WO2021190564A1 - Antibody-drug conjugate and medical use thereof - Google Patents

Antibody-drug conjugate and medical use thereof Download PDF

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Publication number
WO2021190564A1
WO2021190564A1 PCT/CN2021/082735 CN2021082735W WO2021190564A1 WO 2021190564 A1 WO2021190564 A1 WO 2021190564A1 CN 2021082735 W CN2021082735 W CN 2021082735W WO 2021190564 A1 WO2021190564 A1 WO 2021190564A1
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Prior art keywords
antibody
seq
drug conjugate
antigen
pharmaceutically acceptable
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PCT/CN2021/082735
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French (fr)
Chinese (zh)
Inventor
花海清
包如迪
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上海翰森生物医药科技有限公司
江苏豪森药业集团有限公司
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Priority to CN202180021537.4A priority Critical patent/CN115279781A/en
Publication of WO2021190564A1 publication Critical patent/WO2021190564A1/en

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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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link

Definitions

  • the present invention belongs to the field of biomedicine. Specifically, the present invention relates to an anti-BCMA antibody conjugate and its medical use.
  • B cells are lymphocytes, which play an important role in adaptive humoral immunity and the production of antibodies that specifically recognize antigens.
  • the three subtypes of B cells are naive B cells, memory B cells, and plasma cells.
  • two or three fragments of DNA are selected from a genomic library and recombined to produce a combinatorial array of antibody variable domains, which are further changed by the variable domains encoded by B cells of different lineages This results in up to 109 unique B cell lineages that produce antibodies specific to unique targets.
  • Many diseases involve B cells. Malignant transformation of B cells leads to cancer, including some lymphomas such as multiple myeloma and Hodgkin’s lymphoma.
  • B cells including systemic lupus erythematosus (SLE) and IgA nephropathy.
  • SLE systemic lupus erythematosus
  • IgA nephropathy IgA nephropathy.
  • Cancers and autoimmune diseases involving B cells can be considered to be abnormal in B cell function, so a possible strategy to control such diseases is to use antibodies that target pathological B cells.
  • BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily, which is a non-glycosylated inner membrane receptor for the ligands BAFF (B cell activating factor) and APRIL (proliferation inducing ligand). BCMA and its corresponding ligands have different functions in regulating humoral immunity, B cell development and homeostasis. BCMA is expressed by tonsil memory B cells and germinal center B cells, and can be detected in the spleen, lymph nodes, thymus, adrenal glands and liver. The analysis of various B cell lines shows that the expression level of BCMA increases after maturation.
  • Antibodies against BCMA are described in, for example, Gras M-P. et al. Int Immunol. 7 (1995) 1093-1106, WO200124811, WO200124812, WO2010104949 and WO2012163805.
  • Antibodies against BCMA and their use for the treatment of lymphoma and multiple myeloma are described in, for example, WO2002066516 and WO2010104949.
  • WO2013154760 relates to a chimeric antigen receptor comprising a BCMA recognition part and a T-cell activation part.
  • an antigen binding protein capable of internalization that specifically binds to BCMA and inhibits the binding of BAFF and/or APRIL to BCMA is provided, and a conjugate comprising the antigen binding protein and a cytotoxic agent is provided.
  • the purpose of the present invention is to provide an anti-BCMA antibody drug conjugate, or a pharmaceutically acceptable salt or solvent compound thereof, which is an antibody drug conjugate represented by general formula (A) or a pharmaceutically acceptable Salt or solvent compound,
  • D is a cytotoxic drug
  • L 1 and L 2 are joint units
  • y is a number selected from 1-20;
  • Ab is an anti-BCMA antibody or an antigen-binding fragment thereof
  • the anti-BCMA antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and an antibody light chain variable region, wherein the antibody heavy chain variable region contains at least one selected from The HCDR shown in the sequence: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and the antibody light chain variable region contains at least one LCDR selected from the following sequence: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
  • the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof, wherein the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises: SEQ ID NO HCDR1 shown in: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5.
  • the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises : LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and The light chain variable region.
  • the heavy chain variable region of the anti-BCMA antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR2 shown in SEQ ID NO: 5
  • the light chain variable region includes: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8.
  • the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention wherein the CDR sequence of the anti-BCMA antibody or antigen-binding fragment thereof may be the same as the original CDR sequence.
  • 1-3 amino acid mutations that optimize antibody activity, antibody stability, or reduce immunogenicity occur.
  • the anti-BCMA antibody or its antigen-binding fragment is a murine antibody or its antigen-binding Fragments, chimeric antibodies or antigen-binding fragments thereof, human antibodies or antigen-binding fragments thereof, or humanized antibodies or antigen-binding fragments thereof.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises human IgG1 or its variants.
  • the Ab antibody or antigen-binding fragment thereof further comprises a human IgG1, IgG2 or IgG4 heavy chain constant region.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a human IgG1 heavy chain constant region with enhanced ADCC toxicity after amino acid mutation.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 22.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a human antibody ⁇
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody ⁇ chain.
  • the anti-BCMA antibody or antigen-binding fragment thereof further comprises SEQ ID NO: The light chain constant region shown at 23.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a sequence selected from Heavy chain variable region: SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
  • the anti-BCMA antibody or antigen-binding fragment thereof contains at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical heavy chain variable regions: SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the following sequences: SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
  • the anti-BCMA antibody or antigen-binding fragment thereof contains at least A light chain variable region of 70%, 75%, 80%, 85%, 90%, 95% or 99% identity: SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
  • the anti-BCMA antibody or antigen-binding fragment thereof contains a heavy chain shown in the following sequence: SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the anti-BCMA antibody or antigen-binding fragment thereof contains at least 80%, 85%, 90%, 95% or 99% identical heavy chains: SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
  • the anti-BCMA antibody or antigen-binding fragment thereof contains a light chain selected from the following sequences: SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
  • the anti-BCMA antibody or antigen-binding fragment thereof contains at least 80% compared with the following sequence: %, 85%, 90%, 95%, or 99% identical light chains: SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 9 and a light chain variable region shown in SEQ ID NO: 12.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region is selected from SEQ ID NO: 9, or SEQ ID NO: 9 has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity compared to SEQ ID NO: 9, and the light chain variable region is selected from SEQ ID NO: 12 or with SEQ ID NO: 12 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 10 and a light chain variable region shown in SEQ ID NO: 13.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region is selected from SEQ ID NO: 10, or SEQ ID NO: 10 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to SEQ ID NO: 10
  • the light chain variable region is selected from SEQ ID NO: 13 or with SEQ ID NO: 13 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.
  • the anti-BCMA antibody comprises a heavy chain variable region shown in SEQ ID NO: 11 and a light chain variable region shown in SEQ ID NO: 14.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region is selected from SEQ ID NO: 11, or SEQ ID NO: 11 has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity compared to SEQ ID NO: 11.
  • the light chain variable region is selected from SEQ ID NO: 14 or with SEQ ID NO: 14 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.
  • the anti-BCMA antibody comprises a heavy chain shown in SEQ ID NO: 15 and a light chain shown in SEQ ID NO: 18.
  • the anti-BCMA antibody comprising the heavy chain shown in SEQ ID NO: 15 and the light chain shown in SEQ ID NO: 18 is Ab1.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain
  • the heavy chain is selected from SEQ ID NO: 15, or compared with SEQ ID NO: 15 with at least 80%, 85%, 90%, 95%, or 99% identity
  • the light chain is selected from SEQ ID NO: 18, or has at least 80%, 85%, 90%, 95% compared with SEQ ID NO: 18 % Or 99% identity.
  • the anti-BCMA antibody comprises a heavy chain shown in SEQ ID NO: 16 and a light chain shown in SEQ ID NO: 19.
  • the anti-BCMA antibody comprising the heavy chain shown in SEQ ID NO: 16 and the light chain shown in SEQ ID NO: 19 is Ab2.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain
  • the heavy chain is selected from SEQ ID NO: 16, or has at least 80%, 85%, 90%, 95% or 99% identity
  • the light chain is selected from SEQ ID NO: 19, or has at least 80%, 85%, 90%, 95% compared with SEQ ID NO: 19 % Or 99% identity.
  • the anti-BCMA antibody comprises a heavy chain shown in SEQ ID NO: 17 and a light chain shown in SEQ ID NO: 20.
  • the anti-BCMA antibody comprising the heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20 is Ab3.
  • the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain
  • the heavy chain is selected from SEQ ID NO: 17, or has at least 80%, 85%, 90%, 95% or 99% identity
  • the light chain is selected from SEQ ID NO: 20, or has at least 80%, 85%, 90%, 95% compared with SEQ ID NO: 20 % Or 99% identity.
  • the cytotoxic drug is selected from toxins, chemotherapeutics, antibiotics, radioisotopes and nucleolytic enzymes.
  • the cytotoxic drug is selected from tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division; preferably DM1, DM3, DM4, camptothecin, SN-38, MMAF or MMAE; more preferably tubulin inhibitor MMAE or MMAF, or DNA topoisomerase inhibitor SN-38.
  • the cytotoxic drug is selected from:
  • the cytotoxic drug is selected from camptothecin derivatives, preferably exenotecan:
  • the cytotoxic drug is an exenotecan derivative, preferably, the exenotecan derivative is compound 2-A:
  • the antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is an antibody drug conjugate represented by general formula (I) or a pharmaceutically acceptable Salt or solvent compound,
  • L 1 and L 2 are joint units
  • y is an integer selected from 1-8, preferably a number from 2-4;
  • Ab is selected from anti-BCMA antibodies or antigen-binding fragments thereof as defined above.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is a compound represented by general formula (I) or a pharmaceutically acceptable salt or solvent thereof Compound,
  • L 1 and L 2 are joint units:
  • y is a number selected from 1-8, preferably a number from 2-8, further a number from 2-6, more preferably a number from 3-6;
  • Ab is selected from anti-BCMA antibodies or antigen-binding fragments thereof as defined above.
  • the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (IA) or a pharmaceutically acceptable salt or solvent compound thereof.
  • Acceptable salt or solvent compound :
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by general formula (IB) or a pharmaceutically acceptable salt or solvent compound thereof.
  • Acceptable salt or solvent compound is an antibody-drug conjugate represented by general formula (IB) or a pharmaceutically acceptable salt or solvent compound thereof.
  • the anti-BCMA antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as shown in the general formula (II) :
  • L 1 and L 2 are joint units
  • y is a number selected from 1-8, preferably a number from 2-4;
  • Ab is selected from BCMA antibodies or antigen-binding fragments thereof as defined above.
  • the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by general formula (II-1) Conjugate or its pharmaceutically acceptable salt or solvent compound:
  • the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (II-A) or Pharmaceutically acceptable salt or solvent compound:
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by general formula (II-B) or Its pharmaceutically acceptable salt or solvent compound:
  • the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by the general formula (III) or a pharmaceutically acceptable salt thereof or Solvent compound,
  • L 1 and L 2 are joint units
  • y is a number selected from 1-10, preferably a number from 2-8, more preferably a number from 4-8;
  • Ab is selected from BCMA antibodies or antigen-binding fragments thereof as defined above.
  • the L 1 is represented by general formula (B):
  • M 1 is -CR 1 R 2 -;
  • R 1 and R 2 are the same or different, and R 1 and R 2 are each independently selected from hydrogen, alkyl, halogen, hydroxyl, or amino;
  • n is selected from an integer of 0-5, preferably 1, 2 or 3.
  • L 1 and heterocyclic L 2 side is connected.
  • the L 2 is represented by the following general formula (C):
  • M 2 is -CR 4 R 5 -;
  • R 3 is selected from hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy and cycloalkyl:
  • R 4 and R 5 are the same or different, and R 4 and R 5 are each independently selected from hydrogen, alkyl, halogen, hydroxyl, or amino;
  • n is selected from an integer of 0-5, preferably 1, 2 or 3.
  • the S end of L 2 is connected to the joint unit L 1 .
  • the antibody drug conjugate or pharmaceutically acceptable salt or solvent compound thereof as shown in general formula (I), general formula (IA) or general formula (IB), It has a connecting unit L 1 defined by the above general formula (B) and a connecting unit L 2 defined by the above general formula (C).
  • the antibody drug conjugate represented by general formula (II-A) or a pharmaceutically acceptable salt or solvent compound thereof has the formula (C) defined above Connect the unit L 2 .
  • the L 2 is represented by general formula (D):
  • K 1 is s is selected from an integer from 2 to 8, further selected from an integer from 4 to 8, more preferably an integer from 4 to 6;
  • K 2 is -NR 1 (CH 2 CH 2 O) p CH 2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p CH 2 C(O)-, -S(CH 2 ) p C(O)- or a single bond, p is selected from an integer from 1 to 20, preferably an integer from 1 to 6;
  • R 1 is selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl;
  • K 3 is a tetrapeptide residue, preferably, the tetrapeptide residue is selected from two or more of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid , A peptide residue formed by an amino acid in aspartic acid; more preferably a tetrapeptide residue of GGFG;
  • K 4 is -NR 2 (CR 3 R 4 )t-, R 2 , R 3 or R 4 are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkane Base, t is selected from 1 or 2,
  • the K 1 end of the joint unit -L 2 - is connected to Ab, and the K 4 end is connected to L 1 .
  • K 1 is s is 5;
  • K 2 is the key
  • K 3 is the tetrapeptide residue of GGFG
  • K 4 is -NR 2 (CR 3 R 4 )t-, R 2 , R 3 or R 4 are each independently hydrogen, deuterium, hydroxyl, amino, C 1-6 alkyl, halogen, C 1-6 haloalkyl , C 1-6 deuterated alkyl and C 1-6 hydroxyalkyl, t is 1 or 2,
  • the K 1 end is connected to Ab, and the K 4 end is connected to L 1 .
  • L 1 is selected from bond, -O-(CR a R b ) m -CR 5 R 6 -C(O)-, -O-CR 5 R 6 -(CR a R b ) m -, -O-CR 5 R 6 -, -NH-(CR a R b ) m -CR 5 R 6 -C(O)- or -S-(CR a R b ) m -CR 5 R 6- C(O)-;
  • R a and R b are each independently selected from hydrogen, deuterium, halogen or alkyl
  • R 5 is haloalkyl or cycloalkyl
  • R 6 is selected from hydrogen, haloalkyl or cycloalkyl
  • R 5 and R 6 together with the carbon atom to which they are attached form a cycloalkyl group
  • n is selected from 0, 1, 2, 3, or 4.
  • the O end of L 1 is connected to the joint unit L 2 .
  • the L 1 is represented by the general formula (E):
  • R 5 is selected from haloalkyl or cycloalkyl
  • R 6 is selected from hydrogen, haloalkyl or cycloalkyl, or, R 5 and R 6 together with the carbon atom to which they are connected form a cycloalkyl
  • R 5 is selected from C 1-6 haloalkyl or C 3-6 cycloalkyl
  • R 6 is selected from hydrogen, C 1-6 haloalkyl or cycloalkyl, or R 5 and R 6 are connected to it The carbon atoms together form a C 3-6 cycloalkyl group
  • n is selected from an integer from 0 to 4,
  • the -L 2 -L 1 - is selected from the following structures:
  • K 2 is the key
  • K 3 is the tetrapeptide residue of GGFG
  • R 5 is haloalkyl or C 3-6 cycloalkyl
  • R 6 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl
  • R 5 and R 6 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • R 2 , R 3 or R 4 are each independently selected from hydrogen or alkyl
  • s is selected from an integer of 2 to 8; preferably, s is selected from an integer of 4, 5 or 6;
  • n is selected from an integer from 0 to 4.
  • -L 2 -L 1 - is selected from the following structures:
  • the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (IV) or a pharmaceutically acceptable salt or solvent compound thereof.
  • W is selected from a C 1-8 alkyl group, a C 1-8 alkyl-cycloalkyl group or a linear heteroalkyl group of 1 to 8 atoms, and the heteroalkyl group contains 1 to 3 selected from N, O or S
  • the heteroatoms optionally, wherein the C 1-8 alkyl, cycloalkyl and linear heteroalkyl are each independently further substituted by halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, Substituted by one or more substituents of deuterated alkyl, alkoxy and cycloalkyl;
  • K 2 is selected from -NR 1 (CH 2 CH 2 O) p1 CH 2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p1 CH 2 C(O)-, -S(CH 2 ) p1 C(O) -or bond, R 1 is selected from hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl, and p 1 is selected from an integer from 1 to 20;
  • K 3 is a peptide residue composed of 2 to 7 amino acids.
  • Amino acids can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available point of attachment, and the substituent is one Or more are independently selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • R 2 is selected from hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;
  • R 3 and R 4 are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;
  • R 5 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 6 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 5 and R 6 together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;
  • n is selected from an integer from 0 to 4.
  • y is a number selected from 1-10, y is a decimal or integer;
  • Ab is selected from anti-BCMA antibodies or antigen-binding fragments thereof as defined above.
  • the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (IV-A) or Pharmaceutically acceptable salt or solvent compound:
  • the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (IV-A) or A pharmaceutically acceptable salt or solvent compound, wherein:
  • R 5 is selected from haloalkyl or C 3-6 cycloalkyl
  • R 6 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl
  • R 5 and R 6 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group
  • R 2 , R 3 or R 4 are each independently selected from hydrogen or alkyl
  • s is selected from an integer from 2 to 8;
  • n is selected from an integer from 0 to 4.
  • the antibody drug conjugate or pharmaceutically acceptable salt or solvent compound thereof according to the present invention is selected from the following structures:
  • the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is selected from the following compounds,
  • the antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is selected from the following compounds:
  • y is selected from 2-10, preferably 4-8, more preferably 6-8, further preferably 7-8, most preferably 6 or 8, or y is selected from 1-10, preferably 2-10, It is further a number of 2-6, more preferably a number of 3-6, and most preferably 6.
  • the present invention relates to a method for preparing an antibody drug conjugate represented by general formula (IV) or a pharmaceutically acceptable salt or solvate thereof, which comprises the following steps:
  • Ab is an anti-BCMA antibody or antigen-binding fragment thereof as defined above;
  • W, K 2 , K 3 , R 2 to R 6 , m and y are as defined in the general formula (IV).
  • the general formula (F) is a compound represented by the general formula (F-1):
  • K 2 , K 3 , R 2 to R 6 , s and m are as defined in the aforementioned -L 2 -L 1 -.
  • the compound represented by general formula (F) or general formula (F-1) is selected from:
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above-mentioned antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof, and one or more pharmaceutically acceptable excipients, diluents, or Carrier.
  • the present invention provides a medical use.
  • the present invention relates to an anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate or a pharmaceutical composition thereof for use in treatment Or to prevent BCMA-mediated diseases or conditions.
  • the present invention provides a medical use.
  • the present invention relates to an anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate or a pharmaceutical composition thereof for preparing Use in medicine for treating or preventing BCMA-mediated diseases or conditions.
  • the BCMA-mediated disease or disorder is cancer or an autoimmune disease, wherein the cancer is preferably a cancer expressing BCMA, more preferably lymphoma, leukemia or myeloma, and most preferably multiple Myeloma; the autoimmune disease is selected from lupus erythematosus, IgA nephropathy and rheumatoid arthritis.
  • the antibody-drug conjugate and its pharmaceutically acceptable salt or solvent compound of the present invention have obvious tumor inhibitory effects in vitro and in vivo, have low toxicity to normal cells or tissues, have good safety, and are resistant to related ligands and soluble BCMA in serum. It has a good competitive inhibition effect; it has good drug stability in vitro, which is convenient for long-term storage and transportation; at the same time, it has good metabolic activity in the body, a long time in vivo, and has a broad clinical application prospect.
  • antibody in the present invention refers to an immunoglobulin, which 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, so their antigenicity is also different.
  • immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE.
  • the corresponding heavy chains are ⁇ chain, ⁇ chain, and ⁇ chain. , ⁇ chain and ⁇ chain.
  • 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 antibody light chain variable region of the present invention may further include a light chain constant region, and the light chain constant region includes human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or its variants. body.
  • variable region The sequence of about 110 amino acids near the N-terminus of antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. 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 (VL) and heavy chain variable region (VH) 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 VL and VH regions of the antibody or antigen-binding fragment of the present invention comply with the known Kabat numbering rules and Kabat or ABM definition rules (http://bioinf.org.uk/abs /).
  • APC antigen presenting cell
  • DC dendritic cells
  • PBMC peripheral blood mononuclear cells
  • monocytes B lymphoblasts
  • monocyte-derived dendritic cells monocyte-derived dendritic cells
  • antigen presentation refers to the process by which APC captures antigens and enables them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.
  • BCMA includes any variant or isoform of BCMA that is naturally expressed by the cell.
  • the antibodies of the present invention can cross-react with BCMA derived from non-human species.
  • the antibody may also be specific for human BCMA, and may not show cross-reactivity with other species.
  • BCMA or any variants or isoforms thereof can be isolated from the cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques commonly used in the art and those described herein.
  • the anti-BCMA antibody targets human BCMA with a normal glycosylation pattern.
  • recombinant human antibody includes human antibodies prepared, expressed, created or isolated by recombinant methods, the techniques and methods involved are well known in the art, such as:
  • Antibodies isolated from host cells transformed to express antibodies such as transfectomas;
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
  • murine antibody in the present invention refers to a monoclonal antibody to human BCMA prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with BCMA antigen, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.
  • the murine BCMA antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1 and IgG2. , IgG3 or IgG4 or its variant heavy chain constant region.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • the human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies”) .
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody)
  • CDR-grafted antibody refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework.
  • Humanized antibodies can overcome the shortcomings of strong immune responses induced by chimeric antibodies that carry a large amount of mouse protein components.
  • the variable region of the human antibody can be subjected to minimal reverse mutation to maintain activity.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and change the mouse variable region gene.
  • the region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations to enhance ADCC (antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1 heavy chain constant region.
  • ADCC antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • antigen-binding fragment refers to antigen-binding fragments and antibody analogs of antibodies, which usually include at least part of the antigen-binding region or variable region (for example, one or more CDRs) of a parental antibody.
  • Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a mole basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target.
  • antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies.
  • Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.
  • the "Fab fragment” consists of the CH1 and variable regions of one light chain and one heavy chain.
  • the heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.
  • the "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.
  • the "Fab' fragment” contains a light chain and a portion of a heavy chain that contains the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, so that it can be between the two heavy chains of the two Fab' fragments The formation of interchain disulfide bonds to form F(ab')2 molecules.
  • the "F(ab')2 fragment” contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.
  • the "Fv region” contains variable regions from both the heavy and light chains, but lacks the constant region.
  • multispecific antibody is used in its broadest sense to encompass antibodies with polyepitope specificity.
  • These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitope specificity; having two or more VL and VH regions Antibodies, each VH-VL unit binds to a different target or a different epitope of the same target; an antibody with two or more single variable regions, each single variable region with a different target Or binding to different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked together Wait.
  • single-chain antibody is a single-chain recombinant protein formed by connecting the heavy chain variable region VH and the light chain variable region VL of an antibody through a connecting peptide. It is the smallest antibody fragment with a complete antigen-binding site.
  • domain antibody fragment is an immunoglobulin fragment with immunological functions that only contains a heavy chain variable region or a light chain variable region chain.
  • two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment.
  • the two VH regions of the bivalent domain antibody fragment can target the same or different antigens.
  • binding with BCMA in the present invention refers to the ability to interact with human BCMA.
  • antigen-binding site refers to a three-dimensional site recognized by the antibody or antigen-binding fragment of the present invention.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent.
  • Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
  • specific binding and “selective binding” as used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen.
  • the antibody is approximately 10 -7 M or even lower than the equilibrium dissociation smaller dissociation constant ( K D ) binds to a predetermined antigen, and its binding affinity to the predetermined antigen is at least twice its binding affinity to non-specific antigens (such as BSA, etc.) other than the predetermined antigen or closely related antigens.
  • K D equilibrium dissociation smaller dissociation constant
  • the term "antibody that recognizes an antigen” can be used interchangeably with the term “antibody that specifically binds” herein.
  • cross-reactivity refers to the ability of the antibodies of the present invention to bind to BCMA from different species.
  • the antibody of the present invention that binds to human BCMA can also bind to BCMA of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express BCMA.
  • binding assays such as SPR and ELISA
  • Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • Inhibition or “blocking” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with anti-BCMA antibodies compared to ligands not contacted with anti-BCMA antibodies.
  • inhibition of growth is intended to include any measurable decrease in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of the immune response to a specific antigen.
  • induction for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
  • ADCC namely antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • Fc receptors are directly killed by recognizing the Fc segment of antibodies and are coated with antibodies.
  • the target cell The ADCC effect function of the antibody can be enhanced or reduced by modifying the Fc segment of IgG.
  • the modification refers to mutations in the constant region of the heavy chain of the antibody.
