at 10 μg / mL and the highest concentration of the parental monoclonal antibody at 7.5 μg / mL according to the molecular weight. Perform 2-fold dilutions on a vibrating plate machine, with a total of 12 concentration gradients. Add the diluted antibodies to the corresponding wells at 100 μL / well and incubate at 37°C for 1 hour.

[0171] (6) Discard the liquid in the plate, add PBST solution to each well, wash five times, and spin dry using a plate spinner;

[0172] (7) Add Goat Anti-Human IgG / HRP Ab (1:5000 diluted in NRAED enzyme solution), 100 μL / well, and incubate at 37°C for 30 minutes;

[0173] (8) Discard the liquid in the plate, add PBST solution to each well, wash five times, and spin dry using a plate spinner;

[0174] (9) Add AB mixed color development solution, 100 μL / well, and develop color at 37°C for 10 minutes;

[0175] (10) Add 50 μL / well of stop solution to stop color development and immediately read the OD450 using a microplate reader.

[0176] The results are shown in Figure 5 , and the in vitro binding activity of the bispecific antibody is basically consistent with that of the parent antibody.

[0177] Biacore assays

[0178] The affinity of the antibody for the antigen was determined using a BIAcore 8k instrument. Reagents used included the following: commercial PBS-P buffer; 0.5 mM NiSO4 (6.57125 mg of nickel sulfate hexahydrate added to 50 mL of ultrapure water); 350 mM EDTA (ethylenediaminetetraacetic acid); and 3 mM EDTA.

[0179] ① Trop2 or PD-1 protein is captured on the NTA sensor chip by Ni ions.

[0180] ② Condition the chip by adding 30 μL of 350 mM EDTA to the chip for 1 minute, and then activate the chip after washing with buffer.

[0181] ③ Regenerate the chip with 10 μL 0.5 mM NiSO4 and wash it for the first time with 3 mM EDTA.

[0182] ④ Add capture protein and pass it at a flow rate of 8 μL / min for 180 s, then pass the buffer for 120 s.

[0183] ⑤ 350mM EDTA was passed through the chip at a flow rate of 30μL / min for 60s to regenerate the chip, and the buffer was used for the second wash.

[0184] ⑥ Add the analyte to the experiment. After protein capture, the analyte binds for 120 seconds and dissociates for 1200 seconds at a flow rate of 25 μL / min.

[0185] ⑦ KD values ​​were obtained by instrument calculation, and image data processing and plotting were performed using Graphpad Prism 8.

[0186] As shown in Figure 6A to 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. These results demonstrate that h2T81-G4-aPD1 retains the binding activity of both parental mAbs.

[0187] Cell binding assay

[0188] Flow cytometry was used to evaluate the antibody-cell binding activity.

[0189] (1) Cell treatment: Digest CHO-Trop2 and CHO-PD1 cells and count them so that each sample contains 1×10 6 The cells were centrifuged at 1500 rpm for 5 minutes to remove the supernatant, and the cell pellet was resuspended in PBS and washed twice;

[0190] (2) Antibody dilution: dilute the antibody with 1640 medium containing 2% serum, with the first well concentration of the antibody being 10 μg / mL, and perform gradient dilution on the dilution plate; resuspend the cells in 100 μL of the diluted antibody 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, and resuspend the cells in 100 μL of the diluted fluorescent secondary antibody (Goat anti-Human IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488) and incubate at 37°C for 30 minutes in the dark;

[0191] (3) The stained cells were analyzed by flow cytometry. The experimental data were analyzed using FlowJo software to analyze the fluorescence intensity values ​​under different antibody concentrations. The binding of antibodies to cells was evaluated based on the fluorescence values ​​of the antibodies incubated at different concentrations.

[0192] The results are shown in FIG7 , and the binding activity of the bispecific antibody on cells is consistent with that of the parent antibody.

[0193] Example 3: Detection of cell bridging of bispecific antibodies

[0194] Cell bridging assays were performed by detecting antibody binding to CHO cell models engineered to express Trop2 or PD1. CHO-Trop2 and CHO-PD1 cells were infected with lentivirus to express Trop2 or PD1 and cultured in 10% FBS1640. Reagents used: CellTrace TM CFSE cell Proliferation Kit;CellTrace TM Far red cell Proliferation Kit.

[0195] ① According to the kit instructions, use CellTrace to stain CHO-PD1 cells with Far red and CHO-Trop2 cells with CFSE.

[0196] ② Add 200 μL of antibody (20 μg / mL) to 5×10 5 CHO-Trop2 was incubated for 1 h.

[0197] ③ Wash once with PBS and add 5×10 5 CHO-PD1 were co-incubated for 1 h.

[0198] ④Wash twice with PBS and resuspend in an appropriate volume of PBS.

[0199] ⑤ After screening, use LSRFortessaX-20 to analyze the FITC and APC channels and calculate the proportion of cells that are positive in both the FITC and APC channels.

[0200] As shown in Figure 8, 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 indicates that bispecific antibodies can effectively bridge effector cells and tumor cells, demonstrating superior cell bridging efficiency compared to monoclonal antibodies or monoclonal antibody combinations.

[0201] Example 4: In vitro killing results of bispecific antibodies

[0202] The cell killing assay measures the cytotoxicity of human PBMC or T cells against tumor cells that overexpress 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 Human T-Activator CD3 / CD28; CellTrace TM CFSE cell proliferation kit.

