Pharmaceutical composition comprising Anti-CDH6 antibody or ADC thereof

Abstract

A pharmaceutical composition comprising an anti-CDH6 antibody or an antibody-drug conjugate (ADC) thereof. The pharmaceutical composition comprises an anti-CDH6 antibody or an ADC thereof, a buffer, a protein stabilizer, and / or a surfactant. A CDH6 naked antibody formulation has good long-term stability; a CDH6 ADC lyophilized formulation achieves stable preservation at a relatively high concentration and can effectively prevent aggregation and degradation after reconstitution.

Description

A pharmaceutical composition comprising an anti-CDH6 antibody or its ADC

[0001] This disclosure claims priority to Chinese Patent Application No. 202411430854.3, filed on October 14, 2024, entitled "A pharmaceutical composition comprising an anti-CDH6 antibody or its ADC", and Chinese Patent Application No. 202511394418.X, filed on September 26, 2025, entitled "A pharmaceutical composition comprising an anti-CDH6 antibody or its ADC", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of pharmaceutical formulations, and more specifically, to a pharmaceutical composition comprising an anti-CDH6 antibody or its ADC (antibody-drug conjugate). Background Technology

[0003] CDH6 is a type II classical cadherin (Cadherin-6), also known as K-cadherin. CDH6 is a single-transmembrane protein composed of 790 amino acids, with its extracellular region divided into five regions (EC1-EC5). Studies have found that CDH6 is highly expressed in tumor tissues such as renal cell carcinoma, ovarian cancer, and thyroid cancer, while its expression in normal tissues is very low (Cancer Discov; 2017, 7(9):1030-45). CDH6 expression levels are high in fetal kidneys and renal cell carcinoma, while its expression level in normal adult kidneys is very low or negligible. Therefore, CDH6 can serve as a therapeutic target for related solid tumors.

[0004] Antibody-drug conjugates (ADCs) are a class of innovative antibody drugs composed of antibodies or antibody fragments targeting specific antigens and a payload linked together by a linker. ADC products combine the potent effects of traditional small molecule drugs with the targeted specificity of antibody drugs, thereby reducing systemic toxicity and more selectively delivering the payload to tumor cells, the tumor microenvironment, or other target cells. Summary of the Invention

[0005] For CDH6 ADC formulations, the large molecular weight and complex structure of their main active ingredients place higher demands on formulation stability. Developing formulations of macromolecular protein drugs (such as antibody-drug conjugates) first requires addressing the protein stability issue. To address the shortcomings of existing technologies, this invention provides a pharmaceutical composition containing an anti-CDH6 antibody or its ADC. The CDH6 naked antibody formulation of this invention exhibits good long-term stability, and the CDH6 ADC lyophilized formulation achieves stable storage at high concentrations and effectively prevents aggregation and degradation after reconstitution.

[0006] One aspect of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises a pharmaceutically active ingredient and pharmaceutically acceptable excipients; the pharmaceutically active ingredient is selected from: (1) an antibody that specifically binds to CDH6 or an antigen-binding fragment thereof; or (2) an anti-CDH6 antibody-drug conjugate or a pharmaceutically acceptable salt thereof; the excipients comprise buffers, protein stabilizers and / or surfactants.

[0007] In some embodiments, the antibody that specifically binds to CDH6 or its antigen-binding fragment 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, and / or the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1-3 and / or LCDR1-3 are selected from combinations thereof:

[0008] (1) The HCDR1-3 is SEQ ID NO: 11-13; and / or the LCDR1-3 is SEQ ID NO: 14-16;

[0009] (2) The HCDR1-3 is SEQ ID NO: 17-19; and / or the LCDR1-3 is SEQ ID NO: 20-22;

[0010] (3) The HCDR1-3 is SEQ ID NO: 23-25; and / or the LCDR1-3 is SEQ ID NO: 26-28;

[0011] or,

[0012] The HCDR1-3 and / or the LCDR1-3 have at least 80% identity with each CDR in any of the groups (1)-(3), or are sequences with at most 3 insertion, deletion or substitution mutations; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0013] In some embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt has the general structural formula Pc-(LD). n ,

[0014] in,

[0015] D is a cytotoxic drug;

[0016] L represents the connecting subunit;

[0017] Pc is the antibody that specifically binds to CDH6 or its antigen-binding fragment;

[0018] Furthermore, n is a real number from 1 to 16.

[0019] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region and / or light chain variable region are selected from the following:

[0020] (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;

[0021] (2) The heavy chain variable region is the sequence shown in SEQ ID NO.3, and / or the light chain variable region is the sequence shown in SEQ ID NO.4;

[0022] (3) The heavy chain variable region is the sequence shown in SEQ ID NO.5, and / or the light chain variable region is the sequence shown in SEQ ID NO.6;

[0023] or,

[0024] The heavy chain variable region and / or the light chain variable region have at least 80% identity with the heavy chain variable region and / or the light chain variable region in any of the groups (1)-(3) above, or are sequences with at most 3 insertion, deletion or substitution mutations; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0025] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain constant region sequence and / or a light chain constant region sequence. Optionally, the heavy chain constant region and / or light chain constant region are selected from a complete constant region sequence or a fragment thereof, the constant region fragment including CH1, a hinge region, CH2, CH3, or Fc. Optionally, the heavy chain constant region is selected from human or mouse IgG1, IgG2, IgG3, or IgG4 constant regions, and the light chain constant region is selected from human or mouse kappa constant regions or lambda constant regions. Optionally, the antibody or antigen-binding fragment includes a complete heavy chain and a light chain, the heavy chain consisting of the VH and the heavy chain constant region having the sequence shown in SEQ ID NO:9, and the light chain consisting of the VL and the light chain constant region having the sequence shown in SEQ ID NO:10.

[0026] In some embodiments, the antibody or antigen-binding fragment is:

[0027] (1) Chimeric antibodies or fragments thereof;

[0028] (2) Humanized antibodies or fragments thereof; and / or,

[0029] (3) Fully human antibodies or fragments thereof;

[0030] Preferably, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, or single-domain antibodies.

[0031] In some embodiments, the antigen-binding fragment is selected from one or more of F(ab)2, Fab', Fab, Fv, scFv, bispecific antibodies, nanobodies, and antibody minimum recognition units.

[0032] In some embodiments, the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, wherein the cytotoxic drug D is selected from chemotherapeutic drugs or antibiotics.

[0033] In some embodiments, the cytotoxic drug D is selected from DNA topoisomerase inhibitors.

[0034] In some embodiments, the cytotoxic drug D is selected from compounds of formula (DI).

[0035] in,

[0036] R 1 R 2 The atoms connected to them together form a 5-6 membered heterocycle, which contains one or two oxygen atoms as ring atoms, and the 5-6 membered heterocycle may be optionally replaced by one or more D atoms;

[0037] R 4 Selected from H or C1-C3 alkyl groups;

[0038] R 5 Selected from H, halogens, CN, =O, OH, NH2, or C1-C3 alkyl groups;

[0039] R 6 Selected from H or C1-C3 alkyl groups;

[0040] 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 D, halogen, CN, =O, OH, NH2 or C1-C3 alkyl.

[0041] In some implementations, the R 1 R 2 The atoms connected to them together form

[0042] In some implementation schemes, R 4 Selected from H.

[0043] In some implementation schemes, R 5 It is selected from H, halogen, CN, OH, NH2 or C1-C3 alkyl.

[0044] In some implementation schemes, R 5 Selected from H.

[0045] In some implementation schemes, R 6 Selected from H.

[0046] In some implementation schemes, R 7 Selected from cyclopropyl.

[0047] In some embodiments, the compound represented by formula (DI) is selected from one of the following compounds:

[0048] In some embodiments, the aforementioned antibody-drug conjugate of the general formula Pc-(LD)n or a pharmaceutically acceptable salt thereof, wherein the linker unit L is selected from... Its a-terminus is covalently linked to the antibody unit Pc, and its b-terminus is covalently linked to the cytotoxic drug D, where m1 is selected from integers 2 to 8, and L... 1The peptide residues are selected from 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 groups.

[0049] In some implementations, the L 1 It consists of Gly-Gly-Phe-Gly tetrapeptide residues.

[0050] In some implementations, m1 is 5.

[0051] In some implementations, the connection subunit L is Its a-end is covalently linked to the antibody unit Pc, and its b-end is covalently linked to the drug unit D.

[0052] In some implementations, the aforementioned general formula is Pc-(LD). n The antibody-drug conjugate or its pharmaceutically acceptable salt, wherein n is selected from a real number from 1 to 16, for example n is selected from a real number from 2 to 12, for example n is selected from a real number from 4 to 10, for example n is selected from a real number from 5 to 9, for example n is selected from a real number from 6 to 8.

[0053] In some implementations, n is selected from real numbers from 5 to 9, for example, n is 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, 7.7, 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.

[0054] In some implementations, the aforementioned general formula is Pc-(LD). n The antibody-drug conjugate or its pharmaceutically acceptable salt is selected from the following compounds or their pharmaceutically acceptable salts:

[0055] Where Pc and n are as defined in any of the preceding definitions.

[0056] In some embodiments, the concentration of the active pharmaceutical ingredient is from 0.1 mg / ml to 100 mg / ml; preferably 20-40 mg / mL; more preferably 20 mg / mL or 40 mg / mL.

[0057] In some embodiments, the buffer solution is selected from acetate-sodium acetate buffer, citrate-sodium citrate buffer, histidine-histidine hydrochloride buffer, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer; preferably, histidine-histidine hydrochloride buffer.

