WO2022022508A1 - 抗cd79b抗体药物偶联物、其制备方法及其医药用途 - Google Patents

抗cd79b抗体药物偶联物、其制备方法及其医药用途 Download PDF

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WO2022022508A1
WO2022022508A1 PCT/CN2021/108666 CN2021108666W WO2022022508A1 WO 2022022508 A1 WO2022022508 A1 WO 2022022508A1 CN 2021108666 W CN2021108666 W CN 2021108666W WO 2022022508 A1 WO2022022508 A1 WO 2022022508A1
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ligand
alkyl
pharmaceutically acceptable
acceptable salt
solvate
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PCT/CN2021/108666
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English (en)
French (fr)
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任文明
梁金栋
许建烟
黄建
杨昌永
廖成
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上海拓界生物医药科技有限公司
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Priority to CN202180042603.6A priority Critical patent/CN115698079A/zh
Priority to BR112023001359A priority patent/BR112023001359A2/pt
Priority to JP2023505412A priority patent/JP2023535598A/ja
Priority to AU2021317378A priority patent/AU2021317378A1/en
Priority to CA3184724A priority patent/CA3184724A1/en
Priority to MX2023001163A priority patent/MX2023001163A/es
Priority to US18/018,241 priority patent/US20230405138A1/en
Priority to EP21849525.7A priority patent/EP4190812A1/en
Priority to KR1020237006821A priority patent/KR20230044275A/ko
Publication of WO2022022508A1 publication Critical patent/WO2022022508A1/zh

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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present application relates to an anti-CD79B antibody and an antigen-binding fragment, a chimeric antibody comprising the CDR region of the anti-CD79B antibody, a humanized antibody, and a pharmaceutical composition comprising a human anti-CD79B antibody or an antigen-binding fragment thereof, and its use as a Use of anticancer drugs.
  • Lymphoma Malignant tumors (cancer) are the second leading cause of death globally, after heart disease. Lymphoma is a malignant tumor originating from the lymphatic hematopoietic system and is the most common hematological tumor in the world. Lymphomas are divided into two categories: non-Hodgkin's lymphoma (NHL) and Hodgkin's lymphoma (HL). Non-Hodgkin's lymphoma is a general term for a group of lymphoid dysproliferative diseases with strong heterogeneity. Its incidence is much higher than that of Hodgkin's lymphoma, accounting for more than 80% of lymphomas.
  • NHL non-Hodgkin's lymphoma
  • HL Hodgkin's lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • the current standard first-line regimen for diffuse large B-cell lymphoma is rituximab combined with chemotherapy (R-CHOP).
  • R-CHOP chemotherapy
  • the anthracycline-based CHOP cyclophosphamide, doxorubicin, vincristine, prednisone
  • the R-CHOP regimen significantly improved the long-term survival of DLBCL patients.
  • the R-CHOP regimen in the treatment of DLBCL can significantly prolong the median overall survival time of patients by 4.9 years, the median disease-free survival time is over 6.6 years, and the 5-year disease progression-free survival rate Increased from 30% to 54%.
  • 10% to 15% of refractory patients do not respond, and 20% to 30% of patients relapse.
  • R-CHOP regimen such as elderly patients over 80 years old, their physical capacity does not allow for standard R-CHOP treatment.
  • R-CHOP may not be effective.
  • the B cell receptor (BCR) complex is the most important molecule on the surface of B cells.
  • the BCR complex consists of membrane immunoglobulin (mIg) that recognizes and binds antigen and heterodimers of Ig ⁇ (CD79a) and Ig ⁇ (CD79B) that transmit antigen-stimulating signals.
  • Ig ⁇ and Ig ⁇ are glycoproteins of about 47kDa and about 37kDa, respectively, and belong to the immunoglobulin superfamily.
  • the genes encoding Ig ⁇ and Ig ⁇ are called mb-1 and B29, respectively.
  • Both Ig ⁇ and Ig ⁇ have an Ig-like domain at the amino terminus of the extracellular domain. Both Ig ⁇ and Ig ⁇ can act as substrates of protein tyrosine kinases and participate in BCR signal transduction.
  • BCR is widely expressed on B-cell lymphomas as well as on normal B cells.
  • CD20-targeting rituximab Given the clinical success and reliable safety profile of CD20-targeting rituximab, the development of BCR-targeting therapies should also have favorable efficacy and safety profiles.
  • WO2020156439A discloses an anti-CD79B antibody and its use in treating tumors, the contents of which are incorporated in the entire disclosure.
  • the present disclosure relates to anti-CD69B antibodies or antigen-binding fragments and their conjugates with drugs (ADCs) and medical uses, wherein the anti-CD69B antibodies or antigen-binding fragments are provided with cytotoxic substances (MMAE or its derivatives, ixitecan or its derivatives, eribulin or its derivatives) conjugated ADC drugs.
  • ADCs drugs
  • cytotoxic substances MMAE or its derivatives, ixitecan or its derivatives, eribulin or its derivatives
  • WO2020156439A discloses an anti-CD79B antibody and its use in treating tumors, the contents of which are incorporated in the entire disclosure.
  • the present disclosure provides an anti-CD79B antibody and an antigen-binding fragment thereof, comprising:
  • Heavy chain HCDR1 which comprises the sequence shown in GX 1 X 2 FX 3 X 4 Y (SEQ ID NO: 24), wherein X 1 is S or Y, X 2 is S or T, X 3 is T or S, X 4 is S or T;
  • a heavy chain HCDR2 comprising the sequence shown in X 5 PRSGN (SEQ ID NO: 25), wherein X 5 is F or Y;
  • a heavy chain HCDR3 comprising X 6 X 7 X 8 X 9 X 10 GDFX 11 Y (SEQ ID NO: 26 ), wherein X is absent or G, X is absent or S, and X is G or D, X 9 is D or Y, X 10 is L or D, X 11 is D or A;
  • a light chain LCDR1 comprising RSSQSIVHX 12 GNTYX 13 E (SEQ ID NO: 27), wherein X 12 is S or H, and X 13 is F or L;
  • a light chain LCDR2 comprising the sequence shown in SEQ ID NO: 11 or 17;
  • Light chain LCDR3 which comprises the sequence shown in SEQ ID NO: 12 or 18.
  • the above-described anti-CD79B antibody or antigen-binding fragment thereof comprises:
  • An antibody heavy chain variable region comprising at least one HCDR selected from the group consisting of: SEQ ID NO: 23, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO : 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15; and/or
  • An antibody light chain variable region comprising at least one LCDR selected from the group consisting of: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO : 16, SEQ ID NO: 17 or SEQ ID NO: 18.
  • an anti-CD79B antibody or antigen-binding fragment thereof comprising:
  • the aforementioned scheme (a) i.e., an anti-CD79B antibody or antigen-binding fragment thereof, comprises HCDR1, HCDR2, and HCDR3, respectively, as set forth in SEQ ID NOs: 23, 8, and 9, and : LCDR1, LCDR2 and LCDR3 shown in 10, 11, 12
  • VL such as SEQ ID NO: 20 anti-CD79B antibody or its antigen-binding fragment
  • An anti-CD79B antibody or antigen-binding fragment thereof whose full length is as SEQ ID NO: 28 and the full length of the light chain is as SEQ ID NO: 29.
  • the aforementioned anti-CD79B antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody, humanized antibody, human antibody or fragment thereof; eg, a humanized antibody or fragment thereof.
  • an anti-CD79B antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein:
  • the heavy chain variable region (VH) contains:
  • the light chain variable region (VL) contains:
  • the VH of the anti-CD79B antibody or antigen-binding fragment is shown in the sequence SEQ ID NO:3, the VL is shown in the sequence SEQ ID NO:4; or the VH is shown in the sequence SEQ ID NO:5, VL is shown in the sequence SEQ ID NO:6.
  • an anti-CD79B antibody or antigen-binding fragment thereof is provided, wherein:
  • VH contains:
  • the VH of the anti-CD79B antibody or antigen-binding fragment is shown in the sequence of SEQ ID NO: 19, and the VL is shown in the sequence of SEQ ID NO: 20; or the VH is shown in the sequence of SEQ ID NO: 21, VL is shown in the sequence SEQ ID NO:22.
  • the above-mentioned anti-CD79B antibody or antigen-binding fragment thereof comprises a light chain constant region; preferably, the heavy chain constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, the The light chain constant regions are selected from human antibody kappa and lambda chain constant regions and conventional variants thereof.
  • the heavy chain constant region is human IgGl, IgG2.
  • an anti-CD79B antibody or fragment thereof is provided, wherein:
  • the heavy chain is shown in the sequence of SEQ ID NO: 28 or a variant thereof; the variant sequence comprises 0 to 10 amino acid changes in the heavy chain;
  • the light chain is shown in the sequence of SEQ ID NO: 29 or a variant thereof; the variant sequence comprises 0 to 10 amino acid changes in the light chain.
  • an anti-CD79B antibody or fragment thereof is provided, wherein:
  • the heavy chain is shown in the sequence of SEQ ID NO: 30 or a variant thereof; the variant sequence comprises 0 to 10 amino acid changes in the heavy chain;
  • the light chain is shown in the sequence of SEQ ID NO: 31 or a variant thereof; the variant sequence comprises 0 to 10 amino acid changes in the light chain.
  • the aforementioned anti-CD79B antibodies or fragments thereof may be variants having 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) in the VL ) amino acid changes, and/or VH has 0 to 10 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes.
  • the aforementioned variants have the same or similar biological function or effect as the original anti-CD79B antibody or fragment thereof.
  • the antigen-binding fragments of the above-mentioned anti-CD79B antibodies include Fab, Fv, sFv, F(ab') 2 , linear antibodies, single chain antibodies, scFv, sdAb, sdFv, nanobodies, peptibody, domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv).
  • a polynucleotide eg, DNA or RNA, encoding an anti-CD79B antibody or antigen-binding fragment thereof as described above is provided.
  • expression vectors eg, eukaryotic expression vectors, prokaryotic expression vectors, viral vectors, comprising the above-described polynucleotides are provided.
  • host cells eg, eukaryotic cells, prokaryotic cells, transformed with the expression vectors described above are provided.
  • the host cells are bacteria (eg, E. coli), yeast (eg, Pichia), mammalian cells (eg, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells).
  • a method for preparing the above-described anti-CD79B antibody or antigen-binding fragment thereof comprising the steps of: expressing the antibody or antigen-binding fragment thereof in a host cell as previously described, and isolating the antibody or its antigen-binding fragment from the host cell. Antigen-binding fragments.
  • methods of treating or preventing a proliferative disease or delaying the progression of a proliferative disease comprising:
  • anti-human CD79B antibody or antigen-binding fragment thereof or its encoded polynucleotide, pharmaceutical composition is administered to a subject in an effective amount for treating or delaying the disease, and the proliferative disease is cancer or tumor.
  • the cancer or tumor is lymphoma or leukemia
  • the lymphoma is selected from: diffuse large B-cell lymphoma, non-Hodgkin's lymphoma, small lymphocytic lymphoma, and mantle cell lymphoma;
  • the non-Hodgkin's lymphoma is selected from the group consisting of: aggressive NHL, relapsed aggressive NHL, relapsed painless NHL, refractory NHL, refractory painless NHL;
  • the leukemia is selected from: chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia.
  • Ligand -drug conjugates are selected from: chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia.
  • the present disclosure provides a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein:
  • the drug is selected from MMAE or its derivatives, ixitecan or its derivatives, eribulin or its derivatives;
  • the ligand is an anti-CD79B antibody or an antigen-binding fragment thereof, and the anti-CD79B antibody or an antigen-binding fragment thereof is any of the aforementioned anti-CD79B antibodies or antigen-binding fragments thereof in the present disclosure.
  • the present disclosure provides a ligand-ixitecan or its derivative conjugate or a pharmaceutically acceptable salt or solvate thereof, which is the ligand-ixnotecan or its pharmaceutically acceptable salt or solvate represented by formula (I).
  • W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 atoms containing 1 to 3 atoms selected from N, O or S The heteroatom, wherein said C 1-8 alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further selected from halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, Substituted with one or more substituents of deuterated alkyl, alkoxy and cycloalkyl;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- or chemical bond, p 1 is an integer from 1 to 20;
  • L3 is a peptide residue consisting of 2 to 7 amino acids, wherein the amino acids are optionally further selected from halogen, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cyclic substituted with one or more substituents in the alkyl group;
  • R 1 is selected from hydrogen atom, halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R is selected from a hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
  • R 1 and R 2 together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group
  • R 4 and R 5 are the same or different, and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;
  • n is an integer from 0 to 4.
  • n 1 to 10 and can be an integer or a decimal
  • Pc anti-CD79B antibodies or antigen-binding fragments thereof provided by the present disclosure.
  • the present disclosure provides a conjugate of ligand-ixitecan or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, which is the ligand-ixitecan represented by formula (II) Conjugate or its derivative or its pharmaceutically acceptable salt or solvate:
  • s 1 is an integer from 2 to 8; preferably 5;
  • the linking unit -L-Y- of the conjugates of ligands of the present disclosure - ixitecan or derivatives thereof include, but are not limited to:
  • the present disclosure provides ligand-conjugates of ixitecan or derivatives thereof, or pharmaceutically acceptable salts or solvates thereof including, but not limited to:
  • n 1 to 10 and can be an integer or a decimal
  • Pc is the aforementioned anti-CD79B antibody of the present disclosure or an antigen-binding fragment thereof.
  • Pc is the disclosed anti-CD79B antibody or an antigen-binding fragment thereof;
  • W, L 2 , L 3 , R 1 , R 2 , R 5 to R 7 , m and n are as defined in formula (I).
  • Pc is any anti-CD79B antibody or antigen-binding fragment thereof of the disclosure; preferably an antibody comprising a heavy chain as shown in SEQ ID NO: 28 and a light chain as shown in SEQ ID NO: 29, or An antibody comprising a heavy chain as set forth in SEQ ID NO:30 and a light chain as set forth in SEQ ID NO:31.
  • the conjugates of the disclosed ligand-ixitecan or derivatives thereof include tautomers, mesomers, racemates, enantiomers, diastereomers thereof Isomers, deuterated compounds, or mixtures thereof.
  • the present disclosure provides a ligand-eribulin or a derivative thereof conjugate or a pharmaceutically acceptable salt or solvate thereof, which is represented by the formula Pc-(LD) k :
  • the ligand is Pc
  • the Pc is any anti-CD79B antibody or antigen-binding fragment thereof of the disclosure
  • L is a linker that covalently attaches Pc to D
  • k is 1 to 20 (including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20 or any number in between)
  • R 1a is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, each independently optionally selected from alkyl (eg C 1- 6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropyl oxy), halogen (such as fluorine, chlorine, bromine), one or more of deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl Substituent substituted; preferably R 1a is methyl;
  • R 1b is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl, or cyclohexyl), aryl, and heteroaryl, each independently optionally selected from alkyl (eg, C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy , isopropoxy), one of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl or more substituents; preferably R 1b is hydrogen; or
  • R 1a and R 1b together with the atom to which they are attached form a C 5-8 heterocycloalkyl optionally substituted by an alkyl group (such as a C 1-6 alkyl group, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine), Deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocyclyl, aryl substituted with one or more of the substituents in the radical and heteroaryl, and R 1a and R 1b are not both hydrogen.
  • an alkyl group such as a C 1-6 alkyl group, including but not limited
  • R1a in D in the ligand-eribulin or derivative thereof conjugate is methyl.
  • D in the conjugate of ligand-eribulin or a derivative thereof, D is represented by the following formula:
  • k is selected from 1 to 10, and can be an integer or a decimal number.
  • the linker is extracellularly stable such that the ligand-eribulin or derivative thereof conjugate remains intact in the extracellular environment, but is capable of cleavage upon internalization into cancer cells, for example .
  • the drug moiety in the conjugate when the ligand-drug conjugate enters an antigen-expressing cell, the drug moiety in the conjugate is cleaved from the ligand moiety, and the cleavage releases the drug (eg, eribulin or a derivative thereof).
  • the drug eg, eribulin or a derivative thereof.
  • the linker comprises a cleavable moiety; wherein the cleavable moiety is positioned such that no linker and Pc remain in the drug (eg, eribulin derivative) after cleavage.
  • the cleavable moiety in the linker is a cleavable peptide moiety.
  • Ligand Drug Conjugates comprising a cleavable peptide moiety exhibit lower levels of aggregation, improved ligand-to-drug ratios, increased on-target killing of cancer cells, and decreased off-targeting of non-cancer cells kill, and/or higher drug load (p).
  • the addition of a cleavable moiety increases cytotoxicity and/or efficacy relative to a non-cleavable linker.
  • potency and/or cytotoxicity is increased in cancers expressing intermediate levels of antigen (eg, CD79B).
  • the cleavable peptide moiety is cleavable by an enzyme
  • the linker is an enzyme cleavable linker.
  • the linker is a linker that is cleavable by cathepsin.
  • an enzymatically cleavable linker eg, a cathepsin-cleavable linker exhibits one or more of the aforementioned improved properties.
  • the linker comprises an amino acid unit (ie, a peptide residue consisting of 2 to 7 amino acids), preferably the amino acid is selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline , serine, glutamic acid, aspartic acid, more preferably valine-citrulline (Val-Cit), alanine-alanine-asparagine (Ala-Ala-Asn), glycine-glycine- Lysine (Gly-Gly-lys), Valine-Lysine (Val-lys), Valine-Alanine (Val-Ala), Valine-Phenylalanine (Val-Phe) Or Glycine-Glycine-Phenylalanine-Glycine (Gly-Gly-Gly).
  • amino acid unit ie, a peptide residue consisting of 2 to 7 amino acids
  • the amino acid is selected from the group consisting of phenylalanine,
  • the linker in the conjugates of the present disclosure is selected from:
  • the amino acid unit comprises valine-citrulline (Val-Cit).
  • ADCs comprising Val-Cit exhibit increased stability, decreased off-target cell killing, increased on-target cell killing, lower levels of aggregation, and/or higher drug loading.
  • some embodiments provide linkers comprising cleavable sulfonamide moieties that are capable of cleavage under reducing conditions.
  • the linker comprises a cleavable disulfide moiety, and the linker is capable of cleavage under reducing conditions.
  • the linkers of the present disclosure comprise at least one spacer unit that attaches D (eg, an eribulin derivative) to the cleavable moiety.
  • D eg, an eribulin derivative
  • the spacer unit comprises p-aminobenzyloxycarbonyl (PAB),
  • k is selected from 1 to 10, which can be an integer or a decimal;
  • p2 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, which can be an integer or a decimal;
  • p2 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, which can be an integer or a decimal;
  • p2 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, which can be an integer or a decimal;
  • p2 is selected from 2, 4, 6 or 8;
  • k is selected from 1 to 10, which can be an integer or a decimal;
  • p2 is selected from 2, 4, 6 or 8;
  • the conjugate is represented by the formula:
  • k is selected from 1 to 10, and may be an integer or a decimal number; further, in D, R 1a is preferably from methyl, and R 1b is preferably from hydrogen.
  • the present disclosure also provides a compound represented by formula D (Eribulin),
  • R 1a is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cycloalkyl) propyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, each independently optionally selected from alkyl (eg C 1-6 Alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy group), one or more of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl Substituents are substituted, preferably R 1a is methyl;
  • R 1b is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl, or cyclohexyl), aryl, and heteroaryl, each independently optionally selected from alkyl (eg, C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy , isopropoxy), one of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl or more substituents, preferably R 1b is hydrogen, methyl; or
  • R 1a and R 1b are taken together with the carbon atom to which they are attached to form a C 5-8 heterocycloalkyl optionally substituted by an alkyl group (such as a C 1-6 alkyl group, including but not limited to methyl, ethyl , isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine) , deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocyclyl, substituted with one or more substituents in aryl and heteroaryl, and R 1a and R 1b are not both hydrogen.
  • an alkyl group such as a C 1-6 alkyl group, including
  • R 1a and R 1b in the compound represented by formula D are each independently selected from C 1-6 alkyl, including but not limited to methyl, ethyl, and isopropyl.
  • R 1a is selected from C 1-6 alkyl, including but not limited to methyl, ethyl, and isopropyl; R 1b is selected from hydrogen.
  • R 1a and R 1b in the compound of formula D together with the carbon atom to which they are attached form a C 6-8 heterocycloalkyl.
  • the compound represented by formula D (Eribulin) is:
  • the compound represented by formula D (Eribulin) is:
  • the compound represented by formula D (Eribulin) is:
  • the present disclosure also provides a compound represented by formula DZ (Eribulin),
  • R 1a is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to (limited to cyclopropyl, cyclopentyl or cyclohexyl), aryl and heteroaryl, each independently optionally selected from alkyl (eg C1 -6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropyl) propoxy), one of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl or Multiple substituents are substituted, preferably R 1a is methyl,
  • R 1b is selected from hydrogen, alkyl (such as C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cycloalkyl) propyl, cyclopentyl, or cyclohexyl), alkoxy, aryl, and heteroaryl, each independently optionally selected from alkyl (eg, C 1-6 alkyl, including but not limited to methyl, ethyl, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy , isopropoxy), one of halogen (such as fluorine, chlorine, bromine), deuterium, amino, cyano, nitro, hydroxy, hydroxyalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl or more substituents, preferably R 1b is hydrogen;
  • R 1a and R 1b together with the carbon atom to which they are attached form a C 5-8 heterocycloalkyl , which is optionally base, isopropyl), alkoxy (such as C 1-6 alkoxy, including but not limited to methoxy, ethoxy, propoxy, isopropoxy), halogen (such as fluorine, chlorine, bromine ), deuterium, amino, cyano, nitro, hydroxyl, hydroxyalkyl, cycloalkyl (such as C 3-8 cycloalkyl, including but not limited to cyclopropyl, cyclopentyl or cyclohexyl), heterocyclyl , aryl and heteroaryl are substituted with one or more substituents; and R 1a and R 1b are not both hydrogen;
  • Y is selected from -O( CRaRb ) m2 - CR8R9 -C(O)-, -NH- ( CRaRb ) m2 - CR8R9 -C(O) - , -O-CR 8 R 9 (CR a R b ) m2 -, -OCR 8 R 9 -C(O)-, -O(CR a R b ) m2 C(O)- or -S-(CR a R b ) m2 - CR 8 R 9 -C(O)-, wherein R a and R b are the same or different, and are each independently selected from hydrogen, deuterium atom, halogen or alkyl; R 8 is selected from hydrogen, C 3-6 cycloalkyl Alkyl or C 3-6 cycloalkyl; R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen; alternatively,
  • R 1a and R 1b in the compound represented by formula DZ are each independently selected from C 1-6 alkyl groups, including but not limited to methyl, ethyl, and isopropyl.
  • R 1a is selected from C 1-6 alkyl, including but not limited to methyl, ethyl, and isopropyl; R 1b is selected from hydrogen.
  • the compound shown in DZ is:
  • R 8 is selected from hydrogen, C 3-6 cycloalkylalkyl or C 3-6 cycloalkyl
  • R 9 is selected from hydrogen, haloalkyl or C 3-6 cycloalkyl, preferably hydrogen; or, R 8 and R9 together with the carbon atom to which it is attached form a C3-6 cycloalkyl
  • m2 is selected from 0, 1, 2 or 3.
  • the compound shown in DZ is selected from:
  • the compounds shown in DZ (Eribulin) provided by some embodiments may contain one or more asymmetric centers, such as can be
  • Pc is any anti-CD79B antibody or antigen-binding fragment thereof of the disclosure; preferably an antibody comprising a heavy chain as shown in SEQ ID NO: 28 and a light chain as shown in SEQ ID NO: 29, or An antibody comprising a heavy chain as set forth in SEQ ID NO:30 and a light chain as set forth in SEQ ID NO:31.
  • the conjugates of the present disclosure include tautomers, mesomers, racemates, enantiomers, diastereomers, deuterated compounds, or mixtures thereof. .
  • MMAE analog/derivative which is a compound represented by the general formula (D(MMAE)):
  • R 1 -R 6 are selected from hydrogen atom, halogen, hydroxyl, cyano, alkyl, alkoxy and cycloalkyl;
  • R 7 is selected from hydrogen atoms, alkyl groups, alkoxy groups and cycloalkyl groups
  • R 8 -R 11 form a cycloalkyl group, and the remaining two groups are optionally selected from hydrogen atoms, alkyl groups and cycloalkyl groups;
  • R 12 is selected from a hydrogen atom or an alkyl group
  • R 13 -R 15 are selected from a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group or a halogen;
  • R 16 is selected from aryl or heteroaryl optionally further substituted with a substituent selected from hydrogen, halogen, hydroxy, alkyl, alkoxy and cycloalkyl.
  • the compound represented by the general formula (D(MMAE)) is the compound represented by the general formula (D(MMAE) 1 ):
  • R 9 and R 10 form a cycloalkyl
  • R 2 -R 8 , R 11 -R 16 are as defined in general formula (D).
  • the compound represented by the general formula (D(MMAE)) is:
  • Another aspect of the present disclosure relates to a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises a compound of formula (-D(MMAE)) structure:
  • R 2 -R 6 are selected from hydrogen atom, halogen, hydroxyl, cyano, alkyl, alkoxy and cycloalkyl;
  • R 7 is selected from hydrogen atoms, alkyl groups, alkoxy groups and cycloalkyl groups
  • R 8 -R 11 form a cycloalkyl group, and the remaining two groups are optionally selected from hydrogen atoms, alkyl groups and cycloalkyl groups;
  • R 12 is selected from a hydrogen atom or an alkyl group
  • R 13 -R 15 are selected from a hydrogen atom, a hydroxyl group, an alkyl group, an alkoxy group or a halogen;
  • R 16 is selected from aryl or heteroaryl, and said aryl or heteroaryl is optionally further substituted by a substituent selected from hydrogen atom, halogen, hydroxyl, alkyl, alkoxy and cycloalkyl;
  • Wavy lines indicate hydrogen atoms, either covalently attached to linkers or to antibodies.
  • the ligand-MMAE or its derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof comprises a compound represented by formula (-D(MMAE) 1 ) structure:
  • R 9 and R 10 form a cycloalkyl
  • R 2 -R 8 , R 11 -R 16 are as defined in general formula (D(MMAE)).
  • the conjugate of the ligand-MMAE or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the conjugate of the ligand-MMAE or a derivative thereof contains the structure shown below:
  • Wavy lines indicate hydrogen atoms, either covalently attached to linkers or to antibodies.
  • the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate is represented by the general formula (IV):
  • R 2 is C 1 -C 8 alkyl
  • R 3 is C 1 -C 8 alkyl
  • R 4 is C 1 -C 8 alkyl
  • R 5 is H
  • R 6 is C 1 -C 8 alkyl
  • R 7 is C 1 -C 8 alkyl
  • R 8 are independently of each other and each is O-(C 1 -C 8 alkyl);
  • R 9 is H
  • R 10 is phenyl
  • Z is O or NH
  • R 11 is selected from H, C 1 -C 20 alkyl or -(R 13 O)m-R14;
  • n 3;
  • R 13 is C 2 -C 8 alkyl
  • R 14 is C 1 -C 8 alkyl
  • Pc is the disclosed anti-CD79B antibody or an antigen-binding fragment thereof;
  • L is a linker;
  • n is 1 to 10, which can be an integer or a decimal number.
  • the conjugate of the ligand-MMAE or a derivative thereof, or a pharmaceutically acceptable salt or solvate thereof comprises the structure shown below:
  • the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate is represented by the general formula (Pc-LD(MMAE)) :
  • R 2 -R 16 are as defined in general formula (D(MMAE));
  • n 1 to 10 and can be an integer or a decimal
  • Pc is the disclosed anti-CD79B antibody or an antigen-binding fragment thereof; L is a linker.
  • the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate is represented by the general formula (Pc-L-D1).
  • R 2 -R 16 are as defined in the general formula (-D(MMAE));
  • Pc, L, n are as defined in the general formula (Pc-L-D(MMAE)).
  • the ligand-MMAE or its derivative conjugate, or a pharmaceutically acceptable salt or solvate thereof is represented by the general formula:
  • Pc, L, n are as defined in the general formula (Pc-L-D(MMAE)).
  • Y is the stretcher unit, selected from or chemical bond
  • X 1 is selected from hydrogen atom, alkyl group, alkoxy group, aryl group or halogen
  • X 2 is selected from alkylene group, and described alkylene group is optionally further selected from halogen, hydroxyl, cyano group, substituted with one or more substituents of amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 1 is a stretching unit, selected from -(succinimide-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 17 -WC(O)- or -C( O)-WC(O)-, wherein W is selected from C 1-8 alkyl, C 1-8 alkyl-cycloalkyl, or straight chain heteroalkyl of 1 to 8 atoms containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 alkyl, cycloalkyl and straight chain heteroalkyl are each independently optionally further selected from halogen, hydroxy, cyano, substituted with one or more substituents of amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • L 2 is selected from -NR 18 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 18 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- or a chemical bond, wherein p 1 is an integer from 1 to 20; preferably a chemical bond;
  • L 3 is a peptide residue consisting of 2 to 7 amino acids, the amino acids are preferably selected from valine, citrulline, methyl valine; wherein the amino acid is optionally further selected from halogen, hydroxyl, cyano, Substituted with one or more substituents of amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • R 17 and R 18 are the same or different, and are each independently selected from a hydrogen atom, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group;
  • L 4 is an extension unit, preferably a PAB.
  • the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate wherein L 1 is selected from -(succinimide-3- base-N)-(CH 2 )s 1 -C(O)-, wherein s 1 is an integer from 2 to 8; preferably
  • the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate wherein L 3 is a dipeptide amino acid unit, preferably selected from valine -Citrulline.
  • the a-end is connected to the ligand, and the b-end is connected to the drug.
  • the ligand-MMAE or its derivative conjugate or its pharmaceutically acceptable salt or solvate is selected from the following structural formula:
  • n 1 to 10 and can be an integer or a decimal
  • the Pc is an anti-CD79B antibody or antigen-binding fragment thereof of the disclosure; preferably an antibody comprising a heavy chain as set forth in SEQ ID NO:28 and a light chain as set forth in SEQ ID NO:29, or an antibody comprising as SEQ ID NO:30 Antibodies to the heavy chain shown and the light chain shown in SEQ ID NO:31.
  • Another aspect of the present disclosure relates to a method for preparing a compound of general formula (D(MMAE)), or a pharmaceutically acceptable salt thereof, comprising the steps of:
  • R 2 -R 16 are as defined in general formula (D).
  • Another aspect of the present disclosure relates to a compound shown below:
  • Another aspect of the present disclosure relates to a method of preparing Compound 2 (MMAE), or a pharmaceutically acceptable salt thereof, comprising the steps of:
  • Another aspect of the present disclosure relates to a method for preparing a ligand-drug conjugate of general formula (Pc-L-D(MMAE)) or a pharmaceutically acceptable salt or solvate thereof, comprising the steps of:
  • Pc,n is as defined in the general formula (Pc-L-D(MMAE)).
  • the method for the compound represented by the general formula (D(MMAE)) of the present disclosure, or a pharmaceutically acceptable salt thereof, comprises the following steps:
  • R 2 -R 16 are as defined in the general formula (D(MMAE)).
