WO2023186015A1 - B7h4抗体药物偶联物及其用途 - Google Patents

B7h4抗体药物偶联物及其用途 Download PDF

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WO2023186015A1
WO2023186015A1 PCT/CN2023/085097 CN2023085097W WO2023186015A1 WO 2023186015 A1 WO2023186015 A1 WO 2023186015A1 CN 2023085097 W CN2023085097 W CN 2023085097W WO 2023186015 A1 WO2023186015 A1 WO 2023186015A1
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Prior art keywords
antibody
drug conjugate
pharmaceutically acceptable
antibody drug
isomer
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PCT/CN2023/085097
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English (en)
French (fr)
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朱忠远
钟琛
张禹
周蕴华
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映恩生物制药（苏州）有限公司
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Publication of WO2023186015A1 publication Critical patent/WO2023186015A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • the present invention provides antibody drug conjugates that specifically bind to B7H4 and compositions containing the same. Methods and uses of the antibody drug conjugates of the invention are also provided.
  • Immune checkpoint inhibitors are the most studied form of immunotherapy for cancer. Immune checkpoint molecules are often highly expressed in the tumor microenvironment. By inhibiting T cell activation and inducing T cell exhaustion, tumors can evade immune system attack.
  • the B7 family and the TNF family are two major costimulatory molecule families.
  • the B7 family currently has 10 molecules, namely CD80 (B7.1), CD86 (B7.2), B7H1 (PD-L1/CD274), B7- DC(PD-L2/CD273), B7H2(ICOSL), B7H3(CD276), B7H4(B7S1/B7x/Vtcn1), B7H5(VISTA), B7H6 and B7H7(HHLA2).
  • Multiple members of the B7 family and their receptors have been proven to be immune checkpoints, such as PD-L1/PD1, CTLA4, and VISTA.
  • B7H4 is a relatively new member of the B7 family. Although it is widely expressed in cells of the body at the mRNA level, its expression at the protein level is very limited. It is only expressed on some ductal epithelial cells of the body, such as breast ducts and lobules, fallopian tube epithelium, and endometrium. and other tissues have low levels of expression. In contrast, B7H4 is abundantly expressed in a variety of tumor tissues, such as breast cancer, especially triple-negative breast cancer, ovarian cancer, and endometrioma. From the perspective of expression profile, B7H4 can be considered a highly specific tumor-associated antigen. On the other hand, B7H4 is a new immune checkpoint molecule.
  • B7H4 inhibits the proliferation, activation and cytokine production of T cells by interacting with its unknown T cell surface receptor.
  • Tumor cells achieve immune evasion by inhibiting the activation of T cells through high expression of B7H4 molecules and suppressive macrophages that highly express B7H4 molecules in the tumor microenvironment.
  • the expression profile of B7H4 on tumors does not overlap with that of PD-L1.
  • Treatment by targeting B7H4 with antibodies and reactivating the immune system by blocking the negative regulatory effect of B7H4 is a promising method for treating B7H4-positive tumors.
  • Antibody-drug conjugates already on the market include Adcetris and Kadcyla.
  • Monoclonal antibodies or drug conjugates targeting B7-H4 to improve the patient's own immune system response to tumors and achieve the purpose of directly killing tumor cells.
  • Related patents include W02013025779, US20140322129, etc.
  • Anti-B7-H4 monoclonal antibodies from companies such as Medimmune and FivePrime are currently in preclinical development; Genentech's anti-B7-H4 antibody-drug conjugate is also in the preclinical development stage.
  • the technical problem to be solved by the present invention is to overcome the shortcoming of few anti-B7H4 antibody drug conjugates in the prior art and provide An anti-B7H4 antibody drug conjugate and its preparation method and application.
  • the anti-B7H4 antibody-drug conjugate of the present invention has one or more advantageous effects selected from the following group: (1) better inhibitory activity on tumor cell proliferation in vitro; (2) better Endocytic effect; (3) Better anti-tumor effect in vivo; (4) Better affinity to B7H4 in humans and monkeys; (5) Better targeting of B7H4; (6) Better bystander killing effect; (7) better plasma stability; (8) better safety.
  • the present invention mainly solves the above technical problems through the following technical means.
  • the present application provides an anti-B7H4 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof.
  • the structure of the anti-B7H4 antibody drug conjugate is as shown in formula (I): Ab-(LMD) p (I)
  • D is a cytotoxic drug
  • p represents the average number of connections, and p is selected from an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-B7H4 antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region includes amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ respectively.
  • HCDR1, HCDR2 and HCDR3 shown in ID NO:3 and the light chain variable region includes the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
  • the present application provides an anti-B7H4 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof.
  • the structure of the anti-B7H4 antibody drug conjugate is as shown in formula (I): Ab-(LMD) p (I)
  • -M-D is a cytotoxic drug
  • p represents the average number of connections, and p is selected from an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-B7H4 antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region includes amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ respectively.
  • HCDR1, HCDR2 and HCDR3 shown in ID NO:3 and the light chain variable region includes the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
  • the present application provides an anti-B7H4 antibody-drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-B7H4 antibody-drug conjugate has a structure such as formula (I-1) Shown:
  • M is -L 2 -L 1 -C(O)-;
  • L 2 is -O- or -S-;
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -, C 3 -C 6 saturated cycloalkyl or 3-6 membered saturated heterocyclyl, the C 3 -C 6 saturated
  • the cycloalkyl group and the 3-6 membered saturated heterocyclyl group are each independently optionally substituted by one or more R 2a ;
  • n is selected from 1, 2, 3 or 4; the heteroatoms in the 3-6-membered saturated heterocyclic group are selected from N, O and S, and the number of heteroatoms is 1-3;
  • R 1a is each independently selected from hydrogen, halogen, hydroxyl, amino and C 1 -C 6 alkyl, which C 1 -C 6 alkyl is optionally substituted by one or more R;
  • R 1b and R 2a are each independently selected from hydrogen, halogen, hydroxyl, amino and C 1 -C 6 alkyl, which C 1 -C 6 alkyl is optionally substituted by one or more R;
  • R is each independently hydrogen or halogen
  • p represents the average number of connections, and p is selected from an integer or decimal number from 1 to 10;
  • Ab is an anti-B7H4 antibody or an antigen-binding fragment thereof.
  • the average number of connections p is preferably any integer or decimal number from 3 to 8.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein R 1a is each independently selected from halogen, hydroxyl, amino and C 1 -C 6 alkyl, the C 1 -C 6 alkyl is optionally substituted by one or more R, each R is independently hydrogen or halogen.
  • the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention includes a heavy chain variable region and a light chain variable region
  • the heavy chain variable region includes an amino acid sequence such as SEQ ID NO: 1, SEQ HCDR1, HCDR2 and HCDR3 shown in ID NO:2 and SEQ ID NO:3
  • the light chain variable region includes amino acid sequences as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
  • the anti-B7H4 antibody or antigen-binding fragment thereof of the invention comprises: the amino acid sequence is as shown in SEQ ID NO: 7 or has at least 95%, 96%, 97%, 98% or 99% identity thereto.
  • the heavy chain variable region, and the amino acid sequence such as SEQ ID A light chain variable region shown in NO: 8 or having at least 95%, 96%, 97%, 98% or 99% identity thereto.
  • the anti-B7H4 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and a light chain with an amino acid sequence as shown in SEQ ID NO: 8 Variable area.
  • the anti-B7H4 antibody or antigen-binding fragment of the present invention is a murine antibody or fragment thereof, a chimeric antibody or antigen-binding fragment, a humanized antibody or antigen-binding fragment, or a fully human antibody or antigen-binding fragment. .
  • the anti-B7H4 antibody or antigen-binding fragment thereof of the invention is a humanized antibody or fragment thereof.
  • the anti-B7H4 antibody or antigen-binding fragment thereof of the invention is selected from Fab, Fab′, Fab′-SH, Fv, scFv, F(ab′) 2 , sdAb, diabody or linear antibody.
  • the anti-B7H4 antibodies of the invention are monoclonal antibodies.
  • the antibody of the invention is an antibody in the form of IgGl, IgG2, IgG3, or IgG4.
  • the antibodies of the invention are in the form of IgG1.
  • the anti-B7H4 antibody or antigen-binding fragment thereof of the invention comprises: the amino acid sequence is as shown in SEQ ID NO: 9 or has at least 95%, 96%, 97%, 98% or 99% identity therewith.
  • the anti-B7H4 antibody or antigen-binding fragment thereof of the invention includes a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, and a light chain with an amino acid sequence as shown in SEQ ID NO: 10.
  • the anti-B7H4 antibody of the invention is anti-B7H4 antibody DB1001.
  • the amino acid sequence of DB1001 is shown in the sequence listing.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is -(C(R 1a )(R 1b ) ) m -CH 2 -; R 1a is selected from: hydrogen, halogen and C 1 -C 6 alkyl; R 1b is selected from: hydrogen, halogen and C 1 -C 6 alkyl; for example, R 1a is selected from: halogen and C 1 -C 6 alkyl; R 1b is selected from: hydrogen, halogen and C 1 -C 6 alkyl.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is -(C(R 1a )(R 1b ) ) m -CH 2 -; R 1a is hydrogen or -CH 3 ; R 1b is selected from: hydrogen and -CH 3 ; for example, R 1a is -CH 3 ; R 1b is selected from: hydrogen and -CH 3 .
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is -(C(R 1a )(R 1b ) ) m -CH 2 -; m is 1 or 2.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is selected from:
  • the left side of the structural fragment is preferably connected to L2 .
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is a C 3 -C 6 saturated cycloalkyl group or 3-6 membered saturated heterocyclyl, the C 3 -C 6 saturated cycloalkyl and 3-6 membered
  • the saturated heterocyclyl groups are each independently optionally substituted with one or more R 2a , each R 2a being independently selected from: hydrogen, halogen, and C 1 -C 6 alkyl.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 2 and -C(O)- are connected at C 3 -C 6 saturated cycloalkyl or 3-6 membered saturated heterocyclyl on different atoms.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is optionally substituted by one or more R 2a C 3 -C 6 saturated cycloalkyl; R 2a is each independently selected from: hydrogen, halogen and C 1 -C 6 alkyl.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is a C 3 -C 6 saturated cycloalkyl group.
  • L 1 is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, preferably cyclobutyl.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is optionally 1, 2 or 3 R 2a replaced: R 2a is each independently selected from: hydrogen, halogen and C 1 -C 6 alkyl.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is selected from:
  • the anti-B7H4 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • M is -L 2 -L 1 -C(O)-;
  • L 2 is -O-
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 - or C 3 -C 6 saturated cycloalkyl, and the C 3 -C 6 saturated cycloalkyl is optionally replaced by one or more R 2a substitution;
  • n 1 or 2;
  • R 1a is each independently selected from hydrogen, halogen and C 1 -C 6 alkyl, which C 1 -C 6 alkyl is optionally substituted by one or more R;
  • R 1b and R 2a are each independently selected from hydrogen, halogen and C 1 -C 6 alkyl, which C 1 -C 6 alkyl is optionally substituted by one or more R;
  • Each R is independently hydrogen or halogen.
  • the anti-B7H4 antibody drug conjugates of the present invention isomers thereof, pharmaceutically acceptable salts thereof or mixtures thereof, wherein
  • M is -L 2 -L 1 -C(O)-;
  • L 2 is -O-
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 - or optionally substituted by 1, 2 or 3 R 2a :
  • n 1 or 2;
  • R 1a is each independently selected from halogen and C 1 -C 6 alkyl, which C 1 -C 6 alkyl is optionally substituted by one or more R;
  • R 1b and R 2a are each independently selected from hydrogen, halogen and C 1 -C 6 alkyl, which C 1 -C 6 alkyl is optionally substituted by one or more R;
  • Each R is independently hydrogen or halogen.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is:
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is:
  • the anti-B7H4 antibody drug conjugate of the invention wherein the cytotoxic drug choose from any of the following structures:
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is -L a -L b -L c -,
  • the -L a - is
  • W is -(C(R wa )(R wb )) wn -
  • Y is -(OCH 2 CH 2 ) yn -O yp -
  • Z is -(C(R za )(R zb )) zn ;
  • wn 1, 2, 3 or 6
  • 0 or 1 methylene unit of W is each independently -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )-, or -C(O)- substitute;
  • yn 0, 4 or 8
  • yp is 0 or 1
  • Each methylene unit of Z is independently replaced by -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )-, or -C(O)-;
  • -Cyr- is a 3 to 10-membered saturated cycloalkyl group, wherein -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R cx ;
  • each R wa , R wb , R za , R zb , R wx , R zx , and R cx are each independently hydrogen, halogen, -OR r or C 1-6 alkyl optionally substituted by R r ;
  • each R r is independently hydrogen, halogen or C 1-6 alkyl
  • the -L b - is a peptide residue composed of 2 to 7 amino acids
  • the peptide residue of -L b - is a peptide residue composed of amino acids selected from the following group: phenylalanine, glycine, Alanine, valine, citrulline, lysine, serine, glutamic acid, and aspartic acid; preferably, -L b - represents a peptide residue consisting of 2 to 4 amino acids
  • the peptide residue of -L b - is a peptide residue formed from amino acids selected from the group consisting of phenylalanine, glycine, alanine, valine, citrulline and lysine;
  • R L1 and R L2 are each independently selected from the following group: hydrogen, halogen, -OH and C 1-6 alkyl.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is -L a -L b -L c -,
  • the -L a - is
  • W is -(C(R wa )(R wb )) wn -
  • Y is -(OCH 2 CH 2 ) yn -O yp -
  • Z is -(C(R za )(R zb )) zn ;
  • wn 1, 2, 3 or 6
  • 0 or 1 methylene unit of W is each independently -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )-, or -C(O)- substitute;
  • yn 0, 4 or 8
  • yp is 0 or 1
  • Each methylene unit of Z is independently replaced by -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )-, or -C(O)-;
  • -Cyr- is a 3 to 10-membered saturated cycloalkyl group, wherein -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R cx ;
  • each R wa , R wb , R za , R zb , R wx , R zx , and R cx are each independently hydrogen, halogen, -OR r or C 1-6 alkyl optionally substituted by R r ;
  • each R r is independently hydrogen, halogen or C 1-6 alkyl
  • Said -L b - is selected from the following group:
  • R L1 and R L2 are each independently selected from the following group: hydrogen, halogen, -OH and C 1-6 alkyl.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L a - is Preferably
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein -L b - is Preferably
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -Lc- is
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein the linker unit L has its L a end connected to Ab, The L c end is connected to M.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein the linker unit L has its L a end connected to Ab, The L c end is connected to the joint unit M.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is
  • the anti-B7H4 antibody drug conjugate of the present invention wherein the linker unit L is
  • the anti-B7H4 antibody drug conjugate of the present invention has a structure shown in formula (II-A):
  • p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably, it is any integer or decimal from 3 to 8; Ab, L and M are respectively as defined in any embodiment of the present invention.
  • the anti-B7H4 antibody-drug conjugate of the present invention its isomer, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-B7H4 antibody-drug conjugate has a structure such as formula (II- 1) or (II-2):
  • p represents the average number of connections, and p is selected from an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • L 2 is -O- or -S-; preferably -O-;
  • X 1 is selected from C 3 -C 6 saturated cycloalkyl optionally substituted by 1, 2 or 3 R 2a ; preferably, it is optionally substituted by 1, 2 or 3 R 2a :
  • X 2 is selected from -(C(R 1a )(R 1b )) m -CH 2 -;
  • n 1 or 2;
  • R 1a is hydrogen, halogen or C 1 -C 6 alkyl optionally substituted by 1, 2 or 3 R; preferably halogen or C 1 -C 6 alkyl optionally substituted by 1, 2 or 3 R ;
  • R 1b or R 2a can each independently be hydrogen, halogen or C 1 -C 6 alkyl optionally substituted by 1, 2 or 3 R;
  • Each R independently can be hydrogen or halogen.
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-B7H4 antibody drug conjugate is selected from the following structural formula:
  • p represents the average number of connections, and p is selected from an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • the invention provides an anti-B7H4 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-B7H4 antibody drug conjugate is selected from:
  • p represents the average number of connections, and p is selected from an integer or a decimal of 1 to 10, preferably an integer or a decimal of 3 to 8.
  • the amino acid sequence of DB1001 is shown in the sequence listing. The average number of consecutive p is 3.89, or p is 5.4.
  • the average number of connections p in the present invention is selected from an integer or decimal number from 1 to 10.
  • the average number of connections p described in the present invention may be an integer or a decimal number from 2 to 8.
  • the average number of connections p may be an integer or a decimal number from 3 to 8.
  • the average number of connections p can be 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 Integers or decimals to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10.
  • the present invention provides a method for preparing an antibody-drug conjugate, which includes the following steps: under the action of a reducing agent, the antibody dissolved in the buffer and the linker-cell dissolved in the solvent The toxins are mixed to obtain the antibody drug conjugate.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-B7H4 antibody drug conjugate as described herein, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, and a pharmaceutically acceptable Acceptable carrier or excipient.
  • the invention provides an anti-B7H4 antibody-drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture or pharmaceutical composition thereof as described herein for use in the treatment and/or prevention of B7H4 Use in medicines to mediate diseases or disorders, preferably, the disease or disorder is B7H4-positive cancer.
  • the invention provides a method of treating and/or preventing a B7H4-mediated disease or disorder, comprising administering to a subject in need thereof an anti-B7H4 antibody drug conjugate, as described herein, isomer, a pharmaceutically acceptable salt thereof or a mixture or pharmaceutical composition thereof.
  • the disease or disorder is a cancer with high expression of B7H4.
  • the invention provides anti-B7H4 antibody drug conjugates, isomers thereof, pharmaceutically acceptable salts thereof or mixtures or pharmaceutical compositions thereof as described herein for use in treatment and/or prevention.
  • a B7H4-mediated disease or disorder Preferably, the disease or disorder is a B7H4-positive cancer.
  • the cancer of the invention is selected from the group consisting of breast cancer, ovarian cancer, and endometrioma.
  • the invention provides a pharmaceutical combination comprising an antibody drug conjugate as described herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described herein, and one or more additional of therapeutic agents.
  • the invention provides a kit comprising an antibody drug conjugate as described herein, or a pharmaceutical composition as described herein.
  • the term "about” generally refers to a variation within the range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% above or below the specified value. , 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • antibody refers to any form of antibody possessing the desired biological activity. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
  • the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies making up the population may be present except in small amounts. Identical except for naturally occurring mutations. Monoclonal antibodies are highly specific and target a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier “monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • full-length antibody refers to an immunoglobulin molecule that when naturally occurring contains four peptide chains: two heavy (H) chains (approximately 50-70 kDa in full length) and two light (L) chains (full length (approximately 25 kDa) are connected to each other through disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of an antibody may mediate binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • CDR refers to the complementarity determining regions within the variable sequences of an antibody.
  • the precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia (Chothia et al. (1989)) based on the three-dimensional structure of the antibody and the topology of the CDR loops.
  • antibody-binding fragment of an antibody (“parent antibody”) includes fragments or derivatives of the antibody, typically including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody that retains At least some binding specificity of the antibody.
  • antibody-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules, such as scFv; nanobodies and polypeptides formed from antibody fragments specific antibodies.
  • the binding fragment or derivative When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen-binding activity.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or greater of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as “conservative variants” or “functionally conserved variants” of the antibody).
  • chimeric antibody is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, where the first and second antibodies are from different species.
  • the variable domains are obtained from an antibody such as a rodent (a "parent antibody")
  • the constant domain sequences are obtained from a human antibody such that the resulting chimeric antibody induces in human subjects compared to the parent rodent antibody. Possibility of adverse immune response lower.
  • humanized antibody refers to antibody forms containing sequences from human and non-human (eg, mouse, rat) antibodies.
  • a humanized antibody contains substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the The framework (FR) region is the framework region of human immunoglobulin sequences.
  • the humanized antibody optionally can comprise at least a portion of a human immunoglobulin constant region (Fc).
  • halogen generally refers to fluorine, chlorine, bromine, iodine, for example, fluorine, chlorine.
  • alkyl generally refers to the residue derived from an alkane by removal of a hydrogen atom. Alkyl groups may be substituted or unsubstituted, substituted or unsubstituted.
  • alkyl generally refers to a saturated linear or branched aliphatic hydrocarbon radical having a residue derived from the removal of a hydrogen atom from the same carbon atom or two different carbon atoms of the parent alkane, which may contain 1 to Straight or branched chain radicals of 20 carbon atoms, for example containing 1 to 12 carbon atoms, for example alkyl groups containing 1 to 6 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, and the like.
  • Alkyl groups may be substituted or unsubstituted, substituted or unsubstituted, for example when substituted, the substituents may be substituted at any available point of attachment, which substituents may be independently optionally selected from alkyl groups , alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy , substituted by one or more substituents in heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo, for example, it can be hydrogen, protium, deuterium, tritium, halogen, -NO 2 , - CN
  • alkylene generally refers to a saturated linear or branched aliphatic hydrocarbon radical having 2 hydrogen atoms derived from the same carbon atom or two different carbon atoms of the parent alkane. residue, which may be a straight or branched chain group containing 1 to 20 carbon atoms.
  • methylene may refer to a residue derived from a group of 1 carbon atom by removing two hydrogen atoms. base. Methylene groups may be substituted or unsubstituted, substituted or unsubstituted; for example, alkylene groups containing from 1 to 12 carbon atoms, for example, from 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2 -) wait.
  • Alkylene groups may be substituted or unsubstituted, substituted or unsubstituted, for example when substituted, the substituents may be substituted at any available point of attachment, and the substituents may be independently optionally selected from alkanes.
  • substituents among radicals, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups and oxo groups for example, they can be hydrogen, protium, deuterium, tritium, halogen, -NO 2 , -CN, -OH, -SH, -NH 2 , -C(O)H, -CO 2 H, -C(O)C(O)H, -C(O)CH 2 C(O)H, - S(O)H, -S(O) 2 H, -C(O)NH 2 , -SO 2 NH 2 , -OC(O)
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where alkyl or cycloalkyl is as defined herein.
  • alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • the alkoxy group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkyl, Thio group, alkylamino group, halogen, mercapto group, hydroxyl group, nitro group, cyano group, cycloalkane base, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.
  • groups which are independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkyl, Thio group, alkylamino group, halogen, mercapto group, hydroxyl group, nitro group, cyano group, cycloalkane base, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloal
  • alkenyl generally refers to a straight or branched chain hydrocarbon radical containing one or more double bonds.
  • alkenyl groups include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, hexenyl, and the like.
  • C2-6 alkenyl groups with more than one double bond include butadienyl, pentadienyl, hexadienyl and hexatrienyl and branched forms thereof.
  • the position of the unsaturated bond (double bond) can be anywhere in the carbon chain.
  • Alkenyl groups may be substituted or unsubstituted.
  • alkenylene generally refers to a residue derived from the removal of two hydrogen atoms from a carbon atom of an alkene.
  • alkenylene groups may be substituted or unsubstituted.
  • alkynyl generally refers to an unsaturated linear or branched chain alkynyl group, such as ethynyl, 1-propynyl, propargyl, butynyl, etc. Alkynyl groups may be substituted or unsubstituted.
  • alkynylene generally refers to a residue derived from the removal of two hydrogen atoms from a carbon atom of an alkyne.
  • it may be an ethynylene group, a propynylene group, a propargylene group, a butylene group, or the like.
  • Alkynylene groups may be substituted or unsubstituted.
  • aryl generally refers to a residue derived from an aromatic ring by removal of a hydrogen atom.
  • aromatic ring may refer to a 6 to 14 membered all-carbon monocyclic or fused polycyclic ring (that is, a ring sharing adjacent pairs of carbon atoms) with a conjugated ⁇ electron system, which may be 6 to 10 members, such as benzene and Naphthalene.
  • the aromatic ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, where the ring attached to the parent structure is the aryl ring.
  • Aryl groups may be substituted or unsubstituted, and when substituted, the substituents may be one or more of the following groups, independently selected from the following group: alkyl, alkenyl, alkynyl, alkoxy, alkyl Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , and heterocycloalkylthio.
  • Aryl groups may be substituted or unsubstituted.
  • heteroaryl generally refers to a residue having a hydrogen atom removed from a carbon atom of a heteroaromatic ring.
  • heteroaromatic ring refers to a heteroaromatic system containing from 1 to 4 heteroatoms, from 5 to 14 ring atoms, where the heteroatoms may be selected from the group consisting of oxygen, sulfur and nitrogen.
  • the heteroaryl group can be 5 to 10 yuan, and can be 5 yuan or 6 yuan, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazole Key et al.
  • the heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, where the ring attached to the parent structure is the heteroaryl ring.
  • Heteroaryl groups may be optionally substituted or unsubstituted, and when substituted, the substituents may be one or more of the following groups, independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy group, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, ring Alkylthio, and heterocycloalkylthio. Heteroaryl groups may be substituted or unsubstituted.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably containing 3 to 12 carbon atoms, preferably Contains 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, more preferably 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl , cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyl includes spiro ring, fused ring and bridged ring cycloalkyl.
  • Cycloalkyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, and the substituents are preferably independently selected from the group consisting of hydrogen atoms, halogens, alkyl groups, Substituted with one or more substituents from alkoxy, haloalkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • partially unsaturated generally refers to a cyclic structure containing at least one double or triple bond between ring molecules.
  • partially unsaturated encompasses cyclic structures with multiple unsaturations, but is not intended to include aromatic or heteroaromatic rings as defined herein.
  • unsaturated means that a moiety has one or more degrees of unsaturation.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic light substituent, which contains 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen or heteroatoms of sulfur, and the remaining ring atoms are carbon.
  • it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably it contains 3 to 8 ring atoms, of which 1-3 are heteroatoms; more preferably it contains 3 to 6 ring atoms, of which 1-3 are heteroatoms; most preferably contain 5 or 6 ring atoms, of which 1-3 are heteroatoms.
  • Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclyl groups include spirocyclic, fused cyclic and bridged cyclic heterocyclyl groups.
