Antibody or antibody fragment binding to TROP2 and drug conjugate comprising same

Abstract

The invention relates to an antibody or an antibody fragment targeting TROP2, an antibody drug conjugate containing the antibody or the antibody fragment, and a preparation method and application of the antibody or the antibody fragment.

Description

Antibodies or antibody fragments binding to TROP2 and drug conjugates containing the same

[0001] This application requires the applicant to:

[0002] The present application claims priority from a prior application filed with the State Intellectual Property Office of China on January 15, 2024, with patent application number 202410055212.3, entitled “Antibodies or Antibody Fragments Binding to TROP2 and Drug Conjugates Containing Same”, the full text of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of biomedicine, and in particular to an antibody or antibody fragment targeting TROP2, a drug conjugate containing the same, a preparation method and uses thereof, and the like. Background Art

[0004] TROP2, short for Trophoblast cell surface antigen 2, belongs to the TACSTD family. It is a cell surface glycoprotein encoded by the TACSTD2 (Tumor Associated Calcium Signal Transducer 2) gene. It is also known as gastrointestinal tumor-associated antigen (GA733-1), membrane component chromosome 1 surface marker 1 (M1S1), or epithelial glycoprotein-1 (EGP-1). The TROP2 / TACSTD2 gene is 9072 base pairs long and has no introns. Its sole exon encodes a 323-amino acid glycoprotein composed of an extracellular domain, a single transmembrane region, and a short cytoplasmic tail.

[0005] TROP2 is associated with multiple cell signaling pathways, including intracellular calcium transduction, JAK / STAT, Bax&Bcl-2, MAPK / ERK, PI3K / Akt, Wnt / β-catenin, integrin and adhesion tight junction signals, which can promote tumor proliferation and metastasis.

[0006] Under physiological conditions, TROP2 is primarily expressed in epithelial cells and plays an important role in embryogenesis and development. TROP2 mRNA is detectable in many normal tissues, including the breast, cervix, gallbladder, kidney, lung, pancreas, placenta, prostate, salivary glands, skin, stomach, testis, thymus, trachea, and uterus. In particular, TROP2 is expressed at both transcript and protein levels in the skin, trachea, pancreas, kidney, cervix, and uterus. Immunohistochemical analysis of normal human tissues has shown that TROP2 is expressed across a wide range of tissues, including the skin, oral mucosa, and esophagus. It is also expressed in essential organs such as the pancreas, kidney, and liver. Furthermore, TROP2 expression in the cornea has a potential mechanism of action in maintaining vision. TROP2 expression in these normal tissues correlates with preclinical toxicity and clinical side effects of anti-TROP2 ADCs, significantly impacting safety.

[0007] Currently, several TROP2-targeting biopharmaceuticals are in clinical trials both domestically and internationally. The only approved TROP2-targeting drug is Trodelvy, an antibody-drug conjugate developed by Immunomedics. The most advanced clinical candidates are DS-1062a, developed by Daiichi Sankyo, and SKB264, developed by Kelun Biotech, both in Phase 3 clinical trials and in the first tier of pipeline candidates.

[0008] On April 8, 2021, the U.S. Food and Drug Administration (FDA) granted full approval to Trodelvy for the treatment of adult patients with unresectable, locally advanced or metastatic triple-negative breast cancer (TNBC) who have received at least two prior therapies, at least one of which was for metastatic disease. A key feature of Trodelvy is its unstable linker, which releases over 90% of the SN-38 within three days of administration. This design reduces on-target toxicity but potentially increases the risk of systemic toxicity. In clinical practice, significant off-target toxicity has been observed, significantly reducing the clinical benefit for patients. In studies IMMU-132-01, IMMU-132-05 (ASCENT), and IMMU-132-06, 795 patients received TRODELVY. The most common adverse reactions (≥25%) were neutropenia (61%), nausea (66%), diarrhea (65%), fatigue (62%), alopecia (45%), anemia (42%), vomiting (39%), constipation (37%), decreased appetite (34%), rash (32%), and abdominal pain (28%). In IMMU-132-05, 63% of patients discontinued TRODELVY due to adverse reactions. The most common adverse reactions (≥5%) leading to treatment discontinuation were neutropenia (47%), diarrhea (5%), respiratory tract infection (5%), and leukopenia (5%).

[0009] SKB264 and DS-1062a are potential best-in-class drugs for the same target, representing two designs of unstable linkers and stable linkers, respectively.

[0010] SKB264 is a TROP2-targeted ADC developed by Kelun Biotech. It uses a pH-sensitive toxin release mechanism and is coupled to a moderately active toxin (a novel topoisomerase I inhibitor). This drug has been shown to have severe off-target toxicity in clinical practice, with an incidence of grade 3 or higher adverse reactions as high as 67.4%, including anemia (30.2%), neutropenia (32.6%), stomatitis (9.3%), and rash (7.0%). The proportion of patients who had dose reductions due to adverse reactions was as high as 20.9%, seriously affecting the clinical benefits of patients.

[0011] DS-1062a is an ADC targeting a TROP2 antibody coupled to DXd via a GGFG tetrapeptide linker, with an average drug-antibody ratio (DAR) of 4. This drug has been associated with severe on-target toxicities in clinical trials, primarily stomatitis, interstitial pneumonia, and rash. The incidence of grade 3 or higher adverse reactions was 29%, including stomatitis (11%), anemia (4%), and ocular toxicity (2%). Dose-limiting toxicities (DLTs) included on-target toxicities such as mucositis, stomatitis, and systemic maculopapular rash. Several patients have experienced grade 5 interstitial pneumonia and died in the clinic.

[0012] In summary, there is an urgent need to develop new antibodies or antibody fragments that bind to TROP2 and select appropriate linkers and payloads to obtain ADCs that effectively target TROP2, in order to achieve a larger therapeutic window, safety, and efficacy. Summary of the Invention

[0013] In the first aspect, the present invention provides an antibody or antibody fragment that binds to TROP2, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise three CDRs, the heavy chain variable region comprises: CDR1-H, which has a sequence as shown in SEQ ID NO: 1 or a functional variant thereof; CDR2-H, which has a sequence as shown in SEQ ID NO: 2 or a functional variant thereof; and CDR3-H, which has a sequence as shown in SEQ ID NO: 3 or a functional variant thereof; the light chain variable region comprises: CDR1-L, which has a sequence as shown in SEQ ID NO: 4 or a functional variant thereof; CDR2-L, which has a sequence as shown in SEQ ID NO: 5 or a functional variant thereof; and CDR3-L, which has a sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7 or a functional variant thereof.

[0014] In some embodiments of the present invention, the antibody or antibody fragment comprises a heavy chain variable region that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:8 and a light chain variable region that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10.

[0015] In some embodiments of the present invention, the antibody or antibody fragment is selected from the group consisting of a murine antibody, a chimeric antibody, and a humanized antibody.

[0016] In some embodiments of the present invention, the antibody or antibody fragment comprises a heavy chain constant region that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 11 and a light chain constant region that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12.

[0017] In some embodiments of the invention, the antibody or antibody fragment comprises a heavy chain that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13 and a light chain that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0018] In some embodiments of the present invention, the antibody fragment is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a Fv fragment, a dsFv fragment, and a scFv fragment.

[0019] In some embodiments of the invention, the antibody or antibody fragment binds to TROP2 with higher affinity at a pH less than 7.0 compared to at a pH greater than 7.0.

[0020] In some embodiments of the invention, the antibody or antibody fragment binds TROP2 with higher affinity at a pH of about 6.0-6.5 compared to a pH of about 7.2-7.5.

[0021] In some embodiments of the present invention, the antibody or antibody fragment binds to TROP2 protein expressed on the cell surface.

[0022] In some embodiments of the invention, the cells include TROP2-positive cancer cells.

[0023] In some embodiments of the present invention, the cancer cells are selected from breast cancer cells and pancreatic cancer cells; preferably, the breast cancer cells are triple-negative breast cancer cells.

[0024] In some embodiments of the invention, the antibody or antibody fragment is further modified.

[0025] In some embodiments of the present invention, the modification includes N-terminal modification, C-terminal modification, side chain modification, amino acid modification, peptide backbone modification, and conjugation to other polypeptides or proteins.

[0026] In a second aspect, the present invention provides a nucleic acid comprising a nucleic acid sequence encoding the antibody or antibody fragment according to the first aspect of the present invention.

[0027] In a third aspect, the present invention provides a gene vector comprising the nucleic acid described in the second aspect of the present invention.

[0028] In a fourth aspect, the present invention provides a cell comprising the nucleic acid described in the second aspect of the present invention or the gene vector described in the third aspect of the present invention.

[0029] In a fifth aspect, the present invention provides an antibody or antibody fragment-drug conjugate, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled substance thereof, which comprises: (a) the antibody or antibody fragment described in the first aspect of the present invention; (b) a linker and (c) a biologically active molecule.

[0030] The present invention further provides Formula I: Ab-(LD) n ADC, wherein (a) Ab is the antibody or antibody fragment described in the first aspect of the present invention; (b) LD is a linker-bioactive molecule moiety, wherein -L- is a linker and D is a bioactive molecule; (c) n is the molar ratio of the bioactive molecule to the Ab (also known as DAR, i.e., drug-antibody conjugation ratio), selected from an integer from 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) or a decimal. When n is a decimal, it refers to the average number of linker-bioactive molecules (LD) conjugated to each Ab.

[0031] In some embodiments of the present invention, the -L- is selected from -L1-L2-L3-L4-L5- or -L1-L2-L4-L5-, wherein L1 is a covalent linking unit that is covalently linked to Ab, L2 is an extension unit, L3 is selected from amino acid residues optionally substituted with polar hydrophilic groups, L4 is selected from peptide residues consisting of 2-8 amino acids, and L5 is a bond or a self-cleavage fragment.

[0032] In some embodiments, L1 is selected from the following groups: (or its open-loop form ), Wherein * indicates connection with Ab.

[0033] In some embodiments, L1 is formed by reacting the L1' group in the linker with the antibody, for example, when L1 is selected from (where * indicates connection with Ab, and the other end is connected with L2), then correspondingly, L1' is selected from or (** indicates connection with L2);

[0034] In some embodiments of the present invention, L2 is selected from -L 2a -、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each independently selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C2-C6 alkynylene-C1-C6 alkylene-, 1-50 (preferably 9-30, more preferably 9-26, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50) atoms, each of the alkylene, cycloalkylene and heteroalkylene is optionally substituted by one or more substituents independently selected from C1-C6 alkyl, 2-6 atoms of heteroalkyl, hydroxy, amino, carboxyl or C3-C8 cycloalkyl, the heteroalkylene contains 1-12 (preferably 1-8, more preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene and heteroalkylene are selected from one or more of N, O or S (preferably O).

