Anti-LIV-1 antibody-drug conjugate and use thereof

Abstract

An antibody-drug conjugate, the structural formula thereof being Pc-(L-D) n, wherein D is a cytotoxic drug, L is a linker unit, Pc is an antibody specifically binding to LIV-1 or an antigen-binding fragment thereof, and n is a real number from 1 to 16.

Description

Anti-LIV-1 antibody-drug conjugates and uses thereof

[0001] This disclosure claims priority to the Chinese patent application filed with the China Patent Office on January 5, 2024, with application number 202410020541.4, entitled “Anti-LIV-1 Antibody Drug Conjugates and Uses Thereof”, and the Chinese patent application filed with the China Patent Office on June 28, 2024, with application number 202410858956.9, entitled “Anti-LIV-1 Antibody Drug Conjugates and Uses Thereof”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure belongs to the field of biomedicine and relates to a class of antibody-drug conjugates with novel structures, a preparation method thereof, a pharmaceutical composition containing the conjugate, and use of the conjugate as an anti-tumor drug. Background Art

[0003] Antibody-drug conjugates (ADCs), as a new type of targeted drug, link monoclonal antibodies that specifically bind to tumor cell surface antigens with biologically active toxin molecules. These ADCs combine the tumor-targeting properties of the antibody with the highly effective killing properties of the toxin, while avoiding the drawbacks of the former, such as low efficacy, and the latter's excessive side effects and poor drugability. Compared to traditional chemotherapy drugs, ADCs precisely target tumor cells while minimizing their effects on normal cells, resulting in a safer and more effective anti-tumor effect.

[0004] LIV-1, also known as SLC39A6 or ZIP6, is a zinc transporter. Its encoding gene is located on chromosome 18q12.2. It is a multi-transmembrane protein with zinc transporter and metalloprotease activities. LIV-1 is a member of the SLC39A family (ZIP family). Its primary physiological function is to facilitate the transport of extracellular zinc ions into the cytoplasm, thereby collaborating with other family members to maintain intracellular zinc homeostasis. In tumor tissues, LIV-1 has been shown to interact with the transcription factors STAT3 and Snail, thereby downregulating E-cadherin expression and promoting epithelial-mesenchymal transition (EMT), thereby playing a role in the transport and migration of tumor cells.

[0005] LIV-1 is not expressed in most normal tissues and is only positively expressed in the breast, testicles, and prostate. Current studies have shown that LIV-1 is significantly highly expressed in a variety of tumors, especially in breast cancer, with a positive expression rate of up to 93%. In addition to breast cancer, LIV-1 is also highly expressed in a variety of solid tumors, such as melanoma (82%), prostate cancer (72%), ovarian cancer (48%), uterine cancer, lung cancer, etc. Currently, the ADC drug ladiratuzumab vdotin (SGN-LIV1A) targeting LIV-1 is in Phase 2 clinical trials and has been proven to bring clinical benefits to patients with breast cancer, especially triple-negative breast cancer, further verifying that LIV-1 is a potential target for targeting advanced solid tumors. Summary of the Invention

[0006] The present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, the general structural formula of which is Pc-(LD) n ,

[0007] in,

[0008] D is a cytotoxic drug;

[0009] L is a linker unit;

[0010] Pc is an antibody or antigen-binding fragment thereof that specifically binds to LIV-1;

[0011] The antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the HCDR1-3 and / or the LCDR1-3 are selected from the following combinations:

[0012] (1) the HCDR1-3 are SEQ ID NOs: 13-15; or / and the LCDR1-3 are SEQ ID NOs: 16-18;

[0013] (2) the HCDR1-3 are SEQ ID NOs: 19-21; or / and the LCDR1-3 are SEQ ID NOs: 22-24;

[0014] (3) the HCDR1-3 are SEQ ID NOs: 25-27; or / and the LCDR1-3 are SEQ ID NOs: 28-30;

[0015] or,

[0016] The HCDR1-3 and / or the LCDR1-3 have a sequence that is at least 80% identical to each CDR in any of the HCDR1-3 and LCDR1-3 in groups (1) to (3), or a sequence in which at most three insertion, deletion or substitution mutations occur; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity;

[0017] Furthermore, n is a real number from 1 to 16.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), and the heavy chain variable region and / or the light chain variable region are selected from the following:

[0019] (1) the heavy chain variable region is the sequence shown in SEQ ID NO: 1, and / or the light chain variable region is the sequence shown in SEQ ID NO: 2;

[0020] (2) the heavy chain variable region is the sequence shown in SEQ ID NO: 3, or / and the light chain variable region is the sequence shown in SEQ ID NO: 4;

[0021] (3) the heavy chain variable region is the sequence shown in SEQ ID NO: 5, or / and the light chain variable region is the sequence shown in SEQ ID NO: 6;

[0022] or,

[0023] The heavy chain variable region and / or the light chain variable region have a sequence that is at least 80% identical to the heavy chain variable region and / or the light chain variable region in any one of the above-mentioned groups (1) to (3), or a sequence in which at most three insertion, deletion or substitution mutations occur; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region sequence and / or a light chain constant region sequence; optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3 or IgG4 constant region, and the light chain constant region is selected from human or mouse kappa constant region or lamda constant region; optionally, the antibody or antigen-binding fragment thereof comprises a complete heavy chain and a light chain, the heavy chain consists of the VH and the heavy chain constant region, and the heavy chain constant region has the sequence shown in SEQ ID NO: 11, and the light chain consists of the VL and the light chain constant region, and the light chain constant region has the sequence shown in SEQ ID NO: 12.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof is:

[0026] (1) Chimeric antibodies or fragments thereof;

[0027] (2) humanized antibodies or fragments thereof; and / or,

[0028] (3) fully human antibodies or fragments thereof;

[0029] Preferably, the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., a bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, a naked antibody, a conjugated antibody, a humanized antibody, a fully human antibody, Fab, Fab', F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.

[0030] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibody, nanobody and the minimum recognition unit of an antibody.

[0031] In some embodiments, the aforementioned general formula is Pc-(LD) n The antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the cytotoxic drug is selected from a microtubule inhibitor, a DNA damaging agent or a topoisomerase inhibitor, the microtubule inhibitor includes but is not limited to dolastatin, auristatin, maytansine, tubulysins and cryptomycins, the DNA damaging agent includes but is not limited to PBD drugs, and the topoisomerase inhibitor includes but is not limited to camptothecin drugs.

[0032] In some embodiments, the cytotoxic drug is selected from a topoisomerase I inhibitor or an auristatin.

[0033] In some embodiments, the cytotoxic drug is selected from the compound represented by formula (DI),

[0034] in,

[0035] R 1 、R 2 Together with the atoms to which they are attached, they form a 5-6 membered heterocyclic ring containing 1 or 2 oxygen atoms as ring atoms, and the 5-6 membered heterocyclic ring is optionally substituted by one or more deuterium atoms (i.e., D atoms);

[0036] R 4 Selected from H or C1-C3 alkyl;

[0037] R 5 is selected from H, halogen, CN, =O, OH, NH2 or C1-C3 alkyl;

[0038] R 6 Selected from H or C1-C3 alkyl;

[0039] R 7 Selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted with deuterium (D), halogen, CN, ═O, OH, NH2 or C1-C3 alkyl.

[0040] In some embodiments, the R 1 、R 2 Together with the atoms they are connected to form

[0041] In some embodiments, R 4 Selected from H.

[0042] In some embodiments, R 5 Selected from H.

[0043] In some embodiments, R 6 Selected from H.

[0044] In some embodiments, R 7 Selected from cyclopropyl.

[0045] In some embodiments, the compound represented by formula (DI) is selected from

[0046] In some embodiments, the cytotoxic drug is selected from

[0047] In some embodiments, the aforementioned general formula is Pc-(LD) n The antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the linker unit L is selected from Wherein, m1 is selected from integers 2 to 8, L 1 is selected from a peptide residue consisting of 1 to 8 amino acids, which is further optionally substituted with one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl and 4-7 membered heterocyclyl, L 2 Selected from The a-end of the linker unit L is covalently connected to Pc, and the b-end is covalently connected to the cytotoxic drug.

[0048] In some embodiments, the L 1 A peptide residue selected from Val-Cit or Gly-Gly-Phe-Gly.

[0049] In some embodiments, m1 is 5.