  • the heterocyclyl ring can be fused to an aryl, heteroaryl or cycloalkyl ring, and the ring connected to the parent structure is a heterocyclyl.
  • Heterocyclyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, and the substituents are preferably independently selected from the group consisting of hydrogen atoms, halogens, alkyl groups, Substituted with one or more substituents from alkoxy, haloalkyl, hydroxyl, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • ring-forming atoms generally refers to atoms contained in a cyclic structure.
  • the ring-forming atom may be a carbon atom on a benzene ring or a nitrogen atom on a pyridine ring.
  • the ring atom may be substituted or unsubstituted.
  • each independently generally means that the variable applies to any one situation, regardless of the presence or absence of the same or different definitions of the variable in the same compound.
  • the variables may refer to the type and number of substituents of the compound or the types of atoms in the compound.
  • R when R appears 2 times in a compound and R is defined as "independently carbon or nitrogen," both R can be carbon, both R can be nitrogen, or one R can be carbon and the other R nitrogen.
  • a heterocyclic group optionally substituted by an alkyl group means that an alkyl group may but does not have to be present. This description may include the case where the heterocyclic group is substituted by an alkyl group and the case where the heterocyclic group is not substituted by an alkyl group. situation.
  • substituted generally means that one or more hydrogen atoms in a group, eg up to 5, eg 1 to 3 hydrogen atoms, are independently substituted with a corresponding number of substituents.
  • the substituents are only in their possible chemical positions, and a person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort.
  • an amino or hydroxyl group with a free hydrogen may be unstable when combined with a carbon atom with an unsaturated (eg, olefinic) bond.
  • the term 0 or more (e.g., 0 or more, 0 or 1, 0) methylene units are "replaced” generally refers to when the structure contains 1 or more
  • connection of group X to group Y generally can be in any orientation, and any orientation generally refers to the Two or more attachment sites can optionally be attached to the group Y or to the group Z.
  • the term "compound” generally refers to a substance having two or more different elements.
  • the compounds of the present application It can be an organic compound.
  • the compound of the present application can be a compound with a molecular weight of less than 500, a compound with a molecular weight of less than 1,000, a compound with a molecular weight of more than 1,000, or a compound with a molecular weight of more than 10,000 or more than 100,000.
  • a compound may also refer to a compound connected through chemical bonds.
  • it may be a compound in which one or more molecules with a molecular weight of less than 1,000 are connected through chemical bonds to biological macromolecules.
  • the biological macromolecules may be high polysaccharides, proteins, etc.
  • the compounds of this application may include compounds in which a protein is linked to one or more molecules with a molecular weight of less than 1,000, a compound in which a protein is linked to one or more molecules with a molecular weight of less than 10,000, or a compound in which a protein is linked to one or more molecules with a molecular weight of less than 10,000.
  • Compounds with less than 100,000 linked molecules are examples of compounds in which a protein is linked to one or more molecules with a molecular weight of less than 1,000, a compound in which a protein is linked to one or more molecules with a molecular weight of less than 10,000, or a compound in which a protein is linked to one or more molecules with a molecular weight of less than 10,000.
  • alkyl alkenyl
  • cycloalkyl and the like may be preceded by an identifier indicating the presence of the group in the particular case.
  • the number of atoms for example, C 1 -C 4 alkyl, C 3 -C 7 cycloalkoxy, C 1 -C 4 alkylcarbonylamino, etc., the subscript number after "C” indicates the number of atoms present in the group Number of carbon atoms.
  • C alkyl refers to an alkyl group with three carbon atoms (e.g., n-propyl, isopropyl); in C 1-10 , the members of the group can have any number falling within the range 1-10 of carbon atoms.
  • One or more hydrogen atoms in the group are independently substituted with a corresponding number of substituents.
  • the substituents are only in their possible chemical positions, and a person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort.
  • an amino or hydroxyl group with a free hydrogen may be unstable when combined with a carbon atom with an unsaturated (eg, olefinic) bond.
  • the compound or ligand-drug conjugate of the present application includes its tautomers, mesoforms, racemates, enantiomers, and/or diastereomers.
  • the term “diastereomer” generally refers to stereoisomers having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as melting point, boiling point, spectral properties and reactivity.
  • the terms “tautomer” or “tautomeric form” are used interchangeably and generally refer to structural isomers of different energies that are interconvertible through a low energy barrier.
  • protontautomers also known as protontropic tautomers
  • protontautomers include interconversions by proton migration, such as keto-enol isomerization and imine-enol isomerization. Amine isomerization.
  • Valence tautomers involve interconversions through the reorganization of some of the bonding electrons.
  • meso generally refers to atoms that contain asymmetry within the molecule but have an element of symmetry such that the total optical rotation within the molecule is zero.
  • racemate or “racemic mixture” refers to a composition consisting of equimolar amounts of two enantiomeric species.
  • the term "isomer" of a compound or ligand-drug conjugate generally includes tautomers, meso, racemates, enantiomers, diastereomers of the compound. conformation.
  • ligand-drug conjugate generally refers to a ligand linked to a biologically active cytotoxic drug through a stable linker unit.
  • ligand-drug conjugate can be an antibody-drug conjugate (ADC).
  • ADC can refer to the combination of a monoclonal antibody or an antibody fragment with a biological substance through a stable linking unit. active cytotoxic drugs.
  • the term "ligand” generally refers to a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell.
  • the role of the ligand can be to present the drug to the target cell population that binds to the ligand.
  • These ligands include but are not limited to protein hormones, lectins, growth factors, antibodies or others that can bind to cells, receptors and/or antigens. of molecules.
  • the ligand can be represented as Pc, and the ligand antigen forms a connection bond with the connecting unit through the heteroatom on the ligand.
  • the ligand can be an antibody or an antigen-binding fragment thereof (Ab), the antibody may be selected from chimeric antibodies, humanized antibodies, fully human antibodies or murine antibodies; the antibody may be a monoclonal antibody.
  • cytotoxic drug generally refers to toxic drugs that have chemical molecules within tumor cells that are powerful enough to disrupt their normal growth. Cytotoxic drugs can kill tumor cells at high enough concentrations.
  • the "cytotoxic drugs” may include toxins, such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants or animals, radioactive isotopes (such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 or radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and ribolytic enzymes, for example, they can be toxic drugs, including but not limited to camptothecin derivatives, for example, they can be camptothecin derivatives.
  • Dendrine derivative isatecan (chemical name: (1S, 9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H, 12H - Benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione).
  • cytotoxic drugs are also referred to as cytotoxins.
  • linker unit or "linker structure” usually refers to a chemical structural segment or bond that is connected to a ligand at one end and a cytotoxic drug at the other end. It can also be connected to other linkers and then connected to the cytotoxic drug.
  • the direct or indirect connection to the ligand may mean that the group is directly connected to the ligand through a covalent bond, or the ligand may be connected through a linker structure.
  • chemical structural fragments containing acid-labile linker structures eg, hydrazone
  • protease-sensitive linker structures eg, peptidase-sensitive linker structures
  • photolabile linker structures dimethyl linker structures
  • disulfide-containing linker structures can be used, or Bonds serve as joint structures.
  • a structure is "optionally linked to other molecular moieties” generally means that the structure is not linked to any other chemical structure, or that the structure is linked to one or more other chemical structures that are different from the structure (such as those used herein). (e.g., through a chemical bond or through a linker structure).
  • drug loading usually refers to the average amount of cytotoxic drugs loaded on each ligand, and can also be expressed as the ratio of the amount of cytotoxic drugs and antibodies.
  • the range of cytotoxic drug loading can be per ligand (Ab ) connects 0-12, for example 1-10 cytotoxic drugs.
  • the drug loading is expressed as Na, which can be an example of the average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • the drug loading capacity of each ADC molecule after the coupling reaction can be determined by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA tests and HPLC characterization.
  • certain atoms of the compounds of this application may occur in more than one isotopic form.
  • hydrogen may exist as protium ( 1H ), deuterium ( 2H ), and tritium ( 3H ), and carbon may occur naturally as three different isotopes ( 12C , 13C , and 14C ).
  • isotopes that may be incorporated into the compounds of the present application also include, but are not limited to, 15N , 18O , 17O, 18F , 32P , 33P , 129I , 131I , 123I , 124I , 125I , or the like of isotopes.
  • compounds of the present application may be enriched in one or more of these isotopes relative to their natural abundance.
  • isotopically enriched compounds may be used in a variety of applications.
  • substitution with heavy isotopes such as deuterium ( 2H ) may provide certain therapeutic advantages, which may be due to greater metabolic stability.
  • the natural abundance of deuterium ( 2H ) is approximately 0.015%. Therefore, for approximately every 6,500 hydrogen atoms in nature, there is one deuterium atom. Therefore, the deuterium-containing compounds of the present application have a deuterium abundance greater than 0.015% at one or more positions, as appropriate.
  • structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms.
  • compounds whose structures are consistent with the present application, except that the hydrogen atom is replaced by deuterium or tritium, or the carbon atom is replaced by carbon 13 or carbon 14, are within the scope of the present application.
  • the term "pharmaceutical composition” generally refers to a compound containing one or more compounds described in this application or their physiological/pharmaceutical properties. Mixtures of salts or prodrugs with other chemical components are used, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the pharmaceutical composition can facilitate the administration to the living body, facilitate the absorption of the active ingredients, and thereby exert biological activity.
  • the preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.
  • Pharmaceutical compositions may be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. The suspension may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents such as those mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a nontoxic parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol.
  • sterile fixed oil can be conveniently used as the solvent or suspending medium.
  • any blended fixed oil may be used including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid may be used in the preparation of injectables.
  • the term "pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” generally refers to a salt of a compound or ligand-drug conjugate of the present application, or a salt of a compound described in this application, Such salts can be safe and/or effective when used in mammals, and can have appropriate biological activity.
  • the antibody-antibody drug conjugate compound of the present application can form a salt with an acid.
  • Non-limiting examples include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pear Acid, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, acetate Sulfonate, benzenesulfonate, p-toluenesulfonate.
  • the term "pharmaceutically acceptable carrier” generally refers to a vehicle or vehicle for administering therapeutic agents, such as antibodies or polypeptides, genes and other therapeutic agents.
  • the term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and that can be administered without undue toxicity.
  • Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid aggregates and inactivated virus particles. These vectors are well known to those skilled in the art.
  • Pharmaceutically acceptable carriers in therapeutic compositions may include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, etc. may also be present in these carriers.
  • treatment and “treating” generally refer to methods of obtaining beneficial or desired results, including, but not limited to, therapeutic benefits.
  • Therapeutic benefits include, but are not limited to, eradication, suppression, reduction, or amelioration of the underlying disorder being treated. Additionally, therapeutic benefit is achieved by eradicating the suppression, reduction, or amelioration of one or more physiological symptoms associated with the underlying disorder, such that improvement is observed in the patient, but the patient may still suffer from the underlying disorder.
  • prevention and preventing generally refer to methods of obtaining beneficial or desired results, including but not limited to preventive benefits.
  • pharmaceutical compositions may be administered to patients who are at risk of developing a particular disease or to patients who report having one or more physiological symptoms of the disease, even if the disease has not yet been diagnosed.
  • the term "subject” or “patient” generally refers to a human being (i.e., male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent)) or an adult subject (e.g., young adult subject). humans, middle-aged or elderly humans)) and/or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, quail and/or turkeys.
  • a human being i.e., male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent)) or an adult subject (e.g., young adult subject). humans, middle-aged or elderly humans)) and/or other primates (
  • therapeutically effective dose refers to the ligand-drug conjugate of the present invention, when administered alone or in combination with other therapeutic drugs to cells, tissues or subjects, effectively prevent or improve a The amount of symptoms of one or more diseases or conditions or the development of that disease or condition.
  • a therapeutically effective dose also refers to a dose sufficient to result in an improvement in symptoms, such as an amount that treats, cures, prevents, or ameliorates a related medical condition or increases the rate of treatment, cure, prevention, or amelioration of such a condition.
  • active ingredients administered alone are administered to an individual, treatment
  • the therapeutically effective dose refers only to that ingredient.
  • a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether combined, administered sequentially, or administered simultaneously.
  • An effective amount of the therapeutic agent will result in an improvement in diagnostic criteria or parameters of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.
  • cancer is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancers may be benign (also called benign tumors), premalignant, or malignant. The cancer cells may be solid cancer cells or leukemia cancer cells. As used herein, the term “tumor” refers to one or more cells that contain cancer. The term “tumor growth” is used herein to refer to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.
  • B7-H4 or B7H4 refers to a member of the human B7 protein family, also known as CD276, which is a type I transmembrane protein with four Ig-like extracellular domains.
  • B7-H4 is one of the immune checkpoint proteins expressed on the surface of antigen-presenting cells or cancer cells, and has an inhibitory effect on the functional activation of T cells.
  • B7-H4 includes any variant or isoform of B7-H4 that is naturally expressed by a cell.
  • Antibodies of the invention may cross-react with B7-H4 from non-human species. Alternatively, the antibody may be specific for human B7-H4 and may not exhibit cross-reactivity with other species.
  • B7-H4 or any variant or isoform thereof, can be isolated from the cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques common in the art and described herein.
  • the anti-B7-H4 antibody targets human B7-H4 with a normal glycosylation pattern.
  • anti-B7H4 antibody refers to an antibody capable of binding to a B7H4 protein or fragment thereof with sufficient affinity such that the antibody can be used to target B7H4. diagnostic and/or therapeutic agents.
  • the CDR sequence of the antibody used in the drug conjugates, compositions, uses or methods of the invention includes the CDR sequence from the antibody PR008199 described in PCT/CN2021/102952.
  • the variable region sequence of the antibody used in the drug conjugates, compositions or uses of the invention includes the variable region sequence from the antibody PR008199 described in PCT/CN2021/102952.
  • the amino acid sequence of the antibody used in the drug conjugates, compositions, uses or methods of the invention includes the full-length amino acid sequence from antibody PR008199 described in PCT/CN2021/102952.
  • the anti-B7H4 antibody DB1001 (PR008199) or its antigen-binding fragment according to the present invention is prepared as described in PCT/CN2021/102952.
  • the CDR sequences of the antibodies used in the drug conjugates, compositions, uses or methods of the invention comprise the amino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.
  • HCDR1, HCDR2 and HCDR3 are shown, and LCDR1, LCDR2 and LCDR3 including the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively.
  • variable region sequence of the antibody used in the drug conjugates, compositions or uses of the invention includes a heavy chain variable region with an amino acid sequence such as SEQ ID NO: 7, and an amino acid sequence such as SEQ ID NO: 7
  • the antibody used in the drug conjugates, compositions, uses or methods of the invention includes a heavy chain with an amino acid sequence as set forth in SEQ ID NO: 9, and an amino acid sequence as set forth in SEQ ID NO: 10 of light chains.
  • the antibody used in the drug conjugates, compositions, uses or methods of the invention is anti-B7H4 antibody DB1001.
  • variable region of anti-B7H4 antibody DB1001 is as follows, and the CDR region is determined according to the IMGT numbering rules.
  • Antibodies of the invention may be produced using any suitable method for producing antibodies. Any suitable form of B7H4 can be used as an immunogen (antigen) to generate antibodies. By way of example and not limitation, any B7H4 variant or fragment thereof may be used as an immunogen. In some embodiments, hybridoma cells producing murine monoclonal anti-human B7H4 antibodies can be produced by methods well known in the art. Antibodies derived from rodents (e.g., mice) may cause unwanted antibody immunogenicity when used as therapeutics in vivo, and repeated use can lead to an immune response in the body against the therapeutic antibodies. Such immune responses may at least result in the loss of therapeutic efficacy. , and severe cases can lead to potentially fatal allergic reactions.
  • rodents e.g., mice
  • CDR complementarity-determining region
  • chimeric or humanized antibodies of the invention can be prepared based on the sequences of prepared murine monoclonal hybridoma antibodies.
  • DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques.
  • the chimeric B7H4 antibodies of the invention can be operably linked to hybridoma-derived immunoglobulin heavy and light chain variable regions and human IgG constant regions using methods known in the art (see, e.g., No. 4,816,567 belonging to Cabilly et al.), a chimeric heavy chain and a chimeric light chain were obtained.
  • the constant region comprised by the chimeric antibody of the invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.
  • the chimeric B7H4 antibodies of the present invention can be obtained by "mixing and matching" transfection of expression cells with chimeric light chain and chimeric heavy chain expression plasmids, and the B7H4 binding of such "mixed and matched" antibodies Testing can be performed using the binding assays described above and other conventional binding assays (eg, ELISA).
  • the mouse CDR region can be inserted into the human germline framework region using methods known in the art. See Winter et al., U.S. Patent Nos. 5,225,539 and Queen et al, U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370.
  • amino acid changes include amino acid deletions, insertions, or substitutions.
  • the anti-B7H4 antibodies or antigen-binding fragments thereof of the invention include those that have been mutated by amino acid deletions, insertions or substitutions, but are still at least identical to the above-mentioned antibodies (especially in the CDR regions depicted in the above-mentioned sequences). Those antibodies have an amino acid sequence that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.
  • an antibody of the invention has no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR region by amino acid deletion, insertion, or substitution when compared to the CDR region depicted in a specific sequence.
  • Fc region variants can be generated by introducing one or more amino acid modifications into the Fc region of the antibodies provided herein.
  • Fc region variants may comprise human Fc region sequences (eg, human IgG1, IgG2, IgG3, or IgG4 Fc regions) that contain amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are replaced with cysteine residues.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known and readily available in the art.
  • Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers.
  • water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinylpyrrolidone, -1,3-dioxane, poly-1,3,6-triane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol (such as glycerol), polyvinyl
  • PEG poly
  • the present application provides an anti-B7H4 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture form thereof, which may have one or more effects selected from the following group: (1) having Inhibitory activity on the proliferation of tumor cells in vitro; (2) Targeted inhibition; (3) Plasma stability; (4) Anti-tumor effect in vivo; (5) Bystander Effect; (6) has anti-transporter transport ability; (7) has in vivo tumor targeting ability; and (8) has good in vivo safety.
  • the present application provides an anti-B7H4 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the structure of the anti-B7H4 antibody drug conjugate is as shown in formula (I-1):
  • M is -L 2 -L 1 -C(O)-;
  • L 2 is -O- or -S-;
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -, C 3 -C 6 saturated cycloalkyl or 3-6 membered saturated heterocyclyl, the C 3 -C 6 saturated
  • the cycloalkyl group and the 3-6 membered saturated heterocyclyl group are each independently optionally substituted by one or more R 2a ;
  • n is selected from 1, 2, 3 or 4;
  • R 1a is each independently selected from hydrogen, halogen, hydroxyl, amino and C 1 -C 6 alkyl, which is optionally substituted by one or more R; preferably halogen or by 1, 2 Or 3 R optionally substituted C 1 -C 6 alkyl;
  • R 1b and R 2a are each independently selected from hydrogen, halogen, hydroxyl, amino and C 1 -C 6 alkyl, which C 1 -C 6 alkyl is optionally substituted by one or more R;
  • R is each independently hydrogen or halogen
  • p represents the average number of connections, and p is selected from an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-B7H4 antibody or an antigen-binding fragment thereof.
  • the anti-B7H4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence such as SEQ ID NO: 1, SEQ ID NO: 2, respectively. and HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 3, and the light chain variable region includes amino acid sequences such as LCDR1, SEQ ID NO: 5 and SEQ ID NO: 6 respectively.
  • the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention includes: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and an amino acid sequence as shown in SEQ ID NO: 8 Light chain variable region; more preferably, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention includes: a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, and an amino acid sequence as shown in SEQ ID NO: 10 light chain.
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -, R 1a Selected from: halogen, hydroxyl, amino and C 1 -C 6 alkyl optionally substituted by R, R 1b is selected from: hydrogen, halogen, hydroxyl, amino and C 1 -C 6 alkyl optionally substituted by R, m is selected from 1, 2, 3 or 4, each R can be independently hydrogen or halogen; preferably, R 1a is selected from: halogen and C 1 -C 6 alkyl, R 1b Selected from: hydrogen, halogen and C 1 -C 6 alkyl, m is selected from 1 or 2; preferably, R 1a is -CH 3 ; R 1b is selected from: hydrogen and -CH 3 , m is selected from 1 or 2; Preferably, L 1 is selected from:
  • L 1 is a C 3 -C 6 saturated cycloalkyl group or a 3-6 membered saturated heterocyclyl group
  • the C 3 -C 6 saturated cycloalkyl and 3-6 membered saturated heterocyclyl are each independently optionally substituted by one or more R 2a
  • R 2a is each independently selected from: hydrogen, halogen, hydroxyl , amino and C 1 -C 6 alkyl
  • L 1 is a C 3 -C 6 saturated cycloalkyl optionally substituted by one or more R 2a
  • each of R 2a is independently selected from: hydrogen, halogen and C 1 -C 6 alkyl
  • L 1 is C 3 -C 6 saturated cycloalkyl
  • L 1 is selected from
  • L 1 is selected from
  • M is -L 2 -L 1 -C(O)-;
  • L 2 is -O- or -S-; preferably -O-;
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 - or C 3 -C 6 saturated cycloalkyl, and the C 3 -C 6 saturated cycloalkyl is optionally replaced by one or more R 2a is substituted; preferably, L 1 is optionally substituted by 1, 2 or 3 R 2a :
  • n 1 or 2;
  • R 1a is each independently selected from halogen and C 1 -C 6 alkyl, which C 1 -C 6 alkyl is optionally substituted by one or more R;
  • R 1b and R 2a are each independently selected from hydrogen, halogen and C 1 -C 6 alkyl, which is optionally substituted by one or more R; R is each independently hydrogen or halogen .
  • M is selected from:
  • the anti-B7H4 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is
  • the anti-B7H4 antibody-drug conjugate of the present invention its isomer, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-B7H4 antibody-drug conjugate has a structure such as formula (II- 1) or (II-2):
  • L 2 is -O- or -S-; preferably, L 2 is -O-;
  • X 1 is selected from C 3 -C 6 saturated cycloalkyl optionally substituted by 1, 2 or 3 R 2a ; preferably, it is optionally substituted by 1, 2 or 3 R 2a :
  • X 2 is selected from -(C(R 1a )(R 1b )) m -CH 2 -;
  • n 1 or 2;
  • R 1a is halogen or C 1 -C 6 alkyl optionally substituted by 1, 2 or 3 R;
  • R 1b or R 2a may each independently be hydrogen, halogen or C 1 -C 6 alkyl optionally substituted by 1, 2 or 3 R;
  • R can each independently be hydrogen or halogen
  • n represents the average number of connections, and n is selected from an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 8;
  • Ab is an anti-B7H4 antibody or an antigen-binding fragment thereof, which includes a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region includes amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ respectively.
  • HCDR1, HCDR2 and HCDR3 shown in ID NO:3 and the light chain variable region includes the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.
  • the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention includes: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and a light chain with an amino acid sequence as shown in SEQ ID NO: 8 Variable region; more preferably, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention includes: a heavy chain with an amino acid sequence as shown in SEQ ID NO: 9, and a light chain with an amino acid sequence as shown in SEQ ID NO: 10 .
  • the average number of connections p described in the present invention may be an integer or a decimal number from 2 to 8.
  • the average number of connections p may be an integer or a decimal number from 3 to 8.
  • the average number of connections p may be an integer or a decimal number ranging from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.
  • compositions and pharmaceutical preparations are provided.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-B7H4 antibody drug conjugate as described herein, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, and a pharmaceutically acceptable Acceptable carrier or excipient.
  • anti-B7H4 antibody-drug conjugate provided by the present invention can be integrated with suitable carriers, excipients and Other agents are administered in combination to provide improved transfer, delivery, tolerance, etc.
  • composition refers to a formulation that allows the active ingredients contained therein to be present in a biologically active form and that does not contain additional ingredients that would have unacceptable toxicity to the subject administered the formulation.
  • It can be prepared by combining the anti-B7H4 antibody drug conjugate of the present invention or a pharmaceutically acceptable salt thereof with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol , A. Editor (1980)) are mixed to prepare pharmaceutical formulations containing the anti-B7H4 antibodies described herein, preferably in the form of aqueous solutions or lyophilized formulations.
  • compositions or preparations of the present invention may also contain one or more other active ingredients required for the particular indication being treated, preferably those having complementary activities that do not adversely affect each other.
  • the other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics, or various known anti-tumor or anti-cancer agents, in an appropriate amount effective for the intended use. Earth combination exists.
  • pharmaceutical compositions of the invention further comprise a composition encoding a polynucleotide encoding an anti-B7H4 antibody.
  • the invention provides a pharmaceutical combination comprising an antibody drug conjugate as described herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described herein, and one or more additional of therapeutic agents.