  • mice can be immunized with human BCMA or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImmunoGeneTics (IMGT), or from the immunoglobulin journal, 2001ISBN012441351.
  • the engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods.
  • the cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can be stably transfected into CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the FC region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • 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.
  • the antibody of the present invention refers to a monoclonal antibody.
  • the monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage cloned cell line.
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells.
  • administering “administration” and “treatment” also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as containing any one of the antibodies of the present invention, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered in the treated patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent.
  • the amount of the therapeutic agent effective to alleviate the symptoms of any particular disease can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient.
  • any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms it can be evaluated whether the symptoms of the disease have been alleviated.
  • the embodiments of the present invention may be ineffective in alleviating the symptoms of the target disease that each patient has, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
  • naturally occurring refers to the fact that the object can be found in nature.
  • polypeptide sequences or polynucleotide sequences that exist in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified artificially in the laboratory are naturally occurring.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of medical conditions.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • Exogenous refers to substances that are produced outside organisms, cells, or humans according to the background.
  • Endogenous refers to a substance produced in a cell, organism, or human body according to the background.
  • Identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the maximum percent identity.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably, and all such names include their progeny. Therefore, the words “transformant” and “transformed cell” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. "Optional” or “optionally” means that the event or environment described later can but does not have to occur, and the description includes the occasion where the event or environment occurs or does not occur. For example, “optionally comprising 1-3 antibody heavy chain variable regions” means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.
  • “Pharmaceutical composition” means containing one or more antibodies or antigen-binding fragments thereof described herein, and other components such as physiological/pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • “Pharmaceutically acceptable salt” refers to the salt of the antibody-drug conjugate of the present invention. Such salt is safe and effective when used in mammals, and has due biological activity.
  • the antibody-drug conjugate of the present invention 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, hydrogen sulfate Salt, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, Maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
  • solvent compound refers to the antibody-drug conjugate compound of the present invention and one or more solvent molecules to form a pharmaceutically acceptable solvent compound.
  • solvent molecules include: water, ethanol, acetonitrile, isopropanol, Ethyl acetate.
  • Cytotoxic drug when used in the present invention refers to a substance that inhibits the function of cells and/or causes cell death or destruction.
  • Tubulin inhibitor refers to a class of compounds that interfere with the process of cell mitosis by inhibiting the polymerization of tubulin or promoting the aggregation of tubulin, thereby exerting an anti-tumor effect.
  • Non-limiting examples thereof include: maytansinoids, calicheamicin, taxanes, vincristine, colchicine, ceratopsin/auristatin/monomethyl auristatin E (MMAE) /Monomethyl auristatin F (MMAF).
  • Linker refers to a chemical moiety that contains a covalent bond or chain of atoms that is the covalent attachment of an antibody to a drug.
  • Non-limiting examples of linkers include: arylene, heteroarylene, PEG, polymethyleneoxy, succinate, succinamide, diglycolate, malonate, and caproamide.
  • DAR Drug Load
  • y that is, the average number of cytotoxic drugs per antibody in formula (A).
  • the drug loading range in the present invention can be 1-20 cytotoxic drugs (D) per antibody.
  • the antibody-drug conjugate of the general formula (A) is a collection of antibodies conjugated with a certain range (1-20) of cytotoxic drugs.
  • the drug load (DAR) in the antibody-drug conjugate from the coupling reaction can be characterized by conventional means, such as mass spectrometry, HPLC and ELISA. By these means, the quantitative distribution of the antibody-drug conjugate on the y value can be determined.
  • MC 6-maleimidohexanoyl
  • MMAF is a variant of monomethyl auristatin E, which has phenylalanine at the C-terminus of the molecule (MW731.5)
  • the following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention.
  • the experimental methods that do not specify specific conditions in the examples of the present invention usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer.
  • the reagents without specific sources are the conventional reagents purchased on the market.
  • the indirect ELISA method as described in Example 3 was used for the immunized mouse serum to evaluate the serum titer and the ability to bind to cell surface antigens.
  • the detection of the titer (larger than 100,000 times dilution) determines the start of cell fusion.
  • the immunized mice with strong serum titer, affinity and FACS binding were selected for one final immunization and then sacrificed.
  • the spleen cells and SP2/0 myeloma cells were fused and plated to obtain hybridomas.
  • the target hybridomas were screened by indirect ELISA, and The strain was established as a monoclonal cell strain by the limiting dilution method.
  • the obtained positive antibody strains are further screened using indirect ELISA to select hybridomas that bind to the recombinant protein.
  • the logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara #2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-primer set (Novagen, TB326 Rev. B 0503) and then sequenced, and finally the sequence of the mouse antibody M1 was obtained.
  • the heavy chain and light chain variable region sequences of murine monoclonal antibody M1 are as follows:
  • BCMA His protein (Sino Biological Inc., cat#10620-H08H to a concentration of 1 ⁇ g/ml with pH7.4 PBS, add 100 ⁇ l/well to a 96-well high-affinity ELISA plate, and incubate overnight at 4°C in a refrigerator (16-20 hours). After washing the plate 4 times with PBST (pH7.4 PBS containing 0.05% Tween-20), add 150 ⁇ l/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate at room temperature for 1 Blocking was performed in hours. After the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer.
  • BSA bovine serum albumin
  • PE-GAM goat anti-mouse monoclonal antibody
  • PE-GAH goat anti-human monoclonal antibody
  • the humanization of the murine anti-human BCMA monoclonal antibody is carried out according to the methods published in many documents in the field.
  • a human constant domain is used instead of the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the identity of the murine antibody and the human antibody.
  • the present invention humanizes the murine antibody M1.
  • the sequence of the heavy and light chain variable region is compared with the human antibody germline database to obtain a human germline template with high identity.
  • the CDR region of the murine antibody M1 was transplanted to the selected corresponding humanized template. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH are back-mutated, and the CDR region Chemically unstable amino acid residues were optimized. After expression testing and comparison of the number of back mutations, the sequence of the humanized heavy chain variable region HCVR was selected and designed. The sequence is as follows:
  • the sequence of the humanized light chain variable region LCVR was selected and designed, the sequence is as follows:
  • Heavy chain and light chain constant region sequences Connect the designed heavy chain and light chain variable region sequences with the human IgG1 heavy chain and human antibody light chain constant region sequences, respectively.
  • Exemplary heavy chain and light chain constant region sequences are as follows:
  • CDNA fragments were synthesized according to the amino acid sequences of the light and heavy chains of the above humanized antibodies, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20).
  • the expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days.
  • NCI-H929 ATCC deposit number CRL-9068
  • NCI-H929 cells were trypsinized (washed with PBS, 37°C for about 2 minutes), collected and resuspended in pre-cooled FACS buffer, adjusting the cell concentration to 1 ⁇ 10 6 cells/mL.
  • Take the EP tube add 1 mL of cell suspension, centrifuge at 1500 rpm for 5 minutes and remove the supernatant. Add 1 mL of the prepared antibody to be tested to resuspend the cells. The final concentration of the antibody is 20 ⁇ g/ml.
  • the antibody of the present invention has cell affinity activity and endocytosis activity, making the antibody of the present invention suitable for coupling with drugs to form antibody-drug conjugates for the treatment of BCMA-mediated diseases.
  • the coupling process is shown in the following formula, where Ab stands for Ab2 or Ab3 antibody:
  • the compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile, and the 2f solution (concentration 6.17 mg/mL, 3.0 mL) was added to 25 After shaking and reacting at °C for 4 hours, the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5), and filtered under sterile conditions with a filter to obtain the product Ab-MC -MMAF.
  • the DAR average value y of the product ADC2 was determined by HIC-HPLC to be 4, and the antibody-drug conjugate PBS buffer (3.7 mg/mL, 4.7 mL) was refrigerated at 4°C.
  • the product ADC3 was prepared by the above method.
  • the average DAR value y of the product ADC3 (Ab3-MC-MMAF) determined by HIC-HPLC was 4.1, and the antibody-drug conjugate PBS buffer (3.5 mg/mL, 5.0 mL) was refrigerated at 4°C.
  • S-(3-aldehyde propyl) thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile solution for later use.
  • An aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was shaken and reacted at 25° C. for 2 hours.
  • the compound MC-SN-38 (1.3mg, 1.2moL) was dissolved in 0.3ml of acetonitrile, added to the 2h solution (concentration 6.2mg/mL, 3.0mL), and the reaction was shaken at 25°C for 4 hours.
  • the reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5)
  • the product was filtered under sterile conditions with a filter to obtain the product Ab-SN-38 antibody-drug coupling PBS buffer (3.7mg/mL, 4.7mL) of the substance, refrigerated at 4°C.
  • the average value y was determined by the ultraviolet method.
  • the average drug load y CDrug/Cmab.
  • the DAR average value y of the Ab2-SN-38 antibody-drug conjugate determined by the above method is 3.9.
  • 2a (2g, 17.2mmol) was dissolved in 75mL of acetonitrile, and potassium carbonate (9.27g, 67.2mmol), benzyl bromide (20mL, 167.2mmol) and tetrabutylammonium iodide (620mg, 1.68mmol) were added in sequence.
  • the reaction solution was stirred at room temperature for 48 hours, filtered through celite, the filter cake was rinsed with ethyl acetate (20ml), the combined filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with a developing solvent system C to obtain Product 5a (3.2g, yield: 90.1%).
  • reaction solution was filtered with diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude product 5c 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo- 2,9-dioxa-4,7-diazaundec-11-acid (20mg), the product was directly subjected to the next reaction without purification.
  • 5d (30 mg, 35.7 ⁇ mol) was dissolved in 3 mL of dichloromethane, 1.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours.
  • the reaction solution was concentrated under reduced pressure, 1.5 mL of toluene was added and concentrated under reduced pressure, repeated twice.
  • the crude product 5e (20mg, 32.3 ⁇ mol) was dissolved in 1mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5°C in an ice water bath, and 4g (31.8mg, 67.3 ⁇ mol) was added.
  • 4g 31.8mg, 67.3 ⁇ mol
  • 0.5mL N,N-dimethylformamide solution add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine salt ( 27.8 mg, 94.3 ⁇ mol)
  • the reaction was stirred in an ice bath for 10 minutes, the ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour to produce compound 5.
  • 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), collect the corresponding components and concentrate under reduced pressure to obtain products 5-A and 5-B (3.6 mg, 2.6 mg).
  • the average value y was determined by the ultraviolet method. After placing the cuvette containing sodium succinate buffer in the reference absorption cell and the sample determination absorption cell, after deducting the solvent blank, place the cuvette containing the test solution in the sample determination absorption cell Measure the absorbance at 280nm and 370nm.
  • the average drug load y CDrug/Cmab.
  • NCI-H929 cells (ATCC deposit number CRL-9068) were used for evaluation. Collect NCI-H929 cells, after centrifugal counting, adjust the cell density to 0.44 ⁇ 10 6 cells/mL with complete medium, and spread them in the middle 60 wells of a white 96-well plate, each with 90 ⁇ L, the number of cells is 40,000, and add 100 ⁇ L to the remaining side holes PBS, the cell plate was placed in a 37°C, 5% CO2 incubator overnight.
  • the experimental results are shown in Table 10.
  • Tumor inhibition rate was calculated by measuring the tumor volume.
  • Tumor inhibition rate TGI 100%-(Tumor volume of the administration group on the 14th day-Tumor volume of the administration group on the 0th day)/(Tumor volume of the control group on the 14th day-Tumor volume of the control group on the 0th day).
  • the BCMA high expression level cell line NCI-H929 (ATCC deposit number CRL-9068) and the BCMA low expression level cell line RPMI-8226 (ATCC, cat :CCL-155) for evaluation. Collect NCI-H929 and RPMI-8226 cells. After centrifugation and count, adjust the cell density to 1 ⁇ 10 5 cells/mL with complete medium, and spread them in the middle 60 wells of a white 96-well plate. Add 100 ⁇ l/well DPBS to the hole and edge hole, and place the cell plate in a 37°C, 5% CO 2 incubator overnight.
  • the experimental results show that the in vitro killing activity of BCMA-ADC on tumor cells is positively correlated with the expression level of BCMA on the cell surface.
  • the killing effect on NCI-H929 is significantly stronger than that on RPMI-8226; as the number of coupled small molecule toxins increases , The killing activity of tumor cells will increase accordingly.
  • the BALB/c mouse model was used to evaluate the drug metabolism of the anti-BCMA drug conjugate BCMA-ADC in mice.
  • Human BALB/c transgenic mice (Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.) with an average weight of 18-22g and 18-22 weeks of age were randomly divided into 2 groups, each with 3 animals, and the BCMA body drug was tested The conjugates were administered in a single dose of 10 mpk, IV, and PBS was used as a negative control group.
  • ADC1-2 Integrating parameters such as half-life t 1/2 , exposure, and peak blood concentration, ADC1-2 shows good metabolic activity in the body.
  • the BCMA high-expression cell line NCI- H929 (ATCC deposit number CRL-9068) was evaluated.
  • NCI-H929 cells after centrifugal counting, adjust the cell density to 2 ⁇ 10 5 cells/mL with complete medium, and spread them in the middle of the white 96-well plate in 60 wells, 50 ⁇ L per well, the cell number is 10,000 cells/well, edge Add 100 ⁇ L/well DPBS to the wells, and place the cell plate in a 37°C, 5% CO 2 incubator overnight.
  • the antibody-drug conjugate working solution was prepared in a 96-well V-bottom plate with complete medium, so that the exenotecan derivative compound (5-A) and ADC were diluted in equal molar amounts, and the maximum working concentration was 300nM, 5-fold dilution, 9 concentrations, after preparation, add to a white 96-well plate, 100 ⁇ L per well, two replicate wells, put the cell plate in a 37°C, 5% CO 2 incubator and continue to incubate for 72 hours.
  • the experimental results show that: the antibody-drug conjugate of the present invention
  • the in vitro cytotoxic killing activity on tumor cells is obviously stronger than the killing effect of the small molecule toxin compound (5-A) on NCI-H929.
  • the reaction solution was concentrated under reduced pressure, and the obtained crude compound 2-C was purified by high performance liquid chromatography (Separation conditions: Column: XBridge Prep C18 OBD 5um 19*250mm; Mobile phase: A-water (10mmol NH4OAc), 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).
  • the compounds 2-A and 2-B were tested for their inhibitory activity on the in vitro proliferation of U87MG cells (Cell Bank of Chinese Academy of Sciences, Catalog#TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, catalog number HTB-30).
  • the cells were treated in vitro with different concentrations of the compound, and after 6 days of culture, the proliferation of the cells was detected with CTG (Luminescent Cell Viability Assay, Promega, catalog number: G7573) reagent, and the in vitro activity of the compound was evaluated according to the IC50 value.
  • U87MG and SK-BR-3 cells were cultured with 10% FBS in EMEM medium (GE, article number SH30024.01) and McCoy's 5A medium (Gibco, article number 16600-108) containing 10% FBS, respectively.
  • the compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a storage solution with an initial concentration of 10 mM.
  • DMSO dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.
  • the initial concentration of the small molecule compound is 500nM, and the dispensing method is as follows:
  • Adding samples Add 20 ⁇ l of the tested samples of different concentrations to the culture plate, and each sample has two duplicate wells.
  • the culture plate was incubated in an incubator for 6 days (37°C, 5% CO 2 ).
  • Color development Take out the 96-well cell culture plate, add 90 ⁇ l CTG solution to each well, and incubate at room temperature for 10 minutes.
  • Plate reading Take out the 96-well cell culture plate, place it in a microplate reader (BMG labtech, PHERAstar FS), and measure chemiluminescence with the microplate reader.

Abstract

Provided are an anti-BCMA antibody-drug conjugate and medical use thereof. Further, provided are an antibody-drug conjugate comprising an anti-BCMA antibody or an antigen-binding fragment thereof, or a pharmaceutically acceptable salt or a solvate compound thereof, use thereof in preparation of a medicine for treating a BCMA-mediated disease or condition, and use thereof in detection and diagnosis of diseases.

Description

抗体药物偶联物及其医药用途Antibody drug conjugate and its medical use 技术领域Technical field
本发明属于生物医药领域，具体地说，本发明涉及抗BCMA抗体偶联物及其医药用途。The present invention belongs to the field of biomedicine. Specifically, the present invention relates to an anti-BCMA antibody conjugate and its medical use.
背景技术Background technique
B细胞是淋巴细胞，其在自适应体液免疫和特异性识别抗原的抗体的产生中发挥重要的作用。B细胞的三种亚类是幼稚B细胞、记忆B细胞和浆细胞。在VDJ重组的过程，其中DNA的两种或三种片段选自基因组文库，并且重组以产生抗体可变结构域的组合阵列，通过其由不同谱系的B细胞编码的可变结构域进一步的改变导致至多10 9种独特的B细胞谱系，其产生和独特靶具有特异性的抗体。多种疾病涉及B细胞，B细胞的恶性转化导致癌症，包括一些淋巴瘤，如多发性骨髓瘤和霍奇金氏淋巴瘤。自身免疫性疾病也会涉及到B细胞，包括系统性红斑狼疮(SLE)和IgA肾病。涉及B细胞的癌症和自身免疫性疾病可被认为B细胞功能异常，因此控制这类疾病的可能的策略是使用靶向病理B细胞的抗体。 B cells are lymphocytes, which play an important role in adaptive humoral immunity and the production of antibodies that specifically recognize antigens. The three subtypes of B cells are naive B cells, memory B cells, and plasma cells. In the process of VDJ recombination, two or three fragments of DNA are selected from a genomic library and recombined to produce a combinatorial array of antibody variable domains, which are further changed by the variable domains encoded by B cells of different lineages This results in up to 109 unique B cell lineages that produce antibodies specific to unique targets. Many diseases involve B cells. Malignant transformation of B cells leads to cancer, including some lymphomas such as multiple myeloma and Hodgkin’s lymphoma. Autoimmune diseases also involve B cells, including systemic lupus erythematosus (SLE) and IgA nephropathy. Cancers and autoimmune diseases involving B cells can be considered to be abnormal in B cell function, so a possible strategy to control such diseases is to use antibodies that target pathological B cells.
BCMA(CD269或TNFRSF17)是TNF受体超家族的成员，其是对配体BAFF(B细胞激活因子)和APRIL(增殖诱导配体)的非糖基化的内在膜受体。BCMA和它的相应配体具有调节体液免疫、B细胞发育和稳态等的不同作用。BCMA由扁桃体记忆B细胞和由生发中心B细胞表达，在脾脏、淋巴结、胸腺、肾上腺和肝脏均可以检测到，并且多种B细胞系的分析表明在BCMA在成熟后的表达水平增加。BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily, which is a non-glycosylated inner membrane receptor for the ligands BAFF (B cell activating factor) and APRIL (proliferation inducing ligand). BCMA and its corresponding ligands have different functions in regulating humoral immunity, B cell development and homeostasis. BCMA is expressed by tonsil memory B cells and germinal center B cells, and can be detected in the spleen, lymph nodes, thymus, adrenal glands and liver. The analysis of various B cell lines shows that the expression level of BCMA increases after maturation.
针对BCMA的抗体描述于例如Gras M-P.等Int Immunol.7(1995)1093-1106、WO200124811、WO200124812、WO2010104949和WO2012163805中。针对BCMA的抗体及其用于治疗淋巴瘤和多发性骨髓瘤的用途例如叙述于WO2002066516和WO2010104949中。WO2013154760涉及包含BCMA识别部分和T-细胞激活部分的嵌合抗原受体。Antibodies against BCMA are described in, for example, Gras M-P. et al. Int Immunol. 7 (1995) 1093-1106, WO200124811, WO200124812, WO2010104949 and WO2012163805. Antibodies against BCMA and their use for the treatment of lymphoma and multiple myeloma are described in, for example, WO2002066516 and WO2010104949. WO2013154760 relates to a chimeric antigen receptor comprising a BCMA recognition part and a T-cell activation part.
在US9273141提供了一种能够内化的特异性结合BCMA和抑制BAFF和/或APRIL对BCMA的结合的抗原结合蛋白，并提供了包含所述抗原结合蛋白与细胞毒性剂的缀合物。In US9273141, an antigen binding protein capable of internalization that specifically binds to BCMA and inhibits the binding of BAFF and/or APRIL to BCMA is provided, and a conjugate comprising the antigen binding protein and a cytotoxic agent is provided.
发明内容Summary of the invention
本发明的目的在于提供一种抗BCMA抗体药物偶联物，或其药学上可接受的盐或溶剂化合物，其为通式(A)所示的抗体药物偶联物或其药学上可接受的 盐或溶剂化合物，The purpose of the present invention is to provide an anti-BCMA antibody drug conjugate, or a pharmaceutically acceptable salt or solvent compound thereof, which is an antibody drug conjugate represented by general formula (A) or a pharmaceutically acceptable Salt or solvent compound,
Ab-(L 2-L 1-D) y Ab-(L 2 -L 1 -D) y
(A)(A)
其中：in:
D是细胞毒性药物；D is a cytotoxic drug;
L 1、L 2是接头单元； L 1 and L 2 are joint units;
y选自1-20的数；y is a number selected from 1-20;
Ab为抗BCMA抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段包含重链可变区和和抗体轻链可变区，其中抗体重链可变区包含至少1个选自以下序列所示的HCDR：SEQ ID NO:3，SEQ ID NO:4和SEQ ID NO:5；和抗体轻链可变区包含至少1个选自以下序列所述的LCDR：SEQ ID NO:6，SEQ ID NO:7和SEQ ID NO:8。Ab is an anti-BCMA antibody or an antigen-binding fragment thereof, the anti-BCMA antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and an antibody light chain variable region, wherein the antibody heavy chain variable region contains at least one selected from The HCDR shown in the sequence: SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5; and the antibody light chain variable region contains at least one LCDR selected from the following sequence: SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
在本发明一个优选方案中，所述抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，其中抗BCMA抗体或其抗原结合片段的重链可变区包含：SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2和SEQ ID NO:5所示的HCDR3。In a preferred embodiment of the present invention, the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof, wherein the heavy chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises: SEQ ID NO HCDR1 shown in: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述的抗BCMA抗体或其抗原结合片段的轻链可变区包含：SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:8所示的LCDR3。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, the light chain variable region of the anti-BCMA antibody or antigen-binding fragment thereof comprises : LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述的抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，所述抗BCMA抗体或其抗原结合片段的重链可变区包含：SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2和SEQ ID NO:5所示的HCDR3；以及所述轻链可变区包含：SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:8所示的LCDR3。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and The light chain variable region. The heavy chain variable region of the anti-BCMA antibody or its antigen-binding fragment includes: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR2 shown in SEQ ID NO: 5 And the light chain variable region includes: LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，其中抗BCMA抗体或其抗原结合片段的CDR序列可以与原CDR序列相比，发生1-3个优化抗体活性、抗体稳定性或降低免疫原性的氨基酸突变。In a preferred embodiment of the present invention, the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, wherein the CDR sequence of the anti-BCMA antibody or antigen-binding fragment thereof may be the same as the original CDR sequence. Than, 1-3 amino acid mutations that optimize antibody activity, antibody stability, or reduce immunogenicity occur.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述抗BCMA抗体或其抗原结合片段为鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人抗体或其抗原结合片段或人源化抗体或其抗原结合片段。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, the anti-BCMA antibody or its antigen-binding fragment is a murine antibody or its antigen-binding Fragments, chimeric antibodies or antigen-binding fragments thereof, human antibodies or antigen-binding fragments thereof, or humanized antibodies or antigen-binding fragments thereof.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药 学上可接受的盐或溶剂化合物，所述的抗BCMA抗体或其抗原结合片段进一步包含人IgG1或其变体、IgG2或其变体、IgG3或其变体或IgG4或其变体的重链恒定区。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises human IgG1 or its variants. The heavy chain constant region of IgG2 or its variants, IgG3 or its variants, or IgG4 or its variants.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物，所述的Ab抗体或其抗原结合片段进一步包含人IgG1、IgG2或IgG4重链恒定区。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention, the Ab antibody or antigen-binding fragment thereof further comprises a human IgG1, IgG2 or IgG4 heavy chain constant region.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物，所述的抗BCMA抗体或其抗原结合片段进一步包含氨基酸突变后具有增强的ADCC毒性的人IgG1重链恒定区。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a human IgG1 heavy chain constant region with enhanced ADCC toxicity after amino acid mutation.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物，所述的抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO:22所示的重链恒定区。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 22.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述的抗BCMA抗体或其抗原结合片段进一步包含源自人抗体κ链、λ链或其变体的轻链恒定区，优选地，所述的抗BCMA抗体或其抗原结合片段进一步包含源自人抗体κ链的轻链恒定区。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a human antibody κ Preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a light chain constant region derived from a human antibody κ chain.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述的抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO:23所示的轻链恒定区。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof further comprises SEQ ID NO: The light chain constant region shown at 23.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述抗BCMA抗体或其抗原结合片段包含选自以下序列所示的重链可变区：SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or antigen-binding fragment thereof comprises a sequence selected from Heavy chain variable region: SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述抗BCMA抗体或其抗原结合片段包含与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区：SEQ ID NO:9、SEQ ID NO:10或SEQ ID NO:11。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or antigen-binding fragment thereof contains at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical heavy chain variable regions: SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.
