WO2021190586A1 - B7h3抗体-依喜替康类似物偶联物及其医药用途 - Google Patents

B7h3抗体-依喜替康类似物偶联物及其医药用途 Download PDF

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WO2021190586A1
WO2021190586A1 PCT/CN2021/082929 CN2021082929W WO2021190586A1 WO 2021190586 A1 WO2021190586 A1 WO 2021190586A1 CN 2021082929 W CN2021082929 W CN 2021082929W WO 2021190586 A1 WO2021190586 A1 WO 2021190586A1
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antibody
variable region
seq
chain variable
heavy chain
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PCT/CN2021/082929
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English (en)
French (fr)
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应华
张玲
胡齐悦
蒋海侠
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to CN202180021525.1A priority Critical patent/CN115605510A/zh
Publication of WO2021190586A1 publication Critical patent/WO2021190586A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/48—Ergoline derivatives, e.g. lysergic acid, ergotamine
    • 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
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present disclosure relates to anti-B7H3 antibody-exotecan analog conjugates and their applications in medicine. Further, the present disclosure relates to an anti-B7H3 antibody-exitcan analog conjugate or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising the aforementioned conjugate or a pharmaceutically acceptable salt thereof, and the preparation thereof Use in drugs for treating B7H3 mediated diseases or disorders; especially use in preparing anticancer drugs.
  • T cells The immune response mediated by T cells plays an extremely important role in the body's anti-tumor process, and the activation and proliferation of T cells require not only the antigen signal recognized by the TCR, but also the second signal provided by the costimulatory molecule.
  • B7 family molecules belong to the immunoglobulin superfamily of costimulatory molecules. More and more studies have shown that molecules of this family play an important regulatory role in the body's normal immune function and pathological conditions.
  • B7H3 is a member of the B7 family and belongs to the type I transmembrane protein. It contains a signal peptide at the amino terminus, an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region and a Contains a cytoplasmic tail region of about 45 amino acids (Tissue Antigens. 2007 Aug; 70(2): 96-104).
  • IgV immunoglobulin-like variable region
  • IgC constant region
  • a cytoplasmic tail region of about 45 amino acids
  • the extracellular segment of B7H3a is composed of two immunoglobulin domains of IgV-IgC, also known as 2IgB7H3; and the extracellular segment of B7H3b is composed of four immunoglobulin domains of IgV-IgC-IgV-IgC, also known as 4IgB7H3.
  • B7H3 is not expressed or very low expressed in normal tissues and cells, but is highly expressed in a variety of tumor tissues. B7H3 is closely related to tumor progression, patient survival and prognosis. It has been clinically reported that B7H3 is in many cancer types, especially in non-small cell lung cancer, kidney cancer, urinary tract epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreatic cancer. Overexpression (Lung Cancer. 2009 Nov; 66(2): 245-249; Clin Cancer Res. 2008 Aug 15; 14(16): 5150-5157).
  • B7H3 is positively correlated with clinicopathological malignancy (such as tumor volume, extra-prostatic invasion or Gleason score), and is also correlated with cancer progression (Cancer Res. 2007 Aug 15; 67 (16):7893-7900).
  • clinicopathological malignancy such as tumor volume, extra-prostatic invasion or Gleason score
  • cancer progression cancer Res. 2007 Aug 15; 67 (16):7893-7900.
  • B7H3 expression is negatively correlated with event-free survival; and in pancreatic cancer, B7H3 expression is correlated with lymph node metastasis and pathological progression. Therefore, B7H3 is considered as a new tumor marker and potential therapeutic target.
  • Phage display technology phage display technology
  • fusion expression of exogenous protein or polypeptide with phage coat protein thereby expressing the exogenous protein on the surface of the phage.
  • the phage antibody library is an antibody library that combines phage display technology, PCR amplification technology, and protein expression technology using comprehensive technical means.
  • the biggest advantage of the phage antibody library is that fully human antibodies are prepared without immunization in the body, imitating the three processes of antibody production in the body.
  • the phage antibody library also has the following advantages:
  • 2It expresses fully human antibodies with small molecular weight, and is mainly expressed in the form of active fragments Fab and scFv. Compared with intact antibodies, it has obvious advantages in tissue penetration.
  • the screening capacity is large.
  • the hybridoma technology is screened among thousands of clones, and the antibody library technology can select from millions or even hundreds of millions of molecules, and there are many types of antibodies screened.
  • Antibody-drug conjugate connects monoclonal antibodies or antigen-binding fragments with biologically active cytotoxins through linker compounds, making full use of the specificity and specificity of antibody binding to normal cells and tumor cell surface antigens.
  • the high efficiency of cytotoxic substances avoids defects such as low curative effect of antibodies and excessive side effects of toxic substances. This also means that compared with traditional chemotherapy drugs in the past, antibody-drug conjugates can kill tumor cells more accurately and reduce the impact on normal cells.
  • Kadcyla is an ADC drug formed by trastuzumab targeting Her2 and DM1.
  • B7H3 there are also patent reports on antibodies and ADC drugs targeting B7H3, such as WO2008100934, WO2012147713, WO2014061277, WO2015184203, WO2016044383.
  • camptothecin derivatives which have anti-tumor effects by inhibiting topoisomerase I.
  • camptothecin derivative esartecan 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
  • the literature of ADC include WO2014057687; Clinical Cancer Research (2016) 22(20): 5097-5108; Cancer Sci (2016) 107: 1039-1046. However, further development of ADC drugs with better curative effect is still needed.
  • an antibody-drug conjugate represented by the general formula Pc-L-Y-D or a pharmaceutically acceptable salt thereof:
  • Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2- , -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group group, cycloalkyl group and heterocyclic group; and form a cycloalkyl or heterocyclic group, R a and R b are carbon atoms connected thereto;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic group, aryl and heteroaryl;
  • R 2 is selected from hydrogen atom, halogen, Haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; alternatively, R 1 and R 2 form together with the carbon atom to which they are attached Cycloalkyl or heterocyclic group;
  • R a and R 2 together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;
  • n is an integer from 0 to 4; non-limiting examples such as m is selected from 0, 1, 2, 3, and 4;
  • n is from 1 to 10, n is a decimal or integer;
  • Pc is an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody or an antigen-binding fragment thereof comprises:
  • the heavy chain variable region which is a variant of SEQ ID NO: 1, said variant comprising one or more amino acids selected from the group consisting of T16R, Y103F, I28T, A33D, S99E, A100G, A104G, R101K, A104S and A113T Substitution;
  • the variant contains one or more amino acid substitutions selected from T16R, Y103F, I28T and A113T;
  • the light chain variable region which is SEQ ID NO: 2 or a variant thereof, the variant comprises one or two amino acid substitutions selected from R56K and S57G; preferably, the variant comprises an amino acid substitution of S57G ;
  • the amino acid numbering is based on the natural sequence of the variable region sequence.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described above, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises:
  • the heavy chain variable region is a variant of SEQ ID NO: 1, and the variant includes T16R amino acid substitutions; preferably, it includes T16R and A113T amino acid substitutions.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of the preceding items, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises the sequence SEQ ID NO: Light chain variable region shown in 2.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding items, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises a variable light chain
  • the region is a variant of SEQ ID NO: 2, and the variant includes an amino acid substitution of S57G.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding items, wherein the heavy chain variable of the anti-B7H3 antibody or antigen-binding fragment thereof
  • the region is a variant of SEQ ID NO: 1, and the variant contains an amino acid substitution selected from any one of the following groups:
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding items, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises any one of the following :
  • the heavy chain variable region which contains amino acid substitutions of T16R, Y103F and A113T, and
  • the heavy chain variable region which contains amino acid substitutions of T16R, I28T, Y103F and A113T, and
  • the heavy chain variable region which contains amino acid substitutions of T16R, Y103F and A113T, and
  • the light chain variable region which contains amino acid substitutions of S57G;
  • the heavy chain variable region which contains amino acid substitutions of T16R, I28T, Y103F and A113T, and
  • the light chain variable region which contains amino acid substitutions of S57G;
  • the light chain variable region which contains amino acid substitutions of S57G;
  • the heavy chain variable region which contains amino acid substitutions of T16R, I28T and A113T, and
  • the light chain variable region which contains amino acid substitutions of S57G.
  • the present disclosure provides an antibody-drug conjugate represented by the general formula Pc-L-Y-D or a pharmaceutically acceptable salt thereof:
  • Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2- , -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group group, cycloalkyl group and heterocyclic group; and form a cycloalkyl or heterocyclic group, R a and R b are carbon atoms connected thereto;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic group, aryl and heteroaryl;
  • R 2 is selected from hydrogen atom, halogen, Haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; alternatively, R 1 and R 2 form together with the carbon atom to which they are attached Cycloalkyl or heterocyclic group;
  • R a and R 2 together with the carbon atom to which they are connected form a cycloalkyl group or a heterocyclic group;
  • n is an integer from 0 to 4; non-limiting examples such as m is selected from 0, 1, 2, 3, and 4;
  • n is from 1 to 10, n is a decimal or integer;
  • Pc is an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody or an antigen-binding fragment thereof comprises:
  • the heavy chain variable region is a variant of the heavy chain variable region in SEQ ID NO: 1, and the variant includes one or more selected from T16R, Y103F, I28T, A33D, S99E, A100G, A104G, R101K, A104S and A113T amino acid substitutions; preferably, the variant contains one or more amino acid substitutions selected from T16R, Y103F, I28T and A113T; and
  • the light chain variable region which is the light chain variable region in SEQ ID NO: 2 or a variant thereof, said variant comprising one or two amino acid substitutions selected from R56K and S57G; preferably, said The variant contains an amino acid substitution of S57G;
  • the amino acid numbering is based on the natural sequence of the variable region sequence.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described above, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises:
  • the heavy chain variable region is a variant of the heavy chain variable region in SEQ ID NO: 1, and the variant includes amino acid substitutions of T16R; preferably, amino acid substitutions of T16R and A113T.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding items, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises the sequence SEQ ID NO: Light chain variable region shown in 2.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding items, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises a variable light chain
  • the region is a light chain variable variant of SEQ ID NO: 2, and the variant includes an amino acid substitution of S57G.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding items, wherein the heavy chain variable of the anti-B7H3 antibody or antigen-binding fragment thereof
  • the region is a variant of the heavy chain variable region of SEQ ID NO: 1, and the variant includes an amino acid substitution selected from any one of the following groups:
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of the preceding items, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises any one of the following :
  • the heavy chain variable region which contains amino acid substitutions of T16R, Y103F and A113T, and
  • the heavy chain variable region which contains amino acid substitutions of T16R, I28T, Y103F and A113T, and
  • the heavy chain variable region which contains amino acid substitutions of T16R, Y103F and A113T, and
  • the light chain variable region which contains amino acid substitutions of S57G;
  • the heavy chain variable region which contains amino acid substitutions of T16R, I28T, Y103F and A113T, and
  • the light chain variable region which contains amino acid substitutions of S57G;
  • the light chain variable region which contains amino acid substitutions of S57G;
  • the heavy chain variable region which contains amino acid substitutions of T16R, I28T and A113T, and
  • the light chain variable region which contains amino acid substitutions of S57G.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of the preceding items, wherein the anti-B7H3 antibody or antigen-binding fragment thereof is selected from any of the following Group:
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of the preceding items, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises an antibody constant region;
  • the heavy chain constant region of the antibody constant region is derived from human IgG1, IgG2, IgG3 or IgG4.
  • the amino acid sequence of the heavy chain constant region is derived from human IgG1;
  • the light chain constant region of the antibody constant region is derived from Human antibody kappa, lambda chain.
  • the present disclosure provides an antibody-drug conjugate according to any one of the preceding items or a pharmaceutically acceptable salt thereof, wherein the anti-B7H3 antibody comprises any one of the following groups:
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of the preceding items, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab ') 2.
  • Single-chain antibodies scFv
  • dimerized V regions diabodies
  • disulfide stabilized V regions dsFv
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, wherein n is 1 to 8. , Preferably 3-8, more preferably 3-7, n is a decimal or integer.
  • n can be mentioned as 1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof as described in any one of the preceding items,
  • Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-;
  • R a and R b are identical or different and are each independently selected from hydrogen atoms, deuterium atoms, halogen and C1 -6 alkyl;
  • R 1 is a halogenated C 1-6 alkyl group or a C 3-6 cycloalkyl group
  • R 2 is selected from a hydrogen atom, a halogenated C 1-6 alkyl group and a C 3-6 cycloalkyl group;
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • m 0 or 1.
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, wherein Y is selected from any of the following One:
  • the O end of Y is connected to the joint unit L.
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, wherein the linker unit -L- Is -L 1 -L 2 -L 3 -L 4 -,
  • L 1 is selected from -(succinimidyl-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- and -C(O)-WC( O)-; wherein W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl and linear heteroalkyl of 1 to 8 atoms, the heteroalkyl contains 1 to 3 selected from N, O and S heteroatoms, wherein the C 1-8 alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further selected from halogen, hydroxy, cyano, amino, alkyl, Substituted by one or more substituents of chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and chemical bond, where p 1 is an integer from 1 to 20;
  • L 3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( K), citrulline (Cit), serine (S), glutamic acid (E) and aspartic acid (D) formed by amino acid residues, and optionally further selected from halogen, hydroxyl, cyanide Substituted by one or more substituents in the group, amino group, alkyl group, chloroalkyl group, deuterated alkyl group, alkoxy group and cycloalkyl group;
  • L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH 2 ) t -and chemical bonds, wherein t is an integer from 1 to 6;
  • R 3 , R 4 and R 5 are the same or different, and are each independently selected from a hydrogen atom, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group;
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group.
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, wherein the linker unit -L- Is -L 1 -L 2 -L 3 -L 4 -,
  • L 1 is s 1 is an integer from 2 to 8 (specifically, 2, 3, 4, 5, 6, 7 or 8);
  • L 2 is a chemical bond
  • L 3 is a tetrapeptide residue; preferably, L 3 is a tetrapeptide residue of GGFG (SEQ ID NO: 33);
  • L 4 is -NR 5 (CR 6 R 7 )t-, R 5 , R 6 or R 7 are the same or different, and each independently is a hydrogen atom or an alkyl group, and t is 1 or 2;
  • the L 1 terminal is connected to Pc, and the L 4 terminal is connected to Y.
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof as described in any one of the preceding, wherein -L- is:
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any one of the preceding, wherein -LY- is optionally From any of the following:
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any one of the foregoing, which is of the general formula (Pc-LYD) -L a -YD) antibody-drug conjugate or pharmaceutically acceptable salt thereof:
  • Pc, n, m, R 1 and R 2 are as defined in the general formula (Pc-LYD);
  • W, L 2 , L 3 , R 5 , R 6 and R 7 are as defined in the joint unit -L-;
  • Pc is an anti-B7H3 antibody or an antigen-binding fragment thereof, wherein the anti-B7H3 antibody or an antigen-binding fragment thereof comprises:
  • the heavy chain variable region is a variant of the heavy chain variable region in SEQ ID NO: 1, and the variant includes one or more selected from T16R, Y103F, I28T, A33D, S99E, A100G, A104G, R101K, A104S and A113T amino acid substitutions; preferably, the variant contains one or more amino acid substitutions selected from T16R, Y103F, I28T and A113T; and
  • the light chain variable region which is the light chain variable region in SEQ ID NO: 2 or a variant thereof, said variant comprising one or two amino acid substitutions selected from R56K and S57G; preferably, said The variant contains an amino acid substitution of S57G;
  • n is an integer from 0 to 4.
  • n is from 1 to 10, n is a decimal or integer;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclic group, aryl and heteroaryl;
  • R 2 is selected from hydrogen atom, halogen, Haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; alternatively, R 1 and R 2 form together with the carbon atom to which they are attached Cycloalkyl or heterocyclic group;
  • W is selected from a C 1-8 alkyl group, a C 1-8 alkyl-cycloalkyl group, and a linear heteroalkyl group of 1 to 8 atoms, and the heteroalkyl group contains 1 to 3 selected from N, O and S
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and chemical bond, where p 1 is an integer from 1 to 20;
  • L 3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( K), citrulline, serine (S), glutamic acid (E) and aspartic acid (D) formed by amino acid residues, and optionally further selected from halogen, hydroxyl, cyano, amino , Alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl substituted by one or more substituents;
  • the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( K), citrulline, serine (S), glutamic acid (E) and aspartic acid (D) formed by amino acid residues, and optionally further selected from halogen, hydroxyl, cyano, amino , Alkyl, chloroalkyl, deuterated alkyl, alkoxy
  • R 5 is selected from hydrogen atom, alkyl group, haloalkyl group, deuterated alkyl group and hydroxyalkyl group;
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a halogenated alkyl group, a deuterated alkyl group, and a hydroxyalkyl group.
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any one of the foregoing, which is of the general formula (Pc-LYD) -L b -YD) antibody-drug conjugate or pharmaceutically acceptable salt thereof:
  • s 1 is an integer from 2 to 8;
  • Pc, R 1 , R 2 , R 5 to R 7 , m and n are as defined in the general formula (Pc-L a -YD).
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof as described in any one of the preceding, and the antibody-drug conjugate The conjoint is selected from any one of the following:
  • Pc and n are as defined in the general formula (Pc-L a -YD).
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof, and the antibody-drug conjugate is:
  • n is 1 to 8, preferably 3 to 8; more preferably 3 to 7, n is a decimal or integer;
  • h1702-DS-107 is an anti-B7H3 antibody, which contains a heavy chain with the sequence shown in SEQ ID NO: 21 and a light chain with the sequence shown in SEQ ID NO: 22.
  • the present disclosure provides an antibody-drug conjugate represented by the general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof, wherein n is 1 to 8. , Preferably 3-8, more preferably 3-7, n is a decimal or integer.
  • one embodiment of the present disclosure provides an anti-B7H3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region, which is a variant of the heavy chain variable region in SEQ ID NO: 1, said The variant includes one or more amino acid substitutions selected from T16R, Y103F, I28T, A33D, S99E, A100G, A104G, R101K, A104S and A113T; preferably, the variant includes one or more selected from T16R, Y103F , I28T and A113T amino acid substitutions; and
  • the light chain variable region which is the light chain variable region in SEQ ID NO: 2 or a variant thereof, said variant comprising one or two amino acid substitutions selected from R56K and S57G; preferably, said The variant contains an amino acid substitution of S57G.