  • the invention provides a kit comprising an antibody drug conjugate as described herein or, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof or a pharmaceutical combination as described herein.
  • the substance preferably further comprises a drug delivery device.
  • the invention provides an antibody drug conjugate as described herein, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, or a pharmaceutical composition as described herein for use in the treatment of and / Or use in medicine to prevent B7H4-mediated diseases or disorders.
  • the disease or disorder is a B7H4-positive cancer.
  • the invention provides an antibody drug conjugate as described herein, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, or a pharmaceutical composition as described herein for use in the treatment of and /or prevent a B7H4-mediated disease or disorder, preferably, the disease or disorder is a B7H4-positive cancer.
  • the invention provides a method of treating and/or preventing a B7H4-mediated disease or disorder, comprising administering to a subject in need thereof an antibody drug conjugate, an isomer thereof, as described herein. body, a pharmaceutically acceptable salt thereof or a mixture thereof, or a pharmaceutical composition described herein.
  • the disease or disorder is a cancer with high expression of B7H4.
  • the cancer is selected from the group consisting of breast cancer, ovarian cancer, and endometrioma.
  • modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration. wait.
  • the invention provides for co-administering to a subject a therapeutically effective amount of one or more therapies (e.g., formula and/or other therapeutic agents).
  • the therapy includes surgical treatment and/or radiation therapy.
  • the methods or uses provided herein also include administering to the individual one or more therapies (eg, treatment modalities and/or other therapeutic agents).
  • therapies eg, treatment modalities and/or other therapeutic agents.
  • the antibody drug conjugates of the present invention, or pharmaceutically acceptable salts thereof, may be used alone or in combination with other therapeutic agents in therapy. For example, it can be co-administered with at least one additional therapeutic agent.
  • the anti-B7H4 antibody or its antigen-binding fragment is connected to a biologically active cytotoxic drug through a linker unit. After the antibody-drug conjugate is transferred to the tumor cell, the linker is cut off and freed out of the cell.
  • Toxic drugs H-M-D For example, after the antibody drug conjugate DB1001-X1 of the present invention is transferred to cells, the small molecule compound P-II-3 is released; after the antibody drug conjugate DB1001-X2 of the present invention is transferred to cells, the small molecule compound P-II-3 is released. III-30.
  • the cytotoxic drug of the present invention has significantly enhanced proliferation inhibitory activity on NCI-N87 cells, JIMT-1 cells, Colo205 cells and MDA-MB-231 cells, and can exert excellent anti-tumor effects.
  • the anti-B7H4 antibody drug conjugate of the present invention has a good endocytosis effect on B7H4-positive MDA-MB-468 cells, and is better than the reference ADC-1.
  • the antibody drug conjugate of the present application has significant proliferation inhibitory activity on B7H4-positive expressing cells MDA-MB-468, colorectal cancer HT29, human breast cancer MX-1, and endometrial cancer cell RL95-2.
  • the anti-B7H4 antibody-drug conjugate pair of the present invention can block the inhibition of T cells from different donors by B7H4-positive cells, and is better than the reference ADC-1.
  • the anti-B7H4 antibody-drug conjugate of the present invention has good stability in human, rat and monkey plasma in vitro. At the same time, due to the low plasma concentration and short half-life of the cytotoxic drug P-III-30, the systemic exposure of P-III-30 is lower, resulting in higher safety.
  • the anti-B7H4 antibody drug conjugate of the present invention is effective in MDA-MB-468 tumor-bearing mice, breast cancer cell MX-1 tumor-bearing mice, breast cancer cell MCF-7 tumor-bearing mice, and endometrial cancer cells RL95-2.
  • Tumor-bearing mice and ovarian cancer cell OVCAR-3 tumor-bearing mice have significant anti-tumor activity.
  • the present invention has good application prospects in B7H4-positive expression diseases (such as cancer).
  • FIG. 1 Evaluation of the efficacy of antibody drug conjugates on MDA-MB-468 tumor-bearing mice.
  • Figure 2 Species cross-reactivity of antibody-drug conjugates in human, cynomolgus monkey, and mouse B7H4-transfected HEK293T cells.
  • Figure 3 Binding specificity of antibody drug conjugates and cells overexpressing human B7 family proteins.
  • Figure 4 The integrated area of the fluorescence signal generated by the endocytosis of the antibody drug conjugate in MDA-MB-468 cells at different time points.
  • FIG. 5 Endocytosis efficiency of antibody drug conjugates in MDA-MB-468 cells.
  • Figure 6 In vitro proliferation inhibition rate of HT29-B7H4 by antibody drug conjugates.
  • Figure 7 Inhibitory efficiency of antibody drug conjugates, monoclonal antibodies, and loads (cytotoxic drugs) on tumor cell MX-1 proliferation in vitro (3D cell culture method).
  • Figure 8 Antibody drug conjugates, monoclonal antibodies, and loads (cytotoxic drugs) inhibit the proliferation of tumor cells RL95-2 in vitro (3D cell culture method).
  • FIG. 9 Antibody drug conjugates, monoclonal antibodies, and payloads (cytotoxic drugs) inhibit the proliferation of tumor cells JIMT-1 in vitro (3D cell culture method).
  • Figure 10 Activation of PBMC-derived T cells by antibody drug conjugates.
  • Figure 11 Activation of PBMC-derived T cells by antibody drug conjugates.
  • Figure 12 Bystander killing effect of antibody drug conjugate on HT29-Luc cells co-incubated with HT29-B7H4.
  • Figure 13 Release rate of load (cytotoxic drug) after antibody-drug conjugates were incubated in human, monkey, and rat plasma for 21 days in vitro.
  • Figure 14 Evaluation of the efficacy of antibody drug conjugates on breast cancer MX-1 tumor-bearing mice.
  • Figure 15 Evaluation of the efficacy of antibody drug conjugates on endometrial cancer RL95-2 tumor-bearing mice.
  • Figure 16 Evaluation of the efficacy of antibody drug conjugates on ovarian cancer OVCAR-3 tumor-bearing mice.
  • High performance liquid chromatography Waters e2965 high performance liquid chromatography system.
  • Mobile phase B 50mM K 2 HPO 4 (pH 7.0)/isopropyl alcohol (75:25 V/V);
  • Detection conditions Set the mobile phase flow rate to 1ml/min, the detection wavelength to 280nm, and the column temperature to 30°C.
  • Detection conditions Set the mobile phase flow rate to 0.5ml/min, the detection wavelength to 280nm, and the column temperature to 30°C.
  • the antibody of the present invention was prepared with reference to PCT/CN2021/102952.
  • the variable region amino acid sequence of anti-B7H4 antibody DB1001 (PR008199) is as follows, and the CDR region is determined according to Kabat numbering rules.
  • the drug loading capacity (DAR) of the antibody drug conjugate DB1001-X1 was 3.89, and the SEC purity was 100%.
  • Linker-payload X2 Dissolve Linker-payload X2 in dry DMA (N, N-Dimethylacetamide, dimethylacetamide, manufacturer: Sinopharm) to prepare a 10 mg/mL linker-payload DMA solution.
  • the drug loading capacity (DAR) of the antibody drug conjugate DB1001-X2 was 5.4, and the SEC purity was 98.1%.
  • Reference ADC-1 was prepared with reference to compound 34 (hu2F7-ixotecan) in WO2020244657A1.
  • CTG Luminescent Cell Viability Assay, Promega, Cat. No.: G7558
  • NCI-N87/JIMT-1/MBA-MB-231 is cultured in 10% FBS RPMI-1640 medium.
  • Cell preparation Take NCI-N87/JIMT-1/MBA-MB-231 cells in the logarithmic growth phase, wash them once with PBS, add 2-3ml trypsin for digestion for 2-3 minutes, and wait until the cells are completely digested. Add 10-15 ml of cell culture medium to elute the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, then add 10-20 ml of cell culture medium to resuspend the cells to make a single cell suspension.
  • Cell plating Mix the NCI-N87/JIMT-1/MBA-MB-231 single cell suspension, use cell culture medium to adjust the viable cell density to 6x10 4 cells/ml, and mix the density-adjusted cell suspension Mix well and add 50ul/well to a 96-well cell culture plate. The culture plates were incubated in an incubator for 18 hours (37°C, 5% CO2 ).
  • Compound preparation Dissolve the compound in DMSO and prepare a storage solution with an initial concentration of 10mM.
  • Sample addition operation Add the configured samples to be tested at different concentrations into the culture plate, with two duplicate wells for each sample.
  • the culture plates were incubated for 6 days in an incubator (37°C, 5% CO2 ).
  • Color development operation Take out the 96-well cell culture plate, add 50ul CTG reagent to each well, and incubate at room temperature for 10 minutes.
  • Plate reading operation Take out the 96-well cell culture plate, place it in a microplate reader, and use the microplate reader to measure chemiluminescence.
  • Data analysis Use Microsoft Excel and Graphpad Prism 5 to process and analyze the data.
  • Table 1 IC 50 values of the small molecule compounds in this application for inhibiting the proliferation of tumor cells in vitro
  • the toxin of the drug conjugate of the present application has significantly enhanced proliferation inhibitory activity on NCI-N87 cells, JIMT-1 and MDA-MB-231 cells.
  • CTG Luminescent Cell Viability Assay
  • NCI-N87/Colo205 is cultured in 10% FBS RPMI-1640 medium.
  • Cell preparation Take NCI-N87/Colo205 cells in the logarithmic growth phase, wash them once with PBS, add 2-3ml trypsin for digestion for 2-3 minutes, and after the cells are completely digested, add 10-15ml cell culture medium. Elute the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 ml of cell culture medium to resuspend the cells to make a single cell suspension.
  • Cell plating Mix the NCI-N87/Colo205 single cell suspension, use cell culture medium to adjust the viable cell density to 6x10 4 cells/ml, mix the density-adjusted cell suspension, and add 50ul/well. 96-well cell culture plate. Place the culture plate on the Cultivate in an incubator for 18 hours (37°C, 5% CO 2 ).
  • Compound preparation Dissolve the compound in DMSO and prepare a storage solution with an initial concentration of 10mM.
  • Sample addition operation Add the configured samples to be tested at different concentrations into the culture plate, with two duplicate wells for each sample.
  • the culture plates were incubated for 6 days in an incubator (37°C, 5% CO2 ).
  • Color development operation Take out the 96-well cell culture plate, add 50ul CTG reagent to each well, and incubate at room temperature for 10 minutes.
  • Plate reading operation Take out the 96-well cell culture plate, place it in a microplate reader, and use the microplate reader to measure chemiluminescence.
  • Data analysis Use Microsoft Excel and Graphpad Prism 5 to process and analyze the data.
  • the toxin of the drug conjugate of the present application has obvious proliferation inhibitory activity on NCI-N87 and Colo205 cells, and is significantly better than Reference Example 1.
  • the endocytic effect of the antibody drug conjugate of the present application targeting the B7H4 target on B7H4-expressing MDA-MB-468 cells was detected.
  • Cells were co-incubated with a fixed concentration of antibody drug conjugate (ADC) and the endocytosis indicator reagent pHrodo, and the antibody drug was evaluated by observing the fluorescence signal generated by pHrodo in the cells accompanying the entry of the antibody drug conjugate into the cells at different time points. Conjugate endocytosis ability.
  • MDA-MB-468 cells are cultured using 10% FBS Leibovitz's L-15 medium.
  • Cell preparation Take MDA-MB-468 cells in the logarithmic growth phase, wash them once with PBS, and digest them for 2-3 minutes. After the cells are completely digested, add 10-15ml of cell culture medium, and wash the digested cells. Take it off, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells to make a single cell suspension, and adjust the viable cell density to 3x10 5 cells/ml.
  • Cell plating Add 50ul/well to a 96-well cell culture plate. The culture plates were incubated in an incubator for 48 hours (37°C, 5% CO2 ).
  • Sample addition operation Incubate the antibody-drug conjugate to be tested with Fab-pHrodo to form a complex at a concentration of 120nM. Adjust the concentration with 5-fold gradient dilution and add 50ul/well to the cells. There are 8 concentrations in total, and two duplicate wells are set for each concentration.
  • Plate reading operation At the corresponding time point, take out the 96-well cell culture plate, digest the cells, and read the cell number and fluorescence values through FACS.
  • the antibody drug conjugate of this application has an endocytic effect on MDA-MB-468 cells with high B7H4 expression.
  • Example 5 In vitro proliferation inhibition test of tumor cells by antibody drug conjugates
  • CCG method Chemiluminescent cell viability assay
  • Cells in the logarithmic growth phase were collected and plated at a density of 2000 cells/well.
  • the cell plates were cultured overnight in a 37°C, 5% CO2 incubator.
  • DB1001-X1 and DB1001-X2 were diluted 5 times with complete culture medium to obtain 9 concentration gradients (starting with the highest concentration of 300nM).
  • 50 ⁇ L/well was added to the cell culture plate, and the complete culture medium was added.
  • As a blank control set up 2 duplicate wells; continue to incubate in a 37°C incubator for 7 days.
  • Blank control group (control group): saline
  • mice There were 5 mice in each group of vehicle control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.
  • T/C The percentage value of T/C (%) is an indicator that reflects tumor growth inhibition.
  • T and C represent the average tumor volume of the administration group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain administration group on a certain day , T 0 is the average tumor volume of the drug group at the beginning of drug administration; V i is the average tumor volume of the vehicle control group on a certain day (the same day as T i ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration. Average tumor volume.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain administration group on a certain day , T 0 is the average tumor volume of the drug group at the beginning of drug administration; V i is the average tumor volume of the vehicle control group on a certain day (the same day as T i ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration. Average tumor
  • Tumor volume is expressed as mean ⁇ standard error
  • Tumor volume is expressed as mean ⁇ standard error
  • the p value is calculated based on the tumor volume (p ⁇ 0.05 represents a statistical difference, p ⁇ 0.01 represents a significant difference).
  • PE-labeled secondary antibody PE anti-human IgG Fc Antibody
  • DB1001-X2 has similar affinity to human and cynomolgus B7-H4, but does not bind to mouse B7-H4.
  • Isotype ADC means: the antibody is a negative control antibody, the linker-cytotoxin is X2, and it is prepared with reference to DB1001-X2.
  • PE-labeled secondary antibody PE anti-human IgG Fc Anfibody
  • DB1001-X2 binds to human B7-H4, but no binding to other human B7 family proteins B7-H1, B7-DC, B7-H2, B7-H3, B7-H5, B7-1, B7-2 and B7-H7 combine.
  • DB1001-X2 can specifically bind to human B7-H4 protein and has no cross-reactivity with other B7 family proteins.
  • the endocytosis efficiency of the antibody drug conjugate of the present application targeting the B7H4 target on B7H4-expressing MDA-MB-468 cells was compared with other antibody drug conjugates targeting B7H4.
  • Cells and fixed concentrations of antibody drug conjugates and endocytosis indicator reagents Fabfluor-pH was co-incubated, and the endocytosis ability of the antibody drug conjugate was evaluated by continuously observing the fluorescence signal changes of living cells for 24 hours.
  • MDA-MB-468 cells are cultured using 10% FBS Leibovitz's L-15 medium.
  • Cell preparation Take MDA-MB-468 cells in the logarithmic growth phase, wash them once with PBS, and digest them for 2-3 minutes. After the cells are completely digested, add 10-15ml of cell culture medium, and wash the digested cells. Take it off, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells to make a single cell suspension, and adjust the viable cell density to 1x10 5 cells/ml.
  • Cell plating Add 50ul/well to a 96-well cell culture plate. Place the culture plate in an incubator and incubate overnight (37°C, 5% CO 2 ).
  • Capture analysis images Transfer the labeled working solution of the test product to the corresponding well of the experimental plate, transfer the experimental plate to the Incucyte live cell analysis equipment, set up the scanning and photographing program, and use the Incucyte live cell analysis system to obtain the image. Quantitation was performed at 1 hour intervals for 24 hours. The analysis results are expressed as: total fluorescence area ( ⁇ m 2 /image).
  • Figure 4 the integrated area of the fluorescence signal generated by the endocytosis of ADC-1 in MDA-MB-468 cells at different time points of the antibody drug conjugate DB1001-X2 of the present application and the control antibody drug conjugate was compared.
  • Figure 5 is a comparison of the endocytosis efficiency of the antibody drug conjugate DB1001-X2 of the present application and the control antibody drug conjugate in MDA-MB-468 cells.
  • the antibody drug conjugate of the present application has an endocytic effect in MDA-MB-468 cells expressing B7H4, and is better than the reference ADC-1.
  • Example 10 In vitro proliferation inhibition test of tumor cells by antibody drug conjugates (2D cell culture method)
  • Chemiluminescent cell viability detection method detects whether the antibody drug conjugate of the present application targeting the B7H4 target has an effect on the in vitro proliferation of human colorectal cancer HT29 overexpressing B7H4 compared with other antibody drug conjugates targeting B7H4. inhibitory effect.
  • Cells in the logarithmic growth phase were collected and plated at a density of 15,000 cells/well.
  • the cell plates were cultured overnight in a 37°C, 5% CO2 incubator.
  • dilute the test product with complete culture medium to obtain a final concentration of 10 nM.
  • Add 50 ⁇ L/well to the cell culture plate. Use the complete culture medium as a blank control and set up 3 duplicate wells; continue at 37°C. Incubate in the incubator for 7 days.
  • the antibody drug conjugate DB1001-X2 of the present application has significant proliferation inhibitory activity on human colorectal cancer cell HT29 overexpressing B7H4, and is better than the reference ADC-1.
  • Example 11 In vitro proliferation inhibition test of tumor cells by antibody drug conjugates (3D cell culture method)
  • Chemiluminescent cell viability assay evaluates the effects of anti-B7H4 ADC DB1001-X2 and anti-B7H4 monoclonal antibody DB1001 on human breast cell carcinoma MX-1 with high B7H4 expression under 3D culture conditions and in utero with low B7H4 expression.
  • Collect cells in the logarithmic growth phase add cell culture medium to resuspend the cells to make a single cell suspension and adjust the viable cell density to 1x105 cells/ml. Mix 3.5mL of cell suspension and 6.5mL of 1% methylcellulose evenly. Try to avoid the generation of air bubbles, add 90 ⁇ L of cell suspension to each well of a 96-well plate, and incubate overnight in a 37°C, 5% CO2 incubator . On the second day of the experiment, dilute the test product with complete culture medium to obtain a 10-fold solution. Add 10 ⁇ L of the test product solution to each well. The highest final concentration is 100 nM. There are 9 concentrations, 3-fold dilution. The complete culture medium is used as a blank control.
  • IsotypeADC The antibody is a negative control antibody, the linker-cytotoxin is X2, and it is prepared with reference to DB1001-X2.
  • DB1001-X2 has a strong in vitro killing effect on tumor cell lines with high and low expression of B7H4, but has no killing effect on cells that do not express B7H4, indicating that this The killing effect is dependent on B7H4 expression.
  • Example 12 Activation of T cells by antibody drug conjugates
  • the antibody drug conjugate of the present application targeting the B7H4 target is tested for its ability to block the inhibition of T cells by B7H4 and activate T cells to produce IFN-gamma compared with other antibody drug conjugates targeting B7H4.
  • Experimental method Collect logarithmically growing 293T-OS8-humanB7H4 cells (Kangyuan Bochuang Company) and centrifuge at 300g for 5 minutes.
  • Miltenyi T cell isolation kit (Miltenyi, CaT#130-096-535) according to the instruction manual to separate healthy donor 1 (Shanghai Miaoshun, product number PB100C-W, batch number A10Z983077) and healthy donor 2 PBMC (Shanghai Miaoshun, product number PB050C- W, lot number P122010104C) to obtain human primary T cells.
  • Isotype ADC The antibody is a negative control antibody, the linker-cytotoxin is X2, and it is prepared with reference to DB1001-X2.
  • DB1001-X2 can block the inhibition of T cells from different donors by B7H4-positive cells, and is significantly better than the reference ADC-1.
  • the antibody drug conjugates of the present application that target the B7H4 target are detected.
  • the small molecule drugs released after endocytosis diffuse from B7H4-positive cells to nearby B7H4-negative cells, and treat them. Produces cell-killing bystander effect.
  • HT29-B7H4 unlabeled HT29 cells overexpressing B7H4
  • HT29-Luc2 luciferase-transfected HT29 cells not expressing B7H4
  • Example 14 Stability of antibody drug conjugates in human, rat, and monkey plasma in vitro
  • Antibody drug conjugates were incubated with human, mouse, and monkey plasma for 21 days in vitro, and samples were taken at different time points. Liquid chromatography and mass spectrometry were used to measure the amount of small molecule toxins (cytotoxic drugs) released in the plasma to evaluate the antibody drug. Plasma stability of the conjugates.
  • DB1001-X2 was diluted with human, rat or cynomolgus monkey serum to a final concentration of 150 ⁇ g/mL, and incubated at 37°C for 21 days.
  • the intermediate sampling time points were T0, 2 hours, 8 hours, 1 day, 4 days, and 7 Days, 14 days, 21 days.
  • Plasma samples were precipitated with acetonitrile at a volume ratio of 1:1, centrifuged to obtain the supernatant, and then analyzed by LC-MS/MS (liquid phase: Thermo Vanquish; triple quadrupole mass spectrometry: Thermo TSQ Quantis).
  • Antibody drug conjugate release rates were calculated using cytotoxic drug P-III-30 concentrations.
  • DB1001-X2 has good stability in human, rat and monkey plasma in vitro.
  • Cynomolgus monkeys were intravenously administered DB1001-X2 once every 3 weeks for a total of 2 doses. Study its pharmacokinetic characteristics to provide reference for subsequent research.
  • cynomolgus monkeys half female and half male, weighing 2.2-3.6kg when grouped, were divided into 2 groups, namely DB1001-X2 low-dose group and DB1001-X2 high-dose group.
  • the dosages were set to 30 and 80 mg respectively. /kg, administered once every 3 weeks, for a total of 2 administrations, with a administration volume of 5mL/kg.
  • Sampling time Collect TK blood samples once before the first dose and 5min, 0.5h, 2h, 4h, 8h, 24h, 48h, 72h, 120h, 168h, 336h, and 504h after the end of the last dose; TK blood samples were collected once at 0.5h, 2h, 4h, 8h, 24h, 48h and before autopsy.
  • Sampling method Collect about 0.5mL of whole blood from the forelimb vein or other suitable vein.
  • Blood sample processing After collecting blood, place it in labeled blood collection tubes with EDTA ⁇ K2 as anticoagulant. Gently invert the sample tube several times to ensure mixing, and immediately store it in wet ice. And within 1 hour after collection, centrifuge at 3800 rpm for 10 minutes at 4°C, aspirate and aliquot 100 ⁇ L/tube of plasma (2 tubes), temporarily store in dry ice, transfer to ultra-low temperature refrigerator (-60°C and below) within 4 hours, and record in the test record Record the time of whole blood collection and plasma collection.
  • ultra-low temperature refrigerator 60°C and below
  • Sample analysis Use the established ELISA method to analyze the blood concentration of total antibodies and ADC drugs in the serum.
  • the plasma concentration of the cytotoxic drug P-III-30 in plasma was analyzed using an existing LC-MS/MS method.
  • Example 16 Evaluation of the efficacy of antibody drug conjugates on human breast cancer cell MX-1 tumor-bearing mice that highly express B7H4
  • Blank control group (control group): saline
  • mice 6-8 week old female BALB/c Nude mice, purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • mice There were 5 mice in each group in the control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • T/C The percentage value of T/C (%) is an indicator that reflects tumor growth inhibition.
  • T and C represent the average tumor volume of the administration group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain administration group on a certain day , T 0 is the average tumor volume of the drug group at the beginning of drug administration; V i is the average tumor volume of the vehicle control group on a certain day (the same day as T i ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration. Average tumor volume.
  • the independent sample T test (T-Test) was used to compare the two groups of samples, and the data was analyzed using SPSS. P ⁇ 0.05 was considered a significant difference.
  • the drawing software is GraphPad Prism.
  • Tumor volume is expressed as mean ⁇ standard error
  • the p value is calculated based on the tumor volume (p ⁇ 0.05 represents a statistical difference, p ⁇ 0.01 represents a significant difference).
  • the experimental results are shown in Figure 14 and Table 15.
  • the antibody drug conjugate DB1001-X2 showed significant dose-dependent inhibition after administration. tumor activity.
  • Example 17 Evaluation of the efficacy of antibody drug conjugates on human B7H4-positive breast cancer cells MCF-7 tumor-bearing mice
  • Blank control group (control group): saline
  • mice There were 5 mice in each group of vehicle control group or treatment group. The end point of the trial is the 25th day after grouping, and the tumor inhibition rate is calculated by measuring the tumor volume.
  • T/C The percentage value of T/C (%) is an indicator that reflects tumor growth inhibition.
  • T and C represent the average tumor volume of the administration group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain administration group on a certain day , T 0 is the average tumor volume of the drug group at the beginning of drug administration; V i is the average tumor volume of the vehicle control group on a certain day (the same day as T i ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration. Average tumor volume.
  • the independent sample T test (T-Test) was used to compare the two groups of samples, and the data was analyzed using SPSS. P ⁇ 0.05 was considered a significant difference.
  • the drawing software is GraphPad Prism.
  • the antibody drug conjugate DB1001-X2 of the present invention showed significant dose-dependent tumor inhibitory activity after a single administration.
  • Example 18 Evaluation of the efficacy of antibody drug conjugates on human endometrial cancer cell RL95-2 tumor-bearing mice with low expression of B7H4
  • Blank control group (control group): phosphate buffer saline (DPBS)
  • mice 6-8 week old female BALB/c Nude mice were purchased from Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd.
  • RL95-2 cells in the logarithmic growth phase were collected, and 5 ⁇ 106 RL95-2 cells were subcutaneously inoculated on the right side of the neck of each mouse.
  • the inoculation volume was 0.2 mL.
  • the cell suspension was PBS plus Matrigel (volume ratio: 1:1).
  • the drugs were randomly divided into groups according to the tumor volume, with 5 mice in each group.
  • the day of grouping was defined as day D0, and administration started on day D0 of grouping.
  • the test product was injected once every two weeks at a dose of 3 mg/kg for a total of 2 injections.
  • the endpoint of the test was the 28th day after grouping.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • T/C The percentage value of T/C (%) is an indicator that reflects tumor growth inhibition.
  • T and C represent the average tumor volume of the administration group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain administration group on a certain day , T 0 is the average tumor volume of the drug group at the beginning of drug administration; V i is the average tumor volume of the vehicle control group on a certain day (the same day as T i ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration. Average tumor volume.
  • Tumor volume is expressed as mean ⁇ standard error
  • the p value is calculated based on the tumor volume (**** represents p ⁇ 0.0001, * represents p ⁇ 0.05).
  • the experimental results are shown in Figure 15 and Table 16.
  • the antibody drug conjugate DB1001-X2 showed significant dose-dependent tumor inhibitory activity after administration.
  • Example 19 Evaluation of the efficacy of antibody drug conjugates on human ovarian cancer cell OVCAR-3 tumor-bearing mice with low expression of B7H4
  • Blank control group (control group): saline
  • mice 6-8 week old female BALB/c Nude mice, purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • OVCAR-3 cells were inoculated subcutaneously on the right back of 6-8 week old female BALB/c Nude mice, and the cells were resuspended in 1:1 PBS and Matrigel (0.2ml/mouse).
  • the tumor-bearing mice are randomly divided into StudyDirectorTM groups, and the test article is injected intravenously (iv) starting on the same day (day 0), once every two weeks, for a total of 2 injections, DB1001-
  • the doses used for X2 are 1mg/kg, 3mg/kg and 10mg/kg respectively.
  • the end point of the trial is the 27th day after grouping. Tumor volume and body weight are measured twice a week and the data are recorded.
  • mice There were 5 mice in each group in the control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • T/C The percentage value of T/C (%) is an indicator that reflects tumor growth inhibition.
  • T and C represent the average tumor volume of the administration group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain administration group on a certain day , T 0 is the average tumor volume of the drug group at the beginning of drug administration; V i is the average tumor volume of the vehicle control group on a certain day (the same day as T i ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration. Average tumor volume.
  • the independent sample T test (T-Test) was used to compare the two groups of samples, and the data was analyzed using SPSS. P ⁇ 0.05 was considered a significant difference.
  • the drawing software is GraphPad Prism.
  • Tumor volume is expressed as mean ⁇ standard error
  • the p value is calculated based on the tumor volume (p ⁇ 0.05 represents a statistical difference, p ⁇ 0.01 represents a significant difference).
  • the experimental results are shown in Figure 16 and Table 17.
  • the antibody drug conjugate DB1001-X2 showed significant dose-dependent tumor inhibitory activity after administration.