[0058] In some embodiments, the concentration of the buffer solution is 10-100 mM, preferably 15-30 mM, and more preferably 20 mM.

[0059] In some embodiments, the protein stabilizer is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol, sorbitol, or combinations thereof; preferably sucrose, trehalose, glycine, or combinations thereof.

[0060] In some embodiments, the concentration of the protein stabilizer is 0.1-10% (w / v); preferably 0.8% (w / v), 2% (w / v), 4.5% (w / v), 8% (w / v) or 9.5% (w / v); more preferably 0.8% (w / v) or 8% (w / v); most preferably 8% (w / v) sucrose, 8% (w / v) trehalose and 0.8% (w / v) glycine.

[0061] In some embodiments, the surfactant is selected from polysorbate, poloxamer, and glycerol fatty acid esters; preferably polysorbate; more preferably polysorbate 80.

[0062] In some embodiments, the concentration of the surfactant is 0.01 to 0.1% (w / v); preferably 0.02% (w / v), 0.04% (w / v), 0.06% (w / v) or 0.08% (w / v); more preferably 0.04% (w / v).

[0063] In some embodiments, the pH of the pharmaceutical composition is about 4.5-7.5; preferably 4.5-6.5; more preferably 5.5 or 6.

[0064] In some embodiments, the pharmaceutical composition comprises:

[0065] a) The antibody or its antigen-binding fragment that specifically binds to CDH6 at a concentration of 0.1 mg / ml to 100 mg / ml.

[0066] b) A 10-100 mM histidine-histidine hydrochloride buffer solution, pH 4.5-7.5, and

[0067] c) Sucrose with a concentration of 1-10% (w / v);

[0068] Preferably, the pharmaceutical composition comprises:

[0069] a) The antibody or its antigen-binding fragment that specifically binds to CDH6 at a concentration of 10 mg / ml to 50 mg / ml.

[0070] b) A 15-30 mM histidine-histidine hydrochloride buffer solution, pH 5.5-6.5, and

[0071] c) Sucrose with a concentration of 5-9% (w / v);

[0072] More preferably, the pharmaceutical composition comprises:

[0073] a) The antibody or its antigen-binding fragment that specifically binds to CDH6 at a concentration of 20 mg / ml.

[0074] b) A 20 mM histidine-histidine hydrochloride buffer solution at pH 6, and

[0075] c) Sucrose with a concentration of 8% (w / v);

[0076] Preferably, the pharmaceutical composition is in liquid form or a lyophilized powder formulation.

[0077] In some embodiments, the pharmaceutical composition comprises:

[0078] a) The anti-CDH6 antibody-drug conjugate or a pharmaceutically acceptable salt thereof at a concentration of 0.1 mg / ml to 100 mg / ml.

[0079] b) A 10-100 mM histidine-histidine hydrochloride buffer solution, pH 4.5-7.5, and

[0080] c) Trehalose at a concentration of 1-10% (w / v)

[0081] d) 0.1-2% (w / v) glycine,

[0082] f) 0.01-0.1% (w / v) Polysorbate 80;

[0083] Preferably, the pharmaceutical composition comprises:

[0084] a) The anti-CDH6 antibody-drug conjugate or a pharmaceutically acceptable salt thereof at a concentration of 20 mg / ml to 80 mg / ml.

[0085] b) A 15-30 mM histidine-histidine hydrochloride buffer solution, pH 5-6, and

[0086] c) Trehalose at a concentration of 5-10% (w / v),

[0087] d) 0.1-1% (w / v) glycine,

[0088] f) 0.01-0.05% (w / v) Polysorbate 80;

[0089] More preferably, the pharmaceutical composition comprises:

[0090] a) The anti-CDH6 antibody-drug conjugate or a pharmaceutically acceptable salt thereof at a concentration of 40 mg / ml.

[0091] b) A 20 mM histidine-histidine hydrochloride buffer solution, pH 5.5, and

[0092] c) Trehalose at a concentration of 8% (w / v)

[0093] d) 0.8% (w / v) glycine,

[0094] f) 0.04% (w / v) Polysorbate 80;

[0095] Preferably, the pharmaceutical composition is in liquid form or a lyophilized powder formulation.

[0096] In another aspect, the present invention provides a method for preparing the pharmaceutical composition, characterized in that it includes a step of replacing the solution of the active pharmaceutical ingredient with a buffer solution, wherein the buffer solution is preferably a histidine-histidine hydrochloride buffer solution, the concentration of the buffer solution is preferably 20 mM, and the pH of the buffer solution is preferably 6 or 5.5; preferably, the preparation method further includes a freeze-drying step.

[0097] In another aspect, the present invention provides a pharmaceutical composition comprising an anti-CDH6 antibody or an antibody-drug conjugate (ADC) thereof, characterized in that the pharmaceutical composition is prepared by the method described above.

[0098] In another aspect, the present invention provides a lyophilized formulation comprising an anti-CDH6 antibody or an antibody-drug conjugate (ADC) thereof, characterized in that the formulation is obtained by freeze-drying the pharmaceutical composition.

[0099] In another aspect, the present invention provides a reconstituted solution comprising an anti-CDH6 antibody or an antibody-drug conjugate (ADC) thereof, characterized in that the reconstituted solution is prepared by reconstituted the lyophilized formulation.

[0100] In another aspect, the present invention provides the use of the pharmaceutical composition, the pharmaceutical composition prepared by the method, the pharmaceutical composition comprising an anti-CDH6 antibody or its ADC, the lyophilized formulation, or the reconstituted solution, in the preparation of a medicament for the prevention and / or treatment of a disease in an individual; preferably, the disease is a tumor; more preferably, the tumor is a tumor expressing CDH6; most preferably, the tumor is selected from the group consisting of: ovarian cancer, renal cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and bile duct epithelial cancer.

[0101] In another aspect of the present invention, a method for preventing and / or treating a disease in an individual is provided, comprising administering the pharmaceutical composition, the pharmaceutical composition prepared by the method, the pharmaceutical composition comprising an anti-CDH6 antibody or its ADC, the lyophilized formulation, or the reconstituted solution to a patient in need; preferably, the disease is a tumor; more preferably, the tumor is a tumor expressing CDH6; most preferably, the tumor is selected from the group consisting of: ovarian cancer, renal cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and bile duct epithelial cancer.

[0102] The pharmaceutical composition of the present invention comprising an anti-CDH6 antibody or its ADC has the following superior technical effects:

[0103] 1. In response to the challenges posed by the large molecular weight and complex structure of the main active ingredients in CDH6 ADC formulations, this invention has screened and optimized the formulation composition, obtaining a formulation suitable for anti-CDH6 antibodies and their ADCs.

[0104] 2. The formulation of CDH6 naked antibody obtained by screening in this invention is stable within a predetermined time under long-term stability test conditions of -70°C, accelerated stability test conditions of 2-8°C, and pressure test conditions of 25°C, as well as 5 freeze-thaw cycles from -40°C to room temperature.

[0105] 3. The formulation for CDH6 ADC obtained by screening in this invention remains stable under long-term stability test conditions of 2-8°C within a predetermined time, and achieves stable preservation of high-concentration CDH6 ADC lyophilized formulations and effectively prevents aggregation and degradation after reconstitution.

[0106] Terminology Definitions and Explanations

[0107] Unless otherwise stated, the terms used in this disclosure have the following meanings: the definitions of groups and terms recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined and combined with each other. A particular term should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with its ordinary meaning in the art. When trade names appear herein, they are intended to refer to the corresponding product or its active ingredient.

[0108] The term "CDH6" refers to type II classical cadherin (Cadherin-6), also known as K-cadherin. CDH6 is a single transmembrane protein composed of 790 amino acids, with its extracellular region divided into 5 regions (EC1-EC5).

[0109] The term "antibody-drug conjugate" (ADC) refers to an antibody or its antigen-binding fragment linked to a biologically active drug via a stable linker unit. This link can be a covalent bond or a non-covalent interaction, such as through electrostatic forces. Various linkers known in the art can be used to form the immunoconjugate.

[0110] The term "DAR" or "drug-antibody ratio" refers to the average number of small molecule cytotoxic drugs linked to each antibody molecule. In the antibody-drug conjugates disclosed herein, DAR is defined by the variable "n", which can be either an integer or a decimal.

[0111] The term “antibody” in its broadest sense refers to a polypeptide or combination of polypeptides containing sufficient sequences from the variable regions of the immunoglobulin heavy chain and / or from the variable regions of the immunoglobulin light chain, thereby enabling it to specifically bind to an antigen. The term “antibody” as used herein encompasses a wide range of forms and structures, provided they exhibit the desired antigen-binding activity. The term “antibody” as used herein includes alternative protein scaffolds or artificial scaffolds having transplanted complementarity-determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds (which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody) and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may also include non-antibody-derived scaffolds, such as scaffold proteins known in the art for use in transplanting CDRs, including but not limited to tendinins, fibronectins, peptide aptamers, etc.

[0112] The term "antibody" in this article includes a typical "quadruple-chain antibody," which belongs to the immunoglobulin family composed of two heavy chains (HC) and two light chains (LC). The heavy chain refers to a polypeptide chain consisting 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 in the N-to-C-terminal direction. Optionally, when the full-length antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. The light chain is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-to-C-terminal direction. Heavy chains are linked to each other and to each other with disulfide bonds, forming a "Y"-shaped structure. Because the amino acid composition and sequence of the immunoglobulin heavy chain constant region differ, their antigenicity also differs. Based on this, the "immunoglobulins" in this article can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA into IgA1 and IgA2. Light chains are classified as κ or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ or λ chain.