  • the present disclosure provides the anti-B7H3 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region of the anti-B7H3 antibody or antigen-binding fragment thereof is SEQ ID NO: 1 heavy chain may A variant of a variable region, said variant comprising an amino acid substitution selected from any one of the following groups:
  • the present disclosure provides the anti-B7H3 antibody or antigen-binding fragment thereof, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises:
  • the heavy chain variable region which includes the amino acid substitutions of T16R, Y103F and A113T, and the light chain variable region in SEQ ID NO: 2;
  • the heavy chain variable region which includes the amino acid substitutions of T16R, I28T, Y103F and A113T, and the light chain variable region in SEQ ID NO: 2;
  • the heavy chain variable region which contains the amino acid substitutions of T16R, Y103F and A113T, and the light chain variable region, which contains the amino acid substitutions of S57G;
  • the heavy chain variable region which contains amino acid substitutions of T16R, I28T, Y103F and A113T, and the light chain variable region, which contains amino acid substitutions of S57G;
  • the heavy chain variable region which contains amino acid substitutions of T16R, I28T and A113T, and the light chain variable region, which contains amino acid substitutions of S57G.
  • the present disclosure provides the anti-B7H3 antibody or antigen-binding fragment thereof, wherein the anti-B7H3 antibody or antigen-binding fragment thereof is selected from:
  • the present disclosure provides the anti-B7H3 antibody or antigen-binding fragment thereof, wherein the anti-B7H3 antibody or antigen-binding fragment thereof comprises an antibody constant region; the heavy chain constant region of the antibody constant region It is derived from human IgG1, IgG2, IgG3 or IgG4, preferably, the amino acid sequence of the heavy chain constant region is derived from human IgG1, more preferably the heavy chain constant region shown in SEQ ID NO: 15; The light chain constant region is derived from human antibody kappa and lambda chains; preferably the light chain constant region shown in SEQ ID NO: 16.
  • the present disclosure provides the anti-B7H3 antibody or antigen-binding fragment thereof, wherein the anti-B7H3 antibody is selected from:
  • one embodiment of the present disclosure provides a nucleic acid molecule that encodes the anti-B7H3 antibody or antigen-binding fragment thereof as described above.
  • one embodiment of the present disclosure provides a host cell, which contains the nucleic acid molecule as described above.
  • an embodiment of the present disclosure provides a method for preparing a compound represented by the general formula Pc-L a -YD, which includes the following steps:
  • Pc' is reduced Pc, and the compound represented by the general formula (L a -YD) is subjected to a coupling reaction to obtain the compound represented by the general formula (Pc-L a -YD);
  • Pc' is With a reactive group (such as a sulfhydryl group), the reducing agent is preferably TCEP, in particular, the disulfide bond on the antibody is preferably reduced to obtain a sulfhydryl group;
  • Pc is the aforementioned anti-B7H3 antibody or antigen-binding fragment thereof
  • W, L 2 , L 3 , R 1 , R 2 , R 5 to R 7 , m and n are as defined in the general formula Pc-La-YD.
  • an embodiment of the present disclosure provides a method for preparing a compound represented by the general formula Pc-L a -YD, which includes the following steps:
  • the anti-B7H3 antibody h1702-DS-107 was reduced to obtain h1702-DS-107', and h1702-DS-107' was coupled with the compound represented by formula 9-A to obtain the general formula (h1702-DS-107-9-
  • the reducing agent is preferably TCEP, in particular, the disulfide bond on the antibody is preferably reduced to obtain a sulfhydryl group;
  • n is 1 to 8, preferably 3-8, n is a decimal or integer;
  • 1702-DS-107 is an anti-B7H3 antibody, which contains a heavy chain with the sequence shown in SEQ ID NO: 3 and a light chain with the sequence shown in SEQ ID NO: 4.
  • the present disclosure provides a pharmaceutical composition comprising:
  • the unit dose of the pharmaceutical composition contains 0.1 mg to 3000 mg or 1 mg to 1000 mg of the aforementioned anti-B7H3 antibody, antigen-binding fragment thereof, or the aforementioned antibody-drug conjugate.
  • the present disclosure provides the antibody-drug conjugate according to any one of the preceding items or a pharmaceutically acceptable salt thereof, or the anti-B7H3 antibody or antigen-binding fragment thereof according to any one of the preceding items, or a drug containing the same Use of the composition as a medicine.
  • the present disclosure provides the antibody-drug conjugate according to any one of the preceding items or a pharmaceutically acceptable salt thereof, or the anti-B7H3 antibody or antigen-binding fragment thereof according to any one of the preceding items, or comprising the same Use of the pharmaceutical composition of in the preparation of a medicament for the treatment of B7H3-mediated diseases or disorders or tumors, wherein the B7H3-mediated diseases or disorders are B7H3 high-expressing cancers, medium-expressing cancers or low-expressing cancers.
  • the present disclosure provides the antibody-drug conjugate according to any one of the preceding items or a pharmaceutically acceptable salt thereof, or the anti-B7H3 antibody or antigen-binding fragment thereof according to any one of the preceding items, or a pharmaceutical combination comprising the same Use of a substance in the preparation of a medicament for the treatment or prevention of tumors, wherein the tumors and cancers are preferably head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, and glioblastoma multiforme Tumor, neuroblastoma, central nervous system cancer, neuroendocrine tumor, throat cancer, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreas Cancer, stomach cancer, gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell
  • the present disclosure further relates to a method for treating or preventing tumors or cancers, the method comprising administering to a subject in need thereof a therapeutically effective dose or a prophylactically effective dose of the antibody drug pair as described in any one of the preceding items.
  • tumors or cancers suitable for treatment with the active compounds of the application are related to the high, medium or low expression of B7H3.
  • the tumor and cancer are selected from: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central Nervous system cancer, neuroendocrine tumors, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, bowel cancer, Colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer , Chondrosarcoma, Myeloma
  • the lymphoma is selected from: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B- Cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell/histiocytic-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma.
  • the lung cancer is selected from: non-small cell lung cancer and small cell lung cancer.
  • the leukemia is selected from the group consisting of: chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia Cell-like leukemia.
  • the present disclosure further provides the aforementioned anti-B7H3 antibody or its antibody-drug conjugate as a medicine, preferably as a medicine for the treatment of cancer or tumor, and more preferably as a medicine for the treatment of B7H3-mediated cancer.
  • the active compound for example, the ligand-drug conjugate according to the present disclosure, or a pharmaceutically acceptable salt thereof
  • the active compound can be prepared into a form suitable for administration by any appropriate route, and the active compound is preferably in a unit dose Or it is a way that subjects can self-administer in a single dose.
  • the unit dosage form of the compound or composition of the present disclosure can be tablet, capsule, cachet, bottled syrup, powder, granule, lozenge, suppository, rejuvenated powder or liquid preparation.
  • the dosage of the active compound or composition used in the treatment methods of the present disclosure will generally vary with the severity of the disease, the weight of the subject, and the relative efficacy of the compound.
  • a suitable unit dose can be 0.1 mg to 1000 mg.
  • the pharmaceutical composition of the present disclosure may contain one or more excipients selected from the following ingredients: fillers, diluents, binders, wetting agents, disintegrants or excipients, etc. .
  • the composition may contain 0.1% to 99% by weight of the active compound.
  • the anti-B7H3 antibody and antibody-drug conjugate provided in the present disclosure have reduced immunogenicity, higher anti-tumor effect and therapeutic activity, lower toxicity, better pharmacokinetic properties and druggability (such as stability) ).
  • Figure 1 Different ADCs inhibit the proliferation of Detroit562 cell lines with different B7H3 expression levels.
  • Figure 2 The efficacy of different ADCs on Detroit562 xenografts in nude mice.
  • a trade name When a trade name is used in this disclosure, it is intended to include the formulation of the trade name product, the drug and the active drug portion of the trade name product.
  • drug refers to a cytotoxic drug, which can have chemical molecules in the cell that strongly disrupt its normal growth.
  • cytotoxic drugs can kill cells at a sufficient concentration, but due to lack of specificity, while killing tumor cells, they can also cause normal cell apoptosis, leading to serious side effects.
  • the term includes toxins (such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin), radioisotopes (such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and Lu radioisotopes), chemotherapeutics, antibiotics and nucleolytic enzymes.
  • L refers to a chemical structure fragment with one end connected to a ligand (specifically an antibody or antigen-binding fragment) and the other end connected to a drug Or bond, you can also connect to other linkers and then connect to ligands or drugs.
  • Linkers (including extensions, spacers and amino acid units) can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker may be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers such as hydrazone
  • protease-sensitive linkers such as peptidase-sensitive linkers
  • light-labile linkers dimethyl linkers
  • disulfide-containing linkers Chargei et al., Cancer Research 52:127-131
  • antibody-drug conjugate refers to the connection between the antibody and the drug through a connecting unit.
  • antibody-drug conjugate refers to the connection of a monoclonal antibody or an antigen-binding fragment to a toxic drug with biological activity through a stable connecting unit.
  • antibody refers to an immunoglobulin.
  • a complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different.
  • Immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding The heavy chains are respectively ⁇ chain, ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain.
  • the same type of Ig can be divided into different subclasses according to the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • the light chain is divided into a kappa chain or a lambda chain by the difference of the constant region.
  • Each of the five types of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the full-length antibody heavy chain and light chain varies greatly and is a variable region (Fv region); the amino acid sequence near the C-terminus is relatively stable and is a constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment described in the present disclosure comply with the known IMGT rules.
  • fully human antibody means “fully human antibody”, “fully human antibody” or “fully human antibody”, also known as “fully human monoclonal antibody”, the variable and constant regions of the antibody are of human origin, reducing the immunogen Sex and side effects.
  • the related technologies of fully human antibody preparation mainly include: human hybridoma technology, EBV transformed B lymphocyte technology, phage display technology (phage display), transgenic mouse antibody preparation technology (transgenic mouse) and single B cell antibody preparation technology.
  • antigen-binding fragment refers to one or more fragments of an antibody that retain the ability to bind to an antigen. Fragments of the full-length antibody can be used to perform the antigen-binding function of the antibody. Examples of binding fragments contained in “antigen-binding fragments" include:
  • Fab fragment a monovalent fragment composed of VL, VH, CL and CH1 domains
  • CDR Isolated complementarity determining region
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, recombination methods can be used to connect them through a linker so that it can produce a single protein chain in which the VL and VH regions are paired to form a monovalent molecule, called Single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Science USA 85: 5879-5883.
  • Single chain Fv see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Science USA 85: 5879-5883.
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragments" of antibodies.
  • antigen-binding fragments are obtained using conventional techniques known to those skilled in the art, and functional screening is performed in the same manner as for intact antibodies.
  • the antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulins.
  • the antibodies may be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • Fab is a fragment having a molecular weight of about 50,000 and having antigen-binding activity obtained by treating an IgG antibody molecule with the protease papain (for example, cleaving the amino acid residue at position 224 of the H chain), in which the part on the N-terminal side of the H chain The and L chains are joined together by disulfide bonds.
  • protease papain for example, cleaving the amino acid residue at position 224 of the H chain
  • F(ab')2 is obtained by digesting the lower part of the disulfide bond in the hinge region of IgG with the enzyme pepsin. It has a molecular weight of about 100,000 and has antigen-binding activity and contains two Fab regions connected at the hinge position. Fragment.
  • Fab' is a fragment obtained by cleaving the disulfide bond of the hinge region of F(ab')2 above and having a molecular weight of about 50,000 and having antigen-binding activity.
  • the Fab' fragment can be produced by inserting the DNA encoding the Fab' fragment into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector into a prokaryotic organism or eukaryotic organism for expression.
  • single chain antibody means a molecule comprising an antibody heavy chain variable domain (or VH) and an antibody light chain variable domain (or VL) connected by a linker.
  • Such scFv molecules may have the general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated amino acid sequences or variants thereof, for example using 1-4 repeated variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that can be used in the present disclosure are described by Alfthan et al.
  • frame region refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitope or "antigenic determinant” refers to a site on an antigen that is bound by an immunoglobulin or antibody. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular B iology, Volume 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (in KD) of about less than 10 -7 M, for example, about less than 10 -8 M, 10 -9 M, or 10 -10 M or less.
  • nucleic acid molecule refers to a DNA molecule or an RNA molecule.
  • the nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.
  • the nucleic acid is "operably linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the expression vector is a "plasmid”, which refers to a circular double-stranded DNA loop into which additional DNA segments can be joined.
  • the expression vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein can replicate autonomously in the host cell into which they have been introduced (for example, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can be integrated into the genome of the host cell after being introduced into the host cell, so as to follow The host genome replicates together (e.g., a non-episomal mammalian vector).
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacteria, microorganisms, plant or animal cells.
  • Bacteria that are easily transformed include members of the enterobacteriaceae, such as Escherichia coli or Salmonella strains; Bacillaceae such as Bacillus subtilis; Pneumococcus ; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line) and NS0 cells.
  • the antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • the cDNA sequences encoding the heavy and light chains can be cloned and recombined into an expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into host cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the N-terminal site of the Fc region.
  • Positive clones are expanded in the culture medium of the bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by conventional techniques. For example, use A or G Sepharose FF column for purification. Wash away non-specifically bound components.
  • the bound antibody was eluted by the pH gradient method, and the antigen-bound fragments were detected by SDS-PAGE and collected.
  • the antibody can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be frozen immediately, such as -70°C, or lyophilized.
  • peptide refers to a compound fragment with a molecular weight between amino acid and protein, which is composed of two or more amino acid molecules connected to each other through peptide bonds.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atom alkyl group, more preferably an alkyl group containing 1 to 10 carbon atoms, most preferably containing 1 to 6 carbon atoms (including 1, 2, 3 1, 4, 5, or 6 carbon atoms).
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-Dimethylpropyl, 2,2-Dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhe
  • the alkyl group is a lower alkyl group containing 1 to 6 carbon atoms
  • non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, S-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl Group, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2- Dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl Benzylpentyl, 2,3-dimethylbutyl, etc.
  • Alkyl groups can be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment, and the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy , Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane Thio, heterocycloalkylthio and oxo.
  • groups which are independently selected from alkyl, alkenyl, alkynyl, alkoxy , Alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy,
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein the alkyl group is as defined above.
  • alkylene refers to a saturated linear or branched aliphatic hydrocarbon group, which has a residue derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which contains 1 A straight or branched chain group of to 20 carbon atoms, preferably containing 1 to 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, More preferably, an alkylene group containing 1 to 6 carbon atoms (including 1, 2, 3, 4, 5, or 6 carbon atoms).
  • alkylene groups 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.
  • the alkylene group may be substituted or unsubstituted.
  • the substituent When substituted, the substituent may be substituted at any available point of attachment, and the substituent is preferably independently optionally selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino , Halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and One or more substituents in the oxo group are substituted.
  • alkoxy refers to -O-(alkyl) and -O-(cycloalkyl), where alkyl or cycloalkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and 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 alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro Group, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent.
  • the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, most preferably 3 to 8 carbon atoms (including 3, 4, 5, 6, 7, or 8 carbon atoms).
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene Groups, cyclooctyl, etc.; polycyclic cycloalkyls include spiro, fused or bridged cycloalkyls.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O ) m (where m is an integer of 0, 1 or 2) heteroatoms, but does not include the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are carbon. It preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms (1, 2, 3 or 4 heteroatoms); more preferably, the cycloalkyl ring contains 3 to 10 ring atoms (including 3 1, 4, 5, 6, 7, 8, 9, or 10 ring atoms).
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclic groups include spiro, fused or bridged heterocyclic groups.
  • spiroheterocyclic group refers to a 5- to 20-membered polycyclic heterocyclic group that shares one atom (called a spiro atom) between single rings, in which one or more ring atoms are selected from nitrogen, oxygen or S( O) Heteroatoms of m (where m is an integer of 0 to 2), and the remaining ring atoms are carbon. It can contain one or more double bonds, but none of the rings have a fully conjugated ⁇ -electron system. It is preferably 6 to 14 yuan, more preferably 7 to 10 yuan.
  • the spiro heterocyclic group is divided into: a single spiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a single spiro heterocyclic group or a dispiro heterocyclic group . More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered, or 5-membered/6-membered monospiro heterocyclic group.
  • Non-limiting examples of spiroheterocyclic groups include:
  • fused heterocyclic group refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system.
  • One or more rings may contain one or more Double bonds, but none of the rings have a fully conjugated ⁇ -electron system, where one or more of the ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer of 0, 1, or 2), The remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members (7-membered, 8-membered, 9-membered, or 10-membered ring).
  • bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group.
  • fused heterocyclic groups include:
  • bridged heterocyclic group refers to a 5- to 14-membered polycyclic heterocyclic group with any two rings sharing two atoms that are not directly connected. It may contain one or more double bonds, but none of the rings has a complete common A conjugated ⁇ -electron system in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen or S(O) m (where m is an integer of 0, 1 or 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members (7-membered, 8-membered, 9-membered, or 10-membered ring).
  • bridged heterocyclic groups include:
  • the heterocyclic ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, non-limiting examples thereof include:
  • the heterocyclic group may be optionally substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro Group, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.
  • aryl refers to a 6 to 14-membered all-carbon monocyclic or fused polycyclic (that is, rings that share adjacent pairs of carbon atoms) groups with a conjugated ⁇ -electron system, preferably 6 to 10-membered (6-membered) , 7-membered, 8-membered, 9-membered or 10-membered), such as phenyl and naphthyl, preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring, non-limiting examples thereof include:
  • Aryl groups can be substituted or unsubstituted.
  • the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro Group, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.
  • heteroaryl refers to a heteroaromatic system containing 1 to 4 heteroatoms (1, 2, 3 or 4 heteroatoms) and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur And nitrogen.
  • Heteroaryl groups are preferably 5- to 10-membered (5-membered, 6-membered, 7-membered, 8-membered, 9-membered or 10-membered heteroaryl), more preferably 5-membered or 6-membered, such as furyl, thienyl, pyridyl , Pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • Heteroaryl groups may be optionally substituted or unsubstituted.
  • the substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio And heterocycloalkylthio.
  • amino protecting group is to keep the amino group unchanged when other parts of the molecule react, and to protect the amino group with a group that is easy to remove.
  • Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1-3 substituents (1, 2, or 3 substituents) selected from halogen, alkoxy, or nitro.
  • the amino protecting group is preferably 9-fluorenylmethyloxycarbonyl.
  • haloalkyl refers to the replacement of hydrogen on an alkyl group with one or more halogens, where the alkyl group is as defined above.
  • deuterated alkyl refers to the replacement of hydrogen on an alkyl group with one or more deuterium atoms, where the alkyl group is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH 2 .
  • nitro refers to -NO 2 .
  • amido refers to -C(O)N(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.
  • carboxylate group refers to -C(O)O(alkyl) or (cycloalkyl), where alkyl and cycloalkyl are as defined above.