[0035] In some embodiments of the present invention, L3 is selected from amino acid residues optionally substituted with polar hydrophilic groups, wherein the polar hydrophilic groups include sugar residues and their derivatives or polyethylene glycol residues and their derivatives or polysarcosine residues and their derivatives or a combination thereof.

[0036] In some embodiments of the present invention, L4 is selected from a peptide residue consisting of 2-8 amino acids, wherein the amino acids are selected from phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid and glycine.

[0037] In some embodiments of the present invention, L5 is selected from a bond, Among them, * indicates connection with D.

[0038] In some embodiments of the present invention, L2 is selected from -L 2a -、-L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-、-L 2a -NH-C(O)-L 2b -C(O)-, where L 2a and L 2b Each is independently selected from methylene, ethylene, n-propylene, isopropylene, n-pentylene, methylenecyclohexylene, ethynylenemethylene, ethynyleneethylene, ethynylene-n-propylene, ethynylene-n-butylene, ethynylene-n-pentylene, ethynylene-n-hexylene, -(CH2CH2O) s CH2-, -(CH2CH2O) s CH2CH2-、-CH2(OCH2CH2) s -、-CH2CH2(OCH2CH2) s -、-CH2CH2(OCH2CH2) s CH2-, -CH2CH2(OCH2CH2) s CH2CH2-、-CH2(OCH2CH2) s CH2-, wherein s is selected from an integer of 1-12 (preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

[0039] In some embodiments of the present invention, L2 is selected from: -(CH2CH2O) s CH2C(O)-、-(CH2CH2O) s CH2CH2-, Where s is defined as above, and * indicates connection with L1.

[0040] In some embodiments of the present invention, L4 is selected from the group consisting of: Wherein * indicates that L4 is connected to L3 or L2, that is, when -L- is selected from -L1-L2-L3-L4-L5-, * indicates that L4 is connected to L3; when -L- is selected from -L1-L2-L4-L5-, * indicates that L4 is connected to L2.

[0041] In some embodiments of the present invention, -L1-L2- is selected from the group consisting of: Wherein, * indicates connection with Ab.

[0042] In some embodiments of the present invention, -L- is selected from:

[0043] In some embodiments of the present invention, D is a cytotoxic drug.

[0044] In some embodiments of the present invention, D is selected from the group consisting of tubulin inhibitors, DNA intercalators, DNA topoisomerase inhibitors, DNA synthesis inhibitors, RNA polymerase inhibitors, and spliceosome inhibitors.

[0045] In some embodiments of the present invention, the DNA topoisomerase inhibitor is selected from etoposide, including 9-aminocamptothecin, camptothecin, camptothecin derivatives, crinetol, doramycin, etoposide, etoposide phosphate, irinotecan, mitoxantrone, novolin, retinoic acid (retinol), teniposide, topotecan, 9-nitrocamptothecin or RFS2000, mitomycin and its analogues.

[0046] In some embodiments of the present invention, said D is selected from camptothecin derivatives.

[0047] In some embodiments of the present invention, said D is selected from Wherein R1 and R2 are independently selected from H, C 1-3 Alkyl (preferably methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), or R1 and R2 together with the carbon atom to which they are connected form a 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutane, cyclopentyl, cyclohexyl).

[0048] In some embodiments of the present invention, the Ab-(LD) n The structure of the compound is formula I:

[0049] Wherein, Ab is the antibody or antibody fragment described in the first aspect of the present invention, and R1, R2 and n are as described above.

[0050] In some embodiments of the present invention, the compound of Formula I is further Formula Ia or Formula Ib:

[0051] Wherein, in Formula Ia and Formula Ib, Ab and n are as defined in Formula I, and R is R1.

[0052] In some embodiments of the present invention, Formula I is further Formula I-1, Formula Ia-1, or Formula Ib-1:

[0053] Wherein, in Formula I-1, Formula Ia-1 and Formula Ib-1, Ab and n are defined as in Formula I.

[0054] In a sixth aspect, the present invention provides an antibody-drug conjugate according to the fifth aspect of the present invention, and a method for preparing a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled product thereof, the method comprising the following steps: S1: reduction of the antibody; S2: coupling the antibody to a drug linker; S3: purification of the antibody-drug conjugate.

[0055] In some embodiments of the present invention, the method comprises the steps of:

[0056] S1: Antibody reduction:

[0057] Prepare the antibody into an antibody solution (preferably, the antibody concentration is 10 mg / mL), add an appropriate amount (e.g., 10 mM) of TCEP aqueous solution according to the reducing amount, and incubate at 37°C to reduce the disulfide bonds in the antibody;

[0058] S2: Conjugation of Antibodies to Drug Linkers:

[0059] Add an appropriate amount of dimethyl sulfoxide (DMSO) solution dissolved linker-drug compound (preferably 10 mM) to the above solution, mix well, react for a period of time (preferably 0.5-4 hours) to connect the linker-drug compound to the antibody, add N-ethylmaleimide aqueous solution (preferably at a concentration of 100 mM), stir, and terminate the reaction of the linker-drug compound;

[0060] S3: Purification of Antibody-Drug Conjugates:

[0061] A pre-packed chromatography column (preferably 1 mL Mabselect SuRe) is equilibrated with a PBS / EDTA solution (preferably 10 mM PB, 137 mM NaCl, 5 mM EDTA, pH 7.0), and the antibody-drug conjugate is loaded. The column is then re-equilibrated with PBS / EDTA (preferably 10 to 20 mL), eluted with acetate buffer (preferably pH 3.5), and neutralized by adding 1 / 10 volume of sodium citrate neutralizing solution to obtain a purified antibody-drug conjugate.

[0062] In some embodiments of the present invention, the method comprises the steps of:

[0063] S1: Antibody reduction:

[0064] Replace the antibody medium with PBS7.0 / EDTA to prepare a 10 mg / mL antibody solution. Add 10 mM TCEP aqueous solution according to the reducing amount and incubate at 37°C for several hours to reduce the disulfide bonds in the antibody.

[0065] S2: Conjugation of Antibodies to Drug Linkers:

[0066] To the above solution, add 10 mM compound dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) solution at room temperature, mix well, and react at room temperature for 0.5-4 hours to connect the linker-drug compound to the antibody. Then, add 100 mM N-ethylmaleimide aqueous solution and stir at room temperature to terminate the linker-drug compound reaction.

[0067] S3: Purification of Antibody-Drug Conjugates:

[0068] On an AKTA system, a 1 mL Mabselect SuRe prepacked chromatography column was equilibrated with PBS7.0 / EDTA solution (10 mM PB, 137 mM NaCl, 5 mM EDTA, pH 7.0), and the antibody-drug conjugate was loaded. The column was then re-equilibrated with 10 to 20 mL of PBS7.0 / EDTA, eluted with acetate buffer at pH 3.5, and neutralized with 1 / 10 volume of sodium citrate neutralizing solution to obtain the purified antibody-drug conjugate.

[0069] In a seventh aspect, the present invention provides a pharmaceutical composition comprising the antibody-drug conjugate described in the fifth aspect of the present invention, and a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled substance thereof, and a pharmaceutically acceptable carrier or excipient.

[0070] In an eighth aspect, the present invention provides an antibody-drug conjugate according to the fifth aspect of the present invention, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels, or the pharmaceutical composition according to the seventh aspect of the present invention for the preparation of a medicament for treating and / or preventing TROP2-mediated diseases or conditions.

[0071] In some embodiments of the invention, the disease or disorder is cancer.

[0072] In some embodiments of the present invention, the cancer is selected from breast cancer and pancreatic cancer. In some embodiments of the present invention, preferably, the breast cancer is triple-negative breast cancer.

[0073] In a ninth aspect, the present invention provides an antibody-drug conjugate according to the fifth aspect of the present invention, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels, or the pharmaceutical composition according to the seventh aspect of the present invention for use in drugs for treating and / or preventing TROP2-mediated diseases or conditions.

[0074] In the tenth aspect, the present invention provides an antibody-drug conjugate as described in the fifth aspect for treating and / or preventing TROP2-mediated diseases or conditions, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels, or the pharmaceutical composition as described in the seventh aspect of the present invention.

[0075] In the eleventh aspect, the present invention provides a drug combination comprising the antibody-drug conjugate described in the fifth aspect of the present invention, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels, or the pharmaceutical composition described in the seventh aspect of the present invention, and one or more additional therapeutic agents.

[0076] In a twelfth aspect, the present invention provides a kit comprising the antibody-drug conjugate described in the fifth aspect of the present invention, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels, or the pharmaceutical composition described in the seventh aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1A is the binding curve of mut107 at pH 6.0;

[0078] Figure 1B shows the binding curve of mut107 at pH 7.4;

[0079] Figure 1C shows the binding curve of mut1071 at pH 6.0;

[0080] Figure 1D shows the binding curve of mut1071 at pH 7.4;

[0081] Figure 1E is the binding curve of TINA at pH 6.0;

[0082] Figure 1F is the binding curve of TINA at pH 7.4;

[0083] Figure 1G is the binding curve of hRS7 at pH 6.0;

[0084] Figure 1H is the binding curve of hRS7 at pH 7.4;

[0085] Figure 2 shows the binding affinities of mut107, mut1071, TINA, and hRS7 antibodies to TROP2-positive cells at pH 6.0 and pH 7.4;

[0086] FIG3 shows the efficacy of ADC-1, ADC-2, and ADC-3 on CFPAC-1 xenograft tumors in tumor-bearing mice;

[0087] FIG4 shows the efficacy of ADC-2, ADC-3, and ADC-5 on MDA-MB-468 xenografts in tumor-bearing mice;

[0088] FIG5 shows the efficacy of ADC-2, ADC-3, ADC-4, and double-dose ADC-4 on CFPAC-1 xenografts in tumor-bearing mice;

[0089] Figure 6 is a graph showing the body weight change trend of cynomolgus monkeys after intravenous injection of ADC-2. DETAILED DESCRIPTION

[0090] Before describing the disclosed embodiments in detail, it is to be understood that the present invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0091] definition

[0092] Unless otherwise indicated, as used herein, the singular forms "a", "an" and "the" include the plural forms as well. For example, the term "a cell" includes a plurality of cells and mixtures thereof.

[0093] As used herein, the term "about" refers to the usual error range of each value that is readily known to those skilled in the art. The use of "about" values ​​or parameters herein includes (and describes) embodiments involving the value or parameter itself.

[0094] As used herein, the terms "comprising" or "including" are intended to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In the present invention, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the stated elements, integers, or steps is also encompassed. For example, when referring to an antibody variable region "comprising" a specific sequence, it is also intended to encompass an antibody variable region consisting of the specific sequence.

[0095] As used herein, the term "and / or" describes a relationship between associated objects, indicating that three possible relationships exist. For example, "A and / or B" can represent: A alone, A and B together, or B alone. A and B can be singular or plural. The character " / " generally indicates an "or" relationship between the associated objects.

