WO2023143263A1 - 一种针对Her3的抗体，偶联物及其用途 - Google Patents

一种针对Her3的抗体，偶联物及其用途 Download PDF

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WO2023143263A1
WO2023143263A1 PCT/CN2023/072657 CN2023072657W WO2023143263A1 WO 2023143263 A1 WO2023143263 A1 WO 2023143263A1 CN 2023072657 W CN2023072657 W CN 2023072657W WO 2023143263 A1 WO2023143263 A1 WO 2023143263A1
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antibody
seq
her3
antigen
binding fragment
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PCT/CN2023/072657
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English (en)
French (fr)
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蔡家强
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苏州宜联生物医药有限公司
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Publication of WO2023143263A1 publication Critical patent/WO2023143263A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the disclosure belongs to the field of therapeutic biomedicine, and more specifically, the disclosure relates to an antibody against Her3, a conjugate, and the use of the antibody or conjugate in treating diseases.
  • Her3 is a transmembrane receptor tyrosine kinase molecule of the ERBB family, which includes four members: EGFR, Her2, Her3 and Her4.
  • the activation of ERBB receptors requires the formation of homo- or hetero-dimers, and its family members can form dimers with each other.
  • Her3 interacts with the cysteine-rich region to lock its conformation; the non-conservative amino acid variation in the intracellular protein kinase domain makes Her3 retain only a weak kinase activity and needs to interact with other molecules of the EGFR family
  • Formation of heterodimers mediates signal transduction. Her3 plays an important role in embryonic development.
  • Her3 Deletion of Her3 in mice can lead to severe atrioventricular valve dysplasia and mouse embryonic death, which is mainly determined by its NRG1 ligand.
  • Her3 plays an important role in neural crest differentiation (such as Schwann cells) and sympathetic nerve development.
  • Her3 is mutated or overexpressed in a variety of cancers, such as several types of adenocarcinoma (breast cancer, urethral cancer, gastric cancer, colorectal cancer, biliary tract cancer, bladder cancer, endometrial cancer, ovarian cancer, uterine cancer, prostate cancer , lung cancer) and melanoma, glioma, etc.
  • adenocarcinoma breast cancer, urethral cancer, gastric cancer, colorectal cancer, biliary tract cancer, bladder cancer, endometrial cancer, ovarian cancer, uterine cancer, prostate cancer , lung cancer
  • melanoma glioma
  • Antibody-conjugated drugs link biologically active small molecular compounds to monoclonal antibodies or antibody fragments by chemical methods, making full use of the specificity of antibodies binding to surface antigens on normal cells and tumor cells, and anti-tumor biological agents with small molecules.
  • ADC Antibody-conjugated drugs
  • ADCs antibody-drug conjugates
  • ADC drugs have both the target specificity of antibodies to target cells and the The advantage of targeting and the tumor killing ability of loaded drugs, but due to the complex structure of ADC drug molecules, ADC drugs still have the problem of insufficient safety window in practical application, and the existing Her3 antibody-conjugated drugs are still in the early research and development stage , so there is still an urgent need for safer and more effective Her3 antibodies or antibody-drug conjugates to meet unmet clinical needs.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antibodies contemplated by the present disclosure may be fully human antibodies so that they can be safely administered to a human subject without eliciting an immunogenic response. Having a high ability to bind human and non-human Her3, particularly relates to such molecules that are cross-reactive with non-human primate (eg monkey) and mammalian (eg rat) Her3.
  • the present disclosure also relates to an antibody-drug conjugate (ADC) comprising the Her3 antibody, and the antibody or antibody-drug conjugate (ADC) of the present disclosure has great clinical value.
  • the present disclosure provides an antibody or antigen-binding fragment thereof that binds to Her3, said antibody or antigen-binding fragment thereof comprising the following complementarity determining regions (CDRs):
  • HCDR1 or a variant of its sequence HCDR2 or a variant of its sequence, and HCDR3 or a variant of its sequence contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 23, 25 or 27; and/or or
  • LCDR1 or a variant of its sequence LCDR2 or a variant of its sequence
  • the antibody or antigen-binding fragment thereof comprises a variant of HCDR1 or a sequence thereof, a variant of HCDR2 or a sequence thereof, and a variant of HCDR3 or a sequence thereof contained in the VH set forth in SEQ ID NO: 23 and/or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO:24.
  • the antibody or antigen-binding fragment thereof comprises a variant of HCDR1 or a sequence thereof, a variant of HCDR2 or a sequence thereof, and a variant of HCDR3 or a sequence thereof contained in the VH set forth in SEQ ID NO: 25 and/or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO:26.
  • the antibody or antigen-binding fragment thereof comprises a variant of HCDR1 or a sequence thereof, a variant of HCDR2 or a sequence thereof, and a variant of HCDR3 or a sequence thereof contained in the VH set forth in SEQ ID NO: 27 and/or LCDR1 or a variant of its sequence, LCDR2 or a variant of its sequence, and LCDR3 or a variant of its sequence contained in the VL shown in SEQ ID NO:28.
  • the variant of the sequence has one or several amino acid substitutions, deletions or additions compared to its source CDR (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) CDRs.
  • the substitutions are conservative substitutions.
  • said CDRs are defined according to the AbM, Chothia, Kabat or IMGT numbering system definition scheme.
  • the present disclosure provides an antibody or antigen-binding fragment thereof capable of binding to Her3, said antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) and/or a light chain variable region (VL) .
  • VH heavy chain variable region
  • VL light chain variable region
  • an antibody of the present disclosure comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Kabat numbering system:
  • VH heavy chain variable region
  • the sequence is SEQ ID NO: 1 or has one or several amino acid substitutions, deletions or additions (for example 1, 2 or 3 amino acid substitution, deletion or addition) sequence of HCDR1
  • the sequence is SEQ ID NO: 2 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions compared thereto) , deletion or addition) sequence of HCDR2
  • the sequence is SEQ ID NO: 3 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or HCDR3 of the sequence of 3 amino acid substitutions, deletions or additions); and/or,
  • a light chain variable region comprising the following 3 CDRs: the sequence is SEQ ID NO: 7 or has one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2 or 3 amino acid Substitution, deletion or addition) sequence LCDR1, the sequence is SEQ ID NO: 8 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or LCDR2 of the sequence added), the sequence is SEQ ID NO: 9 or a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto LCDR3;
  • the sequence is SEQ ID NO: 7 or has one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2 or 3 amino acid Substitution, deletion or addition) sequence LCDR1
  • the sequence is SEQ ID NO: 8 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or
  • VH heavy chain variable region
  • the sequence is SEQ ID NO: 12 or has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared thereto HCDR1 of a sequence of amino acid substitutions, deletions or additions)
  • the sequence is SEQ ID NO: 13 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions compared thereto) , deletion or addition) sequence of HCDR2
  • the sequence is SEQ ID NO: 14 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions compared thereto) HCDR3 of the sequence of ); and/or,
  • a light chain variable region comprising the following 3 CDRs: the sequence is SEQ ID NO: 18 or has one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2 or 3 amino acid Substitution, deletion or addition) sequence LCDR1, the sequence is SEQ ID NO: 19 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or LCDR2 of the sequence added), the sequence is SEQ ID NO: 20 or a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto LCDR3.
  • the sequence is SEQ ID NO: 18 or has one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2 or 3 amino acid Substitution, deletion or addition) sequence LCDR1
  • the sequence is SEQ ID NO: 19 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or LCD
  • an antibody of the present disclosure comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Kabat numbering system:
  • VH heavy chain variable region
  • a light chain variable region comprising the following 3 CDRs: LCDR1 whose sequence is SEQ ID NO:7, LCDR2 whose sequence is SEQ ID NO:8, and LCDR3 whose sequence is SEQ ID NO:9; or
  • VH heavy chain variable region
  • a light chain variable region comprising the following three CDRs: LCDR1 whose sequence is SEQ ID NO:18, LCDR2 whose sequence is SEQ ID NO:19, and LCDR3 whose sequence is SEQ ID NO:20.
  • an antibody of the present disclosure comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein the CDRs are defined by the IMGT numbering system:
  • VH heavy chain variable region
  • the sequence is SEQ ID NO: 4 or has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3) compared thereto HCDR1 of a sequence of amino acid substitutions, deletions or additions)
  • the sequence is SEQ ID NO: 5 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions compared thereto) , deletion or addition)
  • the sequence is SEQ ID NO: 6 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions compared thereto) HCDR3 of the sequence of ); and/or,
  • a light chain variable region comprising the following 3 CDRs: the sequence is SEQ ID NO: 10 or has one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2 or 3 amino acid Substitution, deletion or addition) sequence LCDR1, the sequence is AAS or a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto LCDR2 whose sequence is SEQ ID NO: 9 or LCDR3 of a sequence having one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto; or
  • VH heavy chain variable region
  • the sequence is SEQ ID NO: 15 or has one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) of the sequence HCDR1
  • the sequence is SEQ ID NO: 16 or compared therewith with one or several amino acid substitutions, deletions or additions (for example 1, 2 or 3 amino acid substitutions, deletions or additions)
  • substitution, deletion or addition) sequence of HCDR2 the sequence is SEQ ID NO: 17 or has one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or HCDR3 of the sequence added); and/or,
  • a light chain variable region comprising the following 3 CDRs: the sequence is SEQ ID NO: 21 or has one or several amino acid substitutions, deletions or additions thereto (e.g. 1, 2 or 3 amino acids) Substitution, deletion or addition) sequence LCDR1, the sequence is AAS or a sequence having one or several amino acid substitutions, deletions or additions (for example, 1, 2 or 3 amino acid substitutions, deletions or additions) compared thereto
  • the LCDR2 whose sequence is SEQ ID NO: 20 or LCDR3 having a sequence of one or several amino acid substitutions, deletions or additions (such as 1, 2 or 3 amino acid substitutions, deletions or additions) compared therewith.
  • an antibody of the present disclosure comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein the CDRs are defined by the IMGT numbering system:
  • VH heavy chain variable region
  • a light chain variable region comprising the following 3 CDRs: LCDR1 whose sequence is SEQ ID NO:10, LCDR2 whose sequence is AAS, and LCDR3 whose sequence is SEQ ID NO:9;
  • VH heavy chain variable region
  • a light chain variable region comprising the following 3 CDRs: LCDR1 whose sequence is SEQ ID NO:21, LCDR2 whose sequence is AAS, and LCDR3 whose sequence is SEQ ID NO:20.
  • an antibody of the present disclosure comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) that is identical to the aforementioned Kabat numbering system or IMGT numbering system
  • the heavy chain variable region (VH) and/or the light chain variable region compared to the defined CDRs At least one CDR in the region (VL) contains a mutation which is a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof combination), and still have Her3-binding activity.
  • substitutions described in this disclosure are conservative substitutions.
  • the antibody or antigen-binding fragment thereof binds human Her3, monkey Her3, and/or rat Her3.
  • the VH of an antibody of the present disclosure or an antigen-binding fragment thereof comprises a framework region (FR) derived from a heavy chain variable region (VH) of a human immunoglobulin, and/or the antibody or antigen thereof
  • the VL of the binding fragment comprises framework regions (FR) derived from the light chain variable region (VL) of a human immunoglobulin. Accordingly, in certain embodiments, the disclosed antibodies or antigen-binding fragments thereof are fully human. In certain embodiments, an antibody or antigen-binding fragment thereof of the disclosure is humanized.
  • an antibody or antigen-binding fragment thereof of the disclosure comprises:
  • a heavy chain framework region of a human immunoglobulin or a variant thereof having up to 20 amino acid conservative substitutions compared to the amino acid sequence encoded by the germline antibody gene from which it is derived e.g. up to 20, Conservative substitutions of up to 15, up to 10, or up to 5 amino acids; for example, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids );and / or
  • a light chain framework region of a human immunoglobulin or a variant thereof having up to 20 amino acid conservative substitutions compared to the amino acid sequence encoded by the germline antibody gene from which it is derived e.g. up to 20, Conservative substitutions of up to 15, up to 10, or up to 5 amino acids; for example, conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids ).
  • the degree of humanization of an antibody or antigen-binding fragment thereof of the disclosure is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, At least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • an antibody or antigen-binding fragment thereof of the disclosure comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • (vi) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% compared to the sequence shown in SEQ ID NO: 24, 26 or 28 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • an antibody of the present disclosure or an antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO:23, and/or, a VL set forth in SEQ ID NO:24.
  • an antibody of the present disclosure or an antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO:25, and/or, a VL set forth in SEQ ID NO:26.
  • an antibody of the present disclosure or an antigen-binding fragment thereof comprises a VH set forth in SEQ ID NO:27, and/or, a VL set forth in SEQ ID NO:28.
  • an antibody or antigen-binding fragment thereof of the disclosure comprises:
  • VH and VL a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) are independently associated with (a) to (c ) VH and VL described in any group have at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, respectively , at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; or
  • VH and VL independently have one or several amino acid substitutions, deletions or additions or any combination thereof (for example, 1, 2, 3, 4, 5, 6) compared to each other. , 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof).
  • said substitution is a conservative substitution.
  • the present disclosure also provides an anti-Her3 antibody or an antigen-binding antibody thereof that competitively binds to an antibody or an antigen-binding fragment thereof as defined above, such as antibody 202-2-1 or an antigen-binding fragment thereof A fragment, characterized in that the antibody or antigen-binding fragment thereof is different from the antibody or antigen-binding fragment thereof that it competes for binding, for example not antibody 202-2-1 or an antigen-binding fragment thereof.
  • the anti-Her3 antibody or antigen-binding fragment thereof competes for binding with antibody 202-2-1, is characterized in that it is not antibody 202-2-1 or an antigen-binding fragment thereof, and binds with 202- 2-1 or an antigen-binding fragment thereof has different CDRs.
  • an antibody of the disclosure binds domain3 of human Her3, eg, one or more amino acids in domain3. Therefore, in certain embodiments, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to HER3, wherein the antibody or antigen-binding fragment thereof binds to domain 3 of human Her3, preferably, to SEQ ID No.31 Amino acids 328 to 499 of human Her3 are shown.
  • the present disclosure provides an antibody or antigen-binding fragment thereof that binds HER3, wherein the antibody or antigen-binding fragment thereof binds to the following steric epitope: SEQ ID The 466th histidine, the 470th tryptophan, the 471th threonine, the 478th threonine, the 483rd to the 487th (aspartic acid-isoleucine- lysine-histidine-asparagine), isoleucine 484, arginine 490 and arginine 491.
  • the anti-Her3 antibody or antigen-binding fragment thereof that competitively binds to antibody 202-2-1 binds to the following spatial epitope: Histidine at position 466 and tryptophan at position 470 of Her3 shown in SEQ ID No.31 , 471 threonine, 478 threonine, 483 to 487 (aspartic acid-isoleucine-lysine-histidine-asparagine), 484 isoleucine, 490 arginine and arginine 491.
  • an antibody or antigen-binding fragment thereof disclosed herein blocks Her3 ligand-dependent and/or Her3 ligand-independent signal transduction.
  • an antibody or antigen-binding fragment thereof disclosed herein inhibits Her3 ligand-dependent and/or Her3 ligand-independent phosphorylation of AKT. In certain embodiments, an antibody or antigen-binding fragment thereof disclosed herein inhibits Her3 ligand-dependent and/or Her3 ligand-independent Her3/Her2 heterodimer formation.
  • antibodies of the present disclosure are chimeric antibodies, humanized antibodies, or fully human antibodies.
  • the disclosed antibodies or antigen-binding fragments thereof are selected from Fab, Fab', (Fab') 2 , Fv fragments such as scFv or disulfide-linked Fv (dsFv), diabodies, and Multispecific antibodies (eg, bispecific antibodies).
  • antibodies of the present disclosure are scFvs.
  • the heavy chain of an antibody of the present disclosure, or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a human immunoglobulin, or a variant thereof that is similar to the wild-type sequence from which it was derived.
  • CH heavy chain constant region
  • Conservative substitutions of at most 50 amino acids e.g., at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids
  • amino acids e.g., at most 45, at most 40, at most 35, at most 30, at most 25, at most 20, at most 15, at most 10, or at most 5 amino acids
  • the light chain of an antibody of the present disclosure, or antigen-binding fragment thereof comprises a light chain constant region (CL) of a human immunoglobulin, or a variant thereof that is similar to the wild-type sequence from which it was derived. than have conservative substitutions of up to 50 amino acids (e.g. up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid conservative substitutions; e.g. 1, 2, 3 1, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions).
  • CL light chain constant region
  • the constant region is altered, e.g., mutated, to modify the properties of the anti-Her3 antibody molecule (e.g., to alter one or more of the following properties: Fc receptor binding, antibody glycosylation, cysteine residue base number, effector cell function or complement function). Altering effector function (e.g., reducing ).
  • the Fc region of an antibody mediates several important effector functions such as ADCC, phagocytosis (ADCP), CDC, etc.
  • an antibody of the disclosure, or antigen-binding fragment thereof has a heavy chain constant region (CH) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE. Region; in particular selected from eg the heavy chain constant region of IgG1, IgG2, IgG3 and IgG4, more particularly selected from the heavy chain constant region of IgG1, eg human IgG1.
  • the human IgG1 heavy chain constant region is set forth in SEQ ID NO:29.
  • an antibody of the disclosure, or antigen-binding fragment thereof has a light chain constant region selected from, for example, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (eg, a human kappa light chain constant region).
  • the light chain constant region has the sequence shown in SEQ ID NO:30.
  • the antibody or antigen-binding fragment thereof comprises CH set forth in SEQ ID NO: 29 or a variant thereof having conservative substitutions of up to 20 amino acids compared to SEQ ID NO: 29 ( For example conservative substitutions of up to 20, up to 15, up to 10 or up to 5 amino acids; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitution), or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.
  • conservative substitutions of up to 20, up to 15, up to 10 or up to 5 amino acids; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitution or at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least
  • the antibody or antigen-binding fragment thereof comprises a light chain constant region or variant thereof.
  • the light chain constant region comprises a kappa light chain constant region. in some real
  • the light chain constant region comprises the light chain constant region (CL) shown in SEQ ID NO: 30 or a variant thereof having at most 20 amino acids conserved compared to SEQ ID NO: 30 Substitutions (e.g. conservative substitutions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO:29 and a light chain constant region (CL) set forth in SEQ ID NO:30.
  • CH heavy chain constant region
  • CL light chain constant region
  • an antibody or antigen-binding fragment thereof of the disclosure comprises:
  • (ii) has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50, up to 45, up to 40, up to 35, up to 30) compared to the sequence shown in (i) , up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, sequences with at least 97%, at least 98%, or at least 99% sequence identity;
  • (v) has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (for example, at most 50, at most 45, at most 40, at most 35, Up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; e.g. 1, 2, 3, 4, 5 , 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • (vi) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences having at least 97%, at least 98%, or at least 99% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • an antibody or antigen-binding fragment thereof of the disclosure comprises:
  • (ii) has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, up to 50, up to 45, up to 40, up to 35, up to 30) compared to the sequence shown in (i) , up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, sequences with at least 97%, at least 98%, or at least 99% sequence identity;
  • (v) has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (for example, at most 50, at most 45, at most 40, at most 35, at most 30 , up to 25, up to 20, up to 15, up to 10 or up to 5 ammonia Substitution, deletion or addition of amino acids or any combination thereof; e.g. substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids or any combination thereof); or
  • (vi) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences having at least 97%, at least 98%, or at least 99% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • an antibody or antigen-binding fragment thereof of the disclosure comprises:
  • (ii) has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, at most 50, at most 45, at most 40, at most 35, at most 30) compared with the sequence shown in (i) , up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, sequences with at least 97%, at least 98%, or at least 99% sequence identity;
  • (v) has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (for example, at most 50, at most 45, at most 40, at most 35, at most 30 , up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • (vi) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, Sequences having at least 97%, at least 98%, or at least 99% sequence identity.
  • substitutions described in (ii) or (v) are conservative substitutions.
  • an antibody of the disclosure, or antigen-binding fragment thereof comprises a heavy chain and a light chain
  • the heavy chain comprises:
  • (ii) has one or several amino acid substitutions, deletions or additions or any combination thereof (for example, at most 50, at most 45, at most 40, at most 35, at most 30) compared with the sequence shown in (i) , up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • (iii) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% of the sequence shown in (i) %, at least 98%, or at least 99% sequence identity;
  • the light chains comprise:
  • (v) has one or several amino acid substitutions, deletions or additions or any combination thereof compared to the sequence shown in (iv) (for example, at most 50, at most 45, at most 40, at most 35, at most 30 , up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions or any combination thereof; for example 1, 2, 3, 4, 5, 6 , 7, 8, 9 or 10 amino acid substitutions, deletions or additions or any combination thereof); or
  • sequences shown in (vi) and (iv) have at least 80%, at least 85%, at least 90%, at least 91%, Sequences with at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;
  • substitutions described in (ii) or (v) are conservative substitutions.
  • the antibody or antigen-binding fragment thereof, wherein the antibody is selected from the group consisting of:
  • antibodies of the present disclosure are multispecific antibodies that bind Her3 as well as one or more other antigens. Accordingly, the present disclosure provides a multispecific antibody.
  • said multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.
  • a multispecific antibody of the disclosure comprises a Her3-binding antigen-binding fragment of the disclosure, and an additional antibody or antigen-binding fragment or antibody analog thereof.
  • an antibody or antigen-binding fragment thereof of the disclosure can be derivatized, eg, linked to another molecule (eg, another polypeptide or protein).
  • derivatization eg, labeling
  • an antibody or antigen-binding fragment thereof does not adversely affect its binding to Her3, particularly human Her3.
  • the disclosed antibodies or antigen-binding fragments thereof are also intended to include such derivatized forms.
  • an antibody of the disclosure can be linked (by chemical coupling, gene fusion, non-covalent linkage, or otherwise) to one or more other molecular moieties, such as another antibody entities (e.g., to form bispecific antibodies), detection reagents, pharmaceutical reagents, and/or proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment thereof to another molecule (e.g., avidin or polyhistidine acid tag).
  • another antibody entities e.g., to form bispecific antibodies
  • detection reagents e.g., to form bispecific antibodies
  • pharmaceutical reagents e.g., to form bispecific antibodies
  • proteins or polypeptides capable of mediating the binding of an antibody or antigen-binding fragment thereof to another molecule (e.g., avidin or polyhistidine acid tag).
  • bispecific antibody is produced by cross-linking two or more antibodies (of the same type or of different types).
  • Methods for obtaining bispecific antibodies are well known in the art, examples of which include, but are not limited to, chemical cross-linking methods, cell engineering methods (hybridoma methods) or genetic engineering methods.
  • a detectable label as described in the present disclosure can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
  • Such labels are well known in the art, examples of which include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.), radionuclide Chlorine (eg, 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (eg, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin ( PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g.
  • enzymes e.g., horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, urease, glucose oxidase, etc.
  • radionuclide Chlorine eg, 3H, 125I, 35
  • acridinium esters such as colloidal gold or colored glass or plastic (eg, polystyrene, polypropylene, latex, etc.) beads, and avidin (eg, streptavidin ) of biotin.
  • Patents that teach the use of such markers include, but are not limited to, US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; Detectable labels as described above can be detected by methods known in the art.
  • radioactive labels can be detected using photographic film or scintillation counters
  • fluorescent labels can be detected using photodetectors to detect emitted light.
  • Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and thermometric labels are detected by simple visualization of a colored label.
  • such labels can be adapted for use in immunological detection (eg, enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescence immunoassay, etc.).
  • different A linker of length attaches a detectable label as described above to an antibody or antigen-binding fragment thereof of the present disclosure to reduce potential steric hindrance.
  • the disclosed antibodies or antigen-binding fragments thereof may also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.
  • chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups, or sugar groups. These groups can be used to improve the biological properties of antibodies, such as increasing serum half-life.
  • the antibodies of the present disclosure can be prepared by various methods known in the art, for example, by genetic engineering and recombination techniques. For example, DNA molecules encoding the heavy and light chain genes of the disclosed antibodies are obtained by chemical synthesis or PCR amplification. The resulting DNA molecule is inserted into an expression vector and then transfected into a host cell. Then, the transfected host cells are cultured under specific conditions and express the antibodies of the present disclosure.
  • Antigen-binding fragments of the present disclosure can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be directly produced by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000 )).
  • Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the culture medium of recombinant host cells. Other techniques for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.
  • the present disclosure provides an isolated nucleic acid molecule comprising an antibody encoding the disclosure, or an antigen-binding fragment thereof, or a heavy chain variable region and/or a light chain variable region thereof, or one of the or a nucleotide sequence of a plurality of CDRs; or a nucleotide sequence encoding a multispecific antibody of the present disclosure.
  • the nucleotide sequence can be substituted according to the codon degeneracy.
  • the nucleotide sequence is codon optimized.
  • the isolated nucleic acid molecule of the present disclosure comprises: (i) respectively encoding The first nucleic acid and the second nucleic acid of the heavy chain variable region and the light chain variable region of the disclosed antibody or antigen-binding fragment thereof, or (ii) respectively encode the heavy chain variable region of the disclosed antibody or antigen-binding fragment thereof and the first nucleic acid of the heavy chain constant region, and the second nucleic acid of the light chain variable region and the light chain constant region, or (iii) the first nucleic acid encoding the heavy chain and the light chain, respectively, of an antibody of the present disclosure or an antigen-binding fragment thereof nucleic acid and a second nucleic acid.
  • the first nucleic acid and the second nucleic acid comprise a degenerate sequence or a nucleic acid having substantially the same sequence as any of the first nucleic acid and the second nucleic acid in (i)-(iii) above.
  • the degenerate sequence or substantially identical sequence refers to having at least about 85%, 90%, 95%, 99% or more of the nucleic acid molecules described in (i)-(iii) Sequences with sequence identity or sequences with one or more nucleotide substitutions, or sequences that differ by no more than 3, 6, 15, 30 or 45 nucleotides.
  • a vector comprising an isolated nucleic acid molecule of the present disclosure.
  • the vectors of the present disclosure are cloning vectors or expression vectors.
  • vectors of the present disclosure are, for example, plasmids, cosmids, phage, lentiviruses, and the like.
  • the vector is capable of expressing an antibody of the present disclosure or an antigen-binding fragment thereof in a subject (eg, a mammal, eg, a human).
  • a host cell comprising the isolated nucleic acid molecule of the present disclosure or the vector of the present disclosure.
  • Host cells can be eukaryotic cells (eg, mammalian cells, insect cells, yeast cells) or prokaryotic cells (eg, E. coli).
  • Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells.
  • Suitable insect cells include, but are not limited to, Sf9 cells.
  • a host cell of the present disclosure is a mammalian cell, such as a CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).
  • a method for preparing an antibody or an antigen-binding fragment thereof of the present disclosure, or a multispecific antibody of the present disclosure comprising, allowing expression of the antibody or an antigen-binding fragment thereof, or the multispecific antibody
  • the host cells of the present disclosure are cultured under conditions, and the antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
  • an antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate wherein the antibody is the above Her3-binding An antibody or antigen-binding fragment thereof linked to a coupling moiety via a linker.
  • the coupling part is selected from: detectable labels, radioactive isotopes, fluorescent substances, luminescent substances, colored substances, enzymes, polyethylene glycol (PEG), nuclides, nucleic acids, small molecule toxins, polypeptides with binding activity, protein , receptors, ligands, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.
  • detectable labels radioactive isotopes, fluorescent substances, luminescent substances, colored substances, enzymes, polyethylene glycol (PEG), nuclides, nucleic acids, small molecule toxins, polypeptides with binding activity, protein , receptors, ligands, and other active substances that inhibit tumor cell growth and promote tumor cell apoptosis or necrosis.
  • the antibody-drug conjugate comprises an anti-Her3 antibody-drug conjugate (Her3-ADC) of the formula:
  • Tb is a Her3-binding antibody or antigen-binding fragment thereof as described above, and;
  • D is the small molecule toxin drug part
  • L is a bond or linker molecule which covalently links Tb and D;
  • q is an integer between 1-16 and represents the number of L-Ds covalently attached to Tb.
  • the antibody-drug conjugate has the structure shown in Formula I:
  • Each Z is independently selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond and an amide group (preferably selected from a direct bond, a carbon-carbon triple bond, a carbon-carbon double bond);
  • Rx, Ry are independently selected from H and C1- 4 alkyl; each m is independently selected from 0, 1, 2, 3, 4, 5 and 6; y1 is selected from any integer between 1-6 (such as 4, 5, 6); each y2 is independently selected from 0 Any integer between -15 (such as 6-15); each y3 is independently selected from 1, 2 and 3; each y4 is independently selected from 0 and 1; the 1 position is connected to Tb through the S atom, and the 2 position is connected to L2 or L 3 connected;
  • L 2 does not exist or exists, when L 2 exists, L 2 is selected from: y1 is selected from any integer between 1-6 (such as 4, 5, 6), each y2 is independently selected from any integer between 0-10 (such as 6-10), each y3 is independently selected from 1 or 2, each y4 is independently selected from 0 or 1, 1 is connected to L 1 , and 2 is connected to L 3 ;
  • L 3 is selected from amino acid residues or short peptides consisting of 2-10 amino acid residues; the amino acid residues are selected from natural amino acid residues, non-natural amino acid residues, or amino acid residues selected from AA 1 or its stereoisomers;
  • any one of R a and R b is H, and the other One is H,
  • R a and R b form a 5-6 membered heterocyclic ring together with the carbon atoms connected to them, and the 5-6 membered heterocyclic ring is a piperidine ring or a piperazine ring,
  • r, r1 , r1a and r1b are each independently 0, 1, 2, 3, 4 or 5;
  • R m1 , R n1 , R m1a , R n1a , R m1b and R n1b are each independently H, C 1-6 alkyl or -COOR x1 , wherein R x1 is C 1-6 alkyl;
  • R m1 and R n1 , R m1a and R n1a , and R m1b and R n1b form a 5-6 membered heterocycle together with the nitrogen atoms connected to them, and in the 5-6 membered heterocycle, the hetero Atoms are selected from 1 or 2 N atoms; the 5-6 membered heterocyclic ring is optionally substituted by one or more R 0' ;
  • R z is selected from C 1-6 alkyl
  • R 0 and R 0' are each independently selected from C 1-6 alkyl, -NR m2 R n2 or a 5-6 membered heterocyclic group optionally substituted by C 1-6 alkyl; the 5-6 membered In the heterocycle, the heteroatom is selected from 1 or 2 N atoms;
  • R m2 and R n2 are each independently selected from H and C 1-6 alkyl;
  • L 4 does not exist or exists, when L 4 exists, L 4 is selected from 1 bit is connected to L 3 , 2 bits are connected to D;
  • R 1 and R 2 are each independently selected from H, halogen and C1-4 alkyl; or, R 1 and R 2 form a 5-6 membered heterocyclic ring together with the carbon atoms connected to them, and the heterocyclic ring contains 1, 2 or 3 O, S or N or any combination thereof;
  • R 3 is selected from H and C1-4 alkyl; alternatively, R 3 and X form together with the carbon atom attached to it 5-6 membered carbon ring;
  • W does not exist or exists, when W exists, W is selected from -O-, -S-, -NR 4 -, 1 bit is connected to X, 2 bits are connected to L 4 or L 3 ;
  • X is selected from optionally substituted -(CH 2 ) n1 -, The 1st position is connected to the parent ring, and the 2nd position is connected to W or L4 ; the substituent is selected from 1 or 2 C1-4 alkyl groups;
  • R 4 , R 5 , and R 7 are each independently selected from H and C1-4 alkyl
  • n, n1, n2, n3 are each independently selected from any integer between 0 and 6.
  • the 1 position of L 1 is connected to Tb through the S atom
  • the 1 position of L 1 is related to the opening of the disulfide bond (for example, through the reducing agent TCEP Reduction of the disulfide bond can open the disulfide bond and generate a sulfhydryl group (SH) after the Tb (such as an antibody) itself contains a sulfhydryl group for connection, that is to say, the -S- between L1 and Tb is not an additional external sulfur atom .