  • the present disclosure also includes compounds of formula (I) in various deuterated forms.
  • Each available hydrogen atom connected to a carbon atom can be independently replaced by a deuterium atom.
  • Those skilled in the art can synthesize the compound of formula (I) in the deuterated form with reference to relevant literature.
  • Commercially available deuterated starting materials can be used when preparing the deuterated form of the compound of formula (I), or they can be synthesized using conventional techniques using deuterated reagents.
  • Deuterated reagents include, but are not limited to, deuterated borane and tri-deuterated. Borane tetrahydrofuran solution, deuterated lithium aluminum hydride, deuterated ethyl iodide and deuterated methyl iodide, etc.
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may but does not have to be present, and the description includes 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.
  • Substituted refers to one or more hydrogen atoms in the group, preferably at most 5, more preferably 1, 2, or 3 hydrogen atoms are independently substituted with substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without too much effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., ethylenic) bond.
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active compound and then exert its biological activity.
  • pharmaceutically acceptable salt refers to the salt of the antibody-drug conjugate of the present disclosure, or the salt of the active compound described in the present disclosure, when such salt is used in a subject It is safe and effective, and has due biological activity.
  • the antibody-antibody drug conjugate compound of the present disclosure contains at least one amino group, so it can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, Sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate Salt, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
  • DAR drug-to-antibody ratio
  • DAR drug-to-antibody ratio
  • It can be in the range of, for example, about 1 to about 10 drugs per antibody; and in certain embodiments, in the range of about 1 to about 8 drugs per antibody, preferably selected from 2- 8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8 and 6-8 range.
  • the drug loading amount may be an arithmetic average value obtained on the basis of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • the ADC general formula of the present disclosure includes a collection of antibody drug conjugates within a certain drug loading range described above.
  • the drug loading is expressed as n, and the drug loading can be determined by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test, and HPLC characteristics.
  • the following non-limiting methods can be used to control the loading of antibody-drug conjugates, including:
  • the conventional preparation method of the pharmaceutical composition can be found in the Chinese Pharmacopoeia.
  • carrier used for the drugs of the present disclosure refers to a system that can change the way the drug enters the subject and the distribution in the body, control the release rate of the drug, and deliver the drug to the target.
  • the drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to contain drug molecules and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • excipient is an additive other than the active ingredient in a pharmaceutical preparation, and can also be referred to as an excipient.
  • excipient Such as binders, fillers, disintegrants, lubricants in tablets; base parts in semi-solid preparations ointments and creams; preservatives, antioxidants, flavors, fragrances, and auxiliary agents in liquid preparations Solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
  • diluent is also called a filler, and its main purpose is to increase the weight and volume of the tablet.
  • the addition of diluent not only guarantees a certain volume, but also reduces the deviation of the dosage of the main components, and improves the compression molding of the drug.
  • an absorbent should be added to absorb the oily substance to keep it in a "dry” state to facilitate the manufacture of tablets.
  • the pharmaceutical composition may be in the form of a sterile injectable aqueous solution.
  • the acceptable solvents and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active compound is dissolved in an oil phase.
  • the active compound is dissolved in a mixture of soybean oil and lecithin, and then the oil solution is added to the mixture of water and glycerin to form a microemulsion.
  • the injection or microemulsion can be injected into the bloodstream of the subject by local large-volume injection.
  • a continuous intravenous delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM. 5400 intravenous pump.
  • the pharmaceutical composition may be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration.
  • the suspension can be formulated according to known techniques using those suitable dispersing or wetting agents and suspending agents mentioned above.
  • the sterile injection preparation may also be a sterile injection solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, for example, a solution prepared in 1,3-butanediol.
  • sterile fixed oil can be conveniently used as a solvent or suspending medium. For this purpose, any blended fixed oil including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be used to prepare injections.
  • the method for preparing antibody-drug conjugate h1702-DS-107 includes the following steps:
  • the anti-B7H3 antibody h1702-DS-107 was reduced to obtain h1702-DS-107', and h1702-DS-107' was coupled with the compound represented by formula 9-A to obtain the general formula (h1702-DS-107-9-
  • the reducing agent is preferably TCEP, in particular, the disulfide bond on the antibody is preferably reduced to obtain a sulfhydryl group; n is 1 to 8, preferably 3-8, and n is a decimal or an integer.
  • WO2019/024911 discloses the preparation of an anti-B7H3 antibody h1702-DS, the full text of which can be referred to.
  • the specific sequence of the antibody h1702-DS includes the amino acid sequence of the heavy chain (IgG1) (as described in the publication WO2018/177393, sequence No. 22) :
  • the above-mentioned anti-B7H3 antibody was mutated to obtain an antibody with lower immunogenicity, and its binding activity to B7H3 was tested (Test Example 1), a new antibody was obtained, and the light chain variable region and heavy chain variable region of the corresponding antibody were obtained. as follows:
  • the antibody variable region is then homologously recombined with the constant region gene (CH1-FC/CL) fragment to construct a complete antibody VH-CH1-FC/VK-CL/VL-CL.
  • the constant region sequence is as follows:
  • Plasmids expressing antibody light and heavy chains were transfected into HEK293E cells. After 6 days, the expression supernatant was collected, centrifuged at high speed to remove impurities, and purified with a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline. Elute the target protein with pH3.0-pH3.5 acidic eluent, and neutralize with 1M Tris-HCl, pH8.0-9.0. After the eluted sample is properly concentrated, it is further purified by PBS-equilibrated gel chromatography Superdex200 (GE) to remove aggregates, collect monomer peaks, and divide them for use.
  • PBS-equilibrated gel chromatography Superdex200 GE
  • the present disclosure uses recombinant cell lines overexpressing B7H3 (CT26/B7H3, where CT26 is derived from the Chinese Academy of Sciences Cell Bank, TCM37) or tumor cells (A498) to detect the ability of the antibodies of the present disclosure to bind to the B7H3 antigen.
  • B7H3 full-length amino acid sequence: B7H3 (SEQ ID NO: 31):
  • the double horizontal line is the signal peptide (Signal peptide:1-28);
  • the underlined part is the extracellular domain of B7H3 (Extracellular domain: 29-466), of which 29-139 are Ig-like V-type 1 domains, and 145-238 are Ig-like C2-type 1 domains; 243-357 Ig-like V-type 2 domains, 363-456 are Ig-like C2-type 2 domains;
  • the dotted line is the transmembrane domain (Transmembrane domain:467-487);
  • the italicized part is the intracellular domain (Cytoplasmic domain: 488-534).
  • the double horizontal line is the signal peptide (Signal peptide:1-28);
  • the cross-lined part is the extracellular domain of B7H3 (Extracellular domain: 29-466), of which 29-139 are Ig-like V-type 1 Domain, 145-238 are Ig-like C2-type 1 Domain; 243-357 are Ig -like V-type 2 Domain, 363–456 are Ig-like C2-type 2 Domain;
  • the dotted line is the transmembrane domain (Transmembrane domain:467-487);
  • the italicized part is the intracellular domain (Cytoplasmic domain: 488-534).
  • the experimental methods without specific conditions in the embodiments of the present disclosure usually follow the conventional conditions or the conditions suggested by the raw material or commodity manufacturers.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR is measured with Bruker AVANCE-400 nuclear magnetic instrument, the solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard is tetramethyl sulfoxide (DMSO-d6) Based on silane (TMS), the chemical shift is given in units of 10 -6 (ppm).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • HPLC determination uses Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150 ⁇ 4.6mm chromatographic column) and Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150 ⁇ 4.6mm chromatographic column).
  • the UV-HPLC measurement uses a Thermonanodrop2000 ultraviolet spectrophotometer.
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate, the size of the silica gel plate used in thin layer chromatography (TLC) is 0.15mm to 0.2mm, and the size of the thin layer chromatography separation and purification product is 0.4mm. To 0.5mm silica gel plate.
  • the known starting materials of the present disclosure can be synthesized by or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals and other companies.
  • the reactions are all carried out under an argon atmosphere or a nitrogen atmosphere.
  • the argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1L.
  • the hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1L.
  • the pressure hydrogenation reaction uses Parr 3916EKX hydrogenator and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenator.
  • the hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.
  • the microwave reaction uses the CEM Discover-S 908860 microwave reactor.
  • the solution in the reaction refers to an aqueous solution.
  • reaction temperature is room temperature. Room temperature is the most suitable reaction temperature, and the temperature range is 20°C to 30°C.
  • the preparation of the PBS buffer with pH 6.5 in the examples: take KH 2 PO 4 8.5g, K 2 HPO 4 .3H 2 O 8.56g, NaCl 5.85g, and EDTA 1.5g in a bottle, dilute the volume to 2L, and ultrasonic Dissolve all of it, shake well and get it.
  • the eluent system of column chromatography and the developing solvent system of thin-layer chromatography used to purify compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, C: petroleum ether and In the ethyl acetate system, the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or alkaline reagents can also be added for adjustment.
  • Q-TOF LC/MS uses Agilent 6530 accurate mass quadrupole-time-of-flight mass spectrometer and Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 ⁇ m, 2.1 ⁇ 75mm column).
  • the reaction solution was concentrated under reduced pressure, and the obtained crude compound 2 was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH 4 OAc), B- Acetonitrile, gradient elution, flow rate: 18 mL/min), collect the corresponding components, and concentrate under reduced pressure to obtain the title product (2-A: 1.5 mg, 2-B: 1.5 mg).
  • reaction was quenched by adding 5 mL of water to the reaction solution, the reaction solution was extracted with ethyl acetate (10 mL ⁇ 3), the organic phases were combined, washed with saturated sodium chloride solution (5 mL ⁇ 2), and the organic phase was dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with the developing solvent system B to obtain the title product 4 (2.5 mg, yield: 80.9%).
  • reaction was quenched by adding 5 mL of water to the reaction solution, the reaction solution was extracted with ethyl acetate (10 mL ⁇ 3), the organic phases were combined, washed with saturated sodium chloride solution (5 mL ⁇ 2), and the organic phase was dried with anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with the developing solvent system B to obtain the title product 6 (2.1 mg, yield: 67.9%).
  • the benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104mg, 0.54mmol; prepared by the method disclosed in the patent application "US2005/20645”) and 2-((((9H-fluoren-9-yl) Methoxy) carbonyl) amino) acetamido) methyl acetate 8b (100 mg, 0.27 mmol; prepared by the method disclosed in the patent application "CN105829346A”) was added to the reaction flask, 5 mL of tetrahydrofuran was added, argon replaced three times, ice The temperature of the water bath was cooled to 0-5°C, potassium tert-butoxide (61mg, 0.54mmol) was added, the ice bath was removed, and the temperature was raised to room temperature and stirred for 10 minutes.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min), the corresponding components were collected, and concentrated under reduced pressure to obtain the title product 8 (2mg, yield: 39.0%).
  • the obtained residue was dissolved in 4 mL of dioxane, 2 mL of water was added, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 20 mL of water was added, extracted with ethyl acetate (10 mL ⁇ 3), the organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography with the developing solvent system C to obtain the title product 9b (48 mg, yield: 19%).
  • the third step 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11- Acid 9c
  • the fifth step 2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl Base-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indole Azino[1,2-b]quinolin-1-yl)acetamide 9e
  • Dissolve 9d (19 mg, 22.6 ⁇ mol) in 2 mL of dichloromethane, add 1 mL of diethylamine, and stir at room temperature for 2 hours.
  • the reaction solution was concentrated under reduced pressure, 1 mL of toluene was added and concentrated under reduced pressure, repeated twice.
  • the solid residue was concentrated under reduced pressure and pulled dry by an oil pump to obtain the crude title product 9e (17 mg), which was directly used in the next reaction without purification.
  • the sixth step N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl Base-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indole Azino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazadeca Hexa-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-A
  • reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 ⁇ m 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min), the corresponding components were collected and concentrated under reduced pressure to obtain the title product (9-A: 2.4 mg, 9-B: 1.7 mg).
  • the ADC load was determined by ultraviolet spectrophotometry (UV-Vis). Instrument: Thermonanodrop2000 ultraviolet spectrophotometer. The principle is that the total absorbance of the ADC at a certain wavelength is equal to the sum of the absorbance of the drug and the monoclonal antibody at that wavelength.
  • a 280nm ⁇ mab-280 bC mab + ⁇ Drug-280 bC Drug formula (1)
  • ⁇ Drug-280 The average molar extinction coefficient of the drug at 280nm is 5100;
  • ⁇ mab-280 The average molar extinction coefficient of the monoclonal antibody at 280nm is 214600;
  • C mab concentration of monoclonal antibody
  • the optical path length is 1 cm.
  • a 370nm ⁇ mab-370 bC mab + ⁇ Drug-370 bC Drug formula (2)
  • ⁇ Drug-370 The average molar extinction coefficient of the drug at 370nm is 19000;
  • ⁇ mab-370 The extinction coefficient of the monoclonal antibody at 370nm is 0;
  • C mab concentration of monoclonal antibody
  • the optical path length is 1 cm.
  • the drug loading in the ADC can be calculated.
  • Drug load C Drug /C mab .
  • ADC conjugate h1702-DS-107-9-A is as follows: humanized antibody (h1702-DS-107) is placed in a 0.05M PBS buffered aqueous solution (antibody concentration 10mg/mL) at pH 6.5 , Add an aqueous solution (Innochem, CAS: 51805-45-9, Cat#B45573) equivalent to the configured 10mM tris(2-carboxyethyl)phosphine (TCEP), place it in a constant temperature shaking incubator at 37°C, and react 3 hours. The above reaction solution was placed in an ice bath and the temperature was lowered to 25°C.
  • antibody-drug conjugates with different DAR values can be obtained.
  • the DAR value is preferably 1-8, more preferably 3-8, and most preferably 3 -7.
  • the ADC compound h1702-DS-107-9-A was specifically prepared as follows:
  • UV-Vis calculated average value: n 6.92.
  • UV-Vis calculated average value: n 4.75.
  • UV-Vis calculated average value: n 3.09.
  • UV-Vis calculated average value: n 6.87.
  • UV-Vis calculated average value: n 4.80.
  • UV-Vis calculated average: n 2.97.
  • ADC-28 is an exemplary product of the general formula of FADC-26, and the structure is as follows:
  • UV-Vis calculated average value: n 7.46.
  • ADC-29 is an exemplary product of the general formula of FADC-25, and the structure is as follows:
  • UV-Vis calculated average value: n 7.24.
  • the h1702-DS has a heavy chain shown in SEQ ID NO:1 and a light chain shown in SEQ ID NO:2.
  • the compound 20 (prepared in Example 20 on page 88 of reference PCT/CN2019/107873) has the structure shown in the following formula.
  • Test Example 1 Expression of h1702-DS mutant and detection of B7H3 binding activity
  • Test Example 2-1 ADC's inhibitory experiment on tumor cell line proliferation
  • the inhibitory effect of the ADC of the present disclosure on cell proliferation was evaluated based on the IC 50 by detecting the content of ATP in the cell.
  • Tumor cells to be tested include: Calu-6 cells (ATCC, Catalog# HTB-56 TM ), Detroit562 cells (ATCC, Catalog# CCL-138 TM ), and CHO-K1 (ATCC, Catalog# CCL-61 TM ).
  • the ADC samples to be tested were diluted with PBS or DMSO at a 3-fold ratio to 9 concentrations (the initial concentration of the samples were all 500 nM).
  • the sample was added to the culture plate, and the culture plate was incubated in an incubator for 6 days (37°C, 5% CO 2 ).
  • CellTiter-Glo reagent Promega, G7571 was used for detection, the chemiluminescence signal value was read in Victor3, and the data was processed by GraphPad software. The measured IC 50 value is shown in Table 5.
  • Test Example 2-2 The proliferation inhibitory effect of ADC on tumor cell lines is positively correlated with the expression level of B7H3
  • B7H3 overexpression and B7H3 knockout were performed on the Detriot562 tumor cell line (Wildtype Detriot562) (to obtain Detriot562 B7H3-/ - cell line).
  • the B7H3 expression cell line (Detriot562 B7H3 medium ) and the B7H3 high overexpression cell line (Detriot562 B7H3 high ) were obtained, as shown in Figure 1 (FACS identification used The antibody is from sino biology, catalog number: 11188-MM06-A).
  • the cell proliferation inhibition test method is as described in Test Example 2-1, and the results are shown in Table 6.
  • the ADC-1 corresponding to antibody h1702-DS-107 and ADC-4 corresponding to h1702-DS have a positive effect on the proliferation of each Detroit562 cell line.
  • the inhibitory effects are similar, and the proliferation inhibitory effects of the two ADCs on Detroit562 are significantly positively correlated with the expression level of B7H3.
  • Test Example 3 Evaluation of the efficacy of ADC on Detroit 562 transplanted tumors in nude mice
  • mice Female, 6-7 weeks old, were subcutaneously inoculated with human pharyngeal carcinoma pleural fluid metastasis cells Detroit 562 cells. On the tenth day after cell inoculation, the animals were randomly divided into groups (D0), each group of 8 animals, starting intraperitoneal injection once a week, a total of 3 doses, two doses of 1mpk and 3mpk, or a single dose of 10mpk, Observe to D28, measure the tumor volume and weight 2-3 times a week, and record the data.
  • the calculation formula of tumor volume (V) is:
  • a and b represent length and width respectively.
  • Relative volume (RTV) VT/V0
  • Tumor inhibition rate (%) (CRTV-TRTV)/CRTV (%)
  • V0 and VT are the tumor volume at the beginning of the experiment and the end of the experiment, respectively.
  • CRTV and TRTV are the relative tumor volumes of the control group (blank) and the experimental group at the end of the experiment, respectively.
  • Test object
  • the tumor inhibition rates of the tested ADCs were: ADC-1 1mg/kg (1mpk) tumor inhibition rate reached 50.68%; ADC-1 3mg/kg (3mpk) tumor inhibition rate reached 78.21% (P ⁇ 0.05); ADC- 1 The tumor inhibition rate of a single dose of 10mg/kg (10mpk) reached 59.7% (P ⁇ 0.05); the tumor inhibition rate of ADC-4 1mg/kg (1mpk) reached 55.91%; ADC-4, 3mg/kg (3mpk) The tumor inhibition rate of ADC-4 reached 72.47% (P ⁇ 0.05); the tumor inhibition rate of ADC-4 10mg/kg (10mpk) single administration reached 86.37% (P ⁇ 0.001).