  • Reagents for providing basic conditions include organic bases and inorganic bases
  • the organic bases include but are not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diethylamine Isopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide
  • the inorganic bases include but are not limited to sodium hydride, potassium phosphate, sodium carbonate, Potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide; preferably diethylamine.
  • MMAE preparation method of compound 2 (MMAE) of the present disclosure or a pharmaceutically acceptable salt or solvate thereof, the method comprising:
  • Reagents for providing basic conditions include organic bases and inorganic bases
  • the organic bases include but are not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diethylamine Isopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide or potassium tert-butoxide
  • the inorganic bases include but are not limited to sodium hydride, potassium phosphate, sodium carbonate, Potassium carbonate, cesium carbonate, sodium hydroxide and lithium hydroxide, preferably N,N-diisopropylethylamine.
  • the condensing agent is selected from 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride, 1-hydroxybenzotriazole and 1- (3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'- Tetramethylurea hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamin
  • the method for the ligand-drug conjugate represented by the general formula (Pc-L-D) of the present disclosure or its pharmaceutically acceptable salt or solvate comprises the following steps:
  • the reducing agent is preferably TCEP, especially, it is preferred to reduce the disulfide bond on the antibody;
  • Pc,n is as defined in the general formula (Pc-L-D(MMAE)).
  • ligand-drug conjugates or pharmaceutically acceptable salts or solvates thereof selected from:
  • Pc is any anti-CD79B antibody or antigen-binding fragment thereof disclosed, and n is 1 to 10, which can be an integer or a decimal number.
  • the Pc is an anti-CD79B antibody or an antigen-binding fragment thereof in the embodiments of the disclosure, for example, a Pc comprising a heavy chain as shown in SEQ ID NO:28 and a light chain as shown in SEQ ID NO:29
  • the average DAR value of the antibody drug conjugates of the present disclosure can be any value between 1-10, such as 2-8, or 2-6, or 1-6, or 4-6 .
  • Tautomers are structural isomers that are readily interconverted by a chemical reaction known as tautomerization. This reaction often results in the transfer of hydrogen atoms or protons, accompanied by the conversion of single bonds and adjacent double bonds. Some common tautomeric pairs are: keto-enol, lactam-lactam. An example of a lactam-lactam equilibrium is between A and B shown below.
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising the conjugate as described above, and a pharmaceutically acceptable excipient, diluent or carrier.
  • the present disclosure further provides use of any one or a combination selected from the following in the preparation of a medicament: an anti-CD79B antibody or an antigen-binding fragment thereof according to the present disclosure, a conjugate according to the present disclosure, and a pharmaceutical composition according to the present disclosure;
  • the antibody or its antigen-binding fragment or its drug conjugate is used for treating proliferative disease or delaying the progression of proliferative disease;
  • the proliferative disorder can be cancer or tumor;
  • the cancer or tumor is selected from lymphoma, Diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic Leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and/or mantle cell
  • the present disclosure provides a method of treating or preventing a proliferative disease or delaying the progression of a proliferative disease, the method comprising administering to a subject an anti-CD79B antibody or antigen-binding fragment thereof according to the present disclosure in an amount effective to treat or delay the disease, or The disclosed pharmaceutical composition, or the antibody-drug conjugate according to the present disclosure; wherein the proliferative disorder is cancer or tumor.
  • the present disclosure provides methods of treating or delaying progression of a B cell proliferative disorder or an autoimmune disorder in a subject in need thereof.
  • the B cell proliferative disorder is cancer or a tumor.
  • the present disclosure provides methods of enhancing immune function in a subject having a cell proliferative disorder or an autoimmune disorder.
  • the cell proliferative disorder is cancer or a tumor.
  • the cancer or tumor in the above regimen can be selected from lymphoma, diffuse large B-cell lymphoma, non-Hodgkin's lymphoma (NHL), aggressive NHL, relapsed aggressive NHL, relapsed painless NHL, refractory NHL, refractory indolent NHL, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and/or mantle cell lymphoma .
  • NHL non-Hodgkin's lymphoma
  • aggressive NHL relapsed aggressive NHL
  • refractory NHL refractory indolent NHL
  • CLL chronic lymphocytic leukemia
  • small lymphocytic lymphoma small lymphocytic lymphoma
  • leukemia hairy cell leukemia
  • HCL hairy cell leukemia
  • ALL acute lymphoblastic leukemia
  • Figure 1 ELISA test results of serum titer of Balb/c mice immunized with human CD79B ECD-hFc protein.
  • Figure 2 FACS detection results of serum titers of Balb/c mice immunized with human CD79B ECD-hFc protein.
  • Figure 3 ELISA test results of serum titer of SJL mice immunized with human CD79B ECD-hFc protein.
  • Figure 4 FACS detection results of serum titers of SJL mice immunized with human CD79B ECD-hFc protein.
  • Figure 5 ELISA test results of serum titer of SJL mice immunized with human CD79B ECD-his protein.
  • Figure 6 FACS detection results of serum titers of SJL mice immunized with human CD79B ECD-his protein.
  • Figure 7 ELISA test results of serum titer of SJL mice immunized with monkey CD79B ECD-his protein.
  • Figure 8 ELISA test results of anti-human CD79B mouse monoclonal antibody.
  • FIG. 9A is the FACS detection result of anti-human CD79B mouse monoclonal antibody.
  • FIG. 9B is the detection result of anti-human CD79B mouse monoclonal antibody FACS, wherein hIgG1 is the negative control antibody and SN8 is the positive control antibody.
  • Figure 10 FACS detection results of cross-reactivity of anti-human CD79B mouse monoclonal antibodies.
  • Figure 11 ELISA test results of anti-monkey CD79B mouse monoclonal antibody.
  • Figures 12A-12G FACS detection of anti-monkey CD79B mouse monoclonal antibody binding to monkey peripheral blood mononuclear cells.
  • Figure 12A is the result of FACS detection of monkey peripheral blood mononuclear cells as blank control;
  • Figure 12B is the FACS detection result of negative control antibody and monkey peripheral blood mononuclear cells;
  • Figure 12C is the FACS detection of positive control antibody and monkey peripheral blood mononuclear cells Results;
  • Figure 12D is the FACS detection result of anti-CD79B antibody mAb018 and monkey peripheral blood mononuclear cells;
  • Figure 12E is the FACS detection result of anti-CD79B antibody mAb019 and monkey peripheral blood mononuclear cells;
  • Figure 12F is anti-CD79B antibody mAb020 and monkey peripheral blood.
  • Figure 12G is the FACS detection results of anti-CD79B antibody mAb021 and monkey peripheral blood mononuclear cells.
  • Figures 13A to 13C show the efficacy of different ADCs on the subcutaneous xenografts of human diffuse large B-cell lymphoma WSU-DLCL2 nude mice.
  • Figure 14 Effects of different ADCs on body weight of tumor-bearing nude mice.
  • Figure 15 Efficacy of different ADCs on subcutaneous xenografts of human diffuse large B-cell lymphoma WSU-DLCL2 nude mice.
  • Figure 16 Effects of different ADCs on body weight of WSU-DLCL2 tumor-bearing nude mice.
  • Figure 17 Tumor photographs of the efficacy of different ADCs on subcutaneous xenografts of human diffuse large B-cell lymphoma WSU-DLCL2 nude mice.
  • Figure 18 Efficacy of different ADCs on subcutaneous xenografts of human follicular lymphoma DOHH-2 nude mice.
  • Figure 19 Effects of different ADCs on body weight of DOHH-2 tumor-bearing nude mice.
  • Antibody is used in the broadest sense to encompass a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), full-length antibodies, and antibody fragments ( or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant regions differ.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains and epsilon chains.
  • IgM immunoglobulins
  • IgD immunoglobulins
  • IgG immunoglobulins
  • IgA immunoglobulins
  • IgE alpha chains and epsilon chains.
  • the same class of Ig can be divided into different subclasses, such as IgG can be divided into IgG1, IgG2, IgG3, IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each class of Ig can have a kappa chain or a lambda chain.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which 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 three hypervariable regions (CDRs) and four relatively conserved framework regions (FRs). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the order from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • the antibody light chain variable region described in the present application may further comprise a light chain constant region comprising human or murine ⁇ , ⁇ chains or variants thereof.
  • the antibody heavy chain variable region described herein may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • murine antibody in this application is a monoclonal antibody to human CD79B or an epitope thereof prepared according to the knowledge and skill in the art. In preparation, test subjects are injected with CD79B antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine CD79B antibody or its antigen-binding fragment may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a murine IgG1, IgG2 , the heavy chain constant region of IgG3 or IgG4 or a variant thereof.
  • Fully human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “fully human antibody” does not include “humanized antibodies”.
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by grafting non-human CDR sequences into the framework of human antibody variable regions. It can overcome the strong immune response induced by chimeric antibodies because they carry a large number of non-human protein components. To avoid a decrease in activity while decreasing immunogenicity, the variable regions of the fully human antibody may be subjected to minimal reverse mutations to maintain activity.
  • chimeric antibody is an antibody obtained by fusing the variable region of the antibody of the first species with the constant region of the antibody of the second species, which can alleviate the immune response induced by the antibody of the first species.
  • a chimeric antibody first create a rabbit that secretes a rabbit-specific monoclonal antibody, isolate the antibody, clone the constant region gene of a fully human antibody as needed, and combine the rabbit variable region gene with the human constant region gene. After linking into a chimeric gene, it is 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 the fully human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising the heavy chain constant region of human IgG1 or IgG4, or using amino acid mutation without ADCC (antibody- dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG1.
  • antigen-binding fragment includes: single chain antibodies (ie, full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab - dsFv, single domain antibodies (eg VH or VL or VHH), scFv, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabody, tribody, triabody, tetrabody and epitope binding fragments of any of the above ( See, eg, Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).
  • Fab-Fv form was first disclosed in WO2009/040562 and its disulfide stabilized form, Fab-dsFv, was first disclosed in WO2010/035012.
  • Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171.
  • Multivalent antibodies may comprise multispecific, eg bispecific, or may be monospecific (see eg WO92/22583 and WO05/113605), an example of the latter being the Tri-Fab (or Tri-Fab) described in WO 92/22583 TFM).
  • single chain antibody is intended to comprise an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker formed molecules.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeats of the GGGGS amino acid sequence or variants thereof, eg using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • CDR1, HCDR2, HCDR3 CDR1, LCDR2, LCDR3
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region
  • LCDR1, LCDR2, LCDR3 three CDRs
  • the amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al. (1991), “Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia” numbering scheme (see Al-Lazikani et al. (1997) JMB 273:927-948) and the ImMunoGenTics (IMGT) numbering scheme (Lefranc MP, Immunologist, 7, 132- 136 (1999); Lefranc, MP et al, Dev. Comp. Immunol., 27, 55-77 (2003) etc.
  • Kabat numbering convention
  • IMGT ImMunoGenTics
  • VH variable domain
  • VL variable domain
  • CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and amino acids in VL Residue numbers are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDRs are defined by amino acid residues 26-35 in human VH (HCDR1 ), 50-65 (HCDR2) and 95-102 (HCDR3) and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • VH The CDR amino acid residue numbers in VL are approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align.
  • antibody framework refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • binding to CD79B refers to the ability to interact with CD79B or an epitope thereof, which may be of human origin.
  • an antigen refers to a molecule of vertebrate origin used to immunize immunocompetent, to generate antibodies recognizing the antigen, or to screen expression libraries (eg, especially phage, yeast or ribosome display libraries).
  • an antigen is defined more broadly to include target molecules specifically recognized by antibodies, as well as including portions or mimetics of molecules used in immunization procedures for the production of antibodies or library screening for the selection of antibodies.
  • Human CD79B as well as truncated and other variants of human CD79B are referred to as antigens for the present disclosure.
  • epitopes refers to the site on an antigen to which an immunoglobulin or antibody binds.
  • Epitopes can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents.
  • Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art and those described in this disclosure, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • the terms “specifically binds” and “selectively binds” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody equilibrates at about less than 10-7 M or even less when measured in an instrument by surface plasmon resonance (SPR) techniques
  • the dissociation constant (KD) binds to a predetermined antigen or its epitope, and its binding affinity to the predetermined antigen or its epitope is a non-specific antigen other than its binding to the predetermined antigen (or its epitope) or a closely related antigen (such as BSA, etc.) binding affinity at least twice.
  • the term "antibody that recognizes an antigen” is used interchangeably with the term “antibody that specifically binds” in this disclosure.
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector.
  • the vectors disclosed herein are capable of replicating autonomously in a host cell (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors) or can integrate into the genome of a host cell, thereby replicating along with the host genome (eg, non- episomal) type mammalian vector).
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems result in the glycosylation of antibodies. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques. For example, use an A or G Sepharose FF column for purification. Non-specifically bound components are washed away.
  • the bound antibody was eluted by pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected.
  • Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.
  • the obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • amino acid sequence identity refers to the alignment of amino acid sequences and the introduction of gaps where necessary to achieve maximum percent sequence identity (and conservative substitutions are not considered part of sequence identity), in the first sequence and in the second sequence Percentage of identical amino acid residues.
  • alignment can be accomplished in a variety of ways that are within the skill in the art, eg, using computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • cross-reactivity refers to the ability of an antibody of the present application to bind CD79B from a different species.
  • an antibody of the present application that binds human CD79B can also bind CD79B of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (eg SPR and ELISA), or binding or functional interaction with cells expressing CD79B.
  • binding assays eg SPR and ELISA
  • Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.
  • Inhibit or “block” are used interchangeably and encompass both partial and complete inhibition/blocking. Inhibition/blocking of CD79B preferably reduces or alters the normal level or type of activity that occurs when CD79B binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in CD79B binding affinity when contacted with an anti-CD79B antibody compared to CD79B not contacted with an anti-CD79B antibody.
  • inhibiting growth eg, in relation to a cell
  • inhibiting growth is intended to include any measurable reduction in cell growth.
  • inducing an immune response and “enhancing an immune response” are used interchangeably and refer to an immune response to stimulation (ie, passive or adaptive) by a particular antigen.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc region of IgG. Said modification refers to mutating the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A of IgG1; IgG2/4 chimera, F235E, or L234A/E235A mutation of IgG4.
  • Antigen-binding fragments are well known and can be found in the art, eg, in Cold Spring Harbor's Technical Guide to Antibody Assays (Chapters 5-8 and 15).
  • animals can be immunized with human CD79B or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced by conventional methods.
  • Antigen-binding fragments can likewise be prepared by conventional methods. Said antibody or antigen-binding fragment is genetically engineered to add one or more human FR regions to non-human CDR regions.
  • Human FR germline sequences can be obtained from ImMunoGeneTics (IMGT), or from J. Immunoglobulins, 2001 ISBN012441351.
  • drug refers to a cytotoxic drug or an immunomodulatory agent.
  • Cytotoxic drugs can have strong chemical molecules inside cells that disrupt their normal growth. In principle, cytotoxic drugs can kill cells at a sufficient concentration, but due to the lack of specificity, while killing tumor cells, they can also lead to apoptosis of normal cells, resulting in serious side effects.
  • toxins eg small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin
  • radioisotopes eg At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P32 and Lu radioisotopes
  • chemotherapeutic drugs antibiotics and nucleolysin.
  • Immunomodulators are inhibitors of immune checkpoint molecules.
  • linker refers to a fragment or bond in which one end is linked to a ligand and the other end is linked to a drug.
  • Other linkers can also be linked to the ligand or drug.
  • a linker may comprise one or more linker members.
  • exemplary linker members include 6-maleimidohexanoyl ("MC"), maleimidopropionyl (“MP”), valine-citrulline (“val-cit” or “vc”) "), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), and those derived from coupling with linker reagents: N-succinimidyl 4- (2-Pyridylthio)valerate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”, in Also referred to herein as "MCC”) and N-succinimidyl(4-iodo-acetyl)aminobenzoate (“SIAB”).
  • MC 6-maleimidohexanoyl
  • MP maleimidopropionyl
  • Linkers can include stretch units, spacer units, amino acid units, and stretch units, and can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers eg, hydrazones
  • protease-sensitive linkers eg, peptidase-sensitive
  • photolabile linkers eg, dimethyl linkers, or disulfide-containing linkers
  • disulfide-containing linkers can be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).
  • amino acid unit refers to an amino acid that can connect the carbonyl group in the following structural formula Y R to the extension unit if there is an extension unit, or directly to the drug if there is no extension unit .
  • amino acid units are represented as -Kk- :
  • -Kk- is a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide or decapeptide, the -K-units each independently have the following structural formula Ka or Kb , and k is An integer between 0-10:
  • R in the above amino acid unit is -H or methyl
  • R 25 is -aryl-, -alkyl-aryl-, -cycloalkyl-, -alkyl-cycloalkyl-, -cycloalkyl-alkyl-, -alkyl-cycloalkyl-alkyl -, -Heterocyclyl-, -Alkyl-Heterocyclyl-, -Heterocyclyl-Alkyl-, -Alkyl-Heterocyclyl-Al-, -Aryl-, -Alkyl-Aryl- , -Aryl-Alkyl-, -Alkyl-Aryl-Al-, -Heteroaryl-, -Alkyl-Heteroaryl-, -Heteroaryl-Al-, -Alkyl-Heteroaryl-Al-, -Alkyl-Heteroaryl base-alkyl-.
  • -Kk- is a dipeptide, preferably -valine-citrulline-, -phenylalanine-lysine- or -N-methylvaline-citrulline- , more preferably -valine-citrulline-.
  • stretch unit refers to a chemical moiety that is covalently linked to a ligand through a carbon atom at one end and to a drug (directly or indirectly) at the other end through a sulfur atom.
  • spacer unit is a bifunctional compound structural fragment that can be used to couple a linker and a drug to form a ligand-drug conjugate, which can selectively link the drug to the linker.
  • amino acid refers to an organic compound containing an amino group and a carboxyl group in the molecular structure, and both the amino group and the carboxyl group are directly connected to the -CH- structure.
  • the general formula is H2NCHRCOOH. According to the position where the amino group is attached to the carbon atom in the carboxylic acid, it can be divided into ⁇ , ⁇ , ⁇ , ⁇ , ⁇ ...-amino acids.
  • the amino acids that constitute natural proteins have their specific structural characteristics, that is, their amino groups are directly connected to the ⁇ -carbon atom, that is, ⁇ -amino acids.
  • extended unit refers to a chemical structure that can couple an amino acid unit to a drug in the presence of an amino acid unit, or to a drug through a carbonyl group on YR when an amino acid unit is absent.
  • the extension unit is PAB
  • the structure is such as a 4-iminobenzylcarbamoyl fragment, and its structure is shown in the following formula, which is connected to D,
  • Connector components include but are not limited to:
  • MC 6-maleimidocaproyl
  • Val-Cit or "vc” valine-citrulline (exemplary dipeptide in a protease cleavable linker);
  • Citrulline 2-amino-5-ureidovaleric acid
  • PAB p-aminobenzyloxycarbonyl (an example of a "self-sacrificing" linker assembly);
  • Me-Val-Cit N-methyl-valine-citrulline (wherein the linker peptide bond has been modified to prevent cleavage by cathepsin B);
  • MC(PEG)6-OH maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine);
  • SPP N-succinimidyl 4-(2-pyridylthio)valerate
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • PBS Phosphate Buffered Saline
  • antibody drug conjugate refers to the attachment of a ligand to a drug through a linker (or linking unit).
  • linker or linking unit
  • antibody-drug conjugate refers to a monoclonal antibody (or antigen-binding fragment) linked to a toxic drug through a linking unit.
  • DAR Drug-to-Antibody Ratio
  • the drug loading may range from 1-20, preferably 1-10 drugs (D) linked per antibody (Ab).
  • the drug load is expressed as k or n, and exemplary can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or the mean of the values in between.
  • Drug loading can be determined by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay, monoclonal antibody size variant assay (CE-SDS) and HPLC characterization.
  • the molecular size variant assay (CE-SDS) of the monoclonal antibody of the present disclosure can use the sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method, under reducing and non-reducing conditions, according to the molecular weight, according to the capillary electrophoresis method (2015 edition of "Chinese Pharmacopoeia” 0542), quantitative determination of the purity of recombinant monoclonal antibody products.
  • CE-SDS sodium dodecyl sulfate capillary electrophoresis
  • the drug is coupled to the N-terminal amino group of the ligand and/or the ⁇ -amino group of the lysine residue through the linking unit, generally, the drug molecule that can be coupled to the antibody in the coupling reaction
  • the number will be less than the theoretical maximum.
  • the loading of the Ligand Drug Conjugate can be controlled by the following non-limiting methods, including:
  • the drug to antibody ratio has an exact value for a particular conjugate molecule (eg, n in formula (I)), it should be understood that when used to describe a sample containing many molecules, this value will often be an average value, due to some degree of heterogeneity typically associated with the conjugation step.
  • the average loading of the conjugate is referred to herein as the drug to antibody ratio or "DAR".
  • the DAR is between about 1 and about 6, and typically about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0, 7.5, 8.0 .
  • at least 50% by weight of the sample is a compound with an average DAR ⁇ 2, and preferably at least 50% of the sample is a conjugate with an average DAR ⁇ 1.
  • Embodiments include wherein the DAR is about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2. 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, Immunoconjugates of 4.8, 4.9, 5.0, 5.1, 5.2, 5.4, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0 .
  • a DAR of 'about x' means that the DAR is measured within 20% of x.
  • DAR detection methods for example, extrapolate DAR values from LC-MS data of reduced and deglycosylated samples.
  • LC/MS allows quantification of the average number of payload (drug moiety) molecules in the ADC linked to the antibody.
  • HPLC separates antibodies into light and heavy chains, and also separates heavy (HC) and light (LC) chains according to the number of linker-payload groups per chain.
  • Mass spectrometry data enables identification of component species in mixtures such as LC, LC+1, LC+2, HC, HC+1, HC+2, etc. From the average loading of the LC and HC chains, the average DAR of the ADC can be calculated.
  • the DAR for a given immunoconjugate sample represents the average number of drug (payload) molecules attached to a tetrameric antibody containing two light chains and two heavy chains. For example, the DAR detection method in WO2018142322.
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably 1 to 12 (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably alkyl groups containing 1 to 10 carbon atoms, most preferably alkyl groups containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhe
  • the alkyl group is a lower alkyl group containing 1 to 6 carbon atoms
  • non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl , 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2- Dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methyl pentyl, 2,3-dimethylbutyl, etc.
  • Alkyl groups can be substituted or unsubstituted. When substituted, the substituents may be substituted at any available point of attachment, and the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy , alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkane Thio, heterocycloalkylthio, oxo.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein alkyl is as defined above.
  • alkylene refers to a saturated straight or branched chain aliphatic hydrocarbon group having a residue derived from the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, which is a residue containing 1 straight or branched chain groups of up to 20 carbon atoms, preferably containing 1 to 12 (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, More preferred are alkylene groups containing 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene groups include, but are not limited to, methylene ( -CH2- ), 1,1-ethylene (-CH( CH3 )-), 1,2-ethylene ( -CH2) CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene ( -CH2CH2CH2- ), 1,4 - Butylene ( -CH2CH2CH2CH2- ) and 1,5 - Butylene ( -CH2CH2CH2CH2CH2- ) Wait.
  • Alkylene may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from alkyl, alkenyl, alkynyl , alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy , cycloalkylthio, heterocycloalkylthio and one or more substituents of oxo.
  • alkoxy refers to -O-(alkyl) and -O-(cycloalkyl), wherein alkyl or cycloalkyl is as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy.
  • Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkoxy Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing from 3 to 20 carbon atoms, preferably from 3 to 12 carbon atoms, more preferably from 3 to 10 carbon atoms, most preferably 3 to 8 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene
  • Polycyclic cycloalkyl groups include spiro, fused or bridged cycloalkyl groups.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O ) m (where m is an integer from 0 to 2) heteroatoms excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • ring atoms excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably the cycloalkyl ring contains 3 to 10 ring atoms.
  • Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like.
  • Polycyclic heterocyclyls include spiro, fused or bridged heterocyclyls.
  • spiroheterocyclyl refers to a polycyclic heterocyclic group with 5 to 20 membered rings sharing one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (where m is an integer from 0 to 2) heteroatoms and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings have a fully conjugated pi electron system. For example, it is 6 to 14 yuan, and another example is 7 to 10 yuan.
  • spiroheterocyclyls are classified into mono-spiroheterocyclyl, bis-spiroheterocyclyl or poly-spiroheterocyclyl, preferably mono-spiroheterocyclyl or bis-spiroheterocyclyl.
  • Non-limiting examples of spiroheterocyclyl include:
  • fused heterocyclyl refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more of the rings may contain one or more Double bonds, but none of the rings have a fully conjugated pi-electron system, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2), the remaining rings Atom is carbon. For example, it is 6 to 14 yuan, and another example is 7 to 10 yuan.
  • fused heterocyclyl groups include:
  • bridged heterocyclyl refers to a 5- to 14-membered, polycyclic heterocyclyl group in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but none of the rings have a complete common A pi-electron system of a yoke in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen, or S(O) m (where m is an integer from 0 to 2) and the remaining ring atoms are carbon. For example, it is 6 to 14 yuan, and another example is 7 to 10 yuan.
  • bridged heterocyclyl groups include:
  • heterocyclyl ring can be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:
  • Heterocyclyl may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , heterocycloalkylthio, oxo.
  • aryl refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, eg, 6 to 10 membered, eg, benzene and naphthyl, such as phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring linked to the parent structure is an aryl ring, non-limiting examples of which include:
  • Aryl may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, Alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycle Alkylthio.
  • heteroaryl refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • Heteroaryl is preferably 5 to 10-membered, more preferably 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetra azolyl, etc.
  • the heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring linked to the parent structure is a heteroaryl ring, non-limiting examples of which include:
  • Heteroaryl groups can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkane Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , Heterocycloalkylthio.
  • cycloalkylalkyl refers to the substitution of a hydrogen on an alkyl group with one or more cycloalkyl groups, preferably with one cycloalkyl group, wherein alkyl is as defined above, wherein cycloalkyl is as defined above.
  • haloalkyl refers to the replacement of a hydrogen on an alkyl group with one or more halogens, wherein alkyl is as defined above.
  • deuterated alkyl refers to the replacement of a hydrogen on an alkyl group with one or more deuterium atoms, wherein alkyl is as defined above.
  • hydroxy refers to the -OH group.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • amino refers to -NH2 .
  • nitro refers to -NO2 .
  • C1-C6 alkyl optionally substituted by halogen or cyano means that halogen or cyano may but need not be present, and the description includes the case where the alkyl is substituted by halogen or cyano and the case where the alkyl is not substituted by halogen and cyano. The case of cyano substitution.
  • Compounds of the present disclosure may contain one or more asymmetric centers and thus may give rise to enantiomers, diastereomers, and may be defined as (R)- or (S)- according to absolute stereochemistry or used for Other stereoisomeric forms of (D)- or (L)-amino acids.
  • the present disclosure includes all possible isomers as well as racemic and optically pure forms thereof.
  • Optically active (+) and (-), (R)- and (S)- or (D)- and (L)-isomers can be prepared using chiral synthons or chiral reagents, or conventional methods can be used For example chromatography and fractional crystallization preparation.
  • the bond when the configuration is not specified, can be or or both and Two configurations.
  • the bond when the configuration is not specified, can be in the Z configuration or the E configuration, or both. For example, can be
  • Stereoisomers refer to compounds of the same atomic composition that are bound by the same bonds but have different three-dimensional structures, which are not interchangeable.
  • Various stereoisomers and mixtures thereof are contemplated in this disclosure, and includes “enantiomers,” which refer to two stereoisomers whose molecules are non-superimposable mirror images of each other.
  • Atoms capable of being isotopically labeled include, but are not limited to, hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, and the like. They can be replaced by isotopic isotopes 2H(D), 3H, 11C, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 36Cl and 125I, respectively.
  • deuterium when a position is specifically designated as deuterium (D), the position is understood to have an abundance of at least 3000 times greater than the natural abundance of deuterium (which is 0.015%) of deuterium (ie, at least 45 % of deuterium incorporated).
  • Substituted means that one or more hydrogen atoms in the group, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently of one another substituted with a substituent. Substituents are only in their possible chemical positions, and those skilled in the art can determine (either experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups with free hydrogens may be unstable when bound to carbon atoms with unsaturated (eg, ethylenic) bonds.
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carriers and Excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • pharmaceutically acceptable salt refers to a salt of a ligand-drug conjugate of the present disclosure, or a salt of a compound described in the present disclosure, when such salt is used in a subject It is safe and effective, and has due biological activity.
  • the antibody-antibody drug conjugate compounds of the present disclosure contain at least one amino group and thus can form salts with acids
  • pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, sulfuric acid Hydrogen, Citrate, Acetate, Succinate, Ascorbate, Oxalate, Nitrate, Pearate, Hydrogen Phosphate, Dihydrogen Phosphate, Salicylate, Hydrogen Citrate, Tartrate , maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
  • solvate refers to a ligand-drug conjugate compound of the present disclosure that forms a pharmaceutically acceptable solvate with one or more solvent molecules, non-limiting examples of which include water, ethanol, acetonitrile, isopropyl alcohol, DMSO, ethyl acetate.
  • the present disclosure relates to a type of cleavable linker with a specific structure and an active substance with a specific structure, and an antibody drug conjugate (ADC) composed of the linker, the active substance and an antibody.
  • ADCs are complexes formed by linking a toxic substance to the antibody via a spacer. The ADC is degraded in vivo to release active molecules, thereby playing an anti-tumor effect.
  • carrier refers to a system that alters how a drug enters a subject and is distributed in the body, controls the rate of drug release, and delivers the drug to a target.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form aggregates in various forms due to their unique amphiphilic structure.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to encapsulate drug molecules, and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • excipient is an addition other than the active ingredient in a pharmaceutical formulation, which may also be referred to as an adjuvant.
  • adjuvant such as binders, fillers, disintegrants, lubricants in tablets; matrix parts in ointments and creams of semi-solid preparations; preservatives, antioxidants, flavoring agents, fragrances, adjuvants in liquid preparations
  • Solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be referred to as excipients.
  • the term "diluent” is also known as filler, the main purpose of which is to increase the weight and volume of the tablet.
  • the addition of the diluent not only ensures a certain volume, but also reduces the dosage deviation of the main components and improves the compression molding of the drug.
  • an absorbent to absorb the oily substances, so as to keep the "dry” state, so as to facilitate the tableting.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present application, or conjugates thereof, to a subject who has, is suspected of having, Presence, predisposition to suffer from one or more diseases or symptoms thereof for which the therapeutic agent is known to have a therapeutic effect.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms in the subject or population being treated, whether by inducing regression of such symptoms or inhibiting the progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to alleviate symptoms of any particular disease may vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the level of the drug that produces the desired effect in the subject. ability. Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms.
  • the embodiments of the present application may be ineffective in alleviating symptoms of a target disease in a subject, according to any statistical test known in the art such as Student's t-test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it reduced target disease symptoms in a statistically significant number of subjects.