  • SH sulfhydryl group
  • -S- is not an additional external sulfur atom, but the sulfhydryl group contained in Tb itself after opening the disulfide bond and L 1 for example The 1 digits are concatenated to form -S-.
  • L is selected from Each Z is independently selected from direct bonds, carbon-carbon triple bonds, carbon-carbon double bonds, C6 aromatic rings and amide groups (preferably selected from direct bonds, carbon-carbon triple bonds, carbon-carbon double bonds); Rx, Ry are independently selected from H and C1-4 alkyl; each m is independently selected from 0, 1, 2, 3, 4, 5 and 6; y1 is selected from any integer between 1-6 (such as 4, 5, 6); each y2 is independently are selected from any integer between 0-15 (such as 6-15); each y3 is independently selected from 1, 2 and 3; each y4 is independently selected from 0 and 1; the 1st position is connected to Tb through the S atom, and the 2nd position Connect to L 2 or L 3 .
  • L is selected from The 1 position is connected to Tb through the S atom, and the 2 position is connected to L2 or L3 .
  • L is selected from The 1 position is connected to Tb through the S atom, and the 2 position is connected to L2 or L3 .
  • L 2 is absent or exists, and when L 2 exists, L 2 is selected from 1 bit is connected to L 1 and 2 bits are connected to L 3 .
  • L2 is absent.
  • L 3 is selected from AA 1 , AA 1 -Gly, Val-Cit, Val-Ala, Val-AA 1 , Val-AA 1 -Gly, AA 1 -Ala-Asn, Ala-Ala-Ala , Ala-Ala-Asn and Gly-Gly-Phe-Gly.
  • L 3 is selected from AA 1 , AA 1 -Gly, Val-Cit, Val-AA 1 -Gly, AA 1 -Ala-Asn and Gly-Gly-Phe-Gly.
  • L 3 is selected from Val-AA 1 -Gly.
  • r, r 1 , r 1a and r 1b are each independently 0 or 4; preferably, r is 0 and r 1 , r 1a and r 1b are 4; or r is 4 and r 1 , r1a and r1b are zero.
  • r, r 1 , r 1a , and r 1b are not all zero at the same time.
  • r is 0 and r is 4 .
  • R m1 , R n1 , R m1a , R n1a , R m1b , and R n1b are each independently H, methyl, ethyl, n-propyl, n-butyl, —COOCH 3 , —COOCH 2 CH3 , -COOCH2CH2CH3 , -COOCH ( CH3 ) 2 , -COOC ( CH3 ) 3 , or -COOCH2CH2CH2CH3 .
  • each of Rm1 , Rn1 , Rm1a , Rn1a , Rm1b , and Rn1b is independently H, methyl, ethyl, n-propyl, —COOCH 3 , —COOCH 2 CH 3 , — COOCH2CH2CH3 , -COOCH ( CH3 ) 2 , -COOC( CH3 ) 3 , or -COOCH2CH2CH2CH3 .
  • R m1 and R n1 , R m1a and R n1a , and R m1b and R n1b form a piperidine ring or a piperazine ring together with their common nitrogen atoms; more preferably No. 1 nitrogen atom is the nitrogen atom jointly connected with R m1 and R n1 .
  • R m1 and R n1 , R m1a and R n1a , and R m1b and R n1b form a piperidine ring together with the nitrogen atom to which they are jointly attached; more preferably No. 1 nitrogen atom is the nitrogen atom jointly connected with R m1 and R n1 .
  • R m1 and R n1 are each independently selected from H, C1-6 alkyl (such as H, methyl); r is 0 , when r 1 is 4, R m1 and R n1 are each independently selected from C1-6 alkyl (such as methyl, ethyl, n-propyl), preferably selected from C2-6 alkyl (such as ethyl, n-propyl).
  • R m1 and R n1 are each independently selected from C1-6 alkyl groups (such as methyl, ethyl, n-propyl).
  • Rz is methyl
  • R is C 1-6 alkyl or piperidinyl or piperazinyl substituted by C 1-6 alkyl; preferably methyl, ethyl or piperidinyl substituted by methyl; such as methyl or
  • each R 01' is independently C 1-6 alkyl or -NR m2 R n2 ; more preferably methyl or -NR m2 R n2 , wherein R m2 and R n2 are each independently preferably H or C 1-6 alkyl, more preferably methyl.
  • R a1 and R b form a piperidine ring together with R b and the carbon atoms connected to them, for example No. 1 carbon atom is the carbon atom that is jointly connected with R a and R b ; for example
  • amino acid residue shown in AA 1 is selected from
  • amino acid residue shown in AA 1 is selected from
  • the amino acid residue shown in AA 1 is selected from In some embodiments, L is selected from 1 bit is connected to L 1 or L 2 , 2 bits are connected to L 4 or D.
  • L is selected from 1 bit is connected to L 1 or L 2 , 2 bits are connected to L 4 or D.
  • L is selected from 1 bit is connected to L 1 or L 2 , 2 bits are connected to L 4 or D. In some embodiments, L 4 is absent or present, and when L 4 is present, L 4 is selected from 1 bit is connected to L 3 and 2 bits are connected to D.
  • L4 is absent.
  • L4 is selected from 1 bit is connected to L 3 and 2 bits are connected to D.
  • L4 is selected from 1 bit is connected to L 3 and 2 bits are connected to D.
  • R 1 is selected from H and halogen
  • R 2 is selected from H and C1-4 alkyl.
  • R 1 and R 2 and the carbon atom to which they are attached form Dashed lines indicate where the heterocycle is fused to the benzene ring.
  • R 1 is H or F
  • R 2 is H or methyl
  • R1 is F
  • R2 is methyl or R1 and R2 together with the carbon atom to which they are attached form
  • R 1 is F and R 2 is methyl.
  • R and R together with the carbon atom to which they are attached form
  • R is H or R and X together with the carbon atom to which it is attached form Dashed lines indicate where the carbocycles are fused to the benzene and pyridine rings.
  • R 3 is H.
  • each R4 is independently selected from H and C1-4 alkyl, and R5 is H.
  • each R4 is independently selected from H, methyl, ethyl, n-propyl, i-propyl, and tert-butyl, and R5 is H.
  • each R 7 is independently selected from H and C1-4 alkyl.
  • R7 is H.
  • n is selected from 1, 2 and 3.
  • n 1
  • n1 is selected from 1, 2, 3 and 4.
  • n2 is 1.
  • n3 is 0.
  • W is selected from -O-, -NR 4 - and 1 bit is connected to X, 2 bits are connected to L 4 or L 3 .
  • W is selected from -O- and -NR 4 -, X is connected at position 1, and L 4 or L 3 is connected at position 2.
  • X is selected from optionally substituted
  • the 1-position is connected to the parent ring, and the 2-position is connected to W or L4 ; the substituent is selected from 1 or 2 C1-4 alkyl groups (such as methyl), or 2 C1-4 alkyl groups (such as methyl ).
  • X is selected from Bit 1 is connected to the mother ring, and bit 2 is connected to W.
  • W is selected from -O-, -NR 4 - and 1 position is connected to X, 2 positions are connected to L4 or L3 ;
  • X is selected from Bit 1 is connected to the mother ring, and bit 2 is connected to W.
  • the structure of is selected from the following structural fragments:
  • the structure of is selected from the following structural fragments:
  • 1 bit is connected to Tb, and 2 bits are connected to W.
  • the structure of is selected from the following structural fragments:
  • 1 bit is connected to Tb, and 2 bits are connected to W.
  • the structure of is selected from the following structural fragments:
  • 1 position is connected to Tb through S atom, and 2 positions are connected to W;
  • the structural fragment shown is 1 bit is connected to L4 .
  • the structure of is selected from the following structural fragments:
  • the structure of is selected from the following structural fragments:
  • the antibody-drug conjugate has the structure shown in Formula I-1:
  • Tb, L 1 , L 2 , L 3 , L 4 , X, R 1 , R 2 , R 3 , R 4 and q have the meanings given above and in any embodiment specifically recited herein.
  • the antibody-drug conjugate has the structure shown in formula I-1A or I-1B:
  • Tb, L2 , L3 , L4 , X, R1 , R2 , R3 , R4 and q have the meanings given above and in any embodiment specifically recited herein.
  • the antibody-drug conjugate has the structure shown in Formula I-2:
  • Tb, L 1 , L 2 , L 3 , L 4 , X, R 1 , R 2 , R 3 and q have the meanings given above and in any embodiment specifically recited herein.
  • the antibody-drug conjugate has the structure shown in Formula I-2A or I-2B:
  • Tb, L 2 , L 3 , L 4 , X, R 1 , R 2 , R 3 and q have the meanings given above and in any embodiment specifically recited herein.
  • the antibody drug conjugate has the structure shown in formula I-3:
  • Tb, L 1 , L 2 , L 3 , L 4 , X, R 1 , R 2 , R 3 , R 4 , R 5 , n and q have the values provided above and in any embodiment specifically recited herein meaning.
  • the antibody drug conjugate has the structure shown in formula I-3A or I-3B:
  • Tb, L2 , L3 , L4 , X, R1 , R2 , R3 , R4 and q have the meanings given above and in any embodiment specifically recited herein.
  • the antibody-drug conjugate has the structure shown in Formula IA:
  • Tb, X , R 1 , R 2 , R 3 , Ra, R b and q have the meanings given above and in any embodiment specifically recited herein.
  • the antibody-drug conjugate has the structure shown in Formula IB:
  • Tb, X, R 1 , R 2 , R 3 , Ra, R b and q have the meanings given above and in any embodiment specifically recited herein .
  • the antibody-drug conjugate is selected from:
  • Tb is the Her3 antibody as described in any scheme above, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, q is preferably 1 , 2, 3, 4, 5, 6, 7, 8;
  • Her3mAb is 202-2-1 antibody.
  • the antibody-drug conjugate is selected from the following:
  • Tb is the Her3 antibody as described in any scheme above, and q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, and q is preferably 1, 2, 3, 4, 5, 6, 7 or 8.
  • the antibody-drug conjugate is selected from the following:
  • Her3mAb is 202-2-1 antibody.
  • the antibody-drug conjugate q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16; q is preferably 1, 2, 3, 4, 5, 6, 7 or 8.
  • an antibody-drug conjugate comprising the aforementioned antibody-drug conjugate of the present disclosure, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or Two or more of the pharmaceutically acceptable solvates thereof, and the antibody-drug conjugate in the antibody-drug conjugate has one, two or more q values.
  • the drug-to-antibody ratio (DAR) in the antibody-drug conjugate is an integer or decimal selected from 1-10.
  • the drug-to-antibody ratio (DAR) in the antibody-drug conjugate is selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.2, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.7, 8.9, and 9.
  • the DAR in the antibody-drug conjugate is selected from: 2 ⁇ 0.5, 4 ⁇ 0.5, 6 ⁇ 0.5, or 8 ⁇ 0.5.
  • the DAR in the antibody-drug conjugate is selected from: 2 ⁇ 0.5, 4 ⁇ 0.5, 5 ⁇ 0.5, 6 ⁇ 0.5, 7 ⁇ 0.5, or 8 ⁇ 0.5.
  • the DAR in the antibody-drug conjugate is 8 ⁇ 0.5, 8 ⁇ 0.4, 8 ⁇ 0.3, 8 ⁇ 0.2, 8 ⁇ 0.1 or 8.0.
  • the antibody-drug conjugate comprises an ADC having a distribution of DARs of 2 to 8, e.g., 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, 7, and 8 type of drug).
  • DARs a distribution of DARs of 2 to 8, e.g., 1.5, 2, 4, 6, and 8 (i.e., 1.5, 2, 4, 6, 7, and 8 type of drug).
  • degradation products can be generated such that DARs 1, 3, 5, and 7 can also be included in antibody-drug conjugates.
  • the ADCs in the mixture may also have a DAR greater than 8.
  • Antibody-drug conjugates are produced by reduction of interchain disulfides followed by conjugation.
  • the antibody-drug conjugate comprises both: an ADC with a DAR of 4 or less (i.e., the drug-loaded species is 4 or less) and a DAR of 6 or more (i.e., the drug-loaded species 6 or higher) ADC.
  • the ninth aspect of the present disclosure provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof of the first aspect of the present disclosure, the multispecific antibody of the second aspect, the nucleic acid molecule of the third aspect, the carrier of the fourth aspect, The host cell of the fifth aspect, the antibody-drug conjugate of the seventh aspect, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate and/or the eighth aspect The antibody-drug conjugate described in the aspect and the pharmaceutically acceptable carrier and/or excipient.
  • the antibody or antigen-binding fragment thereof in the first aspect of the present disclosure, the multispecific antibody in the second aspect, the nucleic acid molecule in the third aspect, the vector in the fourth aspect, the host cell in the fifth aspect, the The antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate and/or the antibody-drug conjugate described in the eighth aspect can be in a therapeutically effective amount.
  • a pharmaceutical composition of the present disclosure comprises an antibody of the present disclosure, or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition of the present disclosure comprises the host cell of the present disclosure, and a pharmaceutically acceptable carrier and/or excipient, wherein the host cell comprises an isolated nucleic acid molecule as described above or carrier.
  • a pharmaceutical composition of the disclosure comprises a multispecific antibody of the disclosure, and a pharmaceutically acceptable carrier and/or excipient.
  • a pharmaceutical composition of the present disclosure comprises an antibody-drug conjugate of the present disclosure, a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. substances and/or the antibody-drug conjugates and pharmaceutically acceptable carriers and/or excipients.
  • the antibody or its antigen-binding fragment, nucleic acid molecule, carrier, host cell, multispecific antibody or antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutical may be in a therapeutically effective amount.
  • antibodies or antigen-binding fragments thereof nucleic acids, vectors, host cells, multispecific antibodies, antibody-drug conjugates, stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable Accepted salts or pharmaceutically acceptable solvates thereof, the antibody-drug conjugate or the use of the pharmaceutical composition in the preparation of medicines for the treatment or prevention of Her3 activity-related disease.
  • antibodies or antigen-binding fragments thereof nucleic acids, vectors, host cells, multispecific antibodies, antibody-drug conjugates, stereoisomers thereof, prodrugs thereof, pharmaceutically acceptable Accepted salts or pharmaceutically acceptable solvates thereof, the antibody-drug conjugate or the use of the pharmaceutical composition in the preparation of medicines for the treatment or prevention of Her3 activity-related tumor.
  • the cancerous disease is selected from the group consisting of esophageal cancer (e.g. esophageal adenocarcinoma and esophageal squamous cell carcinoma), brain tumors, lung cancer (e.g. small cell lung cancer and non-small cell lung cancer), squamous cell carcinoma , bladder cancer, gastric cancer, ovarian cancer, peritoneal cancer, pancreatic cancer, breast cancer, head and neck cancer, cervical cancer, endometrial cancer, colorectal cancer, liver cancer, kidney cancer, urothelial cancer, solid tumors, non-Hodgies Gold lymphoma, central nervous system tumors (e.g. glioma, glioblastoma multiforme cell tumor, glioma, or sarcoma), prostate cancer, or thyroid cancer.
  • esophageal cancer e.g. esophageal adenocarcinoma and esophageal squamous cell carcinoma
  • the cancerous disease is a solid tumor.
  • the tumor is selected from the group consisting of colorectal cancer (such as colon cancer), lung cancer (such as non-small cell lung cancer), breast cancer, and prostate cancer.
  • the cancer disease is breast cancer or lung cancer (preferably non-small cell lung cancer).
  • the disclosure provides a diagnostic or therapeutic kit comprising one or more of the following: an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, Antibody-drug conjugate or pharmaceutical composition.
  • the diagnostic or therapeutic kit also includes instructions for use.
  • HER3 ligand refers to a polypeptide that binds and activates HER3.
  • Examples of HER3 ligands include, but are not limited to, neuregulin 1 (NRG) and neuregulin 2, betacellin, heparin-binding epidermal growth factor, and epiregulin.
  • the present disclosure relates to an isolated antibody or fragment thereof that recognizes a conformational epitope of the HER3 receptor, wherein the conformational epitope comprises amino acid residues in domain 3 of HER3, and wherein the antibody or fragment thereof inhibits phosphorylation of HER3, such as Assessed by HER3 ligand-independent phosphorylation assay.
  • the HER3 ligand-independent phosphorylation assay uses HER2-amplified cells, wherein the HER2-amplified cells are SK-Br-3 cells.
  • ligand-dependent signal transduction refers to signaling that is activated by a ligand Transduction, eg activation of Erbb (eg HER3) by a ligand.
  • Activation of HER3 can be evidenced by increased oligomerization (eg heterodimerization) and/or HER3 phosphorylation of downstream signaling pathways (eg PI3K).
  • the antibody or fragment thereof can statistically significantly reduce phosphorylated HER3 relative to untreated (control) cells in stimulated cells exposed to an antigen binding protein (e.g., antibody) as measured using the assay described in the Examples amount.
  • a cell expressing HER3 may be a naturally occurring cell line (eg, MCF7), or may be a cell recombinantly produced by introducing a nucleic acid encoding a HER3 protein into a host cell.
  • Cell stimulation can be performed by exogenous addition of activating HER3 ligands or by endogenous expression of activating ligands.
  • ligand-independent signal transduction refers to signal transduction that does not require ligand binding, eg, HER3 activation (eg, phosphorylation) that is independent of ligand binding.
  • ligand-independent HER3 activation can be the result of HER2 overexpression or of activating mutations in HER3 heterodimer partners such as EGFR and HER2.
  • the antibody or fragment thereof can statistically significantly reduce the amount of phosphorylated HER3 in cells exposed to the antigen binding protein (eg, antibody) relative to untreated (control) cells.
  • a cell expressing HER3 may be a naturally occurring cell line (eg, SK-Br-3), or may be a cell recombinantly produced by introducing a nucleic acid encoding a HER3 protein into a host cell.
  • examples of the term "pharmaceutically acceptable salt” are organic acid addition salts formed from organic acids that form pharmaceutically acceptable anions, including but not limited to formate, acetate, propionic acid Salt, benzoate, maleate, fumarate, succinate, tartrate, citrate, ascorbate, alpha-ketoglutarate, alpha-glycerophosphate, alkylsulfonate Or arylsulfonate;
  • the alkylsulfonate is methanesulfonate or ethylsulfonate;
  • the arylsulfonate is benzenesulfonate or p-toluenesulfonate.
  • Suitable inorganic salts may also be formed including, but not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate, bicarbonate, and carbonate, sulfate or phosphate salts, and the like.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives.
  • compositions can be obtained using standard procedures well known in the art, for example, by reacting a sufficient amount of a basic compound with a suitable acid to furnish a pharmaceutically acceptable anion.
  • the pharmaceutical excipients refer to the excipients and additives used in the production of drugs and formulation of prescriptions, and refer to the active ingredients that have been reasonably evaluated in terms of safety and are included in pharmaceutical preparations. substances in.
  • pharmaceutical excipients also have important functions such as solubilization, solubility enhancement, and sustained and controlled release. They are important components that may affect the quality, safety, and effectiveness of drugs. According to its source, it can be divided into natural products, semi-synthetics and full synthetics.
  • solvents for oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration and eye administration, etc.
  • solubilizers for oral, injection, mucosal, transdermal or topical administration, nasal or oral inhalation administration and eye administration, etc.
  • the same pharmaceutical excipient can be used in pharmaceutical preparations with different administration routes, and has different functions and uses.
  • the pharmaceutical composition can be made into various suitable dosage forms according to the route of administration.
  • suitable dosage forms such as tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants Agents, aerosols, powders, sprays, etc.
  • the pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the disclosed antibody, antibody-drug conjugate, its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutical
  • the above acceptable solvates suitably contain 0.1 mg to 800 mg, preferably contain 0.5-500 mg, preferably contain 0.5 to 350 mg, particularly preferably 1-250 mg.
  • the pharmaceutical composition can be administered in the form of injection, including injection solution, sterile powder for injection and concentrated solution for injection.
  • the vehicles and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils, such as mono- or diglycerides can be employed as a solvent or suspending medium.
  • treating generally refers to obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of complete or partial prevention of the disease or its symptoms; and/or therapeutic in terms of partial or complete stabilization or cure of the disease and/or side effects due to the disease.
  • Treatment encompasses any treatment of a disease in a patient, including: (a) prophylaxis of a disease or condition in a patient susceptible to the disease or condition but not yet diagnosed; (b) suppressing the symptoms of the disease, ie arresting its development; or (c) alleviating the symptoms of the disease, ie causing regression of the disease or symptoms.
  • the term "individual” includes a human or non-human animal.
  • exemplary human subjects include human subjects suffering from a disease (eg, a disease described herein) (referred to as a patient) or normal subjects.
  • non-human animal in this disclosure includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).
  • the term "effective dose” refers to the amount of a compound which, when administered, alleviates to some extent one or more symptoms of the condition being treated.
  • the term "ligand-drug conjugate” refers to a substance obtained by linking a bioactive molecule (drug molecule) with a targeting moiety.
  • the biologically active molecule is connected to the targeting moiety through a linker.
  • the linker can be cleaved in a specific environment (such as intratumoral hydrolytic enzymes and/or low pH environment) or under a specific action (such as the action of lysosomal proteases), thereby separating the biologically active molecule from the targeting moiety.
  • the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond.
  • the bioactive molecule is directly connected to the targeting moiety through a covalent bond, and the covalent bond can be broken under a specific environment or action, thereby separating the bioactive molecule from the targeting moiety.
  • the ligand drug conjugate comprises a targeting moiety, a linker and a fragment of the compound of formula II of the present disclosure.
  • the term "antibody-drug conjugate” refers to a substance obtained by linking a bioactive molecule (drug molecule) to an antibody or an antigen-binding fragment thereof.
  • the The bioactive molecule is connected to the antibody or its antigen-binding fragment through a linker.
  • the linker can be broken in a specific environment (such as a hydrolytic enzyme and/or a low pH environment in a tumor) or under a specific action (such as the action of a lysosomal protease), thereby allowing the bioactive molecule to bind to the antibody or its antigen fragment separation.
  • the linker comprises a cleavable or non-cleavable unit, such as a peptide or a disulfide bond.
  • the biologically active molecule antibody or its antigen-binding fragment is directly connected by a covalent bond, and the covalent bond can be broken under a specific environment or action, so that the biologically active molecule binds to the antibody or its antigen fragment separation.
  • the antibody drug conjugate comprises an anti-Her3 antibody or an antigen-binding fragment thereof, a linker, and a fragment of the compound of formula II of the present disclosure.
  • biologically active substance refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction.
  • drug molecule refers to a substance that inhibits or prevents the function of cells and/or causes cell death or destruction.
  • conjugated The biologically active substance, biologically active molecule or drug molecule in the drug is a molecule with anti-tumor biological activity.
  • radioisotopes such as those of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and Lu; metal complexes such as metal platinum complexes, metal gold complexes, oxaliplatin etc.; glycopeptide antibiotics, such as bleomycin, pingyangmycin; DNA topoisomerase inhibitors, such as topoisomerase I inhibitors, camptothecin, hydroxycamptothecin, 9-aminocamptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, topoisomerase II inhibitors, actinomycin D, doxorubicin, doxorubicin, docarmycin, Erythromycin, mitoxantrone, podophyllotoxin, etoposide, etc.; drugs that interfere with DNA synthesis, such as methotrexate, 5-fluorouracil, cytar
  • tumor signaling pathway inhibitors such as serine / threonine kinase inhibitors, tyrosine kinase inhibitors, aspartate kinase inhibitors or histidine kinase inhibitors; also include proteasome inhibitors, histone Acetylase inhibitors, tumor angiogenesis inhibitors, cyclin inhibitors, maytansine derivatives, calicheamicin derivatives, auristatin derivatives, pyrrolobenzodiazepines (PBD) derivatives, melphalan, Mitomycin C, Benzene Butyric acid mustard, or other active substances that inhibit tumor cell growth, promote tumor cell apoptosis and necrosis; enzymes and their fragments, such as nucleolysin; antibiotics; toxins, such as small molecule toxins or bacterial, fungal, plant or animal origin Enzyme-active toxins, including fragments and/or variants thereof; growth inhibitors; drug moieties.
  • toxin refers to a
  • small molecule refers to a small molecule drug that has biological activity.
  • small-molecule toxin has cell-damaging activity, that is, the state of causing pathological changes in cells in some form, and cell damage is not limited to direct damage, including all kinds of damage to the structure and function of cells, such as DNA cleavage, base - Dimer formation, cleavage of chromosomes, damage to cell division machinery and decrease of different enzymatic activities.
  • linker refers to a fragment linking a biologically active molecule (drug molecule) with a targeting moiety.
  • targeting moiety refers to a portion of a conjugate capable of specifically binding to a target (or a portion of a target) on a cell surface. Through the interaction of the targeting moiety with the target, the conjugate can be delivered to a specific cell population.
  • the conjugate when the targeting moiety in the conjugate is an antibody, the conjugate may be referred to as a "drug-antibody conjugate".
  • an antibody or antigen-binding fragment thereof includes a derivatized antibody or antigen-binding fragment thereof, such as an antibody or antigen-binding fragment thereof having a sulfhydryl group, wherein the derivatization allows the antibody to have a moiety that is reactive with a drug-linker conjugate. regiment or ability.
  • the thiol-SH can be derivatized by opening disulfide bonds (eg, by reduction with the reducing agent TCEP).
  • cancer and “tumor” are used with the same meaning.
  • gene used herein includes not only DNA but also its mRNA, its cDNA and its cRNA.
  • polynucleotide is used in the same sense as nucleic acid, and also includes DNA, RNA, probes, oligonucleotides and primers.
  • Tb is an abbreviation for “target binding” and includes an antibody or any molecule that binds to a target
  • the term “Ab” is an abbreviation for “antibody” and is used indiscriminately from “antibody or an antigen-binding fragment thereof” .
  • polypeptide As used herein, the terms “polypeptide” and “protein” are used without distinction.
  • cell as used herein also includes cells in an animal subject and cells in culture.
  • her3-binding antibody or antigen-binding fragment thereof in the present disclosure refers to an antibody or antigen-binding fragment thereof that specifically binds to Her3, such as human Her3.
  • Her3 as used herein is also referred to as "ErbB3", which is a transmembrane receptor tyrosine kinase molecule of the ERBB family.
  • the HER3 gene is located on the long arm of chromosome 12 (12q13.2), encoding a 180kDa protein.
  • the extracellular domain of HER3 is divided into four subdomains (domains 1-4): domain1 and domain3 are leucine-rich ⁇ -helical regions responsible for ligand binding. Domain 2 and domain 4 are cysteine-rich regions, and domain 2 also contains a dimer arm necessary for interaction with other receptors.
  • the transmembrane domain is followed by the intracellular domain.
  • the Her3 involved in the present disclosure may be conventional Her3 in the art (eg, human Her3), such as soluble Her3, membrane-form Her3, etc., and may also represent Her3 variant 1 and/or Her3 variant 2.
  • the human Her3 is the Her3 shown in accession number Uniport ID: P21860-1 or NCBI: NP_001973.2.
  • the human Her3 comprises the amino acid sequence shown in SEQ ID NO: 31.
  • Her3 (1-643) as shown in SEQ ID No.31 is from Uniport ID: P21860-1, wherein SEQ ID No.31 is:
  • KD refers to the ratio obtained from Kd (the dissociation rate of a specific binding molecule-target protein interaction) and Ka (the on-rate of a specific binding molecule-target protein interaction) (or Kd/Ka, expressed in molarity (M)) dissociation constant.
  • KD values can be determined using methods well established in the art.
  • a preferred method of determining the K of a binding molecule is through the use of surface plasmon resonance, for example a biosensor system such as the BiacoreTM (GE Healthcare Life Sciences) system.
  • antigen-binding fragment refers to a partial fragment of an antibody that has antigen-binding activity, wherein the fragment has full or partial function of the antibody, including without limitation, Fab, Fab', F(ab')2, Fv For example, single-chain Fv (scFv), disulfide-linked Fv (sdFv) or di-scFv, etc.
  • the term also includes Fab', which is a monovalent fragment of the variable region of an antibody obtained by treatment of F(ab')2 under reducing conditions. However, the term is not limited to these molecules so long as the fragment has binding affinity for the antigen.
  • these functional fragments include not only fragments obtained by treating full-length molecules of antibody proteins with appropriate enzymes, but also proteins produced in appropriate host cells using genetically modified antibody genes.
  • Antibody molecule or “antibody” broadly as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • antibody covers not only whole antibody molecules but also fragments of said antibodies as well as Variants (including derivatives) of the antibodies and antibody fragments.
  • antibody molecule or “antibody” is used in the same context as an antigen-binding fragment, “antibody molecule” or “antibody” refers to an intact antibody molecule or a full-length antibody.
  • single-chain Fv or “scFv” refers to a polypeptide comprising the VL domain of an antibody linked to an antibody VH domain.
  • Antibodies that immunospecifically bind to Her3 may cross-react with other antigens. Preferably, antibodies that immunospecifically bind to Her3 do not cross-react with other antigens.
  • Antibodies that immunospecifically bind Her3 can be identified, for example, by immunoassays or other methods known to those skilled in the art.
  • “Intact” antibody or “full-length” antibody refers to a protein comprising two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, the protein comprising: ( 1) for a heavy chain, comprising a variable region (abbreviated herein as "VH”) and a heavy chain constant region comprising three domains CH1, CH2, CH3; and (2) for a light chain, comprising a light chain
  • VH variable region
  • VL variable region
  • CL light chain constant region
  • Antibodies of the disclosure include, but are not limited to, monoclonal, multispecific, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, anti-idiotypic (anti-Id) antibodies (including, for example, antibodies of the disclosure anti-Id antibody), and epitope-binding fragments of any of the above antibodies.
  • Immunoglobulin molecules of the present disclosure may be of any type (eg, IgG, IgE, IgM, IgD, IgA, and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) or subclass of immunoglobulin.
  • the antibodies of the present disclosure comprise or consist of VH domains, VH CDRs, VL domains, or VL CDRs having any of the amino acid sequences described in Table 1 or fragments or variants thereof.
  • Fab' denotes a monovalent fragment of the variable region of an antibody obtained by treating F(ab')2 under reducing conditions as described above.
  • Fab' of the present disclosure also includes Fab' produced using genetically modified antibody genes.
  • scFv refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker or directly (see, e.g., Bird et al., Science 242 :423-426 (1988); Huston et al., Proc.Natl.Acad.Sci.USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994)).
  • 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 the repeated GGGGS amino acid sequence or variants thereof.
  • a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers 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.
  • di-scFv refers to an antibody fragment formed by linking two scFvs.
  • Her3 variant refers to a polypeptide that has similar or identical functions to Her3 polypeptides, Her3 fragments, anti-Her3 antibodies or antibody fragments thereof, but does not necessarily comprise similar or identical Her3 polypeptides, Her3 fragments, anti-Her3 The amino acid sequence of the antibody or a fragment thereof, or a structure similar or identical to a Her3 polypeptide, a Her3 fragment, an anti-Her3 antibody or a fragment thereof.
  • the antibody or antigen-binding fragment thereof contains variants, amino acid substitutions, deletions or additions, it still has Her3-binding activity.
  • the percent homology between two amino acid sequences is equal to the percent identity between the two sequences.
  • Sequence comparisons and determination of percent identity between sequences can be performed using methods generally known in the art, and can be accomplished using mathematical algorithms. For example, the algorithm of Meyers and Miller, 1988 Comput. Appl. Biosci.
  • the two sequences are of equal length.
  • epitope refers to an epitope that has antigenic or immunogenic activity in an animal body, preferably a mammal. Sexual part of Her3.
  • the epitope with immunogenic activity is the part of Her3 that elicits an antibody response in animals.
  • the epitope with antigenic activity is a part of Her3 to which the antibody immunospecifically binds, which can be determined by methods known in the art. An antigenic epitope need not be immunogenic.