  • the blood collection time points are: 5 minutes, 8 hours, 1 day, 2 days, 4 days, 7 days, 10 days, 14 days, 21 days, and 28 days after administration on the first day, blood is taken from the fundus vein of the rat. 300 ⁇ L each time; the collected blood samples were placed at room temperature for half an hour until they agglutinated, and then centrifuged at 1000 ⁇ g for 15 minutes at 4°C. Collect the supernatant and store immediately at -80°C.
  • the detection methods are coated antitoxin.
  • Anti-Human IgG (HRP) mouse predsorbed, abcam, ab97175) is detected by antibody, or coated with B7H3 antigen.
  • BALB/c-nude nude mice were used as test animals to evaluate the efficacy of ADC compound on human pharyngeal carcinoma pleural fluid metastasis cell Detroit 562 nude mice transplanted tumor.
  • Negative control ADC (3mg/kg): non-B7H3 target antibody conjugated with compound 20 to form a ligand toxin conjugate.
  • BALB/c-nude nude mice purchased from Changzhou Cavins Laboratory Animal Co., Ltd.
  • mice Female, 6-7 weeks old, were subcutaneously inoculated with human pharyngeal carcinoma pleural fluid metastasis cells Detroit 562 cells (ATCC, Catalog# CCL-138 TM ). On the tenth day after cell inoculation, the animals were randomly divided into groups (D0), each group of 8 animals, starting intraperitoneal injection once a week, a total of 3 doses, measuring the tumor volume and body weight 2-3 times a week, and recording the data .
  • the calculation formula of tumor volume (V) is:
  • a and b represent length and width respectively.
  • Relative volume (RTV) V T /V 0
  • Tumor inhibition rate (%) (C RTV -T RTV )/C RTV (%)
  • V 0 and V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • CRTV and TRTV are the relative tumor volumes of the control group (negative control) and the experimental group at the end of the experiment, respectively.
  • ADC-29 The tumor inhibition rate of 3mg/kg (3mpk) reached 72.27% (P ⁇ 0.001 ); ADC-28 3mg/kg (3mpk) tumor inhibition rate reached 56.2% (P ⁇ 0.001). ADC-29 all show stronger anti-tumor efficacy than ADC-28.

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Abstract

提供了B7H3抗体-依喜替康类似物偶联物及其医药用途。具体而言，提供了B7H3抗体-药物偶联物或其药学上可接受的盐，以及其在制备用于治疗B7H3介导的疾病或病症的药物中的用途；尤其在制备抗癌药物中的用途。

Description

B7H3抗体-依喜替康类似物偶联物及其医药用途
本申请要求2020年3月25日提交的中国专利申请(申请号CN 202010218100.7)的优先权。
技术领域
本公开涉及抗B7H3抗体-依喜替康类似物偶联物及其在医药上的应用。进一步地，本公开涉及抗B7H3抗体-依喜替康类似物偶联物或其药学上可接受的盐，以及包含前述偶联物或其药学上可接受的盐的药物组合物，以及其制备用于治疗B7H3介导的疾病或病症的药物中的用途；尤其在制备抗癌药物中的用途。
背景技术
这里的陈述仅是提供与本公开有关的背景信息，而不必然地构成现有技术。
T细胞介导的免疫反应在机体抗肿瘤过程中发挥着极其重要的作用，而T细胞的活化和增殖不仅需要TCR识别的抗原信号，还需要共刺激分子提供的第二信号。B7家族分子属于共刺激分子免疫球蛋白超家族。越来越多的研究表明，该家族分子在机体正常免疫功能和病理状态下均发挥了重要的调节作用。
B7H3是B7家族的成员之一，属于I型跨膜蛋白，包含氨基端的一个信号肽，一个细胞外的免疫球蛋白样可变区(IgV)和恒定区(IgC)、一个跨膜区和一个含有约45个氨基酸的胞质尾区(Tissue Antigens.2007 Aug；70(2):96-104)。目前，B7H3主要存在2种剪切体，B7H3a和B7H3b。B7H3a胞外段由IgV-IgC 2个免疫球蛋白结构域组成，又称为2IgB7H3；而B7H3b胞外段由IgV-IgC-IgV-IgC 4个免疫球蛋白结构域组成，又称为4IgB7H3。
B7H3在正常组织、细胞中不表达或极低表达，却高表达于多种肿瘤组织。B7H3与肿瘤的进展、患者的生存及预后密切相关。临床上已经报道，B7H3在许多癌症类型中、特别是在非小细胞肺癌、肾癌、泌尿道上皮癌、结直肠癌、前列腺癌、多形性胶质母细胞瘤、卵巢癌和胰腺癌中过表达(Lung Cancer.2009 Nov；66(2):245-249；Clin Cancer Res.2008 Aug 15；14(16):5150-5157)。此外，也有文献报道，在前列腺癌中，B7H3的表达强度与临床病理学恶性(诸如肿瘤体积、前列腺外侵袭或Gleason评分)正相关，且也与癌症进展相关(Cancer Res.2007 Aug 15；67(16):7893-7900)。类似地，在多形性胶质母细胞瘤中，B7H3的表达与无事件存活负相关；且在胰腺癌中，B7H3的表达与淋巴结转移和病理学进展相关。因此，B7H3被认为是一种新的肿瘤标志物和潜在的治疗靶点。
目前，已有针对B7H3靶点的治疗策略用于临床前研究，如靶向小鼠B7H3的抗体会增强瘤内的浸润性的CD8-阳性的T细胞和抑制肿瘤生长( Mod Pathol.2010 Aug；23(8):1104-1112)。此外，WO2008/066691专利申请显示，识别B7H3a的抗体 会对腺癌表现出体内抗肿瘤作用。在临床研究中，一种鼠源的B7H3抗体与放射性I 131的偶联药物可显著抑制患者成神经母细胞瘤的生长(J Neufooocol 97(3):409-l 8(2010))。但目前在研的项目都是鼠源抗体经改造的人源化抗体，而人源化抗体在免疫时存在免疫原性相对较高的问题，在人体应用时是一个不利的因素。
噬菌体展示技术(phage display technology)将外源蛋白质或多肽与噬菌体外壳蛋白融合表达，从而将外源蛋白表达在噬菌体的表面。噬菌体抗体库是将噬菌体展示技术、PCR扩增技术、蛋白表达技术相结合的一项运用综合技术手段所建立起来的抗体库。
噬菌体抗体库最大的优点是不经体内免疫，模拟体内抗体生成的三个过程而制备出全人源的抗体。除此之外，噬菌体抗体库还具有以下优势：
①实现了基因型与表型的统一。此外，实验方法简单、快速，传统的通过杂交瘤技术抗体产生方法需历经数月，而抗体库技术只需短短几周的时间。
②表达的是完全人源抗体，且分子量小，主要以活性片段Fab、scFv的形式表达，与完整抗体相比在组织穿透力方面都有明显优势。
③筛选容量大，杂交瘤技术是在上千个克隆内筛选，抗体库技术可以在百万甚至亿万个分子中进行选择，筛选到的抗体种类多。
④用途广泛，采用了原核表达系统，当大规模生产时优势更加明显(Curr Opin Biotechnol.2002 Dec；13(6):598-602；Immunotechnology,2013，48(13)48(13):63-73)。
抗体-药物偶联物(antibody drug conjugate，ADC)把单克隆抗体或者抗原结合片段通过接头化合物与具有生物活性的细胞毒素相连，充分利用了抗体对正常细胞和肿瘤细胞表面抗原结合的特异性和细胞毒性物质的高效性，同时又避免了抗体的疗效偏低和毒性物质毒副作用过大等缺陷。这也就意味着，与以往传统的化疗药物相比，抗体-药物偶联物能更精准地杀伤肿瘤细胞并降低对正常细胞的影响。
目前已有多种ADC药物被用于临床或临床研究；如Kadcyla，是靶向Her2的曲妥珠单抗与DM1形成的ADC药物。同时，也有靶向B7H3的抗体及ADC药物的专利报道，如WO2008100934、WO2012147713、WO2014061277、WO2015184203、WO2016044383。
用于抗体药物偶联物的具有细胞毒性的小分子有几类；其中有一类是喜树碱衍生物，它们通过抑制拓扑异构酶I而具有抗肿瘤作用的。报道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)应用于抗体偶联药物(ADC)的文献有WO2014057687；Clinical Cancer Research(2016)22(20)：5097-5108；Cancer Sci(2016)107:1039-1046。但仍需进一步开发疗效更好的ADC药物。
发明内容
为了降低抗体分子的免疫原性，提供一种更符合需要的B7H3抗体-药物偶联物。本公开提供了一种通式Pc-L-Y-D所示的抗体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2021082929-appb-000001
其中：
Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-和-S-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基和杂环基；或者，R a和R b与其相连接的碳原子一起形成环烷基和杂环基；
R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
或者，R a和R 2与其相连的碳原子一起形成环烷基或杂环基；
m为0至4的整数；非限制性示例如m选自0、1、2、3和4；
n为1至10，n是小数或整数；
L为接头单元；
Pc为抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段包含：
重链可变区，其为SEQ ID NO：1的变体，所述的变体包含一个或多个选自T16R、Y103F、I28T、A33D、S99E、A100G、A104G、R101K、A104S和A113T的氨基酸取代；优选地，所述的变体包含一个或多个选自T16R、Y103F、I28T和A113T的氨基酸取代；和
轻链可变区，其为SEQ ID NO：2或其变体，所述的变体包含一个或两个选自R56K和S57G的氨基酸取代；优选地，所述的变体包含S57G的氨基酸取代；
所述氨基酸编号为依据可变区序列的自然顺序编号。
在另一个实施方式中，本公开提供一种如前所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含：
重链可变区为SEQ ID NO：1的变体，所述的变体包含T16R的氨基酸取代；优选包含T16R和A113T的氨基酸取代。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含如序列SEQ ID NO：2所示的轻链可变区。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含轻链可变区为SEQ ID NO：2的变体，所述的变体包含S57G的氨基酸取代。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段的重链可变区为SEQ ID NO：1的变体，所述的变体包含选自以下任一组的氨基酸取代：
a、T16R、Y103F和A113T；
b、T16R、I28T、Y103F和A113T；
c、T16R和A113T；和
d、T16R、I28T和A113T。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含以下任一组：
e、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和
如SEQ ID NO：2所示的轻链可变区；
f、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和
如SEQ ID NO：2所示的轻链可变区；
g、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和
轻链可变区，其包含S57G的氨基酸取代；
h、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和
轻链可变区，其包含S57G的氨基酸取代；
i、重链可变区，其包含T16R和A113T的氨基酸取代，和
轻链可变区，其包含S57G的氨基酸取代；
j、重链可变区，其包含T16R、I28T和A113T的氨基酸取代，和
轻链可变区，其包含S57G的氨基酸取代。
本公开提供了一种通式Pc-L-Y-D所示的抗体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2021082929-appb-000002
其中：
Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-和-S-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基和杂环基；或者，R a和R b与其相连接的碳原子一起形成环烷基和杂环基；
R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
或者，R a和R 2与其相连的碳原子一起形成环烷基或杂环基；
m为0至4的整数；非限制性示例如m选自0、1、2、3和4；
n为1至10，n是小数或整数；
L为接头单元；
Pc为抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段包含：
重链可变区，其为SEQ ID NO：1中的重链可变区的变体，所述的变体包含一个或多个选自T16R、Y103F、I28T、A33D、S99E、A100G、A104G、R101K、A104S和A113T的氨基酸取代；优选地，所述的变体包含一个或多个选自T16R、Y103F、I28T和A113T的氨基酸取代；和
轻链可变区，其为SEQ ID NO：2中的轻链可变区或其变体，所述的变体包含一个或两个选自R56K和S57G的氨基酸取代；优选地，所述的变体包含S57G的氨基酸取代；
所述氨基酸编号为依据可变区序列的自然顺序编号。
在另一个实施方式中，本公开提供一种如前所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含：
重链可变区为SEQ ID NO：1中的重链可变区的变体，所述的变体包含T16R的氨基酸取代；优选包含T16R和A113T的氨基酸取代。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含如序列SEQ ID NO：2中所示的轻链可变区。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含轻链可变区为SEQ ID NO：2的轻链可变的变体，所述的变体包含S57G的氨基酸取代。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段的重链可变区为SEQ ID NO：1的重链可变区的变体，所述的变体包含选自以下任一组的氨基酸取代：
a、T16R、Y103F和A113T；
b、T16R、I28T、Y103F和A113T；
c、T16R和A113T；和
d、T16R、I28T和A113T。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含以下任一组：
e、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和
SEQ ID NO：2中的轻链可变区；
f、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和
SEQ ID NO：2中的轻链可变区；
g、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和
轻链可变区，其包含S57G的氨基酸取代；
h、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和
轻链可变区，其包含S57G的氨基酸取代；
i、重链可变区，其包含T16R和A113T的氨基酸取代，和
轻链可变区，其包含S57G的氨基酸取代；
j、重链可变区，其包含T16R、I28T和A113T的氨基酸取代，和
轻链可变区，其包含S57G的氨基酸取代。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段选自以下任一组：
如SEQ ID NO：3所示的重链可变区，和
如SEQ ID NO：4所示的轻链可变区；
如SEQ ID NO：5所示的重链可变区，和
如SEQ ID NO：6所示的轻链可变区；
如SEQ ID NO：7所示的重链可变区，和
如SEQ ID NO：8所示的轻链可变区；
如SEQ ID NO：9所示的重链可变区，和
如SEQ ID NO：10所示的轻链可变区；
如SEQ ID NO：11所示的重链可变区，和
如SEQ ID NO：12所示的轻链可变区；
如SEQ ID NO：13所示的重链可变区，和
如SEQ ID NO：14所示的轻链可变区。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含抗体恒定区；所述抗体恒定区的重链恒定区来源于人IgG1、IgG2、IgG3或IgG4，优选地，所述重链恒定区的氨基酸序列源于人IgG1；所述抗体恒定区的轻链恒定区来源于人抗体κ、λ链。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体包含选自以下任一组：
如SEQ ID NO：17所示的重链、和
SEQ ID NO：18所示的轻链；
如SEQ ID NO：19所示的重链、和
SEQ ID NO：20所示的轻链；
如SEQ ID NO：21所示的重链、和
SEQ ID NO：22所示的轻链；和
如SEQ ID NO：23所示的重链、和
SEQ ID NO：24所示的轻链。
在另一个实施方式中，本公开提供一种如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)和二硫键稳定化的V区(dsFv)。
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中n为1至8，优选为3-8，更优选为3-7，n是小数或整数。作为非限制性示例，可以提及的n是1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.6、8.7、8.8、8.9、9.0、9.1、9.2、9.3、9.4、9.5、9.6、9.7、9.8、9.9、10.0、或前述任意数值间的范围；允许技术人员根据需求，确定小数点后的精度。
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，
其中：
Y为-O-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素和C1 -6烷基；
R 1为卤代C 1-6烷基或C 3-6环烷基；
R 2选自氢原子、卤代C 1-6烷基和C 3-6环烷基；
或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
m为0或1。
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中Y选自以下任一项：
Figure PCTCN2021082929-appb-000003
其中Y的O端与接头单元L相连。
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 3-W-C(O)-和-C(O)-W-C(O)-；其中W选自C 1-8烷基、C 1-8烷基-环烷基和1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸(Cit)、丝氨酸(S)、谷氨酸(E)和天冬氨酸(D)中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
L 4选自-NR 5(CR 6R 7) t-、-C(O)NR 5、-C(O)NR 5(CH 2) t-和化学键，其中t为1至6的整数；
R 3、R 4和R 5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基。
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1为
Figure PCTCN2021082929-appb-000004
s 1为2至8的整数(具体而言，2、3、4、5、6、7或8)；
L 2为化学键；
L 3为四肽残基；优选地，L 3为GGFG(SEQ ID NO：33)的四肽残基；
L 4为-NR 5(CR 6R 7)t-，R 5、R 6或R 7相同或不同，且各自独立地为氢原子或烷基，t为1或2；
其中所述的L 1端与Pc相连，L 4端与Y相连。
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中-L-为：
Figure PCTCN2021082929-appb-000005
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中-L-Y-任选自以下任一项：
Figure PCTCN2021082929-appb-000006
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2021082929-appb-000007
其中，
Pc、n、m、R 1、R 2如通式(Pc-L-Y-D)中所定义；
W、L 2、L 3、R 5、R 6和R 7如接头单元-L-中所定义；
具体地，
Pc为抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段包含：
重链可变区，其为SEQ ID NO：1中的重链可变区的变体，所述的变体包含一个或多个选自T16R、Y103F、I28T、A33D、S99E、A100G、A104G、R101K、A104S和A113T的氨基酸取代；优选地，所述的变体包含一个或多个选自T16R、Y103F、I28T和A113T的氨基酸取代；和
轻链可变区，其为SEQ ID NO：2中的轻链可变区或其变体，所述的变体包含一个或两个选自R56K和S57G的氨基酸取代；优选地，所述的变体包含S57G的氨基酸取代；
m为0至4的整数；
n为1至10，n是小数或整数；
R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
W选自C 1-8烷基、C 1-8烷基-环烷基和1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨 酸(E)和天冬氨酸(D)中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
R 5选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基。
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L b-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2021082929-appb-000008
其中：
s 1为2至8的整数；
Pc、R 1、R 2、R 5至R 7、m和n如通式(Pc-L a-Y-D)中所定义。
在另一个实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物选自以下任一项：
Figure PCTCN2021082929-appb-000009
Figure PCTCN2021082929-appb-000010
其中，Pc和n如通式(Pc-L a-Y-D)中所定义。
在另一个实施方式中，本公开提供一种所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物为：
Figure PCTCN2021082929-appb-000011
其中：
n为1至8，优选3至8；更优选3至7，n是小数或整数；
h1702-DS-107为抗B7H3抗体，其包含序列如SEQ ID NO：21所示的重链和序列如SEQ ID NO：22所示的轻链。
在另一些实施方式中，本公开提供一种如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中n为1至8，优选为3-8，更优选为3-7，n是小数或整数。
另一方面，本公开一个实施方式提供一种抗B7H3抗体或其抗原结合片段，其包含重链可变区，其为SEQ ID NO：1中的重链可变区的变体，所述的变体包含一 个或多个选自T16R、Y103F、I28T、A33D、S99E、A100G、A104G、R101K、A104S和A113T的氨基酸取代；优选地，所述的变体包含一个或多个选自T16R、Y103F、I28T和A113T的氨基酸取代；和
轻链可变区，其为SEQ ID NO：2中的轻链可变区或其变体，所述的变体包含一个或两个选自R56K和S57G的氨基酸取代；优选地，所述的变体包含S57G的氨基酸取代。