在本发明一个优选方案中，所述抗BCMA抗体或其抗原结合片段包含选自以下序列所示的轻链可变区：SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14。In a preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a light chain variable region selected from the following sequences: SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述抗BCMA抗体或其抗原结合片段包含与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区：SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or antigen-binding fragment thereof contains at least A light chain variable region of 70%, 75%, 80%, 85%, 90%, 95% or 99% identity: SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 14.
在本发明一个优选方案中，所述的抗BCMA抗体或其抗原结合片段含有如下序列所示的重链：SEQ ID NO：15、SEQ ID NO：16或SEQ ID NO：17。In a preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof contains a heavy chain shown in the following sequence: SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
在本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述抗BCMA抗体或其抗原结合片段包含与以下序列相比具有至少80％，85％，90％，95％或99％同一性的重链：SEQ ID NO： 15、SEQ ID NO：16或SEQ ID NO：17。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate of the present invention or a pharmaceutically acceptable salt or solvent compound thereof, the anti-BCMA antibody or antigen-binding fragment thereof contains at least 80%, 85%, 90%, 95% or 99% identical heavy chains: SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17.
在本发明一个优选方案中，所述的抗BCMA抗体或其抗原结合片段含有选自如下序列所示的轻链：SEQ ID NO：18、SEQ ID NO：19或SEQ ID NO：20。In a preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof contains a light chain selected from the following sequences: SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.
本发明一个优选方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，所述抗BCMA抗体或其抗原结合片段包含与以下序列相比具有至少80％，85％，90％，95％或99％同一性的轻链：SEQ ID NO：18、SEQ ID NO：19或SEQ ID NO：20。In a preferred embodiment of the present invention, according to the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention, the anti-BCMA antibody or antigen-binding fragment thereof contains at least 80% compared with the following sequence: %, 85%, 90%, 95%, or 99% identical light chains: SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.
在本发明一个更优选方案中，所述的抗BCMA抗体或其抗原结合片段包含SEQ ID NO：9所示的重链可变区和SEQ ID NO：12所示的轻链可变区。In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 9 and a light chain variable region shown in SEQ ID NO: 12.
在本发明一个更优选方案中，所述的抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区选自SEQ ID NO：9，或与SEQ ID NO：9相比具有至少70％，75％，80％，85％，90％，95％或99％同一性，所述轻链可变区选自SEQ ID NO：12或与SEQ ID NO：12相比具有至少70％，75％，80％，85％，90％，95％或99％同一性。In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region is selected from SEQ ID NO: 9, or SEQ ID NO: 9 has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity compared to SEQ ID NO: 9, and the light chain variable region is selected from SEQ ID NO: 12 or with SEQ ID NO: 12 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.
在本发明一个更优选方案中，所述的抗BCMA抗体或其抗原结合片段包含SEQ ID NO：10所示的重链可变区和SEQ ID NO：13所示的轻链可变区。In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 10 and a light chain variable region shown in SEQ ID NO: 13.
在本发明一个更优选方案中，所述的抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区选自SEQ ID NO：10，或与SEQ ID NO：10相比具有至少70％，75％，80％，85％，90％，95％或99％同一性，所述轻链可变区选自SEQ ID NO：13或与SEQ ID NO：13相比具有至少70％，75％，80％，85％，90％，95％或99％同一性。In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region is selected from SEQ ID NO: 10, or SEQ ID NO: 10 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared to SEQ ID NO: 10, and the light chain variable region is selected from SEQ ID NO: 13 or with SEQ ID NO: 13 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.
在本发明一个更优选方案中，所述的抗BCMA抗体包含SEQ ID NO：11所示的重链可变区和SEQ ID NO：14所示的轻链可变区。In a more preferred embodiment of the present invention, the anti-BCMA antibody comprises a heavy chain variable region shown in SEQ ID NO: 11 and a light chain variable region shown in SEQ ID NO: 14.
在本发明一个更优选方案中，所述的抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区选自SEQ ID NO：11，或与SEQ ID NO：11相比具有至少70％，75％，80％，85％，90％，95％或99％同一性，所述轻链可变区选自SEQ ID NO：14或与SEQ ID NO：14相比具有至少70％，75％，80％，85％，90％，95％或99％同一性。In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region is selected from SEQ ID NO: 11, or SEQ ID NO: 11 has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity compared to SEQ ID NO: 11. The light chain variable region is selected from SEQ ID NO: 14 or with SEQ ID NO: 14 has at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity.
在本发明一个更优选方案中，所述的抗BCMA抗体包含SEQ ID NO：15所示的重链和SEQ ID NO：18所示的轻链。In a more preferred embodiment of the present invention, the anti-BCMA antibody comprises a heavy chain shown in SEQ ID NO: 15 and a light chain shown in SEQ ID NO: 18.
包含SEQ ID NO：15所示的重链和SEQ ID NO：18所示的轻链的抗BCMA抗体，即为Ab1。The anti-BCMA antibody comprising the heavy chain shown in SEQ ID NO: 15 and the light chain shown in SEQ ID NO: 18 is Ab1.
在本发明一个更优选方案中，所述的抗BCMA抗体或其抗原结合片段包含重链和轻链，所述重链选自SEQ ID NO：15，或与SEQ ID NO：15相比具有至少80％，85％，90％，95％或99％同一性，所述轻链选自SEQ ID NO：18，或与SEQ ID NO：18相比具有至少80％，85％，90％，95％或99％同一性。In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the heavy chain is selected from SEQ ID NO: 15, or compared with SEQ ID NO: 15 with at least 80%, 85%, 90%, 95%, or 99% identity, the light chain is selected from SEQ ID NO: 18, or has at least 80%, 85%, 90%, 95% compared with SEQ ID NO: 18 % Or 99% identity.
在本发明一个更优选方案中，所述的抗BCMA抗体包含SEQ ID NO：16所示的重链和SEQ ID NO：19所示的轻链。In a more preferred embodiment of the present invention, the anti-BCMA antibody comprises a heavy chain shown in SEQ ID NO: 16 and a light chain shown in SEQ ID NO: 19.
包含SEQ ID NO：16所示的重链和SEQ ID NO：19所示的轻链的抗BCMA抗体，即为Ab2。The anti-BCMA antibody comprising the heavy chain shown in SEQ ID NO: 16 and the light chain shown in SEQ ID NO: 19 is Ab2.
在本发明一个更优选方案中，所述的抗BCMA抗体或其抗原结合片段包含重链和轻链，所述重链选自SEQ ID NO：16，或与SEQ ID NO：16相比具有至少80％，85％，90％，95％或99％同一性，所述轻链选自SEQ ID NO：19，或与SEQ ID NO：19相比具有至少80％，85％，90％，95％或99％同一性。In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the heavy chain is selected from SEQ ID NO: 16, or has at least 80%, 85%, 90%, 95% or 99% identity, the light chain is selected from SEQ ID NO: 19, or has at least 80%, 85%, 90%, 95% compared with SEQ ID NO: 19 % Or 99% identity.
在本发明一个更优选方案中，所述的抗BCMA抗体包含SEQ ID NO：17所示的重链和SEQ ID NO：20所示的轻链。In a more preferred embodiment of the present invention, the anti-BCMA antibody comprises a heavy chain shown in SEQ ID NO: 17 and a light chain shown in SEQ ID NO: 20.
包含SEQ ID NO：17所示的重链和SEQ ID NO：20所示的轻链的抗BCMA抗体，即为Ab3。The anti-BCMA antibody comprising the heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20 is Ab3.
在本发明一个更优选方案中，所述的抗BCMA抗体或其抗原结合片段包含重链和轻链，所述重链选自SEQ ID NO：17，或与SEQ ID NO：17相比具有至少80％，85％，90％，95％或99％同一性，所述轻链选自SEQ ID NO：20，或与SEQ ID NO：20相比具有至少80％，85％，90％，95％或99％同一性。In a more preferred embodiment of the present invention, the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, and the heavy chain is selected from SEQ ID NO: 17, or has at least 80%, 85%, 90%, 95% or 99% identity, the light chain is selected from SEQ ID NO: 20, or has at least 80%, 85%, 90%, 95% compared with SEQ ID NO: 20 % Or 99% identity.
在本发明一个实施方案中，所述的细胞毒性药物选自毒素、化疗药物、抗生素、放射性同位素和核溶酶。In one embodiment of the present invention, the cytotoxic drug is selected from toxins, chemotherapeutics, antibiotics, radioisotopes and nucleolytic enzymes.
在本发明优选的实施方案中，所述的细胞毒性药物选自抑制细胞分裂的微管蛋白抑制剂或DNA拓扑异构酶抑制剂；优选DM1、DM3、DM4、喜树碱、SN-38、MMAF或MMAE；更优选微管蛋白抑制剂MMAE或MMAF，或者DNA拓扑异构酶抑制剂SN-38。In a preferred embodiment of the present invention, the cytotoxic drug is selected from tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division; preferably DM1, DM3, DM4, camptothecin, SN-38, MMAF or MMAE; more preferably tubulin inhibitor MMAE or MMAF, or DNA topoisomerase inhibitor SN-38.
在本发明进一步优选的实施方案中，所述的细胞毒性药物选自：In a further preferred embodiment of the present invention, the cytotoxic drug is selected from:
Figure PCTCN2021082735-appb-000001
Figure PCTCN2021082735-appb-000001
在本发明一个优选的实施方案中，所述的细胞毒性药物选自喜树碱衍生物，优选依喜替康：In a preferred embodiment of the present invention, the cytotoxic drug is selected from camptothecin derivatives, preferably exenotecan:
Figure PCTCN2021082735-appb-000002
Figure PCTCN2021082735-appb-000002
在本发明一个更优选的实施方案中，所述的细胞毒性药物为依喜替康衍生物，优选地，所述依喜替康衍生物为化合物2-A：In a more preferred embodiment of the present invention, the cytotoxic drug is an exenotecan derivative, preferably, the exenotecan derivative is compound 2-A:
Figure PCTCN2021082735-appb-000003
Figure PCTCN2021082735-appb-000003
在本发明的一些实施方案中，所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为通式(I)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，In some embodiments of the present invention, the antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is an antibody drug conjugate represented by general formula (I) or a pharmaceutically acceptable Salt or solvent compound,
Figure PCTCN2021082735-appb-000004
Figure PCTCN2021082735-appb-000004
其中：in:
L 1、L 2是接头单元； L 1 and L 2 are joint units;
y为选自1-8的整数，优选为2-4的数；y is an integer selected from 1-8, preferably a number from 2-4;
Ab选自如上述定义的抗BCMA抗体或其抗原结合片段。Ab is selected from anti-BCMA antibodies or antigen-binding fragments thereof as defined above.
在本发明的一些实施方案中，根据本发明抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(I)所示化合物或其药学上可接受的盐或溶剂化合物，In some embodiments of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is a compound represented by general formula (I) or a pharmaceutically acceptable salt or solvent thereof Compound,
Figure PCTCN2021082735-appb-000005
Figure PCTCN2021082735-appb-000005
其中：in:
L 1、L 2是接头单元： L 1 and L 2 are joint units:
y为选自1‐8的数，优选为2-8的数，进一步为2-6的数，更优选3-6的数；y is a number selected from 1-8, preferably a number from 2-8, further a number from 2-6, more preferably a number from 3-6;
Ab选自如上述定义的抗BCMA抗体或其抗原结合片段。Ab is selected from anti-BCMA antibodies or antigen-binding fragments thereof as defined above.
在本发明优选的实施方案中，根据本发明抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(I-A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物：In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (IA) or a pharmaceutically acceptable salt or solvent compound thereof. Acceptable salt or solvent compound:
Figure PCTCN2021082735-appb-000006
Figure PCTCN2021082735-appb-000006
在本发明优选的实施方案中，根据本发明抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(I-B)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物：In a preferred embodiment of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by general formula (IB) or a pharmaceutically acceptable salt or solvent compound thereof. Acceptable salt or solvent compound:
Figure PCTCN2021082735-appb-000007
Figure PCTCN2021082735-appb-000007
根据本发明的抗BCMA抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(II)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化合物：The anti-BCMA antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof as shown in the general formula (II) :
Figure PCTCN2021082735-appb-000008
Figure PCTCN2021082735-appb-000008
其中：in:
L 1、L 2是接头单元； L 1 and L 2 are joint units;
y为选自1-8的数，优选为2-4的数；y is a number selected from 1-8, preferably a number from 2-4;
Ab选自如上述定义的BCMA抗体或其抗原结合片段。Ab is selected from BCMA antibodies or antigen-binding fragments thereof as defined above.
在本发明优选的实施方案中，根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(II-1)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物：In a preferred embodiment of the present invention, the anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by general formula (II-1) Conjugate or its pharmaceutically acceptable salt or solvent compound:
Figure PCTCN2021082735-appb-000009
Figure PCTCN2021082735-appb-000009
在本发明优选的实施方案中，根据本发明抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(II-A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物：In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (II-A) or Pharmaceutically acceptable salt or solvent compound:
Figure PCTCN2021082735-appb-000010
Figure PCTCN2021082735-appb-000010
在本发明优选的实施方案中，根据本发明抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(II-B)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物：In a preferred embodiment of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by general formula (II-B) or Its pharmaceutically acceptable salt or solvent compound:
Figure PCTCN2021082735-appb-000011
Figure PCTCN2021082735-appb-000011
根据本发明的抗BCMA抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(III)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，The anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to the present invention is an antibody-drug conjugate represented by the general formula (III) or a pharmaceutically acceptable salt thereof or Solvent compound,
Figure PCTCN2021082735-appb-000012
Figure PCTCN2021082735-appb-000012
其中：in:
L 1、L 2是接头单元； L 1 and L 2 are joint units;
y为选自1-10的数，优选为2-8的数，更优选4-8的数；y is a number selected from 1-10, preferably a number from 2-8, more preferably a number from 4-8;
Ab选自如上述定义的BCMA抗体或其抗原结合片段。Ab is selected from BCMA antibodies or antigen-binding fragments thereof as defined above.
在本发明一些实施方案中，所述L 1如通式(B)所示： In some embodiments of the present invention, the L 1 is represented by general formula (B):
Figure PCTCN2021082735-appb-000013
Figure PCTCN2021082735-appb-000013
其中：in:
M 1为-CR 1R 2-； M 1 is -CR 1 R 2 -;
R 1和R 2相同或不同，R 1和R 2各自独立的选自氢、烷基、卤素、羟基或氨基； R 1 and R 2 are the same or different, and R 1 and R 2 are each independently selected from hydrogen, alkyl, halogen, hydroxyl, or amino;
n选自0～5的整数，优选1、2或3。n is selected from an integer of 0-5, preferably 1, 2 or 3.
在本发明优选的实施方案中，L 1的杂环端与L 2相连。 In a preferred embodiment of the invention, L 1 and heterocyclic L 2 side is connected.
在本发明一些实施方案中，所述L 2如以下通式(C)所示： In some embodiments of the present invention, the L 2 is represented by the following general formula (C):
Figure PCTCN2021082735-appb-000014
Figure PCTCN2021082735-appb-000014
其中：in:
M 2为-CR 4R 5-； M 2 is -CR 4 R 5 -;
R 3选自氢、卤素、羟基、氨基、烷基、烷氧基和环烷基： R 3 is selected from hydrogen, halogen, hydroxyl, amino, alkyl, alkoxy and cycloalkyl:
R 4和R 5相同或不同，R 4和R 5各自独立的选自氢、烷基、卤素、羟基或氨基； R 4 and R 5 are the same or different, and R 4 and R 5 are each independently selected from hydrogen, alkyl, halogen, hydroxyl, or amino;
m选自0-5的整数，优选1、2或3。m is selected from an integer of 0-5, preferably 1, 2 or 3.
在本发明优选的实施方案中，L 2的S端与接头单元L 1相连。 In a preferred embodiment of the present invention, the S end of L 2 is connected to the joint unit L 1 .
在本发明进一步优选的实施方案中，如通式(I)、通式(I-A)或通式(I-B)，所示的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其具有如上述通式(B)所定义的连接单元L 1和上述通式(C)所定义的连接单元L 2。 In a further preferred embodiment of the present invention, the antibody drug conjugate or pharmaceutically acceptable salt or solvent compound thereof as shown in general formula (I), general formula (IA) or general formula (IB), It has a connecting unit L 1 defined by the above general formula (B) and a connecting unit L 2 defined by the above general formula (C).
在本发明进一步优选的实施方案中，如通式(II-A)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其具有如上述通式(C)所定义的连接单元L 2。 In a further preferred embodiment of the present invention, the antibody drug conjugate represented by general formula (II-A) or a pharmaceutically acceptable salt or solvent compound thereof has the formula (C) defined above Connect the unit L 2 .
在本发明的一些实施方案中，所述L 2如通式(D)所示： In some embodiments of the present invention, the L 2 is represented by general formula (D):
-K 1-K 2-K 3-K 4- -K 1 -K 2 -K 3 -K 4-
(D)(D)
其中：in:
K 1为
Figure PCTCN2021082735-appb-000015
s选自2至8的整数，进一步选为4至8的整数，更优选为4至6的整数； K 1 is
Figure PCTCN2021082735-appb-000015
s is selected from an integer from 2 to 8, further selected from an integer from 4 to 8, more preferably an integer from 4 to 6;
K 2为-NR 1(CH 2CH 2O) pCH 2CH 2C(O)-、-NR 1(CH 2CH 2O) pCH 2C(O)-、-S(CH 2) pC(O)-或单键，p选自1至20的整数，优选1至6的整数； K 2 is -NR 1 (CH 2 CH 2 O) p CH 2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p CH 2 C(O)-, -S(CH 2 ) p C(O)- or a single bond, p is selected from an integer from 1 to 20, preferably an integer from 1 to 6;
R 1选自氢、氘、羟基、氨基、烷基、卤素、卤代烷基、氘代烷基和羟烷基； R 1 is selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl;
K 3为四肽残基，优选地，所述四肽残基由选自两个或多个的苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸中的氨基酸形成的肽残基；更优选为GGFG的四肽残基； K 3 is a tetrapeptide residue, preferably, the tetrapeptide residue is selected from two or more of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid , A peptide residue formed by an amino acid in aspartic acid; more preferably a tetrapeptide residue of GGFG;
K 4为-NR 2(CR 3R 4)t-，R 2、R 3或R 4各自独立地为氢、氘、羟基、氨基、烷基、卤素、卤代烷基、氘代烷基和羟烷基，t选自1或2， K 4 is -NR 2 (CR 3 R 4 )t-, R 2 , R 3 or R 4 are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkane Base, t is selected from 1 or 2,
接头单元-L 2-的K 1端与Ab相连，K 4端与L 1相连。 The K 1 end of the joint unit -L 2 -is connected to Ab, and the K 4 end is connected to L 1 .
在本发明更优选的实施方案中，K 1为
Figure PCTCN2021082735-appb-000016
s为5； In a more preferred embodiment of the present invention, K 1 is
Figure PCTCN2021082735-appb-000016
s is 5;
K 2为键； K 2 is the key;
K 3为GGFG的四肽残基； K 3 is the tetrapeptide residue of GGFG;
K 4为-NR 2(CR 3R 4)t-，R 2、R 3或R 4各自独立地为氢、氘、羟基、氨基、C 1-6烷基、卤素、C 1-6卤代烷基、C 1-6氘代烷基和C 1-6羟烷基，t为1或2， K 4 is -NR 2 (CR 3 R 4 )t-, R 2 , R 3 or R 4 are each independently hydrogen, deuterium, hydroxyl, amino, C 1-6 alkyl, halogen, C 1-6 haloalkyl , C 1-6 deuterated alkyl and C 1-6 hydroxyalkyl, t is 1 or 2,
接头单元-L 2-，其K 1端与Ab相连，K 4端与L 1相连。 In the joint unit -L 2 -, the K 1 end is connected to Ab, and the K 4 end is connected to L 1 .
在本发明一些实施方案中，L 1选自键、-O-(CR aR b) m-CR 5R 6-C(O)-、-O-CR 5R 6-(CR aR b) m-、-O-CR 5R 6-、-NH-(CR aR b) m-CR 5R 6-C(O)-或-S-(CR aR b) m-CR 5R 6-C(O)-； In some embodiments of the present invention, L 1 is selected from bond, -O-(CR a R b ) m -CR 5 R 6 -C(O)-, -O-CR 5 R 6 -(CR a R b ) m -, -O-CR 5 R 6 -, -NH-(CR a R b ) m -CR 5 R 6 -C(O)- or -S-(CR a R b ) m -CR 5 R 6- C(O)-;
R a和R b各自独立地选自氢、氘、卤素或烷基； R a and R b are each independently selected from hydrogen, deuterium, halogen or alkyl;
R 5为卤代烷基或环烷基； R 5 is haloalkyl or cycloalkyl;
R 6选自氢、卤代烷基或环烷基； R 6 is selected from hydrogen, haloalkyl or cycloalkyl;
或者，R 5和R 6与其相连接的碳原子一起形成环烷基； Alternatively, R 5 and R 6 together with the carbon atom to which they are attached form a cycloalkyl group;
m选自0、1、2、3或4。m is selected from 0, 1, 2, 3, or 4.
优选地，L 1的O端与接头单元L 2相连。 Preferably, the O end of L 1 is connected to the joint unit L 2 .
在本发明优选的实施方案中，所述L 1如通式(E)所示： In a preferred embodiment of the present invention, the L 1 is represented by the general formula (E):
Figure PCTCN2021082735-appb-000017
Figure PCTCN2021082735-appb-000017
其中，R 5选自卤代烷基或环烷基，R 6选自氢、卤代烷基或环烷基，或者，R 5和R 6与其相连接的碳原子一起形成环烷基； Wherein, R 5 is selected from haloalkyl or cycloalkyl, R 6 is selected from hydrogen, haloalkyl or cycloalkyl, or, R 5 and R 6 together with the carbon atom to which they are connected form a cycloalkyl;
优选地，R 5选自C 1-6卤代烷基或C 3-6环烷基，R 6选自氢、C 1-6卤代烷基或环烷基，或者，R 5和R 6与其相连接的碳原子一起形成C 3-6环烷基； Preferably, R 5 is selected from C 1-6 haloalkyl or C 3-6 cycloalkyl, R 6 is selected from hydrogen, C 1-6 haloalkyl or cycloalkyl, or R 5 and R 6 are connected to it The carbon atoms together form a C 3-6 cycloalkyl group;
m选自0至4的整数，m is selected from an integer from 0 to 4,
优选地，通式(E)选自以下取代基：Preferably, the general formula (E) is selected from the following substituents:
Figure PCTCN2021082735-appb-000018
Figure PCTCN2021082735-appb-000018
在本发明优选的实施方案中，所述-L 2-L 1-选自以下结构： In a preferred embodiment of the present invention, the -L 2 -L 1 -is selected from the following structures:
Figure PCTCN2021082735-appb-000019
Figure PCTCN2021082735-appb-000019
K 2为键； K 2 is the key;
K 3为GGFG的四肽残基； K 3 is the tetrapeptide residue of GGFG;
R 5为卤代烷基或C 3-6环烷基； R 5 is haloalkyl or C 3-6 cycloalkyl;
R 6选自氢、卤代烷基或C 3-6环烷基； R 6 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl;
或者，R 5和R 6与其相连接的碳原子一起形成C 3-6环烷基； Alternatively, R 5 and R 6 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group;
R 2、R 3或R 4各自独立地选自氢或烷基； R 2 , R 3 or R 4 are each independently selected from hydrogen or alkyl;
s选自2至8的整数；优选地，s选自4、5或6的整数；s is selected from an integer of 2 to 8; preferably, s is selected from an integer of 4, 5 or 6;
m选自0至4的整数；m is selected from an integer from 0 to 4;
优选地，-L 2-L 1-选自以下结构： Preferably, -L 2 -L 1 -is selected from the following structures:
Figure PCTCN2021082735-appb-000020
Figure PCTCN2021082735-appb-000020
Figure PCTCN2021082735-appb-000021
Figure PCTCN2021082735-appb-000021
在本发明优选的实施方案中，根据本发明抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(IV)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化物：In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (IV) or a pharmaceutically acceptable salt or solvent compound thereof. Acceptable salts or solvates:
Figure PCTCN2021082735-appb-000022
Figure PCTCN2021082735-appb-000022
其中：in:
W选自C 1-8烷基、C 1-8烷基-环烷基或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或S的杂原子，任选地，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地进一步被卤素、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； W is selected from a C 1-8 alkyl group, a C 1-8 alkyl-cycloalkyl group or a linear heteroalkyl group of 1 to 8 atoms, and the heteroalkyl group contains 1 to 3 selected from N, O or S The heteroatoms, optionally, wherein the C 1-8 alkyl, cycloalkyl and linear heteroalkyl are each independently further substituted by halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, Substituted by one or more substituents of deuterated alkyl, alkoxy and cycloalkyl;
K 2选自-NR 1(CH 2CH 2O) p1CH 2CH 2C(O)-、-NR 1(CH 2CH 2O) p1CH 2C(O)-、-S(CH 2) p1C(O)-或键，R 1选自氢、烷基、卤代烷基、氘代烷基和羟烷基，p 1选自1至20的整数； K 2 is selected from -NR 1 (CH 2 CH 2 O) p1 CH 2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p1 CH 2 C(O)-, -S(CH 2 ) p1 C(O) -or bond, R 1 is selected from hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl, and p 1 is selected from an integer from 1 to 20;
K 3为由2至7个氨基酸构成的肽残基，氨基酸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，所述取代基为一个或多个独立地选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基； K 3 is a peptide residue composed of 2 to 7 amino acids. Amino acids can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available point of attachment, and the substituent is one Or more are independently selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
R 2选自氢、烷基、卤代烷基、氘代烷基和羟烷基； R 2 is selected from hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;
R 3和R 4各自独立地选自氢、卤素、烷基、卤代烷基、氘代烷基和羟烷基； R 3 and R 4 are each independently selected from hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;
R 5选自卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基； R 5 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
R 6选自氢、卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基； R 6 is selected from hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
或者，R 5和R 6与其相连接的碳原子一起形成环烷基或杂环基； Alternatively, R 5 and R 6 together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;
m选自0至4的整数；m is selected from an integer from 0 to 4;
y为选自1-10的数，y是小数或整数；y is a number selected from 1-10, y is a decimal or integer;
Ab选自如上述定义的抗BCMA抗体或其抗原结合片段。Ab is selected from anti-BCMA antibodies or antigen-binding fragments thereof as defined above.