[0096] As used herein, the term "trophoblast cell surface antigen 2" or "TROP2," also known as tumor-associated calcium signal transducer 2 (TACSTD2), epidermal glycoprotein 1 (EGP-1, EGP1), gastrointestinal tumor-associated antigen (GA733-1, GA7331), surface marker 1 (M1S1). The term "TROP2" also refers to any post-translationally modified variants and conformational variants.

[0097] As used herein, the amino acid positions of all constant regions and domains of heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "using the Kabat numbering system."

[0098] As used herein, the term "antibody" is used in the broadest sense and includes various antibody structures, including but not limited to full-length antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody-antibody fragment-fusions, and compositions thereof. The term "antibody" also includes chimeric, humanized, and other types of engineered antibodies.

[0099] As used herein, the term "chimeric antibody" refers to an engineered antibody that, in the broadest sense, contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, a chimeric antibody comprises VH and VL of an antibody derived from a non-human animal combined with the CH and CL of another antibody (which in some embodiments is a human antibody). As a non-human animal, any animal can be used, such as a mouse, rat, hamster, rabbit, etc. Chimeric antibodies can also represent multispecific antibodies that are specific for at least two different antigens.

[0100] As used herein, the term "humanized antibody" refers to an antibody that is wholly or partially non-human in origin and has been modified to replace, for example, certain amino acids in the framework regions of VH and VL to avoid or minimize an immune response in the human body. The constant regions of a humanized antibody are typically human CH and CL regions.

[0101] As used herein, reference to a particular protein (e.g., an antibody) may include polypeptides having a native amino acid sequence, as well as variants and modified forms, regardless of their source or method of preparation. A protein having a native amino acid sequence is a protein having the same amino acid sequence as obtained from nature. Such native sequence proteins can be isolated from nature or can be prepared using standard recombinant and / or synthetic methods. Native sequence proteins specifically include naturally occurring truncated or soluble forms, naturally occurring variant forms (e.g., alternative splicing forms), naturally occurring allelic variants, and forms including post-translational modifications. Native sequence proteins include proteins that carry post-translational modifications, such as glycosylation or phosphorylation, or other modifications of certain amino acid residues.

[0102] As used herein, "purified" or "isolated" when referring to a polypeptide (e.g., an antibody) or nucleic acid sequence means that the molecule is present in the substantial absence of other biological macromolecules of the same type. As used herein, the term "purified" refers to the presence of at least 75%, 85%, 95%, 96%, 97%, or 98% by weight of a biological macromolecule of the same type. An "isolated" nucleic acid molecule encoding a particular polypeptide refers to a nucleic acid molecule that is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include certain additional bases or moieties that do not adversely affect the essential properties of the composition.

[0103] As used herein, the term "full-length antibody" refers to an immunoglobulin molecule that, when naturally present, comprises four peptide chains: two heavy chains (approximately 50-70 kDa in total length) and two light chains (approximately 25 kDa in total length) interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and highly conserved framework regions (FRs) spaced apart from the complementarity determining regions. The domain arrangement of each VH or VL, from amino terminus to carboxyl terminus, is FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains each contain a binding domain that interacts with an antigen. The constant region of an antibody mediates its binding to host tissues or various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0104] As used herein, the term "heavy chain constant region" or "CH" is used interchangeably in the present invention and comprises at least three heavy chain constant domains (CH1, CH2, and CH3). Exemplary, human heavy chain constant regions include γ, δ, α, ε, and μ, each heavy chain constant region corresponding to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, an antibody comprising an α constant region is an IgA antibody, an antibody comprising a μ constant region is an IgM antibody, and an antibody comprising an ε constant region is an IgE antibody. Certain isotypes can be further divided into subclasses, for example, IgG antibodies include but are not limited to IgG1 (comprising a γ1 constant region), IgG2 (comprising a γ2 constant region), IgG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 constant region); IgA antibodies include but are not limited to IgA1 (comprising an α1 constant region) and IgA2 (comprising an α2 constant region); IgM antibodies include but are not limited to IgM1 and IgM2. Isotypes may also include modifications that alter Fc function, such as enhancing or diminishing effector function or enhancing or diminishing binding to Fc receptors. As described above, one of ordinary skill in the art will appreciate that the heavy chain constant regions may be modified such that they differ in amino acid sequence from naturally occurring immunoglobulin molecules.

[0105] As used herein, the term "light chain constant region" or "CL" is used interchangeably in the present invention and comprises one light chain constant domain CL. Exemplarily, light chains can be divided into two categories, λ and κ, based on the difference in the light chain constant region.

[0106] As used herein, the term "heavy chain variable region (VH)" refers to the amino-terminal variable region domain of an immunoglobulin heavy chain.

[0107] As used herein, the term "light chain variable region (VL)" refers to the amino-terminal variable region domain of an immunoglobulin light chain.

[0108] As used herein, the term "CDR (complementarity determining region)" refers to an amino acid sequence that together defines the binding affinity and specificity of the Fv region of an antibody. Because the specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed mainly of residues from the so-called hypervariable region or complementary determining region (CDR). The light chain (L) and heavy chain (H) of an antibody each have three CDRs, namely CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H. Therefore, the antigen binding site of a conventional antibody includes six CDRs, which include a CDR group from each of the heavy chain variable region and the light chain variable region.

[0109] As used herein, the term "hinge region" includes that portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be divided into three distinct domains: the upper, middle, and lower hinge domains.

[0110] As used herein, the term "Fc region" or "Fc fragment" refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the hinge region, the CH2 domain, and the CH3 domain, and mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or binding to the first component (C1q) of the classical complement system. Fc regions include native sequence Fc regions and variant Fc regions.

[0111] As used herein, the term "antibody fragment" refers to antigen-binding fragments and antibody analogs of antibodies that retain the ability to specifically bind to an antigen, which generally include at least a portion of the antigen-binding region or variable region of the parent antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Typically, when activity is expressed in molar units (KD), the antibody fragment retains at least 10% of the parent binding activity; preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% of the binding affinity of the parent antibody to the target. Antibody fragments include, but are not limited to, Fab fragments (antigen-binding fragments), Fab' fragments, F(ab')2 fragments, Fab'-SH fragments, Fv fragments, Fd fragments, dAb fragments, disulfide-stabilized proteins (dsFv), complementarity-determining region (CDR) fragments, etc.; linear antibodies (Linear Antibody), single-chain antibodies (e.g., scFv single antibodies), scFv-Fc, single antibodies (Unibody, technology from Genmab), bivalent single-chain antibodies, single-chain phage antibodies, single-domain antibodies (Single Domain Antibody) (e.g., VH domain antibodies), single heavy chain antibodies, single light chain antibodies, domain antibodies (Domantis, technology from Domantis), nanobodies (nanobodies, technology from Ablynx); multispecific antibodies formed by antibody fragments (e.g., three-chain antibodies, four-chain antibodies, etc.); and engineered antibodies such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies, etc. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0112] As used herein, the term "Fab fragment," also known as an antigen-binding fragment, refers to the region of an antibody structure that can bind to an antigen. A Fab fragment consists of a complete light chain (VL and CL) and a partial heavy chain (VH and CH1), linked by a disulfide bond. It is relatively small, with a molecular weight of 47-48 kDa. The term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by a disulfide bond in the hinge region. The term "Fab' fragment" refers to the fragment obtained by reducing the disulfide bonds linking the two heavy chain fragments in the (Fab')2 fragment. It consists of an intact light chain and the Fd fragment of the heavy chain (composed of the VH and CH1 domains). Fab fragments can be obtained by protease cleavage of full-length antibodies. For example, human immunoglobulin G (IgG) can be degraded into two Fab fragments and an Fc fragment by papain; IgG can be degraded into a F(ab')2 fragment and an Fc' fragment by pepsin. The F(ab')2 fragment can be further reduced to form two Fab' fragments. Fab fragments can also be produced through expression in prokaryotic systems (such as E. coli) and mammalian cell systems. E. coli expression systems offer low production costs and rapid production, but they are prone to inclusion body formation, making subsequent purification and renaturation more complex. Furthermore, the resulting renatured protein has low or even no activity. Expression of Fab fragments in mammalian cells allows for smooth disulfide bond formation, more closely resembling the natural Fab fragment structure and resulting in higher activity.

[0113] As used herein, the term "Fab'-SH" is the designation herein for Fab' in which one or more cysteine ​​residues of the constant domains bear a free thiol group

[0114] As used herein, the term "Fd" means an antibody fragment consisting of the VH and CH1 domains.

[0115] As used herein, the term "dAb fragment" means an antibody fragment consisting of the VH domain.

[0116] As used herein, the term "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions, and is the minimum fragment that contains a complete antigen recognition and binding site.

[0117] As used herein, the term "dsFv" generally refers to a disulfide-stabilized Fv fragment in which the bond between a single light chain variable domain and a single heavy chain variable domain is a disulfide bond.

[0118] As used herein, the term "single-chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody. It is a recombinant protein of VH and VL connected by a linker. The linker cross-links these two domains to form an antigen-binding site. The linker sequence is generally composed of a flexible peptide, such as, but not limited to, G2(GGGGS)3. The size of an scFv is generally 1 / 6 that of a complete antibody. A single-chain antibody is preferably a single amino acid chain sequence encoded by a single nucleic acid chain.

[0119] As used herein, the term "murine antibody" or "mouse antibody" refers to an antibody in which the framework region and CDR region in the variable region are both derived from mouse germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from mouse germline immunoglobulin sequences. The murine antibodies of the present invention may include amino acid residues that are not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random mutations or point mutations in vitro or by somatic mutations in vivo), but "mouse antibody" or "murine antibody" does not include antibodies in which CDR sequences derived from other mammalian germlines are inserted into mouse framework sequences.

[0120] As used herein, the term "chimeric antibody" refers to an antibody created by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can mitigate the immune response induced by the mouse antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. The variable region genes are then cloned from the hybridoma cells. Furthermore, the human constant region genes are cloned as needed. The mouse variable region genes and human constant region genes are then linked to form a chimeric gene, which is then inserted into a gene vector. Finally, the chimeric antibody is expressed in a eukaryotic or prokaryotic system.

[0121] As used herein, the term "humanized antibody" is an antibody that contains complementarity determining regions (CDRs) derived from non-human antibodies and framework and constant regions derived from human antibodies.

[0122] As used herein, the term "conservative amino acid substitution" is an amino acid substitution in which one amino acid residue is substituted with another amino acid residue having a side chain with similar chemical properties (e.g., charge, size, or hydrophobicity). Typically, a conservative amino acid substitution will not substantially alter the functional properties of the protein. Examples of groups of amino acids having chemically similar side chains include (1) aliphatic side chains: glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), and isoleucine (Ile); (2) aliphatic hydroxyl side chains: serine (Ser) and threonine (Thr); (3) amide-containing side chains: asparagine (Asn) and glutamine (Gln); (4) aromatic side chains: phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp); (5) basic side chains: lysine (Lys), arginine (Arg), and histidine (His); (6) acidic side chains: aspartic acid (Asp) and glutamic acid (Glu); and (7) sulfur-containing side chains: cysteine ​​(Cys) and methionine (Met). Conservative amino acid substitution groups can also be defined based on amino acid size.