  • any statistical test known in the art such as Student's t-test, chi-square test, Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it reduced target disease symptoms in a statistically significant number of subjects.
  • Antibodies including light and heavy chains
  • Antigens were constructed by overlapping extension PCR methods known in the art, and the DNA fragments obtained by overlapping extension PCR were inserted into the expression vector pEE6.4 (Lonza Biologics) using the two restriction sites of HindIII/BstBI. ), expressed in 293F cells (Invitrogen, Cat#R790-07). The resulting recombinant protein is used for immunization or screening.
  • the human CD79B amino acid sequence was derived from NCBI (NP_000617.1) and its extracellular domain (ECD) contains 159 amino acids (Met1-Asp159).
  • the fusion protein of human CD79B extracellular domain (ECD) and human Fc region (human CD79B ECD-hFc) and the fusion protein of human CD79B extracellular domain (ECD) and His tag (human CD79B ECD-His) were used as immunogens, respectively.
  • Balb/c and SJL mice were immunized by intraperitoneal injection to stimulate the production of antibodies against the extracellular domain (ECD) of human CD79B in the mice.
  • Intraperitoneal injection immunization method Calculate the amount of antigen required for this immunization according to the immunization program.
  • the protein antigen was diluted with PBS to the corresponding concentration as required, followed by emulsification of the antigen.
  • the emulsified antigen and adjuvant mixture was transferred into a 2.0 ml sterile syringe and injected into the right abdomen of the mouse.
  • FACS serum titer of immunized mice DoHH2 cells or monkey peripheral blood mononuclear cell suspensions were centrifuged, resuspended in PBS containing 0.1% BSA, and counted. The test serum of each group was added. After incubation at room temperature for 60 minutes, the cells were washed three times and anti-Mouse IgG (Fc IgG) was added. Specificity)-FITC secondary antibody, incubate the cells for 30 minutes at room temperature in the dark, wash the cells three times, gently resuspend the cells with PBS containing 0.1% BSA, and detect on the machine.
  • Fc IgG anti-Mouse IgG
  • mice Five Balb/c mice were co-immunized with human CD79B ECD-hFc protein, numbered 5491, 5492, 5493, 5494, and 5495.
  • the serum titer ELISA test results are shown in Figure 1. The results showed that the titer of mouse immune serum reached more than 1:100K.
  • the results of FACS detection of mouse serum are shown in Figure 2. It can be seen that the antibody produced in the mouse serum can specifically recognize the CD79B protein on the surface of DoHH2 cells.
  • mice Five SJL mice were co-immunized with human CD79B ECD-hFc protein, numbered 5496, 5497, 5498, 5499, and 5500.
  • the serum titer ELISA test results are shown in Figure 3. The results showed that the titer of mouse immune serum reached more than 1:100K.
  • the results of FACS detection of mouse serum are shown in Figure 4. It can be seen that the antibody produced in mouse serum can specifically recognize the CD79B protein on the surface of DoHH2 cells.
  • mice Five SJL mice were co-immunized with human CD79B ECD-his protein, numbered 5726, 5727, 5728, 5729, and 5730.
  • the serum titer ELISA test results are shown in Figure 5. The results showed that the titer of mouse immune serum reached more than 1:10K.
  • the FACS detection results of mouse serum are shown in Figure 6, and it can be seen that the antibody produced in the mouse serum can specifically recognize the CD79B protein on the surface of DoHH2 cells.
  • mice can be used for cell fusion to generate hybridoma cell lines capable of secreting specific antibodies against CD79B.
  • lymphocytes and myeloma cells SP2/0 (ATCC, CCL-121 TM ) of the immunized mice were fused by electrofusion method for subsequent antibody screening.
  • SP2/0 cells were expanded in 10% DMEM medium one week before fusion. Mice spleens and lymph nodes were removed, washed and triturated to collect lymphocytes. The SP2/0 and lymphocytes were mixed in proportion and fused by an electrofusion apparatus. After fusion, the cells were plated in 96-well plates and cultured in a 37°C, 5% CO2 incubator; the cell status was observed every day; 5 days after fusion, the cell fusion rate was counted. The fused hybridoma cells were screened 9-14 days after fusion, and the cells in the positive wells were selected for expansion culture in 24-well plates.
  • the 96-well plate was coated with 1 ⁇ g/ml antigen concentration, 50 ⁇ L per well, and refrigerated at 4°C overnight. The next day, the coated antigen plate was washed once (washing solution: 1 ⁇ PBST). After washing, the cells were blocked with 1% BSA blocking solution prepared in 1 ⁇ PBST at 37°C for 1 hour. After washing the plate 3 times with 1 ⁇ PBST, add 50 ⁇ L of the cell supernatant to be tested in a 37°C incubator and incubate for 1 hour. After washing the plate three times with 1 ⁇ PBST, add 100 ⁇ L of goat anti-mouse secondary antibody diluted 1:5000, and incubate at 37°C for 0.5 hours.
  • TMB color developing solution A and B mixed in a ratio of 1:1 and develop color. After 15 minutes, the color reaction was stopped with 1N hydrochloric acid. Fluorescence values were detected at 450 nm on a Spectra Max M5 plate reader.
  • Antibody number clone number Test result negative control mIgG 0.05 mAb001 12A11-1G1 3.26 mAb002 19F10-1D7 3.69 mAb003 51E5G6 3.02 mAb004 67B10C1 3.41 mAb005 78A9F4 3.73 mAb006 48F11D6 3.34 mAb007 61A11F1 3.40 mAb008 63G2A2 3.56 mAb009 75F1E2 3.57 mAb010 66G3E7 3.83 mAb011 66E12H3 3.41 mAb012 73A8F3 3.45 mAb013 74C4F3 3.31 mAb014 70B8B3 3.10 mAb015 83B2G2 3.41 mAb016 83C2D4 3.46 mAb017 86F11F6 3.80
  • Antibody number clone number mean fluorescence value negative control mIgG 58 mAb001 12A11-1G1 13032 mAb002 19F10-1D7 5943 mAb003 51E5G6 33918 mAb004 67B10C1 26000 mAb005 78A9F4 24454 mAb006 48F11D6 20120 mAb007 61A11F1 18039 mAb008 63G2A2 16453
  • Antibody number clone number mean fluorescence value negative control mIgG 35 mAb018 121H1E9 1973 mAb019 152E5F6 1708 mAb020 159E3E5 1488 mAb021 134H2F5 1225
  • the 96-well plate was coated with 1 ⁇ g/ml antigen concentration, 50 ⁇ L per well, and refrigerated at 4°C overnight. The next day, the coated antigen plate was washed once (washing solution: 1 ⁇ PBST). After washing, the cells were blocked with 1% BSA blocking solution prepared in 1 ⁇ PBST at 37°C for 1 hour. After washing the plate three times with 1 ⁇ PBST, add 50 ⁇ L of antibody diluted 1:10 at 100 nM, put it into a 37°C incubator, and incubate for 1 hour.
  • SPR detection of anti-human CD79B mouse monoclonal antibody was used to detect the affinity between anti-human CD79B antibody and its antigen human CD79B-His.
  • the antigenic human CD79B-His protein was immobilized to a CM5 chip.
  • the coupling level was set at 100RU.
  • the running buffer was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20).
  • the diluted antibody was flowed through the experimental channel and the control channel at a flow rate of 30 ⁇ l/min for 3 minutes, and dissociated for 5 minutes.
  • Regeneration buffer (10 mM glycine, pH 1.5) was then run for 30 seconds at a flow rate of 30 ⁇ l/min. Data were analyzed with Biacore 8K software.
  • Example 1-3 Determination of amino acid sequence of mouse monoclonal antibody variable region
  • the high-affinity hybridoma monoclonal cell line obtained in Example 1-2 was subjected to variable region amino acid sequence determination, and then recombinantly expressed as a human-mouse chimeric antibody (cAb), and further antibody identification was performed.
  • the genes encoding the variable region of the heavy chain and the variable region of the light chain were amplified by reverse transcription PCR, and then connected to the vector; the sequences of the light and heavy chains were obtained by sequencing.
  • the RNA purification kit (Qiagen Company, product number 74134) was used to extract the total RNA of the active single cell line in Example 1-2.
  • the single-stranded cDNA was then prepared using Invitrogen's Cat. No. 18080-051 cDNA Synthesis Kit.
  • the sequences of the light and heavy chain variable regions were synthesized by PCR method, and the PCR products were cloned into the TA vector pMD-18T, and then sent for sequencing.
  • the obtained light and heavy chain sequences were cloned into an expression vector to express the recombinant monoclonal antibody. After verification of activity, the humanization work was carried out.
  • amino acid residues of the VH/VL CDRs of the anti-human CD79B antibody were determined and annotated by the Chothia numbering system.
  • the light and heavy chain sequences of the murine anti-CD79B monoclonal antibodies obtained in Examples 1-3 were compared in the antibody database to establish a humanized antibody model. Select back mutations according to the model, and screen the optimal humanized anti-CD79B monoclonal antibody. Find crystal structures with similar homology to the obtained murine candidate molecule from a database of mouse Fab crystal structure models (such as the PDB database), pick high-resolution (such as PDB) ) of the Fab crystal structure to establish a mouse Fab model.
  • the light and heavy chain sequences of the murine antibody were aligned with the sequences in the model, and the sequences consistent with the sequences of the murine antibody were retained to obtain a structural model of the murine antibody.
  • Discordant amino acids are possible back mutation sites.
  • the murine antibody structure model was run with Swiss-pdb viewer software to optimize energy (minimize). Different amino acid sites in the model except CDRs were backmutated. The activity of the resulting mutant antibody (humanized) was compared with that of the antibody before humanization. Humanized antibodies with good activity are retained. Optimization of CDR regions, including avoidance of glycosylation, deamidation, oxidation sites, etc.
  • VL Humanized Antibody Light Chain Variable Region
  • VH Humanized Antibody Heavy Chain Variable Region
  • VL Humanized Antibody Light Chain Variable Region
  • hAb017-10 humanized antibody heavy chain
  • the HCDR1 sequence of the humanized mAb015 has a T30S mutation, and the mutated HCDR1 is GSSFSSY (SEQ ID NO: 23).
  • the anti-CD79B antibody of the present disclosure has the general formula as shown in Table 7:
  • DOHH-2 cells were cultured according to the conventional method of suspension cells.
  • the composition of the complete medium was: RPMI 1640 medium (GIBCO, Cat No.: 11835-030), plus 10% (v/v) fetal bovine serum (FBS) (GIBCO, Cat No.: 10099-141) and penicillin/streptomycin (GIBCO, Cat No.: 15070-063).
  • FACS buffer phosphate buffered saline
  • FBS fetal bovine serum
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • NMR was measured by Bruker AVANCE-400 nuclear magnetic instrument, and the solvent was deuterated dimethyl sulfoxide (DMSO-d 6 ), deuterated chloroform (CDCl 3 ), deuterated methanol (CD 3 OD), and the internal standard was four Methylsilane (TMS).
  • DMSO-d 6 dimethyl sulfoxide
  • CDCl 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • TMS Methylsilane
  • the MS was measured using an Agilent 1200/1290 DAD-6110/6120 Quadrupole MS LC/MS instrument (manufacturer: Agilent, MS model: 6110/6120 Quadrupole MS).
  • HPLC High performance liquid chromatography
  • Chiral HPLC analysis was determined using an Agilent 1260 DAD high performance liquid chromatograph.
  • HPLC preparations used Waters 2545-2767, Waters 2767-SQ Detector2, Shimadzu LC-20AP and Gilson GX-281 preparative chromatographs.
  • CombiFlash rapid preparation instrument is Combiflash Rf200 (TELEDYNE ISCO).
  • the thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate; the size of the silica gel plate used for thin layer chromatography (TLC) is 0.15mm to 0.2mm; when the product is separated and purified by thin layer chromatography, the size used is 0.4mm to 0.5mm.
  • Silica gel column chromatography generally uses Yantai Huanghai silica gel 200 to 300 mesh silica gel as the carrier.
  • the known starting materials of the present disclosure can be synthesized using or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui chemical companies.
  • the pressure hydrogenation reaction uses Parr 3916EKX hydrogenation apparatus and Qinglan QL-500 hydrogen generator or HC2-SS hydrogenation apparatus.
  • the hydrogenation reaction is usually evacuated and filled with hydrogen, and the operation is repeated 3 times.
  • the microwave reaction used a CEM Discover-S 908860 microwave reactor.
  • the solution refers to an aqueous solution.
  • reaction temperature is room temperature, ranging from 20°C to 30°C.
  • the eluent system of column chromatography and the developing solvent system of thin layer chromatography used for purification include: A: dichloromethane/methanol system, B: n-hexane/ethyl acetate system, C: petroleum ether/ethyl acetate system ;
  • the volume ratio of the solvent is adjusted according to the polarity of the compound, and it can also be adjusted by adding basic or acidic reagents (such as triethylamine and acetic acid, etc.).
  • the crude product 4 in the above-mentioned present embodiment was dissolved in 10 mL of anhydrous ether, silver oxide (34.8 mg, 0.15 mmol) was added, followed by methyl iodide (28.4 mg, 0.2 mmol), and then the reaction was carried out at room temperature for about 10-16 hours, After monitoring that the reaction was basically complete, the solid was removed by filtration, and then concentrated under reduced pressure to obtain the crude product, which was directly carried out to the next step of the reaction.
  • the crude product 5 in the above-mentioned present embodiment was dissolved in 10 mL of tetrahydrofuran, 2 mL of diethylamine was added, and then stirred at room temperature for about 2-4 hours. After the reaction was monitored by HPLC, the crude product was directly concentrated under reduced pressure to obtain the crude product, which was passed through a silica gel column. 40 mg of product was obtained after purification, LC/MS (ESI): m/z 744.2 [M+H] + .
  • Test Example 1 In vitro cytotoxic activity screening:
  • CTG was used to detect the content of ATP, which reflected the survival of tumor cells.
  • Final culture conditions were first determined based on IC50 and maximal inhibition by seeding cells at different densities and culturing for 3 and 5 days. Then according to this condition, the killing effect of the toxin molecule was detected.
  • Eribulin positive control was prepared in a round bottom 96-well plate (3788).
  • the first column of plate 1 was 2 mM (stock solution was diluted 10-fold with DMSO), and thereafter the tenth column was diluted in 10-fold gradient in DMSO, and the 11th column was DMSO.
  • CTG detection The plate was taken out on day 3 and day 5, respectively, and equilibrated to room temperature. Add 90 ⁇ L CTG to each well, react at room temperature for 10 min in the dark, read the luminescence value with a microplate reader and calculate the IC 50 .
  • CTG detection Take out the plate and equilibrate to room temperature. Add 90 ⁇ L CTG to each well, react at room temperature for 10 min in the dark, read the luminescence value with a microplate reader and calculate the IC 50 .
  • Compound D-1 has good killing effect in three tumor cell lines, and is obviously better than the positive drug Eribulin.
  • the measured dichloromethane was dissolved in the product obtained in the previous step (7mg, 0.007mmol), and then 4A molecular sieves (10mg), trimethyloxonium tetrafluoroboric acid (11mg, 0.07mmol) and proton sponge (16mg, were added successively. 0.07 mmol), stirred at room temperature for 1 h.
  • the measured dichloromethane was dissolved in the product obtained in the previous step (7mg, 0.007mmol), and then 4A molecular sieves (10mg), trimethyloxonium tetrafluoroboric acid (11mg, 0.07mmol) and proton sponge (16mg, were added successively. 0.07 mmol), stirred at room temperature for 1 h.
  • Test Example 2 In vitro cytotoxic activity screening
  • Well plate 2 Add 196 ⁇ L of the corresponding culture medium in column 2 to column 11, and pipette 4 ⁇ L from column 3 to column 12 of well plate 1 to column 2 to column 11 of well plate 2. Mix well.
  • CTG assay Remove the plate and equilibrate to room temperature. Add 75 ⁇ L CTG to each well, react at room temperature for 10 min in the dark, read the luminescence value with a microplate reader and calculate the IC50.
  • the compound 1 of this example (30 mg, 0.041 mmol) was added to 1 mL of N,N-dimethylformamide, and 4-((S)-2-((S)-2-(6-(2,5 -Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoamido)-3-methylbutyamido)-5-ureidovaleramido)benzyl(4-nitrobenzene base) carbonate of the present example compound 2a (45 mg, 0.061 mmol, supplier: Ark), then add 0.25 mL of pyridine, replace with argon three times, then add 1-hydroxybenzotriazole (12 mg, 0.089 mmol) and N, N-diisopropylethylamine (16 mg, 0.123 mmol), the reaction was stirred at room temperature for 4 hours, then 2a (45 mg, 0.061 mmol) was added, and stirring was continued for 16 hours.
  • reaction solution was purified by high performance liquid chromatography (separation conditions: chromatographic column: XBridge Prep C18 OBD 5um 19*250mm; mobile phase: A-water (10mmol NH 4 OAc): B-acetonitrile, gradient elution, flow rate: 18mL/ min), the corresponding fractions thereof were collected and concentrated under reduced pressure to obtain the title product Compound 2 of this Example (18 mg, yield: 33.0%).
  • ADC loadings were determined by ultraviolet spectrophotometry (UV-Vis) (Thermo nanodrop 2000 UV spectrophotometer). The principle is that the total absorbance of the ADC at a certain wavelength is equal to the sum of the absorbances of the drug and the antibody at that wavelength.
  • UV-Vis ultraviolet spectrophotometry
  • a 280nm ⁇ mab-280 bC mab + ⁇ Drug-280 bC Drug formula (1)
  • ⁇ Drug-280 The average molar extinction coefficient of the drug at 280nm is 5100;
  • ⁇ mab-280 the average molar extinction coefficient of monoclonal antibody at 280nm is 214600;
  • C mab the concentration of monoclonal antibody
  • the optical path length is 1 cm.
  • a 370nm ⁇ mab-370 bC mab + ⁇ Drug-370 bC Drug formula (2)
  • ⁇ Drug-370 The average molar extinction coefficient of the drug at 370nm is 19000;
  • ⁇ mab-370 The extinction coefficient of monoclonal antibody at 370nm is 0;
  • C mab the concentration of monoclonal antibody
  • the optical path length is 1 cm.
  • An aqueous solution of phosphine (TCEP) (10 mM, 25.3 ⁇ L, 0.253 umol) was placed in a water bath shaker, and the reaction was shaken at 37° C. for 3 hours to stop the reaction. The reaction solution was cooled to 25°C with a water bath.
  • Example 2-2-1 Compound B (1.41 mg, 1.015 umol) in Example 2-2-1 was dissolved in 50 ⁇ L of dimethyl sulfoxide, added to the above reaction solution, placed in a water bath shaker, and shaken at 25°C for 3 hours. Stop the reaction.
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS buffer aqueous solution with pH 6.5, containing 0.001M EDTA) to obtain the title product ADC-1 of this example (that is, hAb015-10-cys -B) in PBS buffer (0.84 mg/mL, 13.5 mL), stored frozen at 4°C.
  • An aqueous solution of phosphine (TCEP) (10 mM, 59.1 ⁇ L, 0.591 umol) was placed in a water bath shaker, and the reaction was shaken at 37° C. for 3 hours to stop the reaction. The reaction solution was cooled to 25°C with a water bath.
  • An aqueous solution of phosphine (TCEP) (10 mM, 85.0 ⁇ L, 0.850 umol) was placed in a water bath shaker, and the reaction was shaken at 37° C. for 3 hours to stop the reaction. The reaction solution was cooled to 25°C with a water bath.
  • An aqueous solution of phosphine (TCEP) (10 mM, 30.4 ⁇ L, 0.304 umol) was placed in a water bath shaker, and the reaction was shaken at 37° C. for 3 hours to stop the reaction. The reaction solution was cooled to 25°C with a water bath.
  • An aqueous solution of phosphine (TCEP) (10 mM, 16.2 ⁇ L, 0.162 umol) was placed in a water bath shaker, and the reaction was shaken at 37° C. for 3 hours to stop the reaction. The reaction solution was cooled to 25°C with a water bath.
  • An aqueous solution of phosphine (TCEP) (10 mM, 25.3 ⁇ L, 0.253 umol) was placed in a water bath shaker, and the reaction was shaken at 37° C. for 3 hours to stop the reaction. The reaction solution was cooled to 25°C with a water bath.
  • Example 3-5 Compound D (1.09 mg, 1.015 umol) in Example 3-5 was dissolved in 45 ⁇ L of dimethyl sulfoxide, added to the above reaction solution, placed in a water bath shaker, and shaken at 25°C for 3 hours to stop the reaction. .
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS buffer aqueous solution with pH 6.5, containing 0.001M EDTA) to obtain the title product ADC-6 of this example (that is, hAb015-10-cys -D, target DAR value about 4) in PBS buffer (0.71 mg/mL, 14.0 mL), stored frozen at 4°C.
  • An aqueous solution of phosphine (TCEP) (10 mM, 50.7 ⁇ L, 0.507 umol) was placed in a water bath shaker, and the reaction was shaken at 37° C. for 3 hours to stop the reaction. The reaction solution was cooled to 25°C with a water bath.
  • Example 3-5 Compound D (1.63 mg, 1.518 umol) in Example 3-5 was dissolved in 68 ⁇ L of dimethyl sulfoxide, added to the above reaction solution, placed in a water bath shaker, and shaken at 25°C for 3 hours to stop the reaction. .
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS buffer solution with pH 6.5, containing 0.001M EDTA) to obtain the title product ADC-7 of this example (that is, hAb015-10-cys -D, DAR value is about 6) in PBS buffer (0.81 mg/mL, 13.5 mL), stored frozen at 4°C.
  • aqueous solution (10 mM, 1.388 mL, 13.88 umol), placed in a water bath shaker, and shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • the compound MC-VC-PAB-MMAE (70.3 mg, 53.40 umol) was dissolved in 3.5 mL of dimethyl sulfoxide, added to the above reaction solution, placed in a water bath shaker, and shaken at 25 ° C for 3 hours to stop the reaction. .
  • the reaction solution was desalted and purified by Sephadex G25 gel column (elution phase: 0.05M PBS buffer aqueous solution with pH 6.5, containing 0.001M EDTA) to obtain the title product ADC-8 of this example (ie SN8-cys-MC).
  • -PAB-MMAE, DAR value about 4 in PBS buffer (5.83 mg/mL, 132 mL), stored frozen at 4°C.
  • Biacore was used to determine the affinity of CD79B antibodies (hAb015-10 and SN8) and ADC to CD79B protein.
  • Biacore T200 (GE); biosensor chip CM5 (Cat.#BR-1005-30, GE); amino coupling kit (Cat.#BR-1000-50, GE); human Anti-capture kit (Cat.#BR-1008-39, GE); Human CD79B-His protein (Cat.#29750-H08H, Sino Biological); HBS-EP+10X buffer solution (Cat.#BR-1006-69 , GE) was diluted to 1X (pH 7.4) with DIWater;
  • the human anti-capture antibody was covalently coupled to the CM5 biochip according to the method in the human anti-capture kit to capture a certain amount of CD79B antibody with affinity.
  • sequence of SN8 (ie the antibody in Polivy) is:
  • DOHH-2 cells were cultured according to the conventional method of suspension cells.
  • the composition of the complete medium was: RPMI 1640 medium (GIBCO, Cat No.: 11835-030), plus 10% (v/v) fetal bovine serum (FBS) (GIBCO, Cat No.: 10099-141) and penicillin/streptomycin (GIBCO, Cat No.: 15070-063).
  • FBS fetal bovine serum
  • GIBCO penicillin/streptomycin
  • cells were collected by low temperature centrifugation at 4°C, 1000 rpm, for 5 minutes. Resuspend cells in 10-15 ml of FACS buffer chilled on ice.
  • the composition of the FACS buffer is: phosphate buffered saline (PBS), pH 7.4, plus 2% fetal bovine serum (FBS).
  • the FACS buffer was pre-cooled on ice. After cell counting and centrifugation, 300,000 cells/well were added to a 96-well plate, the supernatant was discarded by centrifugation, and Fc blocking solution was added, 12.5 ⁇ g/ml (BD, Cat No.: 564220), 100 ⁇ l/well. Block for 10 minutes at room temperature. Then, 20 ⁇ g/ml of ADC to be tested was added to the corresponding wells, and incubated at 4°C for 1 hour in the dark. Unbound ADC was removed by washing 2 times with pre-chilled PBS buffer.
  • Complete cell medium (RPMI 1640 medium with 10% fetal bovine serum) was added and incubated at 37°C, 5% CO2 for 0 and 4 hours. After the supernatant was discarded by centrifugation, 100 ⁇ l/well of 2% PFA buffer was added, and the cells were resuspended and left for 10 minutes. Then wash 3 times with FACS buffer, then add 100 ⁇ L of secondary antibody solution (fluorescently labeled goat anti-human secondary antibody: 1:250 dilution, the concentration is 2 ⁇ g/ml, Biolegend, Cat#409304), incubate at 4°C for half an hour in the dark .
  • secondary antibody solution fluorescently labeled goat anti-human secondary antibody: 1:250 dilution, the concentration is 2 ⁇ g/ml, Biolegend, Cat#409304
  • ADC-1 colorless clear liquid, concentration 0.84mg/mL, purity 99.17%;
  • ADC-2 colorless clear liquid, concentration 2.17mg/mL, purity 97.93%;
  • ADC-3 colorless clear liquid, concentration 2.79mg/mL, purity 98.28%;
  • ADC-4 colorless clear liquid, concentration 1.36mg/mL, purity 98.48%;
  • ADC-5 colorless clear liquid, concentration 0.9mg/mL, purity 98.49%;
  • ADC-6 colorless clear liquid, concentration 0.71mg/mL, purity 98.71%;
  • ADC-7 colorless clear liquid, concentration 0.81mg/mL, purity 98.27%;
  • ADC-8 colorless clear liquid, concentration 5.83mg/mL, purity 97.07%.
  • DoHH2 was purchased from DSMZ
  • WSU-DLCL-2 cells were purchased from American Type Culture Collection (ATCC)
  • Raji cells were purchased from American Type Culture Collection (ATCC).
  • FBS fetal bovine serum
  • RPMI 1640 and FBS were purchased from Gibco; The microplate reader was purchased from BioTek.
  • Inhibition rate (control well OD570nm-OD690nm -administration well OD570nm-OD690nm )/
  • Example 4-4 Therapeutic effect of ADC on subcutaneous transplanted tumor of human diffuse large B-cell lymphoma WSU-DLCL2 nude mice
  • This example evaluates and compares the efficacy of each ADC drug on subcutaneously transplanted tumors in nude mice.
  • hIgG1 (HRP00252): colorless clear liquid, concentration 22.77mg/mL, purity 99.03%, production date 2018.7.24, valid until 2020.01.24 (tentatively 18 months); frozen at -70°C.
  • ADC-1, ADC-2, ADC-3, ADC-4, ADC-6, ADC-8 are the same as in Example 4-3.
  • WSU-DLCL2 cells Human diffuse large B-cell lymphoma WSU-DLCL2 cells were purchased from American Type Culture Collection. WSU-DLCL2 cells were cultured in a 10-cm petri dish, and the culture conditions were RPMI 1640 medium (Gibco) with 10% fetal bovine serum, penicillin and streptomycin, and cultured at 37°C in an incubator with 5% CO2 air. . Passage 2-3 times a week, when cells are in exponential growth phase, harvest cells, count, and seed.
  • mice were subcutaneously inoculated with 2.1 ⁇ 10 7 WSU-DLCL2 cells, and after the tumor grew to 100-150 mm 3 , the animals were grouped according to tumor volume (D0). Mice were administered intravenously (IV), and the administration volume was 10 mL/kg; the specific administration dosage and administration schedule are shown in Table X. The tumor volume was measured twice a week, the mice were weighed, and the data were recorded.
  • the experimental index is to investigate the effect of drugs on tumor growth, and the specific index is T/C% or tumor inhibition rate TGI (%).
  • the tumor diameter was measured twice a week with a vernier caliper, and the formula for calculating the tumor volume (V) was:
  • V 1/2 ⁇ a ⁇ b 2 , where a and b represent length and width, respectively.
  • T/C (%) (TT 0 )/(CC 0 ) ⁇ 100, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. Among them, T is the tumor volume administered with ADC, and C is the tumor volume administered with IgG1 as a control group.
  • Partial tumor regression was defined if the tumor was smaller than the initial volume, that is, T ⁇ T 0 or C ⁇ C 0 ; complete tumor regression (CR) was defined if the tumor disappeared completely.
  • the animals were sacrificed by CO 2 anesthesia, and then the tumors were dissected and photographed.
  • ADC-1 (3mg/kg, 10mg/kg, IV, D0) dose-dependently inhibited the growth of subcutaneously transplanted tumors in WSU-DLCL2 nude mice, the tumor inhibition rates were 75% and 137%, respectively, and all the 10 mg/kg dose groups Partial regression of the tumor;
  • the tumor inhibition rates of ADC-2 (3mg/kg, 10mg/kg, IV, D0) on WSU-DLCL2 were 76% and 123%, respectively, and all tumors partially regressed in the 10 mg/kg dose group;
  • the tumor inhibition rate of ADC-3 (3mg/kg, IV, D0) on WSU-DLCL2 was 76%, and 1/6 of the tumors partially regressed;
  • the tumor inhibition rates of ADC-4 (3mg/kg, 10mg/kg, IV, D0) on WSU-DLCL2 were 95% and 129%, respectively, and 1/6 and 6/6 in the 3 mg/kg and 10 mg/kg groups, respectively Partial regression of the tumor;
  • the tumor inhibition rate of ADC-6 (3mg/kg, IV, D0) on WSU-DLCL2 was 86%, and 2/6 tumors partially regressed;
  • the tumor inhibition rates of ADC-8 (3mg/kg, 10mg/kg, IV, D0) on WSU-DLCL2 were 39% and 93%, respectively, and 4/6 tumors in the 10mg/kg group had partial regression.
  • mice were able to tolerate the above drugs well, and there was no obvious symptoms such as weight loss.
  • IgG1 was used as a negative control. See Table 13 and Figures 13A to 13C and Figure 14 for the results.
  • D0 time of first administration
  • P value refers to comparison with solvent
  • IV intravenous injection
  • Partial regression D21 tumor volume is less than D0.
  • a single intravenous injection of ADC-1, ADC-2, ADC-3, ADC-4, ADC-6 and ADC-8 at 3 mg/kg or 10 mg/kg has obvious curative effect on the subcutaneous xenograft of WSU-DLCL2 nude mice. Partial regression of the tumor was caused; the drug was significantly dose-dependent, and the efficacy of each ADC was better than that of the positive drug ADC-8 (ie Polivy) at the same dose. Tumor-bearing mice can tolerate the above drugs well.
  • Example 4-5 Therapeutic effect of ADC on subcutaneous transplanted tumor of human diffuse large B-cell lymphoma WSU-DLCL2 nude mice
  • This example further evaluates and compares the efficacy of ADC drugs on subcutaneously transplanted tumors in WSU-DLCL2 nude mice.