在本发明优选的实施方案中，根据本发明抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(IV-A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物：In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (IV-A) or Pharmaceutically acceptable salt or solvent compound:
Figure PCTCN2021082735-appb-000023
Figure PCTCN2021082735-appb-000023
在本发明优选的实施方案中，根据本发明抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为如通式(IV-A)所示的抗体-药物偶联物或其药学上可接受的盐或溶剂化合物，其中：In a preferred embodiment of the present invention, the antibody-drug conjugate according to the present invention or a pharmaceutically acceptable salt or solvent compound thereof is an antibody-drug conjugate represented by general formula (IV-A) or A pharmaceutically acceptable salt or solvent compound, wherein:
R 5选自卤代烷基或C 3-6环烷基； R 5 is selected from haloalkyl or C 3-6 cycloalkyl;
R 6选自氢、卤代烷基或C 3-6环烷基； R 6 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl;
或者，R 5和R 6与其相连接的碳原子一起形成C 3-6环烷基； Alternatively, R 5 and R 6 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group;
R 2、R 3或R 4各自独立地选自氢或烷基； R 2 , R 3 or R 4 are each independently selected from hydrogen or alkyl;
s选自2至8的整数；s is selected from an integer from 2 to 8;
m选自0至4的整数。m is selected from an integer from 0 to 4.
在本发明更优选的实施方案中，根据本发明的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，通式(IV-A)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化合物选自以下结构：In a more preferred embodiment of the present invention, the antibody drug conjugate or pharmaceutically acceptable salt or solvent compound thereof according to the present invention, the antibody drug conjugate represented by the general formula (IV-A) or its pharmaceutically acceptable Acceptable salt or solvent compounds are selected from the following structures:
Figure PCTCN2021082735-appb-000024
Figure PCTCN2021082735-appb-000024
Figure PCTCN2021082735-appb-000025
Figure PCTCN2021082735-appb-000025
Figure PCTCN2021082735-appb-000026
Figure PCTCN2021082735-appb-000026
根据本发明的一些实施方案，所述抗体-药物偶联物或其药学上可接受的盐或溶剂化合物选自以下化合物，According to some embodiments of the present invention, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is selected from the following compounds,
Figure PCTCN2021082735-appb-000027
Figure PCTCN2021082735-appb-000027
Figure PCTCN2021082735-appb-000028
Figure PCTCN2021082735-appb-000028
Figure PCTCN2021082735-appb-000029
Figure PCTCN2021082735-appb-000029
Figure PCTCN2021082735-appb-000030
Figure PCTCN2021082735-appb-000030
Figure PCTCN2021082735-appb-000031
Figure PCTCN2021082735-appb-000031
在本发明优选的方案中，所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其选自如下化合物：In a preferred embodiment of the present invention, the antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof is selected from the following compounds:
Figure PCTCN2021082735-appb-000032
Figure PCTCN2021082735-appb-000032
Figure PCTCN2021082735-appb-000033
Figure PCTCN2021082735-appb-000033
Figure PCTCN2021082735-appb-000034
Figure PCTCN2021082735-appb-000034
Figure PCTCN2021082735-appb-000035
Figure PCTCN2021082735-appb-000035
Figure PCTCN2021082735-appb-000036
Figure PCTCN2021082735-appb-000036
Figure PCTCN2021082735-appb-000037
Figure PCTCN2021082735-appb-000037
Figure PCTCN2021082735-appb-000038
Figure PCTCN2021082735-appb-000038
Figure PCTCN2021082735-appb-000039
Figure PCTCN2021082735-appb-000039
Figure PCTCN2021082735-appb-000040
Figure PCTCN2021082735-appb-000040
Figure PCTCN2021082735-appb-000041
Figure PCTCN2021082735-appb-000041
Figure PCTCN2021082735-appb-000042
Figure PCTCN2021082735-appb-000042
Figure PCTCN2021082735-appb-000043
Figure PCTCN2021082735-appb-000043
Figure PCTCN2021082735-appb-000044
Figure PCTCN2021082735-appb-000044
Figure PCTCN2021082735-appb-000045
Figure PCTCN2021082735-appb-000045
Figure PCTCN2021082735-appb-000046
Figure PCTCN2021082735-appb-000046
Figure PCTCN2021082735-appb-000047
Figure PCTCN2021082735-appb-000047
Figure PCTCN2021082735-appb-000048
Figure PCTCN2021082735-appb-000048
Figure PCTCN2021082735-appb-000049
Figure PCTCN2021082735-appb-000049
Figure PCTCN2021082735-appb-000050
Figure PCTCN2021082735-appb-000050
其中，y选自2-10，优选4-8，更优选6-8，进一步优选7-8，最优选6或8；或者y选自1-10的数，优选为2-10的数，进一步为2-6的数，更优选3-6的数，最优选6。Wherein, y is selected from 2-10, preferably 4-8, more preferably 6-8, further preferably 7-8, most preferably 6 or 8, or y is selected from 1-10, preferably 2-10, It is further a number of 2-6, more preferably a number of 3-6, and most preferably 6.
在一个优选的实施方案中，本发明涉及制备如通式(IV)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化物的方法，其包括以下步骤：In a preferred embodiment, the present invention relates to a method for preparing an antibody drug conjugate represented by general formula (IV) or a pharmaceutically acceptable salt or solvate thereof, which comprises the following steps:
Figure PCTCN2021082735-appb-000051
Figure PCTCN2021082735-appb-000051
Ab还原后，与通式(F)偶联反应，得到通式(IV)所示的化合物；After Ab is reduced, it is coupled and reacted with the general formula (F) to obtain the compound represented by the general formula (IV);
其中：in:
Ab为如上述定义的抗BCMA抗体或其抗原结合片段；Ab is an anti-BCMA antibody or antigen-binding fragment thereof as defined above;
W、K 2、K 3、R 2～R 6、m和y如通式(IV)中所定义。 W, K 2 , K 3 , R 2 to R 6 , m and y are as defined in the general formula (IV).
在一个优选的实施方案中，所述的通式(F)为通式(F-1)所示的化合物：In a preferred embodiment, the general formula (F) is a compound represented by the general formula (F-1):
Figure PCTCN2021082735-appb-000052
Figure PCTCN2021082735-appb-000052
或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，Or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof,
其中，K 2、K 3、R 2～R 6、s和m如前述-L 2-L 1-中所定义。 Wherein, K 2 , K 3 , R 2 to R 6 , s and m are as defined in the aforementioned -L 2 -L 1 -.
在一个优选的实施方案中，通式(F)或通式(F-1)所示的化合物选自：In a preferred embodiment, the compound represented by general formula (F) or general formula (F-1) is selected from:
Figure PCTCN2021082735-appb-000053
Figure PCTCN2021082735-appb-000053
Figure PCTCN2021082735-appb-000054
Figure PCTCN2021082735-appb-000054
另一方面，本发明提供一种药物组合物，其包含上述抗体药物偶联物或其药学上可接受的盐或溶剂化合物，和一种或多种可药用的赋形剂、稀释剂或载体。In another aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof, and one or more pharmaceutically acceptable excipients, diluents, or Carrier.
另一方面，本发明提供一种医药用途，本发明涉及抗BCMA抗体－药物偶联物或所述抗体－药物偶联物药学上可接受的盐或溶剂化合物或其药物组合物在用于治疗或预防BCMA介导的疾病或病症的用途。On the other hand, the present invention provides a medical use. The present invention relates to an anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate or a pharmaceutical composition thereof for use in treatment Or to prevent BCMA-mediated diseases or conditions.
另一方面，本发明提供一种医药用途，本发明涉及抗BCMA抗体－药物偶联物或所述抗体－药物偶联物药学上可接受的盐或溶剂化合物或其药物组合物在用于制备治疗或预防BCMA介导的疾病或病症的药物中的用途。On the other hand, the present invention provides a medical use. The present invention relates to an anti-BCMA antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound of the antibody-drug conjugate or a pharmaceutical composition thereof for preparing Use in medicine for treating or preventing BCMA-mediated diseases or conditions.
在本发明一个优选的方案中，所述BCMA介导的疾病或病症为癌症或自身免疫疾病，其中所述癌症优选为表达BCMA的癌症，更优选淋巴瘤、白血病或骨髓瘤，最优选多发性骨髓瘤；所述自身免疫疾病选自红斑狼疮，IgA肾病和风湿性关节炎。In a preferred embodiment of the present invention, the BCMA-mediated disease or disorder is cancer or an autoimmune disease, wherein the cancer is preferably a cancer expressing BCMA, more preferably lymphoma, leukemia or myeloma, and most preferably multiple Myeloma; the autoimmune disease is selected from lupus erythematosus, IgA nephropathy and rheumatoid arthritis.
本发明的抗体药物偶联物及其可药用盐或溶剂化合物体外和体内肿瘤抑制作用明显，对正常细胞或组织的毒性低，具有良好的安全性，对血清中相关配体及可溶性BCMA均具有较好的竞争抑制效果；体外具有较好的药物稳定性，便于长时间保存和运输；同时具有良好的体内代谢活性，体内药效时间长，临床应用前景广阔。The antibody-drug conjugate and its pharmaceutically acceptable salt or solvent compound of the present invention have obvious tumor inhibitory effects in vitro and in vivo, have low toxicity to normal cells or tissues, have good safety, and are resistant to related ligands and soluble BCMA in serum. It has a good competitive inhibition effect; it has good drug stability in vitro, which is convenient for long-term storage and transportation; at the same time, it has good metabolic activity in the body, a long time in vivo, and has a broad clinical application prospect.
发明详述Detailed description of the invention
一、术语1. Terminology
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs.
本发明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。The three-letter codes and one-letter codes of amino acids used in the present invention are as described in J. Biol. Chem, 243, p3558 (1968).
本发明所述的术语“抗体”指免疫球蛋白，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，故其抗原性也不同。据此，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中第每类Ig都可以有κ链或λ链。The term "antibody" in the present invention refers to an immunoglobulin, which 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, so their antigenicity is also different. According to this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE. The corresponding heavy chains are μ chain, δ chain, and γ chain. , Α chain and ε 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. For example, 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.
在本发明中，本发明所述的抗体轻链可变区可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。In the present invention, the antibody light chain variable region of the present invention may further include a light chain constant region, and the light chain constant region includes human or murine κ, λ chains or variants thereof.
在本发明中，本发明所述的抗体重链可变区可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG 3、IgG 4或其变体。In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region comprises human or murine IgG1, IgG2, IgG3, IgG4 or its variants. body.
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(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(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(VL)和重链可变区(VH)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。本发明所述的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则和Kabat或ABM定义规则(http://bioinf.org.uk/abs/)。The sequence of about 110 amino acids near the N-terminus of antibody heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region). The variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) 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 VL and VH regions of the antibody or antigen-binding fragment of the present invention comply with the known Kabat numbering rules and Kabat or ABM definition rules (http://bioinf.org.uk/abs /).
术语“抗原呈递细胞”或“APC”是在其表面上展示与MHC复合的外来抗原的细胞。T细胞利用T细胞受体(TCR)识别这种复合物。APC的实例包括但不限于树突细胞(DC)、外周血单个核细胞(PBMC)、单核细胞、B淋巴母细胞和单核细胞衍生的树突细胞。The term "antigen presenting cell" or "APC" is a cell that displays foreign antigen complexed with MHC on its surface. T cells use the T cell receptor (TCR) to recognize this complex. Examples of APCs include, but are not limited to, dendritic cells (DC), peripheral blood mononuclear cells (PBMC), monocytes, B lymphoblasts, and monocyte-derived dendritic cells.
术语“抗原呈递”是指APC捕获抗原和使它们能够被T细胞识别的过程，例如作为MHC-I/MHC-II偶联物的组分。The term "antigen presentation" refers to the process by which APC captures antigens and enables them to be recognized by T cells, for example as a component of MHC-I/MHC-II conjugates.
术语“BCMA”包括由细胞天然表达的BCMA的任何变体或同种型。本发明的抗体可与得自非人物种的BCMA交叉反应。作为另一种选择，该抗体也可以是人BCMA特异性的，可不表现出与其他物种的交叉反应性。BCMA或其任何变体或同种型可从天然表达它们的细胞或组织中分离而得，或使用本领域通用以及本文所述的那些技术通过重组技术产生。优选地，抗BCMA抗体靶向具有正常糖基化模式的人源BCMA。The term "BCMA" includes any variant or isoform of BCMA that is naturally expressed by the cell. The antibodies of the present invention can cross-react with BCMA derived from non-human species. As another option, the antibody may also be specific for human BCMA, and may not show cross-reactivity with other species. BCMA or any variants or isoforms thereof can be isolated from the cells or tissues in which they are naturally expressed, or produced by recombinant techniques using techniques commonly used in the art and those described herein. Preferably, the anti-BCMA antibody targets human BCMA with a normal glycosylation pattern.
术语“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗体， 所涉及的技术和方法在本领域中是熟知的，诸如：The term "recombinant human antibody" includes human antibodies prepared, expressed, created or isolated by recombinant methods, the techniques and methods involved are well known in the art, such as:
1.从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；1. Antibodies isolated from transgenes of human immunoglobulin genes, transchromosomal animals (such as mice) or hybridomas prepared therefrom;
2.从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；2. Antibodies isolated from host cells transformed to express antibodies, such as transfectomas;
3.从重组组合人抗体文库中分离的抗体；以及3. Antibodies isolated from the recombinant combinatorial human antibody library; and
4.通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。4. Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences to other DNA sequences.
此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。Such recombinant human antibodies contain variable and constant regions, which utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人BCMA的单克隆抗体。制备时用BCMA抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本发明一个优选的实施方案中，所述的鼠源BCMA抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。The term "murine antibody" in the present invention refers to a monoclonal antibody to human BCMA prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with BCMA antigen, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated. In a preferred embodiment of the present invention, the murine BCMA antibody or antigen-binding fragment thereof may further comprise the light chain constant region of murine kappa, lambda chains or variants thereof, or further comprise murine IgG1 and IgG2. , IgG3 or IgG4 or its variant heavy chain constant region.
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本发明的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。The term "human antibody" includes antibodies having variable and constant regions of human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been grafted onto human framework sequences (ie, "humanized antibodies") .
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的免疫应答反应的缺点。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting mouse CDR sequences into a human antibody variable region framework. Humanized antibodies can overcome the shortcomings of strong immune responses induced by chimeric antibodies that carry a large amount of mouse protein components. In order to avoid the decrease of immunogenicity and the decrease of activity at the same time, the variable region of the human antibody can be subjected to minimal reverse mutation to maintain activity.
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变后增强ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1重链恒定区。The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody. To establish a chimeric antibody, it is necessary to select a hybridoma that secretes a murine-specific monoclonal antibody, and then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and change the mouse variable region gene. The region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system. The constant region of a human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising human IgG1, IgG2 or IgG4 heavy chain constant region, or using amino acid mutations to enhance ADCC (antibody -dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1 heavy chain constant region.
术语“抗原结合片段”是指抗体的抗原结合片段及抗体类似物，其通常包括至少部分母体抗体(parental antibody)的抗原结合区或可变区(例如一个或多个 CDR)。抗体片段保留母体抗体的至少某些结合特异性。通常，当基于摩尔来表示活性时，抗体片段保留至少10％的母体结合活性。优选地，抗体片段保留至少20％、50％、70％、80％、90％、95％或100％或更多的母体抗体对靶标的结合亲和力。抗原结合片段实例包括但不限于：Fab、Fab’、F(ab’)2、Fv片段、线性抗体(linear antibody)、单链抗体、纳米抗体、结构域抗体和多特异性抗体。工程改造的抗体变体综述于Holliger和Hudson，2005，Nat.Biotechnol.23：1126-1136中。The term "antigen-binding fragment" refers to antigen-binding fragments and antibody analogs of antibodies, which usually include at least part of the antigen-binding region or variable region (for example, one or more CDRs) of a parental antibody. Antibody fragments retain at least some of the binding specificity of the parent antibody. Generally, when the activity is expressed on a mole basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody to the target. Examples of antigen-binding fragments include, but are not limited to: Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single-chain antibodies, nanobodies, domain antibodies, and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson, 2005, Nat. Biotechnol. 23:1126-1136.
“Fab片段”由一条轻链和一条重链的CH1及可变区组成。Fab分子的重链不能与另一个重链分子形成二硫键。The "Fab fragment" consists of the CH1 and variable regions of one light chain and one heavy chain. The heavy chain of the Fab molecule cannot form a disulfide bond with another heavy chain molecule.
“Fc”区含有包含抗体的CH2和CH3结构域的两个重链片段。两个重链片段由两个或多个二硫键并通过CH3结构域的疏水作用保持在一起。The "Fc" region contains two heavy chain fragments containing the CH2 and CH3 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.
“Fab’片段”含有一条轻链和包含VH结构域和CH1结构域以及CH1和CH2结构域之间区域的一条重链的部分，由此可在两个Fab’片段的两条重链之间形成链间二硫键以形成F(ab’)2分子。The "Fab' fragment" contains a light chain and a portion of a heavy chain that contains the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, so that it can be between the two heavy chains of the two Fab' fragments The formation of interchain disulfide bonds to form F(ab')2 molecules.
“F(ab’)2片段”含有两条轻链和两条包含CH1和CH2结构域之间的恒定区的部分的重链，由此在两条重链间形成链间二硫键。因此，F(ab’)2片段由通过两条重链间的二硫键保持在一起的两个Fab’片段组成。The "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Therefore, the F(ab')2 fragment is composed of two Fab' fragments held together by the disulfide bond between the two heavy chains.
“Fv区”包含来自重链和轻链二者的可变区，但缺少恒定区。The "Fv region" contains variable regions from both the heavy and light chains, but lacks the constant region.
术语“多特异性抗体”按其最广义使用，涵盖具有多表位特异性的抗体。这些多特异性抗体包括但不限于：包含重链可变区VH和轻链可变区VL的抗体，其中该VH-VL单元具有多表位特异性；具有两个或多个VL和VH区的抗体，每个VH-VL单元与不同的靶点或同一个靶点的不同表位结合；具有两个或更多个单可变区的抗体，每个单可变区与不同的靶点或同一个靶点的不同的表位结合；全长抗体、抗体片段、双抗体(diabodies)、双特异性双抗体和三抗体(triabodies)、己共价或非共价连接在一起的抗体片段等。The term "multispecific antibody" is used in its broadest sense to encompass antibodies with polyepitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region VH and a light chain variable region VL, wherein the VH-VL unit has polyepitope specificity; having two or more VL and VH regions Antibodies, each VH-VL unit binds to a different target or a different epitope of the same target; an antibody with two or more single variable regions, each single variable region with a different target Or binding to different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked together Wait.
术语“单链抗体”是由抗体的重链可变区VH和轻链可变区VL通过一段连接肽连接而成的单链重组蛋白，它是具有完全抗原结合位点的最小抗体片段。The term "single-chain antibody" is a single-chain recombinant protein formed by connecting the heavy chain variable region VH and the light chain variable region VL of an antibody through a connecting peptide. It is the smallest antibody fragment with a complete antigen-binding site.
术语“结构域抗体片段”是仅含有重链可变区或轻链可变区链的具有免疫学功能的免疫球蛋白片段。在某些情况下，两个或多个VH区与肽接头共价连接以形成二价结构域抗体片段。二价结构域抗体片段的两个VH区可靶向相同或不同抗原。The term "domain antibody fragment" is an immunoglobulin fragment with immunological functions that only contains a heavy chain variable region or a light chain variable region chain. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment can target the same or different antigens.
本发明的术语“与BCMA结合”，指能与人BCMA相互作用。The term "bind with BCMA" in the present invention refers to the ability to interact with human BCMA.
本发明的术语“抗原结合位点”指本发明抗体或抗原结合片段识别的三维空间位点。The term "antigen-binding site" of the present invention refers to a three-dimensional site recognized by the antibody or antigen-binding fragment of the present invention.
术语“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸、或通过蛋白质的三级折叠而并列的不相邻的氨基酸形成。由相 邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位由给定的抗体结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。The term "epitope" refers to a site on an antigen that specifically binds to an immunoglobulin or antibody. Epitopes can be formed by adjacent amino acids or non-adjacent amino acids that are juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are usually maintained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are usually lost after treatment with the denaturing solvent. Epitopes usually include at least 3-15 amino acids in a unique spatial conformation. Methods to determine what epitope is bound by a given antibody are well known in the art, including immunoblotting and immunoprecipitation detection analysis. Methods for determining the spatial conformation of an epitope include the techniques in the art and the techniques described herein, such as X-ray crystal analysis and two-dimensional nuclear magnetic resonance.
本发明所用的术语“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，当使用人BCMA作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。 The terms "specific binding" and "selective binding" as used in the present invention refer to the binding of an antibody to an epitope on a predetermined antigen. Generally, when using human BCMA used as the analyte and the antibody as the ligand in the instrument by surface plasmon resonance (SPR) measurement technique, the antibody is approximately 10 -7 M or even lower than the equilibrium dissociation smaller dissociation constant ( K D ) binds to a predetermined antigen, and its binding affinity to the predetermined antigen is at least twice its binding affinity to non-specific antigens (such as BSA, etc.) other than the predetermined antigen or closely related antigens. The term "antibody that recognizes an antigen" can be used interchangeably with the term "antibody that specifically binds" herein.
术语“交叉反应”是指本发明的抗体与来自不同物种的BCMA结合的能力。例如，结合人BCMA的本发明的抗体也可以结合另一物种的BCMA。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达BCMA的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。The term "cross-reactivity" refers to the ability of the antibodies of the present invention to bind to BCMA from different species. For example, the antibody of the present invention that binds to human BCMA can also bind to BCMA of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays such as SPR and ELISA, or binding or functional interaction with cells that physiologically express BCMA. Methods of determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
术语“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制/阻断这两者。配体的抑制/阻断优选地降低或改变无抑制或阻断的情况下发生配体结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗BCMA抗体接触时，与未与抗BCMA抗体接触的配体相比，任何可测量的配体结合亲和力降低。The terms "inhibition" or "blocking" are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of the ligand preferably reduces or alters the normal level or type of activity that occurs when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in ligand binding affinity when contacted with anti-BCMA antibodies compared to ligands not contacted with anti-BCMA antibodies.
术语“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。The term "inhibition of growth" (eg, in relation to cells) is intended to include any measurable decrease in cell growth.
术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的剌激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指剌激特定的直接细胞杀伤机制。The terms "inducing an immune response" and "enhancing an immune response" are used interchangeably and refer to the stimulation (ie, passive or adaptive) of the immune response to a specific antigen. The term "induction" for inducing CDC or ADCC refers to stimulating a specific direct cell killing mechanism.