[0113] The “antibody” in this article can be derived from any animal, including but not limited to humans and non-human animals. Non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camels, llamas, guanacos, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g., sharks).

[0114] In this article, "antibody" includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, complete antibodies, fragments of complete antibodies, naked antibodies, conjugated antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0115] The term "anti-CDH6 antibody" refers to a polypeptide or combination of polypeptides that can specifically bind to the antigen CDH6.

[0116] The term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that, apart from possible variants (e.g., containing naturally occurring mutations or generated during the manufacturing process of the formulation, such variants are typically present in small amounts), the individual antibodies comprising this group are identical and / or bind to the same epitopes. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. The modifier "monoclonal" herein should not be construed as requiring the production of the antibody or antigen-binding molecule by any particular method. For example, monoclonal antibodies can be prepared using a variety of techniques, including (but not limited to) hybridoma techniques, recombinant DNA methods, phage library display techniques, methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, and other methods known in the art.

[0117] The term "natural antibody" refers to antibodies produced and paired by the immune system of multicellular organisms. The term "engineered antibody" in this article refers to non-natural antibodies obtained through techniques such as genetic engineering and antibody engineering. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (e.g., scFv), bispecific antibodies, and so on.

[0118] The term "monospecific" refers to having one or more binding sites, where each binding site binds to the same epitope of the same antigen.

[0119] 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 a different epitope of a different antigen. Therefore, terms such as "bispecific," "trispecific," and "quadrispecific" refer to the number of different epitopes that an antibody / antigen binding molecule can bind to.

[0120] The term "valence" indicates the presence of a specified number of binding sites in an antibody / antigen binding molecule. Therefore, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" indicate the presence of one, two, four, and six binding sites in an antibody / antigen binding molecule, respectively.

[0121] In this article, "full-length antibody," "intact antibody," and "complete antibody" are used interchangeably, referring to antibodies with structures that are substantially similar to those of natural antibodies.

[0122] In this document, "antigen-binding fragment" and "antibody fragment" are used interchangeably. They do not possess the complete structure of a full antibody, but only contain a portion or a local variant of the full antibody, which has the ability to bind antigens. "Antigen-binding fragment" or "antibody fragment" in this document includes, but is not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, VHH, and scFv.

[0123] Papain digestion of the intact antibody produces two identical antigen-binding fragments, called "Fab" fragments, each containing variable domains of both the heavy and light chains, as well as a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" in this paper refers to the light chain fragment containing the VL domain and constant domain (CL) of the light chain, and the antibody fragment containing the VH domain and first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is the Fab' fragment in which the cysteine ​​residues of the constant domain carry a free thiol group. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region. The "Fv fragment" is the smallest fragment produced by IgG and IgM, containing an intact antigen-binding site. The Fv fragment has the same binding properties and similar three-dimensional binding properties as the Fab fragment; the VH and VL chains of the Fv fragment are bound together by non-covalent interactions.

[0124] The terms "antigen-binding fragment" and "antibody fragment" in this article include, but are 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. The terms "antigen-binding fragment" and "antibody fragment" in this article can be understood as local or local variants of intact antibodies having the same antigenic determinant (CDR), 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.

[0125] The term "scFv" (single-chain variable fragment) refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between the VH and VL of the scFv, forming a disulfide-linked Fv (dsFv).

[0126] The term "diabody" refers to a single polypeptide chain in which the VH and VL domains are expressed, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0127] The term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain portion is derived from one antibody (which may originate from a specific species or belong to a specific antibody class or subclass), and another portion of its light chain and / or heavy chain is derived from another antibody (which may originate from the same or different species or belong to the same or different antibody class or subclass), but which retains its binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). For example, the term "chimeric antibody" can include antibodies (e.g., human-mouse chimeric antibodies) in which the variable regions of the heavy and light chains of the antibody originate from a first antibody (e.g., a mouse antibody), while the constant regions of the heavy and light chains of the antibody originate from a second antibody (e.g., a human antibody).

[0128] The term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase its homology with that of a human antibody. Typically, all or part of the CDR region of a humanized antibody is derived from a non-human antibody (donor antibody), while all or part of the non-CDR region (e.g., the variable region FR and / or constant region) is derived from a human immunoglobulin (receptor antibody). Humanized antibodies generally retain or partially retain the intended properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, the ability to enhance immune cell activity, and the ability to strengthen the immune response.

[0129] The term "fully human antibody" refers to an antibody having variable regions in which both the FR and CDR are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Fully human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced through random or site-specific mutagenesis in vitro or through somatic mutations in vivo). However, "fully human antibody" as described herein does not include antibodies in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human frame sequence.

[0130] The term "naked antibody" or "naked antibody" as used herein refers to an antibody that is not linked, 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 may be glycosylated by the host cell's glycosylation machinery (e.g., glycosylation enzymes). In some embodiments, naked antibodies are not glycosylated when expressed by host cells that do not possess their own glycosylation machinery (e.g., glycosylation enzymes). In some embodiments, naked antibodies are intact antibodies, while in other embodiments, naked antibodies are antigen-binding fragments of intact antibodies, such as Fab antibodies.

[0131] The term "variable region" refers to a region in the antibody heavy or light chain involved in enabling the antibody to bind to an 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 (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved frame regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p.91 (2007). A single VH or VL domain is sufficient to confer antigen-binding specificity. The terms "complementarity-determining region" and "CDR" are used interchangeably in this article. They typically refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or light chain variable region (VL). This region is called the complementarity-determining region because it can form a precise complementarity with the antigen epitope in its spatial structure. 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, referring to the amino acid residues in the antibody heavy chain variable region or light chain variable region other than the CDR. A typical antibody variable region consists of four FR regions and three CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0132] The term "CDR" in this paper may be labeled and defined in a manner known in the art, including but not limited to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. The tools and 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). The CDR in this paper includes overlaps and subsets of amino acid residues defined in different ways.

[0133] The term "heavy chain constant region" in this document refers to the carboxyl-terminal portion of the antibody heavy chain, which does not directly participate in antibody-antigen binding but exhibits effector functions, such as interaction with the Fc receptor. It has a more conserved amino acid sequence compared to the variable domains of the antibody. A "heavy chain constant region" contains at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof. "Heavy chain constant region" includes a "full-length heavy chain constant region" and a "heavy chain constant region fragment," the former having a structure substantially similar to the natural antibody constant region, while the latter includes only a portion of the full-length heavy chain constant region. Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy chain constant region fragment" may be selected from the CH1, Fc, or CH3 domains.

[0134] The term "light chain constant region" in this article refers to the carboxyl terminus of the antibody light chain, which does not directly participate in the binding of the antibody to the antigen. The light chain constant region can be selected from the constant κ domain or the constant λ domain.

[0135] The term "Fc" in this document refers to the carboxyl-terminal portion of an antibody obtained by papain hydrolysis of an intact antibody, typically containing the CH3 and CH2 domains of the antibody. Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary slightly, the Fc region of the human IgG heavy chain is generally defined as extending from the amino acid residue at Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during antibody production or purification, or through recombinant engineering of the nucleic acid encoding the antibody heavy chain; therefore, the Fc region may or may not include Lys447.

[0136] The term "identity" in this document can be calculated as follows: To determine the percentage of "identity" between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may 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 may be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at that position 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.

[0137] In this article, "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.

[0138] In this article Indicates the connection site.

[0139] The diagrammatic representation of racemic or enantiomerically pure compounds in this article is derived from Maehr, J. Chem. Ed. 1985, 62:114-120. Unless otherwise specified, wedge bonds and virtual wedge bonds are used. The absolute configuration of a solid center is represented by black solid bonds and imaginary bonds. It indicates the relative configuration of a stereocenter (such as the cis-trans configuration of alicyclic compounds).

[0140] The term "stereoisomer" refers to isomers that are produced by different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

[0141] The compounds disclosed herein may have asymmetric atoms such as carbon, sulfur, nitrogen, and phosphorus atoms, or asymmetric double bonds, and therefore may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E- and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof or other mixtures, such as mixtures enriched with enantiomers or diastereomers. All such isomers and mixtures thereof are within the scope of the definition of the compounds disclosed herein. Alkyl groups or other substituents may contain additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms, or asymmetric phosphorus atoms. All such isomers involved in all substituents, and mixtures thereof, are also included within the scope of the definition of the compounds disclosed herein. The compounds containing asymmetric atoms disclosed herein can be isolated in optically active pure form or in racemic form. The optically active pure form can be separated from racemic mixtures or synthesized using chiral starting materials or chiral reagents.

[0142] The term "substituted" refers to the substitution of one or more hydrogen atoms on a specific atom by a substituent, provided that the valence state of the specific atom is normal and the resulting compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are substituted; oxo substitution does not occur on aromatic groups.

[0143] The terms “optional” or “optionally” mean that the event or condition subsequently described may or may not occur, including both the occurrence and non-occurrence of said event or condition. For example, “optionally” substituted with a halogen means that the ethyl group can be unsubstituted (CH2CH3), monosubstituted (CH2CH2F, CH2CH2Cl, etc.), polysubstituted (CHFCH2F, CH2CHF2, CHFCH2Cl, CH2CHCl2, etc.), or fully substituted (CF2CF3, CF2CCl3, CCl2CCl3, etc.). Those skilled in the art will understand that for any group containing one or more substituents, no substitution or substitution pattern that is spatially impossible and / or cannot be synthesized is introduced.