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Abstract

本发明提供了与B7H4特异性结合的抗B7H4抗体药物偶联物和包含其的组合物，其中所述抗B7H4抗体药物偶联物结构如式(I-1)所示。本发明还提供了使用本发明的抗体药物偶联物的方法和用途。本发明的抗B7H4抗体药物偶联物具有更好的对肿瘤细胞的体外增殖的抑制活性以及更好的体内抑瘤效果。

Description

B7H4抗体药物偶联物及其用途
本申请要求申请日为2022/3/30的中国专利申请202210334522X的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明提供了与B7H4特异性结合的抗体药物偶联物和包含其的组合物。还提供了使用本发明的抗体药物偶联物的方法和用途。
背景技术
免疫检查点抑制剂是肿瘤研究最多的免疫治疗形式。在肿瘤微环境中免疫检查点分子往往被高表达，通过抑制T细胞活化以及诱导T细胞耗竭，从而肿瘤可以逃避免疫系统的攻击。B7家族与TNF家族是两类主要的共刺激分子家族，B7家族目前有10个分子，分别为CD80(B7.1)、CD86(B7.2)、B7H1(PD-L1/CD274)、B7-DC(PD-L2/CD273)、B7H2(ICOSL)、B7H3(CD276)、B7H4(B7S1/B7x/Vtcn1)、B7H5(VISTA)、B7H6以及B7H7(HHLA2)。B7家族的多个成员及其受体已经被证明是免疫检查点，如PD-L1/PD1、CTLA4和VISTA等。
B7H4是比较新的B7家族成员，虽然在mRNA水平在机体细胞内广泛表达，但其蛋白水平的表达非常有限，仅在机体的部分导管上皮细胞上如乳腺导管和小叶、输卵管上皮、子宫内膜等组织有低水平表达。相反，B7H4在多种肿瘤组织中大量表达，比如在乳腺癌特别是三阴性乳腺癌、卵巢癌和子宫内膜瘤等肿瘤细胞上。从表达谱这一点上来讲，B7H4可以被认为是一个高特异性的肿瘤相关抗原。另一方面，B7H4是一种新的免疫检查点分子，体外实验证明B7H4通过与其未知的T细胞表面受体相互作用，抑制T细胞的增殖、活化以及细胞因子的产生。肿瘤细胞通过高表达B7H4分子，以及肿瘤微环境里高表达B7H4分子的抑制性巨噬细胞，抑制T细胞的活化从而实现免疫逃避。B7H4在肿瘤上的表达谱与PD-L1不重叠。通过抗体靶向B7H4的治疗，以及阻断B7H4的负调控作用重新活化免疫系统，是一种有前景的治疗B7H4表达阳性肿瘤的手段。
目前有多家制药公司在研发针对B7-H4的单克隆抗体，或与药物偶联物，或双特异性抗体。已经上市的抗体-药物偶联物有Adcetris和Kadcyla。目前有多家跨国制药公司在研发针对B7-H4的单克隆抗体或其药物偶联物，提高患者自身对肿瘤的免疫系统反应，并达到对肿瘤细胞进行直接杀伤的目的。相关专利如W02013025779、US20140322129等。Medimmune、FivePrime等公司的抗B7-H4单克隆抗体目前尚在临床前开发；基因泰克公司的抗B7-H4抗体-药物偶联物也已处在临床前开发阶段。
发明内容
本发明所要解决的技术问题是为了克服现有技术中抗B7H4抗体药物偶联物少的缺陷，而提供了 一种抗B7H4抗体药物偶联物及其制备方法和应用。本发明的抗B7H4抗体药物偶联物相较现有技术具有选自以下组的一种或多种优势效果：(1)更好的对肿瘤细胞的体外增殖的抑制活性；(2)更好的内吞效应；(3)更好的体内抑瘤效果；(4)与人和猴的B7H4亲和力更好；(5)对B7H4更好的靶向性；(6)更好的旁观者杀伤效应；(7)更好的血浆稳定性；(8)更好的安全性。
本发明主要是通过以下技术手段解决上述技术问题的。
一方面，本申请提供一种抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，所述抗B7H4抗体药物偶联物结构如式(I)所示：
Ab-(L-M-D)p   (I)
其中，
L和M是接头单元；
D是细胞毒性药物；
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab为抗B7H4抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。
一方面，本申请提供一种抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，所述抗B7H4抗体药物偶联物结构如式(I)所示：
Ab-(L-M-D)p   (I)
其中，
L和M是接头单元；
-M-D是细胞毒性药物；
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab为抗B7H4抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。
一方面，本申请提供一种抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体药物偶联物结构如式(I-1)所示：
其中，
M为-L2-L1-C(O)-；
L2为-O-或-S-；
L1为-(C(R1a)(R1b))m-CH2-、C3-C6饱和的环烷基或3-6元饱和的杂环基，所述C3-C6饱和的环烷基和3-6元饱和的杂环基各自独立地任选被一个或多个R2a取代；
m选自1、2、3或4；所述的3-6元饱和的杂环基中的杂原子选自N、O和S，杂原子数为1-3个；
R1a各自独立地选自氢、卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
R1b和R2a各自独立地选自氢、卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
R各自独立地为氢或卤素；
L是接头单元；
p表示平均连接数，且p选自1到10的整数或小数；
Ab为抗B7H4抗体或其抗原结合片段。
在一些实施方案中，所述平均连接数p优选为3到8中任一整数或小数。
在本发明某些优选实施方案中，所述的如式(I-1)、(II-1)、(II-2)所示的化合物或其药学上可接受的盐中的某些基团如下定义，未提及的基团同本申请任一方案所述(简称“在一些实施方式中”)。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，R1a各自独立地选自卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代，R各自独立地为氢或卤素。
在一些实施方式中，本发明所述抗B7H4抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述抗B7H4抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：7所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID  NO：8所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链可变区。
在一些实施方式中，本发明所述抗B7H4抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区。
在一些实施方式中，本发明所述抗B7H4抗体或抗原结合片段为鼠源抗体或其片段、嵌合抗体或抗原结合片段、人源化抗体或抗原结合片段、或全人抗体或抗原结合片段。
在一些实施方式中，本发明所述抗B7H4抗体或其抗原结合片段为人源化抗体或其片段。
在一些实施方式中，本发明所述抗B7H4抗体或其抗原结合片段选自Fab、Fab′、Fab′-SH、Fv、scFv、F(ab′)2、sdAb、双抗体或线性抗体。
在一些实施方式中，本发明所述抗B7H4抗体为单克隆抗体。
在一些实施方式中，本发明所述抗体为IgG1形式的抗体、IgG2形式的抗体、IgG3形式的抗体或IgG4形式的抗体。
在一些实施方式中，本发明所述抗体为IgG1形式的抗体。
在一些实施方式中，本发明所述抗B7H4抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：9所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO：10所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链。
在一些实施方式中，本发明所述抗B7H4抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：10所示的轻链。
在一些实施方式中，本发明所述抗B7H4抗体为抗B7H4抗体DB1001。DB1001的氨基酸序列如序列表所示。在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述的M，其L2端与接头单元L相连。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-(C(R1a)(R1b))m-CH2-；R1a选自：氢、卤素和C1-C6烷基；R1b选自：氢、卤素和C1-C6烷基；例如R1a选自：卤素和C1-C6烷基；R1b选自：氢、卤素和C1-C6烷基。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-(C(R1a)(R1b))m-CH2-；R1a为氢或-CH3；R1b选自：氢和-CH3；例如R1a为-CH3；R1b选自：氢和-CH3。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-(C(R1a)(R1b))m-CH2-；m为1或2。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1选自：其中结构片段的左侧优选与L2相连。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为C3-C6饱和的环烷基或3-6元饱和的杂环基，所述C3-C6饱和的环烷基和3-6元 饱和的杂环基各自独立地任选被一个或多个R2a取代，R2a各自独立地选自：氢、卤素和C1-C6烷基。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中，L2和-C(O)-连接在C3-C6饱和的环烷基或3-6元饱和的杂环基的不同原子上。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为任选被一个或多个R2a取代的C3-C6饱和的环烷基；R2a各自独立地选自：氢、卤素和C1-C6烷基。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为C3-C6饱和的环烷基。例如，L1为环丙基、环丁基、环戊基或环己基，优选为环丁基。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为任选被1、2或3个R2a取代的：R2a各自独立地选自：氢、卤素和C1-C6烷基。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1选自：
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
M为-L2-L1-C(O)-；
L2为-O-；
L1为-(C(R1a)(R1b))m-CH2-或C3-C6饱和的环烷基，所述C3-C6饱和的环烷基任选被一个或多个R2a取代；
m选自1或2；
R1a各自独立地选自氢、卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
R1b和R2a各自独立地选自氢、卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
R各自独立地为氢或卤素。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
M为-L2-L1-C(O)-；
L2为-O-；
L1为-(C(R1a)(R1b))m-CH2-或任选被1、2或3个R2a取代的：
m选自1或2；
R1a各自独立地选自卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
R1b和R2a各自独立地选自氢、卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
R各自独立地为氢或卤素。
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-M-选自：
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-M-为：
在一些实施方式中，本发明所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-M-为：
在一些实施方案中，本发明所述的抗B7H4抗体药物偶联物，其中细胞毒性药物选自以下任一结构：

在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为-La-Lb-Lc-，
所述-La-为
其中，W为-(C(Rwa)(Rwb))wn-，Y为-(OCH2CH2)yn-Oyp-，Z为-(C(Rza)(Rzb))zn；
其中wn为1、2、3或6，
W的0个或1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；
其中yn为0、4或8，yp为0或1；
其中zn为1、2或3，
Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代；
-Cyr-为3到10元饱和的环烷基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；
其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx，Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1- 6烷基；
其中每个Rr各自独立地为氢、卤素或C1-6烷基；
所述-Lb-由2到7个氨基酸构成的肽残基，所述-Lb-的肽残基为由选自以下组中的氨基酸形成的肽残基：苯丙氨酸、甘氨酸、丙氨酸、缬氨酸、瓜氨酸、赖氨酸、丝氨酸、谷氨酸、和天冬氨酸；优选地，-Lb-表示由2到4个氨基酸构成的肽残基，所述-Lb-的肽残基为由选自以下组中的氨基酸形成的肽残基：苯丙氨酸、甘氨酸、丙氨酸、缬氨酸、瓜氨酸和赖氨酸；
所述-Lc-为
其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6烷基。
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其 混合物，其中所述L为-La-Lb-Lc-，
所述-La-为
其中，W为-(C(Rwa)(Rwb))wn-，Y为-(OCH2CH2)yn-Oyp-，Z为-(C(Rza)(Rzb))zn；
其中wn为1、2、3或6，
W的0个或1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；
其中yn为0、4或8，yp为0或1；
其中zn为1、2或3，
Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代；
-Cyr-为3到10元饱和的环烷基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；
其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx，Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1- 6烷基；
其中每个Rr各自独立地为氢、卤素或C1-6烷基；
所述-Lb-选自以下组：
所述-Lc-为
其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6烷基。
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-La-为优选为
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其 混合物，其中所述-Lb-为优选为
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-Lc-为
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述的接头单元L，其La端与Ab相连，Lc端与M相连。
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述的接头单元L，其La端与Ab相连，Lc端与接头单元M相连。
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为
在一些实施方案中，本发明所述的抗B7H4抗体药物偶联物，其中所述接头单元L为
在一些实施方案中，本发明所述的抗B7H4抗体药物偶联物，其结构如式(II-A)所示：
其中，p表示平均连接数，且p为1到10中任一整数或小数；优选为3到8中任一整数或小数；Ab、L和M分别如本发明任一实施方案所定义。
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体药物偶联物结构如式(II-1)或(II-2)所示：
其中，
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab如本文任一项所述的抗体或其抗原结合片段；
L2为-O-或-S-；优选为-O-；
X1选自任选被1、2或3个R2a取代的C3-C6饱和的环烷基；优选为任选被1、2或3个R2a取代的：
X2选自-(C(R1a)(R1b))m-CH2-；
m选自1或2；
R1a为氢、卤素或被1、2或3个R任选取代的C1-C6烷基；优选为卤素或被1、2或3个R任选取代的C1-C6烷基；R1b或R2a各自独立可以为氢、卤素或被1、2或3个R任选取代的C1-C6烷基；
R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体药物偶联物选自以下结构式：



其中，
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab如本文任一实施方案所述的抗B7H4抗体或其抗原结合片段。
在又一个方面，本发明提供了一种抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体药物偶联物选自：