[0050] In some embodiments, the linker unit L is Its a-terminal is covalently linked to Pc, and its b-terminal is covalently linked to the cytotoxic drug.

[0051] In some embodiments, the aforementioned general formula is Pc-(LD) n An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein n is selected from a real number of 1 to 16, for example, n is selected from a real number of 2 to 12, for example, n is selected from a real number of 4 to 10, for example, n is selected from a real number of 3 to 9, for example, n is selected from a real number of 4 to 8, for example, n is selected from a real number of 6 to 8, for example, n is selected from a real number of 3 to 5.

[0052] In some embodiments, n is selected from a real number of 3 to 9, for example, n is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.9 7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0053] In some embodiments, n is a real number selected from 6 to 8, for example, n is 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0054] In some embodiments, n is a real number selected from 3 to 5, for example, n is 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0.

[0055] In some embodiments, the disclosed formula is Pc-(LD) n The antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following compounds or a pharmaceutically acceptable salt thereof:

[0056] wherein Pc and n are as defined above.

[0057] In another aspect, the present disclosure provides an isolated nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment thereof.

[0058] In some embodiments, the present disclosure provides an expression vector comprising the aforementioned nucleic acid molecule.

[0059] In some embodiments, the present disclosure provides an isolated host cell of the aforementioned nucleic acid molecule, or the aforementioned expression vector; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

[0060] On the other hand, the present disclosure provides a pharmaceutical composition comprising the antibody-drug conjugate of the aforementioned general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0061] On the other hand, the pharmaceutical composition of the present disclosure further comprises other therapeutic agents; preferably, each part of the pharmaceutical composition, such as the antibody-drug conjugate and other therapeutic agents, can be packaged independently or in combination.

[0062] On the other hand, the present disclosure provides a method for treating mammalian tumors, comprising administering a therapeutically effective amount of the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a mammal, preferably a human, in need of such treatment.

[0063] In another aspect, the present disclosure provides use of the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a drug for treating tumors.

[0064] In another aspect, the present disclosure provides use of the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in treating tumors.

[0065] In another aspect, the present disclosure provides the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for treating tumors.

[0066] In some embodiments, the tumor is an LIV-1 expressing tumor.

[0067] In some embodiments, the tumor is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), prostate cancer, gynecological cancer, ovarian cancer, endometrial cancer, cervical cancer, liver cancer, stomach cancer, intestinal cancer, kidney cancer, squamous cell carcinoma (e.g., bladder, head, neck, and lung), skin cancer (e.g., melanoma), small cell lung cancer, or lung carcinoid.

[0068] In another aspect, the present disclosure provides use of the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a drug for treating tumors.

[0069] In some embodiments, the tumor is an LIV-1 expressing tumor.

[0070] In some embodiments, the tumor is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), prostate cancer, gynecological cancer, ovarian cancer, endometrial cancer, cervical cancer, liver cancer, stomach cancer, intestinal cancer, kidney cancer, squamous cell carcinoma (e.g., bladder, head, neck, and lung), skin cancer (e.g., melanoma), small cell lung cancer, or lung carcinoid.

[0071] In another aspect, the present disclosure provides use of the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in treating tumors.

[0072] In some embodiments, the tumor is an LIV-1 expressing tumor.

[0073] In some embodiments, the tumor is selected from the group consisting of breast cancer (e.g., triple-negative breast cancer), prostate cancer, gynecological cancer, ovarian cancer, endometrial cancer, cervical cancer, liver cancer, stomach cancer, intestinal cancer, kidney cancer, squamous cell carcinoma (e.g., bladder, head, neck, and lung), skin cancer (e.g., melanoma), small cell lung cancer, or lung carcinoid.

[0074] In some embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof can be used in combination with other therapeutic agents; preferably, the therapeutic agent can be a chemotherapeutic agent or an immune checkpoint inhibitor; more preferably, the combined therapy provides a synergistic therapeutic effect in treating tumors.

[0075] In another aspect, the present disclosure provides the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for treating tumors. Beneficial effects:

[0076] The antibody-drug conjugates disclosed herein can specifically recognize the LIV-1 target, have improved homogeneity and good plasma stability in mammalian (e.g., human or monkey) plasma, and / or have stronger tumor inhibition effects, lower toxicity, and longer in vivo half-life; their high safety and efficacy have broad clinical application prospects in the treatment of diseases such as tumors.

[0077] Definitions and Explanations of Terms

[0078] Unless otherwise indicated, the terms used in this disclosure have the following meanings. The definitions of groups and terms described in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions described in tables, and definitions of specific compounds in the examples, may be combined and coupled with each other in any manner. A particular term should not be considered as undefined or unclear unless specifically defined, but should be understood according to its ordinary meaning in the art. When a trade name appears in this document, it is intended to refer to the corresponding commercial product or its active ingredient.

[0079] The term "antibody-drug conjugate" (ADC) refers to an antibody or antigen-binding fragment thereof linked to a biologically active drug via a stable linker unit. The linker unit can be a covalent bond or a non-covalent interaction, such as electrostatic forces. To form an immunoconjugate, various linkers known in the art can be used.

[0080] The term "DAR" or "drug-to-antibody ratio" refers to the average number of small molecule cytotoxic drugs attached to each antibody molecule. In the antibody-drug conjugates of the present disclosure, DAR is defined by the variable "n", which can be either an integer or a decimal.

[0081] The term "antigen binding molecule" is used herein in the broadest sense to refer to a molecule that specifically binds to an antigen. Exemplarily, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetics" refer to organic compounds or binding domains that are capable of specifically binding to an antigen but are unrelated to the structure of an antibody. Exemplarily, antibody mimetics include, but are not limited to, affibodies, affitins, affilins, designed ankyrin repeat proteins (DARPins), nucleic acid aptamers, or Kunitz-type domain peptides.

[0082] The term "antibody" is used in the broadest sense herein and refers to a polypeptide or combination of polypeptides that comprises sufficient sequence from the variable region of the immunoglobulin heavy chain and / or sufficient sequence from the variable region of the immunoglobulin light chain, thereby being able to specifically bind to an antigen. "Antibodies" herein encompass various forms and various structures, as long as they exhibit the desired antigen binding activity. "Antibodies" herein include alternative protein scaffolds or artificial scaffolds with transplanted complementary determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which include mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds comprising, for example, biocompatible polymers. Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art that can be used for transplanting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

[0083] The term "antibody" herein includes a typical "four-chain antibody," which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain that, from the N-terminus to the C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain. Furthermore, when the full-length antibody is of the IgE isotype, it optionally also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain that, from the N-terminus to the C-terminus, consists of a light chain variable region (VL) and a light chain constant region (CL). Heavy chains are linked to each other and to each other through disulfide bonds, forming a "Y"-shaped structure. Due to the different amino acid composition and arrangement order of the constant regions of the heavy chains of immunoglobulins, their antigenicity also varies. Based on this, "immunoglobulins" as used herein can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE. Their corresponding heavy chains are μ, δ, γ, α, and ε, respectively. Igs within the same class are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

[0084] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, guanacos, alpacas, sheep, rabbits, mice, rats, or cartilaginous fish (e.g., sharks).

[0085] "Antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0086] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variants (e.g., containing naturally occurring mutations or produced during the production of the preparation, such variants typically existing in small amounts), the individual antibodies comprising the population are identical and / or bind to the same epitope. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "monoclonal" herein should not be interpreted as requiring the antibody or antigen-binding molecule to be produced by any particular method. For example, monoclonal antibodies can be made by a variety of techniques, including but not limited to hybridoma technology, recombinant DNA methods, phage library display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci and other methods known in the art.

[0087] The term "natural antibody" refers to an antibody produced and paired by the immune system of a multicellular organism. The term "engineered antibody" herein refers to a non-natural antibody obtained by techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv), bispecific antibodies, and the like.

[0088] The term "monospecific" is intended to mean having one or more binding sites, wherein each binding site binds to the same epitope on the same antigen.

[0089] The term "multispecific antibody" refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0090] The term "valent" refers to the presence of a specified number of binding sites in an antibody / antigen-binding molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites, respectively, in an antibody / antigen-binding molecule.

[0091] "Full-length antibody" and "intact antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody.