[0144] C in this article m -C n , refers to having an integer number of carbon atoms in the range mn.

[0145] The term "alkyl" refers to a compound with the general formula C10. n H 2n+1The alkyl group can be straight-chain or branched. The term "C1-C6 alkyl" should be 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.

[0146] The “C1-C6 alkyl” mentioned in this article may further include “C1-C3 alkyl”.

[0147] The term "cycloalkyl" refers to a fully saturated carbon 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, and specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0148] The term "heterocyclic group" refers to a fully saturated or partially saturated monocyclic, fused, spirocyclic, or bridged ring group containing 1-5 heteroatoms or heteroatom groups (i.e., atomic groups containing heteroatoms). These "heteroatoms or heteroatom groups" include, but are not limited to, nitrogen (N), oxygen (O), sulfur (S), phosphorus (P), boron (B), -S(=O)2-, -S(=O)-, -P(=O)2-, -P(=O)-, -NH-, -S(=O)(=NH)-, -C(=O)NH-, or -NHC(=O)NH-, etc. The term "4-7 membered heterocyclic group" refers to a heterocyclic group with 4, 5, 6, or 7 ring atoms, containing 1-3 independently selected heteroatoms or heteroatom groups as described above. The term "5-6 membered heterocyclic group" refers to a heterocyclic group with 5 or 6 ring atoms, and whose ring atoms contain 1-3 independent heteroatoms or heterogroups selected from those described above. Examples of 4-membered heterocyclic groups include, but are not limited to, azirrocyclobutane and oxacyclobutane; examples of 5-membered heterocyclic groups include, but are not limited to, tetrahydrofuranyl, dioxacyclopentenyl, pyrrolyl, imidazoalkyl, pyrazolyl, pyrrolinyl, 4,5-dihydrooxazolyl, or 2,5-dihydro-1H-pyrrolyl; examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithiaalkyl, thiomorpholinyl, piperazine, trithiaalkyl, tetrahydropyridinyl, or 4H-[1,3,4]thiadiazinyl; and examples of 7-membered heterocyclic groups include, but are not limited to, diazacycloheptane. "4-7 membered heterocyclic group" can encompass the ranges of "4-7 membered heterocyclic alkyl," "5-6 membered heterocyclic group," and "5-6 membered heterocyclic alkyl."

[0149] The term "halogen" or "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0150] The term "treatment" refers to surgical or therapeutic treatment aimed at preventing, slowing (reducing) undesirable physiological changes or lesions in the treated individual, such as the progression of cancer, autoimmune diseases, and viral infections. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, disease severity reduction, disease stability (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of disease status, and remission (whether partial or complete), whether detectable or undetectable. Individuals requiring treatment include those already suffering from the condition or disease, those susceptible to the condition or disease, or those intending to prevent the condition or disease. When terms such as slowing, reducing, weakening, mitigating, or remission are used, they also imply elimination, disappearance, or non-occurrence.

[0151] The term "effective dose" refers to the amount of a therapeutic agent, administered alone or in combination with another therapeutic agent to cells, tissues, or subjects, that is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective dose" also refers to the amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating an associated medical condition, or increasing the rate at which such symptoms are treated, cured, prevented, or alleviated. When an active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, consecutively, or simultaneously.

[0152] The term "subject" refers to an organism that receives treatment for a specific disease or condition as described in this disclosure. Examples of subjects and patients include mammals, such as humans, primates (e.g., monkeys), or non-primate mammals receiving treatment for a disease or condition.

[0153] The amount of the disclosed compound constituting a “therapeutic effective amount” varies depending on the compound, the disease state and its severity, the route of administration, and the age of the mammal to be treated, but may routinely be determined by a person skilled in the art based on their own knowledge and the content of this disclosure.

[0154] The term "pharmaceutical acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0155] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable salt of an acid or base, including salts formed by a compound with an inorganic or organic acid, and salts formed by a compound with an inorganic or organic base.

[0156] The term "pharmaceutical composition" refers to a mixture containing one or more compounds described in this disclosure or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the antibody's active ingredient, facilitate administration to the organism, and enhance the absorption of the active ingredient to exert its biological activity. In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0157] The term "lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by a vacuum freeze-drying step, which is in liquid or solution form.

[0158] The term "pharmaceuticalally acceptable excipient" refers to excipients that do not cause significant irritation to the organism and do not impair the biological activity and properties of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and / or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

[0159] The word “comprise” or “include” and its English variants such as comprises or comprising can be understood as having an open, non-exclusive meaning, that is, “including but not limited to”.

[0160] This disclosure also includes compounds of this disclosure that are identical to those described herein, but in which one or more atoms are labeled with isotopes whose atomic weights or mass numbers differ from those commonly found in nature. Examples of isotopes that can be incorporated into compounds of this 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, etc.

[0161] The compounds of the present invention labeled with certain isotopes (e.g., using) 3 H and 14 C-labeling can be used in the analysis of compound and / or substrate tissue distribution. Tritiumization (i.e., 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. The isotopically labeled compounds of this disclosure can typically be prepared by replacing the unlabeled reagent with an isotopically labeled reagent using a procedure similar to those disclosed in the schemes and / or examples below.

[0162] The pharmaceutical compositions disclosed herein are suitable for parenteral administration, such as in suitable unit dosage forms as sterile solutions, suspensions, or lyophilized products. For example, the pharmaceutical compositions disclosed herein may be in the form of sterile aqueous solutions for intramuscular or subcutaneous administration. The pharmaceutical compositions disclosed herein may accept other solvents or media, such as water, Ringer's solution, or isotonic sodium chloride solution, during use.

[0163] In all methods of administration of the compounds described in this disclosure, the daily dose is from 0.001 mg / kg to 600 mg / kg body weight, preferably from 0.05 mg / kg to 200 mg / kg body weight, more preferably from 0.1 mg / kg to 100 mg / kg body weight, in the form of single or separate doses.

[0164] The compounds disclosed herein can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments disclosed herein.

[0165] The chemical reactions in the specific embodiments of this disclosure are carried out in a suitable solvent, which must be suitable for the chemical changes of this disclosure and the reagents and materials required therefor. In order to obtain the compounds of this disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction flow based on existing embodiments.

[0166] An important consideration in synthetic route planning in this field is the selection of appropriate protecting groups for reactive functional groups (such as amino and carboxyl groups in this disclosure). For example, see Greene's Protective Groups in Organic Synthesis (4th Ed). Hoboken, New Jersey: John Wiley & Sons, Inc. All references cited in this disclosure are incorporated herein by reference in their entirety. Attached Figure Description

[0167] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0168] Figure 1. Inhibitory effect of ADC on OVCAR3 cell proliferation.

[0169] Figure 2. Inhibitory effect of ADC on PA-1 cell proliferation.

[0170] Figure 3. Inhibitory effect of ADC on OVCAR3 cell proliferation.

[0171] Figure 4. Inhibitory effect of ADC on PA-1 cell proliferation.

[0172] Figure 5 Tumor growth curves of the PA-1 subcutaneous tumor model (involving ADC-L1-14-P1-12 and ADC-L1-14-P1-5).

[0173] Figure 6 Tumor growth curves of the PA-1 subcutaneous tumor model (involving ADC-L1-19-P1-12 and ADC-L1-19-P1-5).

[0174] Figure 7. Tumor growth curves of the 786-O subcutaneous tumor model (involving ADC-L1-19-P1-12 and ADC-L1-19-P1-5). Detailed Implementation

[0175] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0176] The embodiments of the present invention are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0177] Example 1: Following the methods of Examples 14-1 and 14-2 in patent document WO2023217227A1, cytotoxic drugs 14, 14-P1, and 14-P2 were prepared, with the structures shown below:

[0178] Example 2: Following the methods of Examples 19-1 and 19-2 in patent document WO2023217227A1, cytotoxic drugs 19, 19-P1, and 19-P2 were prepared, with the structures shown below:

[0179] Example 3: Following the method of Example 37 in patent document WO2023217227A1, drug-linker compounds L1-14, L1-14-P1, and L1-14-P2 were prepared, with the structures shown below:

[0180] Example 4: Following the methods of Examples 39-1 and 39-2 in patent document WO2023217227A1, payload-linker compounds L1-19, L1-19-P1, and L1-19-P2 were prepared, with the structures shown below:

[0181] Example 5: Construction and production of anti-human CDH6 antibody

[0182] The heavy and light chain variable region sequences of the anti-human CDH6 monoclonal antibodies (CDH6-Ab-4, CDH6-Ab-3, CDH6-Ab-2, and CDH6-Ab) are shown in Table 1 below, and the CDR sequences divided according to the Kabat format are shown in Table 2 below. The nucleic acid sequences encoding the antibodies VH and VL were recombined into the expression vector pTT5 containing a signal peptide (MGWSWILLFLLSVTAGVHS, SEQ ID NO: 29) and heavy chain constant region / light chain constant region sequences to obtain recombinant plasmids expressing VH-CH / VL-CL. The plasmids and transfection reagent PEI (Polysciences, catalog number: 24765-1) were added to OPTI-MEM (Gibco, catalog number: 11058021), mixed, and incubated for 15 min. The mixture was then added to Expi293F cells (Thermofisher, catalog number: A14527) and cultured in a shaker at 37°C and 120 rpm with 5% CO2. On day 2 after transfection, OPM-293ProFeed (Shanghai AOPMai, catalog number: F081918-001) and 6 g / L glucose (Sigma, catalog number: G7528) were added. On day 6 after transfection, cell supernatant was collected. The supernatant was purified with Protein A (GE, catalog number: 28985254), and the eluted sample was dialyzed onto PBS (pH 7.4) to obtain anti-human CDH6 monoclonal antibody. CDH6-Ab is the positive control human CDH6 antibody, and its sequence is derived from patent WO2018212136A1.