  • ADC-5, ADC-6, ADC-7, ADC-1, ADC-8 drugs were diluted with physiological saline to the concentrations in Example 4-3.
  • each nude mouse was subcutaneously inoculated with 2 ⁇ 10 7 WSU-DLCL2 cells, and after the tumor grew to 100-150 mm 3 , the animals were grouped according to tumor volume (D0). Mice were administered intravenously (IV), and the administration volume was 10 mL/kg; the specific administration dosage and administration schedule are shown in Table 12. The tumor volume was measured twice a week, the mice were weighed, and the data were recorded.
  • the tumor inhibition rates of ADC-5 (3mg/kg, 6mg/kg, 12mg/kg, IV, D0) on the subcutaneous xenografts of WSU-DLCL2 nude mice were 69%, 86% and 88%, respectively, of which 6mg/kg and In the 12mg/kg dose group, 1/6 and 1/6 tumors partially regressed;
  • the tumor inhibition rates of ADC-6 (1.5mg/kg, 3mg/kg, 6mg/kg, IV, D0) on WSU-DLCL2 subcutaneous xenograft tumors were 66%, 108% and 125%, respectively, of which 3mg/kg and 6mg/kg There were 5/6 and 6/6 tumor partial regression in the kg dose group, respectively;
  • the tumor inhibition rate of ADC-7 (1mg/kg, IV, D0) on WSU-DLCL2 subcutaneous xenograft tumors was 91%, and 1/6 of tumors partially regressed;
  • the tumor inhibition rate of ADC-1 (3mg/kg, IV, D0) on WSU-DLCL2 subcutaneous xenograft tumor was 44%;
  • the tumor inhibition rate of ADC-8 (3mg/kg, IV, D0) on WSU-DLCL2 subcutaneous xenograft tumor was 10%.
  • the tumor-bearing mice tolerated the above drugs well, and no symptoms such as weight loss occurred.
  • D0 time of first administration
  • P value refers to comparison with solvent
  • IV intravenous injection.
  • Example 4-6 Therapeutic effect of ADC on human B-cell lymphoma DoHH2 subcutaneously transplanted tumor in nude mice
  • This example further evaluates and compares the efficacy of ADC drugs on subcutaneously transplanted tumors in DoHH2 nude mice.
  • the ADC-1, ADC-6, and ADC-8 drugs were diluted with physiological saline to the concentrations in Example 4-3.
  • DOHH-2 cells were purchased from DSMZ, Germany. DOHH-2 cells were cultured in 10-cm dishes in RPMI 1640 medium (Gibco) with 10% fetal bovine serum and penicillin and streptomycin at 37°C in an incubator with 5% CO 2 air nourish. Passage 2-3 times a week, when cells are in exponential growth phase, harvest cells, count, and seed.
  • mice were subcutaneously inoculated with 3 ⁇ 10 7 DOHH-2 cells, and after the tumor had grown to 100-150 mm 3 , the animals were grouped according to tumor volume (D0). Mice were administered intravenously (IV), and the administration volume was 10 mL/kg; see Table 15 for the specific administration dose and administration schedule. The tumor volume was measured twice a week, the mice were weighed, and the data were recorded.
  • the experimental index is to investigate the effect of drugs on tumor growth, and the specific index is T/C% or tumor inhibition rate TGI (%).
  • the tumor diameter was measured twice a week with a vernier caliper, and the formula for calculating the tumor volume (V) was:
  • V 1/2 ⁇ a ⁇ b 2 where a and b represent length and width, respectively.
  • T/C(%) (TT 0 )/(CC 0 ) ⁇ 100, where T and C are the tumor volumes at the end of the experiment; T0 and C0 are the tumor volumes at the beginning of the experiment. T is the tumor volume administered with ADC, and C is the tumor volume administered with IgG1 as a control group.
  • Partial tumor regression was defined if the tumor was smaller than the initial volume, that is, T ⁇ T 0 or C ⁇ C 0 ; complete tumor regression (CR) was defined if the tumor disappeared completely.
  • the animals were sacrificed by CO 2 anesthesia, and then the tumors were dissected and photographed.
  • the tumor inhibition rates of ADC-1, ADC-6, and ADC-8 (1mg/kg, IV, D0) on DOHH-2 nude mice subcutaneously transplanted tumors were 82% (1/6PR) and 127% (5/6PR), respectively. ), 41%; tumor-bearing mice can tolerate the above drugs well, and there is no obvious symptoms such as weight loss.
  • D0 time of first administration
  • P value refers to comparison with solvent
  • IV intravenous injection
  • Partial regression D21 tumor volume is less than D0.
  • a single intravenous injection of ADC-1 and ADC-6 at 1 mg/kg has obvious curative effect on the subcutaneous xenograft of human follicular lymphoma DoHH2 nude mice, causing partial regression of the tumor; the efficacy is better than that of the positive drug ADC-8 (i.e. Policy).
  • ADC-8 i.e. Policy
  • Tumor-bearing mice can tolerate the above drugs well.
  • the use and welfare of laboratory animals in this disclosure is performed in accordance with the "International Association for the Assessment and Accreditation of Laboratory Animals (AAALAC)".
  • the health status and death of the animals are monitored daily. Routine inspections include observing the effects of the test substances and drugs on the animals' daily behaviors, such as behavioral activities, weight changes, and physical signs.

Abstract

涉及一种抗CD79B抗体药物偶联物、其制备方法及其医药用途。具体而言，提供一种抗体药物偶联物(ADC)，其包含与MMAE或其衍生物、依喜替康或其衍生物、或艾日布林或其衍生物缀合的抗CD79B抗体，含有所述ADC的药物组合物，及其在制备治疗CD79B介导的疾病或病症的药物中的用途，尤其在用于制备抗癌药物中的用途。

Description

抗CD79B抗体药物偶联物、其制备方法及其医药用途
本申请要求2020年07月27日提交的申请号为CN202010730899.8和2020年07月28日提交的CN202010735910.X的专利申请的优先权。
技术领域
本申请涉及一种抗CD79B抗体及抗原结合片段，包含所述抗CD79B抗体CDR区的嵌合抗体、人源化抗体，以及包含人抗CD79B抗体或其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。
背景技术
恶性肿瘤(癌症)是全球第二位死因，仅排在心脏病之后。其中淋巴瘤是起源于淋巴造血系统的恶性肿瘤，是全世界最常见的血液肿瘤。淋巴瘤分为非霍奇金淋巴瘤(non-Hodgkin’s lymphoma，NHL)和霍奇金淋巴瘤(Hodgkin’s lymphoma，HL)两类。非霍奇金淋巴瘤是一组具有较强异质性的淋巴细胞异常增殖性疾病的总称，其发病率远高于霍奇金淋巴瘤，约占淋巴瘤的80％以上。其中弥漫大B细胞淋巴瘤(DLBCL)是成人最常见的淋巴瘤类型，约占所有非霍奇金淋巴瘤的32.5％；在亚洲人群中，这一比例更高，接近40％。好发于老年患者，中位发病年龄为60-64岁，男性患者略多于女性患者。
目前弥漫大B细胞淋巴瘤(DLBCL)的一线标准方案为利妥昔单抗联合化疗方案(R-CHOP)。在利妥昔单抗上市之前，以蒽环类为基础的CHOP(环磷酰胺、多柔比星、长春新碱、泼尼松)方案是DLBCL的一线标准治疗方案。R-CHOP治疗方案使得DLBCL患者的长期存活率得到明显改善。临床试验结果显示：相比于传统的CHOP方案，R-CHOP方案治疗DLBCL能显著延长患者的中位总生存时间达4.9年，中位无病生存时间超过6.6年，5年无疾病进展生存率从30％提高至54％。但仍有10％至15％的难治患者没有反应，并有20％至30％的患者出现复发。而且不是所有DLBCL患者都适合R-CHOP方案，如80岁以上的高龄患者，他们的体能不允许进行标准的R-CHOP治疗。对于侵袭性更高的淋巴瘤类型，以及复发性淋巴瘤，R-CHOP方案可能无效。
根据淋巴细胞的起源分类，DLBCL属于B细胞淋巴瘤。B细胞抗原受体(B cell receptor，BCR)复合物，是B细胞表面最主要的分子。BCR复合物由识别和结合抗原的膜免疫球蛋白(mIg)和传递抗原刺激信号的Igα(CD79a)和Igβ(CD79B)异源二聚体组成。Igα和Igβ分别为约47kDa和约37kDa的糖蛋白，属于免疫球蛋白超家族成员，编码Igα和Igβ的基因分别称为mb-1和B29。Igα和Igβ胞膜外区氨基端处均有一个Ig样结构域。Igα和Igβ均可作为蛋白酪氨酸激酶的底物，参与BCR信号转导。
BCR广泛表达于B细胞淋巴瘤以及正常B细胞上。鉴于靶向CD20的利妥昔单抗在临床上所取得的成功以及可靠的安全性，开发靶向BCR的治疗方法也应该具有良好的疗效和安全性。针对CD79B抗原的治疗性抗体是有益的，其在施用给患者时(尤其是用于长期治疗)产生最低限度的抗原性或不产生抗原性。本领域亟需开发有效的CD79B抗体及其抗体药物偶联物，用于癌症治疗或延缓癌症进展。WO2020156439A公开了一种抗CD79B抗体及其治疗肿瘤的用途，本公开全文引入其内容。
发明内容
本公开涉及抗CD69B抗体或抗原结合片段及其与药物的偶联物(ADC)及医药用途，其中提供与抗CD69B抗体或抗原结合片段与细胞毒性物质(MMAE或其衍生物、依喜替康或其衍生物、艾日布林或其衍生物)偶联的ADC药物。
抗CD79B抗体及其抗原结合片段
WO2020156439A公开了一种抗CD79B抗体及其治疗肿瘤的用途，本公开全文引入其内容。
本公开提供一种抗CD79B抗体及其抗原结合片段，其包含：
重链HCDR1，其包含GX 1X 2FX 3X 4Y(SEQ ID NO：24)所示序列，其中，X 1为S或Y，X 2为S或T，X 3为T或S，X 4为S或T；
重链HCDR2，其包含X 5PRSGN(SEQ ID NO：25)所示序列，其中，X 5为F或Y；
重链HCDR3，其包含X 6X 7X 8X 9X 10GDFX 11Y(SEQ ID NO：26)，其中，X 6为不存在或G，X 7为不存在或S，X 8为G或D，X 9为D或Y，X 10为L或D，X 11为D或A；
轻链LCDR1，其包含RSSQSIVHX 12GNTYX 13E(SEQ ID NO：27)，其中，X 12为S或H，X 13为F或L；
轻链LCDR2，其包含SEQ ID NO：11或17所示序列；和
轻链LCDR3，其包含SEQ ID NO：12或18所示序列。
一些实施方案中，上述抗CD79B抗体或其抗原结合片段包含：
抗体重链可变区，所述的抗体重链可变区包含至少1个选自如以下序列所述的HCDR：SEQ ID NO：23，SEQ ID NO：7，SEQ ID NO：8，SEQ ID NO：9，SEQ ID NO：13，SEQ ID NO：14，SEQ ID NO：15；和/或
抗体轻链可变区，所述的抗体轻链可变区包含至少1个选自如以下序列所示的LCDR：SEQ ID NO：10，SEQ ID NO：11，SEQ ID NO：12，SEQ ID NO：16，SEQ ID NO：17或SEQ ID NO：18。
一些实施方案中，提供抗CD79B抗体或其抗原结合片段，包含：
(a)分别如SEQ ID NO：23、8、9所示的HCDR1、HCDR2和HCDR3，和分 别如SEQ ID NO：10、11、12所示的LCDR1、LCDR2和LCDR3；
(b)分别如SEQ ID NO：7、8、9所示的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO：10、11、12所示的LCDR1、LCDR2和LCDR3；或
(c)分别如SEQ ID NO：13、14、15所示的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO：16、17、18所示的LCDR1、LCDR2和LCDR3。
在一些具体实施方案中，前述方案(a)(即抗CD79B抗体或其抗原结合片段，包含分别如SEQ ID NO：23、8、9所示的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO：10、11、12所示的LCDR1、LCDR2和LCDR3的)中，不包含VH如SEQ ID NO：19、VL如SEQ ID NO：20的抗CD79B抗体或其抗原结合片段，或不包含重链全长如SEQ ID NO：28、轻链全长如SEQ ID NO：29的抗CD79B抗体或其抗原结合片段。
一些实施方案中，上述抗CD79B抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体、人抗体或其片段；例如，为人源化抗体或其片段。
一些实施方案中，提供抗CD79B抗体或其抗原结合片段，其包含重链可变区(VH)和轻链可变区(VL)，其中：
重链可变区(VH)包含：
如SEQ ID NO：3、5所示或与SEQ ID NO：3、5具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；
和/或
轻链可变区(VL)包含：
如SEQ ID NO：4、6所示或与SEQ ID NO：4、6具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列。
一些具体实施方案中，所述抗CD79B抗体或抗原结合片段的VH如序列SEQ ID NO：3所示，VL如序列SEQ ID NO：4所示；或VH如序列SEQ ID NO：5所示，VL如序列SEQ ID NO：6所示。
另一些实施方案中，提供抗CD79B抗体或其抗原结合片段，其中：
VH包含：
如SEQ ID NO：19、21所示或与SEQ ID NO：19、21具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列；
和/或VL，包含：
如SEQ ID NO：20、22所示或与SEQ ID NO：20、22具有至少80％、85％、90％、95％、96％、97％、98％或99％同一性的序列。
一些具体实施方案中，所述抗CD79B抗体或抗原结合片段的VH如序列SEQ ID NO：19所示，VL如序列SEQ ID NO：20所示；或VH如序列SEQ ID NO：21所示，VL如序列SEQ ID NO：22所示。
一些实施方案中，上述抗CD79B抗体或其抗原结合片段包含和轻链恒定区； 优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体。
一些具体实施方案中，所述重链恒定区为人IgG1、IgG2。
一些实施方案中，提供抗CD79B抗体或其片段，其中：
重链为SEQ ID NO：28或其变体序列所示；所述变体序列在重链包含0至10个氨基酸变化；
轻链为SEQ ID NO：29或其变体序列所示；所述变体序列在轻链包含0至10个氨基酸变化。
一些实施方案中，提供抗CD79B抗体或其片段，其中：
重链为SEQ ID NO：30或其变体序列所示；所述变体序列在重链包含0至10个氨基酸变化；
轻链为SEQ ID NO：31或其变体序列所示；所述变体序列在轻链包含0至10个氨基酸变化。
一些具体实施方案中，上述抗CD79B抗体或其片段可以为变体，所述变体在VL有0至10个(1、2、3、4、5、6、7、8、9、10个)氨基酸变化，和/或VH有0至10个(1、2、3、4、5、6、7、8、9、10个)氨基酸变化。
一些具体实施方案中，上述变体具有与原抗CD79B抗体或其片段相同或相似的生物学功能或效果。
一些实施方案中，上述抗CD79B抗体的抗原结合片段包括Fab、Fv、sFv、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)。
一些实施方案中，提供编码如上所述的抗CD79B抗体或其抗原结合片段的多核苷酸，例如DNA或RNA。
一些实施方案中，提供包含上述多核苷酸的表达载体，例如真核表达载体、原核表达载体、病毒载体。
一些实施方案中，提供上述表达载体转化的宿主细胞，例如真核细胞、原核细胞。一些具体实施方案中，宿主细胞为细菌(例如大肠杆菌)、酵母菌(例如毕赤酵母)、哺乳动物细胞(例如中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞)。
一些实施方案中，提供制备上述抗CD79B抗体或其抗原结合片段的方法包括步骤：在如前所述的宿主细胞中表达该抗体或其抗原结合片段，并自该宿主细胞中分离该抗体或其抗原结合片段。
一些实施方案中，提供治疗或预防增殖性疾病或延缓增殖性疾病进展的方法，包括：
向受试者施用治疗或延缓疾病有效量的上述抗人CD79B抗体或其抗原结合片 段或其编码的多核苷酸、药物组合物，所述增殖性疾病是癌症或肿瘤。
一些具体实施方案中，所述癌症或肿瘤是淋巴瘤或白血病；
所述淋巴瘤选自：弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤、小淋巴细胞性淋巴瘤、套细胞淋巴瘤；
所述非何杰金氏淋巴瘤选自：攻击性NHL、复发性攻击性NHL、复发性无痛性NHL、顽固性NHL、顽固性无痛性NHL；
所述白血病选自：慢性淋巴细胞性白血病、毛细胞白血病、急性淋巴细胞性白血病。 配 体-药物偶联物
本公开提供一种配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中：
药物选自MMAE或其衍生物、依喜替康或其衍生物、艾日布林或其衍生物；
配体为抗CD79B抗体或其抗原结合片段，所述抗CD79B抗体或其抗原结合片段为前述本公开任意的抗CD79B抗体或其抗原结合片段。
配体-药物(依喜替康或其衍生物)的偶联物
本公开提供一种配体-依喜替康或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其为式(I)所示的配体-依喜替康或其衍生物的偶联物或其药学上可接受的盐或溶剂化物：
Figure PCTCN2021108666-appb-000001
其中：
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，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-或化学键，p 1为1至20的整数；
L 3为由2至7个氨基酸构成的肽残基，其中氨基酸任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
R 1选自氢原子、卤素、环烷基烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基；
R 2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；
或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
R 4和R 5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基；
m为0至4的整数；
n为1至10，可以为整数或小数；
Pc为本公开提供的抗CD79B抗体或其抗原结合片段。
一些实施方案中，本公开提供配体-依喜替康或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其为式(II)所示的配体-依喜替康或其衍生物的偶联物或其药学上可接受的盐或溶剂化物：
Figure PCTCN2021108666-appb-000002
其中：
s 1为2至8的整数；优选5；
Pc、R 1、R 2、R 5～R 7、m和n如式(I)中所定义。
一些实施方案中，本公开多的配体-依喜替康或其衍生物的偶联物的连接单元-L-Y-包括，但不限于：
Figure PCTCN2021108666-appb-000003
一些实施方案中，本公开提供配体-依喜替康或其衍生物的偶联物或其药学上可接受的盐或溶剂化物包括，但不限于：
Figure PCTCN2021108666-appb-000004
其中：
n为1至10，可以为整数或小数；
Pc为前述本公开的抗CD79B抗体或其抗原结合片段。
一些实施方案中，制备如通式(Pc-L a-Y-D)所示的配体-依喜替康或其衍生物的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：
Figure PCTCN2021108666-appb-000005
Pc还原后，与通式(L a-Y-D)偶联反应，得到通式(Pc-L a-Y-D)所示的化合物；
其中，Pc为本公开的抗CD79B抗体或其抗原结合片段；W、L 2、L 3、R 1、R 2、R 5～R 7、m和n如式(I)中所定义。
以上实施方案中，Pc为本公开任意的抗CD79B抗体或其抗原结合片段；优选为包含如SEQ ID NO：28所示的重链和如SEQ ID NO：29所示的轻链的抗体，或包含如SEQ ID NO：30所示的重链和如SEQ ID NO：31所示的轻链的抗体。
一些具体实施方案中，本公开配体-依喜替康或其衍生物的偶联物包括其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、氘代物、或其混合物形式。
此处全文引入WO2020063673中化合物及其制备方法。
配体-药物(艾日布林或其衍生物)偶联物
本公开提供一种配体-艾日布林或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其如式Pc-(L-D) k所示：
其中，配体为Pc，所述Pc为本公开任意的抗CD79B抗体或其抗原结合片段，
L为将Pc共价附接于D的接头，且k为1至20(包括1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间任意数值)，
D如下式(III)所示：
Figure PCTCN2021108666-appb-000006
其中R 1a选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；优选R 1a是甲基；
R 1b选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代；优选R 1b是氢；或者
R 1a与R 1b与其相连接的原子一起形成C 5-8杂环烷基，所述杂烷基任选被烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、杂环基、芳基和杂芳基中的一个或多个取代基所取代，且R 1a和R 1b不同时为氢。
一些实施方案中，配体-艾日布林或其衍生物的偶联物中D中R1a为甲基。
一些实施方案中，配体-艾日布林或其衍生物的偶联物中D为下式所示：
Figure PCTCN2021108666-appb-000007
一些实施方案中，Pc-(L-D) k所示的配体-艾日布林或其衍生物的偶联物中，k选自1至10，可以为整数或小数。
在一些实施方案中，接头在细胞外是稳定的，使得配体-艾日布林或其衍生物的偶联物在细胞外环境中保持完整，但在例如内化至癌细胞中时能够裂解。
在一些实施方案中，当配体药物偶联物进入表达抗原的细胞时，偶联物中的药物部分从配体部分裂解，且裂解释放药物(如艾日布林或其衍生物)。
在一些实施方案中，接头包含可裂解部分；其中可裂解部分所处的位置，使得裂解之后药物(例如艾日布林衍生物)中没有残留的接头和Pc。
在一些实施方案中，接头中的可裂解部分为可裂解肽部分(moiety)。
在一些实施方案中，包含可裂解肽部分的配体药物偶联物显示较低的聚集水平，改善的配体与药物比率，增加的癌细胞的靶向杀死，减少的非癌细胞的脱靶杀死，和/或较高的药物负载(p)。
在一些实施方案中，相对于不可裂解的接头，添加可裂解部分增加细胞毒性和/或效力。在一些实施方案中，在表达中等水平抗原(例如CD79B)的癌症中增加效力和/或细胞毒性。在一些实施方案中，可裂解肽部分能够由酶裂解，且接头为酶能够裂解的接头。在一些实施方案中，接头为组织蛋白酶能够裂解的接头。在某些实施方案中，酶能够裂解的接头(例如组织蛋白酶能够裂解的接头)显示上述改善特性中的一种或多种。
在一些实施方案中，接头包含氨基酸单元(即，由2至7个氨基酸构成的肽残基)，优选所述氨基酸选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸、天冬氨酸，更优选缬氨酸-瓜氨酸(Val-Cit)、丙氨酸-丙氨酸-天冬酰胺(Ala-Ala-Asn)、甘氨酸-甘氨酸-赖氨酸(Gly-Gly-lys)、缬氨酸-赖氨酸(Val-lys)、缬氨酸-丙氨酸(Val-Ala)、缬氨酸-苯丙氨酸(Val-Phe)或甘氨酸-甘氨酸-苯丙氨酸-甘氨酸(Gly-Gly-Phe-Gly)。
在一些实施方案中，本公开的偶联物中的接头选自：
Figure PCTCN2021108666-appb-000008
Figure PCTCN2021108666-appb-000009
在一些实施方案中，氨基酸单元包含缬氨酸-瓜氨酸(Val-Cit)。
在一些实施方案中，包含Val-Cit的ADC显示增加的稳定性，减少的脱靶细胞杀死，增加的靶向细胞杀死，较低的聚集水平，和/或较高的药物负载。
另一方面，一些实施方案提供的接头包含可裂解磺酰胺部分，所述接头在还原条件下能够裂解。
在一些实施方案中，所述接头包含可裂解二硫化物部分，所述接头在还原条件下能够裂解。
另一方面，本公开的接头包含至少一种将D(如艾日布林衍生物)附接于可裂解部分的间隔单元。
在一些实施方案中，所述间隔单元包含对氨基苯甲氧基羰基(PAB)，
Figure PCTCN2021108666-appb-000010
另一方面，一些实施方案提供的偶联物由下式表示：
Figure PCTCN2021108666-appb-000011
k选自1至10，可以为整数或小数；p1选自2、4、6或8；
Figure PCTCN2021108666-appb-000012
k选自1至10，可以为整数或小数；p1选自2、4、6或8；P3选自0、1或2；
Figure PCTCN2021108666-appb-000013
k选自1至10，可以为整数或小数；p1选自2、4、6或8；
Figure PCTCN2021108666-appb-000014
k选自1至10，可以为整数或小数；p1选自2、4、6或8；P3选自0、1或2；
Figure PCTCN2021108666-appb-000015
k选自1至10，可以为整数或小数；p2选自2、4、6或8；
Figure PCTCN2021108666-appb-000016
k选自1至10，可以为整数或小数；p2选自2、4、6或8；
Figure PCTCN2021108666-appb-000017
k选自1至10，可以为整数或小数；p2选自2、4、6或8；
Figure PCTCN2021108666-appb-000018
k选自1至10，可以为整数或小数；p2选自2、4、6或8；
Figure PCTCN2021108666-appb-000019
k选自1至10，可以为整数或小数；p2选自2、4、6或8；
Figure PCTCN2021108666-appb-000020
k选自1至10，可以为整数或小数；P1选自2、4、6或8；P3选自0、1或2；
Figure PCTCN2021108666-appb-000021
k选自1至10，可以为整数或小数；P1选自2、4、6或8；P3选自0、1或2。
在一些所述方案中，所述的偶联物由下式表示：
Figure PCTCN2021108666-appb-000022
Figure PCTCN2021108666-appb-000023
Figure PCTCN2021108666-appb-000024
其中k选自1至10，可以为整数或小数；进一步地，D中R 1a优选自甲基，R 1b优选自氢。
本公开还提供了式D(Eribulin)所示化合物，
Figure PCTCN2021108666-appb-000025
或其可药用的盐，
其中，
R 1a选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代，优选R 1a是甲基；
R 1b选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、 乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代，优选R 1b是氢、甲基；或者
R 1a与R 1b与其相连接的碳原子一起形成C 5-8杂环烷基，所述杂烷基任选被烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、杂环基、芳基和杂芳基中的一个或多个取代基所取代，且R 1a和R 1b不同时为氢。
在一些实施方案中，式D(Eribulin)所示化合物中R 1a与R 1b各自独立选自C 1-6烷基，包括但不限于甲基、乙基、异丙基。
在一些实施方案中，式D(Eribulin)所示化合物中R 1a选自C 1-6烷基，包括但不限于甲基、乙基、异丙基；R 1b选自氢。
在一些实施方案中，式D(Eribulin)所示化合物中R 1a与R 1b与其相连接的碳原子一起形成C 6-8杂环烷基。
在一些实施方案中，式D(Eribulin)所示化合物为：
Figure PCTCN2021108666-appb-000026
在一些实施方案中，式D(Eribulin)所示化合物为：
Figure PCTCN2021108666-appb-000027
在一些实施方案中，式D(Eribulin)所示化合物为：
Figure PCTCN2021108666-appb-000028
本公开还提供式DZ(Eribulin)所示化合物，
Figure PCTCN2021108666-appb-000029
或其可药用的盐，
其中，R 1a选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代，优选R 1a是甲基；
R 1b选自氢、烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、烷氧基、芳基和杂芳基，所述的烷基、环烷基、芳基和杂芳基各自独立地任选被选自烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代，优选R 1b是氢；
或者R 1a与R 1b与其相连接的碳原子一起形成C 5-8杂环烷基，所述杂烷基任选被烷基(如C 1-6烷基，包括但不限于甲基、乙基、异丙基)、烷氧基(如C 1-6烷氧基，包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基)、卤素(如氟、氯、溴)、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基(如C 3-8环烷基，包括但不限于环丙基、环戊基或环己基)、杂环基、芳基和杂芳基中的一个或多个取代基所取代；且R 1a 和R 1b不同时为氢；
Y选自-O(CR aR b) m2-CR 8R 9-C(O)-、-NH-(CR aR b) m2-CR 8R 9-C(O)-、-O-CR 8R 9(CR aR b) m2-、-OCR 8R 9-C(O)-、-O(CR aR b) m2C(O)-或-S-(CR aR b) m2-CR 8R 9-C(O)-，其中R a和R b相同或不同，且各自独立地选自氢、氘原子、卤素或烷基；R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；或者，R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；m2选自0、1、2或3。
在一些实施方案中，式DZ(Eribulin)所示化合物中R 1a与R 1b各自独立选自C 1-6烷基，包括但不限于甲基、乙基、异丙基。
在一些实施方案中，式DZ(Eribulin)所示化合物中R 1a选自C 1-6烷基，包括但不限于甲基、乙基、异丙基；R 1b选自氢。
在一些实施方案中，式DZ(Eribulin)所示化合物中R 1a与R 1b与其相连接的碳原子一起形成C 6-8杂环烷基。
在一些实施方案中，DZ(Eribulin)所示化合物为：
Figure PCTCN2021108666-appb-000030
或其可药用的盐，
其中：R 8选自氢、C 3-6环烷基烷基或C 3-6环烷基；R 9选自氢、卤代烷基或C 3-6环烷基，优选氢；或者，R 8和R 9与其相连接的碳原子一起形成C 3-6环烷基；m2选自0、1、2或3。
在一些实施方案中，DZ(Eribulin)所示化合物选自：
Figure PCTCN2021108666-appb-000031
Figure PCTCN2021108666-appb-000032
另一方面，一些实施方案提供的DZ(Eribulin)所示化合物可以含有一个或多个不对称中心，如
Figure PCTCN2021108666-appb-000033
可以为
Figure PCTCN2021108666-appb-000034
以上实施方案中，Pc为本公开任意的抗CD79B抗体或其抗原结合片段；优选为包含如SEQ ID NO：28所示的重链和如SEQ ID NO：29所示的轻链的抗体，或包含如SEQ ID NO：30所示的重链和如SEQ ID NO：31所示的轻链的抗体。
一些具体实施方案中，本公开的偶联物包括其互变异构体、内消旋体、外消旋体、对映异构体、非对映异构体、氘代物、或其混合物形式。
此处全文引入CN202010073671.6中化合物及其制备方法。
配体-药物(MMAE或其衍生物)的偶联物
本公开提供一种MMAE类似物/衍生物，其为如通式(D(MMAE))所示的化合物：
Figure PCTCN2021108666-appb-000035
，或其可药用的盐，其中：
R 1-R 6选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
R 7选自氢原子、烷基、烷氧基和环烷基；
R 8-R 11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
R 12选自氢原子或烷基；
R 13-R 15选自氢原子、羟基、烷基、烷氧基或卤素；
R 16选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代。
在本公开的一些实施方案中，所述的通式(D(MMAE))所示的化合物，其为通式(D(MMAE) 1)所示的化合物：
Figure PCTCN2021108666-appb-000036
或其可药用的盐，
R 9与R 10形成环烷基；
R 2-R 8，R 11-R 16如通式(D)中所定义。