其中，
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数。所述DB1001的氨基酸序列如序列表所示。所述平均连续数p为3.89，或p为5.4。
在一些实施方式中，本发明所述平均连接数p选自1到10的整数或小数。
在一些实施方式中，本发明所述平均连接数p可以为2到8的整数或小数。例如，所述平均连接数p可以为3到8的整数或小数。例如，所述平均连接数p可以为1到2、2到3、3到4、4到5、5 到6、6到7、7到8、8到9、9到10的整数或小数。
在又一个方面，本发明提供了一种抗体药物偶联物的制备方法，其包括以下步骤：在还原剂的作用下，溶于缓冲液的所述抗体与溶于溶剂的所述接头-细胞毒素混合，即得所述抗体药物偶联物。
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了如本文所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或药物组合物在制备用于治疗和/或预防B7H4介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为B7H4阳性表达的癌症。
在又一个方面，本发明提供了一种治疗和/或预防B7H4介导的疾病或病症的方法，其包括向有需要的受试者施用如本文所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或药物组合物，优选地，所述疾病或病症为B7H4高表达的癌症。
在又一个方面，本发明提供了如本文所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或药物组合物，其用于治疗和/或预防B7H4介导的疾病或病症，优选地，所述疾病或病症为B7H4阳性表达的癌症。
在一些实施方式中，本发明所述癌症选自乳腺癌、卵巢癌和子宫内膜瘤。
在又一个方面，本发明提供了一种药物组合，其包含如本文所述的抗体药物偶联物或其药学上可接受的盐或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的抗体药物偶联物、或本文所述的药物组合物。
术语定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocolsin Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能 天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“全长抗体”，是指在天然存在时包含四条肽链的免疫球蛋白分子：两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
术语“CDR”是指抗体可变序列内的互补决定区。在重链和轻链的各个可变区中存在3个CDR，其对于各个重链和轻链可变区被命名为HCDR1、HCDR2和HCDR3或LCDR1、LCDR2和LCDR3。本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342：877-883；A1-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997))基于抗体序列可变性的Kabat(Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999 Nucleic Acids Research，27，209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab′，F(ab′)2和Fv片段；双抗体；线性抗体；单链抗体分子，例如scFv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性 较低。
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
在本申请中，术语“卤素”通常是指氟、氯、溴、碘，例如可以是氟、氯。
在本申请中，术语“烷基”通常是指烷除去氢原子所衍生的残基。烷基可以是取代的或非取代的，替代或者非替代的。术语“烷基”通常指饱和的直链或支链脂肪族烃基，其具有从母体烷的相同碳原子或两个不同的碳原子上除去氢原子所衍生的残基，其可以为包含1至20个碳原子的直链或支链基团，例如含有1至12个碳原子，例如含有1至6个碳原子的链烷基。烷基的非限制性实例包括但不限于甲基、乙基、丙基、丙基、丁基等。烷基可以是取代的或非取代的，替代或者非替代的，例如当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基可以独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代，例如可以是氢、氕、氘、氚、卤素、-NO2、-CN、-OH、-SH、-NH2、-C(O)H、-CO2H、-C(O)C(O)H、-C(O)CH2C(O)H、-S(O)H、-S(O)2H、-C(O)NH2、-SO2NH2、-OC(O)H、-N(H)SO2H或C1-6脂肪族基团。例如当被环烷基取代时，为环烷基烷基。
在本申请中，术语“亚烷基”通常指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其可以为包含1至20个碳原子的直链或支链基团，例如，术语“亚甲基”可以是指1个碳原子的基团除去两个氢原子所衍生的残基。亚甲基可以是取代的或非取代的，替代或者非替代的；例如含有1至12个碳原子，例如含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH2-)、1，1-亚乙基(-CH(CH3)-)、1，2-亚乙基(-CH2CH2)-、1，1-亚丙基(-CH(CH2CH3)-)、1，2-亚丙基(-CH2CH(CH3)-)、1，3-亚丙基(-CH2CH2CH2-)、1，4-亚丁基(-CH2CH2CH2CH2-)和1，5-亚丁基(-CH2CH2CH2CH2CH2-)等。亚烷基可以是取代的或非取代的，替代或者非替代的，例如当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基可以独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代，例如可以是氢、氕、氘、氚、卤素、-NO2、-CN、-OH、-SH、-NH2、-C(O)H、-CO2H、-C(O)C(O)H、-C(O)CH2C(O)H、-S(O)H、-S(O)2H、-C(O)NH2、-SO2NH2、-OC(O)H、-N(H)SO2H或C1-6脂肪族基团。亚甲基或亚烷基可以是取代的或非取代的。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基或环烷基的定义如本文所述。烷氧基的非限制性示例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷 基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
在本申请中，术语“烯基”通常是指含有一个或多个双键的直链或支链烃基。烯基的示例性实例包括烯丙基、高烯丙基、乙烯基、巴豆基、丁烯基、戊烯基和己烯基等。具有一个以上双键的C2-6链烯基的示例性实例包括丁二烯基、戊二烯基、己二烯基和己三烯基以及它们的支化形式。不饱和键(双键)的位置可以是在碳链的任何一个位置。烯基可以是取代的或非取代的。
在本申请中，术语“亚烯基”通常是指具有从烯烃的碳原子上除去两个氢原子所衍生的残基。例如，可以是亚烯丙基、亚乙烯基、亚丁烯基、亚戊烯基和亚己烯基等。亚烯基可以是取代的或非取代的。
在本申请中，术语“炔基”通常是指不饱和直链或支链炔基，例如乙炔基、1-丙炔基、炔丙基、丁炔基等。炔基可以是取代的或非取代的。
在本申请中，术语“亚炔基”通常是指具有从炔烃的碳原子上除去两个氢原子所衍生的残基。例如，可以是亚乙炔基、亚丙炔基、亚炔丙基、亚丁炔基等。亚炔基可以是取代的或非取代的。
在本申请中，术语“芳基”通常是指具有芳环上除去氢原子所衍生的残基。术语“芳环”可以指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)，可以为6至10元，例如苯和萘。所述芳环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环。芳基可以是取代的或非取代的，当被取代时，取代基可以为一个或多个以下基团，其独立地选自以下组：烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、和杂环烷硫基。芳基可以是取代的或非取代的。
在本申请中，术语“杂芳基”通常是指具有从杂芳环的碳原子上除去氢原子所衍生的残基。术语“杂芳环”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子可以选自以下组：氧、硫和氮。杂芳基可以为5至10元，可以为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环。杂芳基可以是任选取代的或非取代的，当被取代时，取代基可以为一个或多个以下基团，其独立地选自以下组：烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、和杂环烷硫基。杂芳基可以是取代的或非取代的。
在本申请中，术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，优选包含3至10个碳原子，优选包含3至8个碳原子，更优选包含3至6个碳原子。单环环烷基的非限制性示例包括环丙烷基、环丁烷基、环戊烷基、环戊烯基、环己烷基、环己烯基、环己二烯基、环庚烷基、环庚三烯基、环辛烷基等；多环环烷基包括螺环、稠环和桥环的环烷基。环烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代。
在本申请中，术语“部分不饱和的”通常是指环状结构中环分子间至少含一个双键或三键。术语“部分不饱和”涵盖带有多处不饱和的环状结构，但并非意在包括本申请所定义的芳环或杂芳环。术语″不饱和的″表示部分具有一个或多个不饱和度。
在本申请中，术语“杂环基”指饱和或部分不饱和单环或多环环状轻取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或硫的杂原子，其余环原子为碳。优选包含3至12个环原子，其中1～4个是杂原子；更优选包含3至8个环原子，其中1-3是杂原子；更优选包含3至6个环原子，其中1-3个是杂原子；最优选包含5或6个环原子，其中1-3个是杂原子。单环杂环基的非限制性示例包括吡咯烷基、四氢吡喃基、哌啶基、吗啉基、硫代吗啉基和高哌嗪基的等。多环杂环基包括螺环、稠环和桥环的杂环基。所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其与母体结构连接在一起的环为杂环基。杂环基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自氢原子、卤素、烷基、烷氧基、卤代烷基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代。
在本申请中，术语“成环原子”通常是指环状结构上包含的原子。例如，成环原子可以是苯环上的碳原子，可以是吡啶环上的氮原子。当成环原子上连接氢原子时，成环原子可以是取代的或非取代的。
在本申请中，术语“各自独立地”通常是指变量适用于任何一种情况，而不考虑在相同化合物中具有相同或不同定义的变量存在与否。例如其中的变量可以是指化合物的取代基种类、数量或化合物中原子的种类等。例如，在化合物中出现2次R并且R被定义为“独立地碳或氮”时，两个R可以均为碳，两个R可以均为氮，或一个R可以为碳而另一个R为氮。
在本申请中，术语“任选”或“任选地”通常意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明可以包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
在本申请中，术语“取代的”通常指基团中的一个或多个氢原子，例如为最多5个，例如为1～3个氢原子彼此独立地被相应数目的取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
在本申请中，术语0个或多个(例如，0个或1个以上、0个或1个、0个)亚甲基单元被“替代”通常指当所述结构包含1个或多个亚甲基单元时，所述一个或多个亚甲基单元可以不被替代，或被一个或多个不是亚甲基的基团(例如-NHC(O)-、-C(O)NH-、-C(O)-、-OC(O)-、-C(O)O-、-NH-、-O-、-S-、-SO-、-SO2-、-PH-、-P(＝O)H-、-NHSO2-、-SO2NH-、-C(＝S)-、-C(＝NH)-、-N＝N-、-C＝N-、-N＝C-或-C(＝N2)-)所替代。
在本申请中，基团X与基团Y的“连接”通常可以处于任一定向，任一定向通常是指在基团X用于连接体Y和基团Z时，所述基团X的两个或更多个连接位点可以任意地与基团Y或基团Z连接。
在本申请中，术语“化合物”通常指具有两种或两种以上不同元素的物质。例如，本申请的化合物 可以是有机化合物，例如本申请的化合物可以是分子量500以下的化合物，可以是分子量1000以下的化合物，也可以是分子量1000以上的化合物，也可以是10000以上、100000以上的化合物。在本申请中，化合物还可以是指通过化学键相连的化合物，例如可以是一个或多个分子量1000以下的分子通过化学键与生物大分子相连的化合物，所述生物大分子可以是高聚糖、蛋白、核酸、多肽等。例如本申请的化合物可以包括蛋白质与一个或多个分子量1000以下的分子相连的化合物，可以是包括蛋白质与一个或多个分子量10000以下的分子相连的化合物，可以是包括蛋白质与一个或多个分子量100000以下的分子相连的化合物。
在本申请中，如本领域技术人员可知的，“烷基”、“烯基”、“环烷基”等之类的术语可以在名称前加一个标识表示在特定情况下基团中存在的原子数，例如，C1-C4烷基，C3-C7环烷氧基，C1-C4烷基羰基氨基等，“C”后所跟下标数字表示在基团中存在的碳原子数。例如，C3烷基是指具有三个碳原子的烷基(例如，正丙基，异丙基)；C1-10中，基团的成员可具有落入1-10范围内的任何数目的碳原子。
基团中的一个或多个氢原子，例如为最多5个，例如为1～3个氢原子彼此独立地被相应数目的取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
在本申请中，本申请的化合物或配体-药物偶联物包含其互变异构体、内消旋体、外消旋体、对映异构体、和/或非对映异构体。在本申请中，术语“非对映异构体”通常是指具有两个或更多个手性中心并且其分子不是彼此的镜像的立体异构体。非对映异构体可以具有不同的物理性质，例如、熔点、沸点、波谱性质和反应性。在本申请中，术语“互变异构体”或“互变异构形式”可互换使用，通常是指可通过低能垒(low energy barrier)互相转化的不同能量的结构异构体。例如，质子互变异构体(protontautomer)(也称为质子移变互变异构体(prototropic tautomer))包括通过质子迁移进行的互相转化，诸如酮-烯醇异构化和亚胺-烯胺异构化。价键互变异构体(valence tautomer)包括通过一些成键电子的重组进行的互相转化。在本申请中，术语“内消旋体”通常是指分子内含有不对称性的原子，但具有对称因素而使分子内总旋光度为零。术语″外消旋体″或″外消旋混合物″是指由等摩尔量的两种对映异构体物质构成的组合物。
在本申请中，术语化合物或配体-药物偶联物的“异构体”通常包含化合物的互变异构体、内消旋体、外消旋体、对映异构体非对映异构体。
术语“配体-药物偶联物”通常是指配体通过稳定的连接单元与具有生物活性的细胞毒性药物相连。在本申请中“配体-药物偶联物”可以为抗体-药物偶联物(antibody drug conjugate，ADC)，所述ADC可以是指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的细胞毒性药物相连。
在本申请中，术语“配体”通常指能识别和结合目标细胞相关的抗原或受体的大分子化合物。配体的作用可以是将药物呈递给与配体结合的目标细胞群，这些配体包括但不限于蛋白类激素、凝集素、生长因子、抗体或其他能与细胞、受体和/或抗原结合的分子。在本申请中，配体可以表示为Pc，配体抗原通过配体上的杂原子与连接单元形成连接键。配体可以为抗体或其抗原结合片段 (Ab)，所述抗体可以选自嵌合抗体、人源化抗体、全人抗体或鼠源抗体；所述抗体可以是单克隆抗体。
术语“细胞毒性药物”通常指毒性药物，所述细胞毒性药物可以在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物可以在足够高的浓度下杀死肿瘤细胞。所述“细胞毒性药物”可以包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32或Lu的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶，例如，可以是毒性药物，包括但不限于喜树碱衍生物，例如，可以是喜树碱衍生物依沙替康(化学名：(1S，9S)-1-氨基-9-乙基-5-氟-2，3-二氢-9-羟基-4-甲基-1H，12H-苯并[de]吡喃并[3’，4’：6，7]咪唑并[1，2-b]喹啉-10，13(9H，15H)-二酮)。在本发明的一些实施例中，细胞毒性药物也称为细胞毒素。
术语“接头单元”或“接头结构”通常指指一端与配体连接而另一端与细胞毒性药物相连的化学结构片段或键，也可以连接其他接头后再与细胞毒性药物相连。所述直接或间接连接配体可以是指所述基团通过共价键直接连接配体，也可以是通过接头结构连接配体。例如，可以使用包含酸不稳定接头结构(例如腙)、蛋白酶敏感(例如肽酶敏感)接头结构、光不稳定接头结构、二甲基接头结构、或含二硫化物接头结构的化学结构片段或键作为接头结构。
术语某个结构“任选地与其它分子部分相连接”通常是指该结构不与任何其它化学结构相连接，或者该结构与一个或多个不同于该结构的其它化学结构(例如本申请所述的配体)相连接(例如，通过化学键连接、或通过接头结构连接)。
术语“载药量”通常是指每个配体上加载的细胞毒性药物平均数量，也可以表示为细胞毒性药物和抗体量的比值，细胞毒性药物载量的范围可以是每个配体(Ab)连接0-12个，例如1-10个细胞毒性药物。在本申请的实施方式中，载药量表示为Na，示例性的可以为1，2，3，4，5，6，7，8，9，10的均值。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的载药量。
在本申请中，本申请的化合物的某些原子可能以一种以上的同位素形式出现。例如，氢可能以氕(1H)、氘(2H)和氚(3H)的形式存在，碳可能以三种不同的同位素(12C、13C和14C)自然存在。可并入本申请化合物中的同位素示例还包括但不限于15N、18O、17O、18F、32P、33P、129I、131I、123I、124I、125I，或者类似的同位素。因此，相对于这些同位素的自然丰度，本申请的化合物可富集在一种或多种这些同位素中。如本领域技术人员所知，此类同位素富集化合物可用于多种用途。例如，用重同位素如氘(2H)替代可能会提供某些治疗优势，这可以是由于更高的代谢稳定性。例如，氘(2H)的自然丰度约为0.015％。因此，自然界中大约每6500个氢原子，就有一个氘原子。因此，本申请的含氘化合物在一个或多个位置(视情况而定)的氘丰度大于0.015％。除非另有指明，否则本申请所述的结构还可以包括仅在是否存在一个或多个同位素富集原子方面存在差别的化合物。举例而言，除了氢原子被氘或氚所取代，或碳原子被碳13或碳14所取代之外，其余部分均与本申请结构一致的化合物均在本申请的范围之内。
在本申请中，术语“药物组合物”通常是指含有一种或多种本申请所述化合物或其生理学上/可药 用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物可以是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。常规的药物组合物的制备可以见中国药典。药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1，3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶剂或悬浮介质。例如，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
在本申请中，术语“药学上可接受的盐”或“可药用的盐”通常是指本申请化合物或配体-药物偶联物的盐，或本申请中所述的化合物的盐，这类盐用于哺乳动物体内时可以具有安全性和/或有效性，且可以具有应有的生物活性，本申请抗体-抗体药物偶联化合物可以与酸形成盐，药学上可接受的盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
在本申请中，术语“药学上可接受的载体”通常是指给予治疗剂，例如抗体或多肽、基因和其它治疗剂的载体或载剂。该术语指本身不诱导对接受组合物的个体有害的抗体产生并且可以给予而不产生过度毒性的任何药物载体。合适的载体可以是大的、代谢缓慢的大分子，例如蛋白质、多糖、聚乳酸、聚乙醇酸、多聚氨基酸、氨基酸共聚物、脂质聚集物和灭活的病毒颗粒。本领域技术人员熟知这些载体。治疗组合物中药学上可接受的载体可包括液体，例如水、盐水、甘油和乙醇。这些载体中也可存在辅助物质，例如润湿剂或乳化剂、pH缓冲物质等。
在本申请中，术语“治疗(treatment)”和“治疗(treating)”通常是指获得有益或希望的结果的方法，所述有益或希望的结果包括但不限于治疗益处。治疗益处包括但不限于根除、抑制、减少或改善所治疗的潜在障碍。另外，治疗益处是通过根除抑制、减少或改善与潜在的障碍相关的一种或多种生理症状实现的，从而在患者中观察到改善，但是患者仍然可能患有潜在障碍。
在本申请中，术语“预防(prevention)”和“预防(preventing)”通常是指获得有益或希望的结果的方法，所述有益或希望的结果包括但不限于预防益处。为了预防益处，可以向处于患上特定疾病的风险的患者或向报告具有疾病的一种或多种生理症状的患者施用药物组合物，即使尚未诊断出该疾病。
在本申请中，术语“受试者”或“患者”通常是指人类(即，任何年龄组的男性或女性，例如，小儿对象(例如，婴儿、儿童、青少年)或成人对象(例如，年轻人、中年人或老年人))和/或其他灵长类动物(例如，食蟹猴、恒河猴)；哺乳动物，包括商业上相关的哺乳动物，如牛、猪、马、绵羊、山羊、猫和/或犬；和/或鸟类，包括商业上相关的鸟类，如鸡、鸭、鹅、鹌鹑和/或火鸡。
术语“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明配体-药物偶联物单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的剂量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治 疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。
术语“癌症”在本文中用于指表现出异常高水平的增殖和生长的一组细胞。癌症可能是良性的(也称为良性肿瘤)，恶性前或恶性。癌细胞可以是实体癌细胞或白血病癌细胞。本文使用的术语“肿瘤”是指包含癌症的一个或多个细胞。术语“肿瘤生长”在本文中用于指代包含癌症的一种或多种细胞的增殖或生长，其导致癌症的大小或程度的相应增加。
抗B7H4抗体
术语“B7-H4”或“B7H4”指人B7蛋白质家族的成员，也称为CD276，是具有四个Ig样胞外结构域的I型跨膜蛋白。B7-H4是抗原呈递细胞或癌细胞表面表达的免疫检查点蛋白之一，对T细胞的功能激活有抑制作用。术语“B7-H4“包括由细胞天然表达的B7-H4的任何变体或同种型。本发明的抗体可与得自非人物种的B7-H4交叉反应。作为另一种选择，该抗体也可以是人B7-H4特异性的，可不表现出与其他物种的交叉反应性。B7-H4或其任何变体或同种型可从天然表达它们的细胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。优选地，抗B7-H4抗体靶向具有正常糖基化模式的人源B7-H4。
本发明术语“抗B7H4抗体”、“抗B7H4”、“B7H4抗体”或“结合B7H4的抗体”是指能够以足够的亲合力结合B7H4蛋白或其片段以致所述抗体可以用作靶向B7H4中的诊断剂和/或治疗剂。
在一些方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体的CDR序列包括来自于PCT/CN2021/102952中描述的抗体PR008199的CDR序列。在一些方案中，在本发明的药物偶联物、组合物或用途中使用的抗体的可变区序列包括来自于PCT/CN2021/102952中描述的抗体PR008199的可变区序列。在一些方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体的氨基酸序列包括来自于PCT/CN2021/102952中描述的抗体PR008199的全长氨基酸序列。
本发明所述的抗B7H4抗体DB1001(PR008199)或其抗原结合片段参考PCT/CN2021/102952中描述制备。在一些方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体的CDR序列包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。在一些方案中，在本发明的药物偶联物、组合物或用途中使用的抗体的可变区序列包括氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区。在一些方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体包括氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：10所示的轻链。在一些方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体为抗B7H4抗体DB1001。
抗B7H4抗体DB1001的可变区氨基酸序列如下，CDR区依照IMGT编号规则确定。
DB1001重链可变区：