  • the epitope of the disclosed antibody or its antigen-binding fragment can be determined by existing techniques, such as synthetic peptide method, immunoinformatics prediction, determination of polypeptide activity, epitope peptide scanning method, phage display technology, X-ray diffraction and nuclear magnetic resonance Analysis, antibody homology modeling protein docking prediction method.
  • the antibody or antigen-binding fragment thereof of the present disclosure has an epitope that binds to Her3 domain 3.
  • Antibodies or antigen-binding fragments thereof of the present disclosure include antibodies that bind the same epitope.
  • the phrase "antibodies that bind the same epitope" as used herein means that different antibodies bind a common epitope. If the second antibody binds part of the peptide or part of the tertiary structure to which the first antibody binds, it can be determined that the first antibody and the second antibody bind the same epitope.
  • Epitope 202-2-1 or "202-2-1 epitope” is a region in the extracellular domain of HER3 to which antibody 202-2-1 binds. This epitope is close to the transmembrane domain of HER3 and within domain3 of HER3.
  • the "epitope 202-2-1” consists of the 466th histidine, 470th tryptophan, 471th threonine, 478th threonine, 483th to 487 (aspartic acid-isoleucine-lysine-histidine-asparagine), 484 isoleucine, 490 arginine and 491 arginine.
  • the CDRs contained in the disclosed antibodies or antigen-binding fragments thereof can be identified according to various numbering systems known in the art.
  • the disclosed antibodies or antigen-binding fragments thereof contain CDRs preferably identified by the Kabat, Chothia, AbM or IMGT numbering systems.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • germline antibody gene is a gene encoding an immunoglobulin expressed by a non-lymphoid cell that has not undergone the genetic rearrangement and maturation process leading to the expression of a specific immunoglobulin.
  • amino acid substitutions conservative amino acid substitutions are preferred in this specification.
  • Conservative amino acid substitutions indicate substitutions that occur within a group of amino acids that are related to the amino acid side chain.
  • Preferred sets of amino acids are as follows: acidic set (aspartic acid and glutamic acid); basic set (lysine, arginine, and histidine); nonpolar set (alanine, valine, leucine amino acids, isoleucine, proline, phenylalanine, methionine, and tryptophan); and the uncharged polar family (glycine, asparagine, glutamine, cysteine, serine, threonine and tyrosine).
  • More preferred sets of amino acids are as follows: aliphatic hydroxyl groups (serine and threonine); amide-containing sets (asparagine and glutamine); aliphatic sets (alanine, valine, leucine, and isoleucine) amino acids); and aromatic groups (phenylalanine, tryptophan, and tyrosine).
  • Such amino acid substitutions are preferably made within a set that does not impair the properties of the substance having the original amino acid sequence.
  • IgG isotype control antibodies of the present disclosure are well known and can be purchased or prepared by those of ordinary skill in the art.
  • human anti-Hen Egg Lysozyme IgG anti-HEL, such as human IgG1, referred to as hIgG1
  • anti-HEL anti-HEL
  • hIgG1 human IgG1
  • Variable region sequence of Fab F10.6.6 sequence from the study of anti-protein antibodies (Acierno et al. J Mol Biol. 2007; 374(1):130-46.).
  • the common hIgG1 antibody anti-hel-hIgG1 against chicken egg lysozyme can be purchased from Baiying Biotechnology, product number: B117901. You can also do it yourself preparation.
  • the preparation method is as follows: entrust Nanjing GenScript Biotech Co., Ltd.
  • amino acids are generally referred to by art-recognized one-letter and three-letter abbreviations.
  • alanine can be represented by A or Ala
  • arginine can be represented by R or Arg
  • glycine can be represented by G or Gly
  • glutamine can be represented by Q or Gln.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives.
  • prevention refers to methods performed to prevent or delay the occurrence of a disease or disorder or symptom (eg, tumors and infectious diseases) in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, Reduce the extent of the disease, stabilize (ie, not worsen) the state of the disease, delay or slow the progression of the disease, ameliorate or alleviate the state of the disease, and relieve symptoms (whether partial or total), whether detectable or undetectable .
  • treating can also refer to prolonging survival as compared to expected survival if not receiving treatment.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a non-human primate mammal or a human.
  • the subject eg, human
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing diseases refers to an amount sufficient to prevent, arrest or delay the occurrence of diseases (such as tumors and infectious diseases);
  • an effective amount for treating diseases refers to an amount sufficient to cure or at least partially prevent Amount of disease and its complications in patients with pre-existing disease. Determining such an effective amount is well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease being treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.
  • effector function refers to those attributable to the biological activity of the antibody Fc region (native sequence Fc region or amino acid sequence variant Fc region), and which are associated with the antibody Varies with isotype.
  • pharmaceutically acceptable means that the molecular entities, molecular fragments or compositions will not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • pharmaceutically acceptable carriers or components thereof include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid and the like.
  • In vivo therapeutic effects of antibodies and antibody-drug conjugates on cancer can be determined using experimental animals, for example, administering antibodies to nude mice implanted with a Her3-expressing tumor cell line and measuring any changes in cancer cells.
  • cancer types include lung cancer, colorectal cancer, and the like.
  • direct bond means that the groups on both sides are directly connected, for example, as shown in formula II compound of In , if X is a direct bond, its structural formula is Other direct keys can be understood with reference to the foregoing content.
  • absence means that the group does not exist, for example, the compound shown in formula II , if W does not exist, its structural formula is
  • X is defined, for example, "X is selected from optionally substituted The substituent is selected from 1 or 2 C1-4 alkyl (such as methyl)", then X, for example, can be Other similar definitions of X can be understood with reference to the foregoing.
  • X is defined, for example, "X is selected from optionally substituted The substituent is selected from two C1-4 alkyl groups (such as methyl) and the carbon atoms connected to them at the same time to form a C3-6 cycloalkyl group (such as cyclopropyl)", then X, for example, can be Other similar definitions of X can be understood with reference to the foregoing.
  • the structure of the amino acid residue shown in AA 1 Among them, if R a and R b together with their common carbon atoms form a 4-10 membered heterocyclic ring, the 4-10 membered heterocyclic ring is optionally substituted by one or more R 0 , wherein the term "The 4-10 membered heterocycle is optionally substituted by one or more R 0 " means that the 4-10 membered heterocycle can be unsubstituted or substituted by one or more R 0 , and among the plurality of R 0 , the definition of each R 0 may be the same or different. Other similar definitions can be understood with reference to the foregoing.
  • L is selected from Lys, Val-Cit, Ala-Ala-Asn, Ala-Ala-Asp, Gly-Gly-Phe-Gly, Val-Lys-Gly, Val-Ala, Lys-Ala-Asn
  • the distal amino group of the lysine (Lys) is optionally replaced by 1, 2 or 3 substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl)
  • substituents are substituted.
  • the distal amino group of Lys refers to the lysine residue The exposed amino group-NH 2 .
  • the distal amino group of the lysine (Lys) is optionally substituted by 1, 2 or 3 substituents selected from tert-butoxycarbonyl, C1-6 alkyl (preferably methyl), O
  • the distal amino group of the lysine (Lys) is replaced by O means that the distal amino group of the lysine (Lys) is substituted by oxygen, that is, becomes If the distal amino group is further substituted by two methyl groups, it becomes
  • C1-6 alkyl specifically refers to independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl.
  • C1-6 alkyl means a straight or branched chain alkane containing 1-6 carbon atoms group, including for example “C1-3 alkyl” or “C1-4 alkyl", methyl, ethyl, etc., specific examples include but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl Base, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl.
  • C 1-4 alkyl means a linear or branched alkyl group containing 1-4 carbon atoms, including, for example, "C 1-3 alkyl", methyl, ethyl, etc., specifically Examples include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl.
  • C 2-6 alkenyl refers to a straight-chain, branched or cyclic alkenyl group containing at least one double bond and having 2-6 carbon atoms, including, for example, "C 2-4 alkenyl "wait.
  • Examples include, but are not limited to: vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 1,3-butadienyl, 1-pentenyl, 2-pentenyl Alkenyl, 3-pentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,4-hex Dienyl, cyclopentenyl, 1,3-cyclopentadienyl, cyclohexenyl, 1,4-cyclohexadienyl and the like.
  • C 2-6 alkynyl refers to a straight-chain or branched alkynyl group containing at least one triple bond and having 2-6 carbon atoms, including, for example, "C 2-4 alkynyl” and the like. Examples include, but are not limited to: ethynyl, propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 4-methyl-2-pentynyl, 2-hexynyl, 3 -hexynyl, 5-methyl-2-hexynyl and the like.
  • halogen includes fluorine, chlorine, bromine, iodine.
  • 3-6 membered cycloalkyl or "C 3-6 cycloalkyl” refers to a saturated cyclic alkyl group containing 3-6 carbon atoms, including cyclopropanyl (i.e. cyclopropyl) , Cyclobutanyl (i.e. cyclobutyl), cyclopentyl (i.e. cyclopentyl), cyclohexyl.
  • 3-7 membered carbocycloalkyl or "C 3-7 cycloalkyl” refers to a saturated cyclic alkyl group containing 3-7 carbon atoms, including cyclopropanyl, cyclobutanyl , Cyclopentyl, Cyclohexyl, Cycloheptyl.
  • C1-6 alkoxy means an alkyl group, as defined above, appended to the parent molecular moiety through an oxygen atom. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy base, hexyloxy group, etc.
  • C 1-4 alkoxy refers to the parent molecular moiety through an oxygen atom such as Alkyl as defined above. Specific examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, and the like.
  • the term "4-10 membered heterocyclic group” refers to a cyclic group containing 4-10 ring atoms (wherein at least one ring atom is a heteroatom such as nitrogen atom, oxygen atom or sulfur atom).
  • the term "4-6 membered heterocyclic group” refers to a cyclic group containing 4-6 ring atoms (wherein at least one ring atom is a heteroatom, such as nitrogen atom, oxygen atom or sulfur atom).
  • ring atoms (eg carbon, nitrogen or sulfur atoms) in the ring structure can be oxo substituted.
  • “4-8 membered heterocyclic group” includes, for example, “4-8 membered nitrogen-containing heterocyclic group”, “4-8 membered oxygen-containing heterocyclic group”, “4-7 membered heterocyclic group”, “4-7 membered Oxygen-containing heterocyclic group”, “4-7-membered heterocyclic group”, “4-6-membered heterocyclic group”, “5-7-membered heterocyclic group”, “5-6-membered heterocyclic group”, “5- "6-membered nitrogen-containing heterocyclic group”, including but not limited to oxocyclobutanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, tetrahydropyranyl, homopiperazinyl, etc.
  • the term "4-10 membered heterocyclic ring” refers to a ring containing 4-10 ring atoms (wherein at least one ring atom is a heteroatom such as nitrogen atom, oxygen atom or sulfur atom).
  • the term "5-6 membered heterocyclic ring” refers to a ring containing 5-6 ring atoms (wherein at least one ring atom is a heteroatom, such as nitrogen atom, oxygen atom or sulfur atom), including but not limited to pyrrolidine, tetrahydrofuran, Piperidine, piperazine, tetrahydropyran and other rings.
  • aryl refers to an aromatic monocyclic or polycyclic hydrocarbon group, such as 6-10-membered aryl, 5-8-membered aryl, etc. Specific examples include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, and the like.
  • the "6-10 membered aryl” refers to an aryl group containing 6-10 ring atoms.
  • C6-10 aryl refers to an aryl group containing 6-10 carbon atoms.
  • heteroaryl refers to an aromatic cyclic group in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom or a sulfur atom.
  • ring atoms eg carbon, nitrogen or sulfur atoms
  • ring structure can be oxo substituted.
  • a bond in a structural formula represented by a wavy line " ⁇ " herein is intended to indicate that the structure represents a cis or trans isomer, or a mixture of cis and trans isomers in any proportion.
  • between includes the end value of the interval, such as between 1-16, may refer to 1 or 16.
  • DAR drug to antibody ratio
  • the DAR of an ADC can range from 1 to 16, but depending on the number of attachment sites on the antibody, higher loadings (eg 20) are also possible.
  • the term DAR may be used when referring to the amount of drug loaded onto a single antibody, or alternatively, the average or mean DAR of a group of ADCs.
  • DAR can also be calculated as the average DAR of the population of molecules in the product, i.e., the Ab moieties conjugated to the Ab moieties described herein, measured by detection methods (e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis and/or HPLC).
  • detection methods e.g., by conventional methods such as mass spectrometry, ELISA assays, electrophoresis and/or HPLC.
  • the antibodies involved in the invention are fully human antibodies, so that they can be safely administered to human subjects without triggering immunogenic reactions.
  • the antibody involved in the invention has extremely high affinity and specificity, and can be beneficial to play an anti-tumor effect.
  • the antibody-drug conjugates of the present disclosure have significant anti-tumor effects in vivo.
  • Figure 1A Detection of binding of anti-Her3 antibody to human Her3-his protein ELISA.
  • Figure 1B ELISA detection of anti-Her3 antibody binding to monkey Her3-his protein.
  • Figure 1C ELISA detection of anti-Her3 antibody binding to rat Her3-his protein.
  • Fig. 2A Detection of anti-Her3 antibody binding to A549-human Her3 cells by flow cytometry.
  • Fig. 2B Detection of the binding of anti-Her3 antibody 202-2-1 to T47D cells by flow cytometry.
  • Figure 2C Detection of the binding of anti-Her3 antibody 202-2-1 to MCF-7 cells by flow cytometry.
  • Anti-Her3 antibody inhibits NRG1 phosphorylation of AKT in tumor cells.
  • FIG. 5A Anti-Her3 antibody competition biotinylated control antibody 2 for detection of human Her3 protein binding.
  • Figure 5B-1 The complex of the 202-2-1 antibody Fab and Her3, showing that binding occurs and a complex is formed.
  • Figure 5B-2 202-2-1 View 1 of the complex interface between antibody Fab and Her3, the dotted lines and numbers show the interacting amino acid side chains and their distances (angstroms).
  • FIG. 5B-3 View 2 of the complex interface of 202 antibody Fab and Her3, the dotted lines and numbers show the interacting amino acid side chains and their distances (angstroms).
  • Figure 6A Detection of anti-Her3 antibody 202-2-1ADC drug on PC-9 tumor cell killing.
  • Figure 8 One of the drug efficacy tests of anti-Her3 antibody 202-2-1ADC drug on SW480 tumor model.
  • Figure 9 The second drug efficacy test of anti-Her3 antibody 202-2-1ADC drug on SW480 tumor model.
  • Control antibody 1 is from MediaPharma company patent CN 103189392B (heavy chain SEQ ID NO: 10, light chain SEQ ID NO: 14); control antibody 2 is from Daiichi Sankyo Company, see IMGT database, IMGT/mAb-DB ID: 964, INN number: 11093.
  • the gene of the above-mentioned human Her3 antigen was synthesized and two control antibodies were synthesized by codon optimization (Nanjing GenScript Biotechnology Co., Ltd.), constructed on the PTT5 expression vector, and a large number of plasmids were extracted.
  • the expression vectors prepared by extraction were transiently expressed in HEK293F by PEIMAX for 7 days, and the expression supernatant was purified by Ni column or ProA.
  • the 1-643 amino acid sequence of monkey Her3 (NCBI: XP_001113953.2) was selected, in which a 6xHis tag was added to the C-terminus, and it was named monkey Her3-his.
  • the heavy chain variable regions of clone No. 202-2-1, clone No. 202-3-1 and clone No. 202-3-2 were connected to the heavy chain IgG1 constant region (SEQ NO: 29), and 202-2-
  • the light chain variable regions of No. 1 clone, No. 202-3-1 clone and No. 202-3-2 clone were respectively linked to the Kappa constant region sequence (SEQ NO: 30), through codon optimization and gene synthesis (Anhui General Biology Technology Co., Ltd.), and then respectively constructed on the PTT5 vector.
  • the heavy chain and light chain expression plasmids of the anti-Her3 antibody were co-transfected into HEK293F cells for expression by PEI max reagent, expressed in a 5% CO2 shaker at 37°C for 7 days, and the supernatant was collected and purified by ProteinA magnetic beads to obtain the anti-Her3 antibody
  • Her3 antibodies were named 202-2-1 antibody, 202-3-1 antibody and 202-3-2 antibody, respectively.
  • the heavy and light chain variable regions and corresponding CDRs of the 202-2-1 antibody, 202-3-1 antibody and 202-3-2 antibody are shown in Table 1.
  • Fortibio is a commonly used dynamic affinity detection device, which detects the dynamic affinity between anti-Her3 antibody and human Her3 protein.
  • the method is briefly described as follows: human Her3-His is serially diluted with PBST to obtain 400nM, 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM and OnM. ProteinA biosensors (Pall Life Sciences) were pre-wetted with PBST buffer before use. Anti-Her3 antibody was diluted to 5 ⁇ g/mL with PBST and immobilized on the ProA sensor.
  • Immobilized antibody-immobilized sensors were then equilibrated in PBST buffer for 60 s to obtain a baseline, then transferred to antigen diluent for binding for 60 s, and dissociated in PBST for 180 s. After one analysis cycle, the sensor was regenerated with 10 mM Gly (pH 1.5). Using Date analysis with a 1:1 model analysis, the association (Ka) and dissociation (Kd) rate constants were determined and used to calculate the dissociation equilibrium constant (KD). As shown in Table 3, 202-2-1, 202-3-1 and 202-3-2 have higher dynamic affinity to human Her3, which is comparable to that of control antibody 1.
  • Her3 protein is highly expressed on a variety of solid tumor cells, and the affinity of Her3 antibody to tumor cells is detected by FACS experiments.
  • MCF-7 breast cancer cells, from ATCC, product number HTB-22
  • A549-human Her3 human non-small cell lung cancer, from ATCC, product number CRM-CCL-185)
  • T47D human breast ductal carcinoma, ATCC
  • MCF-7 breast cancer cells, from ATCC, product number HTB-22
  • A549-human Her3 human non-small cell lung cancer, from ATCC, product number CRM-CCL-185
  • T47D human breast ductal carcinoma, ATCC
  • N/A in Table 4 indicates that the affinity is too weak to obtain a fitted EC50 result.
  • NRG1 binds with high affinity through the Her3/Her2 heterodimer, it induces the phosphorylation of the downstream signaling pathway AKT and stimulates the proliferation of tumor cells.
  • Anti-Her3 antibody inhibited NRG1-induced AKT phosphorylation pathway by cytoblotting.
  • FACS was used to detect the endocytosis of A549-human Her3 (human non-small cell lung cancer cells) anti-Her3 antibody.
  • endocytosis rate (%) [1-(the average fluorescence value of the detection sample at this time point-the average fluorescence value of the negative control sample at this time point)/(the average fluorescence value of the detection sample at 0 hours-the average fluorescence value of the negative control sample at 0 hours value)]*100.
  • the results are shown in Figure 4.
  • the anti-Her3 antibody 202-2-1 has a strong endocytic activity on A549-human Her3 tumor cells, which can reach about 26.9% in 4 hours, which is stronger than the 16.6% of the control antibody 2. Compared with the control Antibody 1 is comparable.
  • the anti-Her3 antibody 202-2-1 was endocytized earlier than the control antibody and reached the maximum endocytosis rate, and had a faster endocytosis rate than the control antibody.
  • Antibody 202-3-2 has stronger endocytic activity than control antibody 1 and control antibody 2.
  • complexes were prepared from the Fab of 202-2-1 and the Her3 protein, and the structure of the antibody-antigen complex was analyzed by cryo-electron microscopy.
  • Antigen preparation The amino acid sequence of HER3 extracellular domain (20-643) was obtained from the uniprot database, the DNA was synthesized after DNA sequence optimization (293F was the host), and 6*his was added to the C-terminus of the protein.
  • the gene was constructed into the expression vector of pLVX (Miaoling Biotechnology, Cat. No. P0249) and then transfected into 293F cells (ATCC; PTA-5077), and a stable cell pool was obtained by screening.
  • the cells were cultured by fermentation to obtain the culture supernatant containing the antigen.
  • the antigen was captured by affinity with a nickel column and further purified by molecular sieves.
  • the antigen was dissolved in a Tris-NaCl solution (0.7 mg/ml) at pH 7.5. Characterization of antigen quality: 1. SDS-PAGE experiment showed that there was a uniform band under both non-reducing and reducing conditions. 2. The SEC-HPLC purity is 95.6%. 3. The reverse phase chromatography-HPLC purity is 97.44%. 4. The affinity of the antigen and antibody was tested by ELISA, and the results showed that the affinity of the batch of antigen and antibody 202-2-1 was less than 0.1nM, and the next experiment could be carried out.
  • antibody 202-2-1 was dissolved in 20 mm acetic acid + 150 mm sodium chloride, 20.7 mg/ml. Incubate with pepsin and antibody, incubate at 37 degrees Celsius for 240 minutes, detect the digested product by SDS-PAGE and put it on the SEC (Superdex 200 Increase 10/300 GL column) column to collect the main peak. Use papain to incubate with the above product, incubate at 37 degrees Celsius for 120 minutes, detect the digested product by SDS-PAGE and put it on the SEC (Superdex 200 Increase 10/300 GL column) column, collect the main peak, this product is the Fab of antibody 202-2-1 .
  • the antigen prepared in step (1) was mixed with the Fab, and incubated overnight at 4°C.
  • the incubation product was centrifuged Concentrate, detect the incubation product by SDS-PAGE, put it on a SEC (Superdex 200 Increase 10/300 GL column) column, and collect the main peak.
  • This component is the Fab-antigen complex.
  • negative staining was used to observe the quality of the complex (VitrobotTM MarkIV, easiGlowTM Glow Discharge, 200kV TF20, CCD camera 895).
  • the results of negative staining showed that most complexes were homogeneous in shape, and 80 images were selected for the reconstruction of negative staining, and the PDB model (4LEO) was used for simulation with the density map.
  • the simulation results show that the complex can overlap with most of the PBD model (including the Fab binding domain), and the quality of the complex meets the requirements of cryo-EM observation.
  • Cryo-EM sample preparation/screening/data collection on Cryo-TEM Glacios use 25mM Tris-HCl (pH 7.5), 150mM NaCl to dilute the complex prepared in (2) to 0.2mg/ml, centrifuge at 12000g for 15 minutes ;Prepare relevant equipment consumables, add 3ul samples dropwise to 300mesh R 1.2/1.3holey carbon Film (Quantifoil) grid, and use a 200KV Glacios electron microscope to observe and screen the pictures of the complex.
  • a 300KV Titan Krios electron microscope (equipped with K3 direct electron detector, Gatan brand) was used to further collect high-resolution cryo-EM photos, and a total of 3272 photos were collected for further analysis.
  • Parameter settings magnification 105K, physical pixel size 0.819 angstrom, 56.3e-/angstrom 2 dose of electrons divided into 40 frames, under-focus range is set to -1.0uM to -2.4uM.
  • results of structural analysis show that the antigenic epitope bound by the Fab of the 202-2-1 antibody is located in the domain3 of human Her3, which is within amino acids 328 to 499 in SEQ ID No. 31, and specifically located in the Her3 antigen according to amino acids in SEQ ID No.
  • Step 1 (S)-14-(3-chloropropyl)-7-ethyl-7-hydroxy-10,13-dihydro-11H-[1,3]dioxole[4, Synthesis of 5-g]pyrano[3',4':6,7]indoxazino[1,2-b]quinoline-8,11(7H)-dione (A1B)
  • the reaction solution was stirred at 0° C. for 5 minutes, then warmed to room temperature, and stirred for 3 hours.
  • the reaction solution was diluted with water (50 mL), extracted with ethyl acetate (80 mL ⁇ 2), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain crude product
  • the crude product was further purified by C18 column (acetonitrile/0.05% formic acid in water: 5%-60%) to obtain the target compound A1B (yellow solid, 400 mg, yield: 67%).
  • Step 2 (S)-7-ethyl-7-hydroxy-14-(3-hydroxypropyl)-10,13-dihydro-11H-[1,3]dioxole[4, Synthesis of 5-g]pyrano[3',4':6,7]indoxazino[1,2-b]quinoline-8,11(7H)-dione (A1)
  • the combined organic phases were sequentially dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product.
  • Step 2 B1B (240 mg) was dissolved in DMF (5 ml), piperidine (1 ml) was added, the compound was stirred for 20 minutes, dissolved in reduced pressure to remove low boiling point components, and the residue was directly used in the next step of synthesis.
  • Step 2 B2A (240 mg) was dissolved in DMF (5 ml), piperidine (1 ml) was added, the compound was stirred for 20 minutes, dissolved in reduced pressure to remove low boiling point components, and the residue was directly used in the next step of synthesis. A small amount of crude product was purified by reverse phase chromatography (acetonitrile/0.05% FA in water: 5% to 50%) to obtain the target compound B2.
  • Example 11.6 (S,E)-2-Amino-N-(3-(7-ethyl-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H- [1,3]dioxole[4,5-g]pyrano[3',4':6,7]indoleazino[1,2-b]quinolin-14-yl ) allyl) acetamide (B3)
  • Example 11.7 N 6 ,N 6 -Dimethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)- L-Lysine (C1)
  • 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoic acid (268mg), compound C1A (328mg), triethylamine (322mg) were dissolved in N,N-dimethylformamide (5 mL).
  • Example 11.8 N 6 ,N 6 -Dimethyl-N 2 -((6-(4-(2-(methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazole- 1-yl)hexanoyl)-L-valine)-L-lysine (C2)
  • Example 11.9 N 6 ,N 6 -diethyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)- L-Lysine (C3)
  • Example 11.10 N 6 ,N 6 -dipropyl-N 2 -((6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynoyl)-L-valine)- L-Lysine (C4)
  • Embodiment 12 drug-linker compound synthesis
  • Example 12.1 N-((11S,14S)-11-(4-(dimethylamino)butyl)-1-((S)-4-ethyl-8-fluoro-4-hydroxy-9- Methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indoleazino[1,2-b]quino Lin-11-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecan-14-yl)-6-(2- (Methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (DL-01)
  • reaction solution was filtered and purified by HPLC (acetonitrile/water containing 0.05% formic acid) to obtain yellow solid compound N-((11S,14S)-11-(4-(dimethylamino)butyl)-1-(( S)-4-Ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4 ':6,7]indoxazino[1,2-b]quinolin-11-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12 -Triazahexadecan-14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (DL-01) (12 mg, yield 6.0%).
  • HPLC acetonitrile/water containing 0.05% formic acid
  • Example 12.2 N-((S)-1-(((S)-6-(diethylamino)-1-((2-(((E)-3-((S)-7-ethyl Base-7-hydroxy-8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxole[4,5-g]pyrano[ 3',4':6,7]indoleazino[1,2-b]quinolin-14-yl)allyl)amino)-2-oxoethyl)amino)-1-oxohexane -2-yl)amino)-3-methyl-1-oxobut-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (DL-02 )
  • the reaction solution was prepared and purified by high performance liquid phase (acetonitrile/0.05% trifluoroacetic acid aqueous solution) to obtain the compound N-((S)-1-(((S)-6-(diethylamino)-1-((2- (((E)-3-((S)-7-Ethyl-7-hydroxy- 8,11-dioxo-7,8,11,13-tetrahydro-10H-[1,3]dioxole[4,5-g]pyrano[3',4': 6,7]indoleazino[1,2-b]quinolin-14-yl)allyl)amino)-2-oxoethyl)amino)-1-oxohex-2-yl)amino )-3-methyl-1-oxobut-2-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yne amide (DL-02, 11.1mg, yield 14%)
  • Example 12.3 (S)-6-(Dimethylamino)-N-(2-(((3-((S)-4-ethyl-8-fluoro-4-hydroxy-9-methyl- 3,14-Dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indoleazino[1,2-b]quinoline-11 -yl)propyloxy)methyl)amino)-2-oxoethyl)-2-((S)-3-methyl-2-(6-(4-(2-(methylsulfonyl) Pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)caproylamido)butanamido)caproamide (DL-03)
  • Example 12.4 N-((11S,14S)-11-(4-(di-n-propylamino)butyl)-1-((S)-7-ethyl-7-hydroxy-8,11-di Oxo-7,8,11,13-tetrahydro-10H-[1,3]dioxole[4,5-g]pyrano[3',4':6,7]ind Oxazino[1,2-b]quinolin-14-yl)-15-methyl-7,10,13-trioxo-4-oxa-6,9,12-triazahexadecane -14-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-yneamide (DL-04)
  • Example 12.5 (S)-6-(Dimethylamino)-N-(2-(((3-((S)-7-ethyl-7-hydroxy-8,11-dioxo-7 ,8,11,13-tetrahydro-10H-[1,3]dioxole[4,5-g]pyrano[3',4':6,7]indoleazino[ 1,2-b]quinolin-14-yl)propyloxy)methyl)amino)-2-oxoethyl)-2-((S)-3-methyl-2-(6-( 4-(2-(Methylsulfonyl)pyrimidin-5-yl)-1H-1,2,3-triazol-1-yl)caproylamido)butanamido)caproamide (DL-05)
  • the light chain of the antibody on Her3-ADC-07 was coupled to 0 to 1 toxin molecule (the proportions of LC and DAR1 were 0% and 100%), and the heavy chain was coupled to 0 to 3 toxin molecules ( The ratios of mAb, DAR1, DAR2, and DAR3 are 0%, 0%, 0%, and 100%, respectively), so it can be inferred that the q value is 8.
  • 0 to 1 toxin molecule was coupled to the light chain of the antibody (LC, DAR1 ratios were 93.67%, 6.33%), and 0 to 3 toxin molecules were coupled to the heavy chain (mAb , DAR1, DAR2, and DAR3 are respectively 19.23%, 7.01%, 10.66%, and 63.1%), it can be inferred that the value of q can be 1, 2, 3, 4, 5, 6, 7 or 8.
  • Example 131.2 The compound DL-04 in Example 13.1.2 was replaced by DL-01 to obtain Her3-ADC-05 (DAR4), a coupling product of DL-01 and 202-2-1 antibody.
  • the average DAR value determined by mass spectrometry was 4.0.
  • 0-1 toxin molecule coupled to antibody light chain on Her3-ADC-05(DAR4) as determined by mass spectrometry (LC, DAR1 ratios are 90.89%, 9.11%, respectively), heavy chain coupling 0 to 3 toxin molecules (mAb, DAR1, DAR2, DAR3 ratios are 25.1%, 7.17%, 18.19%, 49.53%), can be It is inferred that the value of q may be 1, 2, 3, 4, 5, 6, 7 or 8.
  • Example 131.1 The compound DL-04 in Example 13.1.1 was replaced by DL-02 to obtain Her3-ADC-15 (DAR8), a coupling product of DL-02 and 202-2-1 antibody.
  • the average DAR value determined by mass spectrometry was 7.9.
  • the light chain of the antibody on Her3-ADC-15 (DAR8) as determined by mass spectrometry is coupled with 0 to 1 toxin molecule (the proportions of LC and DAR1 are 0% and 100%, respectively), and the heavy chain is coupled with 0 to 3 toxin molecules (mAb , DAR1, DAR2, and DAR3 are respectively 0%, 0%, 6.31%, and 93.69%), it can be deduced that the value of q can be 3, 4, 5, 6, 7 or 8.
  • Example 131.1 The compound DL-04 in Example 13.1.1 was replaced by 2-fold molar equivalent of DL-02 to obtain Her3-ADC-15 (DAR2), a coupling product of DL-02 and 202-2-1 antibody.
  • DAR2 Her3-ADC-15
  • the average DAR value determined by mass spectrometry was 2.0.
  • 0 to 1 toxin molecule was coupled to the antibody light chain (LC, DAR1 ratios were 93.82%, 6.18%), and 0 to 3 toxin molecules were coupled to the heavy chain (mAb , DAR1, DAR2, and DAR3 are respectively 61.16%, 5.77%, 11.02%, and 22.05%), and it can be deduced that the value of q can be 1, 2, 3, 4, 5, 6, 7, or 8.