[0123] As used herein, the term "functional variant" refers to a protein that has one or more amino acid substitutions, insertions, or deletions compared to a parent protein, polypeptide, or CDR and retains one or more desired activities of the parent protein. A functional variant can be a protein fragment (i.e., a variant with an N- and / or C-terminal deletion) that retains one or more desired activities of the parent protein.

[0124] As used herein, a sequence that is "at least 85% identical" to a reference sequence is a sequence that has 85% or greater (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, etc.) sequence identity over its entire length to the reference sequence.

[0125] As used herein, the term "cancer cell" includes not only primary cancer cells, but also any cell derived from a cancer cell ancestor. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells.

[0126] As used herein, the term "cancer" or "cancer" is the name for a disease in which the body's cells become abnormal and divide uncontrollably. In some embodiments, the cancer is recurrent, refractory, metastatic, and / or advanced. In some embodiments, the cancer is a solid tumor or a non-solid tumor.

[0127] As used herein, the term "nucleic acid" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term "nucleic acid" also includes nucleic acids containing analogs of known natural nucleotides, which have a binding property similar to that of reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes variants (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences thereof, as well as sequences explicitly indicated.

[0128] As used herein, the term "gene vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, used to transfer an inserted nucleic acid molecule into and / or between host cells. Such gene vectors may include vectors primarily used to insert DNA or RNA into cells, vectors primarily used to replicate DNA or RNA, and vectors primarily used for expression through transcription and / or translation of DNA or RNA. Such gene vectors also include vectors that have multiple of these functions. Such gene vectors may be polynucleotides that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. Generally, by culturing a suitable host cell containing the gene vector, the gene vector can produce the desired expression product. In the present invention, the gene vector may contain one or more of the aforementioned nucleic acid molecules. Furthermore, the gene vector may also contain other genes, such as marker genes that allow selection of the vector in a suitable host cell and under appropriate conditions. Furthermore, the gene vector may also contain expression control elements that allow for proper expression of the coding region in a suitable host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes a 5' non-transcribed sequence and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, a capping sequence, a CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence functionally linked to a nucleic acid for transcriptional control.

[0129] As used herein, "operably linked" means incorporated into a genetic construct so that an expression control sequence effectively controls the expression of the target coding sequence. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence composed of a region of a DNA molecule generally within 100 nucleotides upstream of the transcription start point (generally near the start site of RNA polymerase II). In order for a coding sequence to be under the control of a promoter, the translation start site of the polypeptide translation reading frame must be positioned between 1 and about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, position, and level. Unlike promoters, enhancers can work when located at different distances from the transcription site. Enhancers can also be located downstream of the transcription start site. When RNA polymerase is able to transcribe the coding sequence into mRNA, which can then be translated into a protein encoded by the coding sequence, the coding sequence is "operably linked" to the expression control sequence in the cell and is "under the control" of the expression control sequence.

[0130] As used herein, the term "cell" or "host cell" refers to a cell into which exogenous nucleic acid is introduced, including the offspring of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and offspring derived therefrom, without considering the number of passages. Offspring may not be completely identical to the parent cell in nucleic acid content, but may comprise mutations. Included herein are mutant offspring with the same function or biological activity as screened or selected in the initially transformed cell. The construction of the cell can be by methods known in the art, such as electroporation, chemical transfection (such as DEAE-dextran), conversion, transfection, and infection and / or transduction (such as with a recombinant virus) by which the vector is introduced into the host cell.

[0131] As used herein, the term "drug conjugate" generally refers to a class of aggregates of atoms and groups with a specific structure that can be covalently bound to other molecules through specific functional groups; it contains at least one payload drug and optionally a linker (or "linker"). A variety of payload drugs, linkers, or linker components are known in the art.

[0132] As used herein, the term "drug-antibody coupling ratio," "drug-antibody coupling ratio," "drug-antibody ratio," or "DAR" refers to the number of drugs attached to an antibody of an ADC. The DAR of an ADC can range from 1 to 10, but higher loadings (e.g., 20) are possible depending on the number of attachment sites on the antibody. The term DAR can be used when referring to the number of drugs loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a group of ADCs. When considering the average number of small molecule drugs bound, the average number of drugs bound to the antibody is referred to as the average drug-antibody coupling ratio. The DAR value of an ADC can be determined using ultraviolet-visible absorption spectroscopy (UV-Vis), high performance liquid chromatography-hydrophobic chromatography (HPLC-HIC), high performance liquid chromatography-reverse phase chromatography (RP-HPLC), liquid chromatography-mass spectrometry (LC-MS), and the like. These techniques are described in Ouyang, J. Methods Mol Biol, 2013, 1045: p. 275-83.

[0133] As used herein, the term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with mammalian tissues, particularly human tissues, without excessive toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. For example, pharmaceutically acceptable salts of amines, carboxylic acids, and other types of compounds are well known in the art. The salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or separately by reacting the free base or free acid with a suitable reagent.

[0134] As used herein, the term "isotopically labeled" means that the compounds of the present invention may exist in an isotopically traced or enriched form, containing one or more atoms whose atomic mass or mass number differs from the atomic mass or mass number of the most abundant atom found in nature. Isotopes can be radioactive or non-radioactive. Commonly used isotopic labels include: hydrogen isotopes, 2H and 3H; carbon isotopes, 13C and 14C; chlorine isotopes, 35Cl and 37Cl; fluorine isotope, 18F; iodine isotopes, 123I and 125I; nitrogen isotopes, 13N and 15N; oxygen isotopes, 15O, 17O, and 18O; and sulfur isotope, 35S. These isotopically labeled compounds can be used to study the tissue distribution of pharmaceutical molecules. 2H and 13C are particularly widely used due to their ease of labeling and convenient detection. Substitution with certain heavy isotopes, such as deuterium (2H), can enhance metabolic stability and prolong half-life, thereby reducing dosage and providing therapeutic advantages. Isotope-labeled compounds are generally synthesized from labeled starting materials using known synthetic techniques similar to those used for non-isotope-labeled compounds.

[0135] As used herein, the terms "solvate" and "solvate" refer to the physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain instances, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be capable of isolation. The solvent molecules in the solvate may exist in a regular and / or disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses solution-phase and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0136] As used herein, the term "stereoisomer" refers to compounds having the same chemical constitution but differing in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis / trans) isomers, atropisomers, and the like. Any resulting mixture of stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, and diastereomers based on the differences in the physicochemical properties of the components, for example, by chromatography and / or fractional crystallization.

[0137] As used herein, the term "heteroatom" refers to nitrogen, oxygen, sulfur, or halogen atoms.

[0138] As used herein, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 10 carbon atoms (i.e., C1-10 alkyl), more preferably containing 1 to 8 carbon atoms (C1-8 alkyl), and more preferably containing 1 to 6 carbon atoms (i.e., C1-6 alkyl). For example, "C1-6 alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically, 1, 2, 3, 4, 5, or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, and the like.

[0139] As used herein, the term "-alkyl-" or "alkylene" refers to a saturated straight or branched divalent hydrocarbon group. For example, C1-C8 alkylene refers to a straight or branched alkylene group having 1 to 8 carbon atoms.

[0140] As used herein, the term "heteroalkyl" refers to an alkyl group in which one or more carbon atoms are replaced by a heteroatom, for example, ethoxymethyl, methoxy, etc.

[0141] As used herein, the term "heteroalkylene" refers to a divalent "heteroalkyl" group connected to other groups at both ends.

[0142] As used herein, the term "cycloalkyl" refers to a fully saturated carbocyclic ring that can exist as a monocycle, a bridged ring, or a spirocycle. Preferably, it contains 3-12 carbon atoms (i.e., C3-12 cycloalkyl), more preferably 3-10 carbon atoms (C3-10 cycloalkyl), further preferably 3-7 carbon atoms (C3-7 cycloalkyl), 4-6 carbon atoms (C4-6 cycloalkyl), 5-6 carbon atoms (C5-6 cycloalkyl). Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, norbornyl (bicyclo [2.2.1] heptyl), bicyclo [2.2.2] octyl, adamantyl, etc.

[0143] As used herein, the term "cycloalkylene" refers to a divalent "cycloalkyl" group having other groups attached to both ends.

[0144] As used herein, the term "amino acid residue" refers to the corresponding residue when one hydrogen atom is removed from the amine and / or one hydroxyl group is removed from the carboxyl terminus of an amino acid.

[0145] Part I: Antibodies or Antibody Fragments Binding to TROP2

[0146] This invention utilizes phage display to screen for pH-sensitive antibodies based on the differences in pH between the tumor microenvironment and normal tissue. These antibodies bind to TROP2 with high affinity at the acidic pH of the tumor microenvironment (<6.5), but not at physiological pH (~7.4). This characteristic of the antibodies reduces the distribution and binding of the ADC to normal tissue, thereby reducing on-target toxicity.

[0147] The present invention provides an antibody or antibody fragment that binds to TROP2. The antibody or antibody fragment that binds to TROP2 is an antibody that has sufficient affinity and specificity to bind to TROP2. For example, the antibody or antibody fragment that binds to TROP2 provided by the present invention can be used as a therapeutic agent to target and interfere with diseases or conditions associated with unconventional or abnormal expression and / or activity of TROP2.

[0148] The present invention provides an antibody or antibody fragment that binds to TROP2, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region respectively comprise three CDRs, the heavy chain variable region comprises: CDR1-H, which has a sequence as shown in SEQ ID NO: 1 or a functional variant thereof; CDR2-H, which has a sequence as shown in SEQ ID NO: 2 or a functional variant thereof; and CDR3-H, which has a sequence as shown in SEQ ID NO: 3 or a functional variant thereof; the light chain variable region comprises: CDR1-L, which has a sequence as shown in SEQ ID NO: 4 or a functional variant thereof; CDR2-L, which has a sequence as shown in SEQ ID NO: 5 or a functional variant thereof; and CDR3-L, which has a sequence as shown in SEQ ID NO: 6 or SEQ ID NO: 7 or a functional variant thereof.

[0149] In some embodiments of the present invention, the functional variant comprises one or more amino acid substitutions. In other embodiments of the present invention, the amino acid substitution is preferably a conservative amino acid substitution. The conservative amino acid substitution can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and the substitution has little or substantially no effect on the function, activity or other biological properties of the TROP2 antibody or antibody fragment. In some embodiments, the conservative amino acid substitution is common in the art, for example, a conservative amino acid substitution is a substitution of one or a few amino acids in the following (1)-(7) groups by another or a few amino acids in the same group: (1) aliphatic side chains: Gly, Ala, Val, Leu and Ile; (2) aliphatic hydroxyl side chains: Ser and Thr; (3) amide-containing side chains: Asn and Gln; (4) aromatic side chains: Phe, Tyr and Trp; (5) basic side chains: Lys, Arg and His; (6) acidic side chains: Asp and Glu; and (7) sulfur-containing side chains: Cys and Met. In other embodiments, particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Arg or Lys; Arg is replaced by Lys; Lys is replaced by Arg, His; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser.