在另一个实施方式中，本公开提供一种所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段的重链可变区为SEQ ID NO：1重链可变区的变体，所述的变体包含选自以下任一组的氨基酸取代：
a、T16R、Y103F和A113T；
b、T16R、I28T、Y103F和A113T；
c、T16R和A113T；和
d、T16R、I28T和A113T。
在另一个实施方式中，本公开提供一种所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段包含：
e、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和SEQ ID NO：2中的轻链可变区；
f、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和SEQ ID NO：2中的轻链可变区；
g、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和轻链可变区，其包含S57G的氨基酸取代；
h、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和轻链可变区，其包含S57G的氨基酸取代；
i、重链可变区，其包含T16R和A113T的氨基酸取代，和轻链可变区，其包含S57G的氨基酸取代；
j、重链可变区，其包含T16R、I28T和A113T的氨基酸取代，和轻链可变区，其包含S57G的氨基酸取代。
在另一个实施方式中，本公开提供一种所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段选自：
如SEQ ID NO：3所示的重链可变区，和如SEQ ID NO：4所示的轻链可变区；
如SEQ ID NO：5所示的重链可变区，和如SEQ ID NO：6所示的轻链可变区；
如SEQ ID NO：7所示的重链可变区，和如SEQ ID NO：8所示的轻链可变区；
如SEQ ID NO：9所示的重链可变区，和如SEQ ID NO：10所示的轻链可变区；
如SEQ ID NO：11所示的重链可变区，和如SEQ ID NO：12所示的轻链可变区；和
如SEQ ID NO：13所示的重链可变区，和如SEQ ID NO：14所示的轻链可变区。
在另一个实施方式中，本公开提供一种所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段包含抗体恒定区；所述抗体恒定区的重链恒定区来源于人IgG1、IgG2、IgG3或IgG4，优选地，所述重链恒定区的氨基酸序列源于人IgG1，更优选如SEQ ID NO：15所示的重链恒定区；所述抗体恒定区的轻链恒定区来源于人抗体κ、λ链；优选如SEQ ID NO：16所示的轻链恒定区。
在另一个实施方式中，本公开提供一种所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体选自：
如SEQ ID NO：17所示的重链和SEQ ID NO：18所示的轻链；
如SEQ ID NO：19所示的重链和SEQ ID NO：20所示的轻链；
如SEQ ID NO：21所示的重链和SEQ ID NO：22所示的轻链；和
如SEQ ID NO：23所示的重链和SEQ ID NO：24所示的轻链。
另一方面，本公开一个实施方式提供一种核酸分子，其编码如上所述的抗B7H3抗体或其抗原结合片段。
另一方面，本公开一个实施方式提供一种宿主细胞，其包含如上所述的核酸分子。
另一方面，本公开一个实施方式提供一种制备如通式Pc-L a-Y-D所示的化合物的方法，其包括如下步骤：
Figure PCTCN2021082929-appb-000012
Pc’为经还原后的Pc，与通式(L a-Y-D)所示的化合物进行偶联反应，得到通 式(Pc-L a-Y-D)所示的化合物；作为一个示例，Pc’为带有反应性基团(如巯基)，还原剂优选TCEP，特别地，优选还原抗体上的二硫键，得到巯基；
其中：
Pc为如前所述的抗B7H3抗体或其抗原结合片段；
W、L 2、L 3、R 1、R 2、R 5至R 7、m和n如通式Pc-La-Y-D中所定义。
另一方面，本公开一个实施方式提供一种制备如通式Pc-L a-Y-D所示的化合物的方法，其包括如下步骤：
Figure PCTCN2021082929-appb-000013
抗B7H3抗体h1702-DS-107还原后得到h1702-DS-107’，h1702-DS-107’与式9-A所示的化合物进行偶联反应，得到通式(h1702-DS-107-9-A)所示的化合物；还原剂优选TCEP，特别地，优选还原抗体上的二硫键，得到巯基；
n为1至8，优选3-8，n是小数或整数；
1702-DS-107为抗B7H3抗体，其包含序列如SEQ ID NO：3所示的重链和序列如SEQ ID NO：4所示的轻链。
另一方面，本公开提供了一种药物组合物，其包含：
-如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或如前任一项所述的抗B7H3抗体或其抗原结合片段，以及
-一种或多种药学上可接受的赋形剂、稀释剂或载体。
在一些实施方案中，所述单位剂量的药物组合物中含有0.1mg至3000mg或1mg至1000mg如前所述的抗B7H3抗体、其抗原结合片段、或如前所述的抗体药物偶联物。
另一方面，本公开提供了如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或如前任一项所述的抗B7H3抗体或其抗原结合片段或包含其的药物组合物作为药物的用途。
另一方面，本公开提供了如前任一项所述的抗体-药物偶联物或其药学上可接受的盐、或如前任一项所述的抗B7H3抗体或其抗原结合片段、或包含其的药物组合物在制备用于治疗B7H3介导的疾病或病症或肿瘤的药物中的用途，其中所述B7H3介导的疾病或病症为B7H3高表达癌症，中表达癌症或低表达癌症。
另一方面，本公开提供如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或如前任一项所述的抗B7H3抗体或其抗原结合片段或包含其的药物组合物在制备用于治疗或预防肿瘤的药物中的用途，其中所述肿瘤和癌症优选头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、咽头癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、尿路上皮癌和梅克尔细胞癌；更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
另一方面，本公开进一步涉及一种用于治疗或预防肿瘤或癌症的方法，该方法包括向需要其的受试者施用治疗有效剂量或预防有效剂量的如前任一项所述的抗体药物偶联物或其药学上可接受的盐、或如前任一项所述的抗B7H3抗体或其抗原结合片段、或包含其的药物组合物。
在一些实施方案中，适用于采用本申请的活性化合物进行处理的肿瘤或癌症，与B7H3的高表达、中表达或低表达相关。在一些实施方案中，所述肿瘤和癌症选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、尿路上皮癌和梅克尔细胞癌。
在一些具体的实施方案中，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细 胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤。
在一些具体的实施方案中，所述肺癌选自：非小细胞肺癌和小细胞肺癌。
在一些具体的实施方案中，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病。
另一方面，本公开进一步提供前述的抗B7H3抗体或其抗体-药物偶联物作为药物，优选作为治疗癌症或肿瘤的药物，更优选作为治疗B7H3介导的癌症的药物。
可将活性化合物(例如根据本公开所述的配体-药物偶联物、或其药学上可接受的盐)制成适合于通过任何适当途径给药的形式，活性化合物优选是以单位剂量的方式，或者是以受试者能够以单剂进行自我给药的方式。本公开化合物或组合物的单位剂量的方式可以是片剂、胶囊、扁囊剂、瓶装药水、药粉、颗粒剂、锭剂、栓剂、再生药粉或液体制剂。
本公开治疗方法中所用活性化合物或组合物的施用剂量通常将随疾病的严重性、受试者的体重和化合物的相对功效而改变。作为一般性指导，合适的单位剂量可以是0.1mg至1000mg。
本公开的药物组合物除活性化合物外，可含有一种或多种辅料，所述辅料选自以下成分：填充剂、稀释剂、粘合剂、润湿剂、崩解剂或赋形剂等。根据给药方法的不同，组合物可含有0.1重量％至99重量％的活性化合物。
本公开提供的抗B7H3抗体及抗体药物偶联物具有降低的免疫原性，更高的抑瘤效果和治疗活性，更低的毒性，更好的药物代谢动力学特性和成药性(如稳定性)。
附图说明
图1：不同ADC对不同B7H3表达水平Detroit562细胞系的增殖抑制。
图2：不同ADC对裸鼠Detroit562移植瘤的疗效。
具体实施方式
一、术语
除非另有限定，本文所用的所有技术和科学术语均与本公开所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本公开，但本文描述了优选的方法和材料。描述和要求保护本公开时，依据以下定义使用下列术语。
当本公开中使用商品名时，旨在包括该商品名产品的制剂、该商品名产品的药物和活性药物部分。
除非有相反陈述，在说明书和权利要求书中使用的术语具有下述含义。
术语“药物”是指细胞毒性药物，能在细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够的浓度下都可以杀死细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语包括毒素(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At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)，化疗药物，抗生素和核溶酶。
术语“L”、“接头单元”、“接头”、“连接单元”或“连接片段”是指一端与配体(具体是抗体或抗原结合片段)连接，而另一端与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配体或药物相连。
接头(包括延伸物、间隔物和氨基酸单元)，可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
术语“抗体-药物偶联物”(antibody drug conjugate，ADC)，指抗体通过连接单元与药物相连。在本公开中“抗体-药物偶联物”指把单克隆抗体或者抗原结合片段通过稳定的连接单元与具有生物活性的毒性药物相连。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
术语“抗体”指免疫球蛋白，完整抗体是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
全长抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的框架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的IMGT规则。
术语“完全人源抗体”、“完全人抗体”或“全人抗体”，也称“全人源单 克隆抗体”，其抗体的可变区和恒定区都是人源的，降低了免疫原性和毒副作用。全人源抗体制备的相关技术主要有：人杂交瘤技术、EBV转化B淋巴细胞技术、噬菌体显示技术(phage display)、转基因小鼠抗体制备技术(transgenic mouse)和单个B细胞抗体制备技术等。
术语“抗原结合片段”是指抗体的保持结合抗原的能力的一个或多个片段。可利用全长抗体的片段来进行抗体的抗原结合功能。“抗原结合片段”中包含的结合片段的示例包括：
(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；
(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，
(iii)由VH和CH1结构域组成的Fd片段；
(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；
(v)单结构域或dAb片段(Ward等人,(1989)Nature341：544-546)，其由VH结构域组成；和
(vi)分离的互补决定区(CDR)；或
(vii)通过接头连接的两个或更多个分离的CDR的组合。
此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过接头连接它们，从而使得其能够产生VL和VH区配对形成单价分子的单个蛋白质链，称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。
使用本领域技术人员已知的常规技术获得此类抗原结合片段，并且以与对于完整抗体的方式相同的方式进行功用性筛选。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合片段。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
通常，Fab是通过用蛋白酶木瓜蛋白酶(例如，切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的具有约50,000分子量并具有抗原结合活性的片段，其中H链N端侧的部分和L链通过二硫键结合在一起。
通常，F(ab')2是通过用酶胃蛋白酶消化IgG铰链区中二硫键的下方部分而获得的，分子量约为100,000并具有抗原结合活性并包含在铰链位置相连的两个Fab区的片段。
通常，Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000并具有抗原结合活性的片段。
此外，可以通过将编码Fab'片段的DNA插入到原核生物表达载体或真核生物表 达载体中，并将载体导入到原核生物或真核生物中进行表达，来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或VH)和抗体轻链可变结构域(或VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“框架区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上被免疫球蛋白或抗体结合的部位。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于10 -8M、10 -9M或10 -10M或更小的亲和力(以KD计)结合。
术语“核酸分子”是指DNA分子或RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，表达载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，表达载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌属(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia  pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组的免疫球蛋白表达载体可以稳定地转染宿主细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的N端位点。阳性的克隆在生物反应器的培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗原结合片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
术语“肽”是指分子量介于氨基酸和蛋白质之间的化合物片段，由2个或2个以上氨基酸分子通过肽键相互连接而成。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个(例如1、2、3、4、5、6、7、8、9、10、11和12个)碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子(包含1个、2个、3个、4个、5个或6个碳原子)的烷基。非限制性示例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的，烷基是含有1至6个碳原子的低级烷基，非限制性示例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。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、杂环烷基、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。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”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两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个(例如1、2、3、4、5、6、7、8、9、10、11和12个)碳原子，更优选含有1至6个碳原子(包含1个、2个、3个、4个、5个或6个碳原子)的亚烷基。亚烷基的非限制性示例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚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。
术语“烷氧基”指-O-(烷基)和-O-(环烷基)，其中烷基或环烷基的定义如上所述。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。
术语“环烷基”指饱和或部分不饱和单环或多环的环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子(包含3个、4个、5个、6个、7个或8个碳原子)。单环环烷基的非限制性示例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环或桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环的环状烃取代基，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S(O) m(其中m是整数0、1或2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个环原子，其中1至4个是杂原子(1个、2个、3个或4个杂原子)；更优选，环烷基环包含3至10个环原子(包含3个、4个、5个、6个、7个、8个、9个或10个环原子)。单环杂环基的非限制性示例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环或桥环的杂环基。
术语“螺杂环基”指单环之间共用一个原子(称螺原子)的，5至20元的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂 原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为：单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基或双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。螺杂环基的非限制性示例包括：
Figure PCTCN2021082929-appb-000014
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0、1或2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(7元、8元、9元或10元环)。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。稠杂环基的非限制性示例包括：
Figure PCTCN2021082929-appb-000015
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0、1或2)的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(7元、8元、9元或10元环)。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更优选为双环或三环。桥杂环基的非限制性示例包括：
Figure PCTCN2021082929-appb-000016
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性示例包括：
Figure PCTCN2021082929-appb-000017
等。
杂环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、杂环烷基、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。
术语“芳基”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)基团，优选为6至10元(6元、7元、8元、9元或10元)，例如苯基和萘基，优选苯基。所述芳基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，其非限制性示例包括：
Figure PCTCN2021082929-appb-000018
芳基可以是取代的或非取代的。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。
术语“杂芳基”指包含1至4个杂原子(1个、2个、3个或4个杂原子)、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元(5元、6元、7元、8元、9元或10元杂芳基)，更优选为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，其非限制性示例包括：
Figure PCTCN2021082929-appb-000019
杂芳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。
术语“氨基保护基”是为了使分子其他部位进行反应时氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性示例包含9-芴甲氧羰基、叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基(1个、2个或3个取代基)所取代。所述氨基保护基优选为9-芴甲氧羰基。
术语“卤代烷基”指烷基上的氢被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基上的氢被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。
术语“硝基”指-NO 2。
术语“酰胺基”指-C(O)N(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
术语“羧酸酯基”指-C(O)O(烷基)或(环烷基)，其中烷基、环烷基如上所定义。
本公开还包括各种氘化形式的式(I)化合物。与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的式(I)化合物。在制备氘代形式的式(I)化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。
“任选”或“任选地”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。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1个、2个或3个氢原子彼此独立地被取代基取代。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)键的碳原子结合时可能是不稳定的。
术语“药物组合物”表示含有一种或多种本文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。药物组合物的目的是促进对生物体的给药，利于活性化合物的吸收进而发挥生物活性。
术语“药学上可接受的盐”或“可药用盐”是指本公开抗体-药物偶联物的 盐，或本公开中所述的活性化合物的盐，这类盐用于受试者时具有安全性和有效性，且具有应有的生物活性。作为一个示例，本公开抗体-抗体药物偶联化合物至少含有一个氨基，因此可以与酸形成盐，可药用盐的非限制性示例包括：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。
“载药量”也称药物抗体比例(Drug-to-Antibody Ratio，DAR)，即ADC中每个抗体所偶联的药物的平均数量。其可在例如每个抗体偶联约1至约10个药物的范围内；并且在某些实施例中，在每个抗体偶联约1至约8个药物的范围内，优选选自2-8，2-7，2-6，2-5，2-4，3-4，3-5，5-6，5-7，5-8和6-8的范围。示例性地，载药量可以为在1，2，3，4，5，6，7，8，9，10基础上所得的算术平均值。本披露的ADC通式包括与前述一定载药量范围内的抗体药物偶联物的集合。
在本公开的实施方式中，载药量表示为n，可用常规方法如UV/可见光光谱法、质谱、ELISA试验和HPLC特征测定载药量。
可以用以下非限制性方法控制抗体药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
药物组合物的常规制备方法见中国药典。
术语“载体”用于本公开的药物，是指能改变药物进入受试者的方式和在体内的分布、控制药物的释放速度并将药物输送到靶点的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的示例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除活性成分以外的附加物，也可称为辅料。如片剂中的黏合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐、微晶纤维素等。
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性化合物溶于油 相的无菌注射水包油微乳。例如将活性化合物溶于大豆油和卵磷脂的混合物中，然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入受试者的血流中。或者，最好按可保持本公开化合物恒定循环浓度的方式施用溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的示例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