  • Example 131.2 The compound DL-04 in Example 13.1.2 was replaced by DL-03 to obtain the coupling product of DL-03 and 202-2-1 antibody Her3-ADC-23 (DAR4).
  • the average DAR value determined by mass spectrometry was 4.2.
  • 0 to 1 toxin molecule was coupled to the light chain of the antibody (LC, DAR1 ratios were 91.28%, 8.72%), and 0 to 3 toxin molecules were coupled to the heavy chain (mAb , DAR1, DAR2, and DAR3 are respectively 19.24%, 8.67%, 21.97%, and 50.13%), it can be inferred that the value of q can be 1, 2, 3, 4, 5, 6, 7 or 8.
  • the drug-linker compound is prepared as follows,
  • the above drug-linker compound was used to replace the compound DL-04 in Example 13.1.2, and the control antibody 2 was used to replace the anti-Her3 antibody 202-2-1 in Example 13.1.2 to obtain Her3-C3-ADC-21 (DAR4).
  • the average DAR value determined by mass spectrometry was 4.4.
  • Example 13.7 Using the drug-linker compound in Example 13.7 instead of compound DL-04 in Example 13.1.2, Her3-ADC-21 (DAR4), the coupling product of the drug-linker compound and the 202-2-1 antibody, was obtained.
  • the average DAR value determined by mass spectrometry was 4.1.
  • the light chain of the antibody on Her3-ADC-21 (DAR4) as determined by mass spectrometry is coupled to 0-1 toxin molecule (LC, DAR1 ratios are 100%, 0%), and the heavy chain is coupled to 0-3 toxin molecules (mAb , DAR1, DAR2, and DAR3 are respectively 20.57%, 8.01%, 17.3%, and 54.12%). It can be deduced that the value of q can be 1, 2, 3, 4, 5, or 6.
  • the drug-linker compound was prepared as follows,
  • Example 131.2 The compound DL-04 in Example 13.1.2 was replaced by the above-mentioned drug-linker compound to obtain the coupling product Her3-ADC-33 (DAR4) of the above-mentioned drug-linker compound and the 202-2-1 antibody.
  • the average DAR value determined by mass spectrometry was 5.6.
  • Her3mAb and Her3Ab refer to the anti-Her3-antibody or its antigen-binding fragment. Specifically, in the above examples, Her3mAb is the 202-2-1 antibody, and Her3Ab is the control antibody 2.
  • Compound DL-05 was used instead of compound DL-04 in Example 13.1.3 "Her3-ADC-07 (DAR6) sample preparation" to obtain the coupling product of DL-05 and 202-2-1 antibody Her3-ADC-08 (DAR6), the average DAR value measured by mass spectrometry was 4.9. On Her3-ADC-08 (DAR6) detected by mass spectrometry, 0 to 1 toxin molecule was coupled to the light chain of the antibody (LC, DAR1 ratios were 66.58%, 33.42%), and 0 to 3 toxin molecules were coupled to the heavy chain (mAb , DAR1, DAR2, and DAR3 are respectively 16.51%, 11.27%, 16.63%, and 55.59%). It can be deduced that the value of q can be 1, 2, 3, 4, 5, 6, 7, or 8.
  • Example 131.1 The compound DL-04 in Example 13.1.1 was replaced by 2-fold molar equivalent of DL-05 to obtain the coupling product of DL-05 and 202-2-1 antibody Her3-ADC-08 (DAR2).
  • the average DAR value determined by mass spectrometry was 1.9.
  • 0 to 1 toxin molecule was coupled to the light chain of the antibody (LC, DAR1 ratios were 96.61%, 3.39%), and 0 to 3 toxin molecules were coupled to the heavy chain (mAb , DAR1, DAR2, and DAR3 are respectively 58.72%, 9.05%, 15.41%, and 16.83%), it can be inferred that the value of q can be 1, 2, 3, 4, 5, 6, 7 or 8.
  • Example 14 Determination of the DAR value of the sample after coupling by mass spectrometry
  • Chromatographic column PLRP-S, 2.1*50mm, 5 ⁇ m;
  • Sample treatment Take 50 ⁇ g of samples, add 2 ⁇ l of 1M DTT, add 50 ⁇ l of ultrapure water to dilute to a concentration of about 1.0 mg/ml, mix well, and restore at room temperature for 30 minutes.
  • Mass spectrometry conditions Gas temp: 320°C, Drying Gas: Nebulizer: 35psi; Sheath Gas Temp: 350°C; sheath Gas Flow: 11l/min; m/z 500 ⁇ 3000.
  • mAb represents unconjugated monoclonal antibody
  • LC represents antibody light chain
  • HC represents antibody heavy chain
  • DAR1 represents a conjugate containing light chain or heavy chain coupled to a toxin molecule
  • DAR2 represents light chain Or a conjugate of two toxin molecules coupled to a heavy chain
  • DAR3 represents a conjugate comprising three toxin molecules coupled to a light chain or a heavy chain; wherein, the theoretical molecular weight of the monoclonal antibody is calculated based on the G0F glycoform.
  • mAb, LC, HC, DAR1, DAR2, and DAR3 are as described above.
  • the detection results showed that the antibody light chain on Her3-ADC-07 (DAR8) was coupled with 0 to 1 toxin molecule (the ratios of LC and DAR1 were 0% and 100%), and the heavy chain was coupled with 0 to 3 toxin molecules (mAb , DAR1, DAR2, and DAR3 are respectively 0%, 0%, 0%, and 100%), thus calculating the drug-antibody coupling ratio (DAR value) of Her3-ADC-07 (DAR8) to be 8.0.
  • DAR value drug-antibody coupling ratio
  • PC-9 tumor cells human lung adenocarcinoma, Nanjing Kebai Biotechnology Co., Ltd.
  • NCI-H358 tumor cells human non-small cell lung cancer, ATCC, CRL-5807
  • the specific experimental steps are as follows: digest PC-9 cells and NCI-H358 cells, collect the cells by centrifugation, resuspend the cells to 50,000 cells/mL in 1640+4% FBS medium, spread the cells in 96-well plates at 100 ⁇ L/well; use 1640 Basic training Base gradient dilution of ADC molecules, according to the initial concentration of 150 ⁇ M, 3-fold dilution, 11 concentration points, added to the corresponding wells, 100 ⁇ L per well, the final serum concentration was 2%; 37 ° C, 5% CO 2 incubator for 120 Hours; add CCK8 (Rhinogen), 20 ⁇ L/well, incubate in 37°C, 5% CO 2 incubator for 0.5-2.5 hours, use a microplate reader (MD, model ABS Plus) to read at 450 nm every half hour and import it into Graphpad Prism for curve fit.
  • MD microplate reader
  • test results show that both Her3-ADC-05 and Her3-C3-ADC-05 have a strong tumor cell killing effect.
  • In vitro cell viability detection method use trypsin to digest NCI-H358 tumor cells by conventional methods, collect the number of cells in the tube, resuspend with the corresponding detection medium (containing 2% FBS), add 2000-5000 cells/well to 96-well plates. 100 uL of ADC diluted in 2% FBS medium was added to a 96-well plate, the concentration starting from 150 ⁇ g/ml, 3-fold dilution (11 concentration gradients). Incubate at 37°C in 5% CO2 for 7 days, then add 20 ⁇ L of CCK8 reagent to each well, react for 2-6 hours, and read with a microplate reader (The detection wavelength is 450nm). The samples and test results of each group are shown in Table 8.
  • test results of this example show that the ADCs with various DAR values of the present disclosure have killing effect on tumor cells.
  • mice Female, 5-6 weeks old, were purchased from Weitong Lihua Experimental Animal Co., Ltd.
  • NCI-H358 cells were cultured in 15cm-diameter culture dishes with 1640 medium containing 10% FBS, digested with trypsin-EDTA when they reached about 80-90% confluence, washed twice with PBS, then centrifuged and resuspended in pre-cooled PBS , counted by a cell counter, and diluted with PBS to make the cell concentration 5 ⁇ 10 7 /ml.
  • mice were adapted to the laboratory environment for 7 days, and NCI-H358 cells were inoculated subcutaneously on the right flank, the inoculation volume was 5 ⁇ 10 6 /mouse, and the inoculation volume was 0.2ml (containing 50% Matrigel), and the tumor growth When the size of the tumor reaches about 300mm3 , they are randomly divided into groups according to the size of the tumor, with 5 patients in each group.
  • Rats respectively: anti-chicken egg lysozyme human IgG1 isotype control antibody (negative control, Suzhou Yilian Biology) group, IgG1-ADC-07 (DAR8), Her3-C3-ADC-21 (DAR4), Her3-ADC- 21 (DAR4) and Her3-ADC-07 (DAR8) dose groups. All samples were injected into the tail vein once a week for 3 weeks. After administration, the tumor volume and body weight of the mice were observed and measured regularly. Relative tumor proliferation rate, T/C (%), that is, the percentage value of the relative tumor volume or tumor weight between the treatment group and the control group at a certain time point.
  • T/C Relative tumor proliferation rate
  • Her3-ADC-07 (DAR8) at doses of 5 mg/kg and 1 mg/kg had a significant inhibitory effect on the tumor growth of the NCI-H358 human non-small cell lung cancer xenograft model, and showed that the dose gradient trend.
  • the TGI of Her3-ADC-07 1mg/kg group was 76.26%, slightly stronger than the TGI 75.74% of Her3-ADC-21 5mg/kg group.
  • the TGI of the Her3-ADC-21 1mg/kg group was 29.92%, slightly stronger than the TGI of the Her3-C3-ADC-21 1mg/kg group of 24.11%.
  • the present disclosure demonstrates the drug efficacy in vivo by evaluating the anti-tumor effect of anti-Her3 antibody ADC drug on Balb/c Nude mouse subcutaneous human colon cancer cell line SW480 cells (ATCC source, Her3 expression positive rate is about 30%) transplanted tumor model.
  • the specific steps are as follows: 36 Balb/c nu nude mice, female, 5-6 weeks old, were purchased from Weitong Lihua Experimental Animal Co., Ltd.
  • SW480 cells were cultured in 15cm diameter petri dish with 1640 medium containing 10% FBS, digested with trypsin-EDTA when reaching about 80-90% confluence, washed twice with PBS, then centrifuged and resuspended in pre-cooled PBS, Count with a cell counter and dilute with PBS to make the cell concentration 5 ⁇ 10 7 /ml.
  • Balb/c nu mice were adapted to the laboratory environment for 7 days, and SW480 cells were subcutaneously inoculated on the right flank, the inoculated cell volume was 5 ⁇ 10 6 /mouse, and the inoculation volume was 0.2ml (containing 50% Matrigel), until the tumor grew to 250mm At about 3 , according to the size of the tumor, they were randomly divided into 5 groups, respectively anti-chicken lysozyme human IgG1 isotype control antibody (negative control, Suzhou Yilian Biology) group, IgG1-ADC-07, and Her3-ADC-07 doses Group. All samples were injected into the tail vein once a week for 3 weeks. After administration, the tumor volume and body weight of the mice were observed and measured regularly.
  • T/C Relative tumor proliferation rate
  • Reference Example 16.2 The same model and method were used to measure IgG1, IgG1-ADC-07 (DAR8), Her3-ADC-07 (DAR8), Her3-C3-ADC-21 (DAR8) in human colon cancer cell line SW480 cells ( ATCC source, Her3 expression positive rate is about 30%) anti-tumor effect of xenograft tumor model.
  • Her3-ADC-07 at doses of 10 mg/kg and 3 mg/kg has a significant inhibitory effect on the tumor growth of the SW480 human colorectal cancer xenograft model, and there is a dose gradient trend.
  • the anti-tumor effect of Her3-ADC-07(DAR8) 3mg/kg group was significantly stronger than that of Her3-C3-ADC-21(DAR8) 3mg/kg group, Her3-ADC-07(DAR8) 10mg/kg The antitumor effect of the group was significantly stronger than that of the Her3-C3-ADC-21(DAR8) 10mg/kg group.
  • Her3-ADC-07(DAR8) The stability of Her3-ADC-07(DAR8) in plasma was evaluated by measuring the release of bioactive molecule A1 in human plasma incubated with Her3-ADC-07(DAR8).
  • Her3-ADC-07(DAR8) was incubated with filter-sterilized mouse, rat, dog, monkey and human plasma in triplicate, respectively. The incubation was carried out at 37°C and 5% CO 2 , the final concentration of Her3-ADC-07 (DAR8) was 50 ⁇ g/mL, and the total incubation time was 504 hours. During the incubation, samples were taken at time points 0, 24, 72, 168, 336 and 504 hours, and the reaction was terminated by adding a methanol solution containing an internal standard (0.5 ⁇ M tolbutamide) and 0.1% formic acid. Use UPLC-MS/MS to analyze test sample, analysis condition is:
  • Release percentage concentration of A1 at the measured time/theoretical maximum release concentration
  • Theoretical maximum release concentration ADC incubation concentration/ADC drug molecular weight ⁇ DAR ⁇ molecular weight of bioactive molecule A1.
  • test results show that, at a test concentration of 50 ⁇ g/mL, after Her3-ADC-07 (DAR8) was incubated in mouse, rat, dog, monkey, and human plasma for 504 hours, the release percentage of bioactive molecule A1 was 0.726% respectively , 0.497%, 0.521%, 0.474 and 0.372%.
  • Her3-ADC-07 (DAR8) has good stability in plasma of mice, rats, dogs, monkeys, and humans. After in vitro incubation for 504 hours (21 days), the release of A1 is less than 1% of the theoretical maximum release concentration .
  • the therapeutic window is evaluated based on the highest non-serious toxic dose (HNSTD) in toxicology tests and the minimum effective dose (MED) in drug efficacy tests.
  • the therapeutic window of Her3-ADC-07 (DAR8) is based on the in vivo exposure at HNSTD and MED doses Ratio calculation.
  • Patritumab Deruxtecan is Daiichi Sankyo’s HER3-targeted ADC, and the doses in the cynomolgus monkey toxicity test are 0, 3, 10 and 30 mg/kg, administered every 3 weeks 1 time, 5 consecutive doses.
  • the results showed that reticulocytes, lymphocytes and platelets decreased at a dose of ⁇ 3 mg/kg; transferase increased at a dose of ⁇ 10 mg/kg; body weight was not significantly abnormal at each dose; the toxic target organs were Thymus, bone marrow and skin (Hashimoto Y, et al. Clin Cancer Res. 2019 Dec 1;25(23):7151-7161).
  • thrombocytopenia was one of the dose-limiting toxicities in the dose escalation test ( PA, et al.Cancer Discov.2022Jan; 12(1):74-89.), is also one of the most frequently occurring (30%) treatment adverse events (TEAE) in multi-center clinical studies (Yu-H, et al. al., Abstract#MA21.06, WCLC 2019).
  • Her3-ADC-07 had no significant platelet count abnormality at the highest dose of 20 mg/kg, and no abnormalities in important organs such as liver, kidney, and lung were found at the highest dose of 20 mg/kg. Toxic reaction.
  • NCI-H358 tumor-bearing nude mice were given a single tail vein injection of 0.3mg/kg or 3mg/kg of Her3-ADC-07(DAR8), collected at 0h, 3h, 6h, 24h, 48h, 96h, 168h and 336h Serum and tumor (5 animals at each time point), while analyzing the tumor volume and weight, the ADC concentration in the serum was detected.
  • the ratio of AUC 0-336h or Cmax under the highest non-severe toxic dose of cynomolgus monkeys to AUC 0-336h or Cmax under the lowest onset dose of CDX mice is defined as the therapeutic index (TI), and the treatment of each parameter
  • TI therapeutic index
  • Her3-ADC-07(DAR8) was well tolerated in cynomolgus monkeys at a dose of 10 mg/kg (HNSTD), with a therapeutic index close to 100 times, and no thrombocytopenia occurred at this dose, lung, liver, kidney No toxicity was shown in other important organs, and the above results indicated that Her3-ADC-07 (DAR8) has high safety in long-term medication.

Abstract

本公开涉及一种抗Her3抗体，及其制备方法、其偶联物和应用，特别涉及结合Her3以表现出抗肿瘤活性的抗体及其偶联物。本公开还包括所述抗体和ADC的组合物以及其使用方法。

Description

一种针对Her3的抗体，偶联物及其用途 技术领域
本公开属于治疗性生物医药领域，更具体地，本公开涉及一种针对Her3的抗体，偶联物，还涉及所述抗或偶联物体在治疗疾病中的用途。
背景技术
Her3(ErbB3)为ERBB家族的跨膜受体酪氨酸激酶分子，ERBB家族包括4个成员：EGFR、Her2、Her3和Her4。ERBB受体的激活需要形成同源或异源二聚体，其家族成员间可互相形成二聚体。当配体缺乏时，Her3通过与富半胱氨酸区相互作用使其构象处于锁定状态；胞内蛋白激酶结构域内的非保守氨基酸变异使得Her3仅保留微弱的激酶活性，需与EGFR家族其他分子形成异源二聚体介导信号的转导。Her3在胚胎发育中具有重要作用，小鼠缺失Her3会导致严重的房室瓣发育不良，导致小鼠胚胎死亡，该效应主要由其NRG1配体决定。此外，Her3在神经嵴分化(如施旺细胞)及交感神经发育中起重要作用。Her3在多种癌症中发生突变或过度表达，例如几种类型的腺癌(乳腺癌、尿道癌、胃癌、大肠癌、胆道癌、膀胱癌、子宫内膜癌、卵巢癌、子宫癌、***癌、肺癌)及黑色素瘤、胶质瘤等。
抗体偶联药物(ADC)通过化学方法将具有生物活性的小分子化合物连接到单克隆抗体或抗体片段上，充分利用抗体与正常细胞和肿瘤细胞表面抗原结合的特异性，以小分子抗肿瘤生物活性的高效性，同时避免了前者的特异性疗效低及后者的毒副作用等缺陷。这意味着，与传统化疗或靶向药物相比，抗体偶联药物能更准确地结合肿瘤细胞，减少对正常细胞的影响。
目前已有针对Her3的抗体和ADC药物的专利报告，如Daiichi Sankyo开发的U3-1402，MediaPharma开发的MP-Her3-ADC等。
然而，尽管抗体偶联药物(ADC)兼具抗体对靶细胞的靶点特异性和 靶向性的优势和荷载药物的肿瘤杀伤能力，但由于ADC药物分子的结构复杂性，ADC药物在实际应用中依然存在安全窗不够的问题，已有Her3的抗体偶联药物还处于早期研发阶段，因此目前仍然急需更加安全有效的Her3抗体或抗体偶联药物来满足未满足的临床需求。
发明内容
在本公开中，首先开发了包含结合Her3分子的轻链可变区(VL)和/或重链可变区(VH)结构域，利用所述轻链可变区和/或重链可变区进一步开发了人源抗体。
本公开涉及的抗体可以是全人源抗体，从而可安全地施用给人受试者，而不引发免疫原性反应。具有能够很高的结合人和非人Her3的能力，尤其涉及与非人灵长类动物(例如猴)和哺乳动物(如大鼠)的Her3具有交叉反应性的这样的分子。本公开还涉及包含所述Her3抗体的抗体-药物偶联物(ADC)，本公开的抗体或抗体-药物偶联物(ADC)具有重大的临床价值。
本公开的抗体
在第一方面，本公开提供了一种结合Her3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含如下的互补决定区(CDRs)：
SEQ ID NO:23、25或27所示的重链可变区(VH)中含有的HCDR1或其序列的变体、HCDR2或其序列的变体、以及HCDR3或其序列的变体；和/或
SEQ ID NO:24、26或28所示的轻链可变区(VL)中含有的LCDR1或其序列的变体、LCDR2或其序列的变体、以及LCDR3或其序列的变体。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:23所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:24所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:25所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:26所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:27所示的VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体以及HCDR3或其序列的变体；和/或SEQ ID NO:28所示的VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体以及LCDR3或其序列的变体。
在某些优选实施方案中，所述序列的变体为与其来源CDR相比具有一个或几个氨基酸的置换、缺失或添加(例如1个、2个或3个氨基酸的置换、缺失或添加)的CDR。
在某些优选实施方案中，所述的置换为保守置换。
优选地，所述CDRs根据AbM、Chothia、Kabat或IMGT编号***定义方案所确定。
在一个方面，本公开提供了一种能够结合Her3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：重链可变区(VH)和/或轻链可变区(VL)。
在某些实施方案中，本公开的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDRs按Kabat编号***定义：