[0092] "Antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not have the entire structure of an intact antibody, but only contain a portion or partial variant of an intact antibody that has the ability to bind to an antigen. "Antigen-binding fragment" or "antibody fragment" herein includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0093] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each containing the heavy and light chain variable domains, along with the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" herein refers to an antibody fragment comprising the light chain VL domain and constant domain (CL) of the light chain, and the VH domain and first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domains bear free thiol groups. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites (the two Fab fragments) and a portion of the Fc region.

[0094] "Fv fragment" is the smallest fragment produced by IgG and IgM that contains a complete antigen binding site. The Fv fragment has the same binding properties and similar three-dimensional binding properties as Fab. The VH and VL chains of the Fv fragment are bound together by non-covalent interactions.

[0095] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain comprising a VL and VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers are composed of repeated GGGGS (SEQ ID NO: 31) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO: 32) may be used, but variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv to form a disulfide-linked Fv (dsFv).

[0096] The term "diabody" refers to a protein whose VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites.

[0097] As used herein, the terms "single domain antibody" (sdAb), "VHH," and "nanobody" have the same meaning and are used interchangeably. They refer to a single domain antibody (sdAb) constructed by cloning the variable region of an antibody heavy chain to construct a single heavy chain variable region (VH), which is the smallest fully functional antigen-binding fragment. The heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0098] The term "minimum recognition unit of an antibody" refers to an antibody that contains only one complementarity determining region (CDR) polypeptide, also known as a hypervariable region polypeptide. The molecular mass of this unit is only about 1% of that of a complete antibody, but it can still bind to the corresponding antigen.

[0099] The term "chimeric antibody" refers to an antibody in which a portion of its light chain and / or heavy chain is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains binding activity to the target antigen. For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody), and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).

[0100] The term "humanized antibody" refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0101] The term "fully human antibody" refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0102] The term "naked antibody" herein refers to an antibody that is not connected, fused or conjugated to another agent or molecule (e.g., a label or drug), peptide or polypeptide. In specific embodiments, naked antibodies expressed by mammalian host cells can be glycosylated by the glycosylation machinery (e.g., glycosylase) of the host cell. In certain embodiments, naked antibodies are not glycosylated when expressed by host cells that do not have their own glycosylation machinery (e.g., glycosylase). In certain embodiments, naked antibodies are intact antibodies, while in other embodiments, naked antibodies are antigen-binding fragments of intact antibodies, such as Fab antibodies.

[0103] The term "variable region" refers to the region of an antibody heavy or light chain involved in binding the antibody to the antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR," and "light chain variable region" is used interchangeably with "VL" and "LCVR." The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, each containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). A single VH or VL domain is sufficient to confer antigen-binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably herein and generally refer to the hypervariable regions (HVRs) of the heavy chain variable region (VH) or light chain variable region (VL). This region is also called the complementarity determining region because it forms a precise spatial complementarity with the antigen epitope. The heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region CDR can be abbreviated as LCDR. The terms "framework region" and "FR region" are used interchangeably herein and refer to those amino acid residues in the heavy chain variable region or light chain variable region of an antibody other than the CDRs. A typical antibody variable region is generally composed of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0104] "CDRs" herein can be annotated and defined using methods known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system, and the tool websites used include but are not limited to the AbRSA website (http: / / cao.labshare.cn / AbRSA / cdrs.php), the abYsis website (www.abysis.org / abysis / sequence_input / key_annotation / key_annotation.cgi), and the IMGT website (http: / / www.imgt.org / 3Dstructure-DB / cgi / DomainGapAlign.cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

[0105] The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of an antibody heavy chain, which is not directly involved in antibody-antigen binding but exhibits effector functions, such as interactions with Fc receptors. It has a more conserved amino acid sequence than the variable domains of antibodies. A "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant regions" include "full-length heavy chain constant regions" and "heavy chain constant region fragments." The former has a structure substantially similar to that of a native antibody constant region, while the latter only comprises "a portion of a full-length heavy chain constant region." For example, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is an IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. For example, a typical "heavy chain constant region fragment" can be selected from the CH1, Fc, or CH3 domains.

[0106] The term "light chain constant region" herein refers to the carboxyl terminal portion of the antibody light chain, which is not directly involved in binding the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

[0107] The term "Fc" herein refers to the antibody carboxyl terminal portion formed by papain hydrolysis of an intact antibody, typically comprising the CH3 and CH2 domains of an antibody. The Fc region includes, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine (residue 447 according to the Kabat numbering system) in the Fc region can be, for example, removed during production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the heavy chain of the antibody, and therefore, the Fc region may or may not include Lys447.

[0108] The term "identity" as used herein can be calculated in the following manner: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0109] The term "nucleic acid" herein includes any compound and / or substance comprising a polymer of nucleotides. Each nucleotide is composed of a base, particularly a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is typically expressed as 5' to 3'. In this article, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), particularly messenger RNA (mRNA), synthetic forms of DNA or RNA, and polymers comprising a mixture of two or more of these molecules. Nucleic acid molecules can be linear or cyclic. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein can contain naturally occurring or non-naturally occurring nucleotides. The example of non-naturally occurring nucleotides includes modified nucleotide bases with derived sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules, which are suitable as vectors for directly expressing the antibodies of the present invention in vitro and / or in vivo, for example, in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, thereby allowing the mRNA to be injected into a subject to produce antibodies in vivo.

[0110] As used herein, the term "vector" includes nucleic acid vectors, such as DNA vectors (such as plasmids), RNA vectors, viruses or other suitable replicons (such as viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous proteins into prokaryotic or eukaryotic cells. The expression vector of the present invention contains polynucleotide sequences and, for example, additional sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoters and enhancer regions) that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of the mRNA produced by gene transcription. These sequence elements include, for example, 5- and 3-untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signal sites to direct the efficient transcription of the genes carried on the expression vector. The expression vector of the present invention may also contain the following polynucleotides that encode markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseothricin.

[0111] The term "host cell" herein refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the original transformed cell and its progeny, regardless of the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as that screened or selected for in the initially transformed cell are included herein.

[0112] The percent identity between the two sequences will vary depending on the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0113] Herein, “n is a real number from 1 to 16” means that n is any real number greater than or equal to 1 and less than or equal to 16.

[0114] In this article Indicates the attachment site.

[0115] The diagrammatic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, wedge and dotted wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

[0116] The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0117] The compounds of the present invention may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so that the compounds of the present invention may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E and Z geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the definition of the compounds of the present invention and mixtures thereof. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl groups, and all of which are within the definition of the compounds of the present invention and mixtures thereof. Compounds of the present disclosure containing an asymmetric atom can be isolated in optically pure or racemic forms. Optically pure forms can be resolved from racemic mixtures or synthesized by using chiral starting materials or reagents.

[0118] The term "substituted" refers to the replacement of any one or more hydrogen atoms on a particular atom by a substituent, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxo (i.e., =O), it means that two hydrogen atoms are replaced. Oxo does not occur on aromatic groups.

[0119] The term "optional" or "optionally" refers to that the event or situation described subsequently may or may not occur, and the description includes that the event or situation occurs and that the event or situation does not occur. For example, an ethyl group is "optionally" substituted with halogen, meaning that the ethyl group may be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). It will be appreciated by those skilled in the art that for any group comprising one or more substituents, any sterically impossible and / or incomposable replacement or substitution pattern will not be introduced.

[0120] In this article, C m -C n , means having an integer number of carbon atoms in the range of mn.

[0121] The term "alkyl" refers to a group of the formula C nH 2n+1 The term "C1-C6 alkyl" is understood to mean a straight-chain or branched saturated hydrocarbon group having 1, 2, 3, 4, 5 or 6 carbon atoms. The alkyl group includes, but is not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; the term "C1-C3 alkyl" refers to an alkyl group containing 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.

[0122] The "C1-C6 alkyl group" described herein may further include a "C1-C3 alkyl group".