本发明中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，增强或降低降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变。In the present invention, "ADCC", namely antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity, means that cells expressing Fc receptors are directly killed by recognizing the Fc segment of antibodies and are coated with antibodies. The target cell. The ADCC effect function of the antibody can be enhanced or reduced by modifying the Fc segment of IgG. The modification refers to mutations in the constant region of the heavy chain of the antibody.
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，小鼠可以用人BCMA或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂 志，2001ISBN012441351上获得。The methods of producing and purifying antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Cold Spring Harbor's Antibody Experiment Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human BCMA or fragments thereof, and the obtained antibodies can be renatured, purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared by conventional methods. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions to the non-human CDR regions. The human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImmunoGeneTics (IMGT), or from the immunoglobulin journal, 2001ISBN012441351.
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。相应抗体的cDNA序列可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO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区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清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、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。The engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminus of the FC region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded in the serum-free medium of the bioreactor to produce antibodies. The antibody-secreted culture medium can be purified and collected by conventional techniques. 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.
本发明的抗体指单克隆抗体。本发明所述的单克隆抗体(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术、合成技术(如CDR-grafting)、或其它现有技术进行重组得到。The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) of the present invention refers to an antibody obtained from a single cloned cell line, and the cell line is not limited to a eukaryotic, prokaryotic or phage cloned cell line. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, synthesis technology (such as CDR-grafting), or other existing technologies.
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。"Administration", "administration" and "treatment" when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals , Human, subject, cell, tissue, organ or biological fluid contact. "Administration", "administration" and "treatment" can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. The treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, where the fluids are in contact with cells. "Administration", "administration" and "treatment" also mean the treatment of, for example, cells by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo. "Treatment" when applied to human, veterinary or research subjects, refers to therapeutic treatment, preventive or preventive measures, research and diagnostic applications.
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的任一种抗体，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本发明的实施方案(例如治疗方法或制品)在缓解每个患都有的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。"Treatment" means administering an internal or external therapeutic agent, such as containing any one of the antibodies of the present invention, to a patient who has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Generally, the therapeutic agent is administered in the treated patient or population in an amount effective to alleviate one or more symptoms of the disease, whether by inducing the regression of such symptoms or inhibiting the development of such symptoms to any clinically measured extent. The amount of the therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount") can vary depending on various factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Through any clinical testing methods commonly used by doctors or other professional health care professionals to evaluate the severity or progression of the symptoms, it can be evaluated whether the symptoms of the disease have been alleviated. As far as the embodiments of the present invention (such as treatment methods or products) may be ineffective in alleviating the symptoms of the target disease that each patient has, according to any statistical test methods known in the art such as Student's t test, chi-square test, and basis Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should reduce the symptoms of the target disease in a statistically significant number of patients.
整个说明书和权利要求书中使用的术语“基本上由……组成”或其变形表示包括所有所述元件或元件组，并且任选包括与所述元件类似或不同性质的其它元件，所述其它元件非显著改变指定给药方案、方法或组合物的基本性质或新性质。The term "essentially composed of" or its variants used throughout the specification and claims means that it includes all the elements or element groups, and optionally includes other elements similar to or different in nature from the elements. The element does not significantly change the basic or new properties of a given dosing regimen, method, or composition.
本发明所述的应用于某个对象的术语“天然存在的”是指这样的事实，即该对象可在自然界中发现。例如存在于可从自然界来源分离得到的生物体(包括病毒)、且未经人工在实验室中有意修饰的多肽序列或多核苷酸序列即是天然存在的。The term "naturally occurring" applied to an object in the present invention refers to the fact that the object can be found in nature. For example, polypeptide sequences or polynucleotide sequences that exist in organisms (including viruses) that can be isolated from natural sources and have not been intentionally modified artificially in the laboratory are naturally occurring.
“有效量”包含足以改善或预防医字病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。"Effective amount" includes an amount sufficient to improve or prevent the symptoms or conditions of medical conditions. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's general health, the method of administration and dosage, and the severity of side effects. The effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
“外源性”指要据背景在生物、细胞或人体外产生的物质。"Exogenous" refers to substances that are produced outside organisms, cells, or humans according to the background.
“内源性”指根据背景在细胞、生物或人体内产生的物质。"Endogenous" refers to a substance produced in a cell, organism, or human body according to the background.
“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同一性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同一性百分率时进行比较。"Identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two comparison sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position . The percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, in the optimal sequence alignment, if there are 6 matches or homology in 10 positions in the two sequences, then the two sequences are 60% homologous. Generally speaking, the comparison is made when two sequences are aligned to obtain the maximum percent identity.
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。“任选”或“任选地”意味着随后所描述地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，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Therefore, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that due to deliberate or unintentional mutations, all offspring cannot be exactly the same in terms of DNA content. Including mutant progeny with the same function or biological activity as screened in the original transformed cell. "Optional" or "optionally" means that the event or environment described later can but does not have to occur, and the description includes the occasion where the event or environment occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a specific sequence may but does not have to be present.
“药物组合物”表示含有一种或多种本文所述抗体或其抗原结合片段，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，利于活性成分的吸收进而发挥生物活性。"Pharmaceutical composition" means containing one or more antibodies or antigen-binding fragments thereof described herein, and other components such as physiological/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
“可药用盐”是指本发明抗体-药物偶联物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，其具有应有的生物活性。本发明抗体-药物偶联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实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。"Pharmaceutically acceptable salt" refers to the salt of the antibody-drug conjugate of the present invention. Such salt is safe and effective when used in mammals, and has due biological activity. The antibody-drug conjugate of the present invention 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, hydrogen sulfate Salt, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, Maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
“溶剂化合物”指本发明的抗体-药物偶联物化合物与一种或多种溶剂分子形成可药用的溶剂化合物，溶剂分子的非限制性实例包括：水、乙醇、乙腈、异丙醇、乙酸乙酯。"Solvent compound" refers to the antibody-drug conjugate compound of the present invention and one or more solvent molecules to form a pharmaceutically acceptable solvent compound. Non-limiting examples of solvent molecules include: water, ethanol, acetonitrile, isopropanol, Ethyl acetate.
“细胞毒性药物”在用于本发明时指抑制细胞的功能和/或引起细胞死亡或破坏的物质。"Cytotoxic drug" when used in the present invention refers to a substance that inhibits the function of cells and/or causes cell death or destruction.
“微管蛋白抑制剂”是指通过抑制微管蛋白的聚合或促进微管蛋白的集合而干扰细胞有丝分裂过程，从而发挥抗肿瘤效果的一类化合物。其非限制性实例包括：美登素类、卡利奇霉素、紫杉烷类、长春新碱、秋水仙碱、尾海兔素/奥瑞他汀/单甲基奥瑞他汀E(MMAE)/单甲基奥瑞他汀F(MMAF)。"Tubulin inhibitor" refers to a class of compounds that interfere with the process of cell mitosis by inhibiting the polymerization of tubulin or promoting the aggregation of tubulin, thereby exerting an anti-tumor effect. Non-limiting examples thereof include: maytansinoids, calicheamicin, taxanes, vincristine, colchicine, ceratopsin/auristatin/monomethyl auristatin E (MMAE) /Monomethyl auristatin F (MMAF).
“接头”指包含是抗体共价附着于药物的共价键或原子链的化学模块。接头的非限制性实例包括：亚芳基、亚杂芳基、PEG、聚亚甲基氧基、琥珀酸酯、琥珀酰胺、二乙醇酸酯、丙二酸酯和己酰胺。"Linker" refers to a chemical moiety that contains a covalent bond or chain of atoms that is the covalent attachment of an antibody to a drug. Non-limiting examples of linkers include: arylene, heteroarylene, PEG, polymethyleneoxy, succinate, succinamide, diglycolate, malonate, and caproamide.
“药物载荷”(DAR)由y表示，即通式(A)中每个抗体的平均细胞毒性药物数。本发明中的药物荷载范围可以为每个抗体1-20个细胞毒性药物(D)。通式(A)的抗体-药物偶联物为偶联有一定范围(1-20个)细胞毒性药物的抗体的集合。来自偶联反应的抗体-药物偶联物中的药物载荷(DAR)可通过常规手段表征，诸如质谱，HPLC和ELISA等。通过这些手段可以测定抗体-药物偶联物在y值上的定量分布。"Drug Load" (DAR) is represented by y, that is, the average number of cytotoxic drugs per antibody in formula (A). The drug loading range in the present invention can be 1-20 cytotoxic drugs (D) per antibody. The antibody-drug conjugate of the general formula (A) is a collection of antibodies conjugated with a certain range (1-20) of cytotoxic drugs. The drug load (DAR) in the antibody-drug conjugate from the coupling reaction can be characterized by conventional means, such as mass spectrometry, HPLC and ELISA. By these means, the quantitative distribution of the antibody-drug conjugate on the y value can be determined.
二、缩写2. Abbreviations
MC为6-马来酰亚氨基己酰基MC is 6-maleimidohexanoyl
MMAF为单甲基奥瑞他汀E的变体，其在分子的C-末端处有苯丙氨酸(MW731.5)MMAF is a variant of monomethyl auristatin E, which has phenylalanine at the C-terminus of the molecule (MW731.5)
具体实施方式Detailed ways
以下结合实施例用于进一步描述本发明，但这些实施例并非限制着本发明的范围。本发明实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；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。The following examples are used to further describe the present invention, but these examples do not limit the scope of the present invention. The experimental methods that do not specify specific conditions in the examples of the present invention usually follow conventional conditions, such as Cold Spring Harbor's antibody technology experimental manual, molecular cloning manual; or according to the conditions recommended by the raw material or commodity manufacturer. The reagents without specific sources are the conventional reagents purchased on the market.
实施例1抗原准备Example 1 Antigen preparation
编码带His标签的人BCMA胞外区(BCMA-His)蛋白由SinoBiologics公司合成(Cat No.:10620-H08H)。The protein encoding the extracellular domain of human BCMA (BCMA-His) with a His tag was synthesized by SinoBiologics (Cat No.: 10620-H08H).
BCMA-His序列：BCMA-His sequence:
Figure PCTCN2021082735-appb-000055
Figure PCTCN2021082735-appb-000055
Figure PCTCN2021082735-appb-000056
Figure PCTCN2021082735-appb-000056
实施例2鼠杂交瘤及抗体序列的获得Example 2 Obtaining Murine Hybridoma and Antibody Sequence
用人抗原BCMA-His进行动物免疫，共5只Balb/c和5只A/J小鼠，雌性，10周龄，使用Sigma完全弗氏佐剂(CFA)和Sigma不完全弗氏佐剂(IFA)，免疫原和免疫佐剂以1:1的比例充分混合乳化，制成稳定“油包水”液体；注射剂量25μg/200μL/小鼠。Animals were immunized with human antigen BCMA-His, a total of 5 Balb/c and 5 A/J mice, female, 10 weeks old, using Sigma Complete Freund’s Adjuvant (CFA) and Sigma Incomplete Freund’s Adjuvant (IFA) ), the immunogen and the immune adjuvant are fully mixed and emulsified at a ratio of 1:1 to make a stable "water-in-oil" liquid; the injection dose is 25μg/200μL/mouse.
表1.免疫方案Table 1. Immunization Scheme
第1天Day 1 第一次免疫，完全弗氏佐剂。The first immunization, complete Freund's adjuvant.
第21天Day 21 第二次免疫，不完全弗氏佐剂。The second immunization, incomplete Freund's adjuvant.
第35天Day 35 第三次免疫，不完全弗氏佐剂。The third immunization, incomplete Freund's adjuvant.
第42天Day 42 采血和血清效价检测(3免血)Blood sampling and serum titer test (3 blood exemption)
第49天Day 49 第四次免疫，不完全弗氏佐剂。The fourth immunization, incomplete Freund's adjuvant.
第56天Day 56 采血和血清效价检测(4免血)Blood collection and serum titer test (4 blood exemption)
对免疫小鼠血清使用如实施例3所述的间接ELISA法评估血清效价及结合细胞表面抗原的能力，对照效价检测情况(大于10万倍稀释度)决定启动细胞融合。选择血清效价、亲和力和FACS结合强的免疫小鼠进行一次终免疫后处死小鼠，取脾细胞和SP2/0骨髓瘤细胞融合后铺板获得杂交瘤，通过间接ELISA筛选到目标杂交瘤，并通过有限稀释法建株为单克隆细胞株。得到的阳性抗体株进一步使用间接ELISA进行筛选，从而选定结合重组蛋白的杂交瘤。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,15596-018)提取RNA并反转录(PrimeScript TM Reverse Transcriptase，Takara#2680A)。将反转录得到的cDNA采用小鼠Ig-引物组(Novagen，TB326 Rev.B 0503)进行PCR扩增后测序，最终得到鼠源抗体M1的序列。 The indirect ELISA method as described in Example 3 was used for the immunized mouse serum to evaluate the serum titer and the ability to bind to cell surface antigens. The detection of the titer (larger than 100,000 times dilution) determines the start of cell fusion. The immunized mice with strong serum titer, affinity and FACS binding were selected for one final immunization and then sacrificed. The spleen cells and SP2/0 myeloma cells were fused and plated to obtain hybridomas. The target hybridomas were screened by indirect ELISA, and The strain was established as a monoclonal cell strain by the limiting dilution method. The obtained positive antibody strains are further screened using indirect ELISA to select hybridomas that bind to the recombinant protein. The logarithmic growth phase hybridoma cells were collected, and RNA was extracted with Trizol (Invitrogen, 15596-018) and reverse transcribed (PrimeScript TM Reverse Transcriptase, Takara #2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-primer set (Novagen, TB326 Rev. B 0503) and then sequenced, and finally the sequence of the mouse antibody M1 was obtained.
鼠单抗M1的重链和轻链可变区序列如下：The heavy chain and light chain variable region sequences of murine monoclonal antibody M1 are as follows:
M1 HCVRM1 HCVR
Figure PCTCN2021082735-appb-000057
Figure PCTCN2021082735-appb-000057
M1 LCVRM1 LCVR
Figure PCTCN2021082735-appb-000058
Figure PCTCN2021082735-appb-000058
表2.鼠单抗M1的重链和轻链可变区CDR序列Table 2. Murine monoclonal antibody M1 heavy chain and light chain variable region CDR sequences
名称name 序列sequence 编号serial number
HCDR1HCDR1 GYSFSDYEMHGYSFSDYEMH SEQ ID NO：3SEQ ID NO: 3
HCDR2HCDR2 GIHPGSGGSAYNQKFKGGIHPGSGGSAYNQKFKG SEQ ID NO：4SEQ ID NO: 4
HCDR3HCDR3 TRLDYGYSWAWFPYTRLDYGYSWAWFPY SEQ ID NO：5SEQ ID NO: 5
LCDR1LCDR1 SASSSVIYMNSASSSVIYMN SEQ ID NO：6SEQ ID NO: 6
LCDR2LCDR2 GISNLASGISNLAS SEQ ID NO：7SEQ ID NO: 7
LCDR3LCDR3 QQRSSYPLTQQRSSYPLT SEQ ID NO：8SEQ ID NO: 8
实施例3抗体的体外结合活性检测方法Example 3 In vitro binding activity detection method of antibody
(1)体外间接ELISA结合实验：(1) In vitro indirect ELISA binding experiment:
用pH7.4的PBS将BCMA His蛋白(Sino Biological Inc.，cat#10620-H08H稀释至1μg/ml浓度，以100μl/孔的体积加入96孔高亲和力酶标板中，于4℃冰箱孵育过夜(16-20小时)。用PBST(pH7.4 PBS含0.05％Tween-20)洗板4次后，加入用PBST稀释的3％牛血清白蛋白(BSA)封闭液150μl/孔，室温孵育1小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液洗板4次。Dilute BCMA His protein (Sino Biological Inc., cat#10620-H08H to a concentration of 1μg/ml with pH7.4 PBS, add 100μl/well to a 96-well high-affinity ELISA plate, and incubate overnight at 4°C in a refrigerator (16-20 hours). After washing the plate 4 times with PBST (pH7.4 PBS containing 0.05% Tween-20), add 150μl/well of 3% bovine serum albumin (BSA) blocking solution diluted with PBST, and incubate at room temperature for 1 Blocking was performed in hours. After the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer.
用含3％BSA的PBST稀释待测抗体，1μM起始，10倍梯度，10个剂量，以100μl/孔加到酶标板中，放于室温孵育1小时。孵育结束后用PBST洗板4次，加入100μl/孔用含3％BSA的PBST稀释的HRP标记羊抗人二抗(Abcam，cat#ab97225)，室温孵育1小时。用PBST洗板4次后，加入100μl/孔TMB显色底物(Cell Signaling Technology，cat#7004S)，于室温避光孵育1分钟，加入100μl/孔终止溶液(Cell Signaling Technology，cat#7002S)终止反应，用酶标仪(BioTek，型号Synergy H1)在450nm处读取吸收值，分析数据。做浓度信号值曲线分析结果，如下表所示：Dilute the antibody to be tested with PBST containing 3% BSA, start with 1 μM, 10 times gradient, 10 doses, add 100 μl/well to the microtiter plate, and incubate at room temperature for 1 hour. After the incubation, the plate was washed 4 times with PBST, 100 μl/well of HRP-labeled goat anti-human secondary antibody (Abcam, cat#ab97225) diluted with 3% BSA in PBST was added, and incubated for 1 hour at room temperature. After washing the plate 4 times with PBST, add 100μl/well of TMB chromogenic substrate (Cell Signaling Technology, cat#7004S), incubate at room temperature and dark for 1 minute, and add 100μl/well of stop solution (Cell Signaling Technology, cat#7002S) The reaction was terminated, and the absorbance value was read at 450nm with a microplate reader (BioTek, model Synergy H1), and the data was analyzed. Do concentration signal value curve analysis results, as shown in the following table:
表3.鼠抗体对人BCMA抗原的亲和力(EC 50值) Table 3. Affinity of mouse antibodies to human BCMA antigen (EC 50 value)
鼠抗体Mouse antibody 与人BCMA His抗原结合EC 50(nM) Binding EC 50 with human BCMA His antigen (nM)
M1M1 0.530.53
(2)体外细胞结合实验：(2) In vitro cell binding experiment:
收集培养好的BCMA高表达细胞(过表达BCMA的HEK-293T细胞和表达BCMA的肿瘤细胞，NCI-H929)，调节细胞密度后分铺于96孔U底板，每孔1×10 5至2×10 5个细胞。1200g，5min离心，去上清，添加100ul已梯度稀释的抗体溶液或小鼠免疫血清，4℃度孵育60min；1200g，5min离心，去上清，PBS洗细胞2次后，添加荧光标记二抗(PE-GAM：山羊抗鼠单抗；或PE-GAH：山羊抗人单抗)100ul每孔，4℃度孵育60min。1200g，5min离心去上清。PBS洗细胞2次后，再重悬于PBS，使用流式细胞计数仪检测信号，并作浓度曲线分析结果。 Collect the cultured BCMA highly expressing cells (HEK-293T cells overexpressing BCMA and tumor cells expressing BCMA, NCI-H929), adjust the cell density and spread them on a 96-well U bottom plate, 1×10 5 to 2× per well 10 5 cells. Centrifuge at 1200g for 5min, remove the supernatant, add 100ul of serially diluted antibody solution or mouse immune serum, incubate at 4°C for 60min; centrifuge at 1200g for 5min, remove the supernatant, and wash the cells twice with PBS, add a fluorescently labeled secondary antibody (PE-GAM: goat anti-mouse monoclonal antibody; or PE-GAH: goat anti-human monoclonal antibody) 100ul per well, incubate at 4°C for 60 minutes. Centrifuge at 1200g for 5min to remove the supernatant. After washing the cells twice with PBS, resuspend them in PBS, use a flow cytometer to detect the signal, and make a concentration curve analysis result.
表4.鼠抗体对表达BCMA的细胞的亲和力(EC 50值) Table 4. Affinity (EC 50 value) of mouse antibodies to cells expressing BCMA
Figure PCTCN2021082735-appb-000059
Figure PCTCN2021082735-appb-000059
实施例4小鼠抗体人源化实验Example 4 Mouse antibody humanization experiment
鼠源抗人BCMA单克隆抗体人源化如本领域许多文献公示的方法进行。简言之，使用人恒定结构域替代亲本(鼠源抗体)恒定结构域，根据鼠源抗体和人抗体的同一性选择人种抗体序列，本发明将鼠源抗体M1进行人源化。The humanization of the murine anti-human BCMA monoclonal antibody is carried out according to the methods published in many documents in the field. In short, a human constant domain is used instead of the parental (murine antibody) constant domain, and the human antibody sequence is selected based on the identity of the murine antibody and the human antibody. The present invention humanizes the murine antibody M1.
在所获得的鼠源抗体VH/VL CDR典型结构的基础上，将重、轻链可变区序列与人源抗体种系数据库比较，获得同一性高的人种系模板。On the basis of the obtained typical structure of murine antibody VH/VL CDR, the sequence of the heavy and light chain variable region is compared with the human antibody germline database to obtain a human germline template with high identity.
将鼠源抗体M1的CDR区移植到选择好的相应人源化模板上。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，并对CDR区化学不稳定氨基酸残基优化，经表达测试和回复突变数量对比，选择和设计了人源化重链可变区HCVR的序列，序列如下：The CDR region of the murine antibody M1 was transplanted to the selected corresponding humanized template. Then, based on the three-dimensional structure of the murine antibody, the embedded residues, the residues that directly interact with the CDR region, and the residues that have an important impact on the conformation of VL and VH are back-mutated, and the CDR region Chemically unstable amino acid residues were optimized. After expression testing and comparison of the number of back mutations, the sequence of the humanized heavy chain variable region HCVR was selected and designed. The sequence is as follows:
HCVR1HCVR1
Figure PCTCN2021082735-appb-000060
Figure PCTCN2021082735-appb-000060
HCVR2HCVR2
Figure PCTCN2021082735-appb-000061
Figure PCTCN2021082735-appb-000061
HCVR3HCVR3
Figure PCTCN2021082735-appb-000062
Figure PCTCN2021082735-appb-000062
选择和设计了人源化轻链可变区LCVR的序列，序列如下：The sequence of the humanized light chain variable region LCVR was selected and designed, the sequence is as follows:
LCVR1LCVR1
Figure PCTCN2021082735-appb-000063
Figure PCTCN2021082735-appb-000063
LCVR2LCVR2
Figure PCTCN2021082735-appb-000064
Figure PCTCN2021082735-appb-000064
LCVR3LCVR3
Figure PCTCN2021082735-appb-000065
Figure PCTCN2021082735-appb-000065
将设计的重链和轻链可变区序列分别与人IgG1重链和人抗体轻链恒定区序列连接，示例性的重链和轻链恒定区序列分别如下所示：Connect the designed heavy chain and light chain variable region sequences with the human IgG1 heavy chain and human antibody light chain constant region sequences, respectively. Exemplary heavy chain and light chain constant region sequences are as follows:
IgG1 CIgG1 C
Figure PCTCN2021082735-appb-000066
Figure PCTCN2021082735-appb-000066
Ig kappa CIg kappa C
Figure PCTCN2021082735-appb-000067
Figure PCTCN2021082735-appb-000067
得到重链和轻链序列如下：Obtain the heavy chain and light chain sequences as follows:
Ab1 HCAb1 HC
Figure PCTCN2021082735-appb-000068
Figure PCTCN2021082735-appb-000068
Ab2 HCAb2 HC
Figure PCTCN2021082735-appb-000069
Figure PCTCN2021082735-appb-000069
Ab3 HCAb3 HC
Figure PCTCN2021082735-appb-000070
Figure PCTCN2021082735-appb-000070
Ab1 LCAb1 LC
Figure PCTCN2021082735-appb-000071
Figure PCTCN2021082735-appb-000071
Ab2 LCAb2 LC
Figure PCTCN2021082735-appb-000072
Figure PCTCN2021082735-appb-000072
Ab3 LCAb3 LC
Figure PCTCN2021082735-appb-000073
Figure PCTCN2021082735-appb-000073
表5.抗体及其重链、轻链、可变区的序列编号Table 5. Sequence numbers of antibodies and their heavy chains, light chains and variable regions
Figure PCTCN2021082735-appb-000074
Figure PCTCN2021082735-appb-000074
根据以上各人源化抗体轻链和重链的氨基酸序列合成cDNA片段，插入到pcDNA3.1表达载体(Life Technologies Cat.No.V790-20)中。将表达载体和转染试剂PEI(Polysciences，Inc.Cat.No.23966)以1:2的比例转染HEK293细胞(Life Technologies Cat.No.11625019)，并置于CO 2孵育箱中孵育4-5天。收取细胞培养液，离心过滤后上样到抗体纯化亲和柱，经磷酸缓冲液洗柱、甘氨酸盐酸缓冲液(pH2.7 0.1M Gly-HCl)洗脱、1M Tris盐酸pH 9.0中和、以及磷酸缓冲液透析，得到本发明的人源化抗体蛋白。 CDNA fragments were synthesized according to the amino acid sequences of the light and heavy chains of the above humanized antibodies, and inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20). The expression vector and the transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a ratio of 1:2, and incubated in a CO 2 incubator 4- 5 days. Collect the cell culture fluid, centrifuge and filter, load the sample to the antibody purification affinity column, wash the column with phosphate buffer, elution with glycine hydrochloric acid buffer (pH 2.7 0.1M Gly-HCl), neutralize with 1M Tris hydrochloric acid pH 9.0, and Phosphate buffer solution is dialyzed to obtain the humanized antibody protein of the present invention.