[0183] Table 1. Sequences of the variable regions of the heavy and light chains of anti-human CDH6 monoclonal antibodies.

[0184] Table 2. CDR sequences of anti-human CDH6 monoclonal antibodies (Kabat partitioning)

[0185] Example 6: Preparation method of antibody-drug conjugate

[0186] Conjugation: The antibody prepared in Example 5 was dialyzed and the buffer was changed to a solution of 20 mM PB, 150 mM NaCl, and 1 mM EDTA (pH 6.5). Eight times the volume of 10 mM tris(2-carboxyethyl)phosphine solution (TCEP, Thermo Scientific #77720) was added to the antibody solution, and the mixture was incubated at 37°C on a constant-temperature metal shaker for 2.5 h to reduce the antibody. 15 times the volume of the drug-linker compound (prepared in Examples 3 and 4) was dissolved in DMSO and added to the reaction system, and the reaction solution was conjugated at 25°C for 6 h. The reaction product was desalted using a G25 column and the buffer was changed to phosphate-buffered saline (PBS) to remove unreacted free small molecule toxins. The purity and DAR value of the ADC product were analyzed by SEC and LC-MS methods.

[0187] SEC Purity Analysis: The SEC-HPLC method was used to analyze the protein samples, characterize the molecular size uniformity of the recombinant protein, and determine the purity of the recombinant protein. The HPLC system used was an Agilent 1260, the column was a TSKgel G3000SWXL (purchased from Tosoh Bioscience), the mobile phase was 200 mM phosphate buffer, pH 7.0 / isopropanol (Merck, 1.01040.4008) (v / v 9:1), the detection temperature was 25℃, the flow rate was 0.5 mL / min, the detection wavelength was 280 nm, the target protein loading was 50 μg, and the analysis time was 40 min.

[0188] DAR value determination: The DAR value of ADC molecules 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-sugar modification, and then treated with dithiothreitol (DTT, Sigma#646563) and incubated at 37℃ for 1 h 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 into a Waters ACQUITY Protein BEH size-exclusion 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: spray voltage 3.8 kV, capillary heating temperature 300℃, sheath gas flow rate 35 arb, and precursor ion scan range 800-3000. Finally, the mass spectrometry data was analyzed using the Biopharma Finder software. 4.1 The Respect algorithm is used for deconvolution processing to calculate the molecular weight information of the light and heavy chain mass spectrometry peaks and the mass spectrometry response signals of each component, thereby calculating the DAR value of the ADC sample to be tested.

[0189] Table 3 DAR value and SEC purity of CDH6-ADC

[0190] The isotype control (ISO) anti-FITC-hIgG1 antibody was prepared using the same method described above and conjugated with a Linker-Payload compound to obtain the isotype control of the ADC. Its DAR value and SEC results are shown in Table 4 below.

[0191] Table 4. DAR values ​​and SEC results of ADC isotype controls

[0192] Example 7: In vitro proliferation inhibition test of ADC on tumor cells

[0193] Cells and materials: Human ovarian cancer cell line OVCAR3 (CDH6-overexpressing cell line) was purchased from ATCC (#HTB-161), human ovarian teratoma cell line PA-1 (CDH6-moderately expressing cell line) was purchased from Nanjing Kebai Biotechnology Co., Ltd. (#CBP60800), bovine serum, 1640 medium, MEM medium (Gibco#11095-080), MEM NEAA (Gibco#11140-050), sodium pyruvate (Gibco#11360-070), penicillin-streptomycin and 0.25% Trypsin-EDTA were purchased from Gibco, bovine insulin was purchased from Solarbio, 96-well plates were purchased from Corning, and Cell-Titer Glo reagent was purchased from Promega.

[0194] Cell culture: OVCAR3 cells were cultured in 1640 medium containing 20% ​​fetal bovine serum, 2 μg / mL bovine insulin, and 1% penicillin-streptomycin at 37°C and 5% CO2. PA-1 cells were cultured in MEM medium containing 10% fetal bovine serum, 1% MEM NEAA, 1% sodium pyruvate, and 1% penicillin-streptomycin at 37°C and 5% CO2. Only cells in the logarithmic growth phase were used for experiments.

[0195] Cell proliferation activity assay: The inhibitory activity of ADCs on the proliferation of OVCAR3 and PA-1 cell lines was detected using Cell-Titer Glo reagent. OVCAR3 cells (5000 cells per well) and PA-1 cells (800 cells per well) were seeded in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. ADC molecules were diluted with the corresponding cell culture medium to a concentration of 100 nM, and then serially diluted 3-fold with culture medium to obtain 8 concentrations. 10 μL of the prepared ADC solution was transferred to each well of the 96-well plate to a final concentration of 0-10 nM. After adding the ADC solution to be tested, the plates were cultured at 37°C and 5% CO2 for 5 days. Cell viability was then assessed using Cell-Titer Glo reagent.

[0196] Data analysis: Calculate %inhibition (inhibition rate) and fit IC50. 50 % inhibition = 1 - 100% × (Signal-Bottom) / (Top-Bottom). Signal refers to the signal value of the ADC sample group, Bottom refers to the signal value of the sample without cells but with the same volume of culture medium, and Top refers to the signal value of the sample without ADC but with cells.

[0197] Experimental results: Under the experimental conditions, the ADCs tested showed strong inhibitory activity against the proliferation of both OVCAR3 and PA-1 cells, as detailed in Tables 5-6 and Figures 1-4.

[0198] Table 5. Inhibitory effect of ADCs on the proliferation of OVCAR3 and PA-1 cells. Note: " / " indicates that IC cannot be fitted. 50 .

[0199] Table 6. Inhibitory effect of ADCs on the proliferation of OVCAR3 and PA-1 cells. Note: " / " indicates that IC cannot be fitted. 50 .

[0200] Example 8: Evaluation of the efficacy of PA-1 in a subcutaneous tumor model

[0201] Experimental reagents: Human ovarian cancer PA-1 cells were purchased from Nanjing Kebai Biotechnology Co., Ltd.; MEM culture medium was purchased from Gibco (catalog number 32561-037); fetal bovine serum was purchased from Excell (catalog number FND500); penicillin-streptomycin was purchased from Gibco (catalog number 15140122); NEAA was purchased from Gibco (catalog number 11140-050); sodium pyruvate was purchased from Gibco (catalog number 11360-070); Versene was purchased from Gibco (catalog number 15040-066); D-PBS (calcium and magnesium phosphate buffer) was purchased from Hyclone (catalog number SH30256.01); and Matrigel was purchased from Corning (catalog number 356237).

[0202] Experimental methods:

[0203] Animal Information: Balb / c nude mice, female, 5-6 weeks old, weighing approximately 14-20 grams. The animals were purchased from Beijing Vital River Biotechnology Co., Ltd. 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 food and water.

[0204] Cell culture: Human ovarian cancer PA-1 cell line was cultured in vitro under the following conditions: MEM solution containing 10% fetal bovine serum, 1% penicillin-streptomycin, 1% NEAA, and 1mM sodium pyruvate, in an incubator at 37°C and 5% CO2. Cells were passaged weekly using Versene digestion solution. When cell saturation reached 80%-90% and the desired number was achieved, cells were harvested and counted.

[0205] Cell seeding: Add 0.1 ml of PA-1 cell suspension (containing 1 × 10⁻⁶ cells) to the cell suspension. 7100 cells (MEM:Matrigel volume ratio 1:1) were subcutaneously inoculated into the axilla of each mouse. On day 17 after cell inoculation, mice were randomly assigned to groups based on tumor volume, with the grouping day being Day 0.

[0206] Tumor measurements and experimental indicators:

[0207] The tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume is: V = 0.5axb 2 , where a and b represent the long and short diameters of the tumor, respectively. Mouse body weight was measured twice weekly.

[0208] The tumor-suppressive efficacy of the test drug was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] x 100%.

[0209] Experimental results:

[0210] In the mouse subcutaneous xenograft PA-1 model, both ADC-L1-14-P1-12 and ADC-L1-14-P1-5, at a dose of 3 mg / kg, significantly inhibited tumor growth after a single intravenous administration (P<0.0001). The results are shown in Table 7 and Figure 5.

[0211] Table 7 Tumor volume in PA-1 subcutaneous tumor model

[0212] In the mouse subcutaneous xenograft PA-1 model, ADC-L1-19-P1-12 and ADC-L1-19-P1-5, at doses of 1 mg / kg and 3 mg / kg, respectively, significantly inhibited tumor growth after a single intravenous administration (P<0.0001), and the effect was dose-dependent. The results are shown in Table 8 and Figure 6.

[0213] Table 8 Tumor volume in PA-1 subcutaneous tumor model

[0214] Example 9: Efficacy Evaluation of the 786-O Subcutaneous Tumor Model

[0215] Experimental reagents: Human renal cell carcinoma 786-O cells (CDH6 low-expression cell line) were purchased from ATCC; RPMI-1640 medium was purchased from Gibco (catalog number A104910); fetal bovine serum was purchased from Excel (catalog number FND500); penicillin-streptomycin was purchased from Gibco (catalog number 15140122); 0.25% trypsin-EDTA was purchased from Gibco (catalog number 25200-072); D-PBS (calcium and magnesium ion-free phosphate buffer) was purchased from Hyclone (catalog number SH30256.01); and Matrigel was purchased from Corning (catalog number 356237).