[0123] The term "cycloalkyl" refers to a fully saturated carbocyclic ring that exists in the form of a monocyclic, fused, bridged, or spirocyclic ring. The term "C3-C6 cycloalkyl" should be understood to mean a saturated monocyclic, fused, spirocyclic, or bridged ring having 3 to 6 carbon atoms. Specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0124] The term "heterocyclyl" refers to a fully saturated or partially saturated monocyclic, fused, spiro or bridged ring group, which contains 1-5 heteroatoms or heteroatom groups (i.e., heteroatom-containing atomic groups) in its ring atoms, wherein the "heteroatoms or heteroatom groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH- or -NHC(=O)NH-. The term "4-7 membered heterocyclyl" refers to a heterocyclyl group having 4, 5, 6 or 7 ring atoms, and containing 1-3 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms or heteroatom groups in its ring atoms. The term "5-6 membered heterocyclyl" refers to a heterocyclyl having 5 or 6 ring atoms, wherein the ring atoms contain 1-3 heteroatoms or heteroatom groups independently selected from the above. Examples of 4-membered heterocyclyls include, but are not limited to, azetidinyl and oxetanyl; examples of 5-membered heterocyclyls include, but are not limited to, tetrahydrofuranyl, dioxolyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazole, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclyls include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; examples of 7-membered heterocyclyls include, but are not limited to, diazepanyl. "4-7 membered heterocyclyl" may include, but are not limited to, "4-7 membered heterocyclylalkyl," "5-6 membered heterocyclyl," "5-6 membered heterocyclylalkyl," and the like.

[0125] The term "halo" or "halogen" refers to fluorine, chlorine, bromine and iodine.

[0126] The term "treatment" refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathological changes in the treated subject, such as the progression of cancer, autoimmune diseases and viral infections. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, weakening of the disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial relief or complete relief), whether detectable or undetectable. Objects in need of treatment include objects already suffering from a disease or disease, objects susceptible to a disease or disease, or objects intended to prevent a disease or disease. When referring to terms such as slowing down, alleviating, weakening, alleviating, alleviating, etc., their meanings also include situations such as elimination, disappearance, and non-occurrence.

[0127] The term "effective amount" refers to an amount of a therapeutic agent that, when administered alone or in combination with another therapeutic agent to a cell, tissue, or subject, is effective in preventing or ameliorating a disease symptom or the progression of that disease. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, e.g., to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or alleviation of such a condition. When an active ingredient is administered alone to a subject, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

[0128] The term "subject" refers to an organism that is being treated for a particular disease or condition as described herein. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals, being treated for a disease or condition.

[0129] The amount of a compound of the disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by one skilled in the art based on their own knowledge and this disclosure.

[0130] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0131] The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable acid or base, including a salt formed between a compound and an inorganic acid or organic acid, and a salt formed between a compound and an inorganic base or an organic base.

[0132] The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or their salts and a pharmaceutically acceptable excipient. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present disclosure to an organism.

[0133] The term "pharmaceutically acceptable excipient" refers to an excipient that is non-irritating to organisms and does not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art and include, for example, carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like.

[0134] The word "comprise" or "comprises" and its English variations such as comprises or comprising are to be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0135] The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but where one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as 2 H. 3 H. 11 C. 13 C. 14 C. 13 N. 15 N. 15 O. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F. 123 I. 125 I and 36 Cl et al.

[0136] Certain isotopically labeled compounds of the present disclosure (e.g., 3 H and 14 C-labeled) can be used in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 (i.e. 14 C) isotopes are particularly preferred due to their ease of preparation and detectability. Positron emitting isotopes, such as 15 O. 13 N. 11 C and 18 F can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by following procedures analogous to those disclosed in the Schemes and / or Examples below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0137] The pharmaceutical compositions of the present disclosure may be suitable for parenteral administration, such as sterile solutions, suspensions, or lyophilized products in suitable unit dosage forms. For example, the pharmaceutical compositions of the present disclosure may be in the form of sterile aqueous injection solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions of the present disclosure may be administered in other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution.

[0138] The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining the same with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include, but are not limited to, the examples disclosed herein.

[0139] The chemical reactions of the disclosed embodiments are carried out in suitable solvents that are compatible with the chemical transformations disclosed herein and the reagents and materials required. To obtain the compounds disclosed herein, it may sometimes be necessary for those skilled in the art to modify or select synthetic steps or reaction schemes based on existing embodiments.

[0140] An important consideration in synthetic route planning in the art is the selection of appropriate protecting groups for reactive functional groups (e.g., amino and carboxyl groups in the present disclosure). For example, reference may be made to Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in the present disclosure are hereby incorporated into the present disclosure in their entirety.

[0141] As used herein, the term "cancer" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. As used herein, the term "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used herein.

[0142] The term "EC50" herein refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of its maximal effect is observed and can be measured by methods known in the art.

[0143] The term "LIV-1" in this article, also written as LIV1 or LIV 1, is a zinc transporter, also known as SLC39A6 or ZIP6. Its encoding gene is located on chromosome 18q12.2. It is a multi-transmembrane protein with zinc transporter and metalloproteinase activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings that are understood by those of ordinary skill in the art.

[0145] Figure 1 FACS detection of the binding reaction between LIV1 ADC and monoclonal antibodies and MCF-7 cells.

[0146] Figure 2 In vitro proliferation inhibition test of ADC on tumor cells.

[0147] Fig. 3 Tumor growth curve of OVCAR3 subcutaneous tumor model.

[0148] Figure 4 Body weight change curve of OVCAR3 subcutaneous tumor model mice.

[0149] Figure 5 Tumor growth curve of NCI-H838 subcutaneous tumor model.

[0150] Figure 6 Body weight change curve of NCI-H838 subcutaneous tumor model mice.

[0151] Figure 7 Pharmacokinetic curves of ADC in mice.

[0152] Figure 8 Tumor growth curve of MCF-7 subcutaneous tumor model.

[0153] Figure 9 Body weight change curve of MCF-7 subcutaneous tumor model mice. DETAILED DESCRIPTION

[0154] The present invention will be further described below with reference to specific examples, and the advantages and features of the present invention will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0155] The embodiments of the present invention are merely exemplary and do not limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, and such modifications and replacements shall fall within the scope of protection of the present invention.

[0156] Example 1. Construction and production of anti-human LIV-1 antibodies

[0157] The heavy and light chain variable region sequences and heavy chain constant region / light chain constant region sequences of anti-human LIV-1 monoclonal antibodies (LIV-1-Ab-1, LIV-1-Ab-2, LIV-1-Ab-3, Ladiratuzumab (i.e., LIV-1-Ab-L or Ladiratuzumab-hIgG1)) and an anti-FITC isotype control antibody are shown in Table 1 below. The CDR sequences classified according to the Kabat system are shown in Table 2 below. The nucleic acid sequences encoding the VH and VL of these antibodies were recombined into the expression vector pTT5 containing a signal peptide (MGWSWILLFLLSVTAGVHS, SEQ ID NO: 33) and heavy chain constant region / light chain constant region sequences to generate recombinant plasmids expressing VH-CH / VL-CL. The plasmid and transfection reagent PEI (Polysciences, Catalog No. 24765-1) were added to OPTI-MEM (Gibco, Catalog No. 11058021), mixed thoroughly, and allowed to stand for 15 minutes. The cells were then added to Expi293F cells (Thermofisher, Catalog No. A14527) and incubated in a shaking incubator at 37°C with 5% CO2 and 120 rpm. On the second day of transfection, OPM-293 ProFeed (Shanghai Aopuma, Catalog No. F081918-001) and 6 g / L glucose (Sigma, Catalog No. G7528) were added. On the sixth day of transfection, the cell supernatant was collected and purified using Protein A (GE, Catalog No. 28985254). The eluted sample was dialyzed into PBS (pH 7.4) to obtain anti-human LIV-1 monoclonal antibodies and anti-FITC isotype control antibodies. Ladiratuzumab is a positive control human LIV1 antibody, and the antibody sequence is derived from US20200165335A.

[0158] Table 1 Heavy and light chain variable region sequences of anti-human LIV-1 monoclonal antibodies and isotype control antibodies

[0159] Table 2 Anti-human LIV-1 monoclonal antibody CDR sequences (Kabat classification)

[0160] Example 2: Screening of monoclonal antibodies against drug 001

[0161] Monoclonal antibodies against drug 001 were produced by immunizing mice. The immunogen was drug 001 (structure shown below, preparation method see Example 39 of WO2023020605A1). Hybridoma cells were prepared from spleen lymphocytes of mice with high antibody titers in serum and titers approaching a plateau. Positive hybridoma clones were screened by ELISA and other conventional methods in the art. These clones can specifically recognize antibody-drug conjugates conjugated to drug 001. The hybridoma clones were cultured in serum-free cell culture to further prepare antibodies, which were then purified to obtain anti-drug 001 antibodies.