[0150] In some embodiments of the invention, the antibody or antibody fragment comprises a heavy chain variable region that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 100%, or a range of values ​​between any two points to the amino acid sequence of SEQ ID NO:8.

[0151] In some embodiments of the invention, the antibody or antibody fragment comprises a light chain variable region that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 100%, or a range of values ​​between any two points, to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0152] In some embodiments of the present invention, the antibody or antibody fragment comprises a heavy chain variable region that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100%, or a range of values ​​between any two points to the amino acid sequence of SEQ ID NO: 8, and a light chain variable region that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100%, or a range of values ​​between any two points to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

[0153] In some embodiments of the present invention, the antibody or antibody fragment is selected from a murine antibody, a chimeric antibody, and a humanized antibody. As is well known in the art, chimeric antibodies are prepared by exchanging non-human constant regions (heavy chain, light chain, or both) with human constant region antibodies, for example, referring to U.S. Patent No. 4,816,567 to Cabilly et al., which is incorporated herein by reference in its entirety. Methods for preparing humanized antibodies from non-human antibodies, for example, from murine antibodies, are also known, for example, referring to U.S. Patent No. 5,565,332 to Winter, which is incorporated herein by reference in its entirety.

[0154] In some embodiments of the present invention, the antibody or antibody fragment comprises a heavy chain constant region that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 100%, or a range of values ​​between any two points to the amino acid sequence of SEQ ID NO: 11.

[0155] In some embodiments of the invention, the antibody or antibody fragment comprises a light chain constant region that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 100%, or a range of values ​​between any two values, to the amino acid sequence of SEQ ID NO: 12.

[0156] In some embodiments of the present invention, the antibody or antibody fragment comprises a heavy chain constant region that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100%, or a range of values ​​between any two points to the amino acid sequence of SEQ ID NO: 11 and a light chain constant region that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100%, or a range of values ​​between any two points to the amino acid sequence of SEQ ID NO: 12.

[0157] In some embodiments of the invention, the antibody or antibody fragment comprises a heavy chain that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 100%, or a range of values ​​between any two points, to the amino acid sequence of SEQ ID NO: 13.

[0158] In some embodiments of the invention, the antibody or antibody fragment comprises a light chain that is at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, 100%, or a range of values ​​between any two values, to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0159] In some embodiments of the present invention, the antibody or antibody fragment comprises a heavy chain that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100%, or a range of values ​​between any two points to the amino acid sequence of SEQ ID NO: 13 and a light chain that is at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100%, or a range of values ​​between any two points to the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15.

[0160] In some embodiments of the present invention, the antibody fragment is selected from the group consisting of a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a Fv fragment, a dsFv fragment, and a scFv.

[0161] There are many methods for producing antibody fragments. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies. Illustrative methods for producing antibody fragments are described, for example, in Hudson et al., Nat. Med., 2003, 9:129-134, which is incorporated herein by reference in its entirety. However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries, as described, for example, in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), respectively, describe the isolation of murine and human antibodies using phage libraries. F(ab')2 fragments can also be isolated directly from recombinant host cell cultures. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0162] In some embodiments of the present invention, the antibody or antibody fragment binds to TROP2 with higher affinity at a pH less than 7.0 (e.g., a pH less than 7.0, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, etc., or a range between any two values) as compared to a pH greater than 7.0 (e.g., a pH greater than 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, etc., or a range between any two values).

[0163] In some embodiments of the invention, the antibody or antibody fragment binds to TROP2 with a higher affinity at a pH of about 6.0-6.5 compared to a pH of about 7.2-7.5. In some preferred embodiments of the invention, the antibody or antibody fragment binds to TROP2 with a higher affinity at a pH of about 6.0 compared to a pH of about 7.4.

[0164] In some embodiments of the present invention, the antibody or antibody fragment is further modified. According to the embodiments of the present invention, the antibody modification method can adopt various antibody modification methods known in the art, such as electrostatic adsorption method and chemical coordination method.

[0165] In some embodiments of the present invention, the modifications include N-terminal modification, C-terminal modification, side chain modification, amino acid modification, peptide backbone modification, and conjugation to other polypeptides or proteins. According to embodiments of the present invention, the aforementioned modification methods can employ various antibody modification methods known in the art. For example, they include, but are not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. Compounds produced by modifying the N- and / or C-termini of amino acids. Non-limiting examples are the conversion of carboxyl groups to salts, esters, acylhydrazides, hydroxamic acids, or amides, and the conversion of amino groups to amides, ureas, thioureas, thioamides, sulfonamides, phosphoramides, boric acid amides, or alkylamines. Parts or moieties of a compound produced by modification of an amino acid at the C- and / or N-terminus may also be referred to as amino acid units. Furthermore, the amino acids may also be derivatized at their side chains. If the derivatized amino acid is one whose side chain has been derivatized once or several times, the type of derivatization is generally specifically indicated herein. Preferred derivatizations of the side chains can be carried out, in particular where the side chains have functional groups. Preferred functional groups are, for example, amino, carboxyl, thiol or alcohol groups.

[0166] Part II: Nucleic acids encoding antibodies or antibody fragments that bind to TROP2, gene vectors and cells containing the same

[0167] The present invention provides a nucleic acid comprising a nucleic acid sequence encoding the antibody or antibody fragment described in the first part of the present invention.

[0168] In some embodiments of the present invention, the present invention provides a combination of isolated polynucleic acids, the combination comprising a polynucleic acid encoding a light chain of an antibody or antibody fragment that binds to TROP2 of the present invention and a polynucleic acid encoding a heavy chain of an antibody or antibody fragment that binds to TROP2 of the present invention. In some embodiments, the polynucleic acid is operably linked to a regulatory sequence that can be recognized by a host cell transformed with the vector.

[0169] The present invention also provides a gene vector comprising the nucleic acid as described above in the second part of the present invention.

[0170] In some embodiments of the present invention, the gene vector of the present invention comprises a nucleic acid encoding an antibody or antibody fragment molecule that binds to TROP2 of the present invention or a combination of polynucleic acids of the present invention, and the polynucleic acids are operably linked to regulatory sequences that allow the encoded polypeptide to be expressed in a host cell or a cell-free expression system. The choice of gene vector depends on the choice of host cell, and can be selected so that in the selected host cell, the "gene vector" is a vector that includes one or more expression control sequences, and the "expression control sequence" is a DNA sequence that controls and regulates the transcription and / or translation of another DNA sequence. The nucleic acid in the gene vector can be operably linked to one or more expression control sequences.

[0171] In the present invention, suitable gene vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus, and adeno-associated virus. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (LaJolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0172] The gene vector may include a tag sequence. The tag sequence is typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including the carboxyl or amino termini. Examples of useful tags include, but are not limited to, Fc fragments, polyhistidine, green fluorescent protein (GFP), glutathione S-transferase (GST), c-myc, hemagglutinin, Flag™ tag (Kodak, New Haven, CT), maltose E binding protein, and protein A.

[0173] The present invention also provides a cell comprising the nucleic acid or gene vector as described above in the second part of the present invention.

[0174] In some embodiments, the host cell can be a prokaryotic host cell, a eukaryotic host cell, or a bacteriophage. The prokaryotic host cell can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic host cell can be a fungus such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or Trichoderma, an insect cell such as S. frugiperda, a plant cell such as tobacco, or a mammalian cell such as BHK cells, CHO cells, COS cells, or myeloma cells. In some embodiments, the host cell is preferably a mammalian cell, more preferably a BHK cell, CHO cell, NSO cell, or COS cell.

[0175] Part III: Antibody or Antibody Fragment-Drug Conjugates Binding to TROP2 and Their Pharmaceutically Acceptable Salts, Hydrates, Solvates, Stereoisomers or Isotope Labels

[0176] The present invention provides an antibody or antibody fragment-drug conjugate, and pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled products thereof. The antibody or antibody fragment is the aforementioned antibody or antibody fragment of the present invention. The antibody or antibody fragment-drug conjugate uses a stable linker to reduce off-target toxicity and expand the therapeutic window.

[0177] In some embodiments of the present invention, the drug can be coupled to various groups on the antibody via appropriate linkers to provide an ADC. For example, coupling can be performed through antibody surface lysines, or through oxidized carbohydrates, or through cysteine ​​residues that have been released by reduction of one or more interchain disulfide bonds. Alternatively, the antibody can be modified to include other cysteine ​​residues or non-natural amino acids that provide a reactive handle, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine. Such modifications are well known in the art (see, e.g., U.S. Patent Nos. 7,521,541; 8,455,622 and 9,000,130; Hofer et al., Biochemistry, 48: 12047-12057 (2009); Axup et al., PNAS, 109: 16101-16106 (2012); Wu et al., PNAS, 106: 3000-3005 (2009); Zimmerman et al., Bioconj. Chem., 25: 351-361 (2014)).

[0178] Part IV: Drug conjugates of antibodies or antibody fragments that bind to TROP2, and methods for preparing and using pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, or isotope-labeled products thereof, as well as pharmaceutical combinations and kits containing the same

[0179] The present invention provides an antibody-drug conjugate as described in the third part of the present invention, and a method for preparing a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled product thereof, the method comprising the following steps: S1: reduction of the antibody; S2: conjugation of the antibody to a drug linker; S3: purification of the antibody-drug conjugate.

[0180] The present invention provides a method for preparing the antibody-drug conjugate of the present invention, comprising the following steps:

[0181] S1: reduction of antibodies;

[0182] Specifically, replace the antibody medium with PBS7.0 / EDTA to a concentration of 10 mg / mL. Add an appropriate amount of 10 mM TCEP aqueous solution based on the reduction volume, and incubate at 37°C for several hours to reduce the disulfide bonds within the antibody.

[0183] S2: Conjugation of the antibody to the linker-drug compound;

[0184] Specifically, a 10 mM solution of the compound dissolved in dimethyl sulfoxide (DMSO) at room temperature was added to the above solution, mixed, and allowed to react at room temperature for 0.5-4 hours to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide was added, and the reaction was continued at room temperature to terminate the linker-drug reaction.

[0185] Purification of S3 antibody-drug conjugate;

[0186] Specifically, a 1 mL Mabselect SuRe prepacked chromatography column was equilibrated with PBS7.0 / EDTA solution (10 mM PB, 137 mM NaCl, 5 mM EDTA, pH 7.0) on an AKTA system, and the antibody-drug conjugate was loaded using a sample loop. After equilibration with PBS7.0 / EDTA for another 10 to 20 mL, the column was eluted with acetate buffer at pH 3.5 and neutralized by adding 1 / 10 volume of sodium citrate neutralizing solution to obtain the purified antibody-drug conjugate.