[0216] Experimental methods:

[0217] Animal Information: NOD-SCID mice, female, 8-9 weeks old, weighing approximately 18-25 grams. The animals were purchased from Shanghai Lingchang Biotechnology Co., Ltd. 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 food and water.

[0218] Cell culture: Human renal cell carcinoma 786-O cell line was cultured in vitro under the following conditions: RPMI-1640 solution with 10% fetal bovine serum and 1% penicillin-streptomycin, incubated at 37°C in a 5% CO2 incubator. Cells were passaged once a week using a routine digestion process with 0.25% trypsin-EDTA solution. When cell saturation reached 80%-90% and the required number was achieved, cells were harvested and counted.

[0219] Cell seeding: 0.1 ml of 786-O cell suspension (containing 5 × 10⁶ cells) 6 100 cells (RPMI-1640:Matrigel volume ratio 1:1) were subcutaneously injected into the axilla of each mouse. On day 13 post-inoculation, mice were randomly assigned to groups based on tumor volume, with the grouping day being Day 0.

[0220] Tumor measurements and experimental indicators:

[0221] The tumor diameter was measured twice a week using calipers. The formula for calculating tumor volume is: V = 0.5axb 2 , where a and b represent the long and short diameters of the tumor, respectively. Mouse body weight was measured twice weekly.

[0222] The tumor-suppressive efficacy of the test drug was evaluated using the tumor growth inhibition rate (TGI) (%). TGI (%) = [(1 - (mean tumor volume at the end of treatment - mean tumor volume at the start of treatment) / (mean tumor volume at the end of treatment in the solvent control group - mean tumor volume at the start of treatment in the solvent control group)] x 100%.

[0223] Experimental results:

[0224] In the mouse subcutaneous xenograft tumor 786-O model, both ADC-L1-19-P1-12 and ADC-L1-19-P1-5, at a dose of 3 mg / kg, significantly inhibited tumor growth after a single intravenous administration (P<0.0001). The results are shown in Table 9 and Figure 7.

[0225] Table 9. Tumor volume in the 786-O subcutaneous tumor model.

[0226] Example 10: Screening of pH / buffer system for CDH6 naked cryopreservation formulation

[0227] Screening scheme

[0228] The CDH6 monoclonal antibody (naked antibody) used in Example 10 and Examples 11-12 below is CDH6-Ab-4 from Example 5, whose VH sequence is shown in SEQ ID NO:1 and its VL sequence is shown in SEQ ID NO:2. Specific sequences are shown in Tables 1 and 2. Four different buffer systems (acetic acid, citric acid, histidine, and phosphate, pH range from 4.5 to 7.5) were designed, resulting in 12 formulations F1-F12. Specific information on the candidate formulations is shown in Table 13. The CDH6 monoclonal antibody was replaced in F1-F12 by ultrafiltration concentration and buffer replacement, adjusting the protein concentration to 30 mg / mL. Accelerated stability testing at 40°C was conducted to comprehensively evaluate the stability of the CDH6 monoclonal antibody in the 12 candidate formulations, and the optimal pH / buffer system was selected for further development. The evaluation criteria included appearance, pH, protein concentration, dynamic light scattering (DLS) particle size distribution, thermal stability (DSF), diffusion coefficient (kD), SE-HPLC purity, CE-SDS (NR&R) purity, charge isomerism (iCIEF), and binding activity (ELISA-Binding). The evaluation protocols are detailed in Table 10.

[0229] Table 10 Screening formulations and screening protocols for pH / buffer systems Note: X = appearance, pH, protein concentration, DLS, SE-HPLC, CE-SDS (NR&R) and iCIEF; Y = ELISA relative binding activity; Z = DSF & kD.

[0230] pH / buffer system screening results

[0231] The main results of the pH / buffer system screening are shown in Tables 11-1 and 11-2. The results showed that visible particles appeared in both the citrate and phosphate buffer systems after 4 weeks at 40℃, while the acetate and citrate buffer systems exhibited larger DLS values ​​after 4 weeks at 40℃. Analysis results indicated that the histidine buffer system under accelerated conditions at 40℃ for 4 weeks was superior to other buffer systems in terms of purity, degradation, aggregation, and charge heterogeneity. Furthermore, the CDH6 monoclonal antibody remained relatively stable in all three pH systems (pH 5.5 / pH 6.0 / pH 6.5). Therefore, the histidine buffer system was considered the optimal buffer system, with pH 6.0 as the optimal pH value. The 20mM histidine-histidine hydrochloride buffer system at pH 6.0, i.e., formulation F8, was selected for the next step of excipient screening.

[0232] Table 11-1 Summary of pH / Buffer System Screening Results - 1

[0233] Table 11-2 Summary of pH / Buffer System Screening Results - 2 Note: ND = Not Detected; N / A means this test was not set at this sampling point. In the following tables, ND and N / A have the same meaning. High molecular weight represents aggregated high molecular weight impurities, low molecular weight represents fragmented low molecular weight impurities, and monomer represents monomeric active ingredients. In the following Examples 11-15 tables, high molecular weight, low molecular weight, and monomer have the same meaning as above.

[0234] Example 11: Screening of excipients for CDH6 naked cryopreservation formulation

[0235] Screening scheme

[0236] Based on the pH / buffer screening results, appropriate excipients were added to the optimal buffer system, including glycine, sodium chloride, sucrose, trehalose, mannitol, and sorbitol. Protein samples in liquid form (20 mg / mL) were subjected to 5 freeze-thaw cycles (-40°C to room temperature) or stored at 25°C for 4 weeks. Stability was assessed using appearance, pH, protein concentration, SEC-HPLC, CE-SDS (NR), CE-SDS (R) purity, CEX charge heterogeneity, dynamic light scattering (DLS) particle size distribution, and ELISA activity. See Table 12 for details.

[0237] Table 12 Screening Scheme for Excipients in Naked Antibiotic Formulations (Percentages are all mass-volume fractions) Note: X = Appearance, pH, protein concentration, DLS, SE-HPLC, CEX, CE-SDS (NR&R); Y = ELISA relative binding activity; Z = kD; T0 = starting point of investigation (the meaning of parameter T0 is the same in the tables of the following examples); W = week (the meaning of parameter W is the same in the tables of the following examples).

[0238] Filtering results

[0239] The screening results of excipients for naked antibody cryopreservation formulations are shown in Tables 13, 14-1, and 14-2. Based on the analytical test results, 8% sucrose showed superior performance compared to other formulations at 25°C and in freeze-thaw cycle studies in terms of purity, degradation, aggregation, and charge heterogeneity. The formulation containing 8% sucrose is tentatively selected as the next step for monoclonal antibody formulation confirmation.

[0240] Table 13 Results of the 25℃ stability test for screening excipients for naked antibiotic formulations Note: N / A indicates that this test item was not set for this sampling point.

[0241] Table 14-1 Results of Freeze-Thaw Experiment for Screening Excipients in Naked Antibiotic Formulations - 1

[0242] Table 14-2 Results of Freeze-Thaw Experiment for Screening Excipients in Naked Antibiotic Formulations - 2 (where the "conditions" correspond to those in Table 14-1) Note: N / A indicates that this test item was not set for this sampling point.

[0243] Example 12: Confirmation of CDH6 naked cryopreservation formulation

[0244] Naked cryopreservation formulation confirmation experiment

[0245] Experimental protocol

[0246] Samples containing 20 mg / mL CDH6 monoclonal antibody, 20 mM histidine-histidine hydrochloride (pH 6.0), and 8% (w / v) sucrose were prepared by centrifugation and medium exchange, and filled into 5 mL PC vials, 5 mL per vial. The stability of the CDH6 monoclonal antibody under long-term (-70℃), accelerated (2–8℃), pressure (25℃), and freeze-thaw (-40℃ to 25℃) conditions was studied. Appearance, pH, protein concentration, insoluble microparticles (MFI method), SEC-HPLC, CE-SDS (NR), CE-SDS (R) purity, CEX charge heterogeneity, and relative binding activity by ELISA were used as stability indicators to confirm the stability of the CDH6 monoclonal antibody formulation in a closed container system. The experimental protocol is shown in Table 15.

[0247] Table 15. Experimental Protocol for Validation of Naked Antibody Formulation Note: X = appearance, pH, protein concentration, SE-HPLC, CEX, CE-SDS (NR&R) and ELISA relative binding activity; Y = insoluble microparticles (MFI method) and osmolality; N / A is not set for this sampling point.

[0248] Experimental results

[0249] The results of the formulation confirmation experiment are detailed in Table 16. The results showed no significant changes in appearance, pH, protein concentration, osmolality, insoluble microparticles (MFI method), SEC-HPLC, CE-SDS (NR), CE-SDS (R) purity, CEX charge heterogeneity, and relative binding activity in ELISA. The sample remained stable within the predetermined time under long-term stability testing conditions of -70℃, accelerated testing conditions of 2–8℃, and pressure testing conditions of 25℃, as well as five freeze-thaw cycles from -40℃ to room temperature. Based on the formulation confirmation study, the cryopreservation formulation for the naked CDH6 antibody was determined to be 20 mg / mL CDH6 monoclonal antibody, 20 mM histidine-histidine hydrochloride, 8% sucrose (w / v), and pH 6.0.

[0250] Table 16 Results of stability testing for naked antibody formulations Note: RT = room temperature; ND = not detected; N / A means this test was not set at this sampling point.