[0162] Example 3: Preparation of Antibody-Drug Conjugates

[0163] 3.1 Preparation / Source of Drug-Linker Compounds

[0164] The structure of drug-linker 1 is shown below. Its preparation refers to patent document WO2023217227A1 (Example 39).

[0165] The structure of drug-linker 2 is shown below, and it was purchased from MedChemExpress (MCE) with the product number HY-15575.

[0166] 3.2 Conjugation of Antibodies and Drug-Linkers

[0167] The antibody prepared in Example 1 was dialyzed to a 20mM PB, 150mM NaCl, 1mM EDTA solution (pH 6.5). Eight times the volume of 10mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) was added to the antibody solution, and the mixed solution was reduced on a thermostatic metal shaker at 37°C for 2.5 hours. A 15-fold molar equivalent of the drug-linker 1 compound was dissolved in DMSO and added to the reaction system. The reaction solution was coupled at 25°C for 6 hours. The reaction product was desalted on a G25 column and exchanged to phosphate buffer (PBS) to remove unreacted free small molecule toxins to obtain an antibody-drug conjugate. SEC and LC-MS were used to analyze the purity and DAR value of the ADC product.

[0168] The antibody prepared in Example 1 was dialyzed to a 20 mM PB, 150 mM NaCl, 1 mM EDTA solution (pH 6.5). A 2.4-fold volume of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) was added to the antibody solution, and the mixed solution was reduced on a thermostatic metal shaker at 37°C for 3 h. A 10-fold molar equivalent of the aforementioned drug-linker 2 compound (purchased from MCE, Cat. HY-15575) (dissolved in DMSO to 20 mg / ml) was added to the reaction system, and the reaction solution was coupled at 25°C for 12 h. The reaction product was desalted on a G25 column and exchanged to phosphate-buffered saline (PBS) buffer to remove unreacted free small molecule toxins, yielding an antibody-drug conjugate. The ADC product was analyzed for purity and DAR value using SEC and LC-MS.

[0169] SEC purity analysis: SEC-HPLC method was used to analyze the protein sample to be tested, to characterize the molecular size homogeneity of the recombinant protein, and to measure the purity of the recombinant protein. The HPLC used in this method is an Agilent 1260, and the chromatographic column is a TSKgel G3000SWXL from Tosoh Bioscience, with a mobile phase of 200mM phosphate buffer pH 7.0 containing 10% isopropanol, a detection temperature of 25°C, a flow velocity of 0.5mL / min, a detection wavelength of 280nm, and a target protein loading of 50μg, with an analysis time of 40 minutes. To SEC-HPLC data, manual integration was used to analyze the chromatogram, and protein purity was calculated according to the area normalization method. The main peak was considered to be a monomer, the chromatographic peak before the main peak was called an aggregate, and the chromatographic peak after the main peak was called a fragmentation body.

[0170] DAR value determination: The DAR value of the ADC molecule was measured using ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS). First, the ADC molecule to be tested was treated with PNGase F (NEB#P0705L) to remove the N-glycan modification, and then treated with dithiothreitol (DTT, Sigma#646563) and incubated at 37°C for 1 hour to reduce it to light and heavy chains. Then, it was analyzed using a Thermo Vanquish UHPLC-Q Exactive Plus mass spectrometry system. 2 μg of protein was injected onto a Waters ACQUITY Protein BEH molecular exclusion chromatography column. The mobile phase was an aqueous solution containing 0.1% formic acid, 0.05% TFA, and 25% acetonitrile. The flow rate was 0.2 mL / min and the analysis time was 30 min. The mass spectrometer was a Thermo Q Exactive Plus. The main mass spectrometry parameters were as follows: spray voltage 3.8 kV, capillary heating temperature 300°C, sheath gas flow rate 35 arb, parent ion scan range 800-3000, etc. Finally, the mass spectrometry data analysis software Biopharma Finder was used. 4.1. Deconvolution processing is performed using the Respect algorithm to calculate the molecular weight information of the light and heavy chain mass spectrum peaks and the mass spectrum response signals of each component, respectively, and thus calculate the DAR value of the ADC sample to be tested.

[0171] Table 3 Preparation, DAR value and SEC purity of antibody drug conjugates

[0172] Example 4: Flow cytometry (FACS) assay to detect the binding activity of LIV1 ADC and monoclonal antibodies to endogenous MCF-7 cells

[0173] Human breast cancer MCF-7 cells expressing human LIV-1 protein (from ATCC, LIV-1 highly expressed) were cultured in T-75 cell culture flasks until the logarithmic growth phase, the culture supernatant was discarded by centrifugation, the cell pellet was washed once with PBS, and seeded in a 96-well plate at a density of 2E5 cells / well. The cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. LIV1 ADC and monoclonal antibody were diluted to 100 nM, and 5-fold serial dilutions were made into 8 points. 50 μl of secondary antibody (Alexa Fluor 5000) was added to each well, resuspended, incubated at 4°C for 1 hour, washed twice with PBS, and 50 μl of secondary antibody (Alexa Fluor 5000) was added to each well. 647AffiniPure Goat Anti-Human IgG (H+L) (purchased from Jackson Immuno, catalog number: 109-605-088)), incubated at 4°C for 1 hour, washed twice with PBS, and analyzed by FACS (FACS Canto TM , purchased from BD Company) detection and analysis.

[0174] The results are shown in Table 4 and Figure 1, indicating that LIV1 ADC can specifically bind to MCF-7 and has similar binding activity to the monoclonal antibody, and both exhibit similar binding ability to the positive control ADC. The data in the table are MFI values ​​and EC values. 50 (nM).

[0175] Table 4. FACS detection of binding activity of LIV1-ADC and monoclonal antibodies to endogenous MCF-7 cells

[0176] Example 5: Flow cytometry (FACS) assay to detect the endocytosis of LIV1 ADC and monoclonal antibodies in endogenous cells MCF-7 and OVCAR-3

[0177] LIV-1 high-expressing cells MCF-7 and LIV-1 expressing OVCAR-3 cells in the logarithmic growth phase were collected and seeded into 96-well plates at a density of 2E5 cells / well. After washing with PBS once, LIV1 ADC and monoclonal antibody (final concentration of 100nM) were added respectively and incubated at 4°C for 1 hour. The control group was placed at 4°C, and the endocytosis group was placed at 37°C. After incubation for 2 hours, the cells were washed with PBS three times and the secondary antibody: Alexa Incubate with 647AffiniPure Goat Anti-Human IgG (H+L) (Jackson Immuno, Cat. No. 109-605-088) at 4°C for 1 hour, wash twice with PBS, and analyze by FACS (FACS Canto™, BD Biosciences). Endocytosis efficiency = (control group MFI - endocytosis group MFI) / control group MFI * 100%.

[0178] The results are shown in Table 5, demonstrating that the LIV1 ADC exhibited varying degrees of endocytosis in endogenous MCF-7 and OVCAR-3 cells, with similar endocytosis activity to that of the monoclonal antibody and comparable to the positive control. The data in the table represent MFI values ​​and endocytosis efficiency.

[0179] Table 5. FACS detection of the endocytic effect of LIV1 ADC and mAb on endogenous cells MCF-7 and OVCAR-3

[0180] Example 6: In vitro proliferation inhibition test of ADC on tumor cells

[0181] Cells and Materials: Human ovarian cancer cells OVCAR3 were purchased from ATCC. RPMI-1640 medium (Gibco #A10491-01), penicillin-streptomycin (Gibco #15140-122), and 0.25% trypsin-EDTA (Gibco #25200-056) were purchased from Gibco (USA). Human insulin (Solarbio #40112ES25) was purchased from Solarbio. 96-well plates (Greiner Bio-one #655098) were purchased from Corning Incorporated (USA). Cell-Titer Glo reagent (Promega #G7568) was purchased from Promega (USA).

[0182] Cell culture: OVCAR3 cells were cultured in RPMI-1640 medium supplemented with 20% fetal bovine serum, 10 μg / mL human insulin, and 1% penicillin-streptomycin. All cells were cultured at 37°C in 5% CO2. Cells in the logarithmic growth phase were used for experiments.