(a)包含如下3个CDRs的重链可变区(VH)：序列为SEQ ID NO:1或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:2或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:3或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或 3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDRs的轻链可变区(VL)：序列为SEQ ID NO:7或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:8或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:9或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；
(b)包含如下3个CDRs的重链可变区(VH)：序列为SEQ ID NO:12或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:13或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:14或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDRs的轻链可变区(VL)：序列为SEQ ID NO:18或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为SEQ ID NO:19或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:20或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3。
在某些实施方案中，本公开的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDR按Kabat编号***定义：
(a)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:1的HCDR1，序列为SEQ ID NO:2的HCDR2，序列为SEQ ID NO:3的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:7的LCDR1，序列为SEQ ID NO:8的LCDR2，序列为SEQ ID NO:9的LCDR3；或
(b)包含如下3个CDR的重链可变区(VH)：序列为SEQ ID NO:12的HCDR1，序列为SEQ ID NO:13的HCDR2，序列为SEQ ID NO:14的HCDR3；和/或，
包含如下3个CDRs的轻链可变区(VL)：序列为SEQ ID NO:18的LCDR1，序列为SEQ ID NO:19的LCDR2，序列为SEQ ID NO:20的LCDR3。
在某些实施方案中，本公开的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDRs按IMGT编号***定义：
(a)包含如下3个CDRs的重链可变区(VH)：序列为SEQ ID NO:4或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:5或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:6或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDRs的轻链可变区(VL)：序列为SEQ ID NO:10或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为AAS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:9或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3；或
(b)包含如下3个CDRs的重链可变区(VH)：序列为SEQ ID NO:15或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或 3个氨基酸的置换、缺失或添加)的序列的HCDR1，序列为SEQ ID NO:16或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR2，序列为SEQ ID NO:17或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的HCDR3；和/或，
包含如下3个CDR的轻链可变区(VL)：序列为SEQ ID NO:21或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR1，序列为AAS或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR2，序列为SEQ ID NO:20或与其相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加)的序列的LCDR3。
在某些实施方案中，本公开的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中CDRs按IMGT编号***定义：
(a)包含如下3个CDRs的重链可变区(VH)：序列为SEQ ID NO:4的HCDR1，序列为SEQ ID NO:5的HCDR2，序列为SEQ ID NO:6的HCDR3；和/或，
包含如下3个CDRs的轻链可变区(VL)：序列为SEQ ID NO:10的LCDR1，序列为AAS的LCDR2，序列为SEQ ID NO:9的LCDR3；
(b)包含如下3个CDRs的重链可变区(VH)：序列为SEQ ID NO:15的HCDR1，序列为SEQ ID NO:16的HCDR2，序列为SEQ ID NO:17的HCDR3；和/或，
包含如下3个CDRs的轻链可变区(VL)：序列为SEQ ID NO:21的LCDR1，序列为AAS的LCDR2，序列为SEQ ID NO:20的LCDR3。
在某些实施方案中，本公开的抗体或其抗原结合片段包含下述重链可变区(VH)和/或轻链可变区(VL)，其中，与前述Kabat编号***或IMGT编号***定义的CDRs相比，所述重链可变区(VH)和/或轻链可变 区(VL)中至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个或3个氨基酸的置换、缺失或添加或其任意组合)，且仍具有结合Her3活性。
优选地，本公开所述的置换为保守置换。
在某些实施方案中，所述抗体或其抗原结合片段结合人Her3、猴Her3和/或大鼠Her3。
在某些实施方案中，本公开的抗体或其抗原结合片段的VH包含来源于人免疫球蛋白的重链可变区(VH)的构架区(FR)，和/或所述抗体或其抗原结合片段的VL包含来源于人免疫球蛋白的轻链可变区(VL)的构架区(FR)。因此，在某些实施方案中，本公开的抗体或其抗原结合片段是全人源的。在某些实施方案中，本公开的抗体或其抗原结合片段是人源化的。
在某些实施方案中，本公开的抗体或其抗原结合片段包含：
(a)人免疫球蛋白的重链构架区或其变体，所述变体与其所源自的胚系抗体基因编码的氨基酸序列相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)；和/或
(b)人免疫球蛋白的轻链构架区或其变体，所述变体与其所源自的胚系抗体基因编码的氨基酸序列相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)。
在某些实施方案中，本公开的抗体或其抗原结合片段的人源化程度为至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％。
在某些实施方案中，本公开的抗体或其抗原结合片段包含：
(a)重链可变区(VH)，其包含选自下列的氨基酸序列：
(i)SEQ ID NO:23、25或27所示的序列；
(ii)与SEQ ID NO:23、25或27所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与SEQ ID NO:23、25或27所示的序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
和/或
(b)轻链可变区(VL)，其包含选自下列的氨基酸序列：
(iv)SEQ ID NO:24、26或28所示的序列；
(v)与SEQ ID NO:24、26或28所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与SEQ ID NO:24、26或28所示的序列相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，本公开的抗体或其抗原结合片段包含SEQ ID NO:23所示的VH，和/或,SEQ ID NO:24所示的VL。
在某些实施方案中，本公开的抗体或其抗原结合片段包含SEQ ID NO:25所示的VH，和/或,SEQ ID NO:26所示的VL。
在某些实施方案中，本公开的抗体或其抗原结合片段包含SEQ ID NO:27所示的VH，和/或,SEQ ID NO:28所示的VL。
在某些实施方案中，本公开的抗体或其抗原结合片段包含：
(a)SEQ ID NO:23所示序列的VH和SEQ ID NO:24所示序列的VL；
(b)SEQ ID NO:25所示序列的VH和SEQ ID NO:26所示序列的VL；
(c)SEQ ID NO:27所示序列的VH和SEQ ID NO:28所示序列的VL；
(d)重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)独立地与(a)至(c)任一组中所述的VH和VL相比分别具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或
(e)重链可变区(VH)和轻链可变区(VL)，其中所述重链可变区(VH)和轻链可变区(VL)与(a)至(c)任一组中所述的VH和VL分别相比，独立地具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)。优选地，所述的置换是保守置换。
在某些实施方案中，本公开还提供了一种与上文定义的抗体或其抗原结合片段，例如抗体202-2-1或其抗原结合片段竞争性结合的anti-Her3抗体或其抗原结合片段，其特征在于所述抗体或其抗原结合片段与其所竞争性结合的抗体或其抗原结合片段不同，例如不为抗体202-2-1或其抗原结合片段。
在某些实施方案中，所述anti-Her3抗体或其抗原结合片段与抗体202-2-1竞争性结合，其特征在于不为抗体202-2-1或其抗原结合片段，且与202-2-1或其抗原结合片段具有不同的CDRs。
在某些实施方案中，本公开的抗体结合人Her3的domain3，例如domain3中的一个或多个氨基酸。因此，在某些实施方案中，本公开提供了一种结合HER3的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段结合人Her3的domain 3，优选地，结合SEQ ID No.31所示人Her3第328至499位氨基酸。
在某些实施方案中，本公开提供了一种结合HER3的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段结合如下空间表位：SEQ ID  No.31所示的人Her3的第466位组氨酸，470位色氨酸，471位苏氨酸，478位苏氨酸，483位至487位(天冬氨酸-异亮氨酸-赖氨酸-组氨酸-天冬酰胺)，484位异亮氨酸，490位精氨酸和491位精氨酸。
所述与抗体202-2-1竞争性结合的anti-Her3抗体或其抗原结合片段结合如下空间表位：SEQ ID No.31所示的Her3的第466位组氨酸，470位色氨酸，471位苏氨酸，478位苏氨酸，483位至487位(天冬氨酸-异亮氨酸-赖氨酸-组氨酸-天冬酰胺)，484位异亮氨酸，490位精氨酸和491位精氨酸。
在某些实施方案中，本文公开的抗体或其抗原结合片段阻断Her3配体依赖性和/或Her3配体非依赖性信号转导。
在某些实施方案中，本文公开的抗体或其抗原结合片段抑制Her3配体依赖性和/或Her3配体非依赖性的AKT磷酸化。在某些实施方案中，本文公开的抗体或其抗原结合片段抑制Her3配体依赖和/或Her3配体非依赖性的Her3/Her2异源二聚体形成。
在某些实施方案中，本公开的抗体是嵌合抗体、人源化抗体或全人源抗体。在某些实施方案中，本公开的抗体或其抗原结合片段选自Fab、Fab’、(Fab’)2、Fv片段例如scFv或二硫键连接的Fv(dsFv)、双抗体(diabody)和多特异性抗体(例如双特异性抗体)。在某些实施方案中，本公开的抗体是scFv。
在某些实施方案中，本公开的抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比具有至多50个氨基酸的保守置换(例如至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)。在某些实施方案中，本公开的抗体或其抗原结合片段的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的野生型序列相比具有至多50个氨基酸的保守置换 (例如至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)。
在一些实施方案中，恒定区被改变，例如被突变，以修饰抗Her3抗体分子的性质(例如改变下列中的一个或多个特性：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能)。可以通过将抗体恒定区中的至少一个氨基酸残基替换为不同残基，产生功能改变，例如，改变抗体对效应子配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如降低)。抗体的Fc区介导几种重要的效应子功能，例如ADCC、吞噬作用(ADCP)、CDC等。
在某些实施方案中，本公开的抗体或其抗原结合片段具有重链恒定区(CH)，其选自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重链恒定区；特别地选自例如IgG1、IgG2、IgG3和IgG4的重链恒定区，更特别地选自IgG1(例如是人IgG1)的重链恒定区。在一些实施方案中，人IgG1重链恒定区如SEQ ID NO:29所示。在一些实施方案中，本公开的抗体或其抗原结合片段具有轻链恒定区，其选自例如κ或λ轻链恒定区，优选κ轻链恒定区(例如人κ轻链恒定区)。在一些实施方案中，轻链恒定区具有如SEQ ID NO:30所示的序列。
在一些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:29所示的CH或其变体，所述变体与SEQ ID NO:29相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO:29相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述抗体或其抗原结合片段包含轻链恒定区或其变体。在一些实施方案中，所述轻链恒定区包含κ轻链恒定区。在一些实 施方案中，所述轻链恒定区包含SEQ ID NO:30所示的轻链恒定区(CL)或其变体，所述变体与SEQ ID NO:30相比具有至多20个氨基酸的保守置换(例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的保守置换)，或者与SEQ ID NO:30相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性。
在一些实施方案中，所述抗体或其抗原结合片段包含SEQ ID NO:29所示的重链恒定区(CH)和SEQ ID NO:30所示的轻链恒定区(CL)。
在某些实施方案中，本公开的抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列：
(i)包含SEQ ID NO:23所示的VH序列和SEQ ID NO:29所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO:24所示的VL序列和SEQ ID NO:30所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、 至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本公开的抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列：
(i)包含SEQ ID NO:25所示的VH序列和SEQ ID NO:29所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO:26所示的VL序列和SEQ ID NO:30所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨 基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本公开的抗体或其抗原结合片段包含：
(a)重链，其包含选自下列的氨基酸序列：
(i)包含SEQ ID NO:27所示的VH序列和SEQ ID NO:29所示的CH序列的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；以及
(b)轻链，其包含选自下列的氨基酸序列：
(iv)包含SEQ ID NO:28所示的VL序列和SEQ ID NO:30所示的CL序列的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个， 6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列相比具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列。
在某些实施方案中，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，本公开的抗体或其抗原结合片段包含重链和轻链，
所述重链包含：
(i)SEQ ID NO:11所示的序列；
(ii)与(i)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(iii)与(i)所示的序列具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；和
所述轻链包含：
(iv)SEQ ID NO:22所示的序列；
(v)与(iv)所示的序列相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合(例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个，2个，3个，4个，5个，6个，7个，8个，9个或10个氨基酸的置换、缺失或添加或其任意组合)的序列；或
(vi)与(iv)所示的序列具有至少80％、至少85％、至少90％、至少91％、 至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的序列；
优选地，(ii)或(v)中所述的置换是保守置换。
在某些实施方案中，所述的抗体或其抗原结合片段，其中，所述抗体选自以下组：
(a)包括SEQ ID NO:23所示的VH和SEQ ID NO:29所示的CH的重链，和，包括SEQ ID NO:24所示的VL和SEQ ID NO:30所示的CL的轻链；优选包括SEQ ID NO:11所示的重链和SEQ ID NO:22所示的轻链；
(b)包括SEQ ID NO:25所示的VH和SEQ ID NO:29所示的CH的重链，和，包括SEQ ID NO:26所示的VL和SEQ ID NO:30所示的CL的轻链；
(c)包括SEQ ID NO:27所示的VH和SEQ ID NO:29所示的CH的重链，和，包括SEQ ID NO:28所示的VL和SEQ ID NO:30所示的CL的轻链。
在第二方面，本公开的抗体是多特异性抗体，其结合Her3以及一种或多种其他抗原。因此，本公开提供了一种多特异性抗体。
优选地，所述多特异性抗体是双特异性抗体或三特异性抗体或四特异性抗体。
在一些实施方案中，本公开的多特异性抗体包含本公开的结合Her3的抗原结合片段，和另外的抗体或其抗原结合其片段或抗体类似物。
本公开抗体衍生物
本公开的抗体或其抗原结合片段可进行衍生化，例如被连接至另一个分子(例如另一个多肽或蛋白)。通常，抗体或其抗原结合片段的衍生化(例如，标记)不会对其与Her3(特别是人Her3)的结合产生不利影响。因此，本公开的抗体或其抗原结合片段还意欲包括此类衍生化的形式。例如，可以将本公开的抗体或其抗原结合片段连接(通过化学偶合、基因融合、非共价连接或其它方式)于一个或多个其它分子基团，例如另一个抗 体(例如，形成双特异性抗体)，检测试剂，药用试剂，和/或能够介导抗体或其抗原结合片段与另一个分子结合的蛋白或多肽(例如，抗生物素蛋白或多组氨酸标签)。
一种类型的衍生化抗体(例如，双特异性抗体)是通过交叉连接2个或更多个抗体(属于同一类型或不同类型)而产生的。获得双特异性抗体的方法是本领域公知的，其实例包括但不限于，化学交联法、细胞工程法(杂交瘤法)或基因工程法。
另一种类型的衍生化抗体是标记的抗体。例如，可以将本公开的抗体或其抗原结合片段连接至可检测的标记。本公开所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、藻红蛋白(PE)、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa 750))、吖啶酯类化合物、磁珠(例如，)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。教导该标记物的使用的专利包括，但不限于，美国专利3,817,837；3,850,752；3,939,350；3,996,345；4,277,437；4,275,149；及4,366,241(全部通过引用并入本文)。如上所述的可检测的标记可通过本领域已知的方法检测。例如，放射性标记可使用摄影胶片或闪烁计算器检测，荧光标记物可使用光检测器检测，以检测发射的光。酶标记物一般通过给酶提供底物及检测通过酶对底物的作用产生的反应产物来检测，及测热标记物通过简单可视化着色标记物来检测。在某些实施方案中，此类标记能够适用于免疫学检测(例如，酶联免疫测定法、放射免疫测定法、荧光免疫测定法、化学发光免疫测定法等)。在某些实施方案中，可通过不同 长度的接头(linker)将如上所述的可检测的标记连接至本公开的抗体或其抗原结合片段，以降低潜在的位阻。
此外，本公开的抗体或其抗原结合片段还可以用化学基团进行衍生，例如聚乙二醇(PEG)，甲基或乙基，或者糖基。这些基团可用于改善抗体的生物学特性，例如增加血清半衰期。
抗体的制备
本公开的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本公开抗体的重链和轻链基因的DNA分子。将所得DNA分子***表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本公开的抗体。
本公开的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’)2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’)2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
因此，在第三方面，本公开提供了一种分离的核酸分子，其包含编码本公开的抗体或其抗原结合片段、或其重链可变区和/或轻链可变区、或其一个或多个CDR的核苷酸序列；或编码本公开的多特异性抗体的核苷酸序列。根据本领域已知的密码子简并性，在某些实施方案中，所述核苷酸序列是可以根据密码子简并性进行替换的。在某些实施方案中，所述核苷酸序列是密码子最优化的。
在某些实施方案中，本公开所述分离的核酸分子包含：(i)分别编码本 公开的抗体或其抗原结合片段的重链可变区和轻链可变区的第一核酸和第二核酸，或(ii)分别编码本公开的抗体或其抗原结合片段的重链可变区和重链恒定区的第一核酸，和轻链可变区和轻链恒定区的第二核酸，或(iii)分别编码本公开的抗体或其抗原结合片段的重链和轻链的第一核酸和第二核酸。在某些实施方案中，所述第一核酸和第二核酸包含与上述(i)-(iii)中任一第一核酸和第二核酸的简并序列或基本上相同序列的核酸。在某些实施方案中，所述简并序列或基本上相同序列指与(i)-(iii)中所述核酸分子相比具有至少大约85％、90％、95％、99％或更高序列同一性的序列或具有一个或更多个核苷酸取代的序列，或相差不超过3、6、15、30或45个核苷酸的序列。
第四方面，提供了一种载体(例如克隆载体或表达载体)，其包含本公开的分离的核酸分子。在某些实施方案中，本公开的载体是克隆载体或表达载体。在某些实施方案中，本公开的载体是例如质粒，粘粒，噬菌体，慢病毒等。在某些实施方案中，所述载体能够在受试者(例如哺乳动物，例如人)体内表达本公开的抗体或其抗原结合片段。
第五方面，提供了一种宿主细胞，其包含本公开的分离的核酸分子或本公开的载体。宿主细胞可以是真核细胞(例如哺乳动物细胞、昆虫细胞、酵母细胞)或原核细胞(例如大肠杆菌)。合适的真核细胞包括但不限于NS0细胞、Vero细胞、Hela细胞、COS细胞、CHO细胞、HEK293细胞、BHK细胞、和MDCKII细胞。适宜的昆虫细胞包括但不限于Sf9细胞。在某些实施方案中，本公开的宿主细胞是哺乳动物细胞，例如CHO(例如CHO-K1、CHO-S、CHO DXB11、CHO DG44)。
第六方面，提供了制备本公开的抗体或其抗原结合片段，或本公开的多特异性抗体的方法，其包括，在允许所述抗体或其抗原结合片段、或所述多特异性抗体表达的条件下，培养本公开的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
抗体-药物偶联物
本公开第七方面，提供一种抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其中抗体为上述结合Her3的抗体或其抗原结合片段，其通过接头与偶联部分连接。
所述偶联部分选自：可检测的标记、放射性同位素、荧光物质、发光物质、有色物质、酶、聚乙二醇(PEG)、核素、核酸、小分子毒素、具有结合活性多肽、蛋白、受体、配体，以及其它抑制肿瘤细胞生长、促进肿瘤细胞凋亡或坏死的活性物质。
在一些实施例中，抗体-药物偶联物包含下式的抗Her3抗体药物偶联物(Her3-ADC)：
Tb-(L-D)q,
其中：
Tb是如前所述的结合Her3的抗体或其抗原结合片段，并且；
D是小分子毒素药物部分；
L是键或连接分子，其共价连接Tb和D；
q是1-16之间的整数，且表示共价连接至Tb的L-D的数目。
在一些实施方案中，抗体-药物偶联物具有式I所示结构：
其中，
L1选自 各Z独立地选自直接键、碳碳三键、碳碳双键和酰胺基(优选选自直接键、碳碳三键、碳碳双键)；Rx、Ry独立地选自H和C1-4烷基；各m独立地选自0、1、2、3、4、5和6；y1选自1-6之间任意整数(如4、5、6)；各y2独立地选自0-15(如6-15)之间任意整数；各y3独立地选自1、2和3；各y4独立地选自0和1；1位通过S原子与Tb相连，2位与L2或L3相连；
L2不存在或存在，L2存在时，L2选自： y1选自1-6之间任意整数(如4、5、6)，各y2独立地选自0-10(如6-10)之间任意整数，各y3独立地选自1或2，各y4独立地选自0或1，1位与L1相连，2位与L3相连；
L3选自氨基酸残基或由2-10个氨基酸残基组成的短肽；所述的氨基酸残基选自天然氨基酸残基、非天然氨基酸残基、或选自AA1所示氨基酸残基或其立体异构体；
AA1所示的氨基酸残基中，Ra和Rb中任一个为H，另 一个为H、或者，Ra与Rb和与它们共同相连的碳原子一起形成5-6元杂环，所述的5-6元杂环为哌啶环或哌嗪环，
r、r1、r1a和r1b各自独立地为0、1、2、3、4或5；
Rm1、Rn1、Rm1a、Rn1a、Rm1b和Rn1b各自独立地为H、C1-6烷基或-COORx1，其中，Rx1为C1-6烷基；
或者，Rm1和Rn1、Rm1a和Rn1a、以及Rm1b和Rn1b和与它们共同相连的氮原子一起，形成5-6元杂环，所述的5-6元杂环中，杂原子选自1个或2个N原子；所述的5-6元杂环任选地被一个或多个R0’所取代；
Rz选自C1-6烷基；
R0、R0’各自独立地选自C1-6烷基、-NRm2Rn2或任选被C1-6烷基取代的5-6元杂环基；所述的5-6元杂环中，杂原子选自1个或2个N原子；
Rm2和Rn2各自独立地选自H和C1-6烷基；
L4不存在或存在，L4存在时，L4选自 1位与L3相连，2位与D相连；
R1、R2各自独立地选自H、卤素和C1-4烷基；或者，R1和R2和与其相连的碳原子一起形成5-6元杂环，所述杂环含有1个、2个或3个O，S或N或其任意组合；
R3选自H和C1-4烷基；或者，R3和X和与其相连的碳原子一起形成 5-6元碳环；
W不存在或存在，W存在时，W选自-O-、-S-、-NR4-、 1位与X相连，2位与L4或L3相连；
X选自任选取代的-(CH2)n1-、1位和母环相连，2位和W或L4相连；所述取代基选自1个或2个C1-4烷基；
R4、R5、R7各自独立地选自H和C1-4烷基；
n、n1、n2、n3各自独立地选自0到6之间的任意整数。
另外，还需要说明的是，对于“L1的1位通过S原子与Tb相连”，本领域技术人员可以理解的是，L1的1位是与打开二硫键(例如，通过还原剂TCEP还原二硫键可以打开二硫键，生成巯基-SH)后的Tb(如抗体)自身所含有巯基进行连接，也就是说，L1与Tb之间的-S-并非另外再外接的硫原子。例如，中，-S-并非另外外接的硫原子，而是打开双硫键后的Tb自身所含有巯基与L1例如的1位进行连接后形成的-S-。
在一些实施方案中，L1选自 各Z独立地选自直接键、碳碳三键、碳碳双键、C6芳环和酰胺基(优选选自直接键、碳碳三键、碳碳双键)；Rx、Ry独立地选自H和C1-4烷基；各m独立地选自0、1、2、3、4、5和6；y1选自1-6之间任意整数(如4、5、6)；各y2独立地选自0-15(如6-15)之间任意整数；各y3独立地选自1、2和3；各y4独立地选自0和1；1位通过S原子与Tb相连，2位与L2或L3相连。
在一些实施方案中，L1选自 1位通过S原子与Tb相连，2位与L2或L3相连。
在一些实施方案中，L1选自 1位通过S原子与Tb相连，2位与L2或L3相连。
在一些实施方案中，L2不存在或存在，L2存在时，L2选自1位与L1相连，2位与L3相连。
在一些实施方案中，L2不存在。
在一些实施方案中，L3选自AA1、AA1-Gly、Val-Cit、Val-Ala、Val-AA1、Val-AA1-Gly、AA1-Ala-Asn、Ala-Ala-Ala、Ala-Ala-Asn和Gly-Gly-Phe-Gly。
在一些实施方案中，L3选自AA1、AA1-Gly、Val-Cit、Val-AA1-Gly、 AA1-Ala-Asn和Gly-Gly-Phe-Gly。
在一些实施方案中，L3选自Val-AA1-Gly。
在一些实施方案中，r、r1、r1a和r1b各自独立地为0或4；优选地，r为0，r1、r1a和r1b为4；或者r为4，r1、r1a和r1b为0。
在一些实施方案中，r、r1、r1a和r1b不同时为0。
在一些实施方案中，r为0，r1为4。
在一些实施方案中，Rm1、Rn1、Rm1a、Rn1a、Rm1b和Rn1b各自独立地为H、甲基、乙基、正丙基、正丁基、-COOCH3、-COOCH2CH3、-COOCH2CH2CH3、-COOCH(CH3)2、-COOC(CH3)3或-COOCH2CH2CH2CH3。
在一些实施方案中，Rm1、Rn1、Rm1a、Rn1a、Rm1b和Rn1b各自独立地为H、甲基、乙基、正丙基、-COOCH3、-COOCH2CH3、-COOCH2CH2CH3、-COOCH(CH3)2、-COOC(CH3)3或-COOCH2CH2CH2CH3。在一些实施方案中，Rm1和Rn1、Rm1a和Rn1a、以及Rm1b和Rn1b和与它们共同相连的氮原子一起形成哌啶环或哌嗪环；进一步优选为1号氮原子为与Rm1和Rn1共同相连的氮原子。
在一些实施方案中，Rm1和Rn1、Rm1a和Rn1a、以及Rm1b和Rn1b和与它们共同相连的氮原子一起形成哌啶环；进一步优选为 1号氮原子为与Rm1和Rn1共同相连的氮原子。
在一些实施方案中，r、r1中，r为4，r1为0时，Rm1、Rn1各自独立地选自H、C1-6烷基(如H、甲基)；r为0，r1为4时，Rm1、Rn1各自独立地选自C1-6烷基(如甲基、乙基、正丙基)，优选选自C2-6烷基(如乙基、 正丙基)。
在一些实施方案中，r为0，r1为4时，Rm1、Rn1各自独立地选自C1-6烷基(如甲基、乙基、正丙基)。
在一些实施方案中，Rz为甲基。
在一些实施方案中，R0为C1-6烷基或被C1-6烷基取代的哌啶基或哌嗪基；优选为甲基、乙基或被甲基取代的哌啶基；例如甲基或
在一些实施方案中，R01’各自独立地为C1-6烷基或-NRm2Rn2；更优选为甲基或-NRm2Rn2，其中，Rm2和Rn2各自独立地优选为H或C1-6烷基，更优选为甲基。
在一些实施方案中，AA1所示氨基酸残基中，Ra1与Rb和与它们共同相连的碳原子一起形成哌啶环，例如1号碳原子为与Ra和Rb共同相连的碳原子；例如
在一些实施方案中，AA1所示氨基酸残基选自
在一些实施方案中，AA1所示氨基酸残基选自
在一些实施方案中，AA1所示氨基酸残基选自 在一些实施方案中，L3选自 1位与L1或L2相连，2位与L4或D相连。
在一些实施方案中，L3选自 1位与L1或L2相连，2位与L4或D相连。
在一些实施方案中，L3选自 1位与L1或L2相连，2位与L4或D相连。在一些实施方案中，L4不存在或存在，L4存在时，L4选自 1位与L3相连，2位与D相连。
在一些实施方案中，L4不存在。
在一些实施方案中，L4选自1位与L3相连，2位与D相连。
在一些实施方案中，L4选自1位与L3相连，2位与D相连。
在一些实施方案中，R1选自H和卤素，R2选自H和C1-4烷基。
在一些实施方案中，R1和R2和与其相连的碳原子形成 虚线表示所述杂环与苯环稠合的位置。
在一些实施方案中，R1为H或F，R2为H或甲基。
在一些实施方案中，R1为F，R2为甲基或R1和R2和与其相连的碳原子一起形成
在一些实施方案中，R1为F，R2为甲基。
在一些实施方案中，R1和R2和与其相连的碳原子一起形成
在一些实施方案中，R3为H或R3和X和与其相连的碳原子一起形成虚线表示所述碳环与苯环和吡啶环稠合的位置。
在一些实施方案中，R3为H。
在一些实施方案中，各R4独立地选自H和C1-4烷基，R5为H。
在一些实施方案中，各R4独立地选自H、甲基、乙基、正丙基、异丙基和叔丁基，R5为H。
在一些实施方案中，各R7独立地选自H和C1-4烷基。
在一些实施方案中，R7为H。
在一些实施方案中，n选自1、2和3。
在一些实施方案中，n为1。
在一些实施方案中，n1选自1、2、3和4。
在一些实施方案中，n2为1。
在一些实施方案中，n3为0。
在一些实施方案中，W选自-O-、-NR4-和1位X相连，2位与L4或L3相连。
在一些实施方案中，W选自-O-和-NR4-，1位X相连，2位与L4或L3相连。
在一些实施方案中，X选自任选取代的 1位和母环相连，2位和W或L4相连；所述取代基选自1个或2个C1-4烷基(如甲基)，或者2个C1-4烷基(如甲基)。
在一些实施方案中，X选自1位和母环相连，2位和W相连。
在一些实施方案中，W选自-O-、-NR4-和1位与X相连，2位与L4或L3相连；X选自1位和母环相连，2位和W相连。
W不存在或存在，由此，W不存在时，L4的1位与L3相连，2位与X相连；W存在时，L4的1位与L3相连，2位与W相连。以下关于L4的连接关系可参照前述内容进行理解。
在一些实施方案中，的结构选自以下结构片段：
其中，1位与Tb相连，2位与D相连；AA1如上文所定义。
在一些实施方案中，的结构选自以下结构片段：


其中，1位与Tb相连，2位与W相连。
在一些实施方案中，的结构选自以下结构片段：


其中，1位与Tb相连，2位与W相连。
在一些实施方案中，的结构选自以下结构片段：
其中，1位通过S原子与Tb相连，2位与W相连；
在一些实施方案中，所示的结构片段为 1位与L4连接。
在一些实施方案中，的结构选自以下结构片段：


在一些实施方案中，的结构选自以下结构片段：
在一些实施方案中，抗体-药物偶联物具有式I-1所示结构：
其中，Tb、L1、L2、L3、L4、X、R1、R2、R3、R4和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体-药物偶联物具有式I-1A或I-1B所示结构：
其中，Tb、L2、L3、L4、X、R1、R2、R3、R4和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体-药物偶联物具有式I-2所示结构：
其中，Tb、L1、L2、L3、L4、X、R1、R2、R3和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体-药物偶联物具有式I-2A或I-2B所示结构：
其中，Tb、L2、L3、L4、X、R1、R2、R3和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体药物偶联物具有式I-3所示结构：
其中，Tb、L1、L2、L3、L4、X、R1、R2、R3、R4、R5、n和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体药物偶联物具有式I-3A或I-3B所示结构：
其中，Tb、L2、L3、L4、X、R1、R2、R3、R4和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体-药物偶联物具有式I-A所示结构：
其中，Tb、X、R1、R2、R3、Ra、Rb和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些实施方案中，抗体-药物偶联物具有式I-B所示结构：
其中，Tb、X、R1、R2、R3、Ra、Rb和q具有上文以及本文具体叙述的任何实施方案中所提供的含义。