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。为此目的，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。
二、合成方法
为了完成合成目的，采用如下的合成技术方案：
制备抗体-药物偶联物h1702-DS-107的方法，其包括如下步骤：
Figure PCTCN2021082929-appb-000020
抗B7H3抗体h1702-DS-107还原后得到h1702-DS-107’，h1702-DS-107’与式9-A所示的化合物进行偶联反应，得到通式(h1702-DS-107-9-A)所示的化合物；还原剂优选TCEP，特别地，优选还原抗体上的二硫键，得到巯基；n为1至8，优选3-8，n是小数或整数。
在以上说明书中提出了本公开一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本公开，但是以下描述优选的方法和材料。通过说明书和权利要求书，本公开的其他特点、目的和优点将是显而易见的。
在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括 复数指代物的情况。
除非另有定义，本公开使用的所有技术和科学术语都具有本公开所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。
提出以下实施例是为了更全面地说明本公开的优选实施方式。这些实施例不应以任何方式理解为限制本公开的范围，本公开的范围由权利要求书限定。
一、抗体的制备
实施例1-1抗体的制备、表达与纯化
(一)抗体的设计
WO2019/024911公开了一种抗B7H3抗体h1702-DS的制备，其全文可参考引用，抗体h1702-DS具体序列包括重链(IgG1)氨基酸序列(如公开WO2018/177393中所述第22号序列)：
Figure PCTCN2021082929-appb-000021
和轻链氨基酸序列(如公开WO2018/177393中所述的第26号序列)：
Figure PCTCN2021082929-appb-000022
对上述抗B7H3抗体进行突变，得到具有更低免疫原性的抗体，检测其对B7H3结合活性(测试例1)，获得了新的抗体，相应抗体的轻链可变区及重链可变区如下：
表1.抗B7H3抗体的突变体及其序列
Figure PCTCN2021082929-appb-000023
Figure PCTCN2021082929-appb-000024
抗体可变区再与恒定区基因(CH1-FC/CL)片段进行同源重组，构建完整抗体VH-CH1-FC/VK-CL/VL-CL，所述的恒定区序列如下：
重链恒定区：
Figure PCTCN2021082929-appb-000025
Figure PCTCN2021082929-appb-000026
轻链恒定区：
Figure PCTCN2021082929-appb-000027
得到完整抗体，具体抗体序列包括但不限于：
抗体h1702-DS-105
重链氨基酸序列：
Figure PCTCN2021082929-appb-000028
轻链氨基酸序列：
Figure PCTCN2021082929-appb-000029
抗体h1702-DS-106
重链氨基酸序列：
Figure PCTCN2021082929-appb-000030
轻链氨基酸序列：
Figure PCTCN2021082929-appb-000031
Figure PCTCN2021082929-appb-000032
抗体h1702-DS-107
重链(IgG1)氨基酸序列：
Figure PCTCN2021082929-appb-000033
轻链氨基酸序列：
Figure PCTCN2021082929-appb-000034
抗体h1702-DS-108
重链氨基酸序列：
Figure PCTCN2021082929-appb-000035
轻链氨基酸序列：
Figure PCTCN2021082929-appb-000036
其中上述完整抗体轻重链中CDR序列(IMGT编号规则)如表2所示。
表2.各重链及轻链CDR区序列
Figure PCTCN2021082929-appb-000037
(二)全人抗体的表达与纯化
分别表达抗体轻重链的质粒以转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl，pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。
实施例1-2相关检测细胞系和抗体的制备
(一)B7H3过表达的细胞系
本公开使用过表达B7H3的重组细胞系(CT26/B7H3，其中CT26来源于中科院细胞库，TCM37)或肿瘤细胞(A498)，检测本公开抗体与B7H3抗原的结合能力。
人B7H3全长氨基酸序列：B7H3(SEQ ID NO：31)：
Figure PCTCN2021082929-appb-000038
注释：
双横线部分为信号肽(Signal peptide:1–28)；
划横线部分为B7H3胞外区(Extracellular domain：29-466)，其中29-139为Ig-样V-型1结构域，145–238为Ig-样C2-型1结构域；243-357为Ig-样V-型2结构域，363–456为Ig-样C2-型2结构域；
点划线部分为跨膜区部分(Transmembrane domain:467-487)；
斜体部分为胞内区(Cytoplasmic domain:488-534)。
(二)猴B7H3全长氨基酸序列
Figure PCTCN2021082929-appb-000039
注释：
双横线部分为信号肽(Signal peptide:1–28)；
划横线部分为B7H3胞外区(Extracellular domain：29-466)，其中29-139为Ig-like V-type 1 Domain，145–238为Ig-like C2-type 1 Domain；243-357为Ig-like V-type 2 Domain，363–456为Ig-like C2-type 2 Domain；
点划线部分为跨膜区部分(Transmembrane domain:467-487)；
斜体部分为胞内区(Cytoplasmic domain:488-534)。
二、化合物的制备
本公开实施例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
化合物的结构是通过核磁共振(NMR)或质谱(MS)来确定的。NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d6)、氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)，化学位移是以10 -6(ppm)作为单位给出。
MS的测定用FINNIGAN LCQAd(ESI)质谱仪(生产商：Thermo，型号：Finnigan LCQ advantage MAX)。
UPLC的测定用Waters Acquity UPLC SQD液质联用仪。
HPLC的测定使用安捷伦1200DAD高压液相色谱仪(Sunfire C18 150×4.6mm色谱柱)和Waters 2695-2996高压液相色谱仪(Gimini C18 150×4.6mm色谱柱)。
UV-HPLC的测定使用Thermo nanodrop2000紫外分光光度计。
增殖抑制率及IC 50值的测定用PHERA starFS酶标仪(德国BMG公司)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板，薄层色谱法(TLC)使用的硅胶板采用的规格是0.15mm至0.2mm，薄层层析分离纯化产品采用的规格是0.4mm至0.5mm硅胶板。
柱层析一般使用烟台黄海200至300目硅胶为载体。
本公开的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH&Co.KG，Acros Organnics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中如无特殊说明，反应均在氩气氛或氮气氛下进行。氩气氛或氮气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢化仪。
氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中如无特殊说明，反应中的溶液是指水溶液。
实施例中如无特殊说明，反应的温度为室温。室温为最适宜的反应温度，温度范围是20℃至30℃。
实施例中pH＝6.5的PBS缓冲液的配制：取KH 2PO 4 8.5g，K 2HPO 4.3H 2O 8.56g，NaCl 5.85g，EDTA 1.5g置于瓶中，定容至2L，超声波使其全部溶解，摇匀即得。
纯化化合物采用的柱层析的洗脱剂的体系和薄层色谱法的展开剂的体系包括：A：二氯甲烷和异丙醇体系，B：二氯甲烷和甲醇体系，C：石油醚和乙酸乙酯体系，溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺和酸性或碱性试剂等进行调节。
本公开部分化合物是通过Q-TOF LC/MS来表征的。Q-TOF LC/MS使用安捷伦6530精确质量数四级杆-飞行时间质谱仪和安捷伦1290-Infinity超高效液相色谱仪(安捷伦Poroshell 300SB-C8 5μm,2.1×75mm色谱柱)。
本公开抗体药物偶联物的Y-D药物部分参见PCT/CN2019/107873。全文包括相关的化合物合成及测试例引用至本专利。其中的非限制性示例合成引用如下：
实施例2-1
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环丙烷-1-甲酰胺1
Figure PCTCN2021082929-appb-000040
向依喜替康甲磺酸盐1b(2.0mg，3.76μmol，采用专利申请“EP0737686A1”公开的方法制备而得)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-羟基环丙基甲酸1a(1.4mg，3.7μmol，采用公知的方法“Tetrahedron Letters，25(12)，1269-72；1984”制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(3.8mg，13.7μmol)，加毕，在0-5℃搅拌反应2小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(8mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物1(1.6mg，产率：82.1％)。
MS m/z(ESI):520.2[M+1]
1H NMR(400MHz，CDCl 3):δ7.90-7.84(m，1H)，7.80-7.68(m，1H)，5.80-5.70(m，1H)，5.62-5.54(m，2H)，5.44-5.32(m，2H)，5.28-5.10(m，2H)，3.40-3.15(m，3H)，2.44(s，3H)，2.23(t，1H)，2.06-1.75(m，2H)，1.68-1.56(m，1H)，1.22-1.18(m，2H)，1.04-0.98(m，2H)，0.89(t，3H)。
实施例2-2
(S)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺2-A
(R)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-2-羟基乙酰胺2-B
Figure PCTCN2021082929-appb-000041
向1b(4mg，7.53μmol)中加入2mL乙醇和0.4mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啉，搅拌至反应液变澄清。向反应液中依次加入2-环丙基-2-羟基乙酸2a(2.3mg，19.8μmol，采用专利申请“WO2013106717”公开的方法制备而得)、1-羟基苯并三唑(3mg，22.4μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(4.3mg，22.4μmol)，加毕，在0-5℃搅拌反应1小时。撤去冰水浴，加热至30℃搅拌2小时。反应液减压浓缩，所得到的粗品化合物2用高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um 19*250mm；流动相：A-水(10mmol NH 4OAc)，B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(2-A：1.5mg，2-B：1.5mg)。
MS m/z(ESI):534.0[M+1]。
单一构型化合物2-B(较短保留时间)：
UPLC分析：保留时间1.06分钟，纯度：88％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz，DMSO-d 6):δ8.37(d，1H)，7.76(d，1H)，7.30(s，1H)，6.51(s，1H)，5.58-5.56(m，1H)，5.48(d，1H)，5.41(s，2H)，5.32-5.29(m，2H)，3.60(t，1H)，3.19-3.13(m，1H)，2.38(s，3H)，2.20-2.14(m，1H)，1.98(q，2H)，1.87-1.83(m，1H)，1.50-1.40(m，1H)，1.34-1.28(m，1H)，0.86(t，3H)，0.50-0.39(m，4H)。
单一构型化合物2-A(较长保留时间)：
UPLC分析：保留时间1.10分钟，纯度：86％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz，DMSO-d 6):δ8.35(d，1H)，7.78(d，1H)，7.31(s，1H)，6.52(s，1H)，5.58-5.53(m，1H)，5.42(s，2H)，5.37(d，1H)，5.32(t，1H)，3.62(t，1H)，3.20-3.15(m，2H)，2.40(s，3H)，2.25-2.16(m，1H)，1.98(q，2H)，1.87-1.82(m，1H)，1.50-1.40(m，1H)，1.21-1.14(m，1H)，0.87(t，3H)，0.47-0.35(m，4H)。
实施例2-3
(S)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟-2-羟基丙酰胺3-A
(R)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-3,3,3-三氟-2-羟基丙酰胺3-B
Figure PCTCN2021082929-appb-000042
向1b(5.0mg，9.41μmol)中添加2mL乙醇和0.4mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啡啉，搅拌至反应液变澄清。向反应液中依次加入3,3,3-三氟-2-羟基丙酸3a(4.1mg，28.4μmol，供应商Alfa)、1-羟基苯并三唑(3.8mg，28.1μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(5.4mg，28.2μmol)，加毕，在0-5℃搅拌反应10分钟。撤去冰水浴，加热至30℃搅拌8小时。反应液减压浓缩，所得到的粗品化合物3用高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um 19*250mm；流动相：A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(1.5mg，1.5mg)。
MS m/z(ESI):561.9[M+1]。
单一构型化合物(较短保留时间)：
UPLC分析：保留时间1.11分钟，纯度：88％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz，DMSO-d 6):δ8.94(d，1H)，7.80(d，1H)，7.32(s，1H)，7.20(d，1H)，6.53(s，1H)，5.61-5.55(m，1H)，5.45-5.23(m，3H)，5.15-5.06(m，1H)，4.66-4.57(m，1H)，3.18-3.12(m，1H)，2.40(s，3H)，2.26-2.20(m，1H)，2.16-2.08(m，1H)，2.02-1.94(m，1H)，1.89-1.82(m，1H)，1.50-1.40(m，1H)，0.87(t，3H)。
单一构型化合物(较长保留时间)：
UPLC分析：保留时间1.19分钟，纯度：90％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz，DMSO-d 6):δ8.97(d，1H)，7.80(d，1H)，7.31(s，1H)，7.16(d，1H)，6.53(s，1H)，5.63-5.55(m，1H)，5.45-5.20(m，3H)，5.16-5.07(m，1H)，4.66-4.57(m，1H)，3.18-3.12(m，1H)，2.40(s，3H)，2.22-2.14(m，1H)，2.04-1.95(m，2H)，1.89-1.82(m，1H)，1.50-1.40(m，1H)，0.87(t，3H)。
实施例2-4
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环戊烷-1-甲酰胺4
Figure PCTCN2021082929-appb-000043
向1b(3.0mg，5.64μmol)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-羟基-环戊烷甲酸4a(2.2mg，16.9μmol，采用专利申请“WO2013106717”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(4.7mg，16.9μmol)，加毕，在0-5℃搅拌反应1小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(10mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物4(2.5mg，产率：80.9％)。
MS m/z(ESI):548.0[M+1]。
1H NMR(400MHz，CDCl 3):δ7.73-7.62(m，2H)，5.75-5.62(m，1H)，5.46-5.32(m，2H)，5.26-5.10(m，1H)，3.30-3.10(m，1H)，2.43(s，3H)，2.28-2.20 (m，2H)，2.08-1.84(m，8H)，1.69-1.58(m，2H)，1.04-1.00(m，2H)，0.89(t，3H)。
实施例2-5
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-(羟甲基)环丙烷-1-甲酰胺5
Figure PCTCN2021082929-appb-000044
向1b(2.0mg，3.76μmol)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-(羟甲基)-环戊烷甲酸5a(0.87mg，7.5μmol，采用专利申请“WO201396771”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(2mg，7.24μmol)，加毕，在0-5℃搅拌反应2小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(8mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物5(1.0mg，产率：50％)。
MS m/z(ESI):533.9[M+1]。
1H NMR(400MHz，CDCl 3):δ8.07(s，1H)，7.23-7.18(m，2H)，6.71-6.64(m，1H)，6.55-6.51(m，1H)，5.36-5.27(m，2H)，4.67-4.61(m，2H)，3.53-3.48(m，1H)，3.30-3.22(m，2H)，3.18-3.13(m，1H)，2.71-2.61(m，2H)，2.35-2.28(m，1H)，2.04-1.91(m，4H)，1.53-1.40(m，3H)，0.91-0.75(m，4H)。
实施例2-6
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-(羟基甲基)环丁烷-1-甲酰胺6
Figure PCTCN2021082929-appb-000045
向1b(3.0mg，5.64μmol)中添加1mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加一滴三乙胺，搅拌至反应液变澄清。向反应液中依次加入1-(羟基甲基)环丁烷-1-甲酸6a(2.2mg，16.9μmol；采用文献“Journal of the American Chemical Society，2014，vol.136，#22，p.8138-8142”公开的方法制备而得)和4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(4.7mg，16.9μmol)，加毕，在0-5℃搅拌反应1小时。向反应液中加入5mL水淬灭反应，用乙酸乙酯(10mL×3)萃取反应液，合并有机相，用饱和氯化钠溶液(5mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，将滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物6(2.1mg，产率：67.9％)。
MS m/z(ESI):548.0[M+1]。
1H NMR(400MHz，DMSO-d 6):δ7.85-7.62(m，1H)，6.88(br，1H)，5.87-5.48(m，2H)，5.47-5.33(m，1H)，5.31-5.06(m，1H)，4.25-3.91(m，2H)，3.25(br，1H)，2.60-2.32(m，3H)，2.23(t，1H)，2.15-1.95(m，3H)，1.70-1.56(m，2H)，1.41-1.17(m，9H)，1.03(s，1H)，0.95-0.80(m，2H)。
实施例2-7
N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)-1-羟基环丁烷-1-甲酰胺7
Figure PCTCN2021082929-appb-000046
向1b(3.0mg，5.64μmol)中添加2mL乙醇和0.4mL N,N-二甲基甲酰胺，冰水浴冷却至0-5℃，滴加0.3mL N-甲基吗啡啉，搅拌至反应液变澄清。向反应液中依次加入1-羟基环丁烷甲酸7a(2.0mg，17.22μmol，供应商药石)，1-羟基苯并三唑(2.3mg，17.0μmol)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(3.2mg，16.7μmol)，加毕，在0-5℃搅拌反应10分钟。撤去冰水浴，常温搅拌2小时。反应液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物7(2.5mg，产率：83.1％)。
MS m/z(ESI):534.0[M+1]。
1H NMR(400MHz，DMSO-d 6):δ8.28(d，1H)，7.75(d，1H)，7.29(s，1H)，6.51(s，1H)，6.12(s，1H)，5.59-5.51(m，1H)，5.41(s，2H)，5.20-5.01(m，2H)，3.27-3.17(m，1H)，3.15-3.05(m，1H)，2.71-2.63(m，1H)，2.37(s，3H)，2.12-2.05(m，1H)，2.03-1.94(m，2H)，1.92-1.78(m，4H)，1.50-1.42(m，1H)，0.90-0.83(m，4H)。
实施例2-8
1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺8
Figure PCTCN2021082929-appb-000047
第一步：1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丙烷-1-羧酸苄酯8c
将1-羟基环丙烷-1-羧酸苄酯8a(104mg，0.54mmol；采用专利申请“US2005/20645”公开的方法制备而得)和2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲基乙酸酯8b(100mg，0.27mmol；采用专利申请“CN105829346A”公开的方法制备而得)加入反应瓶，加入5mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(61mg，0.54mmol)，撤去冰浴，升至室温搅拌10分钟，加入20mL冰水，用乙酸乙酯(5mL×2)和氯仿(5mL×5)萃取，合并有机相并浓缩。所得残余物溶于3mL 1,4-二氧六环中，加入0.6mL水，加入碳酸氢钠(27mg，0.32mmol)和氯甲酸-9-芴甲酯(70mg，0.27mmol)，室温搅拌1小时。加入20mL水，用乙酸乙酯(8mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用硅胶柱色谱法以展开剂体系B纯化所得残余物，得到标题产物8c(100mg，产率：73.6％)。
MS m/z(ESI):501.0[M+1]。
第二步：1-((2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲氧基)环丙烷-1-羧酸8d
将8c(50mg，0.10mmol)溶于3mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中， 加入钯碳(25mg，含量10％)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用四氢呋喃淋洗，滤液浓缩，得到标题产物8d(41mg，产率：100％)。
MS m/z(ESI):411.0[M+1]。
第三步：(9H-芴-9-基)甲基(2-(((1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基羰基)环丙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯8e
将1b(7mg，0.013mmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入8d(7mg，0.017mmol)的0.5mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(7mg，0.026mmol)，冰浴搅拌反应35分钟。加入10mL水，用乙酸乙酯(5mL×3)萃取，有机相用饱和氯化钠溶液(10mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩，用薄层层析以展开剂体系B纯化所得残余物，得到标题产物8e(8.5mg，产率78.0％)。