[0183] Cell proliferation activity assay: The inhibitory activity of ADCs against OVCAR3 cell proliferation was assessed using Cell-Titer Glo reagent. Cells were dissociated from the cell culture flask and resuspended in fresh culture medium to adjust the cell density. OVCAR3 cells were seeded at 1500 cells / 90 μL / well in a 96-well plate and cultured overnight at 37°C, 5% CO2. The ADC was diluted to 1000 nM in complete culture medium and serially diluted three-fold over eight steps. Then, 10 μL of the diluted ADC solution was transferred to the 96-well plate, resulting in a starting and ending ADC concentration of 100 nM. The 96-well plate was incubated at 37°C, 5% CO2 for 5 days. Cell-Titer Glo reagent was then added, and cell viability was assessed. Negative and positive controls were set up as bottom and top sections, respectively. The negative control section consisted of the same volume of culture medium as the experimental group, without cells. The positive control section consisted of the same volume of culture medium as the experimental group, without the test antibody.

[0184] Data Analysis:

[0185] Calculate the inhibition percentage (% Inhibition) and fit to obtain the IC of the compound 50 .

[0186] Percent inhibition (% Inhibition) = 1-100% x (Signal-Bottom) / (Top-Bottom).

[0187] Signal refers to the signal value of the experimental group, Bottom refers to the average signal value of the negative control group, and Top refers to the average signal value of the positive control group.

[0188] Experimental results:

[0189] The experimental results are shown in Figure 2. ADC-2, ADC-3, ADC-7, ADC-8 and the positive reference Ladiratuzumab-MMAE all have strong cell inhibitory effects.

[0190] Example 7. Evaluation of drug efficacy in OVCAR3 subcutaneous tumor model

[0191] Experimental reagents:

[0192] Human ovarian cancer OVCAR3 cells: ATCC

[0193] RPMI-1640 culture medium: Gbico; Cat No.: A104910

[0194] Fetal bovine serum: Gibco; Cat No.: 10099-141C

[0195] Bovine insulin: Yeasen, 40107ES60

[0196] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[0197] D-PBS (Calcium- and magnesium-free phosphate buffered saline): Hyclone, Cat. No.: SH30256.01

[0198] Matrigel:Corning,Cat.No.:356237

[0199] Experimental methods:

[0200] Animal information: Balb / c nude female mice, 5-6 weeks old, weighing approximately 14-20 g, were purchased from Beijing Weitonglihua Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to a standard certified commercial laboratory diet and free drinking water.

[0201] Cell Culture: Human ovarian cancer OVCAR3 cells were cultured in RPMI-1640 (cell culture medium) supplemented with 20% fetal bovine serum, 1% Pen-Strep, and 10 μg / ml bovine insulin in a 37°C, 5% CO2 incubator. Cells were routinely digested and passaged weekly using 0.25% trypsin-EDTA. Cells were harvested and counted when cell saturation reached 80%-90% and the desired number of cells was reached.

[0202] Cell inoculation: 0.2 ml / (containing 1×10 7 OVCAR3 cell suspension (RPMI-1640:Matrigel, 1:1 volume ratio) was subcutaneously inoculated in the axilla of each mouse. On day 21 after cell inoculation, mice were randomly divided into groups based on tumor volume for dosing, with grouping designated Day 0.

[0203] Dosing: ADC-2, ADC-3, ADC-1, Ladirazutumab-MMAE, and controls ADC-7 and ADC-8 were administered at a single intraperitoneal dose of 1 mg / kg, 3 mg / kg, or 10 mg / kg. Six mice were included in each group.

[0204] Tumor measurements and experimental parameters:

[0205] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[0206] The anti-tumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[0207] Experimental results:

[0208] In the mouse subcutaneous transplant tumor OVCAR3 model, ADC-2, ADC-3, and ADC-1 of the present invention had a significant inhibitory effect on tumor growth at a single intraperitoneal injection (IP) of 1 mpk, 3 mpk, and 10 mpk (P < 0.0001); and at doses of 1 mpk and 3 mpk, they were significantly better than Ladirazutumab-MMAE (P < 0.001, P < 0.05); at 10 mpk, the efficacy of each molecule was similar; in addition, at a dose of 1 mpk, ADC-3 was significantly better than ADC-2 (P < 0.0001); while the tumor inhibition effects of ADC-7 and ADC-8 were significantly weaker than the corresponding ADC molecules (P < 0.0001). The examples of the present invention were not found to affect the weight of mice at the doses tried, nor did they cause any mouse deaths, and the mice were able to tolerate them. See Table 6, Figures 3 and 4.

[0209] Table 6. Tumor volume of OVCAR3 subcutaneous tumor model

[0210] Note: The table shows only the mean tumor volume without standard error, which is shown in the figure.

[0211] Example 8. Evaluation of drug efficacy in NCI-H838 subcutaneous tumor model

[0212] Experimental reagents:

[0213] Human lung cancer NCI-H838 cells: Kebai

[0214] RPMI-1640 culture medium: Gbico; Cat No.: 61870-036

[0215] Fetal bovine serum: Gibco; Cat No.: 10099-141C

[0216] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[0217] D-PBS (Calcium- and magnesium-free phosphate buffered saline): Hyclone, Cat. No.: SH30256.01

[0218] Matrigel:Corning,Cat.No.:356237

[0219] Experimental methods:

[0220] Animal information: B-NDG mice, female, 5-6 weeks old, weighing approximately 14-20 g, were purchased from Biocytogen. The mice were housed in an SPF-grade environment with individual ventilation in each cage. All animals had free access to standard certified commercial laboratory diet and free drinking water.

[0221] Cell Culture: Human lung cancer NCI-H838 cells were cultured in RPMI-1640 (cell culture medium) supplemented with 10% fetal bovine serum and 1% Pen-Strep in a 37°C, 5% CO2 incubator. Twice weekly, cells were routinely digested and passaged using 0.25% trypsin-EDTA. When cell saturation reached 80%-90% and the required number of cells was reached, cells were harvested and counted.

[0222] Cell inoculation: 0.1 ml / (containing 1×10 7 NCI-H838 cell suspension (RPMI-1640:Matrigel, 1:1 volume ratio) was subcutaneously inoculated in the axilla of each mouse. On day 21 after cell inoculation, mice were randomly divided into groups based on tumor volume for dosing, with group assignment being designated Day 0.

[0223] Dosing: Considering that the clinical doses of ADCs loaded with MMAE and topoisomerase inhibitors typically differ by 2-3 fold, for example, the recommended dose of RC48 (HER2-MMAE ADC) is 2.0 mg / kg and that of DS-8201 (HER2-dxd ADC) is 5.4 mg / kg, to evaluate the in vivo efficacy of different ADC types at clinical doses, ADC-2, ADC-3, ADC-1, and the control ADC-7 in the Examples were administered at doses of 1.7 mg / kg, 5 mg / kg, or both. Ladirazutumab-MMAE was administered at a dose of 3 mg / kg. All doses were administered intraperitoneally every 8 days for a total of four doses. Six mice were included in each group.

[0224] Tumor measurements and experimental parameters:

[0225] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[0226] The anti-tumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[0227] Experimental results:

[0228] In the NCI-H838 subcutaneous xenograft mouse model, ADC-2, ADC-3, and ADC-1, each administered intraperitoneally (IP) at 1.7 mpk and 5 mpk Q8D*4, significantly inhibited tumor growth (P<0.0001). At a dose of 1.7 mpk, ADC-2 was significantly superior to ADC-1 (P<0.05). At a dose of 5 mpk, ADC-2 and ADC-3 had similar efficacy, both significantly superior to ADC-1 and Ladirazutumab-MMAE (P<0.01, P<0.001). ADC-7, however, had a significantly weaker tumor inhibition effect than the corresponding ADC molecules (P<0.0001). The examples of the present invention were not found to affect mouse body weight or cause any mouse mortality at the doses tested, demonstrating that the mice were well tolerated. See Table 7, Figures 5, and 6.

[0229] Table 7. Tumor volume of H838 subcutaneous tumor model

[0230] Note: The table shows only the mean tumor volume without standard error, which is shown in the figure.

[0231] Example 9. Pharmacokinetic study of ADC drugs in mice

[0232] Experimental design

[0233] Three Balb / c mice were intravenously injected with each drug molecule at a dose of 5 mg / kg. Blood samples were collected at 0 hours before dosing and 15 minutes, 2 hours, 8 hours, 24 hours (Day 1), 72 hours (Day 3), 120 hours (Day 5), 168 hours (Day 7), 240 hours (Day 10), 336 hours (Day 14), and 504 hours (Day 21) after dosing. ELISA was used to determine the concentrations of total antibodies and ADC in mouse plasma.