[0187] The present invention also provides a pharmaceutical composition comprising the antibody-drug conjugate described in Part III of the present invention, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels, and a pharmaceutically acceptable carrier or excipient.

[0188] In some embodiments of the present invention, suitable carriers or excipients include any material that maintains the therapeutic function (e.g., anticancer function) of the therapeutic composition when combined with the therapeutic composition and generally cannot react with the patient's immune system. Pharmaceutically acceptable carriers or excipients include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are physiologically compatible. Examples of pharmaceutically acceptable carriers or excipients include one or more of water, saline, phosphate buffered saline (PBS), histidine, dextrose, glycerol, ethanol, mesylate, etc., and combinations thereof. In some embodiments of the present invention, isotonic agents are included in the composition, for example, sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. In some embodiments of the present invention, pharmaceutically acceptable carriers or excipients may further include a small amount of auxiliary substances, such as wetting agents or emulsifiers, preservatives, or buffers, which increase the shelf life or effectiveness of the antibody, Fab, conjugate, and / or ADC.

[0189] The present invention also provides an antibody-drug conjugate as described in Part III of the present invention, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels, or the pharmaceutical composition as described above in Part IV of the present invention for use in the preparation of a medicament for treating and / or preventing TROP2-mediated diseases or conditions.

[0190] In some embodiments of the invention, the disease or disorder is cancer.

[0191] In some embodiments of the present invention, the cancer is selected from breast cancer and pancreatic cancer. In some embodiments of the present invention, preferably, the breast cancer is triple-negative breast cancer.

[0192] The present invention also provides a drug combination comprising the antibody-drug conjugate described in Part III of the present invention, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels, or the pharmaceutical composition as described above in Part IV of the present invention, and one or more additional therapeutic agents.

[0193] In some embodiments of the present invention, the drug conjugates of the present invention can be administered simultaneously or sequentially with other cancer therapeutic agents. The anti-cancer effect can be enhanced accordingly. The other anti-cancer agent used for this purpose can be administered to an individual simultaneously with, separately from, or after the drug conjugate, and the administration can be performed while varying the respective dosing intervals.

[0194] The present invention also provides a kit comprising the antibody-drug conjugate described in Part III of the present invention, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotope labels, or the pharmaceutical composition as described above in Part IV of the present invention.

[0195] In some embodiments of the present invention, the kit further comprises one or more additional therapeutic agents, examples of which are another anticancer agent as described above in Section 4 of the present invention.

[0196] DETAILED DESCRIPTION

[0197] The present invention will be described in detail below with reference to the examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several adjustments and improvements without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0198] Common Procedure A: Purification of Antibody-Drug Conjugates

[0199] 1 mL of Mabselect SuRe was equilibrated with PBS7.0 / EDTA solution (10 mM PB, 137 mM NaCl, 5 mM EDTA, pH 7.0) on an AKTA pure system (Cytiva). TM The antibody-drug conjugate was loaded onto a pre-packed chromatography column using a sample loop. After equilibration with PBS7.0 / EDTA for 10 to 20 mL, the column was eluted with acetate buffer at pH 3.5 and neutralized with 1 / 10 volume of sodium citrate neutralizing solution to obtain the purified antibody-drug conjugate.

[0200] Common Procedure B: Determination of the Drug-Antibody Ratio (DAR) of Antibody-Drug Conjugates

[0201] Dithiothreitol was added to the antibody-drug conjugate to a final concentration of 20 mM, and the sample, which had broken the interchain disulfide bonds of the antibody-drug conjugate, was incubated at 37°C in a water bath for 30 minutes. The sample was then used for HPLC analysis. The HPLC system used was an Agilent Technologies 1260 Infinity HPLC, and the column was a PLRP-S (5 μm particle size; 2.1 mm × 50 mm; Agilent Technologies). The column temperature was 80°C, and the mobile phase A was 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B was 0.1% trifluoroacetic acid (TFA) in acetonitrile. The sample volume was 10 μL, and the gradient program was 27%-27% from 0-3 minutes, 27%-35% from 3-8 minutes, 35%-43% from 8-25 minutes, 43%-95% from 25-26 minutes, 95%-95% from 26-31 minutes, 95%-27% from 31-31.5 minutes, and 27%-27% from 31.5-40 minutes. Compared to unlinked light chains (L0) and heavy chains (H0), the hydrophobicity of drug-linked light chains (light chains linked to one drug, L1) and heavy chains (heavy chains linked to one drug, H1; heavy chains linked to two drugs, H2; and heavy chains linked to three drugs, H3) increases with the number of linked drugs, resulting in elution in the order of L0, L1, H0, H1, H2, and H3. DAR values ​​were calculated based on peak areas at 280 nm.

[0202] Example 1 Antibody modification

[0203] The hRS7 antibody was mutated to obtain a pH-sensitive antibody that binds to Trop2. First, the amino acids of the hRS7 antibody light chain CDR (defined according to the Kabat numbering system) were saturated mutated to establish a phage mutation library. The mutant library was then subjected to three rounds of liquid phase panning, incubated with biotin antigen at pH 6.0 for binding, and after washing away unbound phage, eluted using PBS buffer at pH 7.4. The monoclonal phages selected in the second and third rounds of panning were released and subjected to phage ELISA. Clones with signal values ​​at pH 6.0 exceeding 2 times the signal values ​​at pH 7.4 were selected for sequencing, and then antibody expression was performed to identify pH-sensitive antibodies that bind to Trop2, obtaining mut107 and mut1071 antibodies.

[0204] Example 2 Antibody Preparation

[0205] HEK293 cells were placed in a 5% CO2 constant temperature shaker at 37°C and 120 rpm. The cells were cultured to a density of 2.0×10 6The antibody heavy and light chains were added at a ratio of 0.5 mg of antibody heavy chain (Hc) and 0.5 mg of antibody light chain (Lc) per liter of cells. First, the sterile plasmid was mixed with transfection buffer (KPM). KPM was then mixed with TA-293 transfection reagent (Zhuhai Kairui Biotechnology Co., Ltd.) to prepare the transfection reagent. The transfection reagent was slowly added to the plasmid-containing KPM and gently mixed to form a plasmid-vector complex. After standing for 10 minutes, the plasmid-vector complex was added to the cells. After 24 hours, KE-293 Cell Protein Expression Enhancer (Zhuhai Kairui Biotechnology Co., Ltd.) and KT-Feed Transient Transfection Nutrient Supplement (Zhuhai Kairui Biotechnology Co., Ltd.) were added. Six days after transfection, the cells were harvested and purified. Mut107, mut1071, TINA, hRS7, and palivizumab (anti-RSV antibody) were prepared using this method.

[0206] >mut1071 heavy chain (CDRs are coded using Kabat, with bold and underlined CDR regions)

[0207] Variable region:

[0208] Heavy chain CDRs:

[0209] CDR1-H: NYGMN (SEQ ID NO: 1)

[0210] CDR2-H:WINTYTGEPTYTDDFKG(SEQ ID NO:2)

[0211] CDR3-H:GGFGSSYWYFDV(SEQ ID NO:3)

[0212] Constant region:

[0213] full length:

[0214] >mut1071 light chain (CDRs are coded using Kabat, with bold and underlined CDR regions)

[0215] Variable region:

[0216] Light chain CDRs:

[0217] CDR1-L:KASQDESIAVA (SEQ ID NO:4)

[0218] CDR2-L:SASYRYT (SEQ ID NO:5)

[0219] CDR3-L:HQHYITPLT (SEQ ID NO:6)

[0220] Constant region:

[0221] full length:

[0222] >mut107 heavy chain (CDRs are coded using Kabat, with CDRs in bold and underlined):

[0223] SEQ ID NO:8

[0224] Heavy chain CDRs:

[0225] CDR1-H: SEQ ID NO: 1

[0226] CDR2-H: SEQ ID NO: 2

[0227] CDR3-H: SEQ ID NO: 3

[0228] Constant region:

[0229] SEQ ID NO:11

[0230] full length:

[0231] SEQ ID NO:13

[0232] >mut107 light chain (CDRs are coded using Kabat, with CDRs in bold and underlined):

[0233] Light chain CDRs:

[0234] CDR1-L:KASQDESIAVA (SEQ ID NO:4)

[0235] CDR2-L:SASYRYT (SEQ ID NO:5)

[0236] CDR3-L:EQHYITPLT (SEQ ID NO:7)

[0237] Constant region: SEQ ID NO: 12

[0238] full length:

[0239] >TINA heavy chain (naked antibody for DS-1062a)

[0240] >TINA light chain (naked antibody for DS-1062a)

[0241] >hRS7 heavy chain (naked antibody of Trodelvy)

[0242] >hRS7 light chain (naked antibody of Trodelvy)

[0243] >Palivizumab (an anti-RSV antibody) heavy chain

[0244] >Palivizumab (an anti-RSV antibody) light chain

[0245] Example 3 Preparation of Antibody-Drug Conjugate ADC-1

[0246] Antibody reduction:

[0247] The mut107 antibody medium was replaced with PBS 7.0 / EDTA to prepare a solution with an antibody concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technologies, Inc.) in water (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0248] Conjugation of Antibodies to Drug-Linker Compounds:

[0249] A 10 mM DMSO solution of the drug-linker compound JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd., synthesis method can be found in CN116135232B, Example 1) was added to the above solution at room temperature (68.0 μL; 10 equivalents per antibody molecule), mixed, and reacted at room temperature for 30 minutes to link the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added, and the mixture was allowed to react at room temperature for 20 minutes to terminate the coupling reaction.

[0250] Purification of Antibody-Drug Conjugates:

[0251] The above solution was purified using the common procedure A to obtain a solution containing the ADC-1 compound.

[0252] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0253] The DAR value was measured using the common procedure B, and the DAR value of ADC-1 was 7.3.

[0254] Example 4 Preparation of Antibody-Drug Conjugate ADC-2

[0255] Antibody reduction:

[0256] The mut1071 antibody medium was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 8.16 μL of 100 mM TCEP (J&K Technologies, Inc.) aqueous solution (12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0257] Conjugation of Antibodies to Drug-Linker Compounds:

[0258] A 10 mM DMSO solution of the drug-linker compound JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) in 68.0 μL (10 equivalents per antibody molecule) was added to the above solution at room temperature, mixed, and reacted at room temperature for 30 minutes to link the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) in 10.2 μL (15 equivalents per antibody molecule) was added and allowed to react at room temperature for another 20 minutes to terminate the coupling reaction.

[0259] Purification of Antibody-Drug Conjugates:

[0260] The above solution was purified using the common procedure A to obtain a solution containing the ADC-2 compound.

[0261] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0262] The DAR value was measured using the common procedure B, and the DAR value of ADC-2 was 7.4.