[0251] Example 13: Screening scheme for pH / buffer system of anti-CDH6 antibody drug conjugate (hereinafter referred to as CDH6 ADC) formulation.

[0252] The CDH6 ADC used in Example 13 and Examples 14-15 below is ADC-L1-19-P1-12 from Example 6, with a specific structure shown in Table 3. Two different buffer systems (citric acid and histidine, pH range from 5.0 to 6.5) were designed, resulting in six formulations F1-F6. Specific information on the candidate formulations is shown in Table 17. The CDH6 ADC was replaced with the formulations in F1-F6 through ultrafiltration concentration and buffer replacement, adjusting the protein concentration to 40 mg / mL. Accelerated stability testing at 40°C was conducted to comprehensively evaluate the stability of the CDH6 ADC in the candidate formulations, and the optimal pH / buffer system was selected for further development. Evaluation indicators included appearance, pH, protein concentration, dynamic light scattering (DLS), SE-HPLC purity, CE-SDS (NR&R) purity, drug-antibody conjugation ratio (DAR) of the iCIEF (inductively coupled plasma) method, insoluble microparticles (MFI method), relative binding activity (ELISA-Binding), and osmolality. The detailed investigation plan is shown in Table 17.

[0253] Table 17 Screening schemes for pH and buffer systems in CDH6 ADC formulations Note: X = appearance, pH, protein concentration, DLS, SE-HPLC, iCIEF, CE-SDS(NR), and DAR; Y = insoluble microparticles (MFI method), relative binding activity (ELISA), and osmolar concentration.

[0254] Experimental results

[0255] The results of the screening experiments on pH and buffer systems for the CDH6 ADC formulation are shown in Table 18. Appearance results showed that the citrate buffer system exhibited a more pronounced opalescence than the histidine buffer system. Analytical tests indicated that under accelerated conditions at 40℃ for 4 weeks, both the histidine / histidine hydrochloride buffer system and the citrate buffer system achieved ADC stability in terms of purity, degradation, aggregation, and charge heterogeneity. However, the histidine system at pH 5.5 (i.e., group F4 in Table 17) performed better than other pH values. Therefore, the histidine buffer system at pH 5.5 (i.e., group F4 in Table 17) was selected for the next step of excipient screening.

[0256] Table 18 Results of screening experiments on pH and buffer systems for CDH6 ADC formulations. Note: N / A indicates that this test item was not set for this sampling point.

[0257] Example 14: Screening of excipients for CDH6 ADC formulation

[0258] Screening scheme

[0259] Based on the pH / buffer screening results, appropriate excipients were added to the optimal buffer system, including trehalose, proline, polysorbate 80, glycine, and arginine hydrochloride. Protein samples in liquid form (40 mg / mL) were stored at 25°C for one week. Stability was assessed using appearance, pH value, protein concentration, SEC-HPLC, CE-SDS (NR), CE-SDS (R) purity, iCIEF method for charge heterogeneity, dynamic light scattering (DLS) particle size distribution, and relative binding activity by ELISA. The screening protocol is detailed in Table 19.

[0260] Table 19 CDH6 ADC Formulation Excipient Screening Scheme Note: X = appearance, pH, protein concentration, DLS, SE-HPLC, CE-SDS (NR&R), iCIEF, osmolar concentration; Y = relative binding activity of ELISA; Z = kD and Tg, where Tg is the glass transition temperature.

[0261] Filtering results

[0262] The test results are detailed in Table 20. All the above formulations showed good performance in purity, degradation, aggregation, and charge heterogeneity during the 25℃ stability test. However, the kD values ​​of the four samples were -0.3 mL / g (formulation number F4-1), 1.5 mL / g (formulation number F4-2), 20.5 mL / g (formulation number F4-3), and -10.5 mL / g (formulation number F4-4), respectively. The results show that formulation number F4-3 had the highest kD value, possibly due to its strongest protein-protein repulsion and low tendency to form aggregates. Therefore, the optimal excipient system was selected as 20 mM histidine-histidine hydrochloride, 8% (w / v) trehalose, 0.8% (w / v) glycine, 0.04% (w / v) polysorbate 80, and pH 5.5.

[0263] Table 20 Results of excipient screening for CDH6 ADC formulations Note: N / A indicates that this test item was not set for this sampling point, and Tg' is Tg prime.

[0264] Example 15: CDH6 ADC Formulation Formulation Validation

[0265] Formulation confirmation experiment

[0266] Experimental protocol

[0267] Based on the results of pH / buffer and excipient screening, the target formulation composition for CDH6 ADC preparation was determined to be 20mM histidine-histidine hydrochloride at pH 5.5, 8% (w / v) trehalose, 0.8% (w / v) glycine, and 0.04% (w / v) polysorbate 80.

[0268] The CDH6 ADC was prepared into a sample containing 40 mg / mL CDH6 ADC, 20 mM histidine-histidine hydrochloride at pH 5.5, 8% (w / v) trehalose, 0.8% (w / v) glycine, and 0.04% (w / v) polysorbate 80 using ultrafiltration centrifugation and liquid exchange methods. The sample was filled into 20 mL amber vials, capped with 20 mm rubber stoppers and 20 mm aluminum caps; this was the final selected container closure system. The nominal volume before lyophilization was 5 mL / vial, and the actual volume was 5.42 mL / vial, with an overfill of 0.42 mL. After filling, the vials containing the sample were lyophilized. Stability studies were conducted on the lyophilized powder under long-term (2–8 °C), accelerated (25 °C), and pressure (40 °C) conditions to confirm the stability of the formulation in the final container closure system. See Table 21 for details.

[0269] Table 21 CDH6 ADC Formulation Stability Testing Protocol Note: X = appearance, moisture, appearance after reconstitution and visible foreign matter, pH, protein concentration, SE-HPLC, CE-SDS(NR), iCIEF, DAR and small molecule drug-related impurities (FDRIs); Y = insoluble particles (MFI method) and osmolality.

[0270] Experimental results

[0271] The results of the stability test for the formulation confirmation are shown in Table 22. Except for a slight decrease in the percentage of the icIEF main peak at 40℃, all other stability tests under all conditions, including appearance, moisture content, reconstitution time, reconstituted appearance, pH value, insoluble particles, SEC purity, non-reduced CE-SDS purity, DAR, FDRI, and relative binding activity, showed no significant changes. The sample remained stable within the predetermined long-term stability test conditions of 2–8℃. The final target formulation for the CDH6 ADC was determined to be 40 mg / mL CDH6 ADC, 20 mM histidine-histidine hydrochloride, 8% (w / v) trehalose, 0.8% (w / v) glycine, 0.04% (w / v) polysorbate 80, pH 5.5.

[0272] Table 22 Results of CDH6 ADC Formulation Validation Stability Tests Note: ND = Not detected, N / A means this test item was not set for this sampling point.

Claims

1. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises a pharmaceutically active ingredient and pharmaceutically acceptable excipients; the pharmaceutically active ingredient is selected from: (1) an antibody that specifically binds to CDH6 or an antigen-binding fragment thereof; or (2) an anti-CDH6 antibody-drug conjugate or a pharmaceutically acceptable salt thereof; the excipients comprise buffers, protein stabilizers and / or surfactants.

2. The pharmaceutical composition of claim 1, wherein, The antibody that specifically binds to CDH6 or its antigen-binding fragment 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, and / or the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein HCDR1-3 and / or LCDR1-3 are selected from the following combinations: (1) The HCDR1-3 is SEQ ID NO: 11-13; and / or the LCDR1-3 is SEQ ID NO: 14-16; (2) The HCDR1-3 is SEQ ID NO: 17-19; and / or the LCDR1-3 is SEQ ID NO: 20-22; (3) The HCDR1-3 is SEQ ID NO: 23-25; and / or the LCDR1-3 is SEQ ID NO: 26-28; or, The HCDR1-3 and / or the LCDR1-3 have at least 80% identity with each CDR in any of the groups (1)-(3), or are sequences with at most 3 insertion, deletion or substitution mutations; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that, The antibody-drug conjugate or pharmaceutically acceptable salt thereof has a general structure of Pc-(L-D) n , in, D is a cytotoxic drug; L represents the connecting subunit; Pc is the antibody or antigen-binding fragment thereof that specifically binds to CDH6 as described in claim 1 or 2; Furthermore, n is a real number from 1 to 16.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, The antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain variable region and / or 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, and / or 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, and / or 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, and / or the light chain variable region is the sequence shown in SEQ ID NO.6; or, The heavy chain variable region and / or the light chain variable region have at least 80% identity with the heavy chain variable region and / or the light chain variable region in any of the groups (1)-(3) above, or are sequences with at most 3 insertion, deletion or substitution mutations; preferably, the at least 80% identity is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, The antibody or antigen-binding fragment includes a heavy chain constant region sequence and / or a light chain constant region sequence. Optionally, the heavy chain constant region and / or light chain constant region are selected from a complete constant region sequence or a fragment thereof, and the constant region fragment includes CH1, a hinge region, CH2, CH3, or Fc. Optionally, the heavy chain constant region is selected from the human or mouse IgG1, IgG2, IgG3, or IgG4 constant region, and the light chain constant region is selected from the human or mouse kappa constant region or lambda constant region. Optionally, the antibody or antigen-binding fragment includes a complete heavy chain and a light chain, the heavy chain consisting of the VH and the heavy chain constant region having the sequence shown in SEQ ID NO:9, and the light chain consisting of the VL and the light chain constant region having the sequence shown in SEQ ID NO:

10.