实施例5体外结合亲和力和动力学实验Example 5 In vitro binding affinity and kinetic experiments
使用实施例3(1)中所述的体外间接ELISA结合实验测定的各人源化抗体对人BCMA抗原的亲和力(EC 50)如下表所示： The affinity (EC 50 ) of each humanized antibody to human BCMA antigen determined using the in vitro indirect ELISA binding experiment described in Example 3 (1) is shown in the following table:
表6.各人源化抗体对人BCMA抗原的亲和力(EC 50) Table 6. each humanized antibody affinity for antigen, human BCMA (EC 50)
Figure PCTCN2021082735-appb-000075
Figure PCTCN2021082735-appb-000075
使用实施例3(2)中所述的体外细胞结合实验测定的各人源化抗体对NCI-H929肿瘤细胞的亲和力(EC 50)如下表所示： The affinity (EC 50 ) of each humanized antibody to NCI-H929 tumor cells determined using the in vitro cell binding experiment described in Example 3(2) is shown in the following table:
表7.各人源化抗体对NCI-H929肿瘤细胞的亲和力(EC 50) Table 7. affinity of each humanized antibody NCI-H929 tumor cells (EC 50)
Figure PCTCN2021082735-appb-000076
Figure PCTCN2021082735-appb-000076
实施例6抗体的内吞作用Example 6 Endocytosis of antibody
检测本发明抗体结合BCMA后是否能够和人BCMA共同内吞入细胞内，用NCI-H929(ATCC保藏号CRL-9068)进行评估。NCI-H929细胞使用胰酶消化(先用PBS清洗一遍，37℃、2min左右)，收集细胞并用预冷的FACS缓冲液重悬，调整细胞浓度为1×10 6个/mL。取EP管，加入1mL细胞悬液，1500rpm离心5分钟后去上清，加入1mL已经配制好的待测抗体重悬细胞，抗体的终浓度均为20μg/ml，4度摇床孵育1h，离心弃上清(4℃、1500rpm×5min)，FACS缓冲液洗涤两次，去上清。每管加入100μL荧光二抗工作液重悬细胞，4℃摇床孵育30min，离心弃上清(4℃、1500rpm×5min)，FACS缓冲液洗涤两次，去上清。每管加入1.0mL预热的NCI-H929细胞完全培养基重悬细胞并混匀，分装为4管，每管200μL，分别为0min组，空白组，30min组和2h组，取出0min及blank置于冰上，其余放置于37℃培养箱，分别内吞30min、2h，在相应时间点取出EP管，置于冰上预冷5min，所有处理组离心弃上清(4℃、1500rpm×5min)，用FACS缓冲液洗涤一次，去上清。去除0min组外所有处理组EP管中加入250μL剥离缓冲液(strip buffer)，室温孵育8min，离心弃上清(4℃、1500rpm×5min)，FACS缓冲液洗涤两次，去上清。所有处理组加入100μL免疫染色固定液，4℃放置30min以上，用流式细胞仪DxFlex进行检测。BCMA抗体内吞百分比＝(各个时间点荧光强度值-空白组平均荧光强度值)/零点时的平均荧光轻度值-空白组平均荧光强度值。结果见下表： To test whether the antibody of the present invention can be endocytosed with human BCMA after binding to BCMA, NCI-H929 (ATCC deposit number CRL-9068) is used for evaluation. NCI-H929 cells were trypsinized (washed with PBS, 37°C for about 2 minutes), collected and resuspended in pre-cooled FACS buffer, adjusting the cell concentration to 1×10 6 cells/mL. Take the EP tube, add 1 mL of cell suspension, centrifuge at 1500 rpm for 5 minutes and remove the supernatant. Add 1 mL of the prepared antibody to be tested to resuspend the cells. The final concentration of the antibody is 20 μg/ml. Incubate for 1 hour on a 4 degree shaker, and centrifuge. Discard the supernatant (4°C, 1500rpm×5min), wash twice with FACS buffer, and remove the supernatant. Add 100μL of fluorescent secondary antibody working solution to each tube to resuspend the cells, incubate for 30min in a shaker at 4°C, centrifuge to discard the supernatant (4°C, 1500rpm×5min), wash twice with FACS buffer, and remove the supernatant. Add 1.0mL of pre-warmed NCI-H929 cell complete medium to each tube to resuspend the cells and mix them. Divide into 4 tubes, 200μL per tube, respectively for 0min group, blank group, 30min group and 2h group. Take out 0min and blank. Place on ice, and place the rest in a 37°C incubator. Ingest 30min and 2h respectively. At the corresponding time point, take out the EP tube and place it on ice for 5min. Centrifuge and discard the supernatant in all treatment groups (4°C, 1500rpm×5min). ), wash once with FACS buffer and remove the supernatant. Add 250μL strip buffer to EP tubes of all treatment groups except the 0min group, incubate at room temperature for 8min, centrifuge to discard the supernatant (4℃, 1500rpm×5min), wash twice with FACS buffer, and remove the supernatant. All treatment groups were added with 100 μL of immunostaining fixative, placed at 4°C for more than 30 minutes, and detected by flow cytometer DxFlex. Percentage of BCMA antibody endocytosis=(fluorescence intensity value at each time point-average fluorescence intensity value of blank group)/average light fluorescence value at zero point-average fluorescence intensity value of blank group. The results are shown in the table below:
表8.抗体在NCI-H929肿瘤细胞中的内吞作用(EC 50) Table 8. Antibody endocytosis in NCI-H929 tumor cells (EC 50)
Figure PCTCN2021082735-appb-000077
Figure PCTCN2021082735-appb-000077
ND＝未确定ND=Not determined
结果显示，与抗BCMA抗体J6M0(描述于美国专利9,273,141)相比，本发明抗体具有更高的内吞效率，能够快速内化。The results show that compared with the anti-BCMA antibody J6M0 (described in US Patent 9,273,141), the antibody of the present invention has a higher endocytosis efficiency and can be rapidly internalized.
实施例7抗体偶联MC-MMAFExample 7 Antibody conjugated to MC-MMAF
本发明抗体具有细胞亲和活性且具有细胞内吞活性，使得本发明抗体适合与药物偶联形成抗体-药物偶联物用于治疗BCMA介导的疾病。偶联过程见下式，其中Ab代表Ab2或Ab3抗体：The antibody of the present invention has cell affinity activity and endocytosis activity, making the antibody of the present invention suitable for coupling with drugs to form antibody-drug conjugates for the treatment of BCMA-mediated diseases. The coupling process is shown in the following formula, where Ab stands for Ab2 or Ab3 antibody:
Figure PCTCN2021082735-appb-000078
Figure PCTCN2021082735-appb-000078
第一步将硫代乙酸S-(3-醛丙基)酯(0.7mg，5.3mol)溶解于0.9mL乙腈溶液备用。向抗体pH＝4.3的乙酸/乙酸钠缓冲液(10.35mg/mL,9.0mL，0.97mol)加入上述预制的硫代乙酸S-(3-羟基丙基)酯的乙腈溶液，然后滴加1.0mL的氰基硼氢化钠(14.1mg，224mol)的水溶液，于25℃下振荡反应2小时。反应结束后，用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，得产物1f溶液，浓缩到10mg/mL后直接进行下一步反应。In the first step, S-(3-aldehyde propyl) thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile solution for later use. To the acetic acid/sodium acetate buffer (10.35mg/mL, 9.0mL, 0.97mol) of the antibody pH=4.3 was added the pre-prepared acetonitrile solution of S-(3-hydroxypropyl) thioacetate, and then 1.0mL was added dropwise An aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was shaken and reacted at 25° C. for 2 hours. After the reaction is over, use Sephadex G25 gel column for desalting and purification (elution phase: pH 6.5 0.05M PBS solution) to obtain the product 1f solution, which is concentrated to 10 mg/mL and directly proceed to the next reaction.
第二步，向1f溶液(11.0mL)中加入0.35mL的2.0M盐酸羧胺溶液，于25℃下振荡反应30分钟后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，得到产物2f溶液(浓度6.17mg/mL，14.7mL)。In the second step, 0.35mL of 2.0M carboxyamine hydrochloride solution was added to the 1f solution (11.0mL), and after shaking at 25°C for 30 minutes, the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH6 .5 0.05M PBS solution), the product 2f solution (concentration 6.17 mg/mL, 14.7 mL) was obtained.
第三步，将化合物MC-MMAF(1.1mg，1.2mol，采用PCT专利W02005081711公开的方法制备得到)溶解于0.3mL乙腈中，加入2f溶液(浓度6.17mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，在无菌条件下用滤器过滤后得到产物Ab-MC-MMAF。使用HIC-HPLC测定产物ADC2(Ab2-MC-MMAF)的DAR平均值y为4，将抗体-药物偶联物的PBS缓冲液(3.7mg/mL，4.7mL)于4℃冷藏。采用上述方法制备得到产物ADC3(Ab3-MC-MMAF)。使用HIC-HPLC测定产物ADC3(Ab3-MC-MMAF)的DAR平均值y为4.1，将抗体-药物偶联物的PBS缓冲液(3.5mg/mL，5.0mL)于4℃冷藏。In the third step, the compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile, and the 2f solution (concentration 6.17 mg/mL, 3.0 mL) was added to 25 After shaking and reacting at ℃ for 4 hours, the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5), and filtered under sterile conditions with a filter to obtain the product Ab-MC -MMAF. The DAR average value y of the product ADC2 (Ab2-MC-MMAF) was determined by HIC-HPLC to be 4, and the antibody-drug conjugate PBS buffer (3.7 mg/mL, 4.7 mL) was refrigerated at 4°C. The product ADC3 (Ab3-MC-MMAF) was prepared by the above method. The average DAR value y of the product ADC3 (Ab3-MC-MMAF) determined by HIC-HPLC was 4.1, and the antibody-drug conjugate PBS buffer (3.5 mg/mL, 5.0 mL) was refrigerated at 4°C.
实施例8抗体偶联SN-38Example 8 Antibody coupling SN-38
通过以下偶联过程制备抗体偶联药物，其中Ab代表Ab2：Prepare antibody-conjugated drugs through the following coupling process, where Ab stands for Ab2:
Figure PCTCN2021082735-appb-000079
Figure PCTCN2021082735-appb-000079
第一步将硫代乙酸S-(3-醛丙基)酯(0.7mg，5.3mol)溶解于0.9mL乙腈溶液备用。向抗体pH＝4.3的乙酸/乙酸钠缓冲液(10.35mg/mL,9.0mL，0.97mol)加入上述预制的硫代乙酸S-(3-羟基丙基)酯的乙腈溶液，然后滴加1.0mL的氰基硼氢化钠(14.1mg，224mol)的水溶液，于25℃下振荡反应2小时。反应结束后，用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，得产物1h溶液，浓缩到10mg/mL后直接进行下一步反应。In the first step, S-(3-aldehyde propyl) thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile solution for later use. To the acetic acid/sodium acetate buffer (10.35mg/mL, 9.0mL, 0.97mol) of the antibody pH=4.3 was added the pre-prepared acetonitrile solution of S-(3-hydroxypropyl) thioacetate, and then 1.0mL was added dropwise An aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was shaken and reacted at 25° C. for 2 hours. After the reaction is completed, use Sephadex G25 gel column for desalting and purification (elution phase: pH 6.5 0.05M PBS solution) to obtain a 1h solution of the product, which is concentrated to 10 mg/mL and directly proceed to the next reaction.
第二步，向1h溶液(11.0mL)中加入0.35mL的2.0M盐酸羧胺溶液，于25℃下振荡反应30分钟后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，得到产物2h溶液(浓度6.2mg/mL，15.0mL)，浓缩到约10mg/mL后用于下一步反应。In the second step, 0.35mL of 2.0M carboxyamine hydrochloride solution was added to the 1h solution (11.0mL), and after shaking at 25°C for 30 minutes, the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH6 .5 0.05M PBS solution), the product 2h solution (concentration 6.2mg/mL, 15.0mL) was obtained, which was concentrated to about 10mg/mL and used for the next reaction.
第三步，将化合物MC-SN-38(1.3mg，1.2moL)溶解于0.3ml的乙腈中，加入2h溶液(浓度6.2mg/mL，3.0mL)中，于25℃下振荡反应4小时后，将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH6.5的0.05M的PBS溶液)后，在无菌条件下用滤器过滤后得到产物Ab-SN-38抗体-药物偶联物的PBS缓冲液(3.7mg/mL，4.7mL)，于4℃冷藏。采用紫外法测定平均值y。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。In the third step, the compound MC-SN-38 (1.3mg, 1.2moL) was dissolved in 0.3ml of acetonitrile, added to the 2h solution (concentration 6.2mg/mL, 3.0mL), and the reaction was shaken at 25°C for 4 hours. After the reaction solution was desalted and purified with a Sephadex G25 gel column (elution phase: 0.05M PBS solution with pH 6.5), the product was filtered under sterile conditions with a filter to obtain the product Ab-SN-38 antibody-drug coupling PBS buffer (3.7mg/mL, 4.7mL) of the substance, refrigerated at 4°C. The average value y was determined by the ultraviolet method. After placing the cuvette containing sodium succinate buffer in the reference absorption cell and the sample determination absorption cell, after deducting the solvent blank, place the cuvette containing the test solution in the sample determination absorption cell Measure the absorbance at 280nm and 370nm.
数据处理：data processing:
通过建立标准曲线，测定280nm波长下的吸收，确定抗体含量Cmab，测定370nm波长下的吸收，确定小分子含量CDrug。By establishing a standard curve, measuring the absorption at a wavelength of 280nm to determine the antibody content Cmab, and measuring the absorption at a wavelength of 370nm to determine the small molecule content CDrug.
药物载量平均值y＝CDrug/Cmab。The average drug load y=CDrug/Cmab.
通过上述方法测定Ab2-SN-38抗体-药物偶联物的DAR平均值y为3.9。The DAR average value y of the Ab2-SN-38 antibody-drug conjugate determined by the above method is 3.9.
实施例9抗体偶联依喜替康衍生物Example 9 Antibody Conjugation to Exotecan Derivatives
Figure PCTCN2021082735-appb-000080
Figure PCTCN2021082735-appb-000080
第一步，将2a(2g，17.2mmol溶于75mL乙腈中，依次加入碳酸钾(9.27g，67.2mmol)、溴化苄(20mL，167.2mmol)和四丁基碘化铵(620mg，1.68mmol)。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(20ml)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，得到产物5a(3.2g，产率：90.1％)。In the first step, 2a (2g, 17.2mmol) was dissolved in 75mL of acetonitrile, and potassium carbonate (9.27g, 67.2mmol), benzyl bromide (20mL, 167.2mmol) and tetrabutylammonium iodide (620mg, 1.68mmol) were added in sequence. The reaction solution was stirred at room temperature for 48 hours, filtered through celite, the filter cake was rinsed with ethyl acetate (20ml), the combined filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with a developing solvent system C to obtain Product 5a (3.2g, yield: 90.1%).
第二步，将5a(181.3mg,0.879mmol)和4b(270mg，0.733mmol)加入反应瓶，加入6mL四氢呋喃，氩气置换三次，冰水浴降温至0-5℃，加入叔丁醇钾(164mg，1.46mmol)，撤去冰浴，升至室温搅拌40分钟，加入15mL冰水，用乙酸乙酯(40mL×2)和氯仿(20mL×5)萃取，合并有机相并浓缩。所得残余物溶于6mL二氧六环中，加入3mL水，加入碳酸氢钠(73.8mg，0.879mmol)和氯甲酸-9-芴甲酯(190mg，0.734mmol)，室温搅拌2小时。加入30mL水，用乙酸乙酯(20mL×3)萃取，有机相用饱和氯化钠溶液(30mL)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，得到产物5b 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(73mg，产率：19.4％)。In the second step, add 5a (181.3mg, 0.879mmol) and 4b (270mg, 0.733mmol) to the reaction flask, add 6mL of tetrahydrofuran, replace with argon three times, cool to 0-5℃ in an ice-water bath, add potassium tert-butoxide (164mg , 1.46 mmol), remove the ice bath, warm to room temperature and stir for 40 minutes, add 15 mL ice water, extract with ethyl acetate (40 mL×2) and chloroform (20 mL×5), combine the organic phases and concentrate. The obtained residue was dissolved in 6 mL of dioxane, 3 mL of water was added, sodium bicarbonate (73.8 mg, 0.879 mmol) and 9-fluorene methyl chloroformate (190 mg, 0.734 mmol) were added, and the mixture was stirred at room temperature for 2 hours. 30 mL of water was added, extracted with ethyl acetate (20 mL×3), the organic phase was washed with saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography with developing solvent system C to obtain the product 5b 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-diox Benzyl hetero-4,7-diazaundec-11-acid (73 mg, yield: 19.4%).
MS m/z(ESI):515.0[M+1]。MS m/z(ESI): 515.0[M+1].
第三步，将5b(30mg，0.058mmol)溶于6.75mL四氢呋喃和乙酸乙酯(V:V＝2:1)混合溶剂中，加入钯碳(18mg，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，得到粗品产物5c 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(20mg)，产品不经纯化直接进行下一步反应。The third step is to dissolve 5b (30mg, 0.058mmol) in 6.75mL of tetrahydrofuran and ethyl acetate (V:V=2:1) mixed solvent, add palladium on carbon (18mg, content 10%, dry type), replace with hydrogen Three times, the reaction was stirred at room temperature for 1 hour. The reaction solution was filtered with diatomaceous earth, the filter cake was rinsed with ethyl acetate, and the filtrate was concentrated to obtain the crude product 5c 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo- 2,9-dioxa-4,7-diazaundec-11-acid (20mg), the product was directly subjected to the next reaction without purification.
MS m/z(ESI):424.9[M+1]。MS m/z(ESI): 424.9[M+1].
第四步，将1b(15mg，28.2μmol)加入反应瓶，加入1.5mL 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5c(20mg，47.1μmol)，加入4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐(25.4mg，86.2μmol)，冰浴搅拌反应40分钟。加入15mL水，用乙酸乙酯(20mL×3)萃取，合并有机相。有机相用饱和氯化钠溶液(20mL×2)洗涤，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5d(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(23.7mg，产率：78.9％)。In the fourth step, add 1b (15mg, 28.2μmol) into the reaction flask, add 1.5mL N,N-dimethylformamide, replace with argon three times, cool to 0-5℃ in an ice water bath, and add one drop of triethylamine. Add crude product 5c (20mg, 47.1μmol), add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine salt (25.4mg, 86.2μmol), the reaction was stirred in an ice bath for 40 minutes. Add 15 mL of water, extract with ethyl acetate (20 mL×3), and combine the organic phases. The organic phase was washed with saturated sodium chloride solution (20 mL×2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue obtained was purified by thin layer chromatography with the developing solvent system B to obtain the title product 5d(9H-fluoren-9-yl)methyl(2-(((1-cyclopropyl-2-(((1S,9S) )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[ de]pyrano[3',4':6,7]indolozino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino) -2-oxoethyl)carbamate (23.7 mg, yield: 78.9%).
MS m/z(ESI):842.1[M+1]。MS m/z(ESI): 842.1[M+1].
第五步，将5d(30mg，35.7μmol)溶于3mL二氯甲烷中，加入1.5mL二乙胺，室温搅拌2小时。反应液减压浓缩，加入1.5mL甲苯并减压浓缩，重复两次。向残余物中加入4.5mL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，油泵拉干得到粗品产物5e 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(23mg)，产品不经纯化直接用于下一步反应。In the fifth step, 5d (30 mg, 35.7 μmol) was dissolved in 3 mL of dichloromethane, 1.5 mL of diethylamine was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 1.5 mL of toluene was added and concentrated under reduced pressure, repeated twice. Add 4.5 mL of n-hexane to the residue to make a slurry, and then pour out the supernatant after standing to keep the solid. The solid residue was concentrated under reduced pressure, and the crude product 5e 2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl- 5-Fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3 ',4':6,7]indolozino[1,2-b]quinolin-1-yl)acetamide (23mg), the product was directly used in the next reaction without purification.
MS m/z(ESI):638.0[M+18]。MS m/z(ESI): 638.0[M+18].
第六步，将粗品5e(20mg，32.3μmol)溶于1mL N,N-二甲基甲酰胺，氩气置换三次，冰水浴降温至0-5℃，加入4g(31.8mg，67.3μmol)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(27.8mg，94.3μmol)，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5。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，得到产物5-A和5-B(3.6mg，2.6mg)。 In the sixth step, the crude product 5e (20mg, 32.3μmol) was dissolved in 1mL N,N-dimethylformamide, replaced with argon three times, cooled to 0-5℃ in an ice water bath, and 4g (31.8mg, 67.3μmol) was added. 0.5mL N,N-dimethylformamide solution, add 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine salt ( 27.8 mg, 94.3 μmol), the reaction was stirred in an ice bath for 10 minutes, the ice bath was removed, and the mixture was heated to room temperature and stirred for 1 hour to produce compound 5. The 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), collect the corresponding components and concentrate under reduced pressure to obtain products 5-A and 5-B (3.6 mg, 2.6 mg).
MS m/z(ESI):1074.4[M+1]。MS m/z(ESI): 1074.4[M+1].
单一构型化合物5-A(较短保留时间)：Single configuration compound 5-A (shorter retention time):
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)。 UPLC analysis: retention time 1.14 minutes, purity: 85% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH 4 OAc), B-acetonitrile).
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,2H),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,2H),2.80-2.62(m,2H),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)。 1 H 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,2H),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, 2H), 2.80-2.62 (m, 2H), 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5-B(较长保留时间)：Single configuration compound 5-B (longer retention time):
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)。 UPLC analysis: retention time 1.16 minutes, purity: 89% (chromatographic column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH 4 OAc), B-acetonitrile).
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,4H),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,3H),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,5H),0.91-0.79(m,3H),0.53-0.34(m,4H)。 1 H 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,4H),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, 3H), 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,5H) , 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).
其他中间体的制备方法参考中间体5。Refer to Intermediate 5 for the preparation methods of other intermediates.
在37℃条件下，向抗体Ab2的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；7.3ml，13.8mg/ml，0.681μmol)加入配置好的三(2-羧乙基)膦的水溶液(10mM，0.347mL，3.47μmol)，置于水浴振荡器，于37℃振荡反应3小时，停止反应；将反应液用水浴降温至25℃，稀释至14.0ml，并取出3.3ml溶液往下反应。At 37°C, add the prepared tris (2-carboxyethyl) phosphine to the PBS buffered aqueous solution of antibody Ab2 (0.05M PBS buffered aqueous solution at pH=6.5; 7.3ml, 13.8mg/ml, 0.681μmol) Aqueous solution (10mM, 0.347mL, 3.47μmol), place in a water bath shaker, shake the reaction at 37℃ for 3 hours, stop the reaction; cool the reaction solution to 25℃ with water bath, dilute to 14.0ml, and take out 3.3ml solution down reaction.
将化合物5-A(3.0mg，3.72μmol)溶解于0.15mL DMSO中，加入到上述3.3ml溶液中，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Ab-依喜替康衍生物的示例性产物ADC1(化合物26)的PBS缓冲液(1.35mg/mL，13mL)，于4℃冷冻储存。Compound 5-A (3.0mg, 3.72μmol) was dissolved in 0.15mL DMSO, added to the above 3.3ml solution, placed in a water bath shaker, and reacted with shaking at 25°C for 3 hours to stop the reaction. The reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: pH 6.5 0.05M PBS buffer aqueous solution, containing 0.001M EDTA) to obtain the exemplary product ADC1 (Compound 26) PBS buffer (1.35mg/mL, 13mL), stored frozen at 4°C.
采用紫外法测定平均值y。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。The average value y was determined by the ultraviolet method. After placing the cuvette containing sodium succinate buffer in the reference absorption cell and the sample determination absorption cell, after deducting the solvent blank, place the cuvette containing the test solution in the sample determination absorption cell Measure the absorbance at 280nm and 370nm.