MS m/z(ESI):828.0[M+1]。
第四步：1-((2-氨基乙酰氨基)甲氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺8f
将8e(4mg，4.84μmol)溶于0.2mL二氯甲烷中，加入0.1mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入2mL甲苯减压浓缩，重复两次，加入3mL正己烷打浆，倾倒出上层正己烷，重复三次，减压浓缩得到粗品标题产物8f(2.9mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):606.0[M+1]。
第五步：1-(((S)-7-苄基-20-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)-3,6,9,12,15-五氧代-2,5,8,11,14-五氮杂二十烷基)氧基)-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)环丙烷-1-甲酰胺8
将粗品8f(2.9mg，4.84μmol)溶于0.5mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入(S)-2(-2-(-2-(6-(2,5-二氧代-1H-吡咯-1-基)已酰氨基)乙酰氨基)乙酰氨基)-3-苯基丙酸8g(2.7mg，5.80μmol，采用专利申请“EP2907824”公开的方法制备而得)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(2.7mg，9.67μmol)，冰浴搅拌反应30分钟，撤去冰浴，升至室温搅拌15分钟。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um 19*250mm；流动相:A-水(10mmol NH 4OAc)： B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩得到标题产物8(2mg，产率：39.0％)。
MS m/z(ESI):1060.0[M+1]。
1H NMR(400MHz，DMSO-d 6):δ9.01(d，1H)，8.77(t，1H)，8.21(t，1H)，8.08-7.92(m，2H)，7.73(d，1H)，7.28(s，1H)，7.24-7.07(m，4H)，6.98(s，1H)，6.50(s，1H)，5.61(q，1H)，5.40(s，2H)，5.32(t，1H)，5.12(q，2H)，4.62(t，1H)，4.52(t，1H)，4.40-4.32(m，1H)，3.73-3.47(m，8H)，3.16-3.04(m，2H)，2.89(dd，1H)，2.69-2.55(m，2H)，2.37-2.23(m，4H)，2.12-1.93(m，4H)，1.90-1.74(m，2H)，1.52-1.38(m，4H)，1.33-1.11(m，5H)，0.91-0.81(m，4H)。
实施例2-9
N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-A
N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-B
Figure PCTCN2021082929-appb-000048
Figure PCTCN2021082929-appb-000049
第一步：2-环丙基-2-羟基乙酸苄酯9a
将2a(1.3g，11.2mmol；采用专利申请“WO2013/106717”公开的方法制备而得)溶于50mL乙腈中，依次加入碳酸钾(6.18g，44.8mmol)，溴化苄(1.33mL，11.2mmol)和四丁基碘化铵(413mg，1.1mmol)。将反应液室温搅拌48小时，通过硅藻土过滤，滤饼用乙酸乙酯(10ml)淋洗，合并滤液减压浓缩，用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物9a(2g，产率：86.9％)。
第二步：10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸苄酯9b
将9a(120.9mg，0.586mmol)和8b(180mg，0.489mmol)加入反应瓶，加入4mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(109mg，0.98mmol)，撤去冰浴，升至室温搅拌40分钟，加入10mL冰水，用乙酸乙酯(20mL×2)和氯仿(10mL×5)萃取，合并有机相并浓缩。所得残余物溶于4mL二氧六环中，加入2mL水，加入碳酸氢钠(49.2mg，0.586mmol)和氯甲酸-9-芴甲酯(126mg，0.49mmol)，室温搅拌2小时。加入20mL水，用乙酸乙酯(10mL×3)萃取，有机相用饱和氯化钠溶液(20mL)洗涤，无水硫酸钠干燥，过滤，滤液减压浓缩。用硅胶柱色谱法以展开剂体系C纯化所得残余物，得到标题产物9b(48mg，产率：19％)。
MS m/z(ESI):515.0[M+1]。
第三步：10-环丙基-1-(9H-芴-9-基)-3,6-二氧代-2,9-二氧杂-4,7-二氮杂十一-11-酸9c
将9b(20mg，0.038mmol)溶于4.5mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(12mg，含量10％，干型)，氢气置换三次，室温搅拌反应1小时。反应液用硅藻土过滤，滤饼用乙酸乙酯淋洗，滤液浓缩，得到粗品标题产物9c(13mg)，产品不经纯化直接进行下一步反应。
MS m/z(ESI):424.9[M+1]。
第四步：(9H-芴-9-基)甲基(2-(((1-环丙基-2-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-2-氧代乙氧基)甲基)氨基)-2-氧代乙基)氨基甲酸酯9d
将1b(10mg，18.8μmol)加入反应瓶，加入1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，滴加一滴三乙胺，加入粗品9c(13mg，30.6μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(16.9mg，61.2μmol)，冰浴搅拌反应40分钟。加入10mL水，用乙酸乙酯(10mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(10mL×2)洗涤，有机相用无水硫酸钠干燥，过滤，滤液减压浓缩。用薄层层析以展开剂体系B纯化所得残余物，得到标题产物9d(19mg，产率：73.6％)。
MS m/z(ESI):842.1[M+1]。
第五步：2-((2-氨基乙酰氨基)甲氧基)-2-环丙基-N-((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)乙酰胺9e
将9d(19mg，22.6μmol)溶于2mL二氯甲烷中，加入1mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入1mL甲苯并减压浓缩，重复两次。往残余物中加入3mL正己烷打浆，静置后倾倒出上层清液，保留固体。将固体残余物减压浓缩，油泵拉干得到粗品标题产物9e(17mg)，产品不经纯化直接用于下一步反应。
MS m/z(ESI):638.0[M+18]。
第六步：N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-A
N-((2S,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰胺9-B
将粗品9e(13.9mg，22.4μmol)溶于0.6mL N,N-二甲基甲酰胺，氩气置换三 次，冰水浴降温至0-5℃，加入8g(21.2mg，44.8μmol)的0.3mL N,N-二甲基甲酰胺溶液，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(18.5mg，67.3μmol)，冰浴搅拌反应10分钟，撤去冰浴，升至室温搅拌1小时，反应生成化合物9。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5μm 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物(9-A：2.4mg，9-B：1.7mg)。
MS m/z(ESI):1074.4[M+1]。
单一构型化合物9-A(较短保留时间)：
UPLC分析:保留时间1.14分钟，纯度：85％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz，DMSO-d 6):δ8.60(t，1H)，8.51-8.49(d，1H)，8.32-8.24(m，1H)，8.13-8.02(m，2H)，8.02-7.96(m，1H)，7.82-7.75(m，1H)，7.31(s，1H)，7.26-7.15(m，4H)，6.99(s，1H)，6.55-6.48(m，1H)，5.65-5.54(m，1H)，5.41(s，2H)，5.35-5.15(m，3H)，4.74-4.62(m，1H)，4.54-4.40(m，2H)，3.76-3.64(m，4H)，3.62-3.48(m，2H)，3.20-3.07(m，2H)，3.04-2.94(m，1H)，2.80-2.62(m，1H)，2.45-2.30(m，3H)，2.25-2.15(m，2H)，2.15-2.04(m，2H)，1.93-1.78(m，2H)，1.52-1.39(m，3H)，1.34-1.12(m，5H)，0.87(t，3H)，0.64-0.38(m，4H)。
单一构型化合物9-B(较长保留时间)：
UPLC分析:保留时间1.16分钟，纯度：89％(色谱柱：ACQUITY UPLC BEHC18 1.7um 2.1*50mm，流动相：A-水(5mmol NH 4OAc)，B-乙腈)。
1H NMR(400MHz，DMSO-d 6):δ8.68-8.60(m，1H)，8.58-8.50(m，1H)，8.32-8.24(m，1H)，8.13-8.02(m，2H)，8.02-7.94(m，1H)，7.82-7.75(m，1H)，7.31(s，1H)，7.26-7.13(m，3H)，6.99(s，1H)，6.55-6.48(m，1H)，5.60-5.50(m，1H)，5.41(s，2H)，5.35-5.15(m，2H)，4.78-4.68(m，1H)，4.60-4.40(m，2H)，3.76-3.58(m，4H)，3.58-3.48(m，1H)，3.20-3.10(m，2H)，3.08-2.97(m，2H)，2.80-2.72(m，2H)，2.45-2.30(m，3H)，2.25-2.13(m，2H)，2.13-2.04(m，2H)，2.03-1.94(m，2H)，1.91-1.78(m，2H)，1.52-1.39(m，3H)，1.34-1.12(m，4H)，0.91-0.79(m，3H)，0.53-0.34(m，4H)。
三、ADC的制备
ADC药物载量分析
实验目的及原理
采用紫外分光光度法(UV-Vis)测定ADC载量。仪器：Thermo nanodrop2000紫外分光光度计。其原理是在某波长下ADC的总吸光值等于药物与单克隆抗体在该波长下吸光值的加和。
实验方法
将装有琥珀酸钠缓冲液的比色皿分别置于参比吸收池和样品测定吸收池中后，扣除溶剂空白后，再将装有供试品溶液的比色皿置于样品测定吸收池中，测定280nm和370nm处吸光度。
结果计算：
A 280nm＝ε mab-280bC mab+ε Drug-280bC Drug式(1)
ε Drug-280：药物在280nm平均摩尔消光系数5100；
C Drug：药物的浓度；
ε mab-280：单抗在280nm平均摩尔消光系数214600；
C mab：单抗的浓度；
b：光程长度为1cm。
同理可以得到样品在370nm下的总吸光值方程：
A 370nm＝ε mab-370bC mab+ε Drug-370bC Drug   式(2)
ε Drug-370：药物在370nm平均摩尔消光系数19000；
C Drug：药物的浓度；
ε mab-370：单抗在370nm消光系数为0；
C mab：单抗的浓度；
b：光程长度为1cm。
由式(1)和式(2)结合单克隆抗体和药物在两个检测波长下的消光系数和浓度数据可以计算出ADC中药物的载量。
药物载量＝C Drug/C mab。
实施例3抗体药物偶联的制备
(一)不同DAR值的抗体药物偶联h1702-DS-107-9-A的制备：
Figure PCTCN2021082929-appb-000050
ADC偶联物h1702-DS-107-9-A的制备过程如下：将人源化的抗体(h1702-DS-107)置于pH 6.5的0.05M的PBS缓冲水溶液中(抗体浓度10mg/mL)，加入相当于配置好的10mM的三(2-羧乙基)膦(TCEP)的水溶液(Innochem，CAS:51805-45-9，Cat#B45573)，置于37℃恒温振荡培养箱中，反应3小时。将上述反应液置于冰浴中降温至25℃。
将化合物9-A溶解于二甲基亚砜中，加入到上述反应液中，置于室温下的振荡器上，反应3小时后停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到目标抗体药物偶联分子。
通过对抗体和药物比例、反应量规模、以及其他条件的调整，可以获得不同DAR值(n)的抗体药物偶联物，优选DAR值为1-8，更优先为3-8，最优选3-7。
具体制备获得ADC化合物h1702-DS-107-9-A如下：
实施例3-1 ADC-1(DAR＝6.92)
在37℃条件下，向抗体h1702-DS-107的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，10.7mL，723nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，383μL，3830nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(12.2mg，11358nmol)溶解于600μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-1的PBS缓冲液(1.33mg/mL，70mL)，于4℃储存。
UV-Vis计算平均值：n＝6.92。
实施例3-2 ADC-2(DAR＝4.75)
在37℃条件下，向抗体h1702-DS-107的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.99mL，66.9nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，20.6μL，206nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(1.07mg，996nmol)溶解于28.5μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-2的PBS缓冲液(0.57mg/mL，12.9mL)，于4℃储存。
UV-Vis计算平均值：n＝4.75。
实施例3-3 ADC-3(DAR＝3.09)
在37℃条件下，向抗体h1702-DS-107的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，0.99mL，66.9nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，12.0μL，120nmol)，置于水浴振荡器，于37℃下振 荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(0.71mg，661nmol)溶解于18.9μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-3的PBS缓冲液(0.54mg/mL，13.9mL)，于4℃储存。
UV-Vis计算平均值：n＝3.09。
(二)不同DAR值的参考抗体药物偶联h1702-DS-9-A的制备：
Figure PCTCN2021082929-appb-000051
实施例3-4 ADC-4(DAR＝6.87)
在37℃条件下，向抗体h1702-DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，180mL，12.16μmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，6.20mL，62.0μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(195.9mg，182.4nmol)溶解于乙腈(3.6mL)和DMSO(1.8mL)的混合溶液中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液先后用50mM pH＝6.5PBS缓冲液(含有4％v/v乙腈和2％v/v DMSO)、10mM pH＝5.3琥珀酸缓冲液通过超滤膜包换液纯化，除去小分子，加入蔗糖至60mg/mL、吐温-20至0.2mg/mL，装瓶冻干后得到标题产物ADC-4的冻干粉样品(20mg/瓶)，于4℃储存。
UV-Vis计算平均值：n＝6.87。
实施例3-5 ADC-5(DAR＝4.80)
在37℃条件下，向抗体h1702-DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.20mL，81.1nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，25.1μL，251nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(1.30mg，1210nmol)溶解于34.7μl DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex  G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-5的PBS缓冲液(0.68mg/mL，13.6mL)，于4℃储存。
UV-Vis计算平均值：n＝4.80。
实施例3-6 ADC-6(DAR＝2.97)
在37℃条件下，向抗体h1702-DS的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.20mL，81.1nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，14.6μL，146nmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9-A(0.87mg，931nmol)溶解于23.2μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到标题产物ADC-6的PBS缓冲液(0.73mg/mL，12.2mL)，于4℃储存。
UV-Vis计算平均值：n＝2.97。
参照例
本披露将申请WO2020063676中的全部内容引入本申请。参考引用PCT/CN2019/107873第104页至105页，制备ADC-28，ADC-29。
其中ADC-28为FADC-26通式的示例性产物，结构如下：
Figure PCTCN2021082929-appb-000052
UV-Vis计算平均值：n＝7.46。
ADC-29为FADC-25通式的示例性产物，结构如下：
Figure PCTCN2021082929-appb-000053
UV-Vis计算平均值：n＝7.24。
所述的h1702-DS具有SEQ ID NO：1所示的重链和SEQ ID NO：2所示的轻链。
所述化合物20((参考引用PCT/CN2019/107873第88页实施例20制备)具有如下式所示的结构。
Figure PCTCN2021082929-appb-000054
生物学评价
体外活性生物学评价
测试例1：h1702-DS突变体的表达以及对B7H3结合活性的检测
为了消除h1702-DS潜在的T细胞表位，对h1702-DS进行了一系列突变体的设计、表达以及纯化，并用FACS的方法检测纯化后的突变体对CT26-B7H3细胞系的结合。
结果显示，本公开的突变体h1702-DS-107与CT26-B7H3细胞系存在明显结合信号。随后对该突变体进行梯度稀释后，进一步检测其与CT26-B7H3细胞系结合的EC 50。如表3所示。
表3.h1702-DS各突变体的表达量和与CT26-B7H3细胞系结合的EC 50
Figure PCTCN2021082929-appb-000055
Figure PCTCN2021082929-appb-000056
为了进一步验证突变体与肿瘤细胞系表面的B7H3以及猴B7H3的结合，进一步检测了突变体与A498细胞系以及CHOK1-cynoB7H3的结合。
结果如表4显示，这些突变体均可与A498细胞系以及CHOK1-cynoB7H3的结合。
表4.突变体与肿瘤细胞系表面的B7H3以及猴B7H3的结合
Figure PCTCN2021082929-appb-000057
测试例2：ADC的体外细胞增殖实验
测试例2-1：ADC对肿瘤细胞系增殖抑制实验
本实验通过检测细胞内ATP含量，根据IC 50大小评价本公开ADC对细胞增殖的抑制效果。
待测肿瘤细胞包括：Calu-6细胞(ATCC，Catalog#
Figure PCTCN2021082929-appb-000058
HTB-56 TM)，Detroit562细胞(ATCC，Catalog#
Figure PCTCN2021082929-appb-000059
CCL-138 TM)，和CHO-K1(ATCC，Catalog#
Figure PCTCN2021082929-appb-000060
CCL-61 TM)。
将待测ADC样品用PBS或DMSO以3倍倍比依次稀释成9个浓度(样品的起始浓度均为500nM)。将样品加入培养板中，将培养板在培养箱孵育6天(37℃，5％CO 2)。用CellTiter-Glo试剂(Promega，G7571)进行检测，在Victor3中读取化学发光信号值，数据使用GraphPad软件处理。测得的IC 50值见表5。
结果显示，h1702-DS-107和h1702-DS抗体对应的ADC，在DAR值接近的情况下，对各肿瘤细胞的增殖抑制效果接近，且抑制效果与DAR值正相关，DAR值越大，抑制效果越明显。
表5.不同ADC对细胞增殖的抑制效果
Figure PCTCN2021082929-appb-000061
测试例2-2：ADC对肿瘤细胞系的增殖抑制疗效与B7H3的表达水平呈正相关
为了进一步验证本公开中ADC对细胞的增殖抑制效果是否与B7H3的表达水平呈正相关性，分别在Detriot562肿瘤细胞系(Wildtype Detriot562)上进行了B7H3过表达和B7H3的敲除(得到Detriot562 B7H3-/-细胞系)。对于B7H3过表达的Detroit562细胞系，根据B7H3表达水平的高低，分别得到B7H3中表达细胞系(Detriot562 B7H3中)和B7H3高过表达细胞系(Detriot562 B7H3高)，如图1所示(FACS鉴定所用抗体来自sino biology，货号：11188-MM06-A)。
细胞增殖抑制试验的方法如测试例2-1中描述，结果如表6显示，抗体h1702-DS-107所对应的ADC-1与h1702-DS对应的ADC-4，对各Detroit562细胞系的增殖抑制疗效相近，且两个ADC对Detroit562的增殖抑制疗效与B7H3的表达水平呈现明显的正相关性，B7H3表达水平越高，增殖抑制效果越明显。
表6.ADC对不同B7H3表达水平Detroit562细胞系的增殖抑制
Figure PCTCN2021082929-appb-000062
体内活性生物学评价
测试例3：ADC对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效评价
1.测试方法:
实验用BALB/c-nude裸小鼠，雌性，6-7周，皮下接种人咽头癌胸水转移细胞Detroit 562细胞。接种细胞后第十天，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，1mpk以及3mpk两个剂量，或者10mpk单次给药，观察至D28，每周测2－3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b2
其中a、b分别表示长、宽。
相对体积(RTV)＝VT/V0
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。CRTV、TRTV分别为实验结束时的对照组(空白)及实验组的相对肿瘤体积。
2.测试对象：
ADC-1；
ADC-4；
对照组(PBS)。
3.抗体ADC的抑瘤效果如表7和图2所示：
受试ADC抑瘤率分别是：ADC-1 1mg/kg(1mpk)的抑瘤率达到50.68％；ADC-1 3mg/kg(3mpk)的抑瘤率达到78.21％(P<0.05)；ADC-1 10mg/kg(10mpk)单次给药的抑瘤率达到59.7％(P<0.05)；ADC-4 1mg/kg(1mpk)的抑瘤率达到55.91％；ADC-4，3mg/kg(3mpk)的抑瘤率达到72.47％(P<0.05)；ADC-4 10mg/kg(10mpk)单次给药的抑瘤率达到86.37％(P<0.001)。
给药过程中各组动物体重正常，提示ADC无明显毒副作用。
表7.给药抗体对荷瘤裸鼠Detroit 562移植瘤的疗效(D28)
Figure PCTCN2021082929-appb-000063
vs对照组：*p<0.05；**p<0.001。
测试例4：SD大鼠T1/2评价
SD大鼠4只，雌雄各半，12/12小时光/暗调节，温度24±3℃恒温，湿度50-60％，自由进食饮水。购自杰思捷实验动物有限公司。实验当天对SD大鼠分别尾静脉注射受试药物ADC，给药剂量为3mg/kg，注射体积5mL/kg。
取血时间点为：第1天给药后5分钟、8小时、1天、2天、4天、7天、10天、14天、21天、28天，于大鼠眼底静脉取血，每次300μL；收集的血样在室温下置放半小时至凝集，然后4℃下1000×g离心15分钟。收集上清，立即放置-80℃贮存。
用ELISA检测血清中的B7H3抗体和ADC浓度，分别检测完整ADC(intact ADC)和总抗体(Total antibody，与ADC偶联的抗体和血清中游离的抗体)的PK，检测方法分别为包被抗毒素抗体，或包被B7H3抗原，检测血清中B7H3抗体(Anti-Human IgG(HRP)mouse preadsorbed，abcam，ab97175)。
结果如表8所示，表明ADC-1和ADC-4在总抗体以及整ADC的半衰期非常接近。
表8.B7H3抗体ADC在SD大鼠中的T 1/2
大鼠半衰期(3mpk) ADC-4 ADC-1
总抗体 9.19±1.69 9.47±0.30