在本公开的一些实施方案中，所述的通式(D(MMAE))所示的化合物，其为：
Figure PCTCN2021108666-appb-000037
本公开的另一方面涉及一种配体-药物偶联物或其药学上可接受的盐或溶剂化 物，其中所述配体-药物偶联物包含式(-D(MMAE))所示的结构：
Figure PCTCN2021108666-appb-000038
或其可药用的盐，
其中：
R 2-R 6选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
R 7选自氢原子、烷基、烷氧基和环烷基；
R 8-R 11之中的任意两个形成环烷基，余下的两个基团任选自氢原子、烷基和环烷基；
R 12选自氢原子或烷基；
R 13-R 15选自氢原子、羟基、烷基、烷氧基或卤素；
R 16选自芳基或杂芳基，所述的芳基或杂芳基任选进一步被选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代；
波浪线表示氢原子，或与接头或与抗体共价连接。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其包含式(-D(MMAE) 1)所示的结构：
Figure PCTCN2021108666-appb-000039
其中：
R 9与R 10形成环烷基；
波浪线，R 2-R 8，R 11-R 16如通式(D(MMAE))中所定义。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其中所述配体-MMAE或其衍生物的偶联物包含式如下所示的结构：
Figure PCTCN2021108666-appb-000040
波浪线表示氢原子，或与接头或与抗体共价连接。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其为通式(IV)所示：
Figure PCTCN2021108666-appb-000041
其中，R 2是C 1-C 8烷基；
R 3是C 1-C 8烷基；
R 4是C 1-C 8烷基；
R 5是H；
R 6是C 1-C 8烷基；
R 7是C 1-C 8烷基；
R 8彼此独立，各自是O-(C 1-C 8烷基)；
R 9是H；
R 10是苯基；
Z是O或NH；
R 11选自H、C 1-C 20烷基或-(R 13O)m-R14；
m是3；
R 13是C 2-C 8烷基；
R 14是C 1-C 8烷基；
Pc为本公开的抗CD79B抗体或其抗原结合片段；L为接头；n为1至10，可以为整数或小数。
在一些具体实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其包含下所示的结构：
Figure PCTCN2021108666-appb-000042
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其为通式(Pc-L-D(MMAE))所示：
Figure PCTCN2021108666-appb-000043
其中：
R 2-R 16如通式(D(MMAE))中所定义；
n为1至10，可以为整数或小数；
Pc为本公开的抗CD79B抗体或其抗原结合片段；L为接头。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其为通式(Pc-L-D1)所示的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物：
Figure PCTCN2021108666-appb-000044
其中：
R 2-R 16如通式(-D(MMAE))中所定义；
Pc，L，n如通式(Pc-L-D(MMAE))中所定义。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其为通式下所示：
Figure PCTCN2021108666-appb-000045
Pc，L，n如通式(Pc-L-D(MMAE))中所定义。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其中n为1至8，可以为整数或小数；优选为1至6，可以为整数或小数。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其中接头为-Y-L 1-L 2-L 3-L 4，
Y为拉伸单元(stretcher unit)，选自
Figure PCTCN2021108666-appb-000046
或化学健，X 1选自氢原子、烷基，烷氧基，芳基或卤素，X 2选自亚烷基，所述的亚烷基任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
L 1为拉伸单元，选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 17-W-C(O)-或-C(O)-W-C(O)-，其中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，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
L 2选自-NR 18(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 18(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-或化学键，其中p 1为1至20的整数；优选为化学键；
L 3为由2至7个氨基酸构成的肽残基，所述的氨基酸优选自缬氨酸、瓜氨酸，甲基缬氨酸；其中氨基酸任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
R 17和R 18相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
L 4为延伸单元(extention unit)，优选为PAB。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其中Y为选自
Figure PCTCN2021108666-appb-000047
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其中L 1选自-(琥珀酰亚胺-3-基-N)-(CH 2)s 1-C(O)-，其 中s 1为2至8的整数；优选为
Figure PCTCN2021108666-appb-000048
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其中L 3为二肽氨基酸单元，优选自缬氨酸-瓜氨酸。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其中接头选自：
Figure PCTCN2021108666-appb-000049
其中a端连接配体，b端连接药物。
在本公开的一些实施方案中，所述的配体-MMAE或其衍生物的偶联物或其药学上可接受的盐或溶剂化物，其选自以下结构式：
Figure PCTCN2021108666-appb-000050
其中：
n为1至10，可以为整数或小数；
Pc为本公开的抗CD79B抗体或其抗原结合片段；优选包含如SEQ ID NO：28所示的重链和如SEQ ID NO：29所示的轻链的抗体，或包含如SEQ ID NO：30所示的重链和如SEQ ID NO：31所示的轻链的抗体。
本公开的另一方面涉及一种制备如通式(D(MMAE))所示的化合物，或其可药用的盐的方法，其包括以下步骤：
Figure PCTCN2021108666-appb-000051
通式(-DA(MMAE))脱保护反应，得到通式(-D(MMAE))所示的化合物，
其中：R 2-R 16如通式(D)中所定义。
本公开的另一方面涉及一种如下所示的化合物：
Figure PCTCN2021108666-appb-000052
，或其可药用的盐，可作为制备本公开配体-药物偶联物的中间体。
本公开的另一方面涉及一种制备化合物2(MMAE)，或其可药用的盐的方法，其包括以下步骤：
Figure PCTCN2021108666-appb-000053
化合物1(MMAE)和化合物2a(MMAE)进行缩合反应，得到化合物2(MMAE)。
本公开的另一方面涉及一种制备如通式(Pc-L-D(MMAE))所示的配体-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：
Figure PCTCN2021108666-appb-000054
还原Pc后，与化合物2(MMAE)偶联反应，得到通式(ADC(MMAE)-1)所示的化合物；
其中，Pc，n如通式(Pc-L-D(MMAE))中所定义。
为了完成本公开的MMAE及其衍射物合成目的，本公开采用如下的合成技术方案：
方案一：
本公开通式(D(MMAE))所示的化合物，或其可药用的盐的方法，其包括以下步骤：
Figure PCTCN2021108666-appb-000055
通式(DA(MMAE))在碱性条件下进行脱保护反应，得到通式(D(MMAE))所示的化合物，
其中：R 2-R 16如通式(D(MMAE))中所定义。
提供碱性条件的试剂包括有机碱和无机碱类，所述的有机碱类包括但不限于三乙胺、二乙胺、N-甲基吗啉、吡啶、六氢吡啶、N,N-二异丙基乙胺、正丁基锂、二异丙基氨基锂、醋酸钾、叔丁醇钠或叔丁醇钾，所述的无机碱类包括但不限于氢化钠、磷酸钾、碳酸钠、碳酸钾、碳酸铯、氢氧化钠和氢氧化锂；优选二乙胺。
方案二：
本公开的化合物2(MMAE)或其可药用盐或溶剂合物的制备方法，该方法包括：
Figure PCTCN2021108666-appb-000056
化合物(1(MMAE))和化合物(2a(MMAE))在碱性条件下，加入缩合剂，进行缩合反应，得到化合物2。
提供碱性条件的试剂包括有机碱和无机碱类，所述的有机碱类包括但不限于三乙胺、二乙胺、N-甲基吗啉、吡啶、六氢吡啶、N,N-二异丙基乙胺、正丁基锂、二异丙基氨基锂、醋酸钾、叔丁醇钠或叔丁醇钾，所述的无机碱类包括但不限于氢化钠、磷酸钾、碳酸钠、碳酸钾、碳酸铯、氢氧化钠和氢氧化锂，优选N,N-二异丙基乙胺。
缩合剂选自4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐、1-羟基苯并三唑和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N,N'-二环己基碳化二亚胺、N,N'-二异丙基碳二酰亚胺、O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯、1-羟基苯并三唑、1-羟基-7-偶氮苯并三氮唑、O-苯并三氮唑-N,N,N',N'-四甲脲六氟磷酸酯、2-(7-偶氮苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯、苯并三氮唑-1-基氧基三(二甲基氨基)磷鎓六氟磷酸盐或六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷，优选4-(4,6-二甲氧基-1,3,5-三嗪-2-基)-4-甲基氯化吗啉盐或1-羟基苯并三唑和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐，优选1-羟基苯并三唑。
方案三：
本公开通式(Pc-L-D)所示的配体-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：
Figure PCTCN2021108666-appb-000057
还原Pc后，与化合物2(MMAE)偶联反应，得到通式(ADC(MMAE)-1)所示的化合物；还原剂优选TCEP，特别地，优选还原抗体上的二硫键；
其中，Pc，n如通式(Pc-L-D(MMAE))中所定义。
实施例化合物
本公开提供配体-药物偶联物或其药学上可接受的盐或溶剂化物，选自：
Figure PCTCN2021108666-appb-000058
其中，Pc为本公开任意抗CD79B抗体或其抗原结合片段，n为1至10，可以为整数或小数。
一些具体实施方案中，Pc为本公开实施例中的抗CD79B抗体或其抗原结合片 段，例如为包含如SEQ ID NO：28所示的重链和如SEQ ID NO：29所示的轻链的抗体，或包含如SEQ ID NO：30所示的重链和如SEQ ID NO：31所示的轻链的抗体，n为1至6之间的整数或小数。
在一些具体的实施方案中，本公开的抗体药物偶联物的平均DAR值可以为1-10，例如2-8，或2-6，或1-6，或4-6之间的任意值。
虽然为简便起见将全部上述结构式表示成特定的异构体形式，但是本公开可以包括所有的异构体，如互变异构体、旋转异构体、几何异构体、非对映异构体、外消旋体和对映异构体。
互变异构体是结构异构体，通过被称为互变异构化的化学反应容易相互转化。这种反应常导致氢原子或质子的迁移，伴随着单键和邻近的双键的转换。一些常见的互变异构对为：酮-烯醇、内酰胺-内酰亚胺。内酰胺-内酰亚胺平衡示例是在如下所示的A和B之间。
Figure PCTCN2021108666-appb-000059
本公开中的所有化合物可以被表示成A型或B型。所有的互变异构形式在本公开的范围内。化合物的命名不排除任何互变异构体。
药物组合物
本公开进一步提供一种药物组合物，其含有如上所述的偶联物，以及可药用的赋形剂、稀释或载体。
用途
本公开进一步提供，选自以下的任一项或组合在制备药物中的用途：根据本公开的抗CD79B抗体或其抗原结合片段、根据本公开的偶联物、根据本公开的药物组合物；其中，所述抗体或其抗原结合片段或其药物偶联物用于治疗增殖性疾病或延缓增殖性疾病进展；所述增殖性病症可以是癌症或肿瘤；所述癌症或肿瘤选自淋巴瘤、弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤(NHL)、攻击性NHL、复发性攻击性NHL、复发性无痛性NHL、顽固性NHL、顽固性无痛性NHL、慢性淋巴细胞性白血病(CLL)、小淋巴细胞性淋巴瘤、白血病、毛细胞白血病(HCL)、急性淋巴细胞性白血病(ALL)，和/或套细胞淋巴瘤。
治疗方法
本公开提供一种治疗或预防增殖性疾病或延缓增殖性疾病进展的方法，该方法包括给予受试者治疗或延缓疾病有效量的根据本公开的抗CD79B抗体或其抗原结合片段、或根据本公开的药物组合物、或根据本公开的抗体-药物偶联物；其中，所述增殖性病症是癌症或肿瘤。
本公开提供在有所需要的受试者中治疗B细胞增殖性病症或自身免疫性病症 或延迟B细胞增殖性病症或自身免疫性病症进展的方法。一些实施方案中，B细胞增殖性病症为癌症或肿瘤。
本公开提供在具有细胞增殖性病症或自身免疫性病症的受试者中增强免疫功能的方法。一些实施方案中，所述细胞增殖性病症为癌症或肿瘤。
上述方案中的癌症或肿瘤可以选自淋巴瘤、弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤(NHL)、攻击性NHL、复发性攻击性NHL、复发性无痛性NHL、顽固性NHL、顽固性无痛性NHL、慢性淋巴细胞性白血病(CLL)、小淋巴细胞性淋巴瘤、白血病、毛细胞白血病(HCL)、急性淋巴细胞性白血病(ALL)，和/或套细胞淋巴瘤。
附图说明
图1：人CD79B ECD-hFc蛋白免疫Balb/c小鼠血清效价ELISA检测结果。
图2：人CD79B ECD-hFc蛋白免疫Balb/c小鼠血清效价FACS检测结果。
图3：人CD79B ECD-hFc蛋白免疫SJL小鼠血清效价ELISA检测结果。
图4：人CD79B ECD-hFc蛋白免疫SJL小鼠血清效价FACS检测结果。
图5：人CD79B ECD-his蛋白免疫SJL小鼠血清效价ELISA检测结果。
图6：人CD79B ECD-his蛋白免疫SJL小鼠血清效价FACS检测结果。
图7：猴CD79B ECD-his蛋白免疫SJL小鼠血清效价ELISA检测结果。
图8：抗人CD79B小鼠单克隆抗体ELISA检测结果。
图9A为抗人CD79B小鼠单克隆抗体的FACS检测结果。
图9B为抗人CD79B小鼠单克隆抗体FACS的检测结果，其中hIgG1为阴性对照抗体，SN8为阳性对照抗体。
图10：抗人CD79B小鼠单克隆抗体的交叉反应性的FACS检测结果。
图11：抗猴CD79B小鼠单克隆抗体ELISA检测结果。
图12A至图12G：抗猴CD79B小鼠单克隆抗体与猴外周血单核细胞结合FACS检测。图12A是猴外周血单核细胞FACS检测空白对照的结果；图12B是阴性对照抗体与猴外周血单核细胞的FACS检测结果；图12C是阳性对照抗体与猴外周血单核细胞的FACS检测结果；图12D是抗CD79B抗体mAb018与猴外周血单核细胞的FACS检测结果；图12E是抗CD79B抗体mAb019与猴外周血单核细胞的FACS检测结果；图12F是抗CD79B抗体mAb020与猴外周血单核细胞的FACS检测结果；图12G是抗CD79B抗体mAb021与猴外周血单核细胞的FACS检测结果。所使用的阳性对照是Genentech公司的SN8。
图13A至图13C为不同ADC对人弥漫性大B细胞淋巴瘤WSU-DLCL2裸小鼠皮下移植瘤的疗效。
图14：不同ADC对荷瘤裸小鼠体重的影响。
图15：不同ADC对人弥漫性大B细胞淋巴瘤WSU-DLCL2裸小鼠皮下移植 瘤的疗效。
图16：不同ADC对WSU-DLCL2荷瘤裸小鼠体重的影响。
图17：不同ADC对人弥漫性大B细胞淋巴瘤WSU-DLCL2裸小鼠皮下移植瘤的疗效的肿瘤照片。
图18：不同ADC对人滤泡性淋巴瘤DOHH-2裸小鼠皮下移植瘤的疗效。
图19：不同ADC对DOHH-2荷瘤裸小鼠体重的影响。
具体实施方式
术语
为了更容易理解本申请，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他技术和科学术语都具有本申请所属领域的一般技术人员通常理解的含义。
本申请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。
“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体、多克隆抗体；单特异性抗体、多特异性抗体(例如双特异性抗体)、全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条重链和两条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。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。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，同一类Ig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。每类Ig都可以有κ链或λ链。
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(CDR)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。
在本申请中，本申请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。
在本申请中，本申请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、IgG3、IgG4或其变体。
术语“鼠源抗体”在本申请中为根据本领域知识和技能制备的对人CD79B或其 表位的单克隆抗体。制备时用CD79B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。在本申请一个具体的实施方案中，所述的鼠源CD79B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。
术语“全人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的全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“全人抗体”不包括“人源化抗体”。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量非人蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的全人抗体可变区可进行最少反向突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将第一物种抗体的可变区与第二物种抗体的恒定区融合而成的抗体，可以减轻第一物种抗体诱发的免疫应答反应。作为一个示例，建立嵌合抗体，要先建立分泌兔源性特异性单抗的兔，分离所述抗体，根据需要克隆全人抗体的恒定区基因，将兔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。全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或IgG4重链恒定区，或者使用氨基酸突变后无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。
术语“抗原结合片段”包括：单链抗体(即全长重链和轻链)；Fab、修饰的Fab、Fab’、修饰的Fab’、F(ab’)2、Fv、Fab-Fv、Fab-dsFv、单结构域抗体(例如VH或VL或VHH)、scFv、二价或三价或四价抗体、Bis-scFv、diabody、tribody、triabody、tetrabody和上述任意一种的表位结合片段(参见例如Holliger and Hudson,2005,Nature Biotech.23(9):1126-1136；Adair and Lawson,2005,Drug Design Reviews-Online 2(3),209-217)。
产生和制备这些抗体片段的方法在本领域是公知的(参见例如Verma等人,1998,Journal ofImmunological Methods,216,165-181)。Fab-Fv形式首先公开于WO2009/040562，其二硫键稳定化形式Fab-dsFv首先公开于WO2010/035012。本公开的抗原结合片段还包括描述于WO2005/003169、WO2005/003170和WO2005/003171中的Fab和Fab’片段。多价抗体可包含多特异性例如双特异性或可以是单特异性的(参见例如WO92/22583和WO05/113605)，后者的一个示例是描述于WO 92/22583中的Tri-Fab(或TFM)。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结 构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)形成的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如使用1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immuno l.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunological interest.NIH Publication91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的CDR1、CDR2和CDR3，以及重链可变结构域的CDR2和CDR3。
可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
本申请的术语“与CD79B结合”，指能与CD79B或其表位相互作用，所述CD79B或其表位可以是人源的。
术语“抗原”指用于免疫接种具有免疫活性的源自脊椎动物的分子，以产生识别抗原的抗体，或用于筛选表达文库(例如尤其是噬菌体、酵母或核糖体展示文库)。在本公开中，抗原被更广义地定义，包括由抗体特异性识别的靶分子，以及包括用于产生抗体的免疫接种过程或用于选择抗体的文库筛选中使用的分子的一部分或模拟物。对于本公开的人CD79B以及人CD79B的截短变体和其它变体均被称为抗原。
术语“表位”是指抗原上与免疫球蛋白或抗体结合的位点。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公开所述的技术，例如X射线晶体分析法和二维核磁共振等。
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CD79B或其表位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(KD)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲和力是其与预定抗原(或其表位)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”在本公开中可以与术语“特异性结合的抗体”互换使用。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。
术语“载体”是指能够运输与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体。本文中公开的载体能够在宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至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。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。比如，用A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
氨基酸序列“同一性”指在比对氨基酸序列及必要时引入gap，以达成最大序列同一性百分比(且不将保守性取代视为序列同一性的一部分)，第一序列中与第二序列中相同的氨基酸残基的百分比。为测定氨基酸序列同一性百分比，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
术语“交叉反应”是指本申请的抗体与来自不同物种的CD79B结合的能力。例如，结合人CD79B的本申请的抗体也可以结合另一物种的CD79B。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，或与表达CD79B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分析，或流式细胞术。
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。对CD79B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CD79B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CD79B抗体接触时，与未与抗CD79B抗体接触的CD79B相比，任何可测量的CD79B结合亲和力降低。
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。
术语“诱导免疫应答”和“增强免疫应答”可互换使用，并指对特定抗原剌激(即，被动或适应性的)的免疫应答。
本申请中所述的“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，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，如选自IgG1的N297A、L234A、L235A；IgG2/4嵌合，IgG4的F235E、或L234A/E235A突变。
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，可以用人CD79B或其片段免疫动物，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。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)得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“药物”是指细胞毒性药物或免疫调节剂。细胞毒性药物能在细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物原则上在足够的浓度下都可以杀死细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。该术语包括毒素(如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素)、放射性同位素(例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)、化疗药物、抗生素和核溶酶。免疫调节剂是免疫关卡分子的抑制剂。
术语接头是指一端与配体连接而另一端与药物相连的片段或键，也可以连接其他接头后再与配体或药物相连。
接头可以包含一种或多种接头构件。示例性的接头构件包括6-马来酰亚氨基己酰基(“MC”)、马来酰亚氨基丙酰基(“MP”)、缬氨酸-瓜氨酸(“val-cit”或“vc”)、丙氨酸-苯丙氨酸(“ala-phe”)、对氨基苄氧羰基(“PAB”)、及那样源自与接头试剂的偶联的：N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(“SPP”)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(“SMCC”，在本文中也称作“MCC”)和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(“SIAB”)。接头可以包括拉伸单元、间隔单元、氨基酸单元和延伸单元，可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等,Cancer Research 52:127-131(1992)；美国专利No.5,208,020)。
术语“氨基酸单元”是指如果存在延伸单元的情况下，可以将以下结构式Y R中的羰基与延伸单元相连，如果没有延伸单元的情况下，可以将Y R直接连接在药物上的氨基酸，在本公开实施方案中，氨基酸单元表示为-K k-：
Figure PCTCN2021108666-appb-000060
-K k-是二肽、三肽、四肽、五肽、六肽、七肽、八肽、九肽或十肽，-K-单元各自独立地具有以下结构式K a或K b，k是0-10之间的一个整数：
Figure PCTCN2021108666-appb-000061
其中：
上述氨基酸单元中的R 23为-H或甲基；
R 24为H、甲基、异丙基、异丁基、仲丁基、苄基、对羟基苄基、-CH 2OH、 -CH(OH)CH 3、-CH 2CH 2SCH 3、-CH 2CONH 2、-CH 2COOH、-CH 2CH 2CONH 2、-CH 2CH 2COOH、-(CH 2) 3NHC(＝NH)NH 2、-(CH 2) 3NH 2、-(CH 2) 3NHCOCH 3、-(CH 2) 3NHCHO、-(CH 2) 4NHC(＝NH)NH 2、-(CH 2) 4NH 2、-(CH 2) 4NHCOCH 3、-(CH 2) 4NHCHO、-(CH 2) 3NHCONH 2、-(CH 2) 4NHCONH 2、-CH 2CH 2CH(OH)CH 2NH 2、2-吡啶基甲基-、3-吡啶基甲基-、4-吡啶基甲基-、苯基、环己基，
Figure PCTCN2021108666-appb-000062
R 25为-芳基-、-烷基-芳基-、-环烷基-、-烷基-环烷基-、-环烷基-烷基-、-烷基-环烷基-烷基-、-杂环基-，-烷基-杂环基-、-杂环基-烷基-、-烷基-杂环基-烷基-、-芳基-、-烷基-芳基-、-芳基-烷基-、-烷基-芳基-烷基-、-杂芳基-、-烷基-杂芳基-、-杂芳基-烷基-、-烷基-杂芳基-烷基-。
在一个实施方案中，-K k-为二肽，优选为-缬氨酸-瓜氨酸-、-苯丙氨酸-赖氨酸-或-N-甲基缬氨酸-瓜氨酸-，进一步优选为-缬氨酸-瓜氨酸-。
术语“拉伸单元”指一端通过碳原子与配体共价连接而另一端通过硫原子与药物(直接或间接)相连的化学结构片段。
术语“间隔单元”是一种双功能化合结构片段，可用于偶联接头和药物最终形成配体药物偶联物，这种偶联方式可以将药物选择性的连接到接头上。
术语“氨基酸”是指分子结构中含有氨基和羧基、并且氨基和羧基都直接连接在-CH-结构上的有机化合物。通式是H 2NCHRCOOH。根据氨基连结在羧酸中碳原子的位置，可分为α、β、γ、δ、ε……-氨基酸。在生物界中，构成天然蛋白质的氨基酸具有其特定的结构特点，即其氨基直接连接在α-碳原子上，即α-氨基酸。
术语“延伸单元”是指当氨基酸单元存在的情况下，可以将氨基酸单元与药物偶联，或当氨基酸单元不存在时，可通过与YR上羰基与药物偶联的化学结构。
本公开中延伸单元为PAB，结构如4-亚氨基苄基氨甲酰基片段，其结构如式下式所示，连接在D上，
Figure PCTCN2021108666-appb-000063
缩写
接头组件包括但不限于：
MC＝6-马来酰亚氨基己酰基，结构如下：
Figure PCTCN2021108666-appb-000064
Val-Cit或“vc”＝缬氨酸-瓜氨酸(蛋白酶可切割接头中的例示二肽)；
瓜氨酸＝2-氨基-5-脲基戊酸；
PAB＝对氨基苄氧羰基(“自我牺牲”接头组件的例示)；
Me-Val-Cit＝N-甲基-缬氨酸-瓜氨酸(其中接头肽键已经修饰以防止其受到组织蛋白酶B的切割)；
MC(PEG)6-OH＝马来酰亚氨基己酰基-聚乙二醇(可附着于抗体半胱氨酸)；
SPP＝N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯；
SPDP＝N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯；
SMCC＝琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯；
IT＝亚氨基硫烷；
PBS＝磷酸缓冲盐溶液；
术语“抗体-药物偶联物”(antibody drug conjugate，ADC)，指配体通过接头(或连接单元)与药物相连。在本公开中“抗体-药物偶联物”指把单克隆抗体(或抗原结合片段)通过连接单元与具有毒性药物相连。
术语“载药量”(Drug-to-Antibody Ratio，DAR)是指抗体-药物偶联物群体中，每个抗体所偶联的药物的平均数量，也可以表示为药物量和抗体量的比值。载药量的范围可以是每个抗体(Ab)连接1-20个，优选1-10个药物(D)。在本公开的实施方案中，载药量表示为k或n，示例性的可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或任意两数值之间数值的均值。优选1-10，更优选1-8，或2-8，或2-7，或3-8，或3-7，或3-6，或4-7，或4-6，或4-5的均值。可用常规方法如UV/可见光光谱法、质谱、ELISA试验、单抗分子大小变异体测定法(CE-SDS)和HPLC特征测定载药量。
本公开单抗分子大小变异体测定法(CE-SDS)可采用十二烷基硫酸钠毛细管电泳(CE-SDS)紫外检测方法，在还原和非还原条件下，依据分子量大小，按毛电泳法(2015年版《中国药典》0542)，定量测定重组单克隆抗体产品的纯度。
本公开的一个实施方案中，药物通过连接单元偶联在配体的N端氨基和/或赖氨酸残基的ε-氨基上，一般地，偶联反应中能与抗体偶联的药物分子数将小于理论上的最大值。
可以用以下非限制性方法控制配体药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
虽然对于特定偶联物分子而言，药物对抗体比率具有确切值(例如，在式(I)中为n)，但是应理解当用来描述含有许多分子的样品时，该值将经常是平均值，这归因于典型地与缀合步骤相关的某种程度的非均匀性。偶联物的平均载量在本文中称为药物对抗体比率或“DAR”。在一些实施例中，DAR在约1与约6之间，并且典型地是约1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7.0、7.5、8.0。在一些实施例中，按重量计至少50％的样品是具有平均DAR±2的化合物，并且优选地至少50％的样品是含有平均DAR±1的偶联物。实施例包括其中DAR是约1.1、1.2、1.3、1.4、1.5、1.6、1.7、1.8、1.9、2.0、2.1、2.2、。2.3、2.4、2.5、2.6、2.7、2.8、2.9、3.0、3.1、3.2、3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.4、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0的免疫偶联物。在一些实施例中，‘约为x’的DAR意指DAR的测量值在x的20％内。
DAR的检测方法例如，从还原和去糖基化样品的LC-MS数据外推出DAR值。LC/MS允许定量ADC中连接至抗体的有效负载(药物部分)分子的平均数。HPLC将抗体分离成轻链和重链，并且还根据每条链的接头-有效负载基团的数量分离重链(HC)和轻链(LC)。质谱数据能够鉴定混合物中的组分种类，例如LC、LC+1、LC+2、HC、HC+1、HC+2等。根据LC和HC链的平均负载量，可以计算ADC的平均DAR。给定免疫偶联物样品的DAR表示连接至含有两条轻链和两条重链的四聚体抗体的药物(有效负载)分子的平均数。例如WO2018142322中的DAR检测方法。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个(例如1、2、3、4、5、6、7、8、9、10、11和12个)碳原子的烷基，更优选含有1至10个碳原子的烷基，最优选含有1至6个碳原子的烷基。非限制性示例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的，烷基是含有1至6个碳原子的低级烷基，非限制性实例包括甲基、乙基、正丙基、异丙基、 正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基等。烷基可以是取代的或非取代的。当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个(例如1、2、3、4、5、6、7、8、9、10、11和12个)碳原子，更优选含有1至6个碳原子的亚烷基。亚烷基的非限制性示例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)和1,5-亚丁基(-CH 2CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代。
术语“烷氧基”指-O-(烷基)和-O-(环烷基)，其中烷基或环烷基的定义如上所述。烷氧基的非限制性示例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基、环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基”指饱和或部分不饱和单环或多环的环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，更优选包含3至10个碳原子，最优选包含3至8个碳原子。单环环烷基的非限制性示例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基、环辛基等；多环环烷基包括螺环、稠环或桥环的环烷基。
术语“杂环基”指饱和或部分不饱和单环或多环的环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包 含3至12个环原子，其中1至4个是杂原子；更优选环烷基环包含3至10个环原子。单环杂环基的非限制性示例包括吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉基、高哌嗪基等。多环杂环基包括螺环、稠环或桥环的杂环基。