数据处理：data processing:
通过建立标准曲线，测定280nm波长下的吸收，确定抗体含量Cmab，测定370nm波长下的吸收，确定小分子含量CDrug。By establishing a standard curve, measuring the absorption at a wavelength of 280nm to determine the antibody content Cmab, and measuring the absorption at a wavelength of 370nm to determine the small molecule content CDrug.
药物载量平均值y＝CDrug/Cmab。The average drug load y=CDrug/Cmab.
通过以上方法测定示例性产物ADC1(化合物26，为7.6)。通过UV-HPLC纯化获得ADC1-1(y＝4)、ADC1-2(y＝6)、ADC1-3(y＝8)的样品。The exemplary product ADC1 (Compound 26, 7.6) was determined by the above method. Samples of ADC1-1 (y=4), ADC1-2 (y=6), ADC1-3 (y=8) were obtained by UV-HPLC purification.
其他抗体药物偶联物的制备方法参考ADC1。Refer to ADC1 for preparation methods of other antibody-drug conjugates.
实施例10抗体药物偶联物对NCI-H929肿瘤细胞的杀伤作用Example 10 Killing effect of antibody drug conjugate on NCI-H929 tumor cells
为检测本发明的抗体-药物偶联物对肿瘤细胞的杀伤作用，用NCI-H929细胞(ATCC保藏号CRL-9068)进行评估。收集NCI-H929细胞，离心计数后用完全培养基调整细胞密度为0.44×10 6个/mL，铺于白色96孔板中间60个孔，每孔90μL，细胞数为40000，其余边孔加入100μL PBS，细胞板放入37℃，5％CO2培养箱培养过夜。实验第二天，用PBS在96孔V型底板中配制抗体-药物偶联物溶液，浓度为15μg/mL起始，3倍稀释，9个浓度，配制完成后加入到白色96孔板中，每孔10μL，两复孔，将细胞板放入37℃，5％CO2培养箱中继续培养72小时。实验第五天，检测读数：取出细胞培养板，平衡至室温后，每孔加入50μL CTG溶液(Promega G7573)，振荡混匀后放于暗处静置10分钟后，使用酶标仪的发光程序进行检测。使用GraphPad Prims软件计算EC50值。实验结果如下表所示： To test the killing effect of the antibody-drug conjugate of the present invention on tumor cells, NCI-H929 cells (ATCC deposit number CRL-9068) were used for evaluation. Collect NCI-H929 cells, after centrifugal counting, adjust the cell density to 0.44×10 6 cells/mL with complete medium, and spread them in the middle 60 wells of a white 96-well plate, each with 90 μL, the number of cells is 40,000, and add 100 μL to the remaining side holes PBS, the cell plate was placed in a 37°C, 5% CO2 incubator overnight. On the second day of the experiment, prepare the antibody-drug conjugate solution in a 96-well V-bottom plate with PBS, starting with a concentration of 15 μg/mL, 3 times dilution, and 9 concentrations. After the preparation is completed, add it to the white 96-well plate. 10μL per well, two duplicate wells, put the cell plate in a 37°C, 5% CO2 incubator and continue to incubate for 72 hours. On the fifth day of the experiment, test the reading: Take out the cell culture plate, after equilibrating to room temperature, add 50μL CTG solution (Promega G7573) to each well, shake and mix, place in the dark and stand for 10 minutes, then use the luminescence program of the microplate reader Perform testing. The EC50 value was calculated using GraphPad Prims software. The experimental results are shown in the following table:
表9.抗体偶联药物对NCI-H929肿瘤细胞的杀伤活性(EC 50) Table 9. The killing activity (EC 50 ) of antibody-conjugated drugs on NCI-H929 tumor cells
Figure PCTCN2021082735-appb-000081
Figure PCTCN2021082735-appb-000081
实施例11抗体药物偶联物的抑瘤效果Example 11 Anti-tumor effect of antibody drug conjugate
为进一步研究抗体-药物偶联物对体内形成的肿瘤的杀伤作用，在小鼠体内用NCI-H929细胞形成移植瘤后，评估本发明抗体-药物偶联物的抗肿瘤效果。将9x10 6个NCI-929细胞注射到8周龄的免疫缺陷的裸鼠(NOD-SCID)皮下，8天后开始通过静脉注射注射抗体-药物偶联物ADC2(Ab2-MC-MMAF，实施例7，y＝4)和ADC1-3(实施例9，y＝8)，每1周注射一次，剂量为1mg/kg。对照采用人IgG1蛋白，剂量为1mg/kg。对照组或给药组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。抑瘤率＝100％-(第14天给药组肿瘤体积-第0天给药组肿瘤体积)/(第14天对照组肿瘤体积-第0天对照组肿瘤体积)。实验结果如表10所示。抗体-药物偶联物ADC2(Ab2-MC-MMAF，实施例7，y＝4)和ADC1-3(实 施例9，y＝8)均显示抑瘤活性。 In order to further study the killing effect of the antibody-drug conjugate on the tumor formed in the body, the anti-tumor effect of the antibody-drug conjugate of the present invention was evaluated after the transplanted tumor was formed with NCI-H929 cells in mice. 9 ×10 6 NCI-929 cells were injected subcutaneously into 8-week-old immunodeficient nude mice (NOD-SCID). After 8 days, the antibody-drug conjugate ADC2 (Ab2-MC-MMAF, Example 7) was injected intravenously. , Y=4) and ADC1-3 (Example 9, y=8), injected once every 1 week, with a dose of 1 mg/kg. Human IgG1 protein was used as the control, and the dose was 1 mg/kg. There were 5 mice in each group in the control group or the administration group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate=100%-(Tumor volume of the administration group on the 14th day-Tumor volume of the administration group on the 0th day)/(Tumor volume of the control group on the 14th day-Tumor volume of the control group on the 0th day). The experimental results are shown in Table 10. The antibody-drug conjugate ADC2 (Ab2-MC-MMAF, Example 7, y=4) and ADC1-3 (Example 9, y=8) both showed antitumor activity.
表10.抗体-药物偶联物的抑瘤作用Table 10. Anti-tumor effect of antibody-drug conjugates
给药组G 抑瘤率Tumor inhibition rate
ADC2(实施例7，y＝4)1mg/kgADC2 (Example 7, y=4) 1mg/kg 29.9％29.9%
ADC1-3(实施例9，y＝8)1mg/kgADC1-3 (Example 9, y=8) 1mg/kg 61.1％61.1%
实施例12抗体药物偶联物的体内肿瘤杀伤作用Example 12 In vivo tumor killing effect of antibody drug conjugate
为进一步研究抗体-药物偶联物对体内形成的肿瘤的杀伤作用，在小鼠体内用NCI-H929细胞形成移植瘤后，评估本发明抗体-药物偶联物的抗肿瘤效果。将9x10 6个NCI-929细胞注射到8周龄的免疫缺陷的裸鼠(NOD-SCID)皮下，8天后开始通过静脉注射抗体-药物偶联物ADC2(实施例7，y＝4)和ADC3(实施例7，y＝4.1)，每1周注射2次，剂量为3mg/kg。对照采用人IgG1蛋白，剂量为3mg/kg。对照组或给药组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。抑瘤率TGI＝100％-(第14天给药组肿瘤体积-第0天给药组肿瘤体积)/(第14天对照组肿瘤体积-第0天对照组肿瘤体积)。实验结果如表11所示，ADC2(实施例7，y＝4)和ADC3(实施例7，y＝4.1)均显示对肿瘤的杀伤作用。 In order to further study the killing effect of the antibody-drug conjugate on the tumor formed in the body, the anti-tumor effect of the antibody-drug conjugate of the present invention was evaluated after the transplanted tumor was formed with NCI-H929 cells in mice. 9 ×10 6 NCI-929 cells were injected subcutaneously into 8-week-old immunodeficient nude mice (NOD-SCID). After 8 days, the antibody-drug conjugate ADC2 (Example 7, y=4) and ADC3 were injected intravenously. (Example 7, y=4.1), injected twice every 1 week, with a dose of 3 mg/kg. Human IgG1 protein was used as the control, and the dose was 3 mg/kg. There were 5 mice in each group in the control group or the administration group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate TGI=100%-(Tumor volume of the administration group on the 14th day-Tumor volume of the administration group on the 0th day)/(Tumor volume of the control group on the 14th day-Tumor volume of the control group on the 0th day). The experimental results are shown in Table 11. Both ADC2 (Example 7, y=4) and ADC3 (Example 7, y=4.1) showed killing effects on tumors.
表11.抗体-药物偶联物对体内肿瘤的杀伤活性Table 11. Antibody-drug conjugates killing activity on tumors in vivo
给药组G 抑瘤率TGITumor inhibition rate TGI
ADC2(实施例7，y＝4)3mg/kgADC2 (Example 7, y=4) 3mg/kg 192％192%
ADC3(实施例7，y＝4.1)3mg/kgADC3 (Example 7, y=4.1) 3mg/kg 175％175%
实施例13抗体药物偶联物肿瘤细胞杀伤活性Example 13 Antibody Drug Conjugate Tumor Cell Killing Activity
为检测本发明的抗体-药物偶联物对肿瘤细胞的杀伤作用，采用BCMA高表达水平细胞株NCI-H929(ATCC保藏号CRL-9068)和BCMA低表达水平细胞株RPMI-8226(ATCC，cat:CCL-155)进行评估。收集NCI-H929、RPMI-8226细胞，离心计数后用完全培养基调整细胞密度为1×10 5个/mL，铺于白色96孔板中间60个孔，每孔100μl，细胞数为10000细胞/孔，边缘孔加入100μl/孔DPBS，细胞板放入37℃，5％CO 2培养箱培养过夜。次日，用完全培养基在96孔V型底板中配制抗体-药物偶联物工作溶液，浓度为133uM起始，5倍稀释，9个浓度，配制完成后加入到白色96孔板中，每孔80μl，两复孔，将细胞板放入37℃，5％CO 2培养箱中继续培养72小时。实验第五天，检测读数：取出细胞培养板，平衡至室温后，每孔加入90μl
Figure PCTCN2021082735-appb-000082
细胞活力检测试剂(Promega，Cat#:G7573)，振荡混匀后放于暗处静置10分钟后，使用酶标仪的发光程序进行检测。使用GraphPad Prims软件计算IC 50值。实验结果如下表所示： In order to detect the killing effect of the antibody-drug conjugate of the present invention on tumor cells, the BCMA high expression level cell line NCI-H929 (ATCC deposit number CRL-9068) and the BCMA low expression level cell line RPMI-8226 (ATCC, cat :CCL-155) for evaluation. Collect NCI-H929 and RPMI-8226 cells. After centrifugation and count, adjust the cell density to 1×10 5 cells/mL with complete medium, and spread them in the middle 60 wells of a white 96-well plate. Add 100μl/well DPBS to the hole and edge hole, and place the cell plate in a 37°C, 5% CO 2 incubator overnight. The next day, prepare the antibody-drug conjugate working solution in a 96-well V-bottom plate with complete medium, starting with a concentration of 133uM, 5-fold dilution, and 9 concentrations. After the preparation is complete, add it to the white 96-well plate. Hole 80μl, double hole, put the cell plate into 37°C, 5% CO 2 incubator and continue to incubate for 72 hours. On the fifth day of the experiment, test the reading: take out the cell culture plate, after equilibrating to room temperature, add 90μl to each well
Figure PCTCN2021082735-appb-000082
Cell viability detection reagent (Promega, Cat#: G7573), shake and mix, put it in the dark and let it stand for 10 minutes, then use the luminescence program of the microplate reader for detection. IC 50 values were calculated using GraphPad Prims software. The experimental results are shown in the following table:
表12.抗体偶联药物对肿瘤细胞的杀伤作用Table 12. The killing effect of antibody-conjugated drugs on tumor cells
Figure PCTCN2021082735-appb-000083
Figure PCTCN2021082735-appb-000083
实验结果表明BCMA-ADC对肿瘤细胞的体外杀伤活性与BCMA在细胞表面的表达水平成正相关，对NCI-H929杀伤效果明显强于对RPMI-8226杀伤；随着偶联小分子毒素的数量增加，对肿瘤细胞的杀伤活性随之提高。The experimental results show that the in vitro killing activity of BCMA-ADC on tumor cells is positively correlated with the expression level of BCMA on the cell surface. The killing effect on NCI-H929 is significantly stronger than that on RPMI-8226; as the number of coupled small molecule toxins increases , The killing activity of tumor cells will increase accordingly.
实施例14抗体药物偶联物PK研究Example 14 PK Study of Antibody Drug Conjugate
利用BALB/c小鼠模型评价抗BCMA体药物偶联物BCMA-ADC在小鼠体内的药物代谢情况。将平均体重为18-22g，18-22周龄的人BALB/c转基因小鼠(上海西普尔－必凯实验动物有限公司)随机分为2组，每组3只动物，受试BCMA体药物偶联物均以10mpk，IV，单次的方式给药，PBS溶媒作为阴性对照组，分别在1、2、4、8、24、48、96、144、240、小时采血分离血浆，冻存于-20℃冰箱，之后利用重组人BCMA蛋白包被高亲和力96孔瓶底板，加入稀释的待测血浆样品，再利用HRP标记二抗检测小鼠血浆中BCMA-ADC浓度，并利用WinNonlin软件非房室模型，血管内给药的公式分析其PK参数。实验结果详见下表The BALB/c mouse model was used to evaluate the drug metabolism of the anti-BCMA drug conjugate BCMA-ADC in mice. Human BALB/c transgenic mice (Shanghai Xipuer-Bikai Experimental Animal Co., Ltd.) with an average weight of 18-22g and 18-22 weeks of age were randomly divided into 2 groups, each with 3 animals, and the BCMA body drug was tested The conjugates were administered in a single dose of 10 mpk, IV, and PBS was used as a negative control group. Blood was collected at 1, 2, 4, 8, 24, 48, 96, 144, 240, hours to separate the plasma, and stored frozen In the refrigerator at -20℃, the bottom plate of the high-affinity 96-well vial was coated with recombinant human BCMA protein, and the diluted plasma sample to be tested was added. The HRP-labeled secondary antibody was used to detect the concentration of BCMA-ADC in mouse plasma, and the WinNonlin software was used to detect the concentration of BCMA-ADC in mouse plasma. Atrioventricular model, the formula of intravascular administration analyzes its PK parameters. The experimental results are detailed in the table below
表13.抗体偶联药物在小鼠体内的药代动力学参数Table 13. Pharmacokinetic parameters of antibody-conjugated drugs in mice
参数parameter ADC1-2(实施例9，y＝6)ADC1-2 (Example 9, y=6)
C0(ug/mL)C0(ug/mL) 116.4116.4
AUC 0-t(ug/mL*h)AUC 0-t(ug/mL*h) 1000210002
AUC 0-∞(ug/mL*h)AUC 0-∞(ug/mL*h) 2056820568
t 1/2(h) t 1/2 (h) 258.0258.0
MRT 0-∞(h) MRT 0-∞ (h) 363.3363.3
CL(mL/h/kg)CL(mL/h/kg) 0.150.15
Vss(mL/kg)Vss(mL/kg) 53.053.0
综合半衰期t 1/2、暴露量、血药最高浓度达峰时间等参数，ADC1-2显示出良好的体内代谢活性。 Integrating parameters such as half-life t 1/2 , exposure, and peak blood concentration, ADC1-2 shows good metabolic activity in the body.
实施例15抗体药物偶联物的细胞杀伤活性Example 15 Cell killing activity of antibody drug conjugate
为检测本公开的抗体-药物偶联物(ADC)及依喜替康衍生物(实施例9，化合物(5-A))对肿瘤细胞的细胞毒作用，采用BCMA高表达水平细胞株NCI-H929(ATCC保藏号CRL-9068)进行评估。In order to detect the cytotoxic effects of the antibody-drug conjugates (ADC) and exenotecan derivatives (Example 9, compound (5-A)) of the present disclosure on tumor cells, the BCMA high-expression cell line NCI- H929 (ATCC deposit number CRL-9068) was evaluated.
收集NCI-H929细胞，离心计数后，用完全培养基调整细胞密度为2×10 5个/mL，铺于白色96孔板中间60个孔，每孔50μL，细胞数为10000细胞/孔，边缘孔加入100μL/孔DPBS，细胞板放入37℃，5％CO 2培养箱培养过夜。次日，用完全培养基在96孔V型底板中配制抗体-药物偶联物工作溶液，使依喜替康衍生物化合物(5-A)与ADC按相等摩尔量进行稀释，最高工作浓度为300nM，5倍稀释，9个浓度，配制完成后加入到白色96孔板中，每孔100μL，两复孔，将 细胞板放入37℃，5％CO 2培养箱中继续培养72小时。实验第五天，检测读数：取出细胞培养板，平衡至室温后，每孔加入75μL
Figure PCTCN2021082735-appb-000084
细胞活力检测试剂(Promega，Cat#:G7573)，振荡混匀后放于暗处静置10分钟后，使用酶标仪的发光程序进行检测。使用GraphPad Prims软件计算IC 50值。使用阴性抗体IgG1与依喜替康衍生物化合物(5-A)的抗体药物偶联物(ADC1-4，y＝4)作为阴性对照。实验结果如下表所示： Collect NCI-H929 cells, after centrifugal counting, adjust the cell density to 2×10 5 cells/mL with complete medium, and spread them in the middle of the white 96-well plate in 60 wells, 50μL per well, the cell number is 10,000 cells/well, edge Add 100 μL/well DPBS to the wells, and place the cell plate in a 37°C, 5% CO 2 incubator overnight. The next day, the antibody-drug conjugate working solution was prepared in a 96-well V-bottom plate with complete medium, so that the exenotecan derivative compound (5-A) and ADC were diluted in equal molar amounts, and the maximum working concentration was 300nM, 5-fold dilution, 9 concentrations, after preparation, add to a white 96-well plate, 100μL per well, two replicate wells, put the cell plate in a 37°C, 5% CO 2 incubator and continue to incubate for 72 hours. On the fifth day of the experiment, check the reading: take out the cell culture plate, after equilibrating to room temperature, add 75μL to each well
Figure PCTCN2021082735-appb-000084
Cell viability detection reagent (Promega, Cat#: G7573), shake and mix, put it in the dark and let it stand for 10 minutes, then use the luminescence program of the microplate reader for detection. IC 50 values were calculated using GraphPad Prims software. The antibody-drug conjugate (ADC1-4, y=4) of negative antibody IgG1 and exenotecan derivative compound (5-A) was used as a negative control. The experimental results are shown in the following table:
表14.测试化合物对肿瘤细胞毒杀伤作用Table 14. Toxic and killing effects of test compounds on tumor cells
Figure PCTCN2021082735-appb-000085
Figure PCTCN2021082735-appb-000085
通过比较细胞毒细胞杀伤IC 50，ADC1-1(y＝4)与化合物(5-A)相比，约为1/115(0.88/101)倍，实验结果表明：本发明抗体药物偶联物对肿瘤细胞的体外细胞毒杀伤活性对NCI-H929杀伤效果明显强于小分子毒素化合物(5-A)的杀伤效果。 By comparing the cytotoxic cell killing IC 50 , ADC1-1 (y=4) is about 1/115 (0.88/101) times compared with compound (5-A). The experimental results show that: the antibody-drug conjugate of the present invention The in vitro cytotoxic killing activity on tumor cells is obviously stronger than the killing effect of the small molecule toxin compound (5-A) on NCI-H929.
实施例16依喜替康衍生物的制备Example 16 Preparation of Isinotecan Derivatives
Figure PCTCN2021082735-appb-000086
Figure PCTCN2021082735-appb-000086
向2e(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1e(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-C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 NH4OAc)，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)。Add 2mL ethanol and 0.4mL N,N-dimethylformamide to 2e (4mg, 7.53μmol), replace with argon three times, cool to 0-5℃ in an ice water bath, add 0.3mL N-methylmorpholine dropwise, Stir until the reaction solution becomes clear. Add 2-cyclopropyl-2-hydroxyacetic acid 1e (2.3mg, 19.8μmol, prepared by the method disclosed in the patent application "WO2013106717") and 1-hydroxybenzotriazole (3mg, 22.4μmol) to the reaction solution in sequence. ) And 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3mg, 22.4μmol), after the addition, the reaction was stirred at 0-5°C for 1 hour. Remove the ice-water bath, heat to 30°C and stir for 2 hours. The reaction solution was concentrated under reduced pressure, and the obtained crude compound 2-C was purified by high performance liquid chromatography (Separation conditions: Column: XBridge Prep C18 OBD 5um 19*250mm; Mobile phase: A-water (10mmol NH4OAc), 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).
MS m/z(ESI):534.0[M+1]。MS m/z(ESI): 534.0[M+1].
单一构型化合物2-B(较短保留时间)Single configuration compound 2-B (shorter retention time)
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 NH4OAc),B-乙腈)。UPLC analysis: retention time 1.06 minutes, purity: 88% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).
1HNMR(400MHz,DMSO-d6):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)。 1 HNMR (400MHz, DMSO-d6): 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)Single configuration compound 2-A (longer retention time)
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 NH4OAc),B-乙腈)。UPLC analysis: retention time 1.10 minutes, purity: 86% (column: ACQUITY UPLC BEHC18 1.7um 2.1*50mm, mobile phase: A-water (5mmol NH4OAc), B-acetonitrile).
1HNMR(400MHz,DMSO-d6):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)。 1 HNMR (400MHz, DMSO-d6): 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17依喜替康衍生物对肿瘤细胞体外增殖抑制测试Example 17 Inhibition Test of In Vitro Proliferation of Tumor Cells by Ixinotecan Derivatives
检测化合物2-A和2-B，对U87MG细胞(中科院细胞库，Catalog#TCHu138)和SK-BR-3肿瘤细胞(人乳腺癌细胞，ATCC，货号HTB-30)体外增殖的抑制活性。以不同浓度的化合物体外处理细胞，经6天培养后，采用CTG(Luminescent Cell Viability Assay，Promega，货号：G7573)试剂对细胞的增值进行检测，根据IC50值评价该化合物的体外活性。The compounds 2-A and 2-B were tested for their inhibitory activity on the in vitro proliferation of U87MG cells (Cell Bank of Chinese Academy of Sciences, Catalog#TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, catalog number HTB-30). The cells were treated in vitro with different concentrations of the compound, and after 6 days of culture, the proliferation of the cells was detected with CTG (Luminescent Cell Viability Assay, Promega, catalog number: G7573) reagent, and the in vitro activity of the compound was evaluated according to the IC50 value.
U87MG和SK-BR-3细胞分别用10％FBS的EMEM培养基(GE，货号SH30024.01)和含10％FBS的McCoy's 5A培养基(Gibco，货号16600-108)培养。U87MG and SK-BR-3 cells were cultured with 10% FBS in EMEM medium (GE, article number SH30024.01) and McCoy's 5A medium (Gibco, article number 16600-108) containing 10% FBS, respectively.
取对数生长期的U87MG和SK-BR-3细胞，用PBS(磷酸盐缓冲液，上海源培生物科技股份有限公司)洗涤1次之后，加入2-3ml胰蛋白酶(0.25％Trypsin-EDTA(1x)，Gibico,Life Technologies公司)消化2-3min，待细胞消化完全后，加入10-15ml细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，接着加入10-20ml细胞培养液将细胞重悬，制成单细胞悬液。Take the U87MG and SK-BR-3 cells in the logarithmic growth phase, wash them with PBS (phosphate buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3ml trypsin (0.25% Trypsin-EDTA( 1x), Gibico, Life Technologies) digest for 2-3min, after the cells are digested completely, add 10-15ml cell culture solution, wash the digested cells, centrifuge at 1000rpm for 5min, discard the supernatant, and then add 10-20ml The cell culture fluid resuspends the cells to make a single cell suspension.
将U87MG和SK-BR-3单细胞悬液混匀，用细胞培养液分别调整活细胞密度至2.75×103cells/ml和8.25×103cells/ml，将密度调整过后的细胞悬液混匀，以180μl/孔加入96孔细胞培养板。96孔板外周孔只加入200ul培养基。将培养板在培养箱培养24小时(37℃，5％CO 2)。 Mix the U87MG and SK-BR-3 single cell suspensions, adjust the viable cell density to 2.75×103cells/ml and 8.25×103cells/ml with cell culture medium, and mix the cell suspension after the density adjustment to 180μl /Well add 96-well cell culture plate. Only add 200ul medium to the peripheral wells of the 96-well plate. The culture plate was cultured in an incubator for 24 hours (37°C, 5% CO 2 ).
用DMSO(二甲基亚砜，上海泰坦科技股份有限公司)溶解化合物，配制成初始浓度为10mM的存储液。The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to prepare a storage solution with an initial concentration of 10 mM.