[0263] Example 5 Preparation of Positive Reference Antibody-Drug Conjugate ADC-3

[0264] Antibody reduction:

[0265] The TINA antibody (naked antibody against DS-1062a) medium was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and a 10 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (15.64 μL; 2.3 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0266] Conjugation of Antibodies to Drug-Linker Compounds:

[0267] A 10 mM DMSO solution of the drug-linker compound deruxtecan (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) (30.6 μL; 4.5 equivalents per antibody molecule) was added to the above solution at room temperature, mixed, and reacted at room temperature for 30 minutes to link the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and reacted at room temperature for a further 20 minutes to terminate the coupling reaction.

[0268] Purification of Antibody-Drug Conjugates:

[0269] The above solution was purified using the common procedure A to obtain a solution containing the ADC-3 compound.

[0270] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0271] The DAR value was measured using the common procedure B, and the DAR value of ADC-3 was 4.1.

[0272] Example 6 Preparation of Positive Reference Antibody-Drug Conjugate ADC-4

[0273] Antibody reduction:

[0274] The hRS7 antibody (mut107 and mut1071 mutations) medium was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 100 mM TCEP (J&K Technologies, Inc.) aqueous solution (8.16 μL; 12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0275] Conjugation of Antibodies to Drug-Linker Compounds:

[0276] To this solution, a 10 mM DMSO solution of the drug-linker compound TL001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) (68.0 μL; 10 equivalents per antibody molecule) was added at room temperature, mixed, and allowed to react at room temperature for 30 minutes to link the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) (10.2 μL; 15 equivalents per antibody molecule) was added and allowed to react at room temperature for a further 20 minutes to terminate the coupling reaction.

[0277] Purification of Antibody-Drug Conjugates:

[0278] The above solution was purified using the common procedure A to obtain a solution containing the ADC-4 compound.

[0279] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0280] The DAR value was measured using the common procedure B, and the DAR value of ADC-4 was 7.1.

[0281] Example 7 Preparation of Negative Reference Antibody-Drug Conjugate ADC-5

[0282] Antibody reduction:

[0283] The palivizumab antibody (anti-RSV antibody) medium was replaced with PBS 7.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL EP tube, and 100 mM TCEP (J&K Technologies, Inc.) aqueous solution (8.16 μL; 12 equivalents per antibody molecule) was added. The mixture was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0284] Conjugation of Antibodies to Drug-Linker Compounds:

[0285] A 10 mM DMSO solution of the drug-linker compound JSSW-001 (Shanghai Haoyuan Biopharmaceutical Technology Co., Ltd.) in 68.0 μL (10 equivalents per antibody molecule) was added to the above solution at room temperature, mixed, and reacted at room temperature for 30 minutes to link the drug-linker compound to the antibody. Next, a 100 mM aqueous solution of N-ethylmaleimide (J&K Technology Co., Ltd.) in 10.2 μL (15 equivalents per antibody molecule) was added and allowed to react at room temperature for another 20 minutes to terminate the coupling reaction.

[0286] Purification of Antibody-Drug Conjugates:

[0287] The above solution was purified using the common procedure A to obtain a solution containing the ADC-5 compound.

[0288] Determination of the Drug Antibody Ratio (DAR) of Antibody-Drug Conjugates:

[0289] The DAR value was measured using the common procedure B, and the DAR value of ADC-5 was 7.3.

[0290] Example 8 Biomembrane Interferometry to Determine Antibody Affinity

[0291] The affinity of anti-TROP2 antibodies mut107, mut1071, TINA, and hRS7 for human TROP2 at pH 6.0 and pH 7.4 was determined using a ForteBio Octet Red 96e instrument (Sartorius). Different test samples were immobilized at 5 μg / ml using a Protein A capture sensor (Sartorius). TROP2 antigen was serially diluted from a starting concentration of 100 nM and then diluted two-fold over a total of seven gradients. The buffer system was PBS pH 7.4 + 0.02% Tween 20. The steps were: ① Baseline 60 s, ② Loading 800 s (antibody immobilization, threshold set to 0.50 nM), ③ Baseline 2120 s, ④ Association 120 s, ⑤ Dissociation 300 s, and ⑥ Regeneration 300 s. Blank buffer was used for blank treatment, and the baseline was aligned with the y-axis and fitted using the Savitzky-Golay method in Octet data analysis software. The experimental results are shown in (Figure 1A-1H) and Table 1.

[0292] Table 1 Antibody affinity measured by ForteBio at different pH

[0293] mut107 and mut1071 have high binding affinity to TROP2 at pH 6.0, but do not bind to TROP2 at pH 7.4, and the curves cannot be fitted, indicating good pH-dependent binding activity. However, the reference antibodies TINA and hRS7 did not exhibit pH-dependent binding activity.

[0294] Example 9 Antigen Binding Activity Detection

[0295] Dilute the His-tagged human TROP2 protein to 1 μg / mL with coating solution, add 100 μL to each well, and incubate at 4°C overnight; add 100 μL of blocking solution to each well of the enzyme-labeled plate containing the coating solution, shake at room temperature for 2 hours, and then wash the plate 3 times with washing buffer. Serially dilute the sample and reference product, add 100 μL to each well, shake at room temperature for 2 hours, wash 4 times with washing buffer, add enzyme-labeled antibody, dilute goat anti-human IgG-HRP with sample diluent, add 100 μL to each well, shake at room temperature for 1 hour, wash the plate 4 times with washing buffer, add substrate solution for color development, add 100 μL of freshly prepared TMB reaction solution to each reaction well, react at room temperature for 5-10 minutes, add 100 μL of stop solution to each well and mix gently to terminate the reaction. Use a microplate reader to measure the absorbance at a wavelength of 450 nm, and use software (GraphPad prism) to perform four-parameter fitting, where the C value is the half-effective concentration EC 50 The binding activity of the antibody and protein is shown in Table 2.

[0296] Table 2 Summary of human TROP2 protein binding activity at different pH

[0297] The results showed that mut107 and mut1071 bound to human TROP2 with high affinity at pH 6.0, but hardly bound at pH 7.4, demonstrating good pH-dependent binding activity. However, the reference antibodies TINA and hRS7 did not exhibit pH-dependent binding activity.

[0298] Example 10 Cell Binding Activity Experiment

[0299] The affinity of anti-TROP2 antibodies mut107, mut1071, TINA, and hRS7 was tested using TROP2-positive human triple-negative breast cancer cells (MDA-MB-468). The sample was diluted to 15,000 ng / mL with 2% BSA in PBS and then serially diluted through 11 steps to a concentration range of 0.014 to 15,000 ng / mL. MDA-MB-468 cells were centrifuged at 500 × g for 5 minutes, washed three times with 2% BSA in PBS, and incubated with the various diluted samples for 2 hours at 4°C in the dark. After washing, the cells were incubated with goat anti-human Alexa Fluor 488 fluorescent dye for 1 hour at 4°C in the dark. After washing, the cells were resuspended and the mean fluorescence intensity (MFI) was measured using an Attune NxT flow cytometer (Thermo Fisher Scientific, Inc.). The results are shown in Figure 2 and Table 3.

[0300] Table 3 Antibody cell level binding activity at different pH

[0301] The results showed that mut107 and mut1071 bound to human TROP2-positive MDA-MB-468 cells with high affinity at pH 6.0, but showed little binding to TROP2 at concentrations as high as 15 μg / ml at pH 7.4, demonstrating strong pH-dependent binding activity. In contrast, the positive reference antibodies TINA and hRS7 showed little change in binding to human TROP2-positive MDA-MB-468 cells at both pH 6.0 and pH 7.4, indicating no pH-dependent binding activity.

[0302] Example 11 In vivo efficacy evaluation of CFPAC-1 cell CDX mouse model

[0303] In this experiment, NU / NU mice of appropriate age were inoculated with human pancreatic cancer cells CFPAC-1. When the tumor volume grew to about 100 mm, 3 Around 28 animals with good tumor growth were selected and divided into 4 groups evenly according to tumor volume. The animal groups and dosage regimens are shown in Table 4.

[0304] Table 4 Animal grouping and dosage regimen

[0305] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows: Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0306] On day 19 after administration, the mice were asphyxiated with carbon dioxide, and the tumors were removed and weighed.

[0307] At the experimental endpoint, all antibody-drug conjugates significantly inhibited tumor growth compared to the vehicle group. ADC-2 achieved an inhibition rate of 96.7%. Both ADC-1 and ADC-2 exhibited significantly higher inhibition rates than the positive control, ADC-3. See Figure 3 and Table 5 for details.

[0308] Table 5 ADC efficacy against CFPAC-1 xenografts in tumor-bearing mice

[0309] Example 12 In vivo efficacy evaluation of MDA-MB-468 cell CDX mouse model

[0310] In this experiment, NOD / SCID mice of appropriate age were inoculated with human triple-negative breast cancer cells MDA-MB-468. 3 Around 28 animals with good tumor growth were selected and divided into 4 groups evenly according to tumor volume. The animal groups and dosage regimens are shown in Table 6.

[0311] Table 6 Animal grouping and dosage regimen

[0312] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows: Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0313] On the 16th day after administration, the mice were asphyxiated with carbon dioxide, and the tumors were removed and weighed.

[0314] At the experimental endpoint, compared with the vehicle group, both ADC-2 and ADC-3 could significantly inhibit tumor growth, and the efficacy of ADC-2 was significantly better than that of the positive reference ADC-3. Detailed information is shown in Figure 4 and Table 7.

[0315] Table 7 ADC efficacy against MDA-MB-468 xenografts in tumor-bearing mice

[0316] Example 13 In vivo efficacy evaluation of CFPAC-1 cell CDX mouse model

[0317] In this experiment, NU / NU mice of appropriate age were inoculated with human pancreatic cancer cells CFPAC-1. When the tumor volume grew to about 100 mm, 3 Around 35 animals with good tumor growth were selected and divided into 5 groups evenly according to tumor volume. The animal groups and dosage regimens are shown in Table 8.

[0318] Table 8 Animal grouping and dosage regimen

[0319] After grouping, the mice were dosed and weighed, and the data were recorded. The tumor diameters at different times after administration were measured to dynamically observe the growth of the tumor and calculate the tumor volume. The formula is as follows: Tumor volume (mm 3 )=1 / 2×long diameter (mm)×[short diameter (mm)] 2

[0320] On day 19 after administration, the mice were asphyxiated with carbon dioxide, and the tumors were removed and weighed.

[0321] At the experimental endpoint, all antibody-drug conjugates significantly inhibited tumor growth compared with the vehicle group. ADC-2 was more effective than the positive reference ADC-3 at the same dose and ADC-4 at twice the dose, and one mouse showed complete tumor regression. Detailed information is shown in Figure 5 and Table 9.