DB1001轻链可变区：
DB1001重链氨基酸序列
DB1001轻链氨基酸序列
可采用用于产生抗体的任何合适方法来产生本发明的抗体。任何合适形式的B7H4都可用作产生抗体的免疫原(抗原)。通过举例而非限制，任何B7H4变体或其片段都可用作免疫原。在一些实施方式中，产生鼠源的单克隆抗人B7H4抗体的杂交瘤细胞可通过本领域公知的方法产生。来源于啮齿动物(如小鼠)的抗体在体内用作治疗药物时可能引起不需要的抗体免疫原性，重复使用导致人体产生针对治疗性抗体的免疫应答，这类免疫应答至少导致丧失治疗功效，而严重的则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA 84：3439-43)。然而，嵌合抗体中的完整啮齿动物可变区的保留仍可能在患者中引起有害的免疫原性。将啮齿动物可变结构域的互补决定区(CDR)环移植到人构架上(即人源化)已被用于进一步将啮齿动物序列减至最低(Jones等(1986)Nature 321：522；Verhoeyen等(1988)Science 239：1534)。
在一些实施方式中，本发明的嵌合或人源化抗体可基于所制备的鼠单克隆杂交瘤抗体的序列来制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
在一些实施方式中，本发明所述的嵌合B7H4抗体，可使用本领域已知的方法将杂交瘤来源的免疫球蛋白重链和轻链可变区与人IgG恒定区有效连接(参见例如属于Cabilly等人的美国专利No.4,816,567)，获得嵌合型重链和嵌合型轻链来制备。在一些实施方式中，本发明的嵌合抗体包含的恒定区可选自任何人IgG亚型，如IgG1、IgG2、IgG3、IgG4，优选IgG4。
在一些实施方式中，本发明的嵌合B7H4抗体可由嵌合型轻链与嵌合型重链表达质粒“混合和匹配”转染表达细胞获得，此类“混合和匹配”的抗体的B7H4结合可使用上述结合测定和其它常规结合测定(例如，ELISA)来进行测试。
本发明所述的人源化抗体，可以使用本领域已知的方法将鼠源CDR区插入人种系框架区。参见Winter等人的美国专利No.5,225,539及Queen等人的美国专利No.5,530,101；5,585,089；5,693,762和6,180,370。
在一些实施方式中，氨基酸变化包括氨基酸缺失、插入或置换。在一些实施方式中，本发明的抗B7H4抗体或其抗原结合片段包括具有已通过氨基酸缺失、插入或置换突变的，但仍与上述抗体(特别地在上述序列中描绘的CDR区中)有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方式中，本发明的抗体与具体序列中描绘的CDR区相比较时，在CDR区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。
在一些实施方式中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。
在一些实施方式中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。
在一些实施方式中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
药物偶联物
本申请提供了一种抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物形式，其可以具有选自以下组的一种或多种效果：(1)具有对肿瘤细胞的体外增殖的抑制活性；(2)具有靶向抑制性；(3)具有血浆稳定性；(4)具有体内抑瘤效果；(5)具有旁观杀伤效应(Bystander Effect)； (6)具有抗转运体转运能力；(7)具有体内肿瘤靶向能力；和(8)具有良好的体内安全性。
本申请提供一种抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体药物偶联物结构如式(I-1)所示：
其中，
M为-L2-L1-C(O)-；
L2为-O-或-S-；
L1为-(C(R1a)(R1b))m-CH2-、C3-C6饱和的环烷基或3-6元饱和的杂环基，所述C3-C6饱和的环烷基和3-6元饱和的杂环基各自独立地任选被一个或多个R2a取代；
m选自1、2、3或4；
R1a各自独立地选自氢、卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；优选为卤素或被1、2或3个R任选取代的C1-C6烷基；
R1b和R2a各自独立地选自氢、卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
R各自独立地为氢或卤素；
L是接头单元；
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab为抗B7H4抗体或其抗原结合片段。
在一些实施方式中，抗B7H4抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3；优选地，本发明所述抗B7H4抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区；更优选地，本发明所述抗B7H4抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：10所示的轻链。
在一些实施方式中，在式(I-1)、(II-1)和(II-2)中，L1为-(C(R1a)(R1b))m-CH2-，R1a选自：卤素、羟基、氨基和被R任选取代的C1-C6烷基，R1b选自：氢、卤素、羟基、氨基和被R任选取代的C1-C6烷基，m选自1、2、3或4，R各自独立地可以为氢或卤素；优选地，R1a选自：卤素和C1-C6烷基，R1b 选自：氢、卤素和C1-C6烷基，m选自1或2；优选地，R1a为-CH3；R1b选自：氢和-CH3，m选自1或2；优选地，L1选自：
在一些实施方式中，在式(I-1)、(II-1)和(II-2)中，L1为C3-C6饱和的环烷基或3-6元饱和的杂环基，所述C3-C6饱和的环烷基和3-6元饱和的杂环基各自独立地任选被一个或多个R2a取代，R2a各自独立地选自：氢、卤素、羟基、氨基和C1-C6烷基；优选地，L1为任选被一个或多个R2a取代的C3-C6饱和的环烷基，R2a各自独立地选自：氢、卤素和C1-C6烷基；优选地，L1为C3-C6饱和的环烷基；优选地，L1选自
在一些实施方式中，在式(I-1)、(II-1)和(II-2)中，L1选自
在一些实施方式中，在式(I-1)、(II-1)和(II-2)中，M为-L2-L1-C(O)-；
L2为-O-或-S-；优选为-O-；
L1为-(C(R1a)(R1b))m-CH2-或C3-C6饱和的环烷基，所述C3-C6饱和的环烷基任选被一个或多个R2a取代；优选地，L1为任选被1、2或3个R2a取代的：
m选自1或2；
R1a各自独立地选自卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
R1b和R2a各自独立地选自氢、卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；R各自独立地为氢或卤素。
在一些优选地实施方式中，在式(I-1)、(II-1)和(II-2)中，M选自：
在一些特别优选地实施例中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为
在一些实施方式中，本发明所述抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体药物偶联物结构如式(II-1)或(II-2)所示：
其中，
L2为-O-或-S-；优选地，L2为-O-；
X1选自任选被1、2或3个R2a取代的C3-C6饱和的环烷基；优选为任选被1、2或3个R2a取代的：
X2选自-(C(R1a)(R1b))m-CH2-；
m选自1或2；
R1a为卤素或被1、2或3个R任选取代的C1-C6烷基；
R1b或R2a各自独立可以为氢、卤素或被1、2或3个R任选取代的C1-C6烷基；
R各自独立地可以为氢或卤素；
p表示平均连接数，且n选自1到10的整数或小数，优选3-8的整数或小数；
Ab为抗B7H4抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3；优选地，本发明所述抗B7H4抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区；更优选地，本发明所述抗B7H4抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：9所示的重链，和氨基酸序列如SEQ ID NO：10所示的轻链。
在一些实施方式中，本发明所述平均连接数p可以为2到8的整数或小数。例如，所述平均连接数p可以为3到8的整数或小数。例如，所述平均连接数p可以为1到2、2到3、3到4、4到5、5到6、6到7、7到8、8到9、9到10的整数或小数。
药物组合物和药物制剂
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。
应理解，本发明提供的抗B7H4抗体药物偶联物或、其异构体、其药学上可接受的盐或其混合物，或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的抗B7H4抗体药物偶联物或其药学上可接受的盐与一种或多种任选的药用辅料(Remington′s Pharmaceutical Sciences，第16版，Osol，A.编辑(1980))混合来制备包含本文所述的抗B7H4抗体的药物制剂，优选地以水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。在一些实施方式中，本发明的药物组合物还包含编码抗B7H4抗体的多核苷酸的组合物。
在又一个方面，本发明提供了一种药物组合，其包含如本文所述的抗体药物偶联物或其药学上可接受的盐或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的抗体药物偶联物或、其异构体、其药学上可接受的盐或其混合物或本文所述的药物组合物，优选其进一步包括给药装置。
医药用途
在又一个方面，本发明提供了如本文所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，或本文所述的药物组合物在制备用于治疗和/或预防B7H4介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为B7H4阳性表达的癌症。
在又一个方面，本发明提供了如本文所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，或本文所述的药物组合物，其用于治疗和/或预防B7H4介导的疾病或病症，优选地，所述疾病或病症为B7H4阳性表达的癌症。
在又一个方面，本发明提供了一种治疗和/或预防B7H4介导的疾病或病症的方法，其包括向有需要的受试者施用如本文所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，或本文所述的药物组合物，优选地，所述疾病或病症为B7H4高表达的癌症。
在一些实施方式中，所述癌症选自乳腺癌、卵巢癌和子宫内膜瘤。
在一些实施方式中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
在一些实施方式中，本发明提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方 式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。
在一些实施方式中，本发明提供的方法或用途还包括向个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体药物偶联物或其药学上可接受的盐。例如，可以与至少一种另外的治疗剂共施用。
本发明的积极进步效果在于：
本发明抗B7H4抗体药物偶联物中，抗B7H4抗体或其抗原结合片段通过接头单元与具有生物活性的细胞毒性药物相连，抗体药物偶联物转移至肿瘤细胞后，接头被切断，游离出细胞毒性药物H-M-D。例如，本发明抗体药物偶联物DB1001-X1转移至细胞后，游离出小分子化合物P-II-3；本发明抗体药物偶联物DB1001-X2转移至细胞后，游离出小分子化合物P-III-30。本发明细胞毒性药物对NCI-N87细胞、JIMT-1细胞、Colo205细胞和MDA-MB-231细胞具有明显增强的增殖抑制活性，能发挥优异的抗肿瘤作用。
本发明抗B7H4抗体药物偶联物对B7H4阳性表达MDA-MB-468细胞具有较好的内吞效应，且优于参照ADC-1。
本申请抗体药物偶联物对B7H4阳性表达的细胞MDA-MB-468、结直肠癌HT29、、人乳腺细胞癌MX-1、子宫内膜癌细胞RL95-2具有显著的增殖抑制活性。
本发明抗B7H4抗体药物偶联物对能阻断B7H4阳性细胞对不同供体来源T细胞的抑制，且优于参照ADC-1。
本发明抗B7H4抗体药物偶联物在体外人、大鼠和猴血浆中具有良好的稳定性。同时，由于细胞毒性药物P-III-30的低血浆浓度和短半衰期，P-III-30的全身暴露量较低，从而具有较高的安全性。
本发明抗B7H4抗体药物偶联物对MDA-MB-468荷瘤小鼠、乳腺癌细胞MX-1荷瘤小鼠、乳腺癌细胞MCF-7荷瘤小鼠、子宫内膜癌细胞RL95-2荷瘤小鼠、卵巢癌细胞OVCAR-3荷瘤小鼠具有显著的抗肿瘤活性。
因此，本发明在B7H4的阳性表达疾病(例如癌症)方面有着良好的应用前景。
附图说明
图1：抗体药物偶联物对MDA-MB-468荷瘤小鼠药效评价。
图2：抗体药物偶联物在人、食蟹猴、和小鼠B7H4转染的HEK293T细胞的种属交叉反应。
图3：抗体药物偶联物和过表达人B7家族蛋白细胞的结合特异性。
图4：抗体药物偶联物在MDA-MB-468细胞不同时间点内吞产生荧光信号的积分面积。
图5：抗体药物偶联物在MDA-MB-468细胞的内吞效率。
图6：抗体药物偶联物对HT29-B7H4的体外增殖抑制率。
图7：抗体药物偶联物，单抗，载荷(细胞毒性药物)对肿瘤细胞MX-1体外增殖抑制效率(3D细胞培养法)。
图8：抗体药物偶联物，单抗，载荷(细胞毒性药物)对肿瘤细胞RL95-2体外增殖抑制效率(3D 细胞培养法)。
图9：抗体药物偶联物，单抗，载荷(细胞毒性药物)对肿瘤细胞JIMT-1体外增殖抑制效率(3D细胞培养法)。
图10：抗体药物偶联物对PBMC来源的T细胞激活。
图11：抗体药物偶联物对PBMC来源的T细胞激活。
图12：抗体药物偶联物对与HT29-B7H4共孵育的HT29-Luc细胞旁观者杀伤效应。
图13：抗体药物偶联物在人、猴、大鼠体外血浆孵育21天后载荷(细胞毒性药物)释放率。
图14：抗体药物偶联物对乳腺癌MX-1荷瘤小鼠药效评价。
图15：抗体药物偶联物对子宫内膜癌RL95-2荷瘤小鼠药效评价。
图16：抗体药物偶联物对卵巢癌OVCAR-3荷瘤小鼠药效评价。
具体实施方式
样品检测
1.ADC DAR值分析方法-HIC-HPLC(疏水色谱法)
高效液相色谱仪：沃特世e2965高效液相色谱系统。
色谱柱：MabPacTM HIC-Butyl 5μm 4.6×100mm(厂家：Thermo)；
流动相A：1.5M(NH4)2SO4+50mM K2HPO4(pH 7.0)；
流动相B：50mM K2HPO4(pH 7.0)/异丙醇(75∶25 V/V)；
按照以下洗脱程序进行洗脱，
检测条件：设置流动相流速为1ml/min，检测波长为280nm，柱温30℃。
2.SEC纯度分析-SEC-HPLC(分子排阻色谱法)
高效液相色谱仪：1260安捷伦液相色谱仪。
色谱柱：Waters Xbridge BEH200 SEC(7.8×300mm，3.5μm)
流动相：50mM NaH2PO4+200mM精氨酸(pH 6.80)+10％异丙醇
检测条件：设置流动相流速为0.5ml/min，检测波长为280nm，柱温30℃。
本发明包括所叙述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
实施例
提供以下实施例以证明并进一步解释本发明的一些优选的实施方式和方面，不应被解释为限制其范围。
实施例1、抗B7H4抗体
本发明的抗体参照PCT/CN2021/102952制备，其中，抗B7H4抗体DB1001(PR008199)的可变区氨基酸序列如下，CDR区依照Kabat编号规则确定。
DB1001重链可变区：
DB1001轻链可变区：
根据以上序列，设计引物PCR搭建得到VH/VK基因片段，获得可变区。抗体可变区再与恒定区基因片段进行同源重组，构建DB1001完整抗体序列。经过转染CHO细胞后，按照常规表达纯化方法得到抗体DB1001。
DB1001重链氨基酸序列

DB1001轻链氨基酸序列
实施例2、抗B7H4抗体药物偶联物(ADC)的制备
2.1、细胞毒性药物(payload)的制备
制备例1
第一步：
在氮气保护下，在0℃下向KI4(900mg，1.69mmol)，HATU(691mg，1.88mmol)，3a(320mg，2.00mmol)的DMF(18mL)溶液中加入DIEA(500mg，3.87mmol)。并在25℃搅拌3小时。TLC(EA)显示原料反应完全。将反应液滴加到320mL去离子水中，过滤，得灰色固体850mg，收率：87％
第二步：
向3b(100mg，0.174mmol)的MeOH/DCM(1/1，3mL)中加入NaHCO3(42mg，0.50mmol)固体并在25℃搅拌3小时，TLC(EA)显示反应完全，将反应液过滤，低温旋干后，用aq.HCl(0.5M，10mL)打浆后过滤，经prep-HPLC(0.1％TFA)制备后冻干得到15毫克灰色固体。收率：16％。
MS m/z(ESI)：534[M+1]；
H-NMR(400MHz，DMSO-D)：8.45(d，1H)，7.81(d，1H)，7.32(s，1H)，6.52(m，1H)，5.58-5.56(m，1H)，5.44(s，2H)，5.14(dd，2H)，3.96(m，1H)，3.48(m，1H)，3.19(m，2H)，2.53-2.28(m，3H)，2.48(s，3H)，2.20-2.00(m，4H)，1.95-1.80(m，2H)，0.89(t，3H)。
制备例2
第一步
氮气保护下，向KI4(100mg，0.19mmol)，HATU(85.7mg，0.23mmol)，23a(21.5mg，0.21mmol)的DMF(2mL)溶液中滴加DIEA(60.6mg，0.47mmol)，加完后在0℃反应2小时。LCMS显示原料反应完全。将反应液滴加到20mL水中搅拌，析出固体后过滤，得60.2mg灰色固体P-III-30，收率：61％。MS-ESI：m/z 522.2[M+H]+。
1H NMR(400MHz，DMSO-d6)δ8.42(d，J＝8.7Hz，1H)，7.79(d，J＝11.0Hz，1H)，7.30(s，1H)，6.53(s，1H)，5.62-5.53(m，1H)，5.42(s，2H)，5.30-5.16(m，2H)，4.63(d，J＝4.6Hz，1H)，4.09-3.99(m，1H)，3.22-3.11(m，2H)，2.40(s，3H)，2.28(dd，J＝13.7，7.2Hz，1H)，2.22-2.08(m，3H)，1.94-1.78(m，2H)，1.08(d，J＝6.1Hz，3H)，0.87(t，J＝7.3Hz，3H).
制备例3
第一步
取KI4(50mg，0.094mmol)，25a(11mg，0.094mmol)，HATU(39mg，0.103mmol)加入DMF(3mL)中，氮气置换后加入DIEA(30mg，0.235mmol)，25℃下反应1.5小时后LCMS显示反应结束。将反应液滴入处于搅拌状态下的水(50mL)中，加完后静置5分钟，过滤，滤饼冻干后得20mg灰色固体25，收率：40％。
MS-ESI：m/z 534.3[M+H]+；
1H NMR(400MHz，DMSO-d6)δ8.26(d，J＝9.1Hz，1H)，7.72(d，J＝10.9Hz，1H)，7.28(s，1H)，6.51(s，1H)，6.12(s，1H)，5.59-5.50(m，1H)，5.40(s，2H)，5.15(d，J＝18.8Hz，1H)，4.98(d，J＝19.0Hz，1H)，3.28-3.16(m，1H)，3.15-3.02(m，1H)，2.74-2.52(m，2H)，2.36(s，3H)，2.24-2.04(m，4H)，1.92-1.79(m，4H)，0.86(t，J＝7.3Hz，3H)。
参照例1
第一步
取KI4(50mg，0.094mmol)，26a(18mg，0.094mmol)，HATU(39mg，0.103mmol)加入DMF(3mL)中，氮气置换后加入DIEA(48mg，0.376mmol)，25℃下反应1.5小时后LCMS显示反应结束。将反应液滴入处于搅拌状态下的水(50mL)中，加完后静置5分钟，过滤，滤饼冻干后得30mg灰色固体26b，收率：52％。MS-ESI：m/z 607.4[M+H]+。
第二步
取26b(30mg，0.049mmol)溶于DCM(2mL)中，N2置换后降温至0℃，加入TFA(0.5mL)，0℃下反应1.5小时后LCMS显示反应结束。将反应液低温旋干，用DCM带一次后加乙腈和水冻干得20mg黄色固体，收率：80％。
MS-ESI：m/z 507.1[M+H]+；
1H NMR(400MHz，DMSO-d6)δ8.67(d，J＝8.6Hz，1H)，7.86-7.66(m，4H)，7.32(s，1H)，6.56(brs，1H)，5.63-5.54(m，1H)，5.43(s，2H)，5.29(d，J＝18.9Hz，1H)，5.22(d，J＝18.9Hz，1H)，3.23-3.15(m，2H)，3.13-3.03(m，2H)，2.57-2.51(m，2H)，2.43-2.38(m，3H)，2.27-2.08(m，2H)，1.94-1.78(m，2H)， 0.87(t，J＝7.3Hz，3H)。
2.2、接头-细胞毒素(linker-payload)的制备
接头-细胞毒素X1
第一步
氮气保护下，向27a(5.00g，43.0mmol)，NaHCO3(10.9g，129mmol)的DMF(50mL)溶液中滴加苄溴(11.0g，64.6mmol)，并在25℃反应17小时。TLC(PE/EA＝2/1)显示反应完全，将反应液加入到500mL水中，用EA(250mL)萃取两遍，分液后经饱和氯化钠水溶液(500mL)洗涤，无水Na2SO4干燥，浓缩过柱(PE∶EA＝3∶2)得5.1g无色液体，收率：57.1％。
第二步
氮气保护下，向KI2(4.00g，10.9mmol)，TsOH(800mg，4.65mmol)的THF(30mL)溶液中，在0℃下，滴加27b(4.50g，21.8mmol)的THF(10mL)溶液，并在25℃反应2小时。TLC(PE/EA＝1/2)显示反应完全，将反应液加入到200mL水中，用EA(200mL)萃取两次分液，无水Na2SO4干燥，浓缩过柱(PE/EA＝3/2)得白色固体1.56g，收率：26％。
第三步
氢气环境下，在0℃下，向27c(800mg，1.55mmol)的EtOH(8mL)和EA(8mL)混合溶液中加入Pd/C(80mg)，在0℃搅拌2.5小时。LCMS显示反应完全。反应液经硅藻土过滤，用EA(200mL)洗涤滤饼浓缩后用THF(20mL)溶解旋干得白色固体600mg，收率：91％
第四步
氮气保护下，在0℃下，向27d(220mg，0.515mmol)，HY-13631A(250mg，0.47mmol)和HATU(214mg，0.56mmol)的DMF(6mL)溶液中加入DIEA(152mg，1.18mmol)，并在0℃反应2小时。LCMS显示反应完全。将反应液加入柠檬酸水溶液(pH＝4)(150mL)中，过滤，并用175mL水洗涤滤饼，滤干，用油泵拉干得棕色固体260mg，收率：66％。
第五步
氮气保护下，在0℃下，向27e(260mg，0.309mmol)的DCM(30mL)溶液中滴加二乙胺(8mL)，并在0℃反应3小时。LCMS显示反应完全。将反应液加入0℃的石油醚溶液(600mL)中，有固体析出，静置待固体吸附于瓶底后，倒出溶液，用油泵拉干，得棕色固体90mg，收率：47.1％。
第六步
氮气保护下，在0℃下，向27f(90mg，0.13mmol)，KI-1(92mg，0.19mmol)和DIEA(50mg，0.39mmol)的DMF(2.5mL)溶液中加入HATU(74mg，0.19mmol)并在0℃反应2小时。LCMS显示基本反应结束。在0℃下，将反应液加入PH＝4的柠檬酸水溶液(30mL)中，有絮状固体析出，过滤，经制备板(DCM/MecOH＝10/1)得9.2mg淡黄色固体X1，收率：6％。
MS m/z(ESI)：1074[M+1]
H-NMR(400MHz，MeOD)：7.65(d，1H)，7.62(s，1H)，7.30-7.21(m，5H)，6.79(s，2H)，5.69-5.65(m，1H)，5.57(d，1H)，5.43-5.10(m，3H)，4.70(d，2H)，4.48-4.39(m，2H)，4.10-4.05(m，1H)，4.01-3.75(m，5H)，3.46(t，2H)，3.22-3.15(m，2H)，3.07-3.00(m，1H)，2.75(m，1H)，2.62(m，1H)，2.45(s，3H)，2.37-2.20(m，6H)，2.10-2.02(m，2H)，2.00-1.92(m，2H)1.68-1.57(m，6H)，1.01(t，3H)
接头-细胞毒素X2
第一步
将34a(5g，48.0mmol)、K2CO3(19.9g，144.0mmol)溶于DMF(20mL)中，滴加苄溴(12.3g，72.0mmol)，25℃下反应17小时。TLC(PE/EA＝3/1)检测原料反应完全。将反应液加入水(200mL)中、用EA(250mL)萃取分液，用饱和NaCl洗涤，无水Na2SO4干燥后浓缩过柱(PE∶EA＝2∶1)得8.7g无色液体34b，收率93％。MS-ESI：m/z 195.1[M+H]+。
第二步
取34c(7.3g，19.8mmol)、TsOH(1.46g，8.5mmol)溶于THF(20mL)中，氮气保护并降温至0℃，滴加43b(7.7g，39.6mmol)的THF(10mL)溶液，加完后0℃反应2小时。TLC(PE/EA＝2/1)显示原料大部分反应。将反应液倒入100mL水中，DCM(100mL)萃取，分液并用饱和NaCl洗涤，无水Na2SO4干燥后过柱(PE/EA＝1/1)，得3.9g无色稠状物34d，收率：39％。MS-ESI：m/z503.3[M+H]+。
第三步
氢气环境下，在0℃下，向34d(1.9g，3.78mmol)的EtOH(100mL)和EA(100mL)混合溶液中加入Pd/C(1g，10wt.％)，在0℃反应3小时。TLC(PE/EA＝2/1)显示反应完全。反应液经 硅藻土过滤，用EA/EtOH(1∶1，100mL×3)洗涤滤饼，滤液浓缩，并用THF(50mL×3)溶解旋干并重复三次得1g灰色固体34e，收率：64％。MS-ESI：m/z 435.2[M+Na]+。
第四步
在氮气保护下，0℃下向34e(426mg，1.03mmol)，KI4(500mg，0.94mmol)和HATU(429mg，1.13mmol)的DMF(20mL)溶液中滴加DIEA(303mg，2.35mmol)，加完后在0℃反应2小时。LCMS显示反应结束。将反应液滴到300mL水中，搅拌后静置5分钟，过滤，滤饼用DCM/MeOH(10∶1，100mL)的溶液溶解后，干燥旋干拌样，柱层析(EA∶MeOH＝30∶1)得600mg黄色固体34f，收率：77％。MS-ESI：m/z 830.3[M+H]+。
第五步
氮气保护下，在0℃下，向34f(150mg，0.18mmol)的DCM(5mL)溶液中滴加二乙胺(5mL)，并在0℃反应2小时。LCMS显示反应完全。将石油醚溶液(100mL×6)加入反应液中，有固体析出，静置待固体沉淀后，倒出溶液，再用油泵拉干，得120mg白色粉末34g，LCMS显示产物含量为70％，收率：76％。MS-ESI：m/z 608.3[M+H]+。
第六步
在氮气保护下，向34g(60mg，0.099mmol)、43h(51mg，0.108mmol)、和DIEA(32mg，0.25mmol)的DMF(1mL)溶液中在0℃下加入HATU(45mg，0.118mmol)的DMF(1mL)溶液，并在0℃下反应2小时。LCMS显示原料反应完全。将反应液直接过反相柱，洗脱剂((MeCN/MeOH＝1/1)∶H2O＝60％∶40％)，纯化得14.8mg黄色固体X2，收率14％。
MS-ESI：m/z 1062.4[M+H]+。
1H NMR(400MHz，Methanol-d4)δ7.69-7.61(m，2H)，7.22-7.16(m，2H)，7.16-7.09(m，3H)，6.76(s，2H)，5.70-5.64(m，1H)，5.60(d，J＝16.4Hz，1H)，5.40-5.31(m，2H)，5.26(d，J＝19.0Hz，1H)，4.65-4.50(m，7H)，4.25-4.16(m，1H)，3.87(d，J＝16.7Hz，1H)，3.83-3.76(m，3H)，3.72(d，J＝17.0Hz，2H)，3.44(t，J＝7.1Hz，2H)，3.25-3.17(m，2H)，3.10-3.02(m，1H)，2.92-2.83(m，1H)，2.45-2.39(m，5H)，2.32-2.20(m，5H)，1.97-1.89(m，2H)，1.63-1.50(m，4H)，1.34-1.20(m，6H)，0.99(t，J＝7.3Hz，3H).
接头-细胞毒素X3