完整ADC 8.14±1.23 8.72±0.24
参照测试例：ADC对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效评价(WO2020063673，测试例8)
一、试验目的
本实验以BALB/c-nude裸小鼠为受试动物，评价ADC化合物对人咽头癌胸水转移细胞Detroit 562裸小鼠移植瘤的疗效。
二、受试药物及材料
1、受试药物
ADC-29(3mg/kg)；
ADC-28(3mg/kg)；
阴性对照ADC(3mg/kg)：非B7H3靶点抗体与化合物20偶联形成的配体毒素偶联物。
2、配制方法：均用PBS稀释配制。
3、试验动物
BALB/c-nude裸小鼠：购自常州卡文斯实验动物有限责任公司。
三、试验方法
实验用BALB/c-nude裸小鼠，雌性，6-7周，皮下接种人咽头癌胸水转移细胞Detroit 562细胞(ATCC，Catalog#
Figure PCTCN2021082929-appb-000064
CCL-138 TM)。接种细胞后第十天，将动物随机分组(D0)，每组8只，开始腹腔注射给药1次/周，共给药3次，每周测2－3次瘤体积和体重，记录数据。肿瘤体积(V)计算公式为：
V＝1/2×a×b 2
其中a、b分别表示长、宽。
相对体积(RTV)＝V T/V 0
抑瘤率(％)＝(C RTV-T RTV)/C RTV(％)
其中V 0、V T分别为实验开始时及实验结束时的肿瘤体积。C RTV、T RTV分别为实验结束时的对照组(阴性对照)及实验组的相对肿瘤体积。
四、试验结果
腹腔注射给药每周1次，共给药3次，观察至第28天时，受试ADC抑瘤率分别是：ADC-29 3mg/kg(3mpk)的抑瘤率达到72.27％(P<0.001)；ADC-28 3mg/kg(3mpk)的抑瘤率达到56.2％(P<0.001)。ADC-29均显示出比ADC-28更强的抗肿瘤疗效。
给药过程中各组动物体重正常，提示ADC无明显毒副作用。检测结果如表9所示。所检测抗体能够有效抑制荷瘤裸鼠中Detroit 562移植瘤的生长，并且呈现出剂量依赖性。
表9.给药抗体对荷瘤裸鼠Detroit 562移植瘤的疗效(D28)
Figure PCTCN2021082929-appb-000065

Claims (30)

  1. 一种抗体-药物偶联物或其药学上可接受的盐，其中所述抗体-药物偶联物是通式(Pc-L-Y-D)所示：
    Figure PCTCN2021082929-appb-100001
    其中：
    Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-和-S-(CR aR b) m-CR 1R 2-C(O)-；
    R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基和杂环基；或者，R a和R b与其相连接的碳原子一起形成环烷基或杂环基；
    R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
    或者，R a和R 2与其相连的碳原子一起形成环烷基或杂环基；
    m为0至4的整数；
    n为1至10，n是小数或整数；
    L为接头单元；
    Pc为抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段包含：
    重链可变区，其为SEQ ID NO：1中的重链可变区的变体，所述的变体包含一个或多个选自T16R、I28T、A33D、S99E、A100G、R101K、Y103F、A104G、A104S和A113T的氨基酸取代；优选地，所述的变体包含一个或多个选自T16R、I28T、Y103F和A113T的氨基酸取代；和
    轻链可变区，其为SEQ ID NO：2中的轻链可变区或其变体，所述的变体包含一个或两个选自R56K和S57G的氨基酸取代；优选地，所述的变体包含S57G的氨基酸取代。
  2. 根据权利要求1所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段的重链可变区包含选自以下任一组的氨基酸取代：
    a、T16R、Y103F和A113T；
    b、T16R、I28T、Y103F和A113T；
    c、T16R和A113T；和
    d、T16R、I28T和A113T。
  3. 根据权利要求1所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段包含：
    e、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和
    SEQ ID NO：2中的轻链可变区；
    f、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和
    SEQ ID NO：2中的轻链可变区；
    g、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和
    轻链可变区，其包含S57G的氨基酸取代；
    h、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和
    轻链可变区，其包含S57G的氨基酸取代；
    i、重链可变区，其包含T16R和A113T的氨基酸取代，和
    轻链可变区，其包含S57G的氨基酸取代；
    j、重链可变区，其包含T16R、I28T和A113T的氨基酸取代，和
    轻链可变区，其包含S57G的氨基酸取代。
  4. 根据权利要求1至3中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体或其抗原结合片段选自以下任一组：
    如SEQ ID NO：3所示的重链可变区，和
    如SEQ ID NO：4所示的轻链可变区；
    如SEQ ID NO：5所示的重链可变区，和
    如SEQ ID NO：6所示的轻链可变区；
    如SEQ ID NO：7所示的重链可变区，和
    如SEQ ID NO：8所示的轻链可变区；
    如SEQ ID NO：9所示的重链可变区，和
    如SEQ ID NO：10所示的轻链可变区；
    如SEQ ID NO：11所示的重链可变区，和
    如SEQ ID NO：12所示的轻链可变区；和
    如SEQ ID NO：13所示的重链可变区，和
    如SEQ ID NO：14所示的轻链可变区。
  5. 根据权利要求1至4中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体包含重链恒定区和轻链恒定区；
    所述重链恒定区来源于人IgG1、IgG2、IgG3或IgG4，
    优选地，所述重链恒定区源于人IgG1，更优选如SEQ ID NO：15所示的重链恒定区；
    优选地，所述轻链恒定区来源于人抗体κ、λ链；优选如SEQ ID NO：16所示的轻链恒定区。
  6. 根据权利要求1至5中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗B7H3抗体选自以下任一组：
    如SEQ ID NO：17所示的重链和SEQ ID NO：18所示的轻链；
    如SEQ ID NO：19所示的重链和SEQ ID NO：20所示的轻链；
    如SEQ ID NO：21所示的重链和SEQ ID NO：22所示的轻链；和
    如SEQ ID NO：23所示的重链和SEQ ID NO：24所示的轻链。
  7. 根据权利要求1至6中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述抗原结合片段选自Fab、Fab'、F(ab') 2、单链抗体(scFv)、二聚化的V区(双抗体)和二硫键稳定化的V区(dsFv)。
  8. 根据权利要求1至7中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中n为1至8，优选为3至7，n是小数或整数。
  9. 根据权利要求1至8中任一项所述的抗体-药物偶联物或其药学上可接受的盐，
    其中：
    Y为-O-(CR aR b) m-CR 1R 2-C(O)-；
    R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素和C 1-6烷基；
    R 1为卤代C 1-6烷基或C 3-6环烷基；
    R 2选自氢原子、卤代C 1-6烷基和C 3-6环烷基；
    或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
    m为0或1。
  10. 根据权利要求1至9中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中通式(Pc-L-Y-D)中，Y选自：
    Figure PCTCN2021082929-appb-100002
    其中Y的O端与接头单元L相连。
  11. 根据权利要求1至10中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中通式(Pc-L-Y-D)中，接头单元-L-为-L 1-L 2-L 3-L 4-，
    L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 3-W-C(O)-和-C(O)-W-C(O)-，其中W选自C 1-8烷基、C 1-8烷基-环烷基、和1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
    L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
    L 4选自-NR 5(CR 6R 7) t-、-C(O)NR 5、-C(O)NR 5(CH 2) t-和化学键，其中t为1至6的整数；
    R 3、R 4和R 5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
    R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基。
  12. 根据权利要求1至11中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中通式(Pc-L-Y-D)中，接头单元-L-为-L 1-L 2-L 3-L 4-，
    L 1为
    Figure PCTCN2021082929-appb-100003
    s 1为2至8的整数；
    L 2为化学键；
    L 3为四肽残基；优选地，L 3为SEQ ID NO：33所示的四肽残基；
    L 4为-NR 5(CR 6R 7)t-，其中R 5、R 6或R 7相同或不同，且各自独立地为氢原子 或烷基，t为1或2；
    其中所述的L 1端与Pc相连，L 4端与Y相连。
  13. 根据权利要求1至12中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述-L-为：
    Figure PCTCN2021082929-appb-100004
  14. 根据权利要求1至13中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其中所述--L-Y-是选自以下的任一个所示：
    Figure PCTCN2021082929-appb-100005
  15. 根据权利要求1至10中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
    Figure PCTCN2021082929-appb-100006
    其中，
    Pc为抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段包含：
    重链可变区，其为SEQ ID NO：1中的重链可变区的变体，所述的变体包含一个或多个选自T16R、Y103F、I28T、A33D、S99E、A100G、A104G、R101K、A104S和A113T的氨基酸取代；优选地，所述的变体包含一个或多个选自T16R、Y103F、I28T和A113T的氨基酸取代；和
    轻链可变区，其为SEQ ID NO：2中的轻链可变区或其变体，所述的变体包含一个或两个选自R56K和S57G的氨基酸取代；优选地，所述的变体包含S57G的氨基酸取代；
    m为0至4的整数；
    n为1至10，n是小数或整数；
    R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
    W选自C 1-8烷基、C 1-8烷基-环烷基和1至8个原子的直链杂烷基，所述杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
    L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸(F)、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
    R 5选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
    R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基。
  16. 根据权利要求1至12、15中任一项所述的抗体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L b-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
    Figure PCTCN2021082929-appb-100007
    其中：
    s 1为2至8的整数；
    Pc、R 1、R 2、R 5至R 7、m和n如权利要求15中所定义。
  17. 根据权利要求1至16中任一项所述的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物是选自以下的任一个所示：
    Figure PCTCN2021082929-appb-100008
    Figure PCTCN2021082929-appb-100009
    其中Pc和n如权利要求1中所定义。
  18. 根据权利要求1至17中任一项所述的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物为：
    Figure PCTCN2021082929-appb-100010
    其中：
    n为1至8，优选3至7，n是小数或整数；
    h1702-DS-107为抗B7H3抗体，其包含如SEQ ID NO：21所示的重链和如SEQ ID NO：22所示的轻链。
  19. 一种抗B7H3抗体或其抗原结合片段，其包含：
    重链可变区，其为SEQ ID NO：1中的重链可变区的变体，所述的变体包含一个或多个选自T16R、I28T、A33D、S99E、A100G、R101K、Y103F、A104G、A104S和A113T的氨基酸取代；优选地，所述的变体包含一个或多个选自T16R、I28T、Y103F和A113T的氨基酸取代；和
    轻链可变区，其为SEQ ID NO：2中的轻链可变区或其变体，所述的变体包含一个或两个选自R56K和S57G的氨基酸取代；优选地，所述的变体包含S57G的氨基酸取代。
  20. 根据权利要求19所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段重链可变区包含选自以下任一组的氨基酸取代：
    a、T16R、Y103F和A113T；
    b、T16R、I28T、Y103F和A113T；
    c、T16R和A113T；和
    d、T16R、I28T和A113T。
  21. 根据权利要求19或20所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段包含：
    e、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和
    SEQ ID NO：2中的轻链可变区；
    f、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和
    SEQ ID NO：2中的轻链可变区；
    g、重链可变区，其包含T16R、Y103F和A113T的氨基酸取代，和
    轻链可变区，其包含S57G的氨基酸取代；
    h、重链可变区，其包含T16R、I28T、Y103F和A113T的氨基酸取代，和
    轻链可变区，其包含S57G的氨基酸取代；
    i、重链可变区，其包含T16R和A113T的氨基酸取代，和
    轻链可变区，其包含S57G的氨基酸取代；
    j、重链可变区，其包含T16R、I28T和A113T的氨基酸取代，和
    轻链可变区，其包含S57G的氨基酸取代。
  22. 根据权利要求19至21中任一项所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段选自以下任一组：
    如SEQ ID NO：3所示的重链可变区，和
    如SEQ ID NO：4所示的轻链可变区；
    如SEQ ID NO：5所示的重链可变区，和
    如SEQ ID NO：6所示的轻链可变区；
    如SEQ ID NO：7所示的重链可变区，和
    如SEQ ID NO：8所示的轻链可变区；
    如SEQ ID NO：9所示的重链可变区，和
    如SEQ ID NO：10所示的轻链可变区；
    如SEQ ID NO：11所示的重链可变区，和
    如SEQ ID NO：12所示的轻链可变区；和
    如SEQ ID NO：13所示的重链可变区，和
    如SEQ ID NO：14所示的轻链可变区。
  23. 根据权利要求19至22中任一项所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体或其抗原结合片段包含重链恒定区和轻链恒定区；
    所述重链恒定区来源于人IgG1、IgG2、IgG3或IgG4，
    优选地，所述重链恒定区源于人IgG1，更优选如SEQ ID NO：15所示的重链恒定区；
    所述轻链恒定区来源于人抗体κ、λ链；优选如SEQ ID NO：16所示的轻链恒定区。
  24. 根据权利要求19至23中任一项所述的抗B7H3抗体或其抗原结合片段，其中所述抗B7H3抗体选自以下任一组：
    如SEQ ID NO：17所示的重链和SEQ ID NO：18所示的轻链；
    如SEQ ID NO：19所示的重链和SEQ ID NO：20所示的轻链；
    如SEQ ID NO：21所示的重链和SEQ ID NO：22所示的轻链；和
    如SEQ ID NO：23所示的重链和SEQ ID NO：24所示的轻链。
  25. 一种核酸分子，其编码权利要求19至24中任一项所述的抗B7H3抗体或其抗原结合片段。
  26. 一种宿主细胞，其包含如权利要求25所述的核酸分子。
  27. 一种制备如通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐的方法，其包括以下步骤：
    Figure PCTCN2021082929-appb-100011
    Pc’与通式(L a-Y-D)所示的化合物进行偶联反应，得到通式(Pc-L a-Y-D)所示的化合物；
    其中，
    Pc’为经还原后的Pc，
    Pc、n、m、W、L 2、L 3、R 1、R 2、R 5、R 6和R 7如权利要求15中所定义。
  28. 一种药物组合物，其包含：
    根据权利要求1至18中任一项所述的抗体-药物偶联物或其药学上可接受的盐，或根据权利要求19至24中任一项所述的抗B7H3抗体或其抗原结合片段，以及
    一种或多种药学上可接受的赋形剂、稀释剂或载体。
  29. 根据权利要求1至18中任一项所述的抗体-药物偶联物或其药学上可接受的盐、根据权利要求19至24中任一项所述的抗B7H3抗体或其抗原结合片段、或根据权利要求28所述的药物组合物在制备用于治疗B7H3介导的疾病或病症的药物中的用途。
  30. 根据权利要求1至18中任一项所述的抗体-药物偶联物或其药学上可接受的盐、根据权利要求19至24中任一项所述的抗B7H3抗体或其抗原结合片段、或根据权利要求28所述的药物组合物在制备治疗和/或预防肿瘤和癌症的药物中的用途，其中：
    优选地，所述肿瘤和癌症选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、库肯勃氏瘤、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、尿路上皮癌和梅克尔细胞癌；
    更优选的，所述淋巴瘤选自：何杰金淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤；
    更优选地，所述肺癌选自：非小细胞肺癌和小细胞肺癌；
    更优选地，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细 胞性白血病和髓样细胞白血病。
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023088235A1 (en) * 2021-11-16 2023-05-25 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
WO2023236949A1 (zh) * 2022-06-07 2023-12-14 映恩生物制药（苏州）有限公司 抗b7h3抗体-药物偶联物及其用途
WO2023237050A1 (en) * 2022-06-09 2023-12-14 Beigene, Ltd. Antibody drug conjugates
WO2023241663A1 (zh) * 2022-06-15 2023-12-21 上海翰森生物医药科技有限公司 一种含抗体药物偶联物的药物组合物及其用途
WO2024008102A1 (en) * 2022-07-05 2024-01-11 Wuxi Xdc (Shanghai) Co., Ltd. Linker for conjugation
WO2024022372A1 (zh) * 2022-07-27 2024-02-01 明慧医药（杭州）有限公司 抗体药物偶联物及其应用
WO2024109840A1 (zh) * 2022-11-22 2024-05-30 康诺亚生物医药科技（成都）有限公司 稠环类化合物及其偶联物和用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019024911A1 (zh) * 2017-08-04 2019-02-07 江苏恒瑞医药股份有限公司 B7h3抗体-药物偶联物及其医药用途
CN109563167A (zh) * 2016-06-08 2019-04-02 艾伯维公司 抗b7-h3抗体和抗体药物偶联物
CN110799542A (zh) * 2017-05-12 2020-02-14 纪念斯隆-凯特琳癌症中心 抗-b7h3抗体用于治疗中枢神经系统癌症的用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109563167A (zh) * 2016-06-08 2019-04-02 艾伯维公司 抗b7-h3抗体和抗体药物偶联物
CN110799542A (zh) * 2017-05-12 2020-02-14 纪念斯隆-凯特琳癌症中心 抗-b7h3抗体用于治疗中枢神经系统癌症的用途
WO2019024911A1 (zh) * 2017-08-04 2019-02-07 江苏恒瑞医药股份有限公司 B7h3抗体-药物偶联物及其医药用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OGITANI YUSUKE, ABE YUKI, IGUCHI TAKUMA, YAMAGUCHI JUNKO, TERAUCHI TOMOKO, KITAMURA MICHIKO, GOTO KOICHI, GOTO MAYUMI, OITATE MASA: "Wide application of a novel topoisomerase I inhibitor-based drug conjugation technology", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 26, no. 20, 1 October 2016 (2016-10-01), pages 5069 - 5072, XP055852728, ISSN: 0960-894X, DOI: 10.1016/j.bmcl.2016.08.082 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023088235A1 (en) * 2021-11-16 2023-05-25 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
US11814394B2 (en) 2021-11-16 2023-11-14 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
US11999748B2 (en) 2021-11-16 2024-06-04 Genequantum Healthcare (Suzhou) Co., Ltd. Exatecan derivatives, linker-payloads, and conjugates and thereof
WO2023236949A1 (zh) * 2022-06-07 2023-12-14 映恩生物制药（苏州）有限公司 抗b7h3抗体-药物偶联物及其用途
WO2023237050A1 (en) * 2022-06-09 2023-12-14 Beigene, Ltd. Antibody drug conjugates
WO2023241663A1 (zh) * 2022-06-15 2023-12-21 上海翰森生物医药科技有限公司 一种含抗体药物偶联物的药物组合物及其用途
WO2024008102A1 (en) * 2022-07-05 2024-01-11 Wuxi Xdc (Shanghai) Co., Ltd. Linker for conjugation
WO2024022372A1 (zh) * 2022-07-27 2024-02-01 明慧医药（杭州）有限公司 抗体药物偶联物及其应用
WO2024109840A1 (zh) * 2022-11-22 2024-05-30 康诺亚生物医药科技（成都）有限公司 稠环类化合物及其偶联物和用途

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