[0234] Analytical methods

[0235] Total antibody detection method: hLIV-1 antigen protein (purchased from ACRO, catalog number: LV1-H5223) was diluted to 1 μg / mL in phosphate-buffered saline (Cat: AR0030, Boster Biotech). 100 μL was added to each well of a high-affinity 96-well plate. The plate was sealed with a film and coated overnight at 4°C. After coating, the 96-well plate was removed from the film, and the liquid in the wells was shaken dry. 300 μL of PBST was added to each well and the plate was gently shaken to remove the liquid. The plate was then washed three times. Then, 250 μL of 1% BSA-PBST solution was added to each well of the plate. The plate was placed on an IKA plate shaker (brand: IKA, model: MS3) and shaken at 500 rpm at room temperature for 1 hour for blocking. A standard curve with a concentration range of 2500–19.531 ng / mL (control molecule: 5000–39.062 ng / mL) was prepared using blank mouse plasma (purchased from IPHASE, Catalog No. 032E13.12). After blocking the 96-well plate and washing it three times, the standard curve samples were diluted 100-fold with 1% BSA-PBST solution and 100 μL was added to each well. The plate was incubated on a microplate shaker at 500 rpm for 2 hours at room temperature. After washing the plate three times, 100 μL of Goat Anti-Human IgG Fc HRP (Cat: 109-035-170, Jackson) diluted 500-fold with 1% BSA-PBST solution was added to each well and incubated on a microplate shaker at 500 rpm for 1 hour at room temperature. After incubation, remove the 96-well plate, wash it three times, and add 100 μL of TMB (brand: 34029, purchased from Thermo) colorimetric solution to each well. Develop the color at room temperature in the dark for 7-10 minutes. Terminate the reaction with 1 M H₂SO₄ and read the results with a microplate reader (model Multiskan FC, brand Thermo) at a wavelength of 450 nm to 630 nm. The collected data were analyzed using a four-parameter fitting method.

[0236] Antibody-drug conjugate detection method: Anti-drug 001 antibody (produced in Example 2) was diluted to 1 μg / mL in phosphate buffered saline. A control molecule, Mouse Anti-MMAE Antibody, Mouse IgG1 (Cat. No. MME-M5252, purchased from ACRO), was coated at 100 μL per well in a high-affinity 96-well plate. The plate was sealed with a film and coated overnight at 4°C. The coated 96-well plate was removed and the film removed. After the wells were shaken dry, 300 μL of PBST was added to each well. The plate was gently shaken and the liquid was removed. The plate was washed three times. Then, 250 μL of 1% BSA-PBST solution was added to each well of the plate. The plate was shaken at 500 rpm at room temperature for 1 hour for blocking. A standard curve was prepared using blank mouse plasma with a concentration range of 2500–19.531 ng / mL (control molecule 5000–39.062 ng / mL). After removing the blocked 96-well plate and washing it three times, dilute the standard curve sample 100-fold with 1% BSA-PBST solution, and add 100 μL to each well. Place the loaded 96-well plate on an ELISA plate shaker at 500 rpm and incubate at room temperature for 2 hours. After removing the incubated 96-well plate and washing it three times, add 100 μL of hLIV-1 antigen protein diluted to 0.5 μg / mL with 1% BSA-PBST solution to each well, and incubate on an ELISA plate shaker at 500 rpm and incubate at room temperature for 1 hour. After removing the incubated 96-well plate and washing it three times, add His Tag Antibody-HRP (Cat. No. A00612, purchased from Genscript) diluted 7K-fold with 1% BSA-PBST solution, and incubate on an ELISA plate shaker at 500 rpm and incubate at room temperature for 1 hour. After incubation, remove the 96-well plate, wash three times, and add 100 μL of TMB colorimetric solution to each well. Develop at room temperature in the dark for 7-10 minutes. Terminate the reaction with 1 M H₂SO₄ and read the plate on a microplate reader at a wavelength of 450-630 nm. Collected data were analyzed using four-parameter fitting.

[0237] in conclusion

[0238] As can be seen from the curves in Figure 7, the half-life and blood drug exposure of the total antibodies and ADCs of ADC-1, ADC-2 and ADC-3 are similar, which are significantly better than the control molecule Ladiratuzumab-MMAE.

[0239] Example 10. Evaluation of drug efficacy in MCF-7 subcutaneous tumor model

[0240] Experimental reagents:

[0241] Human breast cancer MCF-7 661 Cells: MCF-7 was originally purchased from ATCC. Its growth rate is slightly slower. 661Primary cells extracted and STR identification has been performed

[0242] RPMI-1640 culture medium: Gbico; Cat No.: A104910

[0243] Fetal bovine serum: Gibco; Cat No.: 10099-141C

[0244] Bovine insulin: Yeasen, 40107ES60

[0245] 0.25% Trypsin-EDTA: Gibco, Cat No.: 25200-072

[0246] D-PBS (Calcium- and magnesium-free phosphate buffered saline): Hyclone, Cat. No.: SH30256.01

[0247] Matrigel:Corning,Cat.No.:356237

[0248] Estrogen patch: 0.72mg / Tablet, 90 days

[0249] Experimental methods:

[0250] Animal information: NPG mice, female, 6-7 weeks old, weighing approximately 14-20 g, were purchased from Beijing Weitongda Biotechnology Co., Ltd. The mice were housed in an SPF-grade environment with separate ventilation in each cage. All animals had free access to standard certified commercial laboratory diet and free drinking water.

[0251] Cell culture: human breast cancer MCF-7 661 Cell lines were cultured in RPMI-1640 (cell culture medium) supplemented with 20% fetal bovine serum, 1% Pen-Strep, and 10 μg / ml bovine insulin in a 37°C, 5% CO2 incubator. Cells were routinely digested and passaged once a week using 0.25% trypsin-EDTA. Cells were harvested and counted when cell saturation reached 80%-90% and the desired number was reached.

[0252] Cell seeding: human breast cancer cells MCF-7 661 (1×10 7 100 μl of 1640 (matrigel = 1:1) was inoculated subcutaneously on the right back of NPG mice treated with estrogen patches. 29 days after inoculation, the tumor grew to approximately 206 mm. 3 Afterwards, the weight, tumor size, and size were eliminated, and the mice were randomly divided into a blank control group according to the tumor volume. The day of grouping was designated as Day 0.

[0253] Dosing: ADC-2, ADC-3, and ladirazutumab-MMAE were administered intraperitoneally at a dose of 1 mg / kg, 3 mg / kg, or 10 mg / kg. The 1 mg / kg and 3 mg / kg doses were administered every four days for a total of four doses during the experimental period. The 10 mg / kg group was administered on days 0 and 4 for a total of two doses during the experimental period. Six mice were included in each group.

[0254] Tumor measurements and experimental parameters:

[0255] Tumor diameter was measured twice a week using a vernier caliper. Tumor volume was calculated using the formula: V = 0.5 axb 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of mice was measured twice a week.

[0256] The anti-tumor efficacy of the compound was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume of a treatment group at the end of dosing - mean tumor volume of the treatment group at the start of dosing) / (mean tumor volume of the solvent control group at the end of treatment - mean tumor volume of the solvent control group at the start of treatment)] x 100%.

[0257] Experimental results:

[0258] MCF-7 subcutaneous transplanted tumor in mice 661 In the (LIV-1 high expression) model, ADC-2 and ADC-3 according to the present invention significantly inhibited tumor growth at intraperitoneal injections (IP) of 1, 3, and 10 mpk (P<0.0001). At a dose of 1 mpk, ADC-2 was significantly superior to Ladirazutumab-MMAE (P<0.05). At a dose of 3 mpk, the tumor inhibition rate of ADC-2 was superior to that of Ladirazutumab-MMAE, and the tumor inhibition rate of ADC-3 was comparable to that of Ladirazutumab-MMAE.

[0259] The examples of the present invention did not affect the body weight of mice at the doses tested, nor did they cause any mortality in mice, indicating that the mice could tolerate the treatment. See Table 8 and Figures 8-9.