在一些优选的实施方案中，所述抗体-药物偶联物，选自：




其中Tb为如上任一方案所述的Her3抗体，q为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16，q优选1、2、3、4、5、6、7、8；
例如，

其中，Her3mAb为202-2-1抗体。
在一些优选的实施方案中，所述抗体-药物偶联物选自如下：
其中，Tb为如上任一方案所述的Her3抗体，q为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15或16，q优选1、2、3、4、5、6、7或8。
在一些优选的实施方案中，所述抗体-药物偶联物选自如下：
其中，Her3mAb为202-2-1抗体。
在一些优选的实施方案中，所述抗体-药物偶联物q为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15或16；q优选1、2、3、4、5、6、7或8。
在本公开的第八方面，提供了一种抗体-药物偶联物，其包含本公开前述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物中的两种或多种，所述抗体-药物偶联物中的抗体-药物偶联物具有一种、两种或多种q值。
在一些实施方案中，所述抗体-药物偶联物中的药物与抗体的比例(DAR)选自1-10中的整数或小数。
在一些实施方案中，所述抗体-药物偶联物中的药物与抗体的比例(DAR)选自2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.2、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.7、8.9和9。
在一些实施方案中，所述抗体-药物偶联物中的DAR选自：2±0.5，4±0.5，6±0.5，或8±0.5。
在一些实施方案中，所述抗体-药物偶联物中的DAR选自：2±0.5，4 ±0.5，5±0.5，6±0.5，7±0.5，或8±0.5。
在一些实施方案中，所述抗体-药物偶联物中的DAR为8±0.5，8±0.4，8±0.3，8±0.2，8±0.1或8.0。
一些实施方案中，抗体-药物偶联物含有具有2至8的DAR的分布的ADC，例如，1.5、2、4、6、和8(即，1.5、2、4、6、7和8的载药种类)。值得注意的是，可以产生降解产物，使得抗体-药物偶联物中也可以包含1、3、5和7的DAR。此外，混合物中的ADC也可具有大于8的DAR。抗体-药物偶联物由链间二硫化物还原然后偶联产生。在一些实施方案中，抗体-药物偶联物包含如下两者：DAR为4或更低(即，载药种类为4或更低)的ADC以及DAR为6或更高(即，载药种类为6或更高)的ADC。
用途、治疗方法和药物组合物
本公开公开第九方面，提供了药物组合物，其包含本公开第一方面的抗体或其抗原结合片段、第二方面的多特异性抗体、第三方面的核酸分子、第四方面的载体、第五方面的宿主细胞、第七方面的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物和/或第八方面所述的抗体-药物偶联物以及药学上可接受的载体和/或赋形剂。
本公开第一方面所述的抗体或其抗原结合片段、第二方面的多特异性抗体、第三方面的核酸分子、第四方面的载体、第五方面的宿主细胞、第七方面所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物和/或第八方面所述的抗体-药物偶联物可为治疗有效量的。
在某些实施方案中，本公开的药物组合物包含本公开的抗体或其抗原结合片段，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，本公开的药物组合物包含本公开的宿主细胞，以及药学上可接受的载体和/或赋形剂，其中所述宿主细胞包含如前所述的分离的核酸分子或载体。
在某些实施方案中，本公开的药物组合物包含本公开的多特异性抗体，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，本公开的药物组合物包含本公开的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物和/或所述的抗体-药物偶联物以及药学上可接受的载体和/或赋形剂。
本公开另一方面，提供本公开的抗体或其抗原结合片段、核酸、载体，宿主细胞，多特异性抗体或者抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物、所述的抗体-药物偶联物或所述药物组合物在制备药物中的用途，所述药物用于调整(抑制或阻断)Her3的活性。所述的抗体或其抗原结合片段、核酸分子、载体、宿主细胞、多特异性抗体或者抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物可为治疗有效量的。
在一些实施例中，提供本公开的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物、所述的抗体-药物偶联物或所述药物组合物在制备药物中的用途，所述药物用于治疗或预防与Her3的活性有关的疾病。
在一些实施例中，提供本公开的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物、所述的抗体-药物偶联物或所述药物组合物在制备药物中的用途，所述药物用于治疗或预防与Her3的活性有关的肿瘤。
在一些实施方案中，所述癌症疾病选自食管癌(例如食管腺癌和食管鳞状细胞癌)、脑瘤、肺癌(例如小细胞性肺癌和非小细胞性肺癌)、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子***、子宫内膜癌、结直肠癌、肝癌、肾癌、尿路上皮癌、实体瘤、非霍奇金淋巴瘤、中枢神经***肿瘤(例如神经胶质瘤、多形性胶质母细 胞瘤、胶质瘤或肉瘤)、***癌或甲状腺癌。
在一些实施方案中，所述癌症疾病为实体肿瘤。
在一些实施方案中，所述肿瘤选自：结直肠癌(如结肠癌)，肺癌(如非小细胞肺癌)，乳腺癌，***癌。
在一些实施方案中，所述癌症疾病为乳腺癌或肺癌(优选非小细胞肺癌)。
在另一个方面，提供了本公开的抗体或其抗原结合片段或多特异性抗体在制备试剂盒中的用途，所述试剂盒用于检测Her3在样品中的存在或其水平。在另一个方面，本公开提供了诊断性或治疗性试剂盒，其包括一个或多个以下物质：本公开所述的抗体或其抗原结合片段、核酸、载体、宿主细胞、多特异性抗体、抗体-药物偶联物或者药物组合物。可选地，所述诊断性或治疗性试剂盒还包括使用说明书。
术语定义
在本文中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
本文中使用的术语“HER3配体”指结合并活化HER3的多肽。HER3配体的实例包括但不限于神经调节蛋白1(NRG)和神经调节蛋白2，乙胞素，肝素结合表皮生长因子和表皮调节素。
本公开涉及识别HER3受体的构象表位的分离的抗体或其片段，其中所述构象表位包含HER3的结构域3中的氨基酸残基，且其中抗体或其片段抑制HER3的磷酸化，如通过HER3配体非依赖性磷酸化测定所评估。在一个实施方案中，HER3配体非依赖性磷酸化测定使用HER2扩增的细胞，其中所述HER2扩增细胞是SK-Br-3细胞。
本文中使用的术语“配体依赖性信号转导”指依赖于配体激活的信号 转导，例如Erbb(例如HER3)通过配体的活化。HER3的活化可以通过使下游信号转导途径(例如PI3K)增加的寡聚化(例如异源二聚化)和/或HER3磷酸化证明。当使用实施例中描述的测定测量时，相对于未处理(对照)细胞，在暴露于抗原结合蛋白(例如抗体)的受刺激的细胞中，抗体或其片段可统计上显著地减少磷酸化HER3的量。表达HER3的细胞可为天然存在的细胞系(例如MCF7)，或可为通过在宿主细胞中引入编码HER3蛋白的核酸重组产生的细胞。可通过外源加入活化HER3配体或通过内源表达活化配体进行细胞刺激。
本文中使用的术语“配体非依赖性信号转导”指不需要配体结合的信号转导，例如不依赖于配体结合的HER3活化(例如磷酸化)。例如，配体非依赖性HER3活化可为HER2过表达的结果或是HER3异源二聚体搭档例如EGFR和HER2的活化突变的结果。相对于未处理(对照)细胞，在暴露于抗原结合蛋白(例如抗体)的细胞中，抗体或其片段可统计上显著地减少磷酸化HER3的量。表达HER3的细胞可为天然存在的细胞系(例如SK-Br-3)，或可为通过在宿主细胞中引入编码HER3蛋白的核酸重组产生的细胞。
如本文所使用，术语“药学上可接受的盐”的例子是由形成药学上可以接受的阴离子的有机酸形成的有机酸加合盐，包括但不限于甲酸盐、乙酸盐、丙酸盐、苯甲酸盐、马来酸盐、富马酸盐、琥珀酸盐、酒石酸盐、柠檬酸盐、抗坏血酸盐、α-酮戊二酸盐、α-甘油磷酸盐、烷基磺酸盐或芳基磺酸盐；优选地，所述烷基磺酸盐为甲基磺酸盐或乙基磺酸盐；所述芳基磺酸盐为苯磺酸盐或对甲苯磺酸盐。也可形成合适的无机盐，包括但不限于盐酸盐、氢溴酸盐、氢碘酸盐、硝酸盐、碳酸氢盐和碳酸盐、硫酸盐或磷酸盐等。
如本文所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于： pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。
药学上可接受的盐可使用本领域熟知的标准程序获得，例如，通过将足量的碱性化合物和提供药学上可以接受的阴离子的合适的酸反应。
本公开中，所述药用辅料是指生产药品和调配处方时，使用的赋形剂和附加剂，是指除活性成分外，在安全性方面已进行了合理的评估，并且包含在药物制剂中的物质。药用辅料除了赋型、充当载体、提高稳定性外，还具有增溶、助溶、缓控释等重要功能，是可能会影响到药品的质量、安全性和有效性的重要成分。根据其来源可分为天然物、半合成物和全合成物。根据其作用与用途可分为：溶剂、抛射剂、增溶剂、助溶剂、乳化剂、着色剂、黏合剂、崩解剂、填充剂、润滑剂、湿润剂、渗透压调节剂、稳定剂、助流剂、矫味剂、防腐剂、助悬剂、包衣材料、芳香剂、抗黏着剂、抗氧剂、螯合剂、渗透促进剂、pH调节剂、缓冲剂、增塑剂、表面活性剂、发泡剂、消泡剂、增稠剂、包合剂、保湿剂、吸收剂、稀释剂、絮凝剂与反絮凝剂、助滤剂、释放阻滞剂等；根据其给药途径可分为口服、注射、黏膜、经皮或局部给药、经鼻或口腔吸入给药和眼部给药等。同一药用辅料可用于不同给药途径的药物制剂，且有不同的作用和用途。
所述药物组合物可根据给药途径制成各种适宜的剂型。例如片剂、胶囊剂、颗粒剂、口服溶液剂、口服混悬剂、口服乳剂、散剂、酊剂、糖浆剂、注射剂、栓剂、软膏剂、乳膏剂、糊剂、眼用制剂、丸剂、植入剂、气雾剂、粉雾剂、喷雾剂等。其中，所述的药物组合物或适宜的剂型可以含有0.01mg至1000mg的本公开的抗体，抗体-药物偶联物其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，适宜含有0.1mg至800mg，优选含有0.5-500mg，优选含有0.5至350mg，特别优选1-250mg。
所述药物组合物可以注射剂形式用药，包括注射液、注射用无菌粉末与注射用浓溶液。其中，可使用的载体和溶剂包括水、林格氏溶液和等渗 氯化钠溶液。另外，灭菌的非挥发油也可用作溶剂或悬浮介质，如单甘油酯或二甘油酯。
本文使用的术语“治疗”一般是指获得需要的药理和/或生理效应。该效应根据完全或部分地预防疾病或其症状，可以是预防性的；和/或根据部分或完全稳定或治愈疾病和/或由于疾病产生的副作用，可以是治疗性的。本文使用的“治疗”涵盖了对患者疾病的任何治疗，包括：(a)预防易感染疾病或症状但还没诊断出患病的患者所发生的疾病或症状；(b)抑制疾病的症状，即阻止其发展；或(c)缓解疾病的症状，即，导致疾病或症状退化。
在本公开中，术语“个体”包括人或非人动物。示例性人个体包括患有疾病(例如本文所述的疾病)的人个体(称为患者)或正常个体。本公开中术语“非人动物”包括所有脊椎动物，例如非哺乳动物(例如鸟类、两栖动物、爬行动物)和哺乳动物，例如非人灵长类、家畜和/或驯化动物(例如绵羊、犬、猫、奶牛、猪等)。
本公开中，术语“有效剂量”指被给药后会在一定程度上缓解所治疗病症的一种或多种症状的化合物的量。
本公开中，术语“配体药物偶联物”是指生物活性分子(药物分子)与靶向部分连接得到的物质。在本公开的部分实施方案中，生物活性分子与靶向部分通过连接体相连。所述连接体在特定环境(例如肿瘤内的水解酶和/或低pH值环境)中或特定作用(例如溶酶体蛋白酶的作用)下能够断裂，从而使生物活性分子与靶向部分分离。在本公开的部分实施方案中，所述连接体包含可切割或不可切割的单元，例如肽或二硫键。在本公开的部分实施方案中，生物活性分子与靶向部分直接通过共价键相连，所述共价键在特定环境或作用下能够断裂，从而使生物活性分子与靶向部分分离。在本公开的部分实施方案中，所述配体药物偶联物包含靶向部分、连接体以及本公开的式II化合物片段。
本公开中，术语“抗体-药物偶联物”是指生物活性分子(药物分子)与抗体或其抗原结合片段连接得到的物质。在本公开的部分实施方案中，生 物活性分子与抗体或其抗原结合片段通过连接体相连。所述连接体在特定环境(例如肿瘤内的水解酶和/或低pH值环境)中或特定作用(例如溶酶体蛋白酶的作用)下能够断裂，从而使生物活性分子与抗体或其抗原结合片段分离。在本公开的部分实施方案中，所述连接体包含可切割或不可切割的单元，例如肽或二硫键。在本公开的部分实施方案中，生物活性分子抗体或其抗原结合片段直接通过共价键相连，所述共价键在特定环境或作用下能够断裂，从而使生物活性分子与抗体或其抗原结合片段分离。在本公开的部分实施方案中，所述抗体体药物偶联物包含抗Her3抗体或其抗原结合片段、连接体以及本公开的式II化合物片段。
本公开中，术语“生物活性物”、“生物活性分子”或“药物分子”指抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质，在本公开的部分实施方案中，偶联物中的生物活性物、生物活性分子或药物分子为具有抗肿瘤生物活性的分子。例如：放射性同位素，例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32、Pb212和Lu的放射性同位素；金属配合物，例如金属铂配合物、金属金配合物，奥沙利铂等；糖肽类抗生素，例如博来霉素、平阳霉素；DNA拓扑异构酶抑制剂，例如拓扑异构酶I抑制剂，喜树碱、羟基喜树碱、9-氨基喜树碱、SN-38、伊立替康、拓扑替康、贝洛替康、卢比替康，拓扑异构酶II抑制剂，放线菌素D、阿霉素、多柔比星、多卡米星，柔红霉素、米托蒽醌、鬼臼毒素、依托泊苷等；干扰DNA合成药物，例如甲氨蝶呤、5-氟尿嘧啶、阿糖胞苷、吉西他滨、巯嘌呤、喷司他丁、氟达拉滨、克拉屈滨、奈拉滨等；作用于结构蛋白的药物，例如微管蛋白抑制剂，长春花生物碱类、长春新碱、长春碱、紫杉醇、多西他赛、卡巴他赛等；肿瘤信号通路抑制剂，例如丝氨酸/苏氨酸激酶抑制剂、酪氨酸激酶抑制剂、天冬氨酸激酶抑制剂或组氨酸激酶抑制剂等；还包括蛋白酶体抑制剂、组蛋白去乙酰化酶抑制剂、肿瘤新生血管生成抑制剂、细胞周期蛋白抑制剂、美登素衍生物、卡里奇霉素衍生物、奥瑞他汀衍生物、Pyrrolobenzodiazepines(PBD)衍生物、美法仑、丝裂霉素C、苯 丁酸氮芥、或其它抑制肿瘤细胞生长、促进肿瘤细胞凋亡和坏死的活性物质；酶及其片段，诸如核溶酶；抗生素；毒素，诸如小分子毒素或者细菌、真菌、植物或动物起源的酶活性毒素，包括其片段和/或变体；生长抑制剂；药物模块。术语“毒素”指能够对细胞的生长或增殖产生有害效果的物质。
本公开中，术语“小分子”是指具有生物活性的小分子药物。术语小分子毒素具有细胞损伤活性，即以某种形式造成细胞的病理学变化的状态，并且细胞损伤不限于直接损伤，包括对细胞的结构和功能的所有种类的损伤，诸如DNA裂解、碱基-二聚体形成、染色体裂解、对细胞***机制的损伤和不同酶促活性的下降。
本公开中，术语“连接体”是指将生物活性分子(药物分子)与靶向部分连接起来的片段。
本公开中，术语“靶向部分”是指偶联物中能够与细胞表面的靶标(或靶标的部分)特异性结合的部分。通过靶向部分与靶标的相互作用，偶联物可以被递送至特定的细胞群。
本公开中，当偶联物中的靶向部分为抗体时，偶联物可被称为“药物-抗体偶联物”。
本公开中，抗体或其抗原结合片段包括衍生化的抗体或其抗原结合片段，例如具有巯基的抗体或其抗原结合片段，其中所述衍生化使得抗体具有与药物连接体偶联物反应的基团或能力。所述巯基-SH可以打开二硫键(例如，通过还原剂TCEP还原)衍生获得。
如本公开所用的并在所附权利要求书中的单数形式“一个”和“所述”等包括复数涵义，除非上下文中清楚地另有指明。因此，单数词语“一个”的涵义包括了“一个或多个”。
本文中使用的术语“癌症”和“肿瘤”以相同的含义使用。
本文中使用的术语“基因”不仅包括DNA，而且包括其mRNA、其cDNA和其cRNA。
本文中使用的术语“多核苷酸”以与核酸相同的含义使用，并且还包括 DNA、RNA、探针、寡核苷酸和引物。
本文中使用的术语“Tb”为“target binding”的缩写，包括抗体或与靶标结合的任意分子，术语“Ab”为“antibody”的缩写，与“抗体或其抗原结合片段”无区别地使用。
本文中使用的术语“多肽”和“蛋白”无区别地使用。
本文中使用的术语“细胞”也包括动物个体内的细胞和培养的细胞。
本公开中所述的“结合Her3的抗体或其抗原结合片段”是指特异性结合Her3，例如人Her3的抗体或其抗原结合片段。
本文中使用的“Her3”也称为“ErbB3”，为ERBB家族的跨膜受体酪氨酸激酶分子。HER3基因定位在12号染色体的长臂上(12q13.2)，编码一个180kDa的蛋白。HER3的细胞外结构域被分为四个子域(domain 1-4)：domain1和domain3是富含亮氨酸的β-螺旋状区域，负责配体结合。而domain 2和domain 4是富含半胱氨酸的区域，此外domain 2还包含一个与其他受体相互作用所必需的二聚体臂。跨膜结构域之后是细胞内结构域。包括一个灵活的Juxta膜区、激酶结构和C端尾部。在没有配体的情况下，domain 2和domain 4之间的结合使HER3处于非活性状态。一旦与配体结合，异源二聚体的另外一半的激酶结构将HER3的C端尾巴上的酪氨酸残基转磷酸化。
本公开涉及的Her3可为本领域常规的Her3(例如人Her3)，例如可溶性Her3、膜形式Her3等，并且还表示Her3变体1和/或Her3变体2。在一些实施方案中，所述人Her3是登录号Uniport ID:P21860-1或NCBI：NP_001973.2所示的Her3。在一些实施方案中，所述人Her3包含SEQ ID NO:31所示的氨基酸序列。
如SEQ ID No.31所示的Her3(1-643)来自Uniport ID:P21860-1，其中SEQ ID No.31为：
KD指获得自Kd(具体结合分子-靶蛋白相互作用的解离速率)与Ka(具体结合分子-靶蛋白相互作用的结合速率)之比(或Kd/Ka，以摩尔浓度(M)表示)的解离常数。可使用本领域充分建立的方法测定KD值。测定结合分子的KD的优选方法是通过使用表面等离子共振，例如生物传感器***，如Biacore TM(GE Healthcare Life Sciences)***。
本文中使用的术语“抗原结合片段”表示抗体的具有抗原结合活性的部分片段，其中所述片段具有抗体的完全或部分功能，包括非限于，Fab，Fab’，F(ab’)2，Fv例如单链Fv(scFv)、二硫键连接的Fv(sdFv)或di-scFv等。该术语也包括Fab’，其为在还原条件下处理F(ab’)2得到的抗体的可变区的单价片段。但是，该术语不限于这些分子，只要所述片段具有与抗原的结合亲和力即可。此外，这些功能片段不仅包括用适当的酶处理抗体蛋白的全长分子得到的片段，而且包括使用遗传修饰的抗体基因在适当宿主细胞中生产的蛋白。
如本文所述的广义上“抗体分子”或“抗体”指免疫球蛋白分子和免疫球蛋白分子的免疫活性部分，即含有免疫特异性结合抗原的抗原结合位点的分子。因此，术语抗体不仅涵盖完整抗体分子，还包括所述抗体的片段以及 所述抗体和抗体片段的变体(包括衍生物)。当“抗体分子”或“抗体”与抗原结合片段在同一语境下使用时，“抗体分子”或“抗体”指完整抗体分子或全长抗体。
术语“单链Fv”或“scFv”是指一种多肽，其包含与抗体VH结构域连接的抗体的VL结构域。免疫特异性结合Her3的抗体可以与其它抗原发生交叉反应。优选地，免疫特异性结合Her3的抗体与其它抗原不发生交叉反应。免疫特异性结合Her3的抗体可以例如通过免疫测定或其它本领域技术人员已知的方法鉴别。“完整”抗体或“全长”抗体指包含两条重链(H)和两条轻链(L)的蛋白，所述重链和轻链通过二硫键相互连接，所述蛋白包含：(1)就重链而言，包含可变区(本文缩写为“VH”)和含有三个结构域CH1、CH2、CH3的重链恒定区；和(2)就轻链而言，包含轻链可变区(本文缩写为″VL″)和含有一个结构域CL的轻链恒定区。本公开的抗体包括但非限于单克隆，多特异性，人或嵌合抗体，单链抗体，Fab片段，F(ab′)片段，抗独特型(抗-Id)抗体(包括例如本公开抗体的抗-Id抗体)，和上述任何抗体的表位结合片段。本公开的免疫球蛋白分子可以是免疫球蛋白的任何类型(例如IgG，IgE，IgM，IgD，IgA和IgY)，类别(例如IgG1，IgG2，IgG3，IgG4，IgA1和IgA2)或亚类。优选地，本公开的抗体包含或由具有表1所述任一氨基酸序列或其片段或变体的VH结构域，VH CDR，VL结构域，或VL CDR组成。
本文中使用的术语“Fab’”表示如上所述在还原条件下处理F(ab’)2得到的抗体的可变区的单价片段。但是，本公开的Fab’也包括使用遗传修饰的抗体基因生产的Fab’。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)或直接相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH- COOH或NH2-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，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。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.Immunol.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。在一些情况下，scFv的VH与VL之间还可以存在二硫键。如本文中所使用的，术语“di-scFv”是指，由两个scFv连接形成的抗体片段。
术语“Her3变体”是指一种多肽，其与Her3多肽，Her3片段，抗Her3抗体或其抗体片段具有相似或相同的功能，但非必需包含相似或相同的Her3多肽，Her3片段，抗Her3抗体或其片段的氨基酸序列，或具有相似或相同的Her3多肽，Her3片段，抗Her3抗体或其片段的结构。
如本文所用，在所述抗体或其抗原结合片段中的即使含有变体，氨基酸的置换、缺失或添加，也仍然具有结合Her3的活性。
如本文所用，两个氨基酸序列之间的百分比同源性等于两个序列之间的百分比同一性(identity)。两个序列之间的序列百分比同一性是序列共享的相同位置的数目的函数(即，％同源性＝相同位置的数目/位置总数目X100)，其中考虑缺口(gap)的数目和每一缺口的长度，需要将其引入用于两个序列的最优对比。可用本领域通常所知的方法进行序列比较和确定序列间的百分比同一性，可用数学算法实现这种序列比较和百分比同一性的确定。例如，可用Meyers和Miller，1988Comput.Appl.Biosci.4：11-17的算法(已整合入ALIGN程序(版本2.0))来确定氨基酸序列之间和/或核苷酸序列之间的百分比同一性。此外，可用从Accelrys在线获得的GCG软件包中的GAP程序(使用其缺省参数)来确定氨基酸序列之间或核苷酸序列之间的百分比同一性。在一个实施方案中，所述两个序列是等长的。
术语“表位”是指在动物体优选哺乳动物体内具有抗原性或免疫原性活 性的Her3的一部分。具有免疫原性活性的表位是Her3的一部分，其在动物体内激发抗体应答。具有抗原性活性的表位是Her3的一部分，抗体与其免疫特异性结合，这可以通过本领域已知的方法测定。抗原性表位非必需是免疫原性的。
本公开的抗体或其抗原结合片段的表位可以通过现有技术测定，例如合成肽法，免疫信息学预测，多肽活性的测定，表位肽扫描法，噬菌体展示技术，X射线衍射和核磁共振分析，抗体同源建模蛋白对接预测法。本公开的抗体或其抗原结合片段的具有结合Her3 domain 3的表位。
本公开的抗体或其抗原结合片段包括结合相同表位的抗体。本文中使用的短语“结合相同表位的抗体”表示，结合共同表位的不同抗体。如果第二抗体结合第一抗体所结合的部分肽或部分三级结构，那么可以确定，所述第一抗体和所述第二抗体结合相同表位。
“表位202-2-1”或“202-2-1表位”是HER3的细胞外域中与抗体202-2-1结合的区域。这个表位接近HER3的跨膜域并且在HER3的domain3内。所述“表位202-2-1”由SEQ ID No.31所示的Her3的第466位组氨酸，470位色氨酸，471位苏氨酸，478位苏氨酸，483位至487位(天冬氨酸-异亮氨酸-赖氨酸-组氨酸-天冬酰胺)，484位异亮氨酸，490位精氨酸和491位精氨酸组成。
在本文中，本公开的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号***确定。在某些实施方案中，本公开的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia、AbM或IMGT编号***确定。
如本文中所使用的，术语“构架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
如本文中所使用的，术语“胚系抗体基因”是非淋巴细胞表达的免疫球蛋白的编码基因，它没有经历导致表达特异免疫球蛋白的遗传学重排及成熟的成熟过程。本公开的各种实施方案所提供的一个优点来源于一种认识， 那就是胚系抗体基因编码的氨基酸序列比成熟抗体基因编码的氨基酸序列更多地保留了动物物种个体的特征性的重要氨基酸序列结构。因此当被治疗性应用于该物种时，更少地被该物种识别为外源物质。
作为氨基酸取代，在本说明书中，保守的氨基酸取代是优选的。保守的氨基酸取代表示，在与氨基酸侧链有关的氨基酸集合内发生的取代。优选的氨基酸集合如下：酸性集合(天冬氨酸和谷氨酸)；碱性集合(赖氨酸、精氨酸和组氨酸)；非极性集合(丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸和色氨酸)；和不带电荷的极性家族(甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸和酪氨酸)。更优选的氨基酸集合如下：脂族羟基(丝氨酸和苏氨酸)；含有酰胺的集合(天冬酰胺和谷氨酰胺)；脂族集合(丙氨酸、缬氨酸、亮氨酸和异亮氨酸)；和芳族集合(苯丙氨酸、色氨酸和酪氨酸)。这样的氨基酸取代优选地在不会损害具有原始氨基酸序列的物质的性能的集合内进行。
此外，已知的是，缺失在培养的哺乳动物细胞中生产的抗体的重链的羧基端的赖氨酸残基(Journal of Chromatography A,705:129-134(1995))，还已知的是，缺失在培养的哺乳动物细胞中生产的抗体的重链的羧基端的2个氨基酸残基(甘氨酸和赖氨酸)，并且新位于羧基端处的脯氨酸残基被酰胺化(Analytical Biochemistry,360:75-83(2007))。但是，重链序列的这样的缺失和修饰不会影响抗体的抗原结合亲和力和效应子功能(补体的活化、抗体依赖性的细胞的细胞毒性等)。
本公开的IgG同型对照抗体是本领域的普通技术人员完全知晓并可以购买或制备获得的。例如人抗鸡卵融菌酶抗体(human anti-Hen Egg Lysozyme IgG，anti-HEL，如human IgG1，简称hIgG1)，其序列来自于Acierno等人发表的Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies研究中Fab F10.6.6序列的可变区序列(Acierno等人.J Mol Biol.2007；374(1):130-46.)。常见的hIgG1针对鸡卵溶菌酶的抗体anti-hel-hIgG1可以购自百英生物，货号：B117901。也可自行 制备。制备方法如下：委托南京金斯瑞生物对human IgG抗体的重轻链(全序列或可变区)基因进行氨基酸的密码子优化和基因合成，参照《分子克隆实验指南(第三版)》介绍的标准技术，采用PCR、酶切、DNA胶回收、连接转化、菌落PCR或酶切鉴定等标准的分子克隆技术将重轻链基因分别亚克隆到哺乳动物表达***的抗体重链表达载体和抗体轻链表达载体，并进一步对重组表达载体的重轻链基因进行测序分析。测序验证正确后，大量制备去内毒素级别的表达质粒并将重轻链表达质粒瞬时共转染HEK293细胞进行重组抗体的表达。培养7天后收集细胞培养液，进行rProtein A柱(GE)亲和纯化，收获的抗体样品用SDS-PAGE和SEC-HPLC标准分析技术对其进行质量鉴定。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本文中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本公开中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示；精氨酸可用R或Arg表示；甘氨酸可用G或Gly表示；谷氨酰胺可用Q或Gln表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状(例如，肿瘤和传染病)在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本公开的目的，有益或所需的临床结果包括但不限于，减轻症状、 缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与期望的存活期相比(如果未接受治疗)，延长存活期。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如非人灵长类哺乳动物或人。在某些实施方式中，所述受试者(例如人)患有肿瘤和传染病，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，肿瘤和传染病)有效量是指，足以预防、阻止或延迟疾病(例如，肿瘤和传染病)的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫***的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“效应子功能(effector function)”是指，那些可归因于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)的生物学活性，且其随抗体同种型而变化。
术语“药学上可接受的”指当分子本体、分子片段或组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其他不良反应。可作为药学上可接受的载体或其组分的一些物质的具体示例包括糖类(如乳糖)、淀粉、纤维素及其衍生物、植物油、明胶、多元醇(如丙二醇)、海藻酸等。
使用实验动物可以确定抗体和抗体-药物偶联物对癌症的体内治疗效果，例如，将抗体施用给植入了表达Her3的肿瘤细胞系的裸鼠，并测量癌细胞的任何变化。
癌症类型的例子包括肺癌、结直肠癌等。
本文中，术语“直接键”表示其两侧的基团直接相连，例如，式II所示 的化合物中，若X为直接键，则其结构式为其余直接键可参照前述内容进行理解。
本文中，术语“不存在”表示该基团不存在，例如，式II所示的化合物中，若W不存在，则其结构式为
式II所示的化合物中，R1和R2和与其相连的碳原子形成“虚线”键表示所述杂环与苯环稠合的位置，例如，形成
本文中，X的定义例如为，“X选自任选取代的所述取代基选自 1个或2个C1-4烷基(如甲基)”，则X例如可以为X其余类似的定义可参照前述内容进行理解。
本文中，X的定义例如为，“X选自任选取代的所述取代基选自2个C1-4烷基(如甲基)和与它们同时相连的碳原子一起形成C3-6环烷基(如环丙基)”，则X例如可以为X其余类似的定义可参照前述内容进行理解。
AA1所示氨基酸残基的结构中，若r为0，则本领域技术人员可以理解的是，AA1所示氨基酸残基的结构将变为
AA1所示氨基酸残基的结构中，若Ra与Rb和与它们共同相连的碳原子一起，形成4-10元杂环，所述4-10元杂环任选地被一个或多个R0所取代，其中，术语“所述4-10元杂环任选地被一个或多个R0所取代”的含义为，所述4-10元杂环可以不被取代，也可以被一个或多个R0所取代，且所述多个R0中，各R0的定义可以相同，也可以不同。其余类似的定义可以参照前述内容进行理解。
本文中，例如，L3选自Lys、Val-Cit、Ala-Ala-Asn、Ala-Ala-Asp、Gly-Gly-Phe-Gly、Val-Lys-Gly、Val-Ala、Lys-Ala-Asn时，“所述赖氨酸(Lys)的远端氨基任选地被1个、2个或3个选自叔丁氧羰基、C1-6烷基(优选甲基)、O的取代基所取代”的含义为，所述L3各选项中的Lys的远端氨基任选地被1个、2个或3个选自叔丁氧羰基、C1-6烷基(优选甲基)、 O的取代基所取代。其中，“Lys的远端氨基”指的是赖氨酸残基中裸露的氨基-NH2。“所述赖氨酸(Lys)的远端氨基任选地被1个、2个或3个选自叔丁氧羰基、C1-6烷基(优选甲基)、O的取代基所取代”表示所述赖氨酸(Lys)的远端氨基可以不被取代，也可以被1个、2个或3个选自叔丁氧羰基、C1-6烷基(优选甲基)、O的取代基所取代，例如，可以被1个叔丁氧羰基取代，即变为或者，可以被2个甲基取代，即变为或者被2个甲基和1个O同时取代，即变为需要说明的是，“所述赖氨酸(Lys)的远端氨基被O取代”指的是所述赖氨酸(Lys)的远端氨基被氧代，即变为若该远端氨基进一步被两个甲基所取代，则其变为
在本说明书的各部分，本公开化合物的取代基按照基团种类或范围公开。特别指出，本公开包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C1-6烷基”特别指独立公开的甲基、乙基、C3烷基、C4烷基、C5烷基和C6烷基。
在本文中，术语“C1-6烷基”表示直链或支链的含有1-6个碳原子的烷 基，包括例如“C1-3烷基”或“C1-4烷基”，甲基，乙基等，具体实例包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基、戊基、己基。
在本文中，术语“C1-4烷基”表示直链或支链的含有1-4个碳原子的烷基，包括例如“C1-3烷基”，甲基，乙基等，具体实例包括但不限于：甲基、乙基、正丙基、异丙基、正丁基、异丁基、仲丁基、叔丁基。
在本文中，术语“C2-6烯基”是指含有至少一个双键且碳原子数为2-6的直链、支链或环状的烯基，包括例如“C2-4烯基”等。其实例包括但不限于：乙烯基、1-丙烯基、2-丙烯基、1-丁烯基、2-丁烯基、1,3-丁二烯基、1-戊烯基、2-戊烯基、3-戊烯基、1,3-戊二烯基、1,4-戊二烯基、1-己烯基、2-己烯基、3-己烯基、1,4-己二烯基、环戊烯基、1,3-环戊二烯基、环己烯基、1,4-环己二烯基等。
在本文中，术语“C2-6炔基”是指含有至少一个三键且碳原子数为2-6的直链或支链的炔基，包括例如“C2-4炔基”等。其实例包括但不限于：乙炔基、丙炔基、2-丁炔基、2-戊炔基、3-戊炔基、4-甲基-2-戊炔基、2-己炔基、3-己炔基、5-甲基-2-己炔基等。
在本文中，术语“卤素”包括氟、氯、溴、碘。
在本文中，术语“3-6元环烷基”或“C3-6环烷基”是指含有3-6个碳原子的饱和环状烷基，包括环丙烷基(即环丙基)、环丁烷基(即环丁基)、环戊烷基(即环戊基)、环己基。
在本文中，术语“3-7元碳环烷基”或“C3-7环烷基”是指含有3-7个碳原子的饱和环状烷基，包括环丙烷基、环丁烷基、环戊烷基、环己基、环庚基。
在本文中，术语“C1-6烷氧基”是指通过氧原子连接至母体分子部分的如上文所定义的烷基。具体实例包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基、戊氧基、己氧基等。
在本文中，术语“C1-4烷氧基”是指通过氧原子连接至母体分子部分的如 上文所定义的烷基。具体实例包括但不限于甲氧基、乙氧基、丙氧基、异丙氧基、正丙氧基、异丙氧基、正丁氧基、异丁氧基、叔丁氧基等。
在本文中，术语“4-10元杂环基”是指含有4-10个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的环状基团。术语“4-6元杂环基”是指含有4-6个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的环状基团。任选地，环状结构中的环原子(例如碳原子、氮原子或硫原子)可以被氧代。“4-8元杂环基”包括例如“4-8元含氮杂环基”、“4-8元含氧杂环基”、“4-7元杂环基”、“4-7元含氧杂环基”、“4-7元杂环基”、“4-6元杂环基”、“5-7元杂环基”、“5-6元杂环基”、“5-6元含氮杂环基”，包括但不限于氧代环丁烷基、吡咯烷基、四氢呋喃基、哌啶基、哌嗪基、四氢吡喃基、高哌嗪基等。
在本文中，术语“4-10元杂环”是指含有4-10个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的环。术语“5-6元杂环”是指含有5-6个环原子(其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子)的环，包括但不限于吡咯烷、四氢呋喃、哌啶、哌嗪、四氢吡喃等环。
在本文中，术语“芳基”是指具有芳香性的单环或多环烃基，例如6-10元芳基、5-8元芳基等。具体的实例包括但不限于苯基、萘基、蒽基、菲基等。所述“6-10元芳基”是指含有6-10个环原子的芳基。所述“C6-10芳基”是指含有6-10个碳原子的芳基。
在本文中，术语“杂芳基”是指具有芳香性的环状基团，其中至少一个环原子为杂原子，例如氮原子、氧原子或硫原子。任选地，环状结构中的环原子(例如碳原子、氮原子或硫原子)可以被氧代。具体实例包括但不限于5-10元杂芳基、5-6元杂芳基、5-10元含氮杂芳基、6-10元含氧杂芳基、6-8元含氮杂芳基、5-8元含氧杂芳基等，例如呋喃基、噻吩基、吡咯基、噻唑基、异噻唑基、噻二唑基、噁唑基、异噁唑基、噁二唑基、咪唑基、吡唑基、1,2,3-***基、1,2,4-***基、1,2,3-噁二唑基、1,2,4-噁二唑基、 1,2,5-噁二唑基、1,3,4-噁二唑基、吡啶基、2-吡啶酮基、4-吡啶酮基、嘧啶基、1,4-二氧杂环己二烯基、2H-1,2-噁嗪基、4H-1,2-噁嗪基、6H-1,2-噁嗪基、4H-1,3-噁嗪基、6H-1,3-噁嗪基、4H-1,4-噁嗪基、哒嗪基、吡嗪基、1,2,3-三嗪基、1,3,5-三嗪基、1,2,4,5-四嗪基、氮杂环庚三烯基、1,3-二氮杂环庚三烯基、氮杂环辛四烯基等。