[0203] ① PBMC isolation: Using 10mL of whole blood as an example, first, evenly mix 10mL of fresh human whole blood with an equal volume of serum-free 1640 and slowly add the mixture to the upper layer of 14mL of Ficoll-Plaque premium 1.084. Centrifuge at 500g for 20min at speed 1. Carefully aspirate the PBMCs between the culture medium and density gradient separation medium into a new centrifuge tube using a pipette. Add serum-free 1640 to 45mL and centrifuge at 1500rpm for 5 minutes. Remove the supernatant and add 3-5mL of red blood cell lysis buffer (Solarbio). Wait 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.

[0204] ② According to the kit instructions, every 1×10 6 25 μL CD3 / CD28 Dynabeads were used to activate PD1 in vitro for about three days.

[0205] ③ According to the kit instructions, use CellTrace to stain PBMC cells with Far red and tumor cells with CFSE.

[0206] ④ Add 5000 tumor cells / well to a 96-well plate, wait for them to adhere to the wall, add 10 times the amount of PBMC and 50 μg / mL of antibody, incubate for 24 hours, and take pictures every 6 hours using a high-content cell screening imaging analysis system (Opera Phenix).

[0207] ⑤ The fluorescence value of each well was calculated using the Columbus website. The final cell lysis (%) of the whole well was calculated using the formula: (fluorescence value of the control group - fluorescence value of the experimental group) / fluorescence value of the control group × 100. Data processing and graphing were performed using Graphpad Prism 8.

[0208] As shown in Figure 9, the bispecific antibody h2T81-G4-aPD1s has a significant killing effect on various human tumor cell lines such as triple-negative breast cancer, colon cancer, and pancreatic cancer.

[0209] Example 5: In vivo tumor inhibition experiment of bispecific antibodies

[0210] In vivo targeted experiments

[0211] (1) Dye labeling: The molecular weights of the bispecific antibody and monoclonal antibody are 200 kDa and 150 kDa, respectively. The Cy5.5 labeling dye is 767.6 Da. The mass volume concentration of the antibody is adjusted to more than 2 mg. Then, the Cy5.5 dye is mixed with the antibody at a 10-fold amount of the substance and kept in the dark at 4 degrees. The mixture is mixed on a rotator for 12 hours. The liquid labeling solution is then transferred to a dialysis bag and dialyzed for 12 hours. The solution is changed every 4 hours. The labeling solution is collected and stored at 4 degrees in the dark for future use.

[0212] (2) In vivo imaging of small animals: In vivo imaging was performed using the Caliper IVIS Lumina II imaging system, with an excitation wavelength of 640 nm, an exposure time of 20 s, and fluorescence imaging mode. Mice were injected with a Cy5.5-labeled bispecific antibody (200 μg / injection / mouse) and a monoclonal antibody (150 μg / injection / mouse) via the tail vein, and in vivo imaging was performed 24 and 48 hours later.

[0213] The results are shown in Figure 10 , indicating that the bispecific antibody can efficiently target the tumor site.

[0214] Tumor inhibition experiment

[0215] In the tumor inhibition experiment, mice were inoculated with a corresponding amount of tumor cells. After the tumor grew to a certain size, the mice were treated with antibody drugs by intraperitoneal injection, and the changes in tumor volume growth after treatment were monitored.

[0216] (1) Tumor cell treatment: The tumor cells to be inoculated were digested with 0.25% trypsin, and the digested cell suspension was collected in a 50 mL / 15 mL centrifuge tube. The cells were counted using trypan blue solution, centrifuged at 1500 rpm for 5 minutes, and the cell supernatant was removed. The cell pellet was resuspended in serum-free cell culture medium, and centrifuged again at 1500 rpm for 5 minutes. The operation was repeated once. The inoculation amount of tumor cells per mouse was 5 × 10 6 cells / 100 μL, and resuspend the cells in the corresponding volume of serum-free cell culture medium according to the cell number.

[0217] (2) Tumor cell inoculation: Hair at the tumor site of hPD1-BALB / c mice was shaved; nude mice did not need to be shaved. Tumor cells were inoculated into the right hind leg of the mouse using a 1 mL syringe.

[0218] (3) Tumor formation observation: The tumor formation status of mice after inoculation of tumor cells was observed based on the tumor formation rate of the tumor cells. The tumor was measured using a vernier caliper and the weight of the mice was monitored using an electronic scale. The tumor volume of the mice was calculated according to the formula:

[0219] Tumor volume = (length × width × width) / 2 mm 3

[0220] Tumors of appropriate size were selected for group enrollment and drug administration (intraperitoneal injection, 200 μL / mouse). The tumor volume of mice was measured and tested every two days to evaluate the inhibitory effect of the drug on mouse tumor growth.

[0221] As shown in Figure 11, the bispecific antibody showed a stronger anti-tumor effect than PD-1 monoclonal antibody or Trop2 monoclonal antibody, and maintained a good tumor regulatory effect after stopping drug administration.

[0222] The foregoing is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art may make numerous improvements and modifications without departing from the principles of the present invention, and such improvements and modifications should be considered within the scope of protection of the present invention. The full scope of the present invention is defined 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 an 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 an IgG antibody), and the other is an 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 an IgG antibody), and the second antigen-binding domain is an 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: a heavy-chain CDR1, CDR2 and CDR3 comprising SEQ ID NOs:1, 2 and 3 respectively, and a light-chain CDR1, CDR2 and CDR3 comprising SEQ ID NOs:4, 5 and 6 respectively; 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, which comprises: (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, which comprises 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, which comprises 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, which comprises culturing the host cell of claim 13 under conditions allowing 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 tumor cell-targeting antibody, 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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