术语“螺杂环基”指5至20元的环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。例如为6至14元，又例如为7至10元。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多螺杂环基，优选为单螺杂环基或双螺杂环基。例如为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。螺杂环基的非限制性示例包括：
Figure PCTCN2021108666-appb-000065
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。例如为6至14元，又例如为7至10元。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，例如为双环或三环，又例如为5元/5元或5元/6元双环稠杂环基。稠杂环基的非限制性示例包括：
Figure PCTCN2021108666-appb-000066
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧或S(O) m(其中m是整数0至2)的杂原子，其余环原子为碳。例如为6至14元，又例如为7至10元。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，例如为双环、三环或四环，更优选为双环或三环。桥杂环基的非限制性示例包括：
Figure PCTCN2021108666-appb-000067
所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性示例包括：
Figure PCTCN2021108666-appb-000068
等。
杂环基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基、氧代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，例如为6至10元，例如苯基和萘基，具体例如苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性示例包括：
Figure PCTCN2021108666-appb-000069
芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性示例包括：
Figure PCTCN2021108666-appb-000070
Figure PCTCN2021108666-appb-000071
杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基。
术语“环烷基烷基”指烷基上的氢被一个或多个环烷基取代，优选被一个环烷基取代，其中烷基如上所定义，其中环烷基如上所定义。
术语“卤代烷基”指烷基上的氢被一个或多个卤素取代，其中烷基如上所定义。
术语“氘代烷基”指烷基上的氢被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟基”指-OH基团。
术语“卤素”指氟、氯、溴或碘。
术语“氨基”指-NH 2。
术语“硝基”指-NO 2。
化学式中简称“Me”为甲基。
“任选地”或“任选”是指意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如“任选的被卤素或者氰基取代的C1-C6烷基”是指卤素或者氰基可以但不必须存在，该说明包括烷基被卤素或者氰基取代的情形和烷基不被卤素和氰基取代的情形。
本公开中化合物可以含有一个或多个不对称中心，因此可以产生对映异构体、非对映异构体、及可以根据绝对立体化学定义为(R)-或(S)-或用于氨基酸的(D)-或(L)-的其他立体异构形式。本公开包括所有可能异构体以及其外消旋的和光学纯的形式。光学活性的(+)和(-)、(R)-和(S)-或(D)-和(L)-异构体可以使用手性合成子或手性试剂制备，或者可以使用常规方法例如色谱法和分级结晶制备。用于制备/分离各个对映体的常规方法包括从合适的光学纯前体手性合成或使用例如手性高压液相色谱法(HPLC)的外消旋物(或盐或衍生物的外消旋物)拆分。当本文描述的化合物含有烯双键或其他几何不对称性中心，除非另有说明，否则其意味着所述化合物包括E和Z几何异构体。而且，所有的互变异构形式也意味着包括在内。
本公开所述化合物的化学结构中，在未指明构型时,键
Figure PCTCN2021108666-appb-000072
可以为
Figure PCTCN2021108666-appb-000073
或
Figure PCTCN2021108666-appb-000074
或者同时包含
Figure PCTCN2021108666-appb-000075
和
Figure PCTCN2021108666-appb-000076
两种构型。本公开所述化合物的化学结构中，在未指明构型时,键
Figure PCTCN2021108666-appb-000077
可以为Z构型或E构型，或者同时包含两种构型。例如，
Figure PCTCN2021108666-appb-000078
可以为
Figure PCTCN2021108666-appb-000079
“立体异构体”指通过相同的键而键合，但具有不同的三维结构的相同原子组成的化合物，其不可互换。本公开中预期各种立体异构体及其混合物，并且包括“对映异构体”，其指其分子彼此为不能重叠的镜像的两种立体异构体。
本公开所述化合物或其可药用盐、或其异构体的任何同位素标记的衍生物都被本公开所覆盖。能够被同位素标记的原子包括但不限于氢、碳、氮、氧、磷、氟、氯、碘等。它们可分别被同位素同位素2H(D)、3H、11C、13C、14C、15N、18F、31P、32P、35S、36Cl和125I等代替。除另有说明，当一个位置被特别地指定为氘(D)时，该位置应理解为具有大于氘的天然丰度(其为0.015％)至少3000倍的丰度的氘(即，至少45％的氘掺入)。
“取代的”指基团中的一个或多个氢原子，优选为最多5个，更优选为1至3个氢原子彼此独立地被取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯)键的碳原子结合时可能是不稳定的。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的盐”或“可药用盐”是指本公开配体-药物偶联物的盐，或本公开中所述的化合物的盐，这类盐用于受试者时具有安全性和有效性，且具有应有的生物活性。本公开抗体-抗体药物偶联化合物至少含有一个氨基，因此可以与酸形成盐，可药用盐的非限制性示例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、 富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
术语“溶剂合物”指本公开的配体-药物偶联化合物与一种或多种溶剂分子形成可药用的溶剂合物，溶剂分子的非限制性示例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
本公开涉及一类可裂解的特定结构的连接臂和特定结构的活性物，及由连接臂、活性物与抗体组成的抗体药物偶联物(ADC)。此类ADC是经由间隔物将一种毒性物质连于抗体而形成的复合物。该ADC在体内经降解而释放出活性分子，从而起到抗肿瘤的作用。
术语“载体”当其用于本公开的药物组合物中时，是指能改变药物进入受试者的方式和在体内的分布、控制药物的释放速度并将药物输送到靶点的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的示例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除活性成分以外的附加物，也可称为辅料。如片剂中的黏合剂、填充剂、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐、微晶纤维素等。
“给予”、“施用”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。“给予”、“施用”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。“给予”、“施用”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。
“治疗”意指给予受试者内用或外用治疗剂，诸如包含本申请的任一种抗体或其抗原结合片段或其偶联物的组合物，所述受试者已经患有、疑似患有、倾向于患有一种或多种疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种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，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其他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。尽管本申请的实施方案(例如治疗方法或制品)在缓解某个受试者中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受试者中减轻目标疾病症状。
实施例
以下结合实施例用于进一步描述，但这些实施例并非限制的范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
一、抗体的制备：
实施例1-1.蛋白抗原的克隆表达
抗体(包含轻、重链)和抗原用本领域公知的重叠延伸PCR方法构建，将重叠延伸PCR得到的DNA片段用HindIII/BstBI这两个酶切位点插入到表达载体pEE6.4(Lonza Biologics)中，在293F细胞(Invitrogen，Cat#R790-07)中表达得到。所得重组蛋白用于免疫或筛选。人CD79B氨基酸序列来源于NCBI(NP_000617.1)，其细胞外区域(ECD)包含159个氨基酸(Met1-Asp159)。
人CD79B细胞外区域(ECD)和人Fc区域融合蛋白(human CD79B ECD-hFc)的氨基酸序列如SEQ ID NO：1所示：
Figure PCTCN2021108666-appb-000080
人CD79B细胞外区域(ECD)和His标签的融合蛋白(human CD79B ECD-His)的氨基酸序列如SEQ ID NO：2所示：
Figure PCTCN2021108666-appb-000081
实施例1-2.小鼠单克隆抗体的制备
1、小鼠免疫和血清效价检测
以人CD79B细胞外区域(ECD)和人Fc区域融合蛋白(human CD79B ECD-hFc)以及人CD79B细胞外区域(ECD)和His标签的融合蛋白(human CD79B ECD-His)分别作为免疫原，通过腹腔注射法对Balb/c和SJL小鼠进行免疫，刺激小鼠体内产生针对人CD79B细胞外区域(ECD)的抗体。同时，以猴CD79B细胞外区域(ECD)和His标签的融合蛋白(cyno CD79B ECD-His)作为免疫原，通过腹腔注射法对SJL小鼠进行免疫，刺激小鼠体内产生针对猴CD79B细胞外区域(ECD)的抗体。
实验步骤如下：
1)腹腔注射免疫法。根据免疫程序计算出该次免疫所需的抗原量。蛋白抗原按照要求用PBS稀释抗原到相应浓度，随后进行抗原的乳化。将乳化好的抗原和佐剂混合物转移到2.0ml无菌注射器中，注射入小鼠右侧腹部。
2)小鼠血清的收集。将每只小鼠对应的血清管做好标记；通过小鼠颌下静脉采取大约100μL全血，将收集的全血样品室温静置大约2h后离心收集血清。将血清存放于4℃冰箱，用于抗体效价等检测。
3)免疫小鼠ELISA血清效价测定。以1μg/ml抗原浓度，每孔50μL，4℃冰箱过夜包被96孔板。次日，将包被好的板清洗一次(清洗液：1×PBST)。清洗后以1×PBST配置的1％BSA封闭液37℃封闭1小时。1×PBST洗板3次后，加入不同稀释浓度的待检血清37℃温箱，孵育1小时。1×PBST洗板3次后，加入100μL1:5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时。洗板后，取TMB显色液A液和B液1：1比例混合后显色。15分钟后，用1N盐酸终止显色反应。在Spectra Max M5读板机上，检测450nm的荧光值。
4)免疫小鼠FACS血清效价测定。DoHH2细胞或猴外周血单核细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入各组的待测血清，室温孵育60分钟后清洗细胞三次后加入抗-Mouse IgG(Fc特异性)-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。
各组小鼠血清效价ELISA和FACS检测结果见图1至图7。
人CD79B ECD-hFc蛋白共免疫5只Balb/c小鼠，编号为5491、5492、5493、5494、5495。血清效价ELISA检测结果见图1。结果表明，小鼠免疫血清效价达 到1:100K以上。小鼠血清FACS检测结果见图2，可见小鼠血清中产生的抗体可特异性识别DoHH2细胞表面的CD79B蛋白。
人CD79B ECD-hFc蛋白共免疫5只SJL小鼠，编号为5496、5497、5498、5499、5500。血清效价ELISA检测结果见图3。结果表明，小鼠免疫血清效价达到1:100K以上。小鼠血清FACS检测结果见图4，可见小鼠血清中产生的抗体可特异性识别DoHH2细胞表面的CD79B蛋白。
人CD79B ECD-his蛋白共免疫5只SJL小鼠，编号为5726、5727、5728、5729、5730。血清效价ELISA检测结果见图5。结果表明，小鼠免疫血清效价达到1:10K以上。小鼠血清FACS检测结果见图6，可见小鼠血清中产生的抗体可特异性识别DoHH2细胞表面的CD79B蛋白。
猴CD79B ECD-his蛋白共免疫5只SJL小鼠，编号为5501、5502、5503、5504、5505。血清效价ELISA检测结果见图7。结果表明，小鼠免疫血清效价达到1:10K以上。
通过以上结果可知，免疫的小鼠中产生了针对CD79B的特异性抗体。以上小鼠可用于细胞融合以产生能够分泌针对CD79B的特异性抗体的杂交瘤细胞系。
2、杂交瘤制备和抗体筛选
利用电融合方法对免疫组小鼠的淋巴细胞和骨髓瘤细胞SP2/0(ATCC，CCL-121 TM)进行融合，用于后续抗体筛选。
1)电融合实验。融合前一周将SP2/0细胞于10％DMEM培养基中进行扩大培养。摘取小鼠脾脏和淋巴结，进行冲洗和研磨以收集淋巴细胞。将SP2/0和淋巴细胞按比例混合，通过电融合仪进行融合。融合后，将细胞铺于96孔板中，37℃，5％CO2培养箱中培养；每天观察细胞状态；融合后5天，统计细胞融合率。融合后9-14天对融合的杂交瘤细胞进行筛选，挑选阳性孔中的细胞于24孔板中扩大培养。
2)有限稀释法进行亚克隆。将需要亚克隆的细胞株自24孔培养孔中重悬，进行计数。稀释每株细胞株的浓度至5-10个细胞/ml；将稀释好的细胞悬液加入96孔培养板中，每孔加0.2ml，每孔含细胞为1-2个。将96孔板放进37°，5％CO 2培养箱中培养。7-10天后，挑选阳性克隆至24孔进行进一步确认。
3)ELISA筛选。以1μg/ml抗原浓度，每孔50μL，4℃冰箱过夜包被96孔板。次日，将包被好的抗原板清洗一次(清洗液：1×PBST)。清洗后以1×PBST配置的1％BSA封闭液37℃封闭1小时。1×PBST洗板3次后，加入50μL待检细胞上清37℃温箱，孵育1小时。1×PBST洗板3次后，加入100μL 1:5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时。洗板后，取TMB显色液A液和B液1：1比例混合后显色。15分钟后，用1N盐酸终止显色反应。在Spectra Max M5读板机上，检测450nm的荧光值。
4)FACS筛选。DoHH2细胞悬液离心后，以含0.1％BSA的PBS重悬细胞，并计数；加入待测上清，室温孵育60分钟，清洗细胞后加入抗-小鼠IgG(Fc特异 性)-FITC二抗，避光室温孵育30分钟；清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。
5)杂交瘤阳性克隆鉴定。获得多个针对人CD79B抗原的特异性抗体；对其中ELISA和FACS测试中结合力最好的17株杂交瘤进行抗体的生产和纯化。抗人CD79B杂交瘤阳性克隆的ELISA检测结果见表1。抗人CD79B杂交瘤阳性克隆的FACS检测结果见表2。也获得了针对猴CD79B抗原的特异性抗体，对其中ELISA和FACS测试中结合力最好的4株杂交瘤进行抗体的生产和纯化。抗猴CD79B杂交瘤阳性克隆细胞的ELISA检测结果见表3。抗猴CD79B杂交瘤阳性克隆细胞的FACS检测结果见表4。均使用mIgG作为阴性对照。
表1.抗人CD79B杂交瘤阳性克隆的ELISA检测结果
抗体编号 克隆号 检测结果(OD450)
阴性对照 mIgG 0.05
mAb001 12A11-1G1 3.26
mAb002 19F10-1D7 3.69
mAb003 51E5G6 3.02
mAb004 67B10C1 3.41
mAb005 78A9F4 3.73
mAb006 48F11D6 3.34
mAb007 61A11F1 3.40
mAb008 63G2A2 3.56
mAb009 75F1E2 3.57
mAb010 66G3E7 3.83
mAb011 66E12H3 3.41
mAb012 73A8F3 3.45
mAb013 74C4F3 3.31
mAb014 70B8B3 3.10
mAb015 83B2G2 3.41
mAb016 83C2D4 3.46
mAb017 86F11F6 3.80
表2.抗人CD79B杂交瘤阳性克隆的FACS检测结果
抗体编号 克隆号 平均荧光值
阴性对照 mIgG 58
mAb001 12A11-1G1 13032
mAb002 19F10-1D7 5943
mAb003 51E5G6 33918
mAb004 67B10C1 26000
mAb005 78A9F4 24454
mAb006 48F11D6 20120
mAb007 61A11F1 18039
mAb008 63G2A2 16453
mAb009 75F1E2 16001
mAb010 66G3E7 15897
mAb011 66E12H3 14688
mAb012 73A8F3 14073
mAb013 74C4F3 12894
mAb014 70B8B3 8776
mAb015 83B2G2 10036
mAb016 83C2D4 9990
mAb017 86F11F6 8132
表3.抗猴CD79B杂交瘤阳性克隆的ELISA检测结果
Figure PCTCN2021108666-appb-000082
表4.抗猴CD79B杂交瘤阳性克隆的FACS检测结果
抗体编号 克隆号 平均荧光值
阴性对照 mIgG 35
mAb018 121H1E9 1973
mAb019 152E5F6 1708
mAb020 159E3E5 1488
mAb021 134H2F5 1225
3、小鼠单克隆抗体生产、纯化及鉴定
1)小鼠单克隆抗体生产和纯化。显微镜下观察需要抗体生产的杂交瘤细胞，长至≥70％以上且细胞状态良好，收集细胞并用Countstar IC1000型细胞计数仪进行计数。将细胞浓度调至1×10 5至5×10 5个/ml，转移至Roller Bottle。将Roller Bottle送到培养箱中37℃培养10-15天，每天观察细胞生长状况；待培养液变橙黄且透明，取出待纯化。按照常规方法，细胞上清通过Protein A柱子进行抗体纯化。
2)抗人CD79B小鼠单克隆抗体ELISA检测。以1μg/ml抗原浓度，每孔50μL，4℃冰箱过夜包被96孔板。次日，将包被好的抗原板清洗一次(清洗液：1×PBST)。清洗后以1×PBST配置的1％BSA封闭液37℃封闭1小时。1×PBST洗板3次后，加入50μL按照100nM，1:10稀释过的抗体，放入37℃温箱，孵育1小时。1×PBST洗板3次后，加入100μL 1:5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时。洗板 后，取TMB显色液A液和B液1：1比例混合后显色。15分钟后，用1N盐酸终止显色反应。在Spectra Max M5读板机上，检测450nm的荧光值。其中有4个抗人CD79B小鼠单克隆抗体的ELISA结合力最强(mAb008、mAb015、mAb016和mAb017)，具体数据见图8。
3)抗人CD79B小鼠单克隆抗体FACS检测。DoHH2细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入100μL按照100nM，1:10稀释过的抗体，室温孵育1小时。清洗细胞三次后加入抗-小鼠IgG(Fc特异性)-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。其中有4个抗人CD79B小鼠单克隆抗体的FACS结合力最强(mAb008、mAb015、mAb016和mAb017)，具体数据见图9A和图9B。其中hIgG1为阴性对照抗体，SN8为阳性对照抗体。SN8是罗氏制药开发的抗体偶联药物波妥珠单抗维多丁(polatuzumab vedotin)中所使用的抗体(序列参考序列来源：US20170362318A)。波妥珠单抗维多丁已被FDA批准上市。从结果可知，在FACS实验中，本公开优选出的三株抗人CD79B小鼠单克隆抗体mAb015、mAb016和mAb017的结合力均优于SN8。
4)抗人CD79B小鼠单克隆抗体交叉反应性FACS检测。采用瞬时转染的方法得到293F-cynoCD79B细胞，细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，加入100μL抗体，浓度分别为10μg/ml和1μg/ml。室温孵育1小时。清洗细胞三次后加入抗-小鼠IgG(Fc特异性)-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。抗人CD79B小鼠单克隆抗体交叉反应性FACS检测结果见图10。
5)抗猴CD79B小鼠单克隆抗体ELISA检测。以μg/ml抗原浓度，每孔50μL，4℃冰箱过夜包被。次日，将包被好的抗原板清洗一次(清洗液：1×PBST)。清洗后以1×PBST配置的1％BSA封闭液37℃封闭1小时。1×PBST洗板3次后，加入50μL按照100nM，1:10稀释过的抗体，放入37℃温箱，孵育1小时。1×PBST洗板3次后，加入100μL 1:5000稀释的羊抗鼠二抗，37℃温箱孵育0.5小时。洗板后，取TMB显色液A液和B液1：1比例混合后显色。15分钟后，用1N盐酸终止显色反应。在Spectra Max M5读板机上，检测450nm的荧光值。检测结果见图11。
6)抗猴CD79B小鼠单克隆抗体与猴外周血单核细胞结合FACS检测。从新鲜猴血中提取猴外周血单核细胞，细胞悬液离心后以含0.1％BSA的PBS重悬细胞后计数，分别加入抗-CD19和抗-cynoCD79B的抗体。室温孵育1小时。清洗细胞三次后加入抗-小鼠IgG(Fc特异性)-FITC二抗，避光室温孵育30分钟后清洗细胞三次，用含0.1％BSA的PBS轻轻重悬细胞，上机检测。抗猴CD79B小鼠单克隆抗体与猴外周血单核细胞结合FACS检测结果见图12A至图12G。CD19为B细胞的标记物。由结果可知，4个抗猴CD79B小鼠单克隆抗体均可与猴B细胞结合。
7)抗人CD79B小鼠单克隆抗体SPR检测。采用表面等离子共振技术(surface plasmon resonance，SPR)检测抗人CD79B抗体与其抗原人CD79B-His之间的亲和力。将抗原人CD79B-His蛋白固定化至CM5芯片。偶联水平设定在100RU。运行缓冲液为HBS-EP+(10mM HEPES，150mM NaCl，3mM EDTA，0.05％表面活性剂P20)。将稀释好的抗体以30μl/min的流速流过实验通道和对比通道3分钟，解离5分钟。然后再生缓冲液(10mM甘氨酸，pH1.5)在30μl/min的流速下运行30秒。数据用Biacore 8K软件进行分析。
实施例1-3.小鼠单克隆抗体可变区氨基酸序列测定
将实施例1-2中得到的高亲和力的杂交瘤单克隆细胞株进行可变区氨基酸序列测定，然后重组表达为人鼠嵌合抗体(cAb)，再进行进一步的抗体鉴定。用逆转录PCR扩增编码重链可变区和轻链可变区的基因，连接到载体；经测序，得到轻重链序列。首先采用RNA纯化试剂盒(Qiagen公司，货号74134)提取实施例1-2中活性好的单细胞株的总RNA。然后使用Invitrogen公司的货号为18080-051cDNA合成试剂盒制备cDNA单链。以此为模版，采用PCR方法合成轻重链可变区的序列，PCR产物克隆到TA载体pMD-18T，然后送去测序。将得到的轻重链序列分别克隆到表达载体，表达重组单克隆抗体。验证活性后，进行人源化工作。
抗人CD79B抗体的VH/VL CDR的氨基酸残基由Chothia编号系统确定并注释。
小鼠杂交瘤细胞单克隆抗体mAb015的序列：
重链可变区：
Figure PCTCN2021108666-appb-000083
轻链可变区：
Figure PCTCN2021108666-appb-000084
小鼠杂交瘤细胞单克隆抗体mAb017的序列：
重链可变区：
Figure PCTCN2021108666-appb-000085
轻链可变区：
Figure PCTCN2021108666-appb-000086
鼠源的CDR序列如表5所示：
表5.鼠源抗人CD79B抗体的CDR序列
Figure PCTCN2021108666-appb-000087
实施例1-4.抗人CD79B抗体人源化
将实施例1-3得到的鼠源抗CD79B单克隆抗体的轻重链序列，在抗体数据库里进行同源性比较后，建立人源化抗体模型。根据模型选择回复突变，筛选最优的人源化抗CD79B单克隆抗体。从小鼠Fab晶体结构模型数据库(比如PDB数据库)中查找与所得鼠源候选分子同源性相似的晶体结构，挑取高分辨率(比如
Figure PCTCN2021108666-appb-000088
)的Fab晶体结构，建立小鼠Fab模型。将鼠源抗体轻重链序列和模型中的序列比对，保留和鼠源抗体序列一致的序列，得到鼠抗体的结构模型。不一致的氨基酸为可能的回复突变位点。用Swiss-pdb viewer软件运行鼠抗体结构模型，优化能量(最小化)。将模型中除CDR外的不同氨基酸位点进行回复突变。将所得的突变抗体(人源化)和人源化之前的抗体进行活性的对比。保留活性好的人源化抗体。对CDR区域优化，包括避免糖基化、脱酰胺化、氧化位点等。
分别克隆、表达、纯化上述抗体；经ELISA、FACS和SPR等检测，选出活性最好的人源化抗体hAb015-10和hAb017-10，数据见表6。人源化抗体hAb015-10和hAb017-10保持了跟小鼠单克隆抗体相似的亲和力和相关功能。
表6.人源化抗CD79B抗体的鉴定结果
Figure PCTCN2021108666-appb-000089
Figure PCTCN2021108666-appb-000090
人源化抗体hAb015-10和hAb017-10的序列见下。
hAb015-10人源化抗体重链可变区(VH)：
Figure PCTCN2021108666-appb-000091
hAb015-10人源化抗体轻链可变区(VL)：
Figure PCTCN2021108666-appb-000092
hAb017-10人源化抗体重链可变区(VH)：
Figure PCTCN2021108666-appb-000093
hAb017-10人源化抗体轻链可变区(VL)：
Figure PCTCN2021108666-appb-000094
hAb015-10人源化抗体重链：
Figure PCTCN2021108666-appb-000095
hAb015-10人源化抗体轻链：
Figure PCTCN2021108666-appb-000096
hAb017-10人源化抗体重链：
Figure PCTCN2021108666-appb-000097
hAb017-10人源化抗体轻链：
Figure PCTCN2021108666-appb-000098
人源化后的mAb015的HCDR1序列中具有T30S突变，突变后的HCDR1为GSSFSSY(SEQ ID NO：23)。本公开的抗CD79B抗体具有如表7通式：
表7.CDR通式结构
Figure PCTCN2021108666-appb-000099
实施例1-5.抗CD79B抗体的内吞作用
为了检测本公开中的CD79B抗体结合人CD79B后，是否能够和人CD79B一起内吞到细胞内，用高表达人CD79B蛋白的DOHH-2细胞(DSMZ，ACC 47)进行细胞内吞实验以评估抗体的内吞能力。
DOHH-2细胞按照常规悬浮细胞的方法进行培养，完全培养基的成分为：RPMI 1640培养基(GIBCO，Cat No.：11835-030)，加10％(v/v)胎牛血清(FBS)(GIBCO，Cat No.：10099-141)和青霉素/链霉素(GIBCO，Cat No.：15070-063)。
实验时4℃低温离心收集细胞，1000rpm，5分钟。将细胞重悬到10-15ml在冰上预冷的FACS缓冲液中。FACS缓冲液的成分为：磷酸盐缓冲液(PBS)，pH7.4，加2％胎牛血清(FBS)。整个实验过程中，FACS缓冲液放在冰上预冷。细胞计数离心后以300,000细胞/孔加入96孔板中，离心弃去上清后加入Fc封闭液，12.5μg/ml(BD，Cat No.：564220)，100μl/孔。在室温下封闭10分钟。然后加入20μg/ml的待测CD79B抗体至对应的孔中，4℃避光孵育1小时。用预冷的PBS缓冲液洗2遍以除去未结合的抗体。加入细胞完全培养基(具有10％胎牛血清的RPMI 1640培养基)，在37℃，5％CO 2下孵育0小时、1小时、2小时、4小时。离心弃去上清后加入100μl/孔2％PFA缓冲液，重悬细胞后放置10分钟。然后用FACS缓冲液清洗3遍，然后加入100μl二抗溶液(荧光标记羊抗人二抗：1：250稀释，浓度为2μg/ml，Biolegend，Cat#409304),在4℃避光孵育半小时。加入预冷的PBS缓冲液，4℃离心弃上清，重复三次。细胞重悬在FACS缓冲液中，200μl/孔，使用流式细胞仪(BD FACS Calibur)检测。
结果表明，在4℃孵育时，SN8、hAb015和hAb017这3个抗体均不能被DOHH-2细胞内吞。而在37℃孵育时，1小时后大部分抗体已经被DOHH-2细胞内吞，4小时后抗体内吞达到最大值。3个抗体均具有较好的内吞作用。
二、化合物的制备：
化合物的结构是通过核磁共振(NMR)或/和质谱(MS)来确定的。NMR位移(δ)以10 -6(ppm)的单位给出。
NMR的测定是用Bruker AVANCE-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d 6)、氘代氯仿(CDCl 3)、氘代甲醇(CD 3OD)，内标为四甲基硅烷(TMS)。
MS的测定用Agilent 1200/1290 DAD-6110/6120 Quadrupole MS液质联用仪(生产商：Agilent，MS型号：6110/6120 Quadrupole MS)。
Waters ACQuity UPLC-QD/SQD(生产商：waters，MS型号：waters ACQuity Qda Detector/waters SQ Detector)THERMO Ultimate 3000-Q Exactive(生产商：THERMO，MS型号：THERMO Q Exactive)。
高效液相色谱法(HPLC)分析使用Agilent HPLC 1200DAD、Agilent HPLC 1200VWD和Waters HPLC e2695-2489高压液相色谱仪。
手性HPLC分析测定使用Agilent 1260 DAD高效液相色谱仪。
高效液相制备使用Waters 2545-2767、Waters 2767-SQ Detecor2、Shimadzu LC-20AP和Gilson GX-281制备型色谱仪。
手性制备使用Shimadzu LC-20AP制备型色谱仪。
CombiFlash快速制备仪为Combiflash Rf200(TELEDYNE ISCO)。
薄层层析硅胶板使用烟台黄海HSGF254或青岛GF254硅胶板；薄层色谱法(TLC)所用硅胶板的规格是0.15mm至0.2mm；薄层层析分离纯化产品时，采用的规格是0.4mm至0.5mm。
硅胶柱色谱法一般使用烟台黄海硅胶200至300目硅胶为载体。
本公开的已知的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH&Co.KG，Acros Organics，Aldrich Chemical Company，韶远化学科技(Accela ChemBio Inc)、达瑞化学品等公司。
实施例中无特殊说明，反应能够均在氩气氛或氮气氛下进行。氢气氛是指反应瓶连接一个约1L容积的氢气气球。
加压氢化反应使用Parr 3916EKX型氢化仪和清蓝QL-500型氢气发生器或HC2-SS型氢化仪。氢化反应通常抽真空，充入氢气，反复操作3次。
微波反应使用CEM Discover-S 908860型微波反应器。
实施例中无特殊说明，溶液是指水溶液。
实施例中无特殊说明，反应的温度为室温，为20℃至30℃。
纯化采用的柱层析的洗脱剂体系和薄层色谱法的展开剂体系包括：A：二氯甲烷/甲醇体系，B：正己烷/乙酸乙酯体系，C：石油醚/乙酸乙酯体系；溶剂的体积比根据化合物的极性不同而进行调节，也可以加入碱性或酸性试剂(如三乙胺和醋酸，等)进行调节。
本公开ADC中的药物部分参见WO2020063676A、US7098308、US6884869、CN202010073671.6、CN201911390425.7，相关的化合物合成及测试全文引用至本公开。其中的非限制性实施例如下：
实施例2-1.化合物A
Figure PCTCN2021108666-appb-000100
将本实施例中的1(10mg,0.014mmol,1.0eq)，本实施例中的2(21mg，0.021mmol，1.5eq)以及催化量的HOBt(0.5mg)溶于无水DMF(2mL)里，氩气保护，开启搅拌，加入2.71mg的DIEA以及0.08mL的吡啶，加热反应到40℃，搅拌2小时。经制备HPLC纯化得A(12.4mg)，收率65.4％，LC/MS(ESI):m/z 1363.4[M+1] +。
HNMR(CDCl 3,400MHz):δ0.63-0.95(m,32H),1.05-1.36(m,27H),1.60-1.98(m,4H),2.1-2.55(m,4H),2.78～3.02(m,3H),3.17-3.79(m,13H),3.93-4.20(m,2H),4.60～4.96(m,3H),5.14-5.36(m,2H),5.67(bs,1H),6.31-6.50(m,2H),6.64(s,2H),6.91(d,J＝14.8Hz,1H),7.19-7.30(m,7H),7.55(bs,2H),8.96(bs,1H),9.13(bs,1H)。
实施例2-2-1.化合物B
Figure PCTCN2021108666-appb-000101
冰水浴下，将本实施例中的1(50mg,0.08mmol)溶解到1.5mL的N,N-二甲基甲酰胺中，然后加入DIPEA(18mg,0.14mmol)，接着加入二(对硝基苯)碳酸酯(49 mg,0.16mmol)，然后在室温下搅拌约2-4小时，HPLC监控反应完全后，加入20mL甲基叔丁基醚，搅拌，过滤收集固体，固体干燥后得到36mg粗品，直接下一步反应，LC/MS(ESI):m/z 784.1[M+H] +。
Figure PCTCN2021108666-appb-000102
冰水浴下，将本实施例中化合物3(72.91mg,0.1mmol)溶于10mL的四氢呋喃中，加入Fmoc-OSu(41mg,0.12mmol)，然后在室温下搅拌3-5小时，HPLC监控反应完成后，减压浓缩得粗品，直接用于下一步反应。
Figure PCTCN2021108666-appb-000103
将上述本实施例中4粗品溶于10mL无水乙醚中，加入氧化银(34.8mg,0.15mmol)，接着加入碘甲烷(28.4mg,0.2mmol)，然后于室温下反应约10-16小时，监控反应基本完全后，过滤除去固体，然后减压浓缩得粗品，直接进行下一步反应。
Figure PCTCN2021108666-appb-000104
将上述本实施例中5粗品溶于10mL的四氢呋喃中，加入2mL的二乙基胺，然后在室温下搅拌约2-4小时，HPLC监控反应完毕后，直接减压浓缩得粗品，经硅胶柱纯化后得40mg产品，LC/MS(ESI):m/z 744.2[M+H] +。
Figure PCTCN2021108666-appb-000105
将上述本实施例中化合物6(13.5mg,0.018mmol)溶于1.5mL的DMF中，加入DIPEA(7mg,0.054mmol)，然后分批加入本实施例中化合物2(18mg,1.3 mmol)，搅拌约24-36小时后，减压浓缩得粗品，经HPLC制备分离得12.5mg化合物B，纯度96.95％，LC/MS(ESI):m/z 1388.3[M+H] +。
HNMR(CDCl 3,400MHz):δ0.85～0.90(m,3H),0.93～1.00(m,3H),1.08～1.10(m,3H),1.20～1.50(m,15H),1.75～2.04(m,6H),2.13～2.55(m,16H),2.70～2.77(m,1H),2.80～2.96(m,2H),3.16～3.97(m,20H),3.99～4.39(m,8H),4.60～4.80(m,6H),4.88～5.10(m,5H),5.24～5.37(m,4H),6.71(s,2H),7.03(d,J＝6.8Hz,1H),7.18～7.30(m,3H),7.63(d,J＝8.0Hz,2H),8.92(bs,1H)。
测试例1：体外细胞毒活性筛选：
1.1.实验原理及方法
本实验利用CTG检测ATP含量，反映肿瘤细胞的存活情况。首先通过种植不同密度的细胞并培养3天和5天，根据IC 50和最大抑制率确定最终培养条件。然后按照此条件检测毒素分子的杀伤作用。
1.2.细胞株的选择
根据实验目的，我们参考文献报道，前期选择乳腺癌、NSCLC两种疾病模型，选出SKBR3(HER2+)、MDA-MB-468(HER2-)和A549三株细胞用于筛选实验。
1.3.细胞培养条件的确定
1)细胞铺板：用胰酶将A549消化后，用培养介质终止，计数后分别取4.3×105，7.2×105，11.5×105个细胞使终体积均为26ml。在96孔板(3903)第2列到第11列每孔加180μL细胞悬液，使得细胞密度分别为每孔3K、5K、8K。第12列为200μL培养介质，剩余孔加入PBS。SKBR3和MDA-MB-468细胞重复上述操作。平行两份。
2)药物配制：在圆底96孔板(3788)中配制艾日布林(Eribulin)阳性对照。孔板1第一列为2mM(用DMSO将储液稀释10倍)，此后到第十列按梯度10倍稀释于DMSO中，第11列为DMSO。孔板2中第二列至第11列每孔加入相应培养介质95μL，从孔板1的第2列至第11列吸取5μL溶液至孔板2，混匀后，吸取20μL加入铺好的细胞中，继续培养3天和5天。
3)CTG检测：分别在第3天和第5天取出板，平衡至室温。每孔加入90μL CTG，避光室温反应10min，酶标仪读取发光值并计算IC 50。
1.4.药效检测
1)细胞铺板：将A549，SKBR3和MDA-MB-468用胰酶消化后，各自培养介质重悬计数，取6.33*10^5cells加培养介质至38ml，每孔180μL铺至96孔板(3903)，使每孔细胞数为3K，于37℃培养24h。
2)药物配制：在圆底96孔板(3788)中配制Eribulin和化合物D-1(实施例2-2-1中的化合物6)。孔板1第一列为2mM(用DMSO将储液稀释10倍)，此后到第十列按梯度10倍稀释于DMSO中，第11列为DMSO。孔板2中第二列至第11列每孔加入相应培养介质95μL，从孔板1的第2列至第11列吸取5μL溶液至 孔板2，混匀后，吸取20μL加入铺好的细胞中，平行2份。继续培养5天。
3)CTG检测：取出板，平衡至室温。每孔加入90μL CTG，避光室温反应10min，酶标仪读取发光值并计算IC 50。
1.5.数据结果
表8
Figure PCTCN2021108666-appb-000106
结论：化合物D-1在三种肿瘤细胞系中都有很好的杀伤效果，且明显优于阳性药艾日布林。
实施例2-2-2.