[0260] Table 8. Tumor volume of MCF-7 subcutaneous tumor model

[0261] Note: The standard error was calculated by dividing the standard deviation by the square root of the sample size, ie, by the Excel formula SEM=STDEV(tumor volume of each group) / SQRT(n, n=number of animals).

Claims

1. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, with the general structural formula Pc-(L-D) n , Wherein, D is a cytotoxic drug; L is a linker unit; Pc is an antibody or an antigen-binding fragment thereof that specifically binds to LIV-1; The antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, or / and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the HCDR1-3 or / and the LCDR1-3 are: (1) The HCDR1-3 are SEQ ID NO: 13-15; or / and the LCDR1-3 are SEQ ID NO: 16-18; (2) The HCDR1-3 are SEQ ID NO: 19-21; or / and the LCDR1-3 are SEQ ID NO: 22-24; (3) The HCDR1-3 are SEQ ID NO: 25-27; or / and the LCDR1-3 are SEQ ID NO: 28-30; Or, The HCDR1-3 or / and the LCDR1-3 have a sequence with at least 80% identity, or a sequence with at most 3 insertions, deletions or substitution mutations, compared to each CDR of the HCDR1-3 and LCDR1-3 in any one of groups (1)-(3); Optionally, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity; And, n is a real number from 1 to 16.

2. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises a heavy chain variable region (VH) or / and a light chain variable region (VL), and the heavy chain variable region or / and the light chain variable region are selected from the following: (1) The heavy chain variable region is the sequence shown in SEQ ID NO: 1, or / and the light chain variable region is the sequence shown in SEQ ID NO: 2; (2) The heavy chain variable region is the sequence shown in SEQ ID NO: 3, or / and the light chain variable region is the sequence shown in SEQ ID NO: 4; (3) The heavy chain variable region is the sequence shown in SEQ ID NO: 5, or / and the light chain variable region is the sequence shown in SEQ ID NO: 6; Or, The heavy chain variable region or / and the light chain variable region have a sequence with at least 80% identity, or a sequence with at most 3 insertions, deletions or substitution mutations, compared to the heavy chain variable region or / and the light chain variable region in any one of the above groups (1)-(3); Preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

3. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-2, wherein, The antibody or its antigen-binding fragment comprises a heavy chain constant region sequence and / or a light chain constant region sequence; optionally, the heavy chain constant region and / or the light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment comprises CH1, hinge region, CH2, CH3 or Fc; optionally, the heavy chain constant region is selected from human or murine IgG1, IgG2, IgG3 or IgG4 constant regions, and the light chain constant region is selected from human or murine kappa constant region or lambda constant region; optionally, the antibody or its antigen-binding fragment comprises a complete heavy chain and a light chain, the heavy chain consists of the VH and the heavy chain constant region, and the heavy chain constant region has the sequence shown in SEQ ID NO:11, and the light chain consists of the VL and the light chain constant region, and the light chain constant region has the sequence shown in SEQ ID NO:

12.

4. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein, The antibody or its antigen-binding fragment described above is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; and / or, (3) a fully human antibody or a fragment thereof; Optionally, the antibody or its antigen-binding fragment is selected from monoclonal antibody, polyclonal antibody, natural antibody, engineered antibody, monospecific antibody, multispecific antibody (such as bispecific antibody), monovalent antibody, multivalent antibody, full-length antibody, antibody fragment, naked antibody, conjugated antibody, humanized antibody, fully human antibody, Fab, Fab’, F(ab’)2, Fd, Fv, scFv, diabody or single domain antibody; optionally, the antibody or its antigen-binding fragment is selected from one or more of F(ab)2, Fab’, Fab, Fv, scFv, bispecific antibody, nanobody and the minimum recognition unit of the antibody.

5. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, wherein, The cytotoxic drug is selected from tubulin inhibitors, DNA damaging agents or topoisomerase inhibitors. The tubulin inhibitors include dolastatin drugs, auristatin drugs, maytansine drugs, Tubulysins drugs and cryptomycins drugs. The DNA damaging agents include PBD drugs. The topoisomerase inhibitors include camptothecin drugs.

6. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein, The cytotoxic drug is selected from the compounds represented by formula (D-I), Wherein, R 1 、R 2 together with the atoms to which they are attached form a 5- or 6-membered heterocyclic group containing 1 or 2 oxygen atoms as ring atoms, said 5- or 6-membered heterocyclic group being optionally substituted by one or more D atoms; R 4 selected from H or C1-C3 alkyl; R 5 selected from H, halogen, CN, =O, OH, NH2 or C1-C3 alkyl; R 6 selected from H or C1-C3 alkyl; R 7 selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, wherein the C1-C3 alkyl or C3-C6 cycloalkyl is optionally substituted by D, halogen, CN, ═O, OH, NH2 or C1-C3 alkyl.

7. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 6, wherein, Said R 1 , R 2 together with the atoms to which they are attached form 8. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 6 or 7, wherein, R 4 、R 5 、R 6 are each selected from H.

9. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 6-8, wherein, R 7 selected from cyclopropyl.

10. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 6-9, wherein, The compound represented by formula (D-I) is selected from one of the following compounds:

11. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, wherein, The cytotoxic drug is selected from 12. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein, The linker unit L is selected from wherein, m1 is selected from the integers 2 to 8, and L 1 is selected from peptide residues composed of 1 to 8 amino acids, and the peptide residues are further optionally substituted by one or more substituents selected from halogen, CN, =O, C1-C6 alkyl, OH, O(C1-C6 alkyl), NH2, NH(C1-C6 alkyl), N(C1-C6 alkyl)2, C3-C6 cycloalkyl, and 4-7 membered heterocyclic group, and L 2 is selected from The a-end of the linker unit L is covalently linked to Pc, and the b-end is covalently linked to the cytotoxic drug.

13. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 12, wherein, The L 1 is a peptide residue of Val-Cit or Gly-Gly-Phe-Gly.

14. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-13, wherein, The linker unit L is Its a-end is covalently linked to Pc, and its b-end is covalently linked to the cytotoxic drug.

15. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein, The antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following antibody-drug conjugates or pharmaceutically acceptable salts thereof:

16. An isolated nucleic acid molecule encoding the antibody or its antigen-binding fragment in the antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of claims 1-4.

17. An expression vector comprising the nucleic acid molecule according to claim 16.

18. A host cell comprising the nucleic acid molecule according to claim 16, and / or the expression vector according to claim 17; preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from mammalian cells, yeast cells, insect cells, Escherichia coli and / or Bacillus subtilis; more preferably, the host cell is selected from Expi293 or CHO cells.

19. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1-15 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

20. The pharmaceutical composition according to claim 19, wherein the pharmaceutical composition further comprises other therapeutic agents; preferably, each component in the pharmaceutical composition, such as the antibody-drug conjugate and the other therapeutic agents, can be independently packaged or packaged in combination.

21. Use of the antibody-drug conjugate according to any one of claims 1-15 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 19-20 in the preparation of a medicament for treating tumors; optionally, the tumor is a tumor expressing LIV-1; optionally, the tumor is selected from breast cancer (such as triple-negative breast cancer), prostate cancer, gynecological tumors, ovarian cancer, endometrial cancer, cervical cancer, liver cancer, gastric cancer, intestinal cancer, renal cancer, squamous cell carcinoma (such as bladder, head, neck and lung), skin cancer (such as melanoma), small cell lung cancer or lung carcinoid.

22. A method for treating tumors in mammals, the method comprising administering to a mammal in need of such treatment, preferably a human, a therapeutically effective amount of the antibody-drug conjugate according to any one of claims 1-15 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 19-20; optionally, the tumor is a tumor expressing LIV-1; optionally, the tumor is selected from breast cancer (such as triple-negative breast cancer), prostate cancer, gynecological tumors, ovarian cancer, endometrial cancer, cervical cancer, liver cancer, gastric cancer, intestinal cancer, renal cancer, squamous cell carcinoma (such as bladder, head, neck and lung), skin cancer (such as melanoma), small cell lung cancer or lung carcinoid.

23. The method for treating mammalian tumors according to claim 22, wherein, The antibody-drug conjugate according to any one of claims 1-15 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 19-20 can be used in combination with other therapeutic agents; preferably, the therapeutic agent can be a chemotherapeutic agent or an immune checkpoint inhibitor; more preferably, the combination therapy provided by the combination use provides a synergistic therapeutic effect in the treatment of tumors.

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