本文用波浪线“～～”表示的结构式中的键意在表示，该结构表示顺式或反式异构体，或任意比例的顺式和反式异构体的混合物。
本文中“之间”包括区间的端值，如1-16之间，可以指1或16。
术语“药物与抗体比”或“DAR”是指药物的数量，例如，与ADC的抗体附接的小分子毒素与抗体的比例平均值。ADC的DAR可以在1到16的范围内，但是取决于抗体上的连接位点的数量，更高的负载(例如20)也是可能的。提及负载到单个抗体上的药物的数量时，或可替代地，提及一组ADC的平均或均值DAR时，可以使用术语DAR。DAR也可被计算为产品中分子群体的平均DAR，即通过检测方法(例如通过常规方法如质谱法、ELISA测定、电泳和/或HPLC)测得的产品中偶联于本文所述的Ab部分的药物部分(D)与Ab部分的总体比例(摩尔比例)。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本公开各较佳实例。本公开所用试剂和原料均市售可得。
本公开的积极进步效果在于：
与现有技术相比，发明涉及的抗体是全人源抗体，从而可安全地施用给人受试者，而不引发免疫原性反应。发明涉及的抗体具有极高的亲和力和特异性，可以有利于发挥抗肿瘤作用。难以预料的，本公开的抗体-药物偶联物具有显著的体内抗肿瘤作用。
缩略词：



附图说明
图1A.抗Her3抗体与人Her3-his蛋白ELISA结合检测。
图1B.抗Her3抗体与猴Her3-his蛋白ELISA结合检测。
图1C.抗Her3抗体与大鼠Her3-his蛋白ELISA结合检测。
图2A.抗Her3抗体与A549-人Her3细胞流式结合检测。
图2B.抗Her3抗体202-2-1与T47D细胞流式结合检测。
图2C.抗Her3抗体202-2-1与MCF-7细胞流式结合检测。
图3.抗Her3抗体抑制NRG1对肿瘤细胞的AKT的磷酸化。
图4.抗Her3抗体在肿瘤细胞A549-人Her3细胞中内吞检测。
图5A.抗Her3抗体竞争生物素化对照抗体2与人Her3蛋白结合检测。
图5B-1.202-2-1抗体Fab与Her3的复合物，显示发生了结合，形成了复合物。
图5B-2.202-2-1抗体Fab与Her3的复合物界面视图1，虚线及数字显示互相作用的氨基酸侧链及其距离(埃)。
图5B-3.202抗体Fab与Her3的复合物界面视图2，虚线及数字显示互相作用的氨基酸侧链及其距离(埃)。
图6A.抗Her3抗体202-2-1ADC药物对PC-9肿瘤细胞杀伤检测。
图6B.抗Her3抗体202-2-1ADC药物对NCI-H358肿瘤细胞杀伤检测。
图7.抗Her3抗体202-2-1ADC药物对NCI-H358肿瘤模型药效测试。
图8.抗Her3抗体202-2-1ADC药物对SW480肿瘤模型药效测试之一。
图9.抗Her3抗体202-2-1ADC药物对SW480肿瘤模型药效测试之二。
具体实施方式
以下通过具体实施方式的描述对本公开作进一步说明，但这并非是对本公开的限制。本领域技术人员根据本公开的教导，可以做出各种修改或改进，而不脱离本公开的基本思想和范围。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1.Her3蛋白以及对照抗体的制备
选取人Her3的1-643位氨基酸(NCBI：NP_001973.2)的核酸序列，其中C端添加检测6xHis标签，命名为人Her3-his；选取鼠Her3(Uniport ID:Q61526-1)的20-641位氨基酸序列，其中C端添加检测6xHis标签，命名为鼠Her3-his。对照抗体1来自于MediaPharma公司专利CN 103189392B(重链SEQ ID NO:10，轻链SEQ ID NO:14)；对照抗体2来自于第一三共公司，见IMGT数据库，IMGT/mAb-DB ID：964，INN编号：11093。分别合成上述人Her3抗原的基因以及密码子优化合成两种对照抗体(南京金斯瑞生物技术有限公司)，构建到PTT5表达载体上，并大量抽提质粒。将抽提制备好的表达载体分别通过PEIMAX在HEK293F中瞬转表达7天，表达上清经Ni柱或ProA纯化制备。选取猴Her3(NCBI：XP_001113953.2)的1-643位氨基酸序列，其中C端添加检测6xHis标签，命名为猴Her3-his。
实施例2.用噬菌体库筛选抗Her3抗体
采集健康人静脉血，通过Ficol分离PBMC细胞，抽提RNA进行反转录，通过设计特定引物扩增重链可变区以及轻链可变区，通过同源重组将ScFv构建到噬菌体载体上，与辅助载体一起电转化至TG1中，获得全人源噬菌体库，库容量1.5×1010。经过人Her3-his初次富集，然后猴Her3-His (购自北京义翘神州生物技术有限公司)二次富集，挑选96个噬菌体，通过ELISA以及T47D肿瘤细胞流式细胞获得最优克隆202-2-1号克隆和202-3-1号克隆。其中为改善202-3-1的理化性质，对其FR进行定点突变，命名为202-3-2号克隆。
实施例3抗Her3抗体表达
将202-2-1号克隆、202-3-1号克隆和202-3-2号克隆的重链可变区分别与重链IgG1恒定区(SEQ NO:29)连接，将202-2-1号克隆、202-3-1号克隆和202-3-2号克隆的轻链可变区分别与Kappa恒定区序列(SEQ NO:30)连接，通过密码子优化及基因合成(安徽通用生物技术有限公司)，然后分别构建到PTT5载体上。通过PEI max试剂将抗Her3抗体重链和轻链表达质粒分别共转染HEK293F细胞进行表达，37℃5％CO2摇床表达7天，收集上清液，通过ProteinA磁珠进行纯化，获得抗Her3抗体分别命名为202-2-1抗体、202-3-1抗体以及202-3-2抗体。202-2-1抗体、202-3-1抗体和202-3-2抗体的重轻链可变区以及对应CDRs见表1。
表1抗人Her3抗体的氨基酸序列




实施例4抗Her3抗体蛋白亲和力检测
通过ELISA实验方法检测抗Her3抗体与Her3蛋白的亲和力。具体实验操作：将人Her3-his、猴Her3-his蛋白以及大鼠Her3-his用pH值为9.6的碳酸盐缓冲液稀释至终浓度为1μg/mL按100μL/孔分别加入96孔酶标孔中，4℃包被过夜；弃包被液，PBST洗涤1次，每孔加入100μL PBS(含2％BSA)，37℃封闭1小时；然后每孔加入100μL PBS(含2％BSA)稀释好的抗Her3抗体(1μg起始，3倍稀释，2复孔)，37℃，孵育2h；PBST洗涤3次，加入辣根过氧化物酶标记的抗人Fc抗体(用PBS按1:10000稀释)，100μL/孔，37℃作用1小时；PBST洗板5次。加入TMB显色液显色，100μL/孔，37℃，7min，加入终止液终止反应，50μL/孔，于450nm下测吸光度。将原始数据导入Graph-prism计算EC50值，结果见图1A-1C，详细结果如表2所示，202-2-1与人Her3蛋白以及猴Her3蛋白有强的结合且与对照抗体1相当，均不与大鼠Her3蛋白结合；202-3-1、202-3-2与人Her3蛋白以及猴Her3蛋白有强的结合弱于对照抗体1，但均与大鼠Her3蛋白结合。
表2抗Her3抗体蛋白亲和力检测结果
实施例5抗Her3抗体动态亲和力检测
Fortibio是常用的动态亲和力检测设备，通过其检测抗Her3抗体与人Her3蛋白的动态亲和力。简述方法如下：对人Her3-His采用PBST进行系列稀释，得到400nM、200nM、100nM、50nM、25nM、12.5nM、6.25nM、3.125nM和0nM。ProteinA生物传感器(Pall生命科学)在使用前用PBST缓冲液预湿。将抗Her3抗体用PBST稀释到5μg/mL并固化在ProA传感器上。然后将固化了抗体的传感器在PBST缓冲液中平衡60s以获得基线，然后转移到抗原稀释液结合60s，再在PBST中解离180s。在一个分析循环后，传感器用10mM Gly(pH 1.5)再生。使用Date analysis用1：1模型分析，确定结合(Ka)和解离(Kd)速率常数并使用其来计算解离平衡常数(KD)。如表3所示，202-2-1、202-3-1以及202-3-2与人Her3有较高的动态亲和力，与对照抗体1相当。
表3：抗Her3抗体分子与人Her3蛋白的动态亲和力分析
抗体 KD(M) ka(1/Ms) kdis(1/s)
202-2-1 1.31E-08 1.67E+05 2.18E-03
202-3-1 7.79E-09 1.82E+05 1.42E-03
202-3-2 6.81E-09 1.82E+05 1.24E-03
对照抗体1 5.30E-09 5.62E+05 2.97E-03
实施例6抗Her3抗体细胞亲和力检测
Her3蛋白在多种实体瘤细胞上高表达，通过FACS实验方法检测Her3抗体与肿瘤细胞亲和力。具体实验操作：将生长至对数期的MCF-7(乳腺癌细胞，来自ATCC，货号HTB-22)、A549-人Her3(人非小细胞肺癌，来自ATCC，货号CRM-CCL-185)以及T47D(人乳腺导管癌，ATCC)通过胰酶消化，用含2％BSA的PBS缓冲液重悬到5*10^5细胞每毫升，加入100uL细胞悬液至96孔板中；用含有2％BSA的PBS缓冲液稀释对照抗体1或对照抗体2、202-2-1、202-3-1以及202-3-2，起始浓度150μg/mL或50μg/mL,3倍稀释，10个浓度点左右，然后加入100uL抗体悬液至96孔板中混匀；4℃孵育1小时，采用预冷的PBS清洗2次，每次300uL/孔，500g离心5分钟；加入1：50荧光二抗APC anti-Human IgG(购买自Biolegend)100uL/每孔，混匀；4℃孵育0.5小时，采用预冷的PBS清洗2次，每次300uL/孔，500g离心5分钟，用500ul PBS重悬，采用贝克曼流式细胞仪进行检测平均荧光信号值。结果如图2A-2C以及表4所示，202-2-1细胞亲和力与对照抗体1相当，优于对照抗体2，但最大MFI高于对照抗体1。202-3-1和202-3-2细胞亲和力比对照抗体1稍弱。
表4抗Her3抗体细胞亲和力检测结果
表4中的N/A表示亲和力太弱，无法获得拟合的EC50结果。
实施例7抗Her3抗体抑制NRG1诱导的AKT磷酸化检测
NRG1通过Her3/Her2异源二聚体高亲和力结合后，诱导下游信号通路AKT的磷酸化，刺激肿瘤细胞的增殖。通过细胞印记法检测抗Her3抗体抑制NRG1诱导的AKT磷酸化通路。具体实验步骤：提前一天在96孔板铺50000个/孔MCF7肿瘤细胞(人乳腺癌细胞)过夜，无血清RPMI1640培养基饥饿约24小时，将202-2-1与202-3-1抗体100nM起始3倍稀释后加入至细胞37℃共孵育30分钟，再加NRG1(义翘神州，货号：11609-HNCH)10ng/ml(终浓度)37℃孵育30分钟，结束弃上清。PBS洗一次，加入100uL/孔甲醛室温固定30分钟，PBS洗2次，加入预冷100uL/孔甲醇透膜30分钟，PBS洗3次，加入PBS(含2％BSA)封闭2小时，加入100uL抗1：500Phospho-Akt(Ser473)抗体(CST，货号9271-S)4℃孵育过夜，然后PBST洗3次，加1:1000HRP羊驼抗兔IgG抗体(四川阿帕克)37℃孵育1小时，PBST清洗5次，加100uL ECL显影液(诺维赞，货号E412-01)，通过酶标仪(MD，型号i3x)读发光值。通过Graph-prism计算IC50值，结果如图3所示，抗Her3抗体202-2-1与202-3-1均有显著抑制NRG1诱导的AKT磷酸化，但202-2-1显著强于202-3-1，IC50分别是0.2434nM和1.037nM。
实施例8抗Her3抗体特异性检测
通过ELISA实验方法检测Her3抗体与Her3蛋白结合特异性，而与其同家族蛋白EGFR、Her2以及Her4不结合。具体实验操作：将人EGFR-His、人Her2-His、人Her4-his蛋白(购买自义翘神州生物技术有限公司)以及人Her3-his用pH值为9.6的碳酸盐缓冲液稀释至终浓度为1μg/mL按100μL/孔分别加入96孔的酶标板中，4℃包被过夜；弃包被液，PBST洗涤1次，每孔加入100μL PBS(含2％BSA)，37℃封闭1h；然后每孔加入 100μL PBS(含2％BSA)稀释好的Her3抗体1μg/mL、200ng/mL、40ng/mL,37℃，孵育2h；PBST洗涤3次，加入辣根过氧化物酶标记的抗人Fc抗体(用PBS按1:10000稀释)，100μL/孔，37℃作用1h；PBST洗板5次。加入TMB显色液显色，100μL/孔，37℃，7min，加入终止液终止反应，50μL/孔，于450nm下酶标仪读数。结果见表5所示，202-2-1、202-3-1以及202-3-2与人Her3特异性结合，而不结合人EGFR、人Her2以及人Her4蛋白。
表5抗Her3抗体与Her3家族蛋白结合检测
实施例9抗Her3抗体内吞检测
采用FACS方式检测A549-人Her3(人非小细胞肺癌细胞)对抗Her3抗体被细胞内吞的强弱。将生长至对数生长期的A549-人Her3细胞采用胰酶进行消化，离心收集细胞，预冷PBS洗三次；用PBS(含1％BSA)重悬细胞，分别加入待检测抗Her3抗体，浓度为10μg/mL，4℃孵育1小时；离心收集细胞，PBS洗三次，用DMEM+10％FBS重悬细胞，将重悬后细胞分成4份，分别在37℃孵育0、1、2、4小时；孵育相完成后，离心收集细胞，预冷PBS洗三次，用50uL 1％BSA(in PBS)重悬细胞，每孔加入1uL荧光二抗APC anti-Human IgG(Biolegend)，混匀，4℃孵育0.5小时；离心收集细胞，预冷PBS洗三次，用200uL PBS重悬细胞，流式细胞仪(贝克曼，型号CytoFlex)上机检测。按照公式：内吞率(％)＝【1-(该时间点检测样品平均荧光值-该时间点阴性对照样品平均荧光值)/(0小时检测样品平均荧光值-0小时阴性对照样品平均荧光值)】*100。结果如图4所示，抗Her3抗体202-2-1在A549-人Her3肿瘤细胞上具有较强的内吞活性，4小时能达到26.9％左右，强于对照抗体2的16.6％,与对照抗体1相当。但抗Her3抗体202-2-1内吞先于对照抗体达到最大内吞率，具有快于对照抗体的内吞速率。抗体202-3-2的内吞活性强于对照抗体1和对照抗体2。
实施例10.抗原结合表位检测
10.1抗Her3抗体表位竞争检测
采用竞争ELISA方式检测抗Her3抗体202-2-1与对照抗体1、对照抗体2是否表位竞争。具体步骤如下：采用CBS包被液包被人Her3-his抗原，100ng/孔，4度过夜孵育；去除包被液，用300μL PBS洗孔一次，用PBS(含2％BSA)溶液封闭每个孔，100μL每孔，37度孵育2小时；弃去除封闭液，将202-2-1、对照抗体1以及对照抗体2用PBS(含2％BSA)进行稀释，15μg/mL起始，3倍稀释，总共11个稀释点，第12个点用稀释液代替，2复孔，50μL每孔加入孔中；将生物素标记的对照抗体1稀释至10ng/mL或对照抗体2稀释至88ng/mL，50μL每孔加入孔中，25度孵育2 小时；用300μL PBST洗孔，重复3次，加入用PBS(含2％BSA)进行1：5000倍数稀释HRP抗生物素二抗100μL/孔，25度孵育1小时；用300μL PBST洗孔，重复5次；每孔加入100μL TMB显色液(安徽湖州英创)，室温显色5分钟后每孔加入50μL H2SO4终止反应，立即用酶标仪读取OD450nm。将原始数据导入Graph-prism计算EC50值，结果见图5A，抗Her3抗体202-2-1与对照抗体2不存在表位竞争。
10.2抗Her3抗体的抗原表位测定
为了进一步研究Her3抗体具体的作用方式及识别表位，将202-2-1的Fab与Her3蛋白分别制备复合物并通过冷冻电镜来解析抗体抗原复合物的结构。
(1)抗原制备：自uniprot数据库获取HER3胞外域(20-643)氨基酸序列，经DNA序列优化(293F为宿主)后合成DNA，蛋白C末端加入6*his。基因构建至pLVX(淼灵生物，货号P0249)表达载体后转染293F细胞(ATCC；PTA-5077)，筛选得到稳定细胞pool。细胞经发酵培养得到含抗原的培养上清，使用镍柱亲和捕获抗原并使用分子筛进行进一步纯化抗原，最终抗原溶解于PH7.5的Tris-NaCl溶液中(0.7mg/ml)。抗原质量表征：1.SDS-PAGE实验显示，在非还原及还原条件下均为均一条带。2.SEC-HPLC纯度为95.6％。3.反相色谱-HPLC纯度为97.44％。4.ELISA检测抗原抗体亲和力，结果显示该批次抗原和抗体202-2-1亲和力小于0.1nM，可以进行下一步实验。
(2)Fab-抗原复合物制备：抗体202-2-1溶解于20mm醋酸+150mm氯化钠，20.7mg/ml。使用胃蛋白酶与抗体孵育，37摄氏度孵育240分钟，SDS-PAGE检测消化产物并上SEC(Superdex 200 Increase 10/300 GL column)柱，收取主峰。使用木瓜蛋白酶与上述产物孵育，37摄氏度孵育120分钟，SDS-PAGE检测消化产物并上SEC(Superdex 200 Increase 10/300 GL column)柱，收取主峰，该产物即为抗体202-2-1的Fab。取步骤(1)中制备的抗原与Fab混合，于4摄氏度孵育过夜。孵育产物经离心 浓缩，SDS-PAGE检测孵育产物，上SEC(Superdex 200 Increase 10/300 GL column)柱，收取主峰。该组分即为Fab-抗原复合物。冷冻电镜制样前，使用负染的方法观察复合物质量(VitrobotTM MarkIV，easiGlowTM Glow Discharge，200kV TF20，CCD camera 895)。负染结果显示多数复合物形态均一，选取80个图像用于负染的重构，PDB模型(4LEO)用于与密度图的模拟。模拟结果显示复合物可与PBD模型的大部分重叠(包含Fab结合结构域)，认为复合物质量符合冷冻电镜观测的要求。
(3)冷冻电镜样品制备/在冷冻透射电镜Glacios上的筛选/数据搜集：使用25mMTris-HCl(pH 7.5),150mM NaCl稀释(2)中制备的复合物至0.2mg/ml，12000g离心15分钟；准备各相关设备耗材，3ul样品滴加至300mesh R 1.2/1.3holey carbon Film(Quantifoil)grid，使用200KV Glacios电镜观察筛选复合物图片。使用300KV Titan Krios电镜(装备K3直接电子探测器，Gatan品牌)进一步搜集高分辨率冷冻电镜照片，共搜集3272个照片供继续分析。参数设置：放大倍数105K，物理像素尺寸0.819埃，共56.3e-/埃2剂量的电子分为40帧，欠焦范围设置为-1.0uM至-2.4uM。
(4)图像处理/原子模型建立及表位分析：使用cryoSPARC v3.3.1处理所有3272个图像数据，包括衬度传递函数评估与修正，2维分类，异质性精修，同质性精修。为建立Her3-Fab复合物结构模型，使用UCSF Chimera软件将Her3结构(PDB 1M6B)及和抗体202-2-1同源性高的Fab结构(轻链：PDB 7CDJ，重链：PDB 6D01)与冷冻电镜图进行比对。在Coot(Emsley P et al.,Acta Crystallogr D Biol Crystallogr.2010Apr；66(Pt4):486-501.)中及在冷冻电镜图的引导下，结合使用CCP-EM进行实际空间优化，手动构建了该模型。结果如图5B-1,5B-2,5B-3所示。
分析结构结果可知：202-2-1抗体Fab结合的抗原表位位于人Her3的domain3，为SEQ ID No.31中第328至499位氨基酸内，具体的位于Her3抗原按SEQ ID No.31氨基酸依次编号的第466位组氨酸，470位至471位(色氨酸-苏氨酸)，478位苏氨酸，483位至487位(天冬氨酸-异亮氨酸- 赖氨酸-组氨酸-天冬酰胺)，484位异亮氨酸，490位至491位(精氨酸-精氨酸)；Fab重链按SEQ ID No.23氨基酸依次编号的第30位(丝氨酸)，53位至54位(酪氨酸-天冬氨酸)，56位至57位(丝氨酸-组氨酸)，59位酪氨酸，100位色氨酸参与了抗原的结合；Fab轻链按SEQ ID No.24氨基酸依次编号的第92位天冬酰胺参与了抗原的结合。
实施例11生物活性分子和合成“药物-连接体化合物”过程使用的中间体的合成
实施例11.1：(S)-7-乙基-7-羟基-14-(3-羟基丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1)的合成
步骤一：(S)-14-(3-氯丙基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1B)的合成
冰浴条件下，向化合物(S)7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环[4,5-g]吡喃并[3'，4'：6,7]茚并[1,2-b]喹啉-8,11(7H)-二酮(A1.5-A，500mg)的75％硫酸溶液(5mL)中加入七水合硫酸亚铁(570mg七水合硫酸亚铁溶于1mL水中)，和4,4-二甲氧基氯丁烷(3.89g)，反应液搅拌三分钟后滴加双氧水(29％，2.5mL)。反应液在0℃下搅拌反应5分钟后升至室温，并搅拌反应3小时。反应液加入水(50mL)稀释，乙酸乙酯(80mL×2)萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩得到粗产 品，粗产品用C18柱(乙腈/0.05％甲酸的水溶液：5％-60％)进一步纯化得到目标化合物A1B(黄色固体，400mg，收率：67％)。
LCMS(ESI)[M+H]+：468.9；
1H NMR(400MHz,DMSO-d6)δ7.65(s,1H),7.51(s,1H),7.24(s,1H),6.50(s,1H),6.30(s,2H),5.42(s,2H),5.26(s,2H),3.81(d,J＝5.9Hz,2H),3.22(s,2H),1.98(d,J＝6.7Hz,4H),0.88(t,J＝7.2Hz,3H)。
步骤二：(S)-7-乙基-7-羟基-14-(3-羟基丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1)的合成
将化合物(S)-7-乙基-7-羟基-14-(3-氯丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(100mg，0.213mmol)溶于10％硫酸(5mL)溶液中，反应在110℃下反应48小时。向反应液中加入饱和碳酸氢钠(30mL)溶液，用二氯甲烷(10mL X 5)萃取，无水硫酸钠干燥，抽滤，减压浓缩得到粗产品。经高效液相制备纯化(乙腈/水含0.05％甲酸)得到(S)-7-乙基-7-羟基-14-(3-羟基丙基)-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A1，1.78mg)。
LCMS(ESI)[M+H]+：451.0；
1H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.47–5.37(m,2H),5.32–5.19(m,2H),3.51–3.46(m,2H),3.17–3.13(m,2H),1.92–1.76(m,4H),0.90–0.84(m,3H)。
实施例11.2：(S)-4-乙基-8-氟-4-羟基-11-(3-羟基丙基)-9-甲基-1,12-二氢- 14H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-3,14(4H)-二酮(A2)的合成
步骤一：
0℃下，向化合物A2A(10g)的1,2-二氯乙烷(200mL)溶液，滴加1mol/L三氯化硼(96mL)，4-氯丁腈(9.9g)。80℃下搅拌反应2小时。反应液降至室温，加入2mol/L盐酸(90mL)并在80℃下回流搅拌0.5小时。反应液降至室温,加入少量水稀释，用二氯甲烷(200mL X 3)萃取，有机相用无水硫酸钠干燥，抽滤，浓缩得到粗品，粗产品经柱层析分离纯化(石油醚：乙酸乙酯＝10/1)得到目标化合物A2B(4g)。
LCMS(ESI)[M+H]+：230.0。
步骤二：
向化合物A2B(50mg)中加入(S)-4-乙基-4-羟基-7,8-二氢-1H-吡喃并[3,4-f]吲哚嗪-3,6,10(4H)-三酮(35mg)，一水合对甲苯磺酸(41.4mg),溶于二氯甲烷(30mL)溶液中，溶液澄清混匀后减压浓缩，用油泵抽至真空，反应在120℃真空下反应3小时。LCMS显示反应完全。将反应液降至室温，加入水(20mL)，用二氯甲烷(20mL X 3)萃取。合并有机相依次并无水硫酸钠干燥、过滤，滤液减压浓缩得到粗产品。粗产品经柱层析分离纯化(二氯甲烷比甲醇＝20比1)得到目标化合物A2C(80mg)为白色固体。
LCMS(ESI)[M+H]+：457.0。
步骤三：
将化合物A2C(75mg)溶于六甲基磷酰三胺，加入纯水(0.8mL)反应液在100℃下搅拌72小时。LCMS检测反应完成。经制备色谱(0.01％TFA in water，MeCN)纯化得到目标化合物A2(10mg)。
LCMS(ESI)[M+H]+：439.2；
1H NMR(400MHz,DMSO-d6)δ8.22(d,J＝8.4Hz,1H),7.87(d,J＝10.9Hz,1H),7.31(s,1H),6.50(s,1H),5.43(s,2H),5.30(s,2H),4.67(t,J＝4.9Hz,1H),3.55–3.47(m,2H),3.28–3.20(m,2H),2.51(s,3H),1.93–1.81(m,4H),0.88(t,J＝7.3Hz,3H)。
实施例11.3：(S,E)-14-(3-氨基-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A3)的合成
步骤一：
将化合物A3A(200mg，0.39mmol)，化合物A3B(112mg，0.39mmol)，氟化铯(152mg，0.975mmol)和四三苯基膦钯(45mg，0.039mmol)加入1,4-二氧六环溶液(8mL)。在氮气气氛下120℃微波反应0.5小时。LCMS显示反应完全。向反应液加入二氯甲烷(20mL)和甲醇(10mL)混合溶液稀释，过滤。滤液浓缩，粗品通过制备TLC纯化(二氯甲烷：甲醇＝30:1)得到目标化合物(S,E)-14-(3-((叔丁氧羰基)氨基)-1-丙烯-1-基)-7-乙基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-7-基乙酸酯(A3C，60mg，收率：26％)为 棕色固体。
LCMS(ESI)[M+H]+＝590.3；
1H NMR(400MHz,DMSO-d6)δ7.63(s,1H),7.49(s,1H),7.35(s,1H),7.09(d,J＝16.7Hz,1H),6.93(s,1H),6.45(d,J＝16.6Hz,1H),6.30(s,2H),5.47(s,2H),5.34–5.24(m,2H),3.94(s,2H),2.21(br s,3H),2.03–1.96(m,2H),1.45(s,9H),0.91(t,J＝6.7Hz,3H).
步骤二：
向化合物(S,E)-14-(3-((叔丁氧羰基)氨基)-1-丙烯-1-基)-7-乙基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-7-基乙酸酯(A3C，50mg，0.085mmol)的甲醇溶液(15mL)和加入甲醇钠(9.2mg，0.17mmol)，在50℃下搅拌反应2小时。LCMS显示反应完全。将反应液浓缩得到目标化合物(S,E)-14-(3-((叔丁氧羰基)氨基)-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A3D，50mg)粗品为棕色固体。LCMS(ESI)[M+H]+＝548；
步骤三：
向化合物(S,E)-14-(3-((叔丁氧羰基)氨基)-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A3D，50mg，0.091mmol)的二氯甲烷溶液(2mL)中，加入三氟乙酸(1mL)，在室温下搅拌反应30分钟。LCMS显示反应完全。将反应液浓缩，粗品经高效液相制备纯化(乙腈/0.05％甲酸的水溶液)得到目标化合物(S,E)-14-(3-氨基-1-丙烯-1-基)-7-乙基-7-羟基-10,13-二氢-11H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-8,11(7H)-二酮(A3)(8.1mg，收率21％)为棕色固体。
LCMS(ESI)[M+H]+＝448.3；
1H NMR(400MHz,DMSO-d6)δ8.20(s,2H),7.72(s,1H),7.54(s,1H),7.40(d,J＝16.5Hz,1H),7.27(s,1H),6.52–6.46(m,2H),6.31(s,2H),5.42(s, 2H),5.27(s,2H),3.86(br s,2H),1.90–1.83(m,2H),0.88(t,J＝7.1Hz,3H).
实施例11.4：(S)-2-氨基-N-((4-(4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙氧基)甲基)乙酰胺(B1)的合成
步骤一：
将化合物A2(160mg,0.365mmol)溶在N,N-二甲基甲酰胺(3mL)中，加入(2-((((9H-芴-9-基)甲氧基)羰基)氨基)乙酰氨基)甲基乙酸酯((B1A，672mg,1.83mmol)，然后加入盐酸乙酸乙酯(0.073mL,3M)于反应液中，反应液在室温下搅拌过夜。LCMS检测反应完成。该反应液直接经反相色谱(乙腈/0.05％FA的水溶液:5％到50％)纯化后得目标化合物B1B(80mg,收率29.0％)为白色固体。
LCMS(ESI)[M+H]+＝747.4；
1H NMR(400MHz,DMSO-d6)δ8.74(t,J＝6.4Hz,1H),8.28–8.17(m,1H),7.99–7.88(m,3H),7.73(d,J＝7.3Hz,2H),7.63(t,J＝5.7Hz,1H),7.44(t,J＝7.4Hz,2H),7.35(t,J＝7.2Hz,3H),6.58(s,1H),5.48(s,2H),5.29(s,2H),4.66(d,J＝6.3Hz,2H),4.32(d,J＝6.9Hz,2H),4.26(d,J＝6.1Hz,1H),3.71(d,J＝5.8Hz,2H),3.57(t,J＝5.7Hz,2H),3.29–3.20(m,2H),2.55(s,3H),2.00–1.86(m,4H),0.93(t,J＝7.2Hz,3H).
步骤二：B1B(240mg)溶于DMF(5ml)，加入哌啶(1ml)，化合物搅拌20分钟，溶于减压除去低沸点组分，残留物直接用于下步合成。
ESI-MS(m/z):525.2[M+H]+。
少量粗产物经反相色谱(乙腈/0.05％FA的水溶液:5％到50％)纯化后得 目标化合物。
ESI-MS(m/z):525.1[M+H]+；
1H NMR(400MHz,DMSO)δ9.13(t,J＝6.6Hz,1H),8.21(d,J＝8.1Hz,1H),8.02(brs,2H),7.89(d,J＝10.8Hz,1H),7.32(s,1H),6.54(s,1H),5.44(s,2H),5.28(s,2H),4.66(d,J＝6.5Hz,2H),3.64(s,2H),3.53(t,J＝6.1Hz,2H),3.25-3.18(m,2H),2.52(s,3H),1.98-1.84(m,4H),0.88(t,J＝7.3Hz,3H)。
实施例11.5：(S)-2-氨基-N-((3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙氧基)甲基)乙酰胺(B2)的合成
步骤一：化合物(B1A)(368mg)，化合物(A1)(440mg)和对甲苯磺酸吡啶盐(PPTS)(25mg)在二氯甲烷(20ml)中回流20小时，然后用碳酸氢钠水溶液和盐酸水溶液分别洗涤，减压除去有机溶剂，得到粗产物，粗产品经柱层析分离纯化(二氯甲烷：甲醇＝10/1)得到目标化合物化合物B2A(240mg)。
LCMS(ESI)[M+H]+：759。
步骤二：B2A(240mg)溶于DMF(5ml)，加入哌啶(1ml)，化合物搅拌20分钟，溶于减压除去低沸点组分，残留物直接用于下步合成。少量粗产物经反相色谱(乙腈/0.05％FA的水溶液:5％到50％)纯化后得目标化合物B2。
ESI-MS(m/z):537[M+H]+；
1H NMR(400MHz,DMSO-d6)δ9.13(t,1H)，8.04(br,2H),7.58(s，1H)，7.51(s，1H)，7.25(s，1H)，6.29(s，2H)，5.43(S，2H)，5.21(s,2H)，4.65(d，2H)，3.63(m，2H)，3.53(m，2H)，3.11(m，2H)， 1.87(m，4H)，0.88(t，3H)。
实施例11.6：(S,E)-2-氨基-N-(3-(7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)乙酰胺(B3)
步骤一：
室温下，向化合物A3(200mg，0.447mmol)的N,N-二甲基甲酰胺溶液(5mL)中加入三乙胺(135mg，1.341mmol)和N-羟基-2,5-二氧吡咯烷叔丁氧羰基甘氨酸酯(183mg，0.671mmol)，在室温下反应1小时。LCMS显示反应完全。向反应液中加入水(20mL)，用乙酸乙酯(20mL×3)萃取，有机相用饱和食盐水洗涤，无水硫酸钠干燥，浓缩得到目标化合物B3A(130mg，收率：48％)为棕色固体。
LCMS(ESI)[M+H]+＝605.6。
步骤二：
室温下，将化合物B3A(130mg，0.215mmol)用盐酸1，4-二氧六环溶液(5mL)溶解，在室温下反应30分钟。LCMS显示反应完全。反应液通过反向纯化得到目标化合物B3(60mg，收率：52％)为棕色固体。
LCMS(ESI)[M+H]+＝505.2；
1H NMR(400MHz,DMSO-d6)δ8.85(s,1H),8.15(s,2H),7.70(s,1H),7.54(s,1H),7.26(s,1H),7.21(d,J＝16.2Hz,1H),6.51(d,J＝16.2Hz,1H),6.31(s,2H),5.42(s,2H),5.28(s,2H),4.19(s,2H),1.86–1.82(m,4H),0.87(t,J＝7.3Hz,3H).
实施例11.7：N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C1)
将6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(268mg),化合物C1A(328mg)，三乙胺(322mg)溶于N,N-二甲基甲酰胺(5mL)。然后再加入1-羟基苯并***(HOBT，162mg)和1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI，229mg),反应液室温搅拌16小时，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到目标化合物N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C1，白色固体，327mg)。
LCMS(ESI)[M+H]+：524.4。
1H NMR(400MHz,)δ9.13(s,2H),7.95(t,J＝8.8Hz,2H),4.21(dd,J＝8.8,6.9Hz,1H),4.08–4.03(m,1H),3.41(s,3H),2.55(t,J＝7.0Hz,2H),2.42–2.32(m,4H),2.27(s,6H),1.98(dd,J＝13.6,6.8Hz,1H),1.86–1.77(m,2H),1.74–1.55(m,2H),1.47–1.37(m,2H),1.31–1.23(m,2H),0.85(dd,J＝12.8,6.8Hz,6H)。
实施例11.8：N6,N6-二甲基-N2-((6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰基)-L-缬氨酸)-L-赖氨酸(C2)
步骤一：
将化合物6-(4-(2-(甲硫基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸(C2A，1.0g，3.3mmol)溶在四氢呋喃和水(40mL，3:1)混合溶液中，加入过氧 单磺酸钾(10.0g，16.3mmol)。室温搅拌反应3小时，LCMS检测反应完成。反应液过滤，滤饼用DMSO洗涤，合并滤液。经反相纯化(C18，乙腈：0.1％甲酸＝5％-55％)纯化，得到6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸(C2B，750mg，收率67.9％)白色固体。
LCMS(ESI)[M+H]+＝340.1；
1H NMR(400MHz,DMSO-d6)δ9.48(s,2H),8.95(s,1H),4.49(t,J＝7.0Hz,2H),3.45(s,3H),2.22(t,J＝7.3Hz,2H),1.95–1.84(m,2H),1.61–1.50(m,2H),1.36–1.26(m,2H)。
步骤二：
将化合物6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酸(C2B，300mg，0.88mmol)溶于N,N-二甲基甲酰胺(6mL)，加入HATU(337mg，0.88mmol)，DIPEA(286mg，2.21mmol)，室温搅拌反应30分钟，然后加入二肽C1.16-D(243mg，0.88mmol)，室温搅拌反应2小时。LCMS检测反应完成，反应液通过C18柱分离纯化(乙腈/0.01％FA的水溶液：5％-50％)得到目标化合物N6,N6-二甲基-N2-((6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰基)-L-缬氨酸)-L-赖氨酸(C2，300mg，收率57％)白色固体。
LCMS(ESI)[M+H]+＝595.5,tR＝2.024min。
1H NMR(400MHz,DMSO-d6)δ9.49(s,2H),9.01(s,1H),7.85(br s,1H),7.84(d,J＝8.9Hz,1H),4.48(t,J＝6.8Hz,2H),4.17–4.15(m,1H),4.02(br s,1H),3.44(s,3H),2.42(br s,2H),2.30(s,6H),2.18–2.14(m,2H),1.99–1.96(m,1H),1.88(dd,J＝14.6,7.1Hz,2H),1.67(br s,1H),1.59–1.53(m,2H),1.43(br s,2H),1.28–1.26(m,5H),0.83–0.89(m,6H).