6. The pharmaceutical composition according to any one of claims 1-5, characterized in that, The antibody or antigen-binding fragment is: (1) Chimeric antibody or fragment thereof; (2) Humanized antibodies or fragments thereof; and / or, (3) Fully human antibodies or fragments thereof; Optionally, the antibody or antigen-binding fragment is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, full-length antibodies, antibody fragments, naked antibodies, conjugated antibodies, humanized antibodies, fully human antibodies, Fab, Fab', F(ab')2, Fd, Fv, scFv, diabody, or single-domain antibodies.

7. The pharmaceutical composition according to any one of claims 3-6, characterized in that, The cytotoxic drug D is selected from chemotherapy drugs or antibiotics, and optionally, the cytotoxic drug D is selected from DNA topoisomerase inhibitors.

8. The pharmaceutical composition according to any one of claims 3-7, characterized in that, the cytotoxic drug D is selected from the group consisting of compounds of formula (D-I), in, R 1 , R 2 with the atom to which they are attached form a 5-6 membered heterocyclic ring containing 1 or 2 oxygen atoms as ring atoms, which is optionally substituted by one or more D atoms; R 4 selected from H or C1-C3alkyl; R 5 selected from H, halogen, CN, OH, NH2, or Ci-C3alkyl; R 6 selected from H or C1-C3alkyl; R 7 is selected from H, C1-C3 alkyl or C3-C6 cycloalkyl, said C1-C3 alkyl or C3-C6 cycloalkyl being optionally substituted by D, halogen, CN, =0, OH, NH2or C1-C3 alkyl.

9. The pharmaceutical composition according to claim 8, characterized in that, R 1 R 2 with the atom to which they are attached 10. The pharmaceutical composition according to claim 8 or 9, characterized in that, R 4 , R 5 , R 6 are each selected from H.

11. The pharmaceutical composition according to any one of claims 8-10, characterized in that, R 7 selected from cyclopropyl.

12. The pharmaceutical composition according to any one of claims 8-11, characterized in that, The compound of formula (D-I) is selected from one of the following compounds:

13. The pharmaceutical composition according to any one of claims 3-12, characterized in that, The connecting subunit L is selected from Its a-terminus is covalently linked to the antibody unit Pc, and its b-terminus is covalently linked to the cytotoxic drug D, where m1 is selected from integers 2 to 8, and L... 1 The peptide residues are selected from 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 groups.

14. The pharmaceutical composition according to claim 13, characterized in that, The L 1 is a Gly-Gly-Phe-Gly tetrapeptide residue.

15. The pharmaceutical composition according to any one of claims 3-14, characterized in that, The connecting subunit L is Its a-end is covalently linked to the antibody unit Pc, and its b-end is covalently linked to the drug unit D.

16. The pharmaceutical composition according to claim 3, characterized in that, The antibody-drug conjugate or its pharmaceutically acceptable salt is selected from the following antibody-drug conjugates or their pharmaceutically acceptable salts:

17. The pharmaceutical composition according to any one of claims 1-16, characterized in that, The concentration of the active pharmaceutical ingredient is from 0.1 mg / ml to 100 mg / ml; preferably 20-40 mg / mL; more preferably 20 mg / mL or 40 mg / mL.

18. The pharmaceutical composition according to any one of claims 1-17, characterized in that, The buffer solution is selected from acetate-sodium acetate buffer, citrate-sodium citrate buffer, histidine-histidine hydrochloride buffer, and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer; preferably, histidine-histidine hydrochloride buffer.

19. The pharmaceutical composition according to any one of claims 1-18, characterized in that, The concentration of the buffer solution is 10-100 mM, preferably 15-30 mM, and more preferably 20 mM.

20. The pharmaceutical composition according to any one of claims 1-19, characterized in that, The protein stabilizer is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol, sorbitol or a combination thereof; preferably sucrose, trehalose, glycine or a combination thereof.

21. The pharmaceutical composition according to any one of claims 1-20, characterized in that, The concentration of the protein stabilizer is 0.1-10% (w / v); preferably 0.8% (w / v), 2% (w / v), 4.5% (w / v), 8% (w / v) or 9.5% (w / v); more preferably 0.8% (w / v) or 8% (w / v); most preferably 8% (w / v) sucrose, 8% (w / v) trehalose and 0.8% (w / v) glycine.

22. The pharmaceutical composition according to any one of claims 1-21, characterized in that, The surfactant is selected from polysorbate, poloxamer, and glycerol fatty acid esters; preferably polysorbate; more preferably polysorbate 80.

23. The pharmaceutical composition according to any one of claims 1-22, characterized in that, The concentration of the surfactant is 0.01 to 0.1% (w / v); preferably 0.02% (w / v), 0.04% (w / v), 0.06% (w / v) or 0.08% (w / v); more preferably 0.04% (w / v).

24. The pharmaceutical composition according to any one of claims 1-23, characterized in that, The pH of the pharmaceutical composition is about 4.5-7.5; preferably 4.5-6.5; more preferably 5.5 or 6.

25. The pharmaceutical composition according to any one of claims 1-24, characterized in that, The pharmaceutical composition comprises: a) The antibody or its antigen-binding fragment that specifically binds to CDH6 at a concentration of 0.1 mg / ml to 100 mg / ml. b) A 10-100 mM histidine-histidine hydrochloride buffer solution, pH 4.5-7.5, and c) Sucrose with a concentration of 1-10% (w / v); Preferably, the pharmaceutical composition comprises: a) The antibody or its antigen-binding fragment that specifically binds to CDH6 at a concentration of 10 mg / ml to 50 mg / ml. b) A 15-30 mM histidine-histidine hydrochloride buffer solution, pH 5.5-6.5, and c) Sucrose with a concentration of 5-9% (w / v); More preferably, the pharmaceutical composition comprises: a) The antibody or its antigen-binding fragment that specifically binds to CDH6 at a concentration of 20 mg / ml. b) A 20 mM histidine-histidine hydrochloride buffer solution at pH 6, and c) Sucrose with a concentration of 8% (w / v); Preferably, the pharmaceutical composition is in liquid form or a lyophilized powder formulation.

26. The pharmaceutical composition according to any one of claims 1-24, characterized in that, The pharmaceutical composition comprises: a) The anti-CDH6 antibody-drug conjugate or a pharmaceutically acceptable salt thereof at a concentration of 0.1 mg / ml to 100 mg / ml. b) A 10-100 mM histidine-histidine hydrochloride buffer solution, pH 4.5-7.5, and c) Trehalose at a concentration of 1-10% (w / v) d) 0.1-2% (w / v) glycine, f) 0.01-0.1% (w / v) Polysorbate 80; Preferably, the pharmaceutical composition comprises: a) The anti-CDH6 antibody-drug conjugate or a pharmaceutically acceptable salt thereof at a concentration of 20 mg / ml to 80 mg / ml. b) A 15-30 mM histidine-histidine hydrochloride buffer solution, pH 5-6, and c) Trehalose at a concentration of 5-10% (w / v), d) 0.1-1% (w / v) glycine, f) 0.01-0.05% (w / v) Polysorbate 80; More preferably, the pharmaceutical composition comprises: a) The anti-CDH6 antibody-drug conjugate or a pharmaceutically acceptable salt thereof at a concentration of 40 mg / ml. b) A 20 mM histidine-histidine hydrochloride buffer solution, pH 5.5, and c) Trehalose at a concentration of 8% (w / v) d) 0.8% (w / v) glycine, f) 0.04% (w / v) Polysorbate 80; Preferably, the pharmaceutical composition is in liquid form or a lyophilized powder formulation.

27. A method for preparing the pharmaceutical composition according to any one of claims 1-26, characterized in that, The method includes a step of replacing the solution of the active pharmaceutical ingredient with a buffer solution, wherein the buffer solution is preferably histidine-histidine hydrochloride buffer, the concentration of the buffer solution is preferably 20 mM, and the pH of the buffer solution is preferably 6 or 5.5; preferably, the preparation method further includes a freeze-drying step.

28. A pharmaceutical composition comprising an anti-CDH6 antibody or an antibody-drug conjugate (ADC) thereof, characterized in that, The pharmaceutical composition is prepared by the method of claim 27.

29. A lyophilized formulation comprising an anti-CDH6 antibody or an antibody-drug conjugate (ADC) thereof, characterized in that, The formulation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1-26, 28.

30. A reconstituted solution comprising an anti-CDH6 antibody or an antibody-drug conjugate (ADC) thereof, characterized in that, The reconstituted solution is prepared by reconstituted the lyophilized formulation of claim 29.

31. Use of the pharmaceutical composition according to any one of claims 1-26, 28, the pharmaceutical composition prepared by the method of claim 27, the lyophilized formulation of claim 29, or the reconstituted solution of claim 30 in the preparation of a medicament for the prevention and / or treatment of a disease in an individual; preferably, the disease is a tumor; more preferably, the tumor is a tumor expressing CDH6; most preferably, the tumor is selected from the group consisting of: ovarian cancer, renal cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and bile duct epithelial cancer.

32. A method for preventing and / or treating a disease in an individual, comprising administering to a patient in need a pharmaceutical composition according to any one of claims 1-26, 28, a pharmaceutical composition prepared by the method of claim 27, a lyophilized formulation according to claim 29, or a reconstituted solution according to claim 30; preferably, the disease is a tumor; more preferably, the tumor is a tumor expressing CDH6; most preferably, the tumor is selected from the group consisting of: ovarian cancer, renal cancer, liver cancer, soft tissue cancer, central nervous system cancer, thyroid cancer, and bile duct epithelial cancer.

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