[0322] Table 9 ADC efficacy against CFPAC-1 xenografts in tumor-bearing mice

[0323] Example 16 Safety Evaluation Study

[0324] In this experiment, two crab-eating macaques of appropriate age were selected and injected intravenously with ADC-2. The dosage design is shown in Table 10. After administration, there was no obvious abnormality in the weight and food intake of the animals. The weight change trend is shown in Figure 6. Compared with the toxicity test results of DS-1062a reported in the literature (Mol Cancer Ther. 2021 Dec; 20 (12): 2329-2340.), ADC-2 did not find severe toxicity after three consecutive doses of 30 mg / kg. Only one animal's thymus was found to be smaller, and there were no other toxic target organs. However, DS-1062a showed multiple organ toxicity after administration. Intestinal damage occurred after administration of 10 mg / kg or more, lung, cornea, skin, thymus, and liver damage occurred after administration of 30 mg / kg or more, and kidney and articular cartilage damage occurred after administration of 80 mg / kg. Compared with the preclinical results of DS-1062a, ADC-2 significantly reduced on-target toxicity, with the highest non-severe toxic dose (HNSTD) being 30 mg / kg, which is three times that of DS-1062a, significantly improving the tolerable dose.

[0325] Table 10 Pre-toxicity dosing regimen for cynomolgus monkeys

[0326] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. An antibody or antibody fragment that binds to TROP2, which comprises a heavy chain variable region and a light chain variable region, and both the heavy chain variable region and the light chain variable region comprise 3 CDRs, and is characterized in that, the heavy chain variable region comprises: CDR1-H, which has the sequence shown in SEQ ID NO:1 or a functional variant thereof; CDR2-H, which has the sequence shown in SEQ ID NO:2 or a functional variant thereof; and CDR3-H, which has the sequence shown in SEQ ID NO:3 or a functional variant thereof; the light chain variable region comprises: CDR1-L, which has the sequence shown in SEQ ID NO:4 or a functional variant thereof; CDR2-L, which has the sequence shown in SEQ ID NO:5 or a functional variant thereof; and CDR3-L, which has the sequence shown in SEQ ID NO:6 or SEQ ID NO:7 or a functional variant thereof.

2. The antibody or antibody fragment according to claim 1, wherein The antibody or antibody fragment comprises a heavy chain variable region having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identity to the amino acid sequence of SEQ ID NO:8 and a light chain variable region having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identity to the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:

10.

3. The antibody or antibody fragment according to claim 1 or 2, characterized in that, The antibody or antibody fragment is selected from murine antibodies, chimeric antibodies and humanized antibodies; Preferably, the antibody fragment is selected from Fab, F(ab')2, Fab', Fd, Fv, dAb, dsFv and scFv.

4. The antibody or antibody fragment according to any one of claims 1-3, characterized in that, The antibody or antibody fragment comprises a heavy chain constant region having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identity to the amino acid sequence of SEQ ID NO:11 and a light chain constant region having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identity to the amino acid sequence of SEQ ID NO:

12.

5. The antibody or antibody fragment according to any one of claims 1-4, characterized in that, The antibody or antibody fragment comprises a heavy chain having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identity to the amino acid sequence of SEQ ID NO:13 and a light chain having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, 100% identity to the amino acid sequence of SEQ ID NO:14 or SEQ ID NO:

15.

6. The antibody or antibody fragment according to any one of claims 1-5, characterized in that, Compared with the condition where the pH is greater than 7.0, the antibody or antibody fragment binds to TROP2 with higher affinity under the condition where the pH is less than 7.0; Preferably, compared with the condition where the pH is about 7.2 - 7.5, the antibody or antibody fragment binds to TROP2 with higher affinity under the condition where the pH is about 6.0 - 6.5; More preferably, compared with the condition where the pH is about 7.4, the antibody or antibody fragment binds to TROP2 with higher affinity under the condition where the pH is about 6.

0.

7. The antibody or antibody fragment according to any one of claims 1-6, characterized in that, The antibody or antibody fragment binds to the TROP2 protein expressed on the cell surface; Preferably, the cell includes Trop2-positive cancer cells; More preferably, the cancer cells are selected from breast cancer cells and pancreatic cancer cells; Most preferably, the breast cancer cells are triple-negative breast cancer cells.

8. The antibody or antibody fragment according to any one of claims 1-7, characterized in that, The antibody or antibody fragment is further modified; Preferably, the modification includes N-terminal modification, C-terminal modification, side chain modification, amino acid modification, peptide backbone modification, and binding to other polypeptides or proteins.

9. A nucleic acid, characterized in that, The nucleic acid contains a nucleic acid sequence encoding the antibody or antibody fragment of any one of claims 1-8.

10. A gene vector, characterized in that, The gene vector contains the nucleic acid of claim 9.

11. A cell, characterized in that, The cell contains the nucleic acid of claim 9 or the gene vector of claim 10.

12. An antibody or antibody fragment-drug conjugate represented by the following formula, and its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled compound: Ab-(L-D) n Wherein: (a) Ab is the antibody or antibody fragment of any one of claims 1-8; (b) L-D is a linker-bioactive molecule moiety, where -L- is a linker and D is a bioactive molecule; (c) n is the molar ratio of the bioactive molecule to Ab, selected from an integer from 1 to 12 (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) or a decimal; when n is a decimal, it refers to the average number of linker-bioactive molecule (L-D) molecules conjugated to each Ab; Preferably, the -L- is selected from -L1-L2-L3-L4-L5- or -L1-L2-L4-L5-, where L1 is a covalent linking unit that covalently links to Ab, L2 is an extension unit, L3 is selected from amino acid residues optionally substituted with polar hydrophilic groups, L4 is selected from peptide residues composed of 2-8 amino acids, and L5 is a bond or a self-cleaving fragment; More preferably, the L1 is selected from: (or its open-ring form )、 Where * represents the connection to Ab; L2 is selected from -L 2a -, -L 2a -C(O)-, -C(O)-L 2a -C(O)-NH-L 2b -C(O)-, -L 2a -NH-C(O)-L 2b -C(O)-, wherein L 2a and L 2b are each independently selected from -C1-C8 alkylene-, -C1-C8 alkylene-C3-C8 cycloalkylene-, -C2-C6 alkynylene-C1-C6 alkylene-, a straight-chain or branched-chain heteroalkylene of 1-50 (preferably 9-30, more preferably 9-26, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) atoms, wherein the alkylene, cycloalkylene, and heteroalkylene are each optionally substituted by one or more substituents independently selected from C1-C6 alkyl, heteroalkyl of 2-6 atoms, hydroxy, amino, carboxy, or C3-C8 cycloalkyl, and the heteroalkylene contains 1-12 (preferably 1-8, more preferably 3-8, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) heteroatoms, and the heteroatoms of the heteroalkylene and heteroalkyl are selected from one or more of N, O, or S (preferably O); L3 is selected from amino acid residues optionally substituted with polar hydrophilic groups, and the polar hydrophilic groups include sugar residues and their derivatives or polyethylene glycol residues and their derivatives or poly(sarcosine) residues and their derivatives or a combination thereof; L4 is selected from peptide residues composed of 2-8 amino acids, and the amino acids are selected from phenylalanine, isoleucine, leucine, tryptophan, valine, methionine, tyrosine, alanine, threonine, histidine, serine, glutamine, arginine, lysine, asparagine, glutamic acid, proline, citrulline, aspartic acid, and glycine; L5 is selected from keys, Where, * represents the connection to D.

13. The antibody or antibody fragment-drug conjugate according to claim 12, and its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled substance, characterized in that, The D is a cytotoxic drug; preferably, the D is selected from tubulin inhibitors, DNA intercalating agents, DNA topoisomerase inhibitors, DNA synthesis inhibitors, RNA polymerase inhibitors, and splicesome inhibitors; more preferably, the D is selected from camptothecin derivatives.

14. The antibody or antibody fragment-drug conjugate according to claim 12 or 13, and its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotopically labeled compound, characterized in that, D is selected from wherein each of R1 and R2 is independently selected from H, C1-3 alkyl (preferably methyl, ethyl, n-propyl, isopropyl), 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl), or R1 and R2 together with the carbon atom connecting them form a 3-6 membered cycloalkyl (preferably cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl).

15. The antibody or antibody fragment-drug conjugate according to claim 14, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotopically labeled compounds, wherein the structure of the drug conjugate is shown in Formula I: Among them, Ab is the antibody or antibody fragment of any one of claims 1-8, and R1, R2, and n are as described in claim 14; Preferably, the conjugate of formula I is of formula Ia or formula Ib: Wherein, in formula Ia and formula Ib, R is R1, and the definitions of R1, Ab, and n are as described in claim 14; More preferably, the conjugate of formula I is of formula I-1, formula Ia-1 or formula Ib-1: Wherein, in formula I-1, formula Ia-1, and formula Ib-1, the definitions of Ab and n are as described in claim 14.

16. The method for preparing the antibody-drug conjugate according to any one of claims 12-15, and its pharmaceutically acceptable salt, hydrate, solvate, stereoisomer or isotope-labeled substance, characterized in that, The method includes the following steps: S1: Reduction of the antibody; S2: Conjugation of the antibody with the drug linker; S3: Purification of the antibody-drug conjugate; Preferably, the method comprises the following steps: S1: Reduction of the antibody: Prepare an antibody solution from the antibody, add an appropriate amount of TCEP aqueous solution according to the reduction amount, incubate, and reduce the disulfide bonds in the antibody; S2: Conjugation of the antibody with the drug linker: Add an appropriate amount of the linker-drug compound dissolved in dimethyl sulfoxide (DMSO) solution to the above solution, mix well, react for 0.5 - 4 hours, connect the linker-drug compound to the antibody, add an aqueous solution of N-ethylmaleimide, stir, and terminate the reaction of the linker-drug compound; S3: Purification of the antibody-drug conjugate: Equilibrate a pre-packed chromatography column with PBS / EDTA solution, load the antibody-drug conjugate, re-equilibrate with PBS / EDTA, elute with acetate buffer, and add sodium citrate neutralizing solution for neutralization to obtain the purified antibody-drug conjugate.

17. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody-drug conjugate according to any one of claims 12 - 15, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotopically labeled compounds, and a pharmaceutically acceptable carrier or excipient.

18. Use of the antibody-drug conjugate according to any one of claims 12 - 15, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotopically labeled compounds, or the pharmaceutical composition according to claim 17 in the preparation of a drug for the treatment and / or prevention of TROP2-mediated diseases or disorders; Preferably, the disease or disorder is cancer; More preferably, the cancer is selected from breast cancer and pancreatic cancer.

19. A drug combination, characterized in that, The pharmaceutical combination comprises the antibody-drug conjugate according to any one of claims 12 - 15, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotopically labeled compounds, or the pharmaceutical composition according to claim 17, and one or more additional therapeutic agents.

20. A kit, characterized in that, The kit comprises the antibody-drug conjugate according to any one of claims 12 - 15, and its pharmaceutically acceptable salts, hydrates, solvates, stereoisomers or isotopically labeled compounds, or the pharmaceutical composition according to claim 17.