Figure PCTCN2021108666-appb-000107
室温下，量取0.3mL的1,4-二氧六环和0.3mL的水溶解化合物E-305(31mg，0.042mmol，根据文献Bioorg.Med.Chem.Lett.14(2004)5551–5554.合成得到)，再依次加入芴甲氧羰酰琥珀酰亚胺(17mg，0.050mmol)和碳酸钠固体(18mg，0.168mmol)。室温搅拌过夜，检测原料基本转化完全。反应中加入水淬灭，乙酸乙酯提取，减压浓缩，经硅胶柱纯化后得15mg产物，LC/MS(ESI):m/z 965.64[M+H] +。
Figure PCTCN2021108666-appb-000108
室温下，量取的二氯甲烷溶解上步所得产物(7mg，0.007mmol)，再依次加入4A分子筛(10mg)、三甲基氧鎓四氟硼酸(11mg，0.07mmol)和质子海绵(16mg，0.07mmol)，室温搅拌1h。检测原料基本转化完全，反应中加入水淬灭，甲 基叔丁基醚提取，再用1N稀盐酸洗，减压浓缩，经硅胶柱纯化后得7mg产物，LC/MS(ESI):m/z 979.68[M+H] +。
冰水浴下，量取1mL的四氢呋喃溶解上步所得产物(10mg，0.01mmol)，再滴加入DBU(6μL，0.04mmol)，搅拌搅拌至反应完全。反应中加入水淬灭，二氯甲烷提取，减压浓缩，经HPLC制备分离得5mg产物D-2，LC/MS(ESI):m/z757.85[M+H] +。
实施例2-2-3.
Figure PCTCN2021108666-appb-000109
冰水浴下，量取2mL的四氢呋喃溶解本实施例中化合物3(6mg，0.008mmol，根据文献Bioorg.Med.Chem.Lett.21(2011)1639–1643.合成得到)，再滴加入四氢锂铝溶液(80μL，1M in THF，0.08mmol)，搅拌并将反应温度缓慢升至40℃，LCMS检测原料基本转化完全，反应用十水合硫酸钠淬灭，冰水浴下搅拌半小时，过滤，滤液减压浓缩得粗品，直接用于下一步反应，LC/MS(ESI):m/z 758.4[M+H] +。
Figure PCTCN2021108666-appb-000110
室温下，量取的二氯甲烷溶解上步所得产物(7mg，0.007mmol)，再依次加入4A分子筛(10mg)、三甲基氧鎓四氟硼酸(11mg，0.07mmol)和质子海绵(16mg，0.07mmol)，室温搅拌1h。检测原料基本转化完全，反应中加入水淬灭，甲基叔丁基醚提取，再用1N稀盐酸洗，减压浓缩，经硅胶柱纯化后得7mg产物，LC/MS(ESI):m/z 979.68[M+H] +。
Figure PCTCN2021108666-appb-000111
室温下，量取0.5mL的1,4-二氧六环和0.5mL的水溶解上步所得产物，再依次加入芴甲氧羰酰琥珀酰亚胺(6.5mg，0.019mmol)和碳酸钠(6.8mg，0.064mmol)，室温搅拌过夜，检测原料基本转化完全。反应中加入水淬灭，乙酸乙酯提取，减压浓缩，粗品经硅胶柱纯化后得产物14mg，LC/MS(ESI):m/z 980.4[M+H] +。
Figure PCTCN2021108666-appb-000112
冰水浴下，量取1mL的二氯甲烷溶解上步所得产物(14mg，0.014mmol)，再加入戴斯-马丁氧化剂(18.2mg，0.042mmol)，搅拌并允许反应缓慢升至室温，搅拌至LCMS检测原料基本转化完全。加入碳酸氢钠水溶液淬灭反应，二氯甲烷提取，浓缩，粗品经硅胶柱纯化后得产物8mg，LC/MS(ESI):m/z 978.4[M+H] +。
Figure PCTCN2021108666-appb-000113
冰水浴下，量取1mL的四氢呋喃溶解上步所得产物(8mg，0.008mmol)，再滴加入DBU(6μL，0.032mmol)，搅拌1小时，中控检测原料基本转化完全，反应中加入水淬灭，二氯甲烷提取，浓缩，粗品经HPLC制备分离得目标产物D-3(1.3mg)，LC/MS(ESI):m/z 755.93[M+H] +。
测试例2：体外细胞毒活性筛选
2.1.实验原理及方法
本实验利用CTG检测ATP含量，反映肿瘤细胞的存活情况。
2.2.细胞培养条件的确定
1)细胞铺板：
用胰酶将A549，SKBR3和MDA-MB-468消化后，各自培养介质重悬计数，将细胞密度调整为2.2×104个/ml，在96孔板的列2至列11每孔加入135μl细胞悬液，列12为空白对照。在5％CO2的37℃培养箱中培养24h。
2)药物配制：
a)储液准备：用DMSO将药物溶解，使储液浓度为5mM。
b)孔板1：起始列1将储液稀释40倍，列2至列11依次3倍梯度稀释。列 12为DMSO。
c)孔板2：在列2至列11加入196μL相应培养介质，从孔板1的列3至列12吸4μL至孔板2的列2到列11。混匀。
2.3.处理细胞
从孔板2吸取15μL加入细胞。在5％CO 2的37℃培养箱中继续培养5天。
2.4.CTG检测：取出板，平衡至室温。每孔加入75μL CTG，避光室温反应10min，酶标仪读取发光值并计算IC50。
2.5.数据结果
表9
Figure PCTCN2021108666-appb-000114
Figure PCTCN2021108666-appb-000115
实施例2-2-4.
Figure PCTCN2021108666-appb-000116
冰水浴下，将艾日布林(9mg,0.012mmol)溶于0.3mL DMF中，加入DIPEA(3.5mg,0.028mmol)，然后分批加入本实施例中化合物2(7.8mg,0.011mmol)，搅拌至反应基本完全，减压浓缩得粗品，经HPLC制备分离得4.95mg化合物L-2，纯度97％，LC/MS(ESI):m/z 1374.3[M+H] +。
实施例2-2-5.
Figure PCTCN2021108666-appb-000117
称取本实施例中化合物4(13.4mg,0.0316mmol,1.7eq)和艾日布林甲磺酸盐(15mg,0.0182mmol,1eq)溶于DMF(0.5ml)中，冰浴冷却下加入三乙胺(10mg,0.0988mmol,5.4eq)、DMTMM(4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐，9.8mg,0.0332mmol,1.8eq)，反应液自然升到室温搅拌至基本完成。加入水(2ml)和乙酸乙酯(3ml)稀释分液，乙酸乙酯提取水相，合并有机相无水硫酸钠干燥，浓缩，粗品经制备板纯化得到16mg，收率86.7％，LC/MS(ESI):m/z 1136.3[M+H] +。
Figure PCTCN2021108666-appb-000118
在冰浴冷却下，称取上步所得产物(16mg,0.0141mmol,1eq)溶于THF中(0.4ml)，加入三乙胺(4.2mg,0.057mmol,4eq)，保持冰浴下搅拌至基本完成。加入二氯甲烷(5ml)稀释，水(2ml X 3)洗，有机相用无水硫酸钠干燥，浓缩得到粗品直接用于下一步，LC/MS(ESI):m/z 914.3[M+H] +。
Figure PCTCN2021108666-appb-000119
称取上步所得产物(16mg,0.0175mmol,1eq)和本实施例化合物6(11.6mg,0.0246mmol,1.4eq)溶于DMF中(0.5ml)，加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(9.9mg,0.026mmol,1.5eq)和N,N-二异丙基乙胺(5.5mg,0.0426mmol,2.4eq)，保持冰浴下搅拌至基本完成。加入水(2ml)和乙酸乙酯(3ml)稀释分液，乙酸乙酯提取水相，合并有机相无水硫酸钠干燥，浓缩，粗品经制备HPLC纯化得到10mg产物L-3，LC/MS(ESI):m/z 1368.3[M+H] +。
实施例2-2-6.
Figure PCTCN2021108666-appb-000120
称取本实施例中化合物4(11.6mg,0.0273mmol,1.5eq)和实施例2-2-1中的化合物D-1(艾日布林衍生物，13.5mg,0.0181mmol,1eq)溶于N,N-二甲基甲酰胺(0.5ml)中，冰浴冷却下加入DMTMM(10.1mg,0.0343mmol,1.3eq)，反应液至基本反应完成。加入水(2ml)和乙酸乙酯(3ml)中止和稀释反应，分液，乙酸乙酯提取水相，合并有机相无水硫酸钠干燥，浓缩，粗品经制备板纯化得到10mg，收率47.9％，LC/MS(ESI):m/z 1150.2[M+H] +。
Figure PCTCN2021108666-appb-000121
称取上步产物(10mg,0.0087mmol,1eq)溶于THF(1ml)中，加入1,8-二氮杂二环十一碳-7-烯(5.2mg,0.034mmol,4eq)，冰浴下搅拌至基本反应完成。加入二氯甲烷(5ml)稀释，水(2ml X 3)洗，有机相用无水硫酸钠干燥，浓缩得到粗品直接用于下一步，LC/MS(ESI):m/z 928.2[M+H] +。
Figure PCTCN2021108666-appb-000122
称取上步产物(16mg,0.0087mmol,1eq)和化合物6(7.8mg,0.0165mmol,1.9eq)溶于DMF中(0.5ml)，加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯(6.2mg,0.0163mmol,1.9eq)和DIEA(5.7mg,0.0441mmol,5eq)，冰浴下搅拌至基本反应基本完成。加入水(2ml)和乙酸乙酯(3ml)稀释分液，乙酸乙酯提取水相，合并有机相无水硫酸钠干燥，浓缩，粗品经制备HPLC纯化得到3.5mg产物L-4，两步收率29.1％，LC/MS(ESI):m/z 1382.2[M+H] +。
实施例2-3.
(S)-N-((3R,4S,5S)-1-((1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)-2-氮杂双环[3.1.0]己-2-基)-3-甲氧基-5-甲基-1-氧代庚-4-基)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰氨基)丁酰胺本实施例化合物1
Figure PCTCN2021108666-appb-000123
第一步
(9H-芴-9-基)甲基(1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)-2-氮杂双环[3.1.0]己烷-2-羧酸酯本实施例化合物1c将(2R,3R)-3-((1S,3S,5S)-2-(((9H-芴-9-基)甲氧基)羰基)-2-氮杂双环[3.1.0]己-3-基)-3-甲氧基-2-甲基丙酸本实施例化合物1a(1.05g，2.49mmol，采用专利申请“US2019/55223中说明书第20页的步骤7”公开的方法制备而得)和(1S,2R)-2-氨基 -1-苯基丙-1-醇本实施例化合物1b(0.42g，2.78mmol，采用公知的方法“Journal of Organic Chemistry,2012,vol.77,#12,p.5454-5460”制备而得)加入反应瓶，加入10mL二氯甲烷和2mL N,N-二甲基甲酰胺，氩气置换三次，搅拌下加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(1.14g，3.00mmol)和N,N-二异丙基乙胺(0.97g，7.47mmol)，室温搅拌1小时，加入30mL水，用二氯甲烷(15mL×4)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A纯化所得残余物，得到标题产物本实施例化合物1c(1.38g，产率：99.8％)。
MS m/z(ESI):555.2[M+1]
第二步
(2R,3R)-3-((1S,3S,5S)-2-氮杂双环[3.1.0]己-3-基)-N-((1S,2R)-1-羟基-1-苯基丙-2-基)-3-甲氧基-2-甲基丙酰胺本实施例化合物1d
将本实施例化合物1c(1.38g，2.49mmol)溶于10mL二氯甲烷中，加入20mL二乙胺，氩气置换三次，室温搅拌反应1小时。反应液浓缩，用硅胶柱色谱法以展开剂体系A纯化所得残余物，得到标题产物本实施例化合物1d(805mg，产率：97.3％)。
MS m/z(ESI):333.2[M+1]
第三步
(9H-芴-9-基)甲基((S)-1-(((S)-1-(((3R,4S,5S)-1-((1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)-2-氮杂双环[3.1.0]己-2-基)-3-甲氧基-5-甲基-1-氧代庚-4-基)(甲基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-3-甲基-1-氧代丁-2-基)(甲基)氨基甲酸酯本实施例化合物1f
将(5S,8S,11S,12R)-11-((S)-仲丁基)-1-(9H-芴-9-基)-5,8-二异丙基-12-甲氧基-4,10-二甲基-3,6,9-三氧代-2-氧杂-4,7,10-三氮杂十四-14-酸本实施例化合物1e(1.54g，2.41mmol，供应商：皓元)加入反应瓶，加入30mL乙腈，氩气置换三次，冰水浴降温至0-5℃，加入2-(7-氧化苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸盐(1.10g，2.89mmol)和N,N-二异丙基乙胺(0.94g，7.27mmol)，冰浴搅拌10分钟。加入本实施例化合物1d(805mg，2.42mmol)的10mL乙腈混悬液，冰浴搅拌反应40分钟。加入60mL水，用乙酸乙酯(20mL×4)萃取，有机相用饱和氯化钠溶液(60mL)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A纯化所得残余物，得到粗品产物本实施例化合物1f(2.9g)。
MS m/z(ESI):952.3[M+1]
第四步
(S)-N-((3R,4S,5S)-1-((1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)-2-氮杂双环[3.1.0]己-2-基)-3-甲氧基-5-甲基-1-氧代庚-4-基)-N,3-二甲基-2-((S)-3-甲基-2-(甲基氨基)丁酰氨基)丁酰胺本实施例化合
物1
将粗品本实施例化合物1f(510mg，0.53mmol)溶于2mL二氯甲烷中，加入4mL二乙胺，氩气置换三次，室温搅拌反应1小时。反应液浓缩，用硅胶柱色谱法以展开剂体系A纯化所得残余物，得到标题产物本实施例化合物1(266mg，产率：68.0％)。
MS m/z(ESI):730.4[M+1]
1H NMR(400MHz,CD 3OD):δ7.36-7.40(m,2H),7.31(t,2H),7.24(d,1H),4.69(d,1H),4.56(d,1H),4.17-4.28(m,2H),4.06-4.14(m,1H),3.91(d,1H),3.78(t,1H),3.27-3.44(m,7H),3.15(s,3H),2.84-2.93(m,1H),2.60-2.67(m,2H),2.30-2.37(m,3H),2.02-2.10(m,2H),1.79-1.95(m,4H),1.38-1.53(m,2H),1.25-1.36(m,2H),1.21(d,1H),1.13-1.17(m,2H),1.07-1.11(m,2H),0.93-1.05(m,15H),0.83-0.89(m,4H),0.70-0.79(m,1H)。
实施例2-4.
4-((S)-2-((S)-2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰氨基)-3-甲基丁酰氨基)-5-脲基戊酰氨基)苄基
((S)-1-(((S)-1-(((3R,4S,5S)-1-((1S,3S,5S)-3-((1R,2R)-3-(((1S,2R)-1-羟基-1-苯基丙-2-基)氨基)-1-甲氧基-2-甲基-3-氧代丙基)-2-氮杂双环[3.1.0]己-2-基)-3-甲氧基-5-甲基-1-氧代庚-4-基)(甲基)氨基)-3-甲基-1-氧代丁-2-基)氨基)-3-甲基-1-氧代丁-2-基)(甲基)氨基甲酸酯本实施例化合物2
Figure PCTCN2021108666-appb-000124
将本实施例化合物1(30mg，0.041mmol)加入到1mL N,N-二甲基甲酰胺中，加入4-((S)-2-((S)-2-(6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基)己酰氨)-3-甲基丁酰氨)-5-脲基戊酰氨)苄基(4-硝基苯基)碳酸酯本实施例化合物2a(45mg，0.061mmol，供应商：Ark)，再加入0.25mL吡啶，氩气置换三次，再加入1-羟基苯并三唑(12mg，0.089mmol)和N,N-二异丙基乙胺(16mg，0.123mmol)，室温搅拌反应4小时后补加2a(45mg，0.061mmol)，继续搅拌16小时。反应液进行高效液相色谱法纯化(分离条件：色谱柱：XBridge Prep C18 OBD 5um 19*250mm；流动相:A-水(10mmol NH 4OAc)：B-乙腈，梯度洗脱，流速：18mL/min)，收集其相应组分，减压浓缩，得到标题产物本实施例化合物2(18mg，产率：33.0％)。
MS m/z(ESI):1329.3[M+1]
1H NMR(400MHz,CD 3OD):δ7.58(d,2H),7.29-7.42(m,6H),7.20-7.26(m,1H),6.79(s,2H),5.04-5.20(m,4H),4.47-4.61(m,3H),4.13-4.28(m,3H),4.06-4.12(m,1H),3.91(d,1H),3.75-3.82(m,1H),3.48(t,3H),3.27-3.41(m,7H),3.16-3.25(m,2H),3.18(s,3H),2.91-2.97(m,2H),2.60-2.65(m,2H),2.27(t,2H),2.20(t,1H),2.01-2.10(m,3H),1.69-1.94(m,6H),1.63-1.68(m,6H),1.46-1.51(m,1H),1.27-1.37(m,5H),1.12-1.21(m,3H),1.09(d,2H),0.93-1.04(m,11H),0.80-0.92(m,11H),0.70-0.77(m,2H)。
三、抗CD79B抗体药物偶联物的制备：
ADC载药量分析的实验目的及原理：
采用紫外分光光度法(UV-Vis)测定ADC载量(Thermo nanodrop2000紫外分光光度计)。原理是在某波长下ADC的总吸光值等于药物与抗体在该波长下吸光值的加和。
实验方法
将装有琥珀酸钠缓冲液的比色皿分别置于参比吸收池和样品测定吸收池中后，扣除溶剂空白后，再将装有待测试溶液的比色皿置于样品测定吸收池中，测定280nm和370nm处吸光度。
结果计算：
A 280nm＝ε mab-280bC mab+ε Drug-280bC Drug       式(1)
ε Drug-280：药物在280nm平均摩尔消光系数5100；
C Drug：药物的浓度；
ε mab-280：单抗在280nm平均摩尔消光系数214600；
C mab：单抗的浓度；
b：光程长度为1cm。
同理可以得到样品在370nm下的总吸光值方程：
A 370nm＝ε mab-370bC mab+ε Drug-370bC Drug       式(2)
ε Drug-370：药物在370nm平均摩尔消光系数19000；
C Drug：药物的浓度；
ε mab-370：单抗在370nm消光系数为0；
C mab：单抗的浓度；
b：光程长度为1cm。
由式(1)和式(2)两种方程结合单克隆抗体和药物在两个检测波长下的消光系数和浓度数据可以计算出ADC的载药量：载药量＝C Drug/C mab。
实施例3-1.ADC-1
Figure PCTCN2021108666-appb-000125
在37℃条件下，向抗体hAb015-10的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.5mL，0.101μmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，25.3μL，0.253umol)，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℃。
将实施例2-2-1中的化合物B(1.41mg，1.015umol)溶解于50μL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到本实施例标题产物ADC-1(即hAb015-10-cys-B)的PBS缓冲液(0.84mg/mL，13.5mL)，于4℃冷冻储存。
HIC计算平均值：n＝3.06。
实施例3-2.ADC-2
Figure PCTCN2021108666-appb-000126
在37℃条件下，向抗体hAb015-10的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，3.5mL，0.236umol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，59.1μL，0.591umol)，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℃。
将化合物A(3.2mg，2.348umol)溶解于150μL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到本实施例标题产物ADC-2(即hAb015-10-cys-Malei-PEG2-vc-PAB-MMAE)的PBS缓冲液(2.17mg/mL，16.4mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝3.68。
实施例3-3.ADC-3
Figure PCTCN2021108666-appb-000127
在37℃条件下，向抗体hAb015-10的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，5.0mL，0.338umol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，85.0μL，0.850umol)，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℃。
将化合物MC-vc-PAB-MMAE(4.45mg，3.380umol)溶解于250μL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到本实施例标题产物ADC-3(即hAb015-10-cys-MC-vc-PAB-MMAE)的PBS缓冲液(2.79mg/mL，17.4mL)，于4℃冷冻储存。
CE-SDS计算平均值：n＝3.09。
实施例3-4.ADC-4
Figure PCTCN2021108666-appb-000128
在37℃条件下，向抗体hAb015-10的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.8mL，0.122umol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，30.4μL，0.304umol)，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℃。
Figure PCTCN2021108666-appb-000129
将本实施例化合物C(1.62mg，1.220umol)溶解于90μL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到本实施例标题产物ADC-4(即hAb015-10-cys-C)的PBS缓冲液(1.37mg/mL，12.0mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝4.52。
实施例3-5.ADC-5
Figure PCTCN2021108666-appb-000130
在37℃条件下，向抗体hAb015-10的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.5mL，0.101umol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，16.2μL，0.162umol)，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℃。
Figure PCTCN2021108666-appb-000131
将本实施例化合物D(0.87mg，0.810umol)溶解于37μL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到本实施例标题产物ADC-5(即HAB015-10-cys-D, DAR值约为2)的PBS缓冲液(0.90mg/mL，14.0mL)，于4℃冷冻储存。
化合物D的制备方法引自WO2020063676A，例如其实施例9。
RP-HPLC计算平均值：n＝1.81。
实施例3-6.ADC-6
Figure PCTCN2021108666-appb-000132
在37℃条件下，向抗体hAb015-10的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.5mL，0.101umol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，25.3μL，0.253umol)，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℃。
将实施例3-5中的化合物D(1.09mg，1.015umol)溶解于45μL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到本实施例标题产物ADC-6(即hAb015-10-cys-D，目标DAR值为约4)的PBS缓冲液(0.71mg/mL，14.0mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝3.46。
实施例3-7.ADC-7
Figure PCTCN2021108666-appb-000133
在37℃条件下，向抗体hAb015-10的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，1.5mL，0.101umol)加入配置好的三(2-羧乙基) 膦(TCEP)的水溶液(10mM，50.7μL，0.507umol)，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℃。
将实施例3-5中的化合物D(1.63mg，1.518umol)溶解于68μL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到本实施例标题产物ADC-7(即hAb015-10-cys-D,DAR值约为6)的PBS缓冲液(0.81mg/mL，13.5mL)，于4℃冷冻储存。
RP-HPLC计算平均值：n＝5.84。
实施例3-8.ADC-8
Figure PCTCN2021108666-appb-000134
在37℃条件下，向抗体SN8的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，79mL，5.338umol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，1.388mL，13.88umol)，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℃。
将化合物MC-VC-PAB-MMAE(70.3mg，53.40umol)溶解于3.5mL二甲亚砜中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到本实施例标题产物ADC-8(即SN8-cys-MC-PAB-MMAE，DAR值约为4)的PBS缓冲液(5.83mg/mL，132mL)，于4℃冷冻储存。
CE-SDS计算平均值：n＝3.59。
四、生物学评价：
实施例4-1.Biacore亲和力实验
本实施例用Biacore测定CD79B抗体(hAb015-10和SN8)和ADC与CD79B蛋白的亲和力。
实验仪器、材料与试剂：Biacore T200(GE)；生物传感芯片CM5(Cat.#BR-1005-30，GE)；氨基偶联试剂盒(Cat.#BR-1000-50，GE)；人抗捕获试剂盒(Cat.#BR-1008-39，GE)；人CD79B-His蛋白(Cat.#29750-H08H，Sino  Biological)；HBS-EP+10X缓冲溶液(Cat.#BR-1006-69，GE)用D.I.Water稀释至1X(pH 7.4)；
实验方法：按照人抗捕获试剂盒说明书中的方法将人抗捕获抗体共价偶联于CM5生物芯片上，从而亲和捕获一定量的CD79B抗体。
数据的统计和分析：用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型拟合数据，得出亲和力数值。
实验结果和结论：CD79B抗体和ADC与CD79B蛋白相结合的亲和力检测结果见表10。针对人CD79B蛋白，裸抗和不同ADC具有类似的结合力，且均高于阳性药Polivy。
其中，SN8(即Polivy中的抗体)序列为：
>SN8重链氨基酸序列
Figure PCTCN2021108666-appb-000135
>SN8轻链氨基酸序列
Figure PCTCN2021108666-appb-000136
表10.抗体及不同ADC与人CD79B蛋白的结合力
ADC/抗体 人CD79B EC 50(nM)
hAb015-10 0.38
ADC-1 0.51
ADC-2 0.48
ADC-3 0.40
ADC-4 0.32
ADC-5 0.80
ADC-6 0.43
ADC-7 0.53
ADC-8 5.98
实施例4-2.体外细胞内吞实验
用高表达人CD79B蛋白的DOHH-2细胞(DSMZ，ACC 47)进行细胞内吞实验，以评估ADC的内吞能力。
DOHH-2细胞按照常规悬浮细胞的方法进行培养，完全培养基的成分为：RPMI 1640培养基(GIBCO，Cat No.:11835-030)，加10％(v/v)胎牛血清(FBS)(GIBCO，Cat No.:10099-141)和青霉素/链霉素(GIBCO，Cat No.:15070-063)。实验时4℃低温离心收集细胞，1000rpm，5分钟。将细胞重悬到10-15ml在冰上预冷的FACS缓冲液中。FACS缓冲液的成分为：磷酸盐缓冲液(PBS)，pH7.4，加2％胎牛血清(FBS)。整个实验过程中，FACS缓冲液放在冰上预冷。细胞计数离心后以300,000细胞/孔加入96孔板中，离心弃去上清后加入Fc封闭液，12.5μg/ml(BD，Cat No.:564220)，100μl/孔。在室温下封闭10分钟。然后加入20μg/ml的待测ADC至对应的孔中，4℃避光孵育1小时。用预冷的PBS缓冲液洗2遍以除去未结合的ADC。加入细胞完全培养基(具有10％胎牛血清的RPMI 1640培养基)，在37℃，5％CO2下孵育0小时和4小时。离心弃去上清后加入100μl/孔2％PFA缓冲液，重悬细胞后放置10分钟。然后用FACS缓冲液清洗3遍，然后加入100μL二抗溶液(荧光标记羊抗人二抗:1:250稀释，浓度为2μg/ml，Biolegend，Cat#409304),在4℃避光孵育半小时。加入预冷的PBS缓冲液，4℃离心弃上清，重复三次。细胞重悬在FACS缓冲液中，200μl/孔，使用流式细胞仪(BD FACS Calibur)检测。
结果见表11。不同ADC与DoHH2细胞孵育4h的内吞率均大于65％，具有良好的内吞能力。各ADC的内吞率与阳性药Polivy相当。
表11.不同ADC在DoHH2细胞中的内吞作用
EC 50 内吞率(4h)
ADC-1 73％
ADC-2 66％
ADC-3 70％
ADC-4 81％
ADC-5 76％
ADC-6 75％
ADC-7 68％
ADC-8 76％
实施例4-3.细胞增殖实验
本实施例评价不同ADC对体外培养DoHH2、WSU-DLCL2和Raji细胞增殖的影响。根据文献报道(Leukemia.2015 Jul；29(7):1578-86；Blood.2007 Jul 15；110(2):616-23)，DoHH2是CD79B高表达细胞，WSU-DLCL2是CD79B低表达细胞，Raji为CD79B表达阴性细胞。
实验材料：
ADC-1：无色澄明液体，浓度0.84mg/mL，纯度99.17％；
ADC-2：无色澄明液体，浓度2.17mg/mL，纯度97.93％；
ADC-3：无色澄明液体，浓度2.79mg/mL，纯度98.28％；
ADC-4：无色澄明液体，浓度1.36mg/mL，纯度98.48％；
ADC-5：无色澄明液体，浓度0.9mg/mL，纯度98.49％；
ADC-6：无色澄明液体，浓度0.71mg/mL，纯度98.71％；
ADC-7：无色澄明液体，浓度0.81mg/mL，纯度98.27％；
ADC-8：无色澄明液体，浓度5.83mg/mL，纯度97.07％。
经实际检测，ADC-1的DAR＝3.06；ADC-2的DAR＝3.68；ADC-3的DAR＝3.09；ADC-4的DAR＝4.52；ADC-5的DAR＝1.81；ADC-6的DAR＝3.46；ADC-7的DAR＝5.84；ADC-8的DAR＝3.59。
以上药物均遮光，4℃密闭保存。
细胞株：DoHH2购自DSMZ，WSU-DLCL-2细胞购自American Type Culture Collection(ATCC)，Raji细胞购自American Type Culture Collection(ATCC)。
细胞用含10％胎牛血清(FBS)的RPMI 1640培养液培养。
试剂及仪器：RPMI 1640和FBS购自Gibco公司；四甲基偶氮唑兰(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide，MTT)购自上海生工。酶标仪购自BioTek公司。
实验方法：
接种一定数量的对数生长期细胞于96孔培养板，加入不同浓度的药物作用72小时。药物作用结束后，加入MTT工作液作用4小时，然后加入三联液溶解蓝紫色结晶甲瓒。酶标仪570nm和690nm波长下测定OD值，以下列公式计算细胞生长抑制率：
抑制率＝(对照孔 OD570nm-OD690nm－给药孔 OD570nm-OD690nm)/
对照孔 OD570nm-OD690nm×100％
根据各浓度抑制率，用PrismGraph 8计算半数抑制浓度IC50。
结果如表12所示。
表12.不同ADC的体外增殖抑制活性
Figure PCTCN2021108666-appb-000137
实施例4-4.ADC对人弥漫性大B细胞淋巴瘤WSU-DLCL2裸小鼠皮下移植瘤的疗效
本实施例评价并比较各ADC药物对裸小鼠皮下移植瘤的疗效。
hIgG1(HRP00252)：无色澄明液体，浓度22.77mg/mL，纯度99.03％，生产日期2018.7.24，有效期至2020.01.24(暂定18个月)；-70℃冻存。
1.药物：ADC-1、ADC-2、ADC-3、ADC-4、ADC-6、ADC-8同实施例4-3。
2.细胞和小鼠：人弥漫性大B细胞淋巴瘤WSU-DLCL2细胞购自American Type Culture Collection。WSU-DLCL2细胞用10-cm培养皿培养，培养条件为RPMI 1640培养基(Gibco)中加10％胎牛血清以及青、链霉素，于37℃、含5％CO2空气的培养箱中培养。一周2-3次传代,当细胞呈指数生长期时，收集细胞，计数，接种。
裸小鼠，BALB/c-nu，35日，♀，购自北京华阜康生物科技股份有限公司。生产许可证号：SCXK(京)2019-0008，动物合格证号No.1103222011004014。饲养环境：SPF级。
3.实验步骤：每只裸小鼠皮下接种2.1×10 7WSU-DLCL2细胞，待肿瘤生长至100-150mm 3后，根据肿瘤体积将动物分组(D0)。小鼠静脉注射(IV)给药，给药体积10mL/kg；具体给药剂量和给药方案见表X。每周测2次肿瘤体积，称小鼠体重，记录数据。
4.实验指标及统计学分析：
实验指标为考察药物对肿瘤生长的影响，具体指标为T/C％或抑瘤率TGI(％)。
每周二次用游标卡尺测量肿瘤直径,肿瘤体积(V)计算公式为：
V＝1/2×a×b 2，其中a、b分别表示长、宽。