小分子化合物的起始浓度为500nM，配药方法如下：The initial concentration of the small molecule compound is 500nM, and the dispensing method is as follows:
在96孔U型底配药板第一列中分别加入30μl不同待测样品，样品浓度为100uM；第2列至第11列每孔中加入20ul DMSO。取第一列样品10ul至第二列20ul DMSO中，混匀，取10ul至第三列中，以此类推至第10列。将配药板中的药每孔取5ul至95ul EMEM培养基中，混匀，待用。Add 30μl of different samples to be tested into the first column of the 96-well U-bottomed dispensing plate, with a sample concentration of 100uM; add 20ul DMSO to each well in the second column to the 11th column. Take 10ul of the sample from the first column to 20ul in the second column and mix well, then take 10ul to the third column, and so on to the 10th column. Take 5ul to 95ul of EMEM medium from each well of the medicine in the dispensing plate, mix well, and set aside.
加样：向培养板中加入20μl配置的不同浓度的待测样品，每个样品两复孔。将培养板在培养箱孵育6天(37℃，5％CO 2)。 Adding samples: Add 20μl of the tested samples of different concentrations to the culture plate, and each sample has two duplicate wells. The culture plate was incubated in an incubator for 6 days (37°C, 5% CO 2 ).
显色：取出96孔细胞培养板，向每孔加入90μl CTG溶液，室温孵育10分钟。Color development: Take out the 96-well cell culture plate, add 90μl CTG solution to each well, and incubate at room temperature for 10 minutes.
读板：取出96孔细胞培养板，置于酶标仪(BMG labtech，PHERAstar FS)中，用酶标仪测定化学发光。Plate reading: Take out the 96-well cell culture plate, place it in a microplate reader (BMG labtech, PHERAstar FS), and measure chemiluminescence with the microplate reader.
数据分析：用Microsoft Excel，Graphpad Prism 5对数据进行处理分析。实验结果参见表15。Data analysis: Use Microsoft Excel, Graphpad Prism 5 to process and analyze the data. See Table 15 for the experimental results.
表15.依喜替康衍生物对肿瘤细胞对肿瘤细胞的杀伤作用Table 15. The killing effect of exenotecan derivatives on tumor cells against tumor cells
Figure PCTCN2021082735-appb-000087
Figure PCTCN2021082735-appb-000087

Claims (23)

  1. 一种通式(A)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，An antibody drug conjugate represented by general formula (A) or a pharmaceutically acceptable salt or solvent compound thereof,
    Figure PCTCN2021082735-appb-100001
    Figure PCTCN2021082735-appb-100001
    其中：in:
    D是细胞毒性药物；D is a cytotoxic drug;
    L 1、L 2是接头单元； L 1 and L 2 are joint units;
    y选自1-20的数；y is a number selected from 1-20;
    Ab为抗BCMA抗体或其抗原结合片段，所述抗BCMA抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，重链可变区包含SEQ ID NO:3所示的HCDR1、SEQ ID NO:4所示的HCDR2和SEQ ID NO:5所示的HCDR3；以及轻链可变区包含SEQ ID NO:6所示的LCDR1、SEQ ID NO:7所示的LCDR2和SEQ ID NO:8所示的LCDR3。Ab is an anti-BCMA antibody or antigen-binding fragment thereof. The anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 3 HCDR1, HCDR2 shown in SEQ ID NO: 4 and HCDR3 shown in SEQ ID NO: 5; and the light chain variable region includes LCDR1 shown in SEQ ID NO: 6 and LCDR2 shown in SEQ ID NO: 7 and SEQ ID NO: 7 ID NO: LCDR3 shown in 8.
  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或溶剂化合物，其中所述抗BCMA抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段，嵌合抗体或其抗原结合片段，人抗体或其抗原结合片段或人源化抗体或其抗原结合片段。The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 1, wherein the anti-BCMA antibody or antigen-binding fragment thereof is selected from murine antibody or antigen-binding fragment thereof, chimeric antibody or Its antigen-binding fragment, human antibody or its antigen-binding fragment or humanized antibody or its antigen-binding fragment.
  3. 根据权利要求2所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述抗BCMA抗体或其抗原结合片段进一步包含人IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 2, wherein the anti-BCMA antibody or antigen-binding fragment thereof further comprises human IgG1, IgG2, IgG3, or IgG4 or a variant thereof Heavy chain constant region,
    优选地，所述抗BCMA抗体或其抗原结合片段进一步包含氨基酸突变后具有增强的ADCC毒性的IgG1重链恒定区；Preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises an IgG1 heavy chain constant region with enhanced ADCC toxicity after amino acid mutation;
    或者，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO:22所示的重链恒定区。Alternatively, the anti-BCMA antibody or antigen-binding fragment thereof further comprises a heavy chain constant region as shown in SEQ ID NO: 22.
  4. 根据权利要求2所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述抗BCMA抗体或其抗原结合片段进一步包含人抗体κ链、λ链或其变体的轻链恒定区；优选地，所述抗BCMA抗体或其抗原结合片段进一步包含人抗体κ链的轻链恒定区；进一步优选地，所述抗BCMA抗体或其抗原结合片段进一步包含如SEQ ID NO:23所示的轻链恒定区。The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 2, wherein the anti-BCMA antibody or antigen-binding fragment thereof further comprises a human antibody kappa chain, lambda chain or a variant thereof. Chain constant region; preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises the light chain constant region of a human antibody kappa chain; further preferably, the anti-BCMA antibody or antigen-binding fragment thereof further comprises SEQ ID NO: The light chain constant region shown at 23.
  5. 根据权利要求2所述的抗体药物偶联物或其药学上可接受的盐或溶剂化 合物，其中所述抗BCMA抗体或其抗原结合片段包含选自以下序列所示的重链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的重链可变区：SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11；The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 2, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain variable region selected from the following sequences, or A heavy chain variable region with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared with the following sequences: SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO:11;
    和/或，选自以下序列所示的轻链可变区，或与以下序列相比具有至少70％，75％，80％，85％，90％，95％或99％同一性的轻链可变区：SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14。And/or, selected from the light chain variable region shown in the following sequence, or light chain having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% identity compared with the following sequence Variable regions: SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14.
  6. 根据权利要求5所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其中，所述抗BCMA抗体或其抗原结合片段包含：The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 5, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises:
    SEQ ID NO：9所示的重链可变区和SEQ ID NO：12所示的轻链可变区；或，The heavy chain variable region shown in SEQ ID NO: 9 and the light chain variable region shown in SEQ ID NO: 12; or,
    SEQ ID NO：10所示的重链可变区和SEQ ID NO：13所示的轻链可变区；或，SEQ ID NO：11所示的重链可变区和SEQ ID NO：14所示的轻链可变区。The heavy chain variable region shown in SEQ ID NO: 10 and the light chain variable region shown in SEQ ID NO: 13; or the heavy chain variable region shown in SEQ ID NO: 11 and the heavy chain variable region shown in SEQ ID NO: 14 The variable region of the light chain shown.
  7. 根据权利要求5所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述抗BCMA抗体或其抗原结合片段包含选自如下序列所示的重链，或与以下序列相比具有至少80％，85％，90％，95％或99％同一性的重链：SEQ ID NO：15、SEQ ID NO：16和SEQ ID NO：17；The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 5, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises a heavy chain selected from the following sequence, or is combined with the following sequence Compared with heavy chains with at least 80%, 85%, 90%, 95% or 99% identity: SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17;
    和/或，选自以下序列所示的轻链，或与以下序列相比具有至少80％，85％，90％，95％或99％同一性的轻链：SEQ ID NO：18、SEQ ID NO：19和SEQ ID NO：20。And/or, selected from the light chains shown in the following sequences, or light chains with at least 80%, 85%, 90%, 95% or 99% identity compared with the following sequences: SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20.
  8. 根据权利要求7所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述抗BCMA抗体或其抗原结合片段包含：The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 7, wherein the anti-BCMA antibody or antigen-binding fragment thereof comprises:
    SEQ ID NO：15所示的重链和SEQ ID NO：18所示的轻链；或，The heavy chain shown in SEQ ID NO: 15 and the light chain shown in SEQ ID NO: 18; or,
    SEQ ID NO：16所示的重链和SEQ ID NO：19所示的轻链；或，The heavy chain shown in SEQ ID NO: 16 and the light chain shown in SEQ ID NO: 19; or,
    SEQ ID NO：17所示的重链和SEQ ID NO：20所示的轻链。The heavy chain shown in SEQ ID NO: 17 and the light chain shown in SEQ ID NO: 20.
  9. 根据权利要求1-8任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其中所述的细胞毒性药物选自毒素、化疗药物、抗生素、放射性同位素和核溶酶；优选抑制细胞分裂的微管蛋白抑制剂或DNA拓扑异构酶抑制剂；进一步优选DM1、DM3、DM4、喜树碱、SN-38、MMAF或MMAE；更优选MMAE或MMAF，或SN-38：The antibody-drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to any one of claims 1-8, wherein the cytotoxic drug is selected from the group consisting of toxins, chemotherapeutic drugs, antibiotics, radioisotopes and nucleolytic compounds. Enzyme; preferably a tubulin inhibitor or DNA topoisomerase inhibitor that inhibits cell division; more preferably DM1, DM3, DM4, camptothecin, SN-38, MMAF or MMAE; more preferably MMAE or MMAF, or SN- 38:
    Figure PCTCN2021082735-appb-100002
    Figure PCTCN2021082735-appb-100002
    或者，所述的细胞毒性药物选自喜树碱衍生物，优选依喜替康或依喜替康衍生物：Alternatively, the cytotoxic drug is selected from camptothecin derivatives, preferably exenotecan or exenotecan derivatives:
    Figure PCTCN2021082735-appb-100003
    Figure PCTCN2021082735-appb-100003
  10. 根据权利要求9所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为通式(III)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化合物：The antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 9, which is an antibody drug conjugate represented by the general formula (III) or a pharmaceutically acceptable salt or solvent compound thereof :
    Figure PCTCN2021082735-appb-100004
    Figure PCTCN2021082735-appb-100004
    其中：in:
    L 1、L 2是接头单元； L 1 and L 2 are joint units;
    y为选自1-10的数，优选为2-8的数，更优选4-8的数；y is a number selected from 1-10, preferably a number from 2-8, more preferably a number from 4-8;
    Ab选自权利要求1-8中任一项所述的抗BCMA抗体或其抗原结合片段。Ab is selected from the anti-BCMA antibody or antigen-binding fragment thereof according to any one of claims 1-8.
  11. 根据权利要求10所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，所述L 2如通式(D)所示： The antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 10, wherein the L 2 is represented by the general formula (D):
    -K 1-K 2-K 3-K 4- -K 1 -K 2 -K 3 -K 4-
    (D)(D)
    其中：in:
    K 1为
    Figure PCTCN2021082735-appb-100005
    s选自2至8的整数；     K 1 is
    Figure PCTCN2021082735-appb-100005
    s is selected from an integer from 2 to 8;
    K 2选自-NR 1(CH 2CH 2O) pCH 2CH 2C(O)-、-NR 1(CH 2CH 2O) pCH 2C(O)-、-S(CH 2) pC(O)-或单键，p选自1至20的整数，优选1至6的整数； K 2 is selected from -NR 1 (CH 2 CH 2 O) p CH 2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p CH 2 C(O)-, -S(CH 2 ) p C(O)- or a single bond, p is selected from an integer from 1 to 20, preferably an integer from 1 to 6;
    R 1选自氢、氘、羟基、氨基、烷基、卤素、卤代烷基、氘代烷基和羟烷基； R 1 is selected from hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl;
    K 3为四肽残基，优选地，所述四肽残基由选自两个或多个的苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸中的氨基酸形成的肽残基；更优选为GGFG的四肽残基； K 3 is a tetrapeptide residue, preferably, the tetrapeptide residue is selected from two or more of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid , A peptide residue formed by an amino acid in aspartic acid; more preferably a tetrapeptide residue of GGFG;
    K 4为-NR 2(CR 3R 4) t-，R 2、R 3或R 4各自独立地为氢、氘、羟基、氨基、烷基、卤素、卤代烷基、氘代烷基和羟烷基，t选自1或2， K 4 is -NR 2 (CR 3 R 4 ) t -, R 2 , R 3 or R 4 are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkane Base, t is selected from 1 or 2,
    接头单元L 2的K 1端与Ab相连，K 4端与L 1相连。 The K 1 end of the joint unit L 2 is connected to Ab, and the K 4 end is connected to L 1 .
  12. 根据权利要求11所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，所述L 1选自键、-O-(CR aR b) m-CR 5R 6-C(O)-、-O-CR 5R 6-(CR aR b) m-、-O-CR 5R 6-、-NH-(CR aR b) m-CR 5R 6-C(O)-或-S-(CR aR b) m-CR 5R 6-C(O)-， The antibody drug conjugate according to claim 11 or a pharmaceutically acceptable salt or solvent compound thereof, wherein L 1 is selected from bond, -O-(CR a R b ) m -CR 5 R 6 -C( O)-, -O-CR 5 R 6 -(CR a R b ) m -, -O-CR 5 R 6 -, -NH-(CR a R b ) m -CR 5 R 6 -C(O) -Or-S-(CR a R b ) m -CR 5 R 6 -C(O)-,
    其中：in:
    R a和R b各自独立地选自氢、氘、卤素或烷基； R a and R b are each independently selected from hydrogen, deuterium, halogen or alkyl;
    R 5为卤代烷基或环烷基； R 5 is haloalkyl or cycloalkyl;
    R 6选自氢、卤代烷基或环烷基； R 6 is selected from hydrogen, haloalkyl or cycloalkyl;
    或者，R 5和R 6与其相连接的碳原子一起形成环烷基； Alternatively, R 5 and R 6 together with the carbon atom to which they are attached form a cycloalkyl group;
    m选自0、1、2、3或4，m is selected from 0, 1, 2, 3 or 4,
    L 1的O端与接头单元L 2相连。 The O end of L 1 is connected to the joint unit L 2 .
  13. 根据权利要求12所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，所述L 1如通式(E)所示： The antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 12, wherein the L 1 is represented by the general formula (E):
    Figure PCTCN2021082735-appb-100006
    Figure PCTCN2021082735-appb-100006
    其中：in:
    R 5选自卤代烷基或环烷基，R 6选自氢、卤代烷基或环烷基，或者，R 5和R 6 与其相连接的碳原子一起形成环烷基；优选地，R 5选自C 1-6卤代烷基或C 3-6环烷基，R 6选自氢、C 1-6卤代烷基或C 3-6环烷基，或者，R 5和R 6与其相连接的碳原子一起形成C 3-6环烷基， R 5 is selected from haloalkyl or cycloalkyl, R 6 is selected from hydrogen, haloalkyl or cycloalkyl, or R 5 and R 6 together with the carbon atom to which they are attached form a cycloalkyl; preferably, R 5 is selected from C 1-6 haloalkyl or C 3-6 cycloalkyl, R 6 is selected from hydrogen, C 1-6 haloalkyl or C 3-6 cycloalkyl, or, R 5 and R 6 are together with the carbon atom to which they are attached To form a C 3-6 cycloalkyl group,
    m选自0至4的整数，m is selected from an integer from 0 to 4,
    优选地，通式(E)选自以下取代基：Preferably, the general formula (E) is selected from the following substituents:
    Figure PCTCN2021082735-appb-100007
    Figure PCTCN2021082735-appb-100007
  14. 根据权利要求1-13中任一项所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其中-L 2-L 1-选自以下结构： The antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to any one of claims 1-13, wherein -L 2 -L 1 -is selected from the following structures:
    Figure PCTCN2021082735-appb-100008
    Figure PCTCN2021082735-appb-100008
    K 2为键； K 2 is the key;
    K 3为GGFG的四肽残基； K 3 is the tetrapeptide residue of GGFG;
    R 5为卤代烷基或C 3-6环烷基； R 5 is haloalkyl or C 3-6 cycloalkyl;
    R 6选自氢、卤代烷基或C 3-6环烷基； R 6 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl;
    或者，R 5和R 6与其相连接的碳原子一起形成C 3-6环烷基； Alternatively, R 5 and R 6 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl group;
    R 2、R 3或R 4各自独立地选自氢或烷基； R 2 , R 3 or R 4 are each independently selected from hydrogen or alkyl;
    s选自2至8的整数；s is selected from an integer from 2 to 8;
    m选自0至4的整数，m is selected from an integer from 0 to 4,
    优选地，-L 2-L 1-选自以下结构： Preferably, -L 2 -L 1 -is selected from the following structures:
    Figure PCTCN2021082735-appb-100009
    Figure PCTCN2021082735-appb-100009
    Figure PCTCN2021082735-appb-100010
    Figure PCTCN2021082735-appb-100010
  15. 根据权利要求10所述的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，其为通式(IV)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化物：The antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof according to claim 10, which is an antibody drug conjugate represented by the general formula (IV) or a pharmaceutically acceptable salt or solvate thereof :
    Figure PCTCN2021082735-appb-100011
    Figure PCTCN2021082735-appb-100011
    其中：in:
    W选自C 1-8烷基、C 1-8烷基-环烷基或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或S的杂原子，任选地，所述的C 1-8烷基、环烷基和直链杂烷基各自独立地进一步被卤素、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； W is selected from a C 1-8 alkyl group, a C 1-8 alkyl-cycloalkyl group or a linear heteroalkyl group of 1 to 8 atoms, and the heteroalkyl group contains 1 to 3 selected from N, O or S Optionally, the C 1-8 alkyl group, cycloalkyl group and linear heteroalkyl group are each independently further substituted by halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterium Substituted by one or more substituents of alkyl, alkoxy and cycloalkyl;
    K 2选自-NR 1(CH 2CH 2O) p1CH 2CH 2C(O)-、-NR 1(CH 2CH 2O) p1CH 2C(O)-、-S(CH 2) p1C(O)-或键，R 1选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基，p 1选自1至20的整数； K 2 is selected from -NR 1 (CH 2 CH 2 O) p1 CH 2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p1 CH 2 C(O)-, -S(CH 2 ) p1 C(O) -or bond, R 1 is selected from hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl, and p 1 is selected from an integer of 1 to 20;
    K 3为由2至7个氨基酸构成的肽残基，氨基酸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，所述取代基为一个或多个独立地选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基； K 3 is a peptide residue composed of 2 to 7 amino acids. Amino acids can be substituted or unsubstituted. When substituted, the substituent can be substituted at any available point of attachment, and the substituent is one Or more are independently selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
    R 2选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基； R 2 is selected from hydrogen atom, alkyl group, halogenated alkyl group, deuterated alkyl group and hydroxyalkyl group;
    R 3和R 4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； R 3 and R 4 are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;
    R 5选自卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基； R 5 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
    R 6选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基； R 6 is selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclic, aryl or heteroaryl;
    或者，R 5和R 6与其相连接的碳原子一起形成环烷基或杂环基； Alternatively, R 5 and R 6 together with the carbon atom to which they are attached form a cycloalkyl group or a heterocyclic group;
    m选自0至4的整数；m is selected from an integer from 0 to 4;
    y选自1至10的数，y是小数或整数；y is a number selected from 1 to 10, y is a decimal or an integer;
    Ab为抗BCMA抗体或其抗原结合片段。Ab is an anti-BCMA antibody or an antigen-binding fragment thereof.
  16. 根据权利要求15所述的抗体药物偶联物，其为通式(IV-A)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化合物：The antibody drug conjugate according to claim 15, which is an antibody drug conjugate represented by the general formula (IV-A) or a pharmaceutically acceptable salt or solvent compound thereof:
    Figure PCTCN2021082735-appb-100012
    Figure PCTCN2021082735-appb-100012
    优选地，通式(IV-A)选自以下结构：Preferably, the general formula (IV-A) is selected from the following structures:
    Figure PCTCN2021082735-appb-100013
    Figure PCTCN2021082735-appb-100013
    Figure PCTCN2021082735-appb-100014
    Figure PCTCN2021082735-appb-100014
    Figure PCTCN2021082735-appb-100015
    Figure PCTCN2021082735-appb-100015
  17. 根据权利要求1所述的通式(A)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化合物，所述抗体药物偶联物或其药学上可接受的盐或溶剂化合物选自如下结构：The antibody drug conjugate represented by the general formula (A) or a pharmaceutically acceptable salt or solvent compound thereof according to claim 1, the antibody drug conjugate or a pharmaceutically acceptable salt or solvent compound thereof Selected from the following structures:
    Figure PCTCN2021082735-appb-100016
    Figure PCTCN2021082735-appb-100016
    Figure PCTCN2021082735-appb-100017
    Figure PCTCN2021082735-appb-100017
    Figure PCTCN2021082735-appb-100018
    Figure PCTCN2021082735-appb-100018
    Figure PCTCN2021082735-appb-100019
    Figure PCTCN2021082735-appb-100019
    Figure PCTCN2021082735-appb-100020
    Figure PCTCN2021082735-appb-100020
    Figure PCTCN2021082735-appb-100021
    Figure PCTCN2021082735-appb-100021
    Figure PCTCN2021082735-appb-100022
    Figure PCTCN2021082735-appb-100022
    Figure PCTCN2021082735-appb-100023
    Figure PCTCN2021082735-appb-100023
    其中，y选自2-10，优选4-8，更优选6-8，最优选6或8。Among them, y is selected from 2-10, preferably 4-8, more preferably 6-8, and most preferably 6 or 8.
  18. 一种制备如通式(IV)所示的抗体药物偶联物或其药学上可接受的盐或溶剂化物的方法，其包括以下步骤：A method for preparing the antibody drug conjugate represented by general formula (IV) or a pharmaceutically acceptable salt or solvate thereof, which comprises the following steps:
    Figure PCTCN2021082735-appb-100024
    Figure PCTCN2021082735-appb-100024
    Ab还原后，与通式(F)偶联反应，得到通式(IV)所示的化合物；After Ab is reduced, it is coupled and reacted with the general formula (F) to obtain the compound represented by the general formula (IV);
    其中：in:
    Ab为抗BCMA抗体或其抗原结合片段；Ab is an anti-BCMA antibody or an antigen-binding fragment thereof;
    W、K 2、K 3、R 2～R 6、m和y如权利要求15中所定义。 W, K 2 , K 3 , R 2 to R 6 , m, and y are as defined in claim 15.
  19. 根据权利要求18所述的方法，其中所述的通式(F)为通式(F-1)所示的化合物：The method according to claim 18, wherein the general formula (F) is a compound represented by the general formula (F-1):
    Figure PCTCN2021082735-appb-100025
    Figure PCTCN2021082735-appb-100025
    或其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、或其混合物形式，或其可药用的盐，Or its tautomers, mesosomes, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof,
    其中K 2、K 3、R 2～R 6、s和m如权利要求14中所定义。 Wherein K 2 , K 3 , R 2 to R 6 , s and m are as defined in claim 14.
  20. 通式(F)或通式(F-1)所示的化合物选自：The compound represented by general formula (F) or general formula (F-1) is selected from:
    Figure PCTCN2021082735-appb-100026
    Figure PCTCN2021082735-appb-100026
    Figure PCTCN2021082735-appb-100027
    Figure PCTCN2021082735-appb-100027
  21. 一种药物组合物，其包含如权利要求1-17任一项所述的抗体药物偶联物或所述抗体药物偶联物药学上可接受的盐或溶剂化合物，和一种或多种可药用的赋形剂、稀释剂或载体。A pharmaceutical composition comprising the antibody drug conjugate according to any one of claims 1-17 or a pharmaceutically acceptable salt or solvent compound of the antibody drug conjugate, and one or more Pharmaceutical excipients, diluents or carriers.
  22. 权利要求1-17任一项所述的抗体药物偶联物或权利要求21所述的药物组合物在制备用于治疗或预防BCMA介导的疾病或病症的药物中的用途。Use of the antibody drug conjugate according to any one of claims 1-17 or the pharmaceutical composition according to claim 21 in the preparation of a medicament for the treatment or prevention of BCMA-mediated diseases or disorders.
  23. 根据权利要求22所述的用途，其特征在于，所述BCMA介导的疾病或病症为癌症或自身免疫疾病，其中所述癌症优选为表达BCMA的癌症，更优选淋巴瘤、白血病或骨髓瘤，最优选多发性骨髓瘤；所述自身免疫疾病选自红斑狼疮，IgA肾病和风湿性关节炎。The use according to claim 22, wherein the BCMA-mediated disease or disorder is cancer or an autoimmune disease, wherein the cancer is preferably a cancer expressing BCMA, more preferably lymphoma, leukemia or myeloma, Most preferred is multiple myeloma; the autoimmune disease is selected from lupus erythematosus, IgA nephropathy and rheumatoid arthritis.
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WO2023237050A1 (en) * 2022-06-09 2023-12-14 Beigene, Ltd. Antibody drug conjugates

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