第一步
向32a(2.00g，6.6mmol)，K2CO3(1.82g，13.2mmol)的MeCN(20mL)中加入溴丙烯(960mg，7.92mmol)，在20℃下搅拌5小时。TLC(PE/EA＝1/2)显示反应结束。将反应液倒入水100mL中，将pH值调至5，用EA(100mL)萃取三次，无水硫酸钠干燥，旋干过柱纯化(PE/EA＝2/1)得1.83g白色固体32b，收率：81％。
第二步
向32b(1.38g，4.02mmol)的DCM(10mL)中加入TFA(10mL)，在25℃下搅拌17小时。TLC(PE/EA＝1/3)显示反应结束。将反应液旋干得0.91g黄色粘状物32c，收率不计。
第三步
向32c(910mg，4.87mmol)，NaHCO3(613mg，7.3mmol)的DME/H2O(20mL/10mL)中加入41d(1.92g，4.87mmol)，在25℃下搅拌3小时。TLC(DCM/MeOH＝1/1)显示反应结束。将反应液倒入水100mL中，用aq.HCl(1N)将pH值调至5，用EA(150mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(DCM/MeOH＝20/1)得1.53g白色固体32e，收率：67％。MS-ESI：m/z 467.4[M+H]+。
第四步
向32f(3g，5.83mmol)的MeOH(50mL)中加入Pd/C(600mg)，在25℃在氢气球下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.9g白色固体32g，收率：77％。
第五步
向32g(789mg，1.86mmol)，KI4(900mg，1.69mmol)，三乙胺(342mg，3.38mmol)的DMF(10mL)中加入HATU(707mg，1.86mmol)，在0℃下搅拌3.5小时。TLC(EA)显示反应结束。将反应液倒入H2O(80mL)，用EA(100mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(EA)得1.186g白色固体32h，收率：83％。MS-ESI：m/z 842.3[M+H]+。
第六步
将32h(1.186g，1.41mmol)的DCM/二乙胺(20mL，20/1)在25℃下搅拌17小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液倒入石油醚(200mL)中过滤得768mg白色固体32i，收率：88％。MS-ESI：m/z 620.3[M+H]+。
第七步
向32i(676mg，1.09mmol)，32e(508mg，1.09mmol)，DIEA(423mg，3.27mmol)的DMF(10mL)中加入HATU(414mg，1.09mmol)，在20℃下搅拌17小时。TLC(PE/EA＝1/5)显示反应结束。将反应液倒入水中(30mL)过滤，滤饼过柱纯化(DCM/MeOH＝50/1)511mg白色固体32j，收率：44％。MS-ESI：m/z 1068.3[M+H]+。
第八步
将32j(482mg，0.451mmol)的二乙胺/DCM(10mL，1/5)的溶液在10℃下搅拌17小时。TLC(EA)显示反应结束。将反应液倒入PE(300mL)中过滤得301mg白色固体32k，收率不计。
第九步
向32k(301mg，0.356mmol)，Pd(PPh3)4(82mg，0.071mmol)的THF(5mL)中加入吗啡啉(93mg，1.07mmol)，在25℃下搅拌5小时。LCMS显示反应结束。将反应液制备得108mg白色固体321，收率：38％。MS-ESI：m/z 806.3[M+H]+。
第十步
向321(108mg，0.134mmol)，三乙胺(41mg，0.402mmol)的THF(2mL)和DMF(2mL)中加入溴乙酰溴(27mg，0.134mmol)，在0℃下搅拌1小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液直接制备得15mg白色固体X3，收率：12％。
MS-ESI：m/z 926.3[M+H]+。
1H NMR(400MHz，DMSO-d6)δ12.11(s，1H)，8.54-8.42(m，3H)，8.27-8.16(m，2H)，7.78(d，J＝11.0Hz，1H)，7.30(s，1H)，6.53(s，1H)，5.61-5.51(m，1H)，5.42(s，2H)，5.20-5.05(m，2H)，4.56-4.42(m，2H)，4.32-4.22(m，1H)，3.96-3.87(m，3H)，3.79(d，J＝5.6Hz，2H)，3.70(d，J＝5.9Hz，2H)，3.25-3.08(m，2H)，2.61-2.53(m，2H)，2.45-2.36(m，4H)，2.36-2.22(m，3H)，2.20-2.03(m，4H)，1.99-1.68(m，4H)，0.87(t，J＝7.3Hz，3H).
接头-细胞毒素X4
第一步
向33a(2.00g，2.58mmol)的MeOH(20mL)中加入Pd/C(400mg，10wt.％)，在20℃下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.3g白色固体33b，收率：74％。
第二步
向33b(0.55g，0.802mmol)，KI4(427mg，0.802mmol)和DIPEA(310mg，2.40mmol)的DMF(5mL)中加入HATU(305mg，0.802mmol)，在0℃下搅拌2小时。TLC(DCM/MeOH＝1/10)显示反应结束。将反应液倒入水(40mL)中，过滤得粗品，经柱纯化(DCM/MeOH＝20/1)得360mg黄色固体33c，收率41％。
第三步
向33c(360mg，0.326mmol)的DCM(10mL)中加入二乙胺(2mL)。在25℃下搅拌17小时。TLC(DCM/MeOH＝5/1)显示反应结束。将反应液倒入PE(100Ml)中，过滤得205mg白色固体33d，收率：71％。MS-ESI：m/z 881.3[M+H]+。
第四步
向33d(205mg，0.233mmol)和三乙胺(118mg，1.17mmol)的DMF(1mL)和水(1mL)中加入溴乙酰溴(94mg，0.446mmol)的THF(2mL)溶液，并在0度搅拌1小时，反应液直接制备得15mg白色固体X4，收率：6％。
MS-ESI：m/z 1001.2[M+H]+。
1H NMR(400MHz，DMSO-d6)δ8.57-8.50(m，1H)，8.50-8.43(m，2H)，8.35-8.29(m，1H)，8.19-8.12(m，2H)，7.80(d，J＝10.8Hz，1H)，7.27-7.14(m，7H)，6.53(s，1H)，5.59-5.51(m，1H)，5.44-5.39(m，2H)，5.20-5.07(m，2H)，4.56-4.44(m，3H)，3.92(s，3H)，3.80-3.68(m，5H)，3.41(s，1H)，3.21-3.12(m，2H)，2.83-2.74(m，1H)，2.58-2.55(m，3H)，2.39(s，4H)，2.18-2.03(m，4H)，1.93-1.78(m，2H)，0.87(t，J＝7.3Hz，3H).
2.3、抗B7H4抗体药物偶联物的制备
抗体药物偶联物ADC DB1001-X1的制备
用超纯水分别配制还原剂和保护剂如下：2mg/ml TCEP(Tris-2-carboxyethyl-phosphine，厂家：Thermo)水溶液和100mmol/L EDTA(乙二胺四乙酸二钠厂家：Sigma)水溶液。
取160mg 17.99mg/ml的DB1001(PR08199)单抗置于600ml离心瓶，加入30mM His-HAc，pH5.5缓冲液稀释抗体浓度至10mg/ml，按照反应液总体积5％加入100mM EDTA水溶液，震荡混匀后加入2mg/ml TCEP水溶液进行抗体还原，TCEP与抗体的摩尔比2.3∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。按照药物与抗体终浓度摩尔比12∶1加入前述Linker-payload的DMA溶液，按照反应液总体积10％补充DMA，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO 30KD，厂家：密理博)置换样品保存buffer，先用含有10％DMSO的30mM His-HAc，pH5.5缓冲液超滤3次，再用无DMSO的30mM His-HAc，pH5.5超滤6次，得到抗体药物偶联物DB1001-X1 125.6mg，浓度12.854mg/mL，收率78.5％。
经HIC检测，抗体药物偶联物DB1001-X1的载药量(DAR)为3.89，SEC纯度为100％。
抗体药物偶联物ADC DB1001-X2的制备
用超纯水分别配制还原剂和保护剂如下：2mg/ml TCEP(Tris-2-carboxyethyl-phosphine，厂家：Thermo)水溶液和100mmol/L EDTA(乙二胺四乙酸二钠厂家：Sigma)水溶液。
将Linker-payload X2溶于干燥的DMA(N，N-Dimethylacetamide，二甲基乙酰胺，厂家：国药集团)，配制成10mg/mL的linker-payload DMA溶液。
取160mg 17.99mg/ml的DB1001(PR08199)单抗置于600ml离心瓶，加入30mM His-HAc，pH5.5缓冲液稀释抗体浓度至10mg/ml，按照反应液总体积5％加入100mM EDTA水溶液，震荡混匀后加入2mg/ml TCEP水溶液进行抗体还原，TCEP与抗体的摩尔比2.3∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。按照药物与抗体终浓度摩尔比12∶1加入前述Linker-payload的DMA溶液，按照反应液总体积10％补充DMA，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO 30KD，厂家：密理博)置换样品保存buffer，先用含有10％DMSO的30mM His-HAc，pH5.5缓冲液超滤3次，再用无DMSO的30mM His-HAc，pH5.5超滤6次，得到抗体药物偶联物DB1001-X2 128.3mg，浓度10.522mg/mL，收率80.18％。
经HIC检测，抗体药物偶联物DB1001-X2的载药量(DAR)为5.4，SEC纯度为98.1％。
参照ADC-1的制备
参照WO2020244657A1中化合物34(hu2F7-依喜替康)，制备参照ADC-1。
实施例3.小分子化合物(细胞毒性药物)的体外增殖抑制测试
3.1、小分子化合物(细胞毒性药物)对肿瘤细胞体外增殖抑制测试
测试目的
为了检测药物化合物，对NCI-N87，JIMT-1和MBA-MB-231肿瘤细胞体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经6天培养后，采用CTG(Luminescent Cell Viability Assay，Promega，货号：G7558)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
1、细胞培养：NCI-N87/JIMT-1/MBA-MB-231用10％FBS RPMI-1640培养基培养。
2、细胞准备：取对数生长期的NCI-N87/JIMT-1/MBA-MB-231细胞，用PBS洗涤1次之后，加入2-3ml胰蛋白酶消化2-3min，待细胞消化完全后，加入10-15ml细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，接着加入10-20ml细胞培养液将细胞重悬，制成单细胞悬液。
3、细胞铺板：将NCI-N87/JIMT-1/MBA-MB-231单细胞悬液混匀，用细胞培养液分别调整活细胞密度至6x104cells/ml，将密度调整过后的细胞悬液混匀，以50ul/孔加入96孔细胞培养板。将培养板在培养箱培养18小时(37℃，5％CO2)。
4、化合物准备：用DMSO溶解化合物，配制成初始浓度为10mM的存储液。
小分子化合物共8个浓度，分别为：300，100，30，10，3，1，0.3，0.1nM。
5、加样操作：向培养板中加入配置的不同浓度的待测样品，每个样品两复孔。将培养板在培养箱孵育6天(37℃，5％CO2)。
6、显色操作：取出96孔细胞培养板，向每孔加入50ul CTG试剂，室温孵育10分钟。
7、读板操作：取出96孔细胞培养板，置于酶标仪中，用酶标仪测定化学发光。
数据分析：用Microsoft Excel，Graphpad Prism 5对数据进行处理分析。
表1本申请中的小分子化合物对肿瘤细胞体外增殖抑制的IC50值
结论：根据表1的结果，本申请药物偶联物的毒素对NCI-N87细胞、JIMT-1和MDA-MB-231细胞具有明显增强的增殖抑制活性。
3.2、小分子化合物(细胞毒性药物)对肿瘤细胞体外增殖抑制测试
测试目的
为了检测药物化合物，对NCI-N87细胞和Colo205肿瘤细胞体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经6天培养后，采用CTG(Luminescent Cell Viability Assay，Promega，货号：G7558)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。
1、细胞培养：NCI-N87/Colo205用10％FBS RPMI-1640培养基培养。
2、细胞准备：取对数生长期的NCI-N87/Colo205细胞，用PBS洗涤1次之后，加入2-3ml胰蛋白酶消化2-3min，待细胞消化完全后，加入10-15ml细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，接着加入10-20ml细胞培养液将细胞重悬，制成单细胞悬液。
3、细胞铺板：将NCI-N87/Colo205单细胞悬液混匀，用细胞培养液分别调整活细胞密度至6x104cells/ml，将密度调整过后的细胞悬液混匀，以50ul/孔加入96孔细胞培养板。将培养板在培 养箱培养18小时(37℃，5％CO2)。
4、化合物准备：用DMSO溶解化合物，配制成初始浓度为10mM的存储液。
小分子化合物共8个浓度，分别为：300，100，30，10，3，1，0.3，0.1nM。
5、加样操作：向培养板中加入配置的不同浓度的待测样品，每个样品两复孔。将培养板在培养箱孵育6天(37℃，5％CO2)。
6、显色操作：取出96孔细胞培养板，向每孔加入50ul CTG试剂，室温孵育10分钟。
7、读板操作：取出96孔细胞培养板，置于酶标仪中，用酶标仪测定化学发光。
数据分析：用Microsoft Excel，Graphpad Prism 5对数据进行处理分析。
表2本申请中的小分子化合物对NCI-N87和Colo205细胞体外增殖抑制的IC50值
结论：根据表2的结果，本申请药物偶联物的毒素对NCI-N87和Colo205细胞具有明显的增殖抑制活性，且显著优于参照例1。
实施例4.抗体药物偶联物的细胞内吞活性
测试目的
检测本申请针对B7H4靶标的抗体药物偶联物，在B7H4表达的MDA-MB-468细胞上的内吞效应。将细胞和固定浓度的抗体药物偶联物(ADC)和内吞指示试剂pHrodo进行共孵育，通过观察不同时间点细胞内伴随抗体药物偶联物进入细胞的pHrodo所产生的荧光信号来评价抗体药物偶联物内吞能力。
实验方法
1、细胞培养：MDA-MB-468细胞使用10％FBS Leibovitz′s L-15培养基培养。
2、细胞准备：取对数生长期的MDA-MB-468细胞，用PBS洗涤1次之后，消化2-3min，待细胞消化完全后，加入10-15ml细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，加入细胞培养液将细胞重悬制成单细胞悬液并调整活细胞密度至3x105cells/ml。
3、细胞铺板：以50ul/孔加入96孔细胞培养板。将培养板在培养箱培养48小时(37℃，5％CO2)。
4、加样操作：将待测抗体药物偶联物与Fab-pHrodo共同孵育形成复合物，浓度为120nM，以5倍梯度稀释调整浓度，以50ul/孔加到细胞。共8个浓度，每个浓度设置两个复孔。
5、细胞培养：将培养板在培养箱孵育48小时(37℃)。
6、读板操作：到达对应时间点，取出96孔细胞培养板，将细胞消化后通过FACS读取细胞数量及荧光数值。
表3本申请抗体药物偶联物在MDA-MB-468细胞不同时间点内吞产生荧光信号
结论：根据表3的结果，本申请针抗体药物偶联物对B7H4高表达的MDA-MB-468细胞具有内吞效应。
实施例5：抗体药物偶联物对肿瘤细胞体外增殖抑制测试
采用化学发光细胞活率检测法(即CTG方法)评估anti-B7H4 ADC DB1001-X1、DB1001-X2在B7H4阳性表达的细胞MDA-MB-468和B7H4阴性表达的MDA-MB-231中孵育处理7天，对细胞增殖的抑制作用。
收集对数生长期细胞，以2000个细胞/孔的密度铺板，细胞板放入37℃、5％CO2培养箱培养过夜。实验第二天，将DB1001-X1、DB1001-X2用完全培养基按5倍稀释，获得9个浓度梯度(以300nM的最高浓度开始)药物后，50μL/孔加入细胞培养板中，完全培养基作为空白对照，设置2个复孔；继续于37℃培养箱内孵育7天。孵育结束，取出细胞培养板，平衡至室温后，每孔加入50μL CTG检测试剂(Promega，Cat#：G7573)，震荡混匀后放置于暗处静置10分钟后，利用酶标仪检测读取信号值。应用GraphPad Prism软件，使用非线性回归模型绘制S型剂量-反应曲线并计算IC50值。细胞存活率计算公式＝(Lum待测药-Lum空白对照)/(Lum溶剂空白对照-Lum空白对照)×100％。
实验结果如下表所示：
表4：抗体偶联药物体外增殖抑制活性
实验结论：根据表4的结果，本申请抗体药物偶联物DB1001-X1和DB1001-X2对B7H4阳性表达的MDA-MB-468具有显著增强的增殖抑制活性。
实施例6：抗体药物偶联物对细胞MDA-MB-468荷瘤小鼠药效评价
为研究DB1001-X1、DB1001-X2对体内形成肿瘤的抑制作用，在小鼠体内用B7H4阳性表达的细胞MDA-MB-468形成移植瘤后，评估DB1001-X1、DB1001-X2的抗肿瘤效果。
1.受试药物及材料
空白对照组(对照组)：生理盐水
DB1001-X1(治疗组)：3mg/kg
DB1001-X1(治疗组)：10mg/kg
DB1001-X2(治疗组)：3mg/kg
DB1001-X2(治疗组)：10mg/kg
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：6-8周龄的雌性CB-17 SCID小鼠，购自北京维通利华实验动物技术有限公司。
4.试验方法：
将10×106个MDA-MB-468细胞接种于6-8周龄的雌性NOD/SCID小鼠右侧背部皮下，当肿瘤长至约172mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射DB1001-X1或DB1001-X2，共注射1次，采用剂量分别为3mg/kg和10mg/kg。每周测量2次瘤体积和体重，记录数据。
溶媒对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。受试物组和Vehicle组的组间显著性分析通过one-way ANOVA法进行。
表5各组不同时间点的瘤体积1(mm3)

注：
1.肿瘤体积用平均值±标准误表示；
2.开始给药后的天数。
表6受试物对MDA-MB-468细胞皮下异种移植肿瘤模型的抑瘤药效评价(基于给药后21天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T21/V21×100)和TGI(TGI(％)＝[1-(T21-T0)/(V21-V0)]×100)反映；
3.p值根据肿瘤体积计算(p＜0.05代表有统计学差异，p＜0.01代表有显著性差异)。
实验结果如图1及表5和表6所示。抗体药物偶联物DB1001-X1和DB1001-X2单次给药后表现出显著增强的剂量依赖性抑瘤活性。
实施例7.抗体药物偶联物的种属交叉
测试目的
检测抗体药物偶联物DB1001-X2在人、食蟹猴、和小鼠B7H4转染的HEK293T细胞的种属交叉反应。
实验方法
1、所有细胞株于37℃，5％CO2条件下培养于完全培养基。
2、收获处于对数生长期的细胞并用台盼蓝排斥法检测细胞活力，确保细胞活力在90％以上，1000r/min离心5min后，弃上清。使用PBS洗涤细胞一次，用FACS Buffer重悬制成单细胞悬液并调整细胞密度至5×106cells/mL；
3、向96孔板中分别加入50μL的细胞悬液，使受试品工作液最高浓度为100nM，依次3倍稀释，共8个浓度；混匀后，放置于4℃，孵育40min。
4、使用FACS Buffer洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用100μL FACS Buffer重悬细胞；
5、加入2μL的PE标记的二抗(PE anti-human IgG Fc Antibody)，混匀后，放于4℃避光孵育40min；
6、使用FACS Buffer洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用250μL FACS Buffer重悬细胞；
7、流式细胞仪检测荧光值。
结果表7 DB1001-X2和人，猴，小鼠B7H4的亲和力
如表7和图2所示，DB1001-X2与人和食蟹猴的B7-H4有相似的亲和力，但与小鼠的B7-H4没有结合。
以下实施例中，Isotype ADC是指：抗体为阴性对照抗体，接头-细胞毒素为X2，参照DB1001-X2制备。
实施例8.抗体药物偶联物的靶向特异性
测试目的
通过流式检测抗体药物偶联物DB1001-X2和过表达人B7家族蛋白细胞的结合特异性。
实验方法
1、建立稳定表达人PD-L1(B7-H1)，PD-L2(B7-DC)，ICOSLG(B7-H2)，CD276(B7-H3)，B7H4，VISTA(B7-H5)，CD80(B7-1)，CD86(B7-2)的HEK293T单克隆细胞株，及稳定表达人B7-H7(HHLA2)的CHO-K1单克隆细胞株；
2、所有细胞株于37℃，5％CO2条件下培养于完全培养基；
3、收获处于对数生长期的细胞并用台盼蓝排斥法检测细胞活力，确保细胞活力在90％以上，1000r/min离心5min后，弃上清。使用PBS洗涤细胞一次，用FACS Buffer重悬制成单细胞悬液并调整细胞密度至5×106cells/mL；
4、向96孔板中分别加入50μL的细胞悬液，使受试品工作液浓度为100nM或者300nM，共1个浓度；混匀后，放置于4℃，孵育40min；
5、使用FACS Buffer洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用100μL FACS Buffer重悬细胞；
6、加入2μL的PE标记的二抗(PE anti-human IgG Fc Anfibody)，混匀后，放于4℃避光孵育40min；
7、使用FACS Buffer洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用250 μL FACS Buffer重悬细胞；
8、流式细胞仪检测荧光值。
实验结果
如图3所示，DB1001-X2与人B7-H4结合，但未检测到与其他人B7家族蛋白B7-H1、B7-DC、B7-H2、B7-H3、B7-H5、B7-1、B7-2和B7-H7结合。
实验结论
DB1001-X2能与人B7-H4蛋白特异性结合，与其他B7家族蛋白无交叉反应。
实施例9：抗体药物偶联物的细胞内吞活性
测试目的
检测本申请针对B7H4靶标的抗体药物偶联物与其他针对B7H4的抗体药物偶联物相比，在表达B7H4的MDA-MB-468细胞上的内吞效率。将细胞和固定浓度的抗体药物偶联物和内吞指示试剂Fabfluor-pH进行共孵育，通过连续观察24小时活细胞的荧光信号变化来评价抗体药物偶联物的内吞能力。
实验方法
1、细胞培养：MDA-MB-468细胞使用10％FBS Leibovitz′s L-15培养基培养。
2、细胞准备：取对数生长期的MDA-MB-468细胞，用PBS洗涤1次之后，消化2-3min，待细胞消化完全后，加入10-15ml细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，加入细胞培养液将细胞重悬制成单细胞悬液并调整活细胞密度至1x105cells/ml。
3、细胞铺板：以50ul/孔加入96孔细胞培养板。将培养板放在培养箱培养过夜(37℃，5％CO2)。
4、标记待测抗体药物偶联物：将供试品储液稀释为240nM的4×工作液(终浓度60nM)，将Fabfluor-pH储液稀释为720nM的4×工作液(终浓度180nM)，将两者充分混合，37℃避光孵育15分钟。
5、俘获分析图像：转移标记后的供试品工作液至实验板的相应孔中，将实验板转移至Incucyte活细胞分析设备中，设置扫描拍照程序，使用Incucyte活细胞分析系统获取图像。以1小时的间隔进行定量，持续24小时。分析结果表示为：总荧光面积(μm2/图像)。
实验结果
如图4所示，本申请抗体药物偶联物DB1001-X2和对照抗体药物偶联物参照ADC-1在MDA-MB-468细胞不同时间点内吞产生荧光信号的积分面积。图5为本申请抗体药物偶联物DB1001-X2和对照抗体药物偶联物在MDA-MB-468细胞的内吞效率比较。
表8抗体药物偶联物在MDA-MB-468的内吞