T/C(％)＝(T-T 0)/(C-C 0)×100，其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。其中，T为给药ADC的肿瘤体积，C为给药IgG1作为对照组的肿瘤体积。
肿瘤生长抑制率％(TGI％)＝100-T/C(％)；
当肿瘤出现消退时，肿瘤生长抑制率％(TGI％)＝100-(T-T 0)/T 0×100；
如果肿瘤比起始体积缩小，即T<T 0或C<C 0时，即定义为肿瘤部分消退(PR)；如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。
实验结束、达到实验终点、或溶剂组平均肿瘤体积达到1500mm 3，CO 2麻醉处死动物，随后解剖取瘤并拍照。
除非特别说明，二组肿瘤体积之间比较采用双因素ANOVA检验，P<0.05定义为有统计学显著性差异。
5.结果：
ADC-1(3mg/kg、10mg/kg,IV,D0)剂量依赖性地抑制WSU-DLCL2裸小鼠皮下 移植瘤的生长，抑瘤率分别为75％和137％，10mg/kg剂量组全部肿瘤部分消退；
ADC-2(3mg/kg、10mg/kg,IV,D0)对WSU-DLCL2的抑瘤率分别为76％和123％,10mg/kg剂量组有全部肿瘤部分消退；
ADC-3(3mg/kg,IV,D0)对WSU-DLCL2的抑瘤率为76％，有1/6肿瘤部分消退；
ADC-4(3mg/kg、10mg/kg,IV,D0)对WSU-DLCL2的抑瘤率分别为95％和129％,3mg/kg和10mg/kg剂量组分别有1/6和6/6肿瘤部分消退；
ADC-6(3mg/kg,IV,D0)对WSU-DLCL2的抑瘤率为86％，有2/6肿瘤部分消退；
ADC-8(3mg/kg、10mg/kg,IV,D0)对WSU-DLCL2的抑瘤率分别为39％和93％,10mg/kg剂量组有4/6肿瘤部分消退。
荷瘤小鼠对以上药物均能较好耐受，没有明显体重减轻等症状发生。使用IgG1作为阴性对照。结果请参见表13以及图13A至图13C、图14。
表13.ADC对WSU-DLCL2裸小鼠皮下移植瘤的疗效
Figure PCTCN2021108666-appb-000138
D0:第一次给药时间；P值指与溶剂相比；IV：静脉注射；部分消退：D21肿瘤体积小于D0。
6.结论
ADC-1、ADC-2、ADC-3、ADC-4、ADC-6和ADC-8的3mg/kg或10mg/kg单次静脉注射均对WSU-DLCL2裸小鼠皮下移植瘤有明显疗效，引起肿瘤部分消退；药物有明显的剂量依赖性，且同等剂量下各ADC药效均优于阳性药ADC-8(即Polivy)。荷瘤小鼠对以上药物均能较好耐受。
实施例4-5.ADC对人弥漫性大B细胞淋巴瘤WSU-DLCL2裸小鼠皮下移植瘤的疗效
本实施例进一步评价并比较ADC药物对WSU-DLCL2裸小鼠皮下移植瘤的疗效。
1.药物：用生理盐水将ADC-5、ADC-6、ADC-7、ADC-1、ADC-8药物稀释至实施例4-3中浓度。
2.细胞和小鼠：同实施例4-4。
3.实验步骤：每只裸小鼠皮下接种2×10 7WSU-DLCL2细胞，待肿瘤生长至100-150mm 3后，根据肿瘤体积将动物分组(D0)。小鼠静脉注射(IV)给药，给药体积10mL/kg；具体给药剂量和给药方案见表12。每周测2次肿瘤体积，称小鼠体重，记录数据。
4.实验指标同实施例4-4，统计学分析为：
除非特别说明，二组肿瘤体积之间比较采用双尾Student’s t检验，P<0.05定义为有统计学显著性差异。
5.结果
ADC-5(3mg/kg、6mg/kg、12mg/kg,IV,D0)对WSU-DLCL2裸小鼠皮下移植瘤的抑瘤率分别为69％、86％和88％，其中6mg/kg和12mg/kg剂量组分别有1/6和1/6肿瘤部分消退；
ADC-6(1.5mg/kg、3mg/kg、6mg/kg,IV,D0)对WSU-DLCL2皮下移植瘤的抑瘤率分别为66％、108％和125％，其中3mg/kg和6mg/kg剂量组分别有5/6和6/6肿瘤部分消退；
ADC-7(1mg/kg,IV,D0)对WSU-DLCL2皮下移植瘤的抑瘤率为91％,有1/6肿瘤部分消退；
ADC-1(3mg/kg,IV,D0)对WSU-DLCL2皮下移植瘤的抑瘤率为44％；
ADC-8(3mg/kg,IV,D0)对WSU-DLCL2皮下移植瘤的抑瘤率为10％。
荷瘤小鼠对以上药物均能很好耐受，没有体重减轻等症状发生。
具体结果请参见表14和图15至图17。
表14.给药方案和实验结果
Figure PCTCN2021108666-appb-000139
其中，D0：第一次给药时间；P值指与溶剂相比；IV：静脉注射。
实施例4-6.ADC对人B细胞淋巴瘤DoHH2裸小鼠皮下移植瘤的疗效
本实施例进一步评价并比较ADC药物对DoHH2裸小鼠皮下移植瘤的疗效。
1.药物：用生理盐水将ADC-1、ADC-6、ADC-8药物稀释至实施例4-3中浓度。
2.细胞和小鼠：DOHH-2细胞购自德国DSMZ。DOHH-2细胞用10-cm培养皿培养，培养条件为RPMI 1640培养基(Gibco)中加10％胎牛血清以及青、链霉素，于37℃、含5％CO 2空气的培养箱中培养。一周2-3次传代,当细胞呈指数生长期时，收集细胞，计数，接种。
裸小鼠，BALB/c-nu，4-5周，♀，购自上海灵畅生物科技有限公司。生产许可证号：SCXK(沪)2018-0003，动物合格证号20180003010222。饲养环境：SPF级。
3.实验步骤：每只裸小鼠皮下接种3×10 7DOHH-2细胞，待肿瘤生长至100-150mm 3后，根据肿瘤体积将动物分组(D0)。小鼠静脉注射(IV)给药，给药体积10mL/kg；具体给药剂量和给药方案见表15。每周测2次肿瘤体积，称小鼠体重，记录数据。
4.实验指标及统计学分析：
实验指标为考察药物对肿瘤生长的影响，具体指标为T/C％或抑瘤率TGI(％)。
每周二次用游标卡尺测量肿瘤直径,肿瘤体积(V)计算公式为：
V＝1/2×a×b 2其中a、b分别表示长、宽。
T/C(％)＝(T-T 0)/(C-C 0)×100其中T、C为实验结束时的肿瘤体积；T0、C0为实验开始时的肿瘤体积。T为给药ADC的肿瘤体积，C为给药IgG1作为对照组的肿瘤体积。
肿瘤生长抑制率％(TGI％)＝100-T/C(％)；
当肿瘤出现消退时，肿瘤生长抑制率％(TGI％)＝100-(T-T 0)/T 0×100；
如果肿瘤比起始体积缩小，即T<T 0或C<C 0时，即定义为肿瘤部分消退(PR)；如果肿瘤完全消失，即定义为肿瘤完全消退(CR)。
实验结束、达到实验终点、或溶剂组平均肿瘤体积达到1500mm 3，CO 2麻醉处死动物，随后解剖取瘤并拍照。
除非特别说明，二组肿瘤体积之间比较采用双因素ANOVA检验，P<0.05定义为有统计学显著性差异。
5.结果：
ADC-1、ADC-6、ADC-8(1mg/kg,IV,D0)对DOHH-2裸小鼠皮下移植瘤的抑瘤率分别为82％(1/6PR)、127％(5/6PR)、41％；荷瘤小鼠对以上药物均能很好耐受，没有明显体重减轻等症状发生。
结果请参见表15以及图18、图19。
表15.不同ADC对人滤泡性淋巴瘤DOHH-2裸小鼠皮下移植瘤的疗效
Figure PCTCN2021108666-appb-000140
D0:第一次给药时间；P值指与溶剂相比；IV：静脉注射；部分消退：D21肿瘤体积小于D0。
6.结论
ADC-1和ADC-6的1mg/kg单次静脉注射均对人滤泡型淋巴瘤DoHH2裸小鼠皮下移植瘤有明显疗效，引起肿瘤部分消退；药效均优于阳性药ADC-8(即Polivy)。荷瘤小鼠对以上药物均能较好耐受。
本公开中实验动物的使用及福利遵照“国际实验动物评估和认可委员会(AAALAC)”的规定执行。每天监测动物的健康状况及死亡情况，例行检查包括观察受试物和药物对动物日常行为表现的影响如行为活动，体重变化，外观体征等。

Claims (51)

  1. 一种配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中：
    所述药物选自：微管聚集抑制剂、Topo I抑制剂、MMAE或其衍生物；优选药物选自MMAE或其衍生物、依喜替康或其衍生物、艾日布林或其衍生物；
    所述配体为抗CD79B抗体或其抗原结合片段，其包含：
    重链HCDR1，其包含SEQ ID NO：24所示序列；
    重链HCDR2，其包含SEQ ID NO：25所示序列；
    重链HCDR3，其包含SEQ ID NO：26所示序列；
    轻链LCDR1，其包含SEQ ID NO：27所示序列；
    轻链LCDR2，其包含SEQ ID NO：11或SEQ ID NO：17所示序列；和
    轻链LCDR3，其包含SEQ ID NO：12或SEQ ID NO：18所示序列。
  2. 如权利要求1所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗CD79B抗体或其抗原结合片段包含：
    a)分别如SEQ ID NO：23、8、9所示的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO：10、11、12所示的LCDR1、LCDR2和LCDR3；
    b)分别如SEQ ID NO：7、8、9所示的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO：10、11、12所示的LCDR1、LCDR2和LCDR3；或
    c)分别如SEQ ID NO：13、14、15所示的HCDR1、HCDR2和HCDR3，和分别如SEQ ID NO：16、17、18所示的LCDR1、LCDR2和LCDR3。
  3. 如权利要求1-2任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗CD79B抗体或其抗原结合片段为鼠源抗体、嵌合抗体、人源化抗体、人抗体或其片段；
    优选地，其为人源化抗体或其片段。
  4. 如前述权利要求任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗CD79B抗体或其抗原结合片段包含：
    如SEQ ID NO：3所示或与之具有至少90％同一性的重链可变区，和
    如SEQ ID NO：4所示或与之具有至少90％同一性的轻链可变区；
    或
    如SEQ ID NO：5所示或与之具有至少90％同一性的重链可变区，和
    如SEQ ID NO：6所示或与之具有至少90％同一性的轻链可变区；
    或
    如SEQ ID NO：19所示或与之具有至少90％同一性的重链可变区，和
    如SEQ ID NO：20所示或与之具有至少90％同一性的轻链可变区；
    或
    如SEQ ID NO：21所示或与之具有至少90％同一性的重链可变区，和
    如SEQ ID NO：22所示或与之具有至少90％同一性的轻链可变区。
  5. 如前述权利要求任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗CD79B抗体或其抗原结合片段包含恒定区Fc，
    优选地，所述Fc为IgG1、IgG2或IgG4；
    更优选地，所述Fc为IgG1、IgG2。
  6. 如前述权利要求任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗CD79B抗体的抗原结合片段为scFv、Fv、Fab或Fab’片段。
  7. 如前述权利要求任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述抗CD79B抗体包含：
    如SEQ ID NO：28所示或与之具有至少90％同一性的重链，和
    如SEQ ID NO：29所示或与之具有至少90％同一性的轻链；
    或
    如SEQ ID NO：30所示或与之具有至少90％同一性的重链，和
    如SEQ ID NO：31所示或与之具有至少90％同一性的轻链。
  8. 如权利要求1-7任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其为式(I)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2021108666-appb-100001
    其中：
    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，其中所述的C 1-8烷基、环烷基和直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-或化学键，p 1为1至20的整数；
    L 3为由2至7个氨基酸构成的肽残基，其中任选地氨基酸被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
    R 1选自氢原子、卤素、环烷基烷基、氘代烷基、环烷基、杂环基、芳基或杂芳基；
    R 2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；
    或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
    R 4和R 5相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
    R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基；
    m为0至4的整数；
    n为1至10，可以为整数或小数；
    Pc为权利要求1-7任一项所限定的抗CD79B抗体或其抗原结合片段。
  9. 如权利要求8所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其为式(II)所示的配体-药物偶联物或其药学上可接受的盐或溶剂化物：
    Figure PCTCN2021108666-appb-100002
    其中：
    s 1为2至8的整数；优选5；
    Pc、R 1、R 2、R 5、R 6、R 7、m和n如权利要求8中所定义。
  10. 如权利要求8或9所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中-L a-Y-或-L b-Y-选自：
    Figure PCTCN2021108666-appb-100003
  11. 如权利要求1-10任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其是选自以下结构式所示：
    Figure PCTCN2021108666-appb-100004
    其中：
    n为1至10，可以为整数或小数，优选n为1至6的整数或小数；
    Pc为权利要求1-7中任一项所限定的抗CD79B抗体或其抗原结合片段。
  12. 一种制备权利要求1-11中任一项所述的配体-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：
    Figure PCTCN2021108666-appb-100005
    Pc还原后，与通式L a-Y-D所示化合物偶联，得到通式Pc-L a-Y-D所示的配体-药物偶联物；
    其中：
    Pc为权利要求1-7任一项所限定的抗CD79B抗体或其抗原结合片段；
    W、L 2、L 3、R 1、R 2、R 5、R 6、R 7、m和n如权利要求8中所定义。
  13. 如权利要求1-7任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其是通式Pc-(L-D) k所示，其中：
    Pc为权利要求1-7任一项所限定的抗CD79B抗体或其抗原结合片段，
    L为接头，
    k为1至20的整数或小数，
    D是式(III)所示：
    Figure PCTCN2021108666-appb-100006
    其中，
    R 1a选自氢、烷基、环烷基、芳基和杂芳基，
    任选地，所述的烷基、环烷基、芳基和杂芳基各自独立地被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代，优选R 1a是甲基；
    R 1b选自氢、烷基、烷氧基、环烷基、芳基和杂芳基，
    任选地，所述的烷基、环烷基、芳基和杂芳基各自独立地被选自烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代，优选R 1b是氢；或者
    R 1a与R 1b与其相连接的原子一起形成C 5-8杂环烷基；任选地，所述杂烷基被烷基、烷氧基、卤素、氘、氨基、氰基、硝基、羟基、羟烷基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基所取代，且R 1a和R 1b不同时为氢。
  14. 如权利要求13所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中k选自1至10，可以为整数或小数。
  15. 如权利要求13所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述接头包含可裂解肽部分。
  16. 如权利要求15所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述可裂解肽部分能由酶裂解，优选所述酶是组织蛋白酶，更优选组织蛋白酶B。
  17. 如权利要求15或16所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中：
    所述接头包含由2至7个氨基酸构成的肽残基，
    所述氨基酸选自：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；
    更优选，所述肽残基选自：缬氨酸-瓜氨酸、丙氨酸-丙氨酸-天冬酰胺、甘氨 酸-甘氨酸-赖氨酸、缬氨酸-赖氨酸、缬氨酸-丙氨酸、缬氨酸-苯丙氨酸、甘氨酸-甘氨酸-苯丙氨酸-甘氨酸。
  18. 如权利要求13所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述接头包含可裂解的磺酰胺部分或可裂解的二硫化物部分。
  19. 如权利要求18所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，所述接头在还原条件下能够裂解。
  20. 如权利要求13-19任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述接头包含附接于D的间隔单元。
  21. 如权利要求20所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述间隔单元包含对氨基苯甲氧基羰基(PAB)。
  22. 如权利要求13-21任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其由选自以下的任一结构表示：
    Figure PCTCN2021108666-appb-100007
    k选自1至10，可以为整数或小数；p1选自2、4、6或8；
    Figure PCTCN2021108666-appb-100008
    k选自1至10，可以为整数或小数；p1选自2、4、6或8；P3选自0、1或2；
    Figure PCTCN2021108666-appb-100009
    k选自1至10，可以为整数或小数；p1选自2、4、6或8；
    Figure PCTCN2021108666-appb-100010
    k选自1至10，可以为整数或小数；p1选自2、4、6或8；P3选自0、1或2；
    Figure PCTCN2021108666-appb-100011
    k选自1至10，可以为整数或小数；p2选自2、4、6或8；
    Figure PCTCN2021108666-appb-100012
    k选自1至10，可以为整数或小数；p2选自2、4、6或8；
    Figure PCTCN2021108666-appb-100013
    k选自1至10，可以为整数或小数；p2选自2、4、6或8；
    Figure PCTCN2021108666-appb-100014
    k选自1至10，可以为整数或小数；p2选自2、4、6或8；
    Figure PCTCN2021108666-appb-100015
    k选自1至10，可以为整数或小数；p2选自2、4、6或8；
    Figure PCTCN2021108666-appb-100016
    k选自1至10，可以为整数或小数；p1选自2、4、6或8；P3选自0、1或2；
    Figure PCTCN2021108666-appb-100017
    k选自1至10，可以为整数或小数；p1选自2、4、6或8；P3选自0、1或2。
  23. 如权利要求22所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其由选自以下的任一结构表示：
    Figure PCTCN2021108666-appb-100018
    其中k选自1至10，可以为整数或小数；
    优选地，D中R 1a是甲基，R 1b是氢。
  24. 如权利要求1-7任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其是式(IV)所示：
    Figure PCTCN2021108666-appb-100019
    其中，
    R 2是C 1-C 8烷基；
    R 3是C 1-C 8烷基；
    R 4是C 1-C 8烷基；
    R 5是H；
    R 6是C 1-C 8烷基；
    R 7是C 1-C 8烷基；
    R 8彼此独立地是O-(C 1-C 8烷基)；
    R 9是H；
    R 10是苯基；
    Z是O或NH；
    R 11选自H、C 1-C 20烷基或-(R 13O) 3-R14；
    R 13是C 2-C 8烷基；
    R 14是C 1-C 8烷基；
    Pc为权利要求1-7中所定义的抗CD79B抗体或其抗原结合片段；
    L为接头；
    n为1至10，可以为整数或小数。
  25. 如权利要求24所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其包含下所示的结构：
    Figure PCTCN2021108666-appb-100020
  26. 如权利要求1-7任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其包含如式(V)所示的结构：
    Figure PCTCN2021108666-appb-100021
    其中，R 2-R 6选自氢原子、卤素、羟基、氰基、烷基、烷氧基和环烷基；
    R 7选自氢原子、烷基、烷氧基和环烷基；
    R 8-R 11之中的任意两个形成环烷基，余下的两个任选自氢原子、烷基和环烷基；
    R 12选自氢原子或烷基；
    R 13-R 15选自氢原子、羟基、烷基、烷氧基或卤素；
    R 16选自芳基或杂芳基，任选地所述的芳基或杂芳基被选自氢原子、卤素、羟基、烷基、烷氧基和环烷基的取代基所取代；
    n为1至10，可以为整数或小数；
    Pc为权利要求1-7中任一项所限定的抗CD79B抗体或其抗原结合片段；
    L为接头。
  27. 如权利要求1-7任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其包含如式(VI)所示的结构：
    Figure PCTCN2021108666-appb-100022
    其中，n、R 2-R 16如权利要求26中所定义；
    Pc为权利要求1-7中任一项所述的抗CD79B抗体或其抗原结合片段；L为接头。
  28. 如权利要求1-7任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其为式(VII)所示：
    Figure PCTCN2021108666-appb-100023
    n为1至10，可以为整数或小数；
    Pc为权利要求1-7中任一项所限定的抗CD79B抗体或其抗原结合片段；
    L为接头。
  29. 如权利要求24-28任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中：
    n为1至8，可以为整数或小数；
    优选，n为1至6，可以为整数或小数。
  30. 如权利要求24-29任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述接头为-Y-L 1-L 2-L 3-L 4，
    Y选自
    Figure PCTCN2021108666-appb-100024
    或化学健，X 1选自氢原子、烷基，烷氧基，芳基或卤素，X 2选自亚烷基，任选所述的亚烷基被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 17-W-C(O)-或-C(O)-W-C(O)-，其中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，其中任选所述的C 1-8烷基、环烷基和直链杂烷基各自独立地被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L 2选自-NR 18(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 18(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-或化学键，其中p 1为1至20的整数；优选L 2为化学键；
    L 3为由2至7个氨基酸构成的肽残基，所述的氨基酸优选自缬氨酸、瓜氨酸，甲基缬氨酸；其中任选氨基酸被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
    R 17和R 18相同或不同，且各自独立地选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
    L 4为延伸单元，优选L 4为PAB。
  31. 如权利要求30所述的配体-药物偶联物或其药学上可接受的盐或溶剂化 物，其中Y为
    Figure PCTCN2021108666-appb-100025
  32. 如权利要求30所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中L 1选自-(琥珀酰亚胺-3-基-N)-(CH 2)s 1-C(O)-，其中s 1为2至8的整数；
    优选L 1为
    Figure PCTCN2021108666-appb-100026
  33. 如权利要求30-32任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中L 3为二肽氨基酸单元，优选L 3是缬氨酸-瓜氨酸。
  34. 如权利要求24-33任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其中所述接头选自：
    Figure PCTCN2021108666-appb-100027
    其中a端连接所述Pc，b端连接所述药物。
  35. 如权利要求24-34任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，其选自以下结构式：
    Figure PCTCN2021108666-appb-100028
    Figure PCTCN2021108666-appb-100029
    其中：
    n为1至10，可以为整数或小数；
    Pc为如权利要求1-7任一项所限定的抗CD79B抗体或其抗原结合片段。
  36. 一种制备权利要求35所述的配体-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：
    Figure PCTCN2021108666-appb-100030
    其中，Pc，n如权利要求35中所定义。
  37. 一种配体-药物偶联物或其药学上可接受的盐或溶剂化物，其是选自以下任一项所示：
    Figure PCTCN2021108666-appb-100031
    Figure PCTCN2021108666-appb-100032
    其中，Pc为权利要求1-7所限定的抗CD79B抗体或其抗原结合片段，n为1至10，可以为整数或小数；
    优选地，n为1至6之间的整数或小数。
  38. 一种配体-药物偶联物或其药学上可接受的盐或溶剂化物，其为权利要求1-11、13-35、37中任意一项所述的配体-药物偶联物的氘代物、或其混合物形式。
  39. 一种药物组合物，其含有：
    药学上可接受的载体、稀释剂或赋形剂；和
    治疗有效量的根据权利要求1-11、13-35、37-38中任意一项所述的配体-药物 偶联物或其药学上可接受的盐或溶剂化物。
  40. 根据权利要求1-11、13-35、37-38中任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，或根据权利要求39所述的药物组合物在制备药物中的用途，其中：
    所述药物用于治疗增殖性疾病或延缓增殖性疾病进展，
    优选地，所述增殖性病症是癌症或肿瘤；
    更优选地，所述癌症或肿瘤选自淋巴瘤、B细胞淋巴瘤、弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤(NHL)、攻击性NHL、复发性攻击性NHL、复发性无痛性NHL、顽固性NHL、顽固性无痛性NHL、慢性淋巴细胞性白血病(CLL)、小淋巴细胞性淋巴瘤、白血病、毛细胞白血病(HCL)、急性淋巴细胞性白血病(ALL)，和/或套细胞淋巴瘤。
  41. 一种治疗或预防增殖性疾病或延缓增殖性疾病进展的方法，所述方法包括：
    向受试者施用治疗有效量或延缓疾病有效量的权利要求1-11、13-35、37-38任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，或权利要求39-40所述的药物组合物，
    优选地，所述增殖性病症是癌症或肿瘤；
    更优选地，所述癌症或肿瘤选自淋巴瘤、B细胞淋巴瘤、弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤(NHL)、攻击性NHL、复发性攻击性NHL、复发性无痛性NHL、顽固性NHL、顽固性无痛性NHL、慢性淋巴细胞性白血病(CLL)、小淋巴细胞性淋巴瘤、白血病、毛细胞白血病(HCL)、急性淋巴细胞性白血病(ALL)，和/或套细胞淋巴瘤。
  42. 一种在具有B细胞增殖性病症或自身免疫性病症的受试者中增强免疫功能的方法，包括：
    向受试者施用治疗或延缓疾病有效量的权利要求1-11、13-35、37-38任一项所述的配体-药物偶联物或其药学上可接受的盐或溶剂化物，或权利要求39-40所述的药物组合物，
    优选地，该B细胞增殖性病症为癌症或肿瘤；
    更优选地，该B细胞增殖性病症为淋巴瘤、B细胞淋巴瘤、弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤(NHL)，攻击性NHL，复发性攻击性NHL，复发性无痛性NHL，顽固性NHL，顽固性无痛性NHL，慢性淋巴细胞性白血病(CLL)，小淋巴细胞性淋巴瘤，白血病，毛细胞白血病(HCL)，急性淋巴细胞性白血病(ALL)，和/或套细胞淋巴瘤。
  43. 一种抗CD79B抗体或其抗原结合片段，包含：
    重链可变区，其包含分别如SEQ ID NO：23、SEQ ID NO：8和SEQ ID NO：9所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含分别如SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的LCDR1、LCDR2和LCDR3。
  44. 如权利要求43所述的抗CD79B抗体或其抗原结合片段，包含：
    如SEQ ID NO：19所示或与之具有至少90％同一性的重链可变区，和
    如SEQ ID NO：20所示或与之具有至少90％同一性的轻链可变区。
  45. 如权利要求43或44所述的抗CD79B抗体或其抗原结合片段，包含恒定区Fc，
    优选地，所述Fc为IgG1、IgG2或IgG4。
  46. 如权利要求43至45任一项所述的抗CD79B抗体或其抗原结合片段，包含如SEQ ID NO：28所示或与之具有至少90％同一性的重链，和
    如SEQ ID NO：29所示或与之具有至少90％同一性的轻链。
  47. 一种多核苷酸，其编码权利要求43至46任一项所述的抗CD79B抗体或其抗原结合片段。
  48. 一种宿主细胞，其包含权利要求47所述的多核苷酸，
    优选地，所述宿主细胞为细菌、酵母、哺乳动物细胞；
    更优选地，所述宿主细胞为大肠杆菌、毕赤酵母、中国仓鼠卵巢细胞或人胚肾293细胞。
  49. 一种制备抗CD79B抗体或其抗原结合片段的方法，包括：
    在权利要求48所述的宿主细胞中表达抗人CD76B抗体或其抗原结合片段，以及从培养物中分离所述抗CD79B抗体或其抗原结合片段。
  50. 一种药物组合物，其含有选自以下的任一项或其任意组合：
    权利要求43至46任一项中所述的抗CD79B抗体或其抗原结合片段或权利要求47所述的多核苷酸；以及，
    任选地，可药用的赋形剂、稀释剂或载体。
  51. 一种治疗或预防增殖性疾病或延缓增殖性疾病进展的方法，包括：
    向受试者施用治疗或延缓疾病有效量的权利要求43至46任一项中所述的抗CD79B抗体或其抗原结合片段或权利要求47所述的多核苷酸，或权利要求50所 述的药物组合物，
    优选地，该B细胞增殖性病症为癌症或肿瘤；
    更优选地，该B细胞增殖性病症为淋巴瘤、B细胞淋巴瘤、弥漫大B细胞淋巴瘤、非何杰金氏淋巴瘤(NHL)，攻击性NHL，复发性攻击性NHL，复发性无痛性NHL，顽固性NHL，顽固性无痛性NHL，慢性淋巴细胞性白血病(CLL)，小淋巴细胞性淋巴瘤，白血病，毛细胞白血病(HCL)，急性淋巴细胞性白血病(ALL)，和/或套细胞淋巴瘤。
PCT/CN2021/108666 2020-07-27 2021-07-27 抗cd79b抗体药物偶联物、其制备方法及其医药用途 WO2022022508A1 (zh)

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WO2023041007A1 (zh) * 2021-09-16 2023-03-23 正大天晴药业集团股份有限公司 抗her2抗体药物偶联物及其组合物和用途
WO2023041006A1 (zh) * 2021-09-16 2023-03-23 正大天晴药业集团股份有限公司 抗her3抗体药物偶联物及其组合物和用途
WO2023143347A1 (zh) * 2022-01-26 2023-08-03 上海迈晋生物医药科技有限公司 一种包含抗CD79b抗体药物偶联物的药物组合物及其用途
WO2023155808A1 (zh) * 2022-02-16 2023-08-24 苏州宜联生物医药有限公司 抗体-艾日布林或其衍生物的偶联物、其中间体、制备方法、药物组合物和用途
WO2023160651A1 (zh) * 2022-02-24 2023-08-31 苏州信诺维医药科技股份有限公司 一种抗体及其药物偶联物和用途
WO2024033362A1 (en) * 2022-08-08 2024-02-15 Atb Therapeutics Humanized antibodies against cd79b
WO2024078612A1 (en) * 2022-10-14 2024-04-18 Genequantum Healthcare (Suzhou) Co., Ltd. Linker-payload compound, conjugates and applications thereof

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