实验结论
根据表8、图4和图5的结果，本申请抗体药物偶联物在表达B7H4的MDA-MB-468细胞具有内吞效应，且优于参照ADC-1。
实施例10：抗体药物偶联物对肿瘤细胞体外增殖抑制测试(2D细胞培养法)
测试目的
采用化学发光细胞活率检测法(即CTG方法)检测本申请针对B7H4靶标的抗体药物偶联物与其他针对B7H4的抗体药物偶联物相比，对过表达B7H4的人结直肠癌HT29体外增殖的抑制作用。
实验方法
收集对数生长期细胞，以15000个细胞/孔的密度铺板，细胞板放入37℃、5％CO2培养箱培养过夜。实验第二天，将受试品用完全培养基稀释，获得终浓度为10nM的药物后，50μL/孔加入细胞培养板中，完全培养基作为空白对照，设置3个复孔；继续于37℃培养箱内孵育7天。孵育结束，取出细胞培养板，平衡至室温后，每孔加入50μL CTG检测试剂(Promega，Cat#：G7573)，震荡混匀后放置于暗处静置10分钟后，利用酶标仪检测读取信号值。应用GraphPad Prism软件，细胞存活率计算公式＝(Lum待测药-Lum空白对照)/(Lum溶剂空白对照-Lum空白对照)×100％。
实验结果
表9抗体偶联物对HT29-B7H4体外增殖抑制作用
实验结论
根据表9和图6实验结果，本申请抗体药物偶联物DB1001-X2对过表达B7H4的人结直肠癌细胞HT29具有显著的增殖抑制活性，且优于参照ADC-1。
实施例11：抗体药物偶联物对肿瘤细胞体外增殖抑制测试(3D细胞培养法)
测试目的
采用化学发光细胞活率检测法(即CTG方法)评估anti-B7H4 ADC DB1001-X2及anti-B7H4单抗DB1001对3D培养条件下B7H4高表达的人乳腺细胞癌MX-1，B7H4低表达的子宫内膜癌细胞RL95-2，及B7H4阴性表达的乳腺癌细胞JIMT-1的增殖抑制作用。
实验方法
收集对数生长期细胞，加入细胞培养液将细胞重悬制成单细胞悬液并调整活细胞密度至1x105cells/ml，将3.5mL细胞悬液和6.5mL的1％甲基纤维素混合均匀，尽量避免产生气泡，在96孔板中每孔加入90μL细胞悬液，在37℃、5％CO2培养箱培养过夜。实验第二天，用完全培养基稀释受试品得到10倍溶液，每孔加入10μL受试品溶液，最高终浓度为100nM，9个浓度，3倍稀释，完全培养基作为空白对照，设置3个复孔；继续于37℃培养箱内孵育6天。孵育结束，取出细胞培养板，平衡至室温后，每孔加入100μL CTG检测试剂(Promega，Cat#：G7573)，震荡混匀后放置于暗处静置10分钟后，把细胞板放置于室温30分钟以稳定发光信号，用EnVision读取Luminescence。应用GraphPad Prism软件，使用非线性回归模型绘制S型剂量-反应曲线并计算IC50值。细胞存活率计算公式＝(Lum待测药-Lum空白对照)/(Lum溶剂空白对照-Lum空白对照)×100％。
IsotypeADC：抗体为阴性对照抗体，接头-细胞毒素为X2，参照DB1001-X2制备。
实验结果
表10：抗体偶联药物对肿瘤细胞株增殖抑制活性
实验结论
如表10以及图7、图8和图9所示，DB1001-X2对高表达和低表达B7H4的肿瘤细胞株都有强的体外杀伤作用，对不表达B7H4的细胞没有杀伤作用，说明这种杀伤作用是依赖于B7H4表达的。
实施例12：抗体药物偶联物对T细胞激活
测试目的
检测本申请针对B7H4靶标的抗体药物偶联物与其他针对B7H4的抗体药物偶联物相比，阻断B7H4对T细胞的抑制，激活T细胞产生IFN-gamma的能力。
1、实验方法收集对数生长的293T-OS8-humanB7H4细胞(康源博创公司)，300g离心5分钟。
2、把细胞重悬至1x105cell/ml，按照1x104/100μL/孔的密度铺板，培养过夜。
3、使用美天妮T细胞分离试剂盒(Miltenyi，CaT#130-096-535)按说明书的方法分离健康供体1(上海妙顺，货号PB100C-W，批号A10Z983077)和健康供体2的PBMC(上海妙顺，货号PB050C- W，批号P122010104C)得到人原代T细胞。
4、配制2×终浓度的待测受试品，终浓度为0.02nM。
5、移除实验孔内的培养基，然后将供体1和供体2的人原代T细胞按照2x105/100μL/孔的密度加入含有293T-OS8-humanB7H4细胞的实验孔中。
6、随后加入100μL/孔待测抗体，设置两个重复。
7、培养3天后，收集上清，ELISA方法检测IFN-gamma的浓度。
Isotype ADC：抗体为阴性对照抗体，接头-细胞毒素为X2，参照DB1001-X2制备。
实验结果
表11抗体药物偶联物激活T细胞分泌IFN-γ
实验结论
如表11、图10和图11所示，DB1001-X2能阻断B7H4阳性细胞对不同供体来源T细胞的抑制，且显著优于参照ADC-1。
实施例13：抗体药物偶联物旁观者杀伤
测试目的
检测本申请针对B7H4靶标的抗体药物偶联物与其他针对B7H4的抗体药物偶联物相比，内吞后释放的小分子药物从B7H4阳性的细胞扩散到附近B7H4表达阴性的细胞，并对其产生细胞杀伤的旁观者效应。
将不带标记的过表达B7H4的HT29细胞(HT29-B7H4)和转染荧光素酶的不表达B7H4的HT29细胞(HT29-Luc2)按照比例混合，和受试品共孵育一段时间后，加入荧光素酶底物，活细胞产生的荧光素酶催化底物产生荧光素，通过荧光素的数值来检测荧光素酶的含量，即反映活细胞数。
实验方法
1、收集对数生长期细胞，加入含2％血清1640培养基重悬细胞，调整HT29-B7H4和HT29-Luc2的细胞数目，分别加入45ul细胞至96孔板中，使两细胞比例分别为3∶1，即HT29-B7H4细胞数11250个/孔，HT29-Luc2细胞数3750个/孔，在37℃、5％CO2培养箱培养过夜。
2、配制10×浓度的受试品溶液，在96孔板的细胞中每孔加入10uL药物溶液，使受试品终浓度为10nM，3个复孔。检测day0的冷光值。
3、将已加药的96孔板中的细胞置于37℃、5％CO2条件下继续培养7天。
4、孵育结束，取出细胞培养板，平衡至室温后，HT29-B7H4单孵育的每孔加入50μL CTG检测试剂(Promega，Cat#：G7573)，震荡混匀后放置于暗处静置10分钟后，利用酶标仪检测读取信号值。
5、HT29-Luc2单孵育和共孵育的每孔加入等体积的Bright-Glo溶液，在定轨摇床上振动15分钟使细胞裂解。然后在多功能酶标仪上读取冷光值。
5、数据分析：应用GraphPadPrism软件，细胞存活率计算公式＝(Lum待测药-Lum空白对照)/(Lum溶剂空白 对照-Lum空白对照)×100％。实验结果
表12抗体药物偶联物对B7H4阴性的HT29-Luc2旁观者杀伤效应
实验结论，如表12和图12所示，DB1001-X2对和B7H4阳性细胞一起孵育的B7H4阴性细胞有很好的旁观者杀伤效应。
实施例14：抗体药物偶联物在人、大鼠、猴体外血浆的稳定性
测试目的
抗体药物偶联物在体外和人、鼠、猴血浆孵育21天，在不同时间点取样，用液相色谱和质谱测量血浆中释放的小分子毒素(细胞毒性药物)的量，来评估抗体药物偶联物的血浆稳定性。
实验方法
将DB1001-X2用人、大鼠或食蟹猴血清稀释到终浓度150μg/mL，在37℃下孵育持续21天，中间取样时间点为T0，2小时，8小时，1天，4天，7天，14天，21天。血浆样品用乙腈按体积比1∶1沉淀，离心取上清，然后通过LC-MS/MS(液相：Thermo Vanquish；三重四极杆质谱：Thermo TSQ Quantis)进行分析。使用细胞毒性药物P-III-30浓度计算抗体药物偶联物释放速率。
实验结果
见表13和图13，孵育21天后，150μg/mL的DB1001-X2的载荷(细胞毒性药物)P-III-30在人、大鼠和猴血浆中释放率不到1％。
表13细胞毒性药物P-III-30在人、大鼠和猴血浆21天后的释放率
实验结论
DB1001-X2在体外人、大鼠和猴血浆中具有良好的稳定性。
实施例15：抗体偶联药物的药代动力学
测试目的
食蟹猴静脉给予DB1001-X2，每3周给药1次，共给药2次。研究其药代动力学特征，为后续研究提供参考。
实验方法
4只食蟹猴，雌、雄各半，分组时体重2.2～3.6kg，共分2组，分别为DB1001-X2低剂量组、DB1001-X2高剂量组，给药剂量分别设置为30、80mg/kg，每3周给药1次，共给药2次，给药容积5mL/kg。
采样时间：首次给药前及给药结束后5min、0.5h、2h、4h、8h、24h、48h、72h、120h、168h、336h、504h各采集1次TK血样；末次给药结束后5min、0.5h、2h、4h、8h、24h、48h及解剖前各采集1次TK血样。采样方法：前肢静脉或其它适宜静脉，采集约0.5mL全血。
血样处理：采集血液后，置于以EDTA·K2作为抗凝剂的贴有标签的采血管中。将样品管轻轻颠倒数次以确保混匀后，即刻放入湿冰中保存。并在采集后1h内，4℃3800rpm离心10min，吸取并分装血浆100μL/管(2管)，暂存于干冰中，4h内转移至超低温冰箱(-60℃及以下)，在试验记录中记录全血采集、收集血浆的时间。
所有样本采集完成后，干冰条件下运送至检测部门。
样本分析：使用建立的ELISA法方法分析血清中总抗体及ADC药物的血药浓度。使用已有的LC-MS/MS方法分析血浆中细胞毒性药物P-III-30的血药浓度。
实验结果见表14。
表14抗体药物偶联物在食蟹猴血浆的药代动力学
实验结论
根据表14的结果，在DB1001-X2和总抗体之间没有观察到药代动力学差异，这表明DB1001-X2在体内血浆中稳定性好。同时，在剂量为30mg/kg和80mg/kg的食蟹猴中，检测到P-III-30的低血浆浓度，这表明由于DB1001-X2的稳定性和P-III-30的短全身半衰期，P-III-30的全身暴露量较低，从而具有较高的安全性。
实施例16：抗体药物偶联物对人高表达B7H4的乳腺癌细胞MX-1荷瘤小鼠药效评价
为研究DB1001-X2对体内形成肿瘤的抑制作用，在小鼠体内用B7H4阳性高表达的人乳腺癌细胞MX-1形成移植瘤后，评估DB1001-X2的抗肿瘤效果。
1.受试药物及材料
空白对照组(对照组)：生理盐水
DB1001-X2(治疗组)：1mg/kg
DB1001-X2(治疗组)：3mg/kg
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：6-8周龄的雌性BALB/c Nude小鼠，购自集萃药康生物科技有限公司。
4.试验方法：
将5×106个MX-1细胞接种于6-8周龄的雌性BALB/c Nude小鼠右侧背部皮下，当肿瘤长至约160.82mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(iv.)注射受试品，每两周一次，共注射2次，DB1001-X2采用剂量分别为1mg/kg和3mg/kg，ISO-ADC采用剂量为3mg/kg。试验终点时间为分组后第27天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P＜0.05为具有显著性差异。作图软件为GraphPad Prism。
表15.受试物对MX-1细胞皮下异种移植肿瘤模型的抑瘤药效评价(基于给药后27天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T27/V27×100)和TGI(TGI(％)＝[1-(T27-T0)/(V27-V0)]×100)反映；
3.p值根据肿瘤体积计算(p＜0.05代表有统计学差异，p＜0.01代表有显著性差异)。
实验结果如图14及表15所示，抗体药物偶联物DB1001-X2给药后表现出显著的剂量依赖性抑 瘤活性。
实施例17：抗体药物偶联物对人B7H4阳性的乳腺癌细胞MCF-7荷瘤小鼠药效评价
为比较DB1001-X2与同类竞品对体内形成肿瘤的抑制作用，在小鼠体内用B7H4阳性表达的人乳腺癌细胞MCF-7形成移植瘤后，评估DB1001-X2的抗肿瘤效果。
1.受试药物及材料
空白对照组(对照组)：生理盐水
DB1001-X2(治疗组)：3mg/kg
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：6-7周龄的雌性NCG小鼠，来自江苏集萃药康生物科技股份有限公司。
4.试验方法：
收集对数生长期的MCF-7细胞(接种代次为N+12)，去除培养液并用PBS洗两次后接种(荷瘤前及荷瘤后细胞存活率分别为：96.03％及94.81％)，接种量：1×107cells/75μL/只(1∶1添加基质胶)。接种后第19天，平均肿瘤体积达到107.44mm3时，挑选50只小鼠根据肿瘤体积随机分成10组，每组5只。分组当天定义为D0天，并于分组当天D0天开始给药。分剩小鼠安乐处理。受试品共注射1次，采用剂量为3mg/kg。每周测量2次瘤体积和体重，记录数据。
溶媒对照组或治疗组每组5只小鼠。试验终点时间为分组后第25天，通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P＜0.05为具有显著性差异。作图软件为GraphPad Prism。
实验结果：本发明抗体药物偶联物DB1001-X2单次给药后表现出显著的剂量依赖性抑瘤活性。
实施例18：抗体药物偶联物对人低表达B7H4的子宫内膜癌细胞RL95-2荷瘤小鼠药效评价
为研究DB1001-X2对体内形成肿瘤的抑制作用，在小鼠体内用B7H4阳性低表达的人子宫内膜癌细胞RL95-2形成移植瘤后，评估DB1001-X2的抗肿瘤效果。
1.受试药物及材料
空白对照组(对照组)：磷酸盐缓冲液(DPBS)
DB1001-X2(治疗组)：1mg/kg
DB1001-X2(治疗组)：3mg/kg
DB1001-X2(治疗组)：10mg/kg
2.配制方法：所有样品均用磷酸盐缓冲液(DPBS)稀释配制。
3.试验动物：6-8周龄的雌性BALB/c Nude小鼠，购自浙江维通利华实验动物技术有限公司。
4.试验方法：
收集对数生长期的RL95-2细胞，在每只小鼠的右侧颈背部皮下接种5×106个RL95-2细胞，接种体积为0.2mL，细胞悬液为PBS加基质胶(体积比为1∶1)。体内药效实验在细胞接种后第20天，肿瘤平均体积达到134mm3时，根据肿瘤体积随机化分组给药，每组5只小鼠。分组当天定义为D0天，并于分组当天D0天开始给药。受试品每两周注射一次，采用剂量为3mg/kg，共注射2次，试验终点时间为分组后第28天，每周测量2次瘤体积和体重，记录数据。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
统计分析，基于分组后第28天数据，利用GraphPad Prism软件进行统计学分析评估组间差异。三组或多组间比较用one-way ANOVA(Dunnett多重比较检验和Tukey多重比较检验)进行分析，p＜0.05认为有显著性差异。
表16.受试物对RL95-2细胞皮下异种移植肿瘤模型的抑瘤药效评价(基于给药后28天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T28/V28×100)和TGI(TGI(％)＝[1-(T28-T0)/(V28-V0)]×100)反映；
3.p值根据肿瘤体积计算(****代表p＜0.0001，*代表p＜0.05)。
实验结果如图15及表16所示，抗体药物偶联物DB1001-X2给药后表现出显著的剂量依赖性抑瘤活性。
实施例19：抗体药物偶联物对人低表达B7H4的卵巢癌细胞OVCAR-3荷瘤小鼠药效评价
为研究DB1001-X2对体内形成肿瘤的抑制作用，在小鼠体内用B7H4阳性低表达的人卵巢癌细 胞OVCAR-3形成移植瘤后，评估DB1001-X2的抗肿瘤效果。
1.受试药物及材料
空白对照组(对照组)：生理盐水
DB1001-X2(治疗组)：1mg/kg
DB1001-X2(治疗组)：3mg/kg
DB1001-X2(治疗组)：10mg/kg
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：6-8周龄的雌性BALB/c Nude小鼠，购自集萃药康生物科技有限公司。
4.试验方法：
将1×107个OVCAR-3细胞接种于6-8周龄的雌性BALB/c Nude小鼠右侧背部皮下，细胞重悬在1∶1的PBS与基质胶中(0.2ml/只)，当肿瘤长至约142.85mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射受试品，每两周一次，共注射2次，DB1001-X2采用剂量分别为1mg/kg，3mg/kg和10mg/kg。试验终点时间为分组后第27天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P＜0.05为具有显著性差异。作图软件为GraphPad Prism。
表17.受试物对OVCAR-3细胞皮下异种移植肿瘤模型的抑瘤药效评价(基于给药后27天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T27/V27×100)和TGI(TGI(％)＝[1-(T27-T0)/(V27-V0)]×100)反映；
3.p值根据肿瘤体积计算(p＜0.05代表有统计学差异，p＜0.01代表有显著性差异)。
实验结果如图16及表17所示，抗体药物偶联物DB1001-X2给药后表现出显著的剂量依赖性抑瘤活性。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (30)

  1. 一种抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，所述抗B7H4抗体药物偶联物结构如式(I-1)所示：
    其中，
    M为-L2-L1-C(O)-；
    L2为-O-或-S-；
    L1为-(C(R1a)(R1b))m-CH2-、C3-C6饱和的环烷基或3-6元饱和的杂环基，所述C3-C6饱和的环烷基和3-6元饱和的杂环基各自独立地任选被一个或多个R2a取代；
    m选自1、2、3或4；所述的3-6元饱和的杂环基中的杂原子选自N、O和S，杂原子数为1-3个；
    R1a各自独立地选自氢、卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
    R1b和R2a各自独立地选自氢、卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
    R各自独立地为氢或卤素；
    L是接头单元；
    p表示平均连接数，且p选自1到10的整数或小数；
    Ab为抗B7H4抗体或其抗原结合片段。
  2. 如权利要求1所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含氨基酸序列分别如SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的LCDR1、LCDR2和LCDR3。
  3. 如权利要求1或2所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：7所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID NO：8所示或与其具有 至少95％、96％、97％、98％或99％同一性的轻链可变区。
  4. 如权利要求1或2所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体或其抗原结合片段为鼠源抗体或其片段、嵌合抗体或其片段、人源化抗体或其片段。
  5. 如权利要求1或2所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体或其抗原结合片段选自Fab、Fab′、Fab′-SH、Fv、scFv、F(ab′)2、sdAb、双抗体或线性抗体。
  6. 如权利要求1或2所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗体为IgG1形式的抗体、IgG2形式的抗体、IgG3形式的抗体或IgG4形式的抗体。
  7. 如权利要求1或2所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体或其抗原结合片段包含：氨基酸序列如SEQ ID NO：9所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO：10所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链。
  8. 如权利要求1-7中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中p为3-8的整数或小数。
  9. 如权利要求1-7中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-(C(R1a)(R1b))m-CH2-；R1a选自：氢、卤素和C1-C6烷基；R1b选自：氢、卤素和C1-C6烷基。
  10. 如权利要求9所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-(C(R1a)(R1b))m-CH2-；R1a为-CH3；R1b选自：氢和-CH3。
  11. 如权利要求1-10中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为-(C(R1a)(R1b))m-CH2-；m为1或2。
  12. 如权利要求1-11中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1选自：
  13. 如权利要求1-7中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为C3-C6饱和的环烷基或3-6元饱和的杂环基，所述C3-C6饱和的环烷基和3-6元饱和的杂环基各自独立地任选被一个或多个R2a取代，R2a各自独立地选自：氢、卤素和C1-C6烷基。
  14. 如权利要求13所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为任选被一个或多个R2a取代的C3-C6饱和的环烷基；R2a各自独立地选自：氢、卤素和C1-C6烷基。
  15. 如权利要求14所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1为任选被1、2或3个R2a取代的：R2a各自独立地选自：氢、卤素和C1-C6烷基。
  16. 如权利要求1-15中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1选自：
  17. 如权利要求1-16中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
    M为-L2-L1-C(O)-；
    L2为-O-；
    L1为-(C(R1a)(R1b))m-CH2-或C3-C6饱和的环烷基，所述C3-C6饱和的环烷基任选被1、2或3个R2a取代；
    m选自1或2；
    R1a各自独立地选自氢、卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
    R1b和R2a各自独立地选自氢、卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
    R各自独立地为氢或卤素。
  18. 如权利要求17中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中
    M为-L2-L1-C(O)-；
    L2为-O-；
    L1为-(C(R1a)(R1b))m-CH2-或任选被1、2或3个R2a取代的：
    m选自1或2；
    R1a各自独立地选自卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
    R1b和R2a各自独立地选自氢、卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个R取代；
    R各自独立地为氢或卤素。
  19. 如权利要求1-18中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-M-选自：
  20. 如权利要求1-19中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为-La-Lb-Lc-，
    所述-La-为
    其中，W为-(C(Rwa)(Rwb))wn-，Y为-(OCH2CH2)yn-Oyp-，Z为-(C(Rza)(Rzb))zn；
    其中wn为1、2、3或6，
    W的0个或1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；
    其中yn为0、4或8，yp为0或1；
    其中zn为1、2或3，
    Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代；
    -Cyr-为3到10元饱和的环烷基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；
    其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx，Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1-6烷基；
    其中每个Rr各自独立地为氢、卤素或C1-6烷基；
    所述-Lb-表示由2到4个氨基酸构成的肽残基，所述-Lb-的肽残基为由选自以下组中的氨基酸形成的肽残基：苯丙氨酸、甘氨酸、丙氨酸、缬氨酸、瓜氨酸和赖氨酸；
    所述-Lc-为
    其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6烷基。
  21. 如权利要求20所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-La-为
  22. 如权利要求20所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-Lb-选自以下组：
    优选地，所述-Lb-为
  23. 如权利要求20所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述-Lc-为
  24. 如权利要求20-23中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述L为
  25. 如权利要求1-24中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体药物偶联物结构如式(II-1)或(II-2)所示：
    其中，
    p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
    Ab如权利要求1-7中任一项所述；
    L2为-O-或-S-；优选为-O-；
    X1选自任选被1、2或3个R2a取代的C3-C6饱和的环烷基；优选为任选被1、2或3个R2a取代的：
    X2选自-(C(R1a)(R1b))m-CH2-；
    m选自1或2；
    R1a为氢、卤素或被1、2或3个R任选取代的C1-C6烷基；
    R1b或R2a各自独立可以为氢、卤素或被1、2或3个R任选取代的C1-C6烷基；
    R各自独立地可以为氢或卤素。
  26. 如权利要求1-25中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体药物偶联物选自以下结构式：



    其中，
    p如权利要求1或8所述；
    Ab如权利要求1-7中任一项所述。
  27. 一种抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗B7H4抗体药物偶联物选自：


    其中，
    p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数。
  28. 一种药物组合物，其包含如权利要求1-27中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。
  29. 如权利要求1-27中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或权利要求28所述的药物组合物在制备用于治疗和/或预防B7H4介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为B7H4阳性表达的癌症；更优选地，所述癌症选自乳腺癌、卵巢癌和子宫内膜瘤。
  30. 一种治疗和/或预防B7H4介导的疾病或病症的方法，其包括向有需要的受试者施用如权利要求1-27中任一项所述的抗B7H4抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或权利要求28所述的药物组合物，优选地，所述疾病或病症为B7H4阳性表达的癌症；更优选地，所述癌症选自乳腺癌、卵巢癌和子宫内膜瘤。
PCT/CN2023/085097 2022-03-30 2023-03-30 B7h4抗体药物偶联物及其用途 WO2023186015A1 (zh)

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