实施例11.9：N6,N6-二乙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C3)
步骤一：
将化合物C3A(5.0g,12.05mmol)溶在二氯甲烷(100mL)中，向反应液中加入乙醛(3.2g,72.3mmol)，室温下搅拌反应10分钟，再向反应液中加入三乙酰氧基硼氢化钠(12.8g,60.25mmol)，反应在室温下搅拌反应1小时。LCMS显示反应完全。向反应液中加入氯化铵饱和水溶液搅拌一个小时，旋干，过滤后滤液用C18柱反向分离纯化(乙腈比0.05％的甲酸水溶液:5％到55％)得到目标化合物C3B(4.57g,收率82.0％)为白色固体。
LCMS(ESI)[M+H]+＝436.4；
1H NMR(400MHz,DMSO-d6)δ7.70(d,J＝7.0Hz,1H),7.41(d,J＝9.0Hz,1H),7.38–7.26(m,5H),5.08–4.99(m,2H),4.00(dd,J＝12.6,6.5Hz,1H),3.86(dd,J＝8.6,6.8Hz,1H),2.74(dd,J＝14.0,6.9Hz,4H),2.64–2.54(m,2H),2.05–1.94(m,1H),1.72–1.52(m,2H),1.52–1.38(m,2H),1.38–1.18(m,2H),1.04(t,J＝7.1Hz,6H),0.87–0.81(m,6H)。
步骤二：
室温下，将化合物C3B(1.6g，3.68mmol)溶于甲醇(80mL)中，然后向反应液中加入Pd/C(0.16g)，氢气下室温下搅拌反应12小时。LCMS显示反应完成。将反应液过滤，滤液减压浓缩得到目标化合物C3C(900mg,收率82％)为米白色固体。
1H NMR(400MHz,DMSO-d6)δ8.04(br s,1H),4.02–3.99(m,1H),3.10 (d,J＝4.5Hz,1H),2.65(q,J＝7.1Hz,4H),2.55–2.51(m,2H),2.06–1.93(m,1H),1.73–1.54(m,2H),1.47–1.38(m,2H),1.30–1.21(m,2H),1.01(t,J＝7.1Hz,6H),0.89(d,J＝6.9Hz,3H),0.79(d,J＝6.8Hz,3H)。
步骤三：
将化合物6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(268mg,1mmol)溶于DMF(8mL)中，依次加入HATU(380mg,1mmol)和三乙胺(322mg,2.5mmol)，室温搅拌20分钟后加入化合物C3C(301mg,1mmol)，室温继续搅拌30分钟。LCMS检测反应完成后，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到目标化合物C3(280mg，收率51％)为白色固体。
LCMS(ESI)[M+H]+＝552.3；
1H NMR(400MHz,DMSO-d6)δ9.13(s,2H),7.95(d,J＝8.9Hz,1H),7.86(d,J＝7.2Hz,1H),4.18(dd,J＝8.8,6.8Hz,1H),4.02(dd,J＝12.8,7.2Hz,1H),3.41(s,3H),2.74–2.69(m,4H),2.62–2.52(m,4H),2.44–2.29(m,2H),2.04–1.94(m,1H),1.86–1.77(m,2H),1.72–1.54(m,2H),1.51–1.39(m,2H),1.33–1.23(m,2H),1.02(t,J＝7.2Hz,6H),0.87–0.82(m,6H)。
实施例11.10：N6,N6-二丙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C4)
步骤一：
将化合物C3A(5.0g，12mmol)溶在二氯甲烷(100mL)中，向反应液中加入正丙醛(4.2g，72.3mmol)，室温下搅拌反应10分钟，再向反应液中加入三乙酰氧基硼氢化钠(12.8g，60.25mmol)，反应在室温下搅拌反应1小时。LCMS显示反应完全。向反应液中加入氯化铵饱和水溶液搅拌一个小时，旋干，过滤后滤液用C18柱反向分离纯化(乙腈比0.05％的甲酸水溶液:5％到55％)得到目标化合物C4A(4.57g,收率82.0％)为白色固体。
LCMS(ESI)[M+H]+＝464.0；
1H NMR(400MHz,DMSO-d6)δ7.81(d,J＝7.3Hz,1H),7.38–7.28(m,5H),5.04(d,J＝1.7Hz,2H),4.12–4.02(m,1H),3.94–3.82(m,1H),2.65–2.52(m,6H),2.06–1.94(m,1H),1.76–1.64(m,1H),1.64–1.53(m,1H),1.52–1.40(m,6H),1.34–1.18(m,2H),0.92–0.80(m,12H)。
步骤二：
室温下，将化合物C4A(2.0g，4.32mmol)溶于甲醇(80mL)中，然后向反应液中加入Pd/C(0.16g)，氢气下室温下搅拌反应12小时。LCMS显示反应完成。将反应液过滤，滤液减压浓缩得到目标化合物C4B(1.2g,收率85.5％)为白色固体。
步骤三：
将化合物6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酸(100mg,0.373mmol)，溶于N,N-二甲基甲酰胺(1mL),然后加入HATU(142mg,0.373mmol)，N,N-二异丙基乙胺(120mg,0.93mmol)，该体系搅拌30分钟，然后加入化合物C4B(122mg，0.371mmol)，反应液室温搅拌1小时，LCMS检测反应完成后，反应液直接经过C18柱反相(乙腈和0.05％的甲酸水溶液体系)提纯得到目标化合物N6,N6-二丙基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(C4，50mg，收率28％)为淡黄色固体。
LCMS(ESI)[M+H]+＝580.0；
1H NMR(400MHz,DMSO-d6)δ8.24(s,2H),7.98–7.93(m,2H),4.24– 4.16(m,1H),4.10(d,J＝5.2Hz,1H),3.41(s,3H),2.79–2.64(m,6H),2.55(t,J＝7.1Hz,2H),2.45–2.26(m,2H),2.06–1.91(m,1H),1.89–1.78(m,2H),1.76–1.66(m,1H),1.64–1.57(m,1H),1.57–1.42(m,6H),1.37–1.24(m,2H),0.93–0.78(m,12H)。
实施例12药物-连接体化合物合成
实施例12.1：N-((11S,14S)-11-(4-(二甲基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-01)
将化合物N6,N6-二甲基-N2-((6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰基)-L-缬氨酸)-L-赖氨酸(126mg)(C1)，B1(150mg)，1-羟基苯并***(49mg)，1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(69mg)和二异丙基乙胺(156mg,1.21mmol)溶在N,N-二甲基甲酰胺(10mL)溶液中，反应在40℃下搅拌12小时。LCMS显示反应完成。反应液过滤经高效液相制备纯化(乙腈/水含0.05％甲酸)得到黄色固体化合物N-((11S,14S)-11-(4-(二甲基氨基)丁基)-1-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺 (DL-01)(12mg，收率6.0％)。
LCMS(ESI)[M+H]+＝1030.6；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),8.62(t,J＝6.4Hz,1H),8.23–8.13(m,2H),8.07(d,J＝7.2Hz,1H),7.95–7.83(m,2H),7.31(s,1H),6.53(s,1H),5.44(s,2H),5.29(s,2H),4.64–4.53(m,2H),4.28–4.20(m,1H),4.19–4.11(m,1H),3.76–3.69(m,2H),3.50(t,J＝5.7Hz,2H),3.41(s,3H),3.23–3.19(m,2H),2.67–2.62(m,2H),2.57–2.54(m,2H),2.54(s,3H),2.42–2.26(m,3H),2.02–1.77(m,6H),1.68–1.66(m,1H),1.58–1.45(m,3H),1.36–1.25(m,2H),0.88(t,J＝7.4Hz,3H),0.82(t,J＝6.3Hz,6H)。
实施例12.2：N-((S)-1-(((S)-6-(二乙基氨基)-1-((2-(((E)-3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-02)
向化合物B3(40mg，0.074mmol)和化合物C3(40mg，0.072mmol)的N,N-二甲基甲酰胺溶液(4mL)溶解中依次加入HBTU(30mg，0.079mmol)、DIPEA(26mg，0.2mmol)。反应液室温下反应1小时。LCMS显示反应完全。反应液经高效液相制备纯化(乙腈/0.05％三氟乙酸水溶液)得到化合物N-((S)-1-(((S)-6-(二乙基氨基)-1-((2-(((E)-3-((S)-7-乙基-7-羟基- 8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)烯丙基)氨基)-2-氧代乙基)氨基)-1-氧代己-2-基)氨基)-3-甲基-1-氧代丁-2-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-02，11.1mg，收率14％)。
LCMS(ESI)[M+H]+＝1038.6；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),9.02(s,1H,TFA),8.33–8.25(m,2H),8.12(d,J＝7.3Hz,1H),7.92(d,J＝8.4Hz,1H),7.68(d,J＝4.1Hz,1H),7.52(d,J＝2.8Hz,1H),7.26(s,1H),7.15(d,J＝16.2Hz,1H),6.52–6.50(m,2H),6.30(s,2H),5.43(s,2H),5.28–5.24(m,2H),4.27(d,J＝6.2Hz,2H),4.12(br s,2H),3.81(br s,2H),3.41(s,3H),3.11–3.07(m,6H),2.97(br s,2H),2.53–2.50(m,1H),2.39–2.24(m,4H),1.85–1.79(m,8H),1.16(t,J＝6.4Hz,6H),0.88(t,J＝6.4Hz,3H),0.78(d,J＝6.4Hz,6H)。
实施例12.3：(S)-6-(二甲基氨基)-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-03)
将化合物C2(113mg,0.19mmol),化合物B1(100mg,0.19mmol)溶于DMF(1mL),然后加入HBTU(72mg,0.19mmol)和N,N-二异丙基乙胺(61mg,0.48mmol),加完后反应液室温搅拌1小时，LCMS检测反应完成后，将反应液经制备色谱(0.01％TFA的水溶液，乙腈)纯化得到目标化合物(S)-6-(二甲基氨基)-N-(2-(((3-((S)-4-乙基-8-氟-4-羟基-9-甲基-3,14-二氧代-3,4,12,14-四氢-1H-吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-11-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H- 1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-03，16mg,收率8％)为白色固体。
LCMS(ESI)[M+H]+＝1101.6；
1H NMR(400MHz,DMSO)δ9.46(s,2H),8.93(s,1H),8.63(t,J＝6.3Hz,1H),8.20–8.28(m,2H),8.04(d,J＝7.2Hz,1H),7.88(d,J＝10.8Hz,1H),7.81(d,J＝8.3Hz,1H),7.31(s,1H),6.53(s,1H),5.44(s,2H),5.29(s,2H),4.63–4.54(m,2H),4.46(t,J＝7.0Hz,2H),4.29–4.19(m,1H),4.15–4.08(m,1H),3.73(d,J＝6.9Hz,2H),3.50(t,J＝5.8Hz,2H),3.44(s,3H),3.24–3.14(m,2H),3.01–2.91(m,2H),2.72(s,6H),2.53(s,3H),2.23–2.07(m,2H),1.93–1.84(m,6H),1.72–1.64(m,1H),1.61–1.45(m,6H),1.34–1.22(m,4H),0.87(t,J＝7.4Hz,3H),0.82–0.76(m,6H).
实施例12.4：N-((11S,14S)-11-(4-(二正丙基氨基)丁基)-1-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)-15-甲基-7,10,13-三氧代-4-氧杂-6,9,12-三氮杂十六烷-14-基)-6-(2-(甲磺酰基)嘧啶-5-基)己-5-炔酰胺(DL-04)
将化合物C4(43mg,0.074mmol),化合物B2(40mg，0.075mmol)，溶于N,N-二甲基甲酰胺(1mL)，然后依次加入HBTU(28mg,0.075mmol)，N,N-二异丙基乙胺(24mg,0.187mmol)，反应液室温搅拌1小时，LCMS检测反应完成后，反应液直接经制备色谱(0.01％三氟乙酸水溶液,乙腈)纯化得到目标化合物(DL-04，8.5mg,收率10％)为黄色固体。
LCMS(ESI)[M+H]+＝1098.6；
1H NMR(400MHz,DMSO-d6)δ9.10(s,2H),9.03(s,1H),8.64(t,J＝6.4Hz,1H),8.19(t,J＝5.9Hz,1H),8.08(d,J＝7.4Hz,1H),7.92(d,J＝8.5Hz, 1H),7.59(s,1H),7.51(s,1H),7.24(s,1H),6.49(s,1H),6.29(s,2H),5.42(s,2H),5.24(s,2H),4.66–4.52(m,2H),4.31–4.21(m,1H),4.19–4.10(m,1H),3.74(d,J＝5.5Hz,2H),3.49–3.48(m,2H),3.40(s,3H),3.15–3.06(m,2H),3.02–2.95(m,6H),2.59–2.52(m,3H),2.41–2.29(m,2H),2.05–1.90(m,2H),1.91–1.77(m,6H),1.63–1.57(m,6H),1.31–1.29(m,2H),0.92–0.80(m,15H)。
实施例12.5：(S)-6-(二甲基氨基)-N-(2-(((3-((S)-7-乙基-7-羟基-8,11-二氧代-7,8,11,13-四氢-10H-[1,3]二氧杂环戊烯并[4,5-g]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-14-基)丙基氧基)甲基)氨基)-2-氧代乙基)-2-((S)-3-甲基-2-(6-(4-(2-(甲磺酰基)嘧啶-5-基)-1H-1,2,3-***-1-基)己酰胺基)丁酰胺基)己酰胺(DL-05)
将化合物C2(40mg，0.06mmol)溶于N,N-二甲基甲酰胺(1.5mL)，依次加入HBTU(26mg，0.06mmol)、B2(36mg，0.06mmol)和DIPEA(66mg，0.17mmol，室温搅拌反应1小时。LCMS检测反应完成，反应液通过制备(乙腈/0.05％三氟乙酸水溶液)分离纯化得到目标化合物DL-05(13.88mg，TFA盐收率16％),为米黄色固体。
LCMS(ESI)[M+H]+＝1113.7；
1H NMR(400MHz,DMSO-d6)δ9.46(s,2H),9.29(s,1H,TFA盐),8.92(s,1H),8.63(t,J＝6.6Hz,1H),8.18(br s,1H),8.03(d,J＝7.6Hz,1H),7.81(d,J＝8.3Hz,1H),7.59(s,1H),7.50(s,1H),7.24(s,1H),6.48(s,1H),6.28(s,2H),5.42(s,2H),5.24(s,2H),4.64–4.55(m,2H),4.46(t,J＝7.0Hz,2H),4.28–4.20(m,1H),4.15–4.09(m,1H),3.76–3.71(m,4H),3.53–3.48(m,4H),3.44(s,3H),3.10–3.08(m,2H),2.97–2.95(m,2H),2.75(d,J＝4.9Hz,6H),2.20–2.09(m,2H),1.92–1.83(m,5H),1.68–1.65(m,1H),1.63–1.50(m,5H),0.90–0.74(m,9H)。
实施例13抗Her3抗体-药物偶联物的制备
实施例13.1：Her3-ADC-07的制备
q可由DAR测定中提供数据所推定。
13.1.1 Her3-ADC-07(DAR8)样品制备
将抗Her3抗体202-2-1 30mg，用稀释液(20mM PB+105mM NaCl，pH 7.7)稀释，加入终浓度为5mM的依地酸钠溶液，混匀；加入抗体4.5倍摩尔当量的20mmol/L的TCEP溶液，混匀，室温放置30分钟；向上述溶液体系加入抗体10倍摩尔当量的10mmol/L的溶解于二甲基亚砜中的DL-04，混匀，室温静置2小时，得到偶联后样品，反应结束后使用30KDa的超滤管将样品置换到pH为5.5的10mM组氨酸缓冲液中并去除低分子物质，最后将样品浓缩以获得含有202-2-1抗体ADC组合物的溶液Her3-ADC-07(DAR8)。根据实施例14质谱法测定平均DAR值为8.0。
根据质谱法测定的Her3-ADC-07(DAR8)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为0％，100％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为0％，0％，0％，100％)，可推知q值为8。
13.1.2 Her3-ADC-07(DAR4)样品制备
将抗Her3抗体202-2-1 30mg，用稀释液(20mM PB+105mM NaCl，pH 7.7)稀释，加入终浓度为5mM的依地酸钠溶液，混匀；加入抗体4.5倍摩尔当量的20mmol/L的TCEP溶液，混匀，室温放置30分钟；向上述 溶液体系加入抗体4.8倍摩尔当量的10mmol/L的溶解于二甲基亚砜中的DL-04，混匀，室温静置2小时，得到偶联后样品，反应结束后使用30KDa的超滤管将样品置换到pH为5.5的10mM组氨酸缓冲液中并去除低分子物质，最后将样品浓缩以获得含有202-2-1抗体ADC组合物的溶液Her3-ADC-07(DAR4)。质谱法测定平均DAR值为4.5。
质谱法测定的Her3-ADC-07(DAR4)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为93.67％，6.33％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为19.23％，7.01％，10.66％，63.1％)，可推知q值可为1、2、3、4、5、6、7或8。
13.1.3 Her3-ADC-07(DAR6)样品制备
将抗Her3抗体202-2-1 30mg，用稀释液(20mM PB+105mM NaCl，pH 7.7)稀释，加入终浓度为5mM的依地酸钠溶液，混匀；加入抗体4.5倍摩尔当量的20mmol/L的TCEP溶液，混匀，室温放置30分钟；向上述溶液体系加入抗体7.2倍摩尔当量的10mmol/L的溶解于二甲基亚砜中的DL-04，混匀，室温静置2小时，得到偶联后样品，反应结束后使用30KDa的超滤管将样品置换到pH为5.5的10mM组氨酸缓冲液中并去除低分子物质，最后将样品浓缩以获得含有202-2-1抗体ADC组合物的溶液Her3-ADC-07(DAR6)。质谱法测定平均DAR值为5.8。
质谱法测定的Her3-ADC-07(DAR6)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为81.31％，18.69％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为7.17％，2.36％，4.44％，86.03％)，可推知q值可为1、2、3、4、5、6、7或8。
实施例13.2：Her3-ADC-05(DAR4)的制备
利用DL-01代替实施例13.1.2的化合物DL-04，得到DL-01与202-2-1抗体的偶联产物Her3-ADC-05(DAR4)。质谱法测定平均DAR值为4.0。
质谱法测定的Her3-ADC-05(DAR4)上抗体轻链偶联0～1个毒素分子 (LC，DAR1比例分别为90.89％，9.11％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为25.1％，7.17％，18.19％，49.53％)，可推知q值可为1、2、3、4、5、6、7或8。
实施例13.3：Her3-C3-ADC-05(DAR4)(DAR4)样品制备
利用DL-01代替实施例13.1.2的化合物DL-04，对照抗体2代替实施例13.1.2的抗Her3抗体202-2-1，得到DL-01与对照抗体2的偶联产物Her3-C3-ADC-05(DAR4)。质谱法测定平均DAR值为4.7。
实施例13.4：Her3-ADC-15的制备
q可由DAR测定中提供数据所推定。
13.4.1 Her3-ADC-15(DAR8)样品制备
利用DL-02代替实施例13.1.1的化合物DL-04，得到DL-02与202-2-1抗体的偶联产物Her3-ADC-15(DAR8)。质谱法测定平均DAR值为7.9。
质谱法测定的Her3-ADC-15(DAR8)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为0％，100％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为0％，0％，6.31％，93.69％)，可推知q值可为为3、4、5、6、7或8。
13.4.2Her3-ADC-15(DAR2)样品制备
利用2倍摩尔当量的DL-02代替实施例13.1.1的化合物DL-04，得到DL-02与202-2-1抗体的偶联产物Her3-ADC-15(DAR2)。质谱法测定平均DAR值为2.0。
质谱法测定的Her3-ADC-15(DAR2)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为93.82％，6.18％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为61.16％，5.77％，11.02％，22.05％)，可推知q值可为1、2、3、4,、5、6、7或8。
实施例13.5：Her3-ADC-23(DAR4)的制备
利用DL-03代替实施例13.1.2的化合物DL-04，得到DL-03与202-2-1抗体的偶联产物Her3-ADC-23(DAR4)。质谱法测定平均DAR值为4.2。
质谱法测定的Her3-ADC-23(DAR4)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为91.28％，8.72％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为19.24％，8.67％，21.97％，50.13％)，可推知q值可为1、2、3、4、5、6、7或8。
实施例13.6：Her3-C3-ADC-23(DAR4)样品制备
利用DL-03代替实施例13.1.2的化合物DL-04，用对照抗体2代替13.1.2的Her3抗体202-2-1，得到DL-03与对照抗体2的偶联产物Her3-C3-ADC-23(DAR4)。质谱法测定平均DAR值为4.9。
实施例13.7：Her3-C3-ADC-21(DAR4)样品制备
参照CN104755494B制备获得药物-连接体化合物如下，
利用上述药物-连接体化合物代替实施例13.1.2的化合物DL-04，对照抗体2代替实施例13.1.2的抗Her3抗体202-2-1，得到Her3-C3-ADC-21(DAR4)。质谱法测定平均DAR值为4.4。
实施例13.8：Her3-C3-ADC-21(DAR8)样品制备
利用实施例13.7药物-连接体化合物代替实施例13.1.1的化合物DL-04，对照抗体2代替实施例13.1.1的抗Her3抗体202-2-1，得到Her3-C3-ADC-21(DAR8)。质谱法测定平均DAR值为9.5。
实施例13.9：Her3-ADC-21(DAR4)样品制备
利用实施例13.7药物-连接体化合物代替实施例13.1.2的化合物DL-04，得到上述药物-连接体化合物与202-2-1抗体的偶联产物Her3-ADC-21(DAR4)。质谱法测定平均DAR值为4.1。
质谱法测定的Her3-ADC-21(DAR4)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为100％，0％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为20.57％，8.01％，17.3％，54.12％)，可推知q值可为1、2、3、4、5或6。
实施例13.10：Her3-ADC-33的制备
参照WO2019195665实施例4-1制备获得药物-连接体化合物如下，
用上述药物-连接体化合物代替实施例13.1.2的化合物DL-04，得到上述药物-连接体化合物与202-2-1抗体的偶联产物Her3-ADC-33(DAR4)。质谱法测定平均DAR值为5.6。
质谱法测定的Her3-ADC-33(DAR4)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为49％，51％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为18.01％，3.25％，9.13％，69.62％)，可推知q值可为1、2、3、4、5、6、7或8。
实施例13.11：IgG-ADC-07(DAR8)的制备
用IgG1同型抗体(抗鸡卵融菌酶抗体，苏州宜联生物医药有限公司)代替实施例13.1.1的202-2-1抗体，得到DL-04与IgG1抗体的偶联产物IgG1-ADC-07。质谱法测定平均DAR值为8.0。
注：Her3mAb和Her3Ab均指抗Her3-的抗体或其抗原结合片段，具体在上述实施例中，Her3mAb为202-2-1抗体，Her3Ab为对照抗体2。
实施例13.12：Her3-ADC-08的制备
q可由DAR测定中提供数据所推定。
(1)Her3-ADC-08(DAR6)样品制备
用化合物DL-05代替实施例13.1.3“Her3-ADC-07(DAR6)样品制备”中的化合物DL-04，得到DL-05与202-2-1抗体的偶联产物Her3-ADC-08(DAR6)，质谱法测定平均DAR值为4.9。质谱法测定的Her3-ADC-08(DAR6)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为66.58％，33.42％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为16.51％，11.27％，16.63％，55.59％)，可推知q值可为1、2、3、4、5、6、7或8。
(2)Her3-ADC-08(DAR2)样品制备
利用2倍摩尔当量的DL-05代替实施例13.1.1的化合物DL-04，得到DL-05与202-2-1抗体的偶联产物Her3-ADC-08(DAR2)。质谱法测定平均DAR值为1.9。质谱法测定的Her3-ADC-08(DAR2)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为96.61％，3.39％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为58.72％，9.05％，15.41％，16.83％)，可推知q值可为1、2、3、4、5、6、7或8。
实施例14采用质谱法进行偶联后样品的DAR值测定
检测：对Her3-ADC-07(DAR8)进行LC-MS分子量和DAR值分析，条件如下：
色谱条件：
色谱柱：PLRP-S，2.1*50mm,5μm；
流动相A：0.1％FA/H2O；流动相B：0.1％FA/ACN
柱温：30℃样品室温度：8℃流速：0.6ml/min进样量：2μl
样品处理：分别取样品50μg，加入1M DTT2μl，加超纯水至50μl稀释至约1.0mg/ml浓度，混匀，室温还原30min。
LC/MS型号：Agilent 1290-6545XT Q-TOF
质谱条件：Gas temp：320℃，Drying Gas：Nebulizer：35psi；Sheath Gas Temp：350℃；sheath Gas Flow：11l/min；m/z 500～3000。
结果如下所示：
表6理论分子量及实测分子量
表6中，mAb表示未偶联的单克隆抗体；LC代表抗体轻链；HC代表抗体重链；DAR1代表包含轻链或重链偶联1个毒素分子的偶联物；DAR2代表包含轻链或重链偶联2个毒素分子的偶联物；DAR3代表包含轻链或重链偶联3个毒素分子的偶联物；其中，单抗理论分子量以G0F糖型计算。下文中mAb、LC、HC、DAR1、DAR2、DAR3如上说明。
检测结果显示，Her3-ADC-07(DAR8)上抗体轻链偶联0～1个毒素分子(LC，DAR1比例分别为0％，100％)、重链偶联0～3个毒素分子(mAb、DAR1、DAR2、DAR3的比例分别为0％，0％，0％，100％)，由此计算Her3-ADC-07(DAR8)的药物抗体偶联比(DAR值)为8.0。
实施例15抗Her3ADC药物对肿瘤细胞系的体外杀伤活性检测
A.Her3 ADC的PC-9和NCI-H358肿瘤抑制活性测定
选取PC-9肿瘤细胞(人肺腺癌，南京科佰生物科技有限公司)和NCI-H358肿瘤细胞(人非小细胞肺癌,ATCC，CRL-5807)检测Her3-ADC-07对内源表达人Her3肿瘤细胞系的杀伤活性。具体实验步骤如下：消化PC-9细胞和NCI-H358细胞，离心收集细胞，用1640+4％FBS培养基重悬细胞至50000个/mL，100μL/孔铺细胞至96孔板中；用1640基础培养 基梯度稀释ADC分子，按照终浓度150μM起始，3倍稀释，11个浓度点，加入对应孔中，每孔100μL，最终血清浓度为2％；37℃、5％CO2培养箱中培养120小时；加入CCK8(Rhinogen)，20μL/孔，37℃、5％CO2培养箱中孵育0.5-2.5小时，每半小时用酶标仪(MD，型号ABS Plus)450nm读数并导入Graphpad Prism进行曲线拟合。
实验结果如图6A和6B所示，Her3-ADC-07对PC-9肿瘤细胞杀伤活性EC50＝49.26nM，对NCI-H358肿瘤细胞杀伤活性EC50＝183.3nM。
B.Her3 ADC的KYSE520肿瘤抑制活性测定
使用胰酶通过常规方法对KYSE520(人食管鳞癌肿瘤细胞，ATCC)进行消化，收集细胞并对管中细胞进行计数，用对应的检测培养基(含2％FBS)重悬，将2000-5000细胞/孔加至96孔板。2％FBS培养基稀释的ADC(如表7)加入100uL至96孔板，浓度从50μg/ml起始，3倍稀释(11个浓度梯度)。37℃5％CO2培养5天，然后每孔加入CCK8试剂20μL，反应至1-4小时，酶标仪读数(检测波长为450nm)。实验细胞和检测结果如表7所示。
表7
ADC样品 KYSE520(EC50,ng/mL)
Her3-ADC-05(DAR4) 19.01
Her3-C3-ADC-05(DAR4) 32.94
测试结果表明，Her3-ADC-05和Her3-C3-ADC-05都具有很强的肿瘤细胞杀伤作用。
C.Her3 ADC的NCI-H358肿瘤抑制活性测定
体外细胞活性检测方法：使用胰酶通过常规方法对NCI-H358肿瘤细胞进行消化，收集管细胞数，用对应的检测培养基(含2％FBS)重悬，将2000-5000细胞/孔加至96孔板。2％FBS培养基稀释的ADC加入100uL至96孔板，浓度从150μg/ml起始，3倍稀释(11个浓度梯度)。37℃5％CO2培养7天，然后每孔加入CCK8试剂20μL，反应至2-6小时，酶标仪读数 (检测波长为450nm)。各组样品和检测结果如表8所示。
表8
本实例测试结果表明，本公开的多种DAR值的ADC对肿瘤细胞均具有杀伤作用。
实施例16 anti-Her3抗体-药物偶联物和IgG-ADC体内活性测试
16.1 Her3抗体药物偶联物对NCI-H358移植瘤药效测试
通过评价抗Her3抗体ADC药物对Balb/c Nude小鼠皮下人非小细胞肺癌细胞株NCI-H358细胞(ATCC来源，Her3表达阳性率在10％左右)移植瘤模型的抗肿瘤作用来展示体内药效。
具体步骤如下：Balb/c nu裸小鼠，雌性，5-6周龄，购自维通利华实验动物有限公司。NCI-H358细胞用含10％FBS的1640培养基培养于15cm直径培养皿，达80-90％左右融合度时用胰蛋白酶-EDTA消化，用PBS洗两次，然后离心重悬于预冷PBS中，细胞计数仪计数，PBS稀释使细胞浓度为5×107/ml。Balb/c nu小鼠实验室环境适应7天，于右肋部皮下接种NCI-H358细胞，接种细胞量为5×106/只，接种体积为0.2ml(含50％Matrigel)，待肿瘤生长至300mm3左右时，根据肿瘤大小随机分组，每组5 只，分别为：抗鸡卵溶菌酶人IgG1同型对照抗体(阴性对照，苏州宜联生物)组、IgG1-ADC-07(DAR8)、Her3-C3-ADC-21(DAR4)，Her3-ADC-21(DAR4)以及Her3-ADC-07(DAR8)剂量组。所有样品均为尾静脉注射，每周1次，共给药3周。给药后观察并定期测量小鼠肿瘤体积及体重。相对肿瘤增殖率，T/C(％)，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。相对肿瘤抑制率，TGI(％)，计算公式为：TGI％＝(1-T/C)×100％(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV)或瘤重(TW))。
结果见表9及图7。
表9.Her3-ADC-07对人非小细胞肺癌NCI-H358裸小鼠皮下移植瘤的疗效
由实验结果可知，和IgG1组相比，5mg/kg和1mg/kg剂量Her3-ADC-07(DAR8)对NCI-H358人非小细胞肺癌移植瘤模型的肿瘤生长有显著抑制作用，并且呈现剂量梯度趋势。Her3-ADC-07 1mg/kg组TGI为76.26％，略强于Her3-ADC-21 5mg/kg剂量组的TGI 75.74％。Her3-ADC-21 1mg/kg组TGI为29.92％，略强于Her3-C3-ADC-21 1mg/kg剂量组的TGI 24.11％。
另外，动物体重结果显示，整个给药期以及恢复期间，各给药组动物耐受良好。
16.2.anti-Her3抗体-药物偶联物对SW480移植瘤药效测试之一
本公开通过评价抗Her3抗体ADC药物对Balb/c Nude小鼠皮下人结肠癌细胞株SW480细胞(ATCC来源，Her3表达阳性率在30％左右)移植瘤模型的抗肿瘤作用来展示体内药效。具体步骤如下：36只Balb/c nu裸小鼠，雌性，5-6周龄，购自维通利华实验动物有限公司。SW480细胞用含10％FBS的1640培养基培养于15cm直径培养皿，达80-90％左右融合度时用胰蛋白酶-EDTA消化，用PBS洗两次，然后离心重悬于预冷PBS中，细胞计数仪计数，PBS稀释使细胞浓度为5×107/ml。Balb/c nu小鼠实验室环境适应7天，于右肋部皮下接种SW480细胞，接种细胞量为5×106/只，接种体积为0.2ml(含50％Matrigel)，待肿瘤生长至250mm3左右时，根据肿瘤大小随机分组，每组5只，分别为抗鸡溶菌酶人IgG1同型对照抗体(阴性对照，苏州宜联生物)组、IgG1-ADC-07、以及Her3-ADC-07剂量组。所有样品均为尾静脉注射，每周1次，共给药3周。给药后观察并定期测量小鼠肿瘤体积及体重。
1)相对肿瘤增殖率，T/C(％)，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。
2)相对肿瘤抑制率，TGI(％)，计算公式为：TGI％＝(1-T/C)×100％(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV)或瘤重(TW))。
具体结果见表10及图8。由实验结果可知，10mg/kg和3mg/kg剂量Her3-ADC-07对SW480人结直肠癌移植瘤模型的肿瘤生长有显著一定抑制作用，并且呈现剂量梯度趋势。
表10 SW480+Balb/c Nude小鼠模型药效结果

16.3.anti-Her3抗体-药物偶联物对SW480移植瘤药效测试之二
参考实施例16.2.相同的模型和方法测定IgG1，IgG1-ADC-07(DAR8),Her3-ADC-07(DAR8),Her3-C3-ADC-21(DAR8)在人结肠癌细胞株SW480细胞(ATCC来源，Her3表达阳性率在30％左右)移植瘤模型的抗肿瘤作用。
具体测试结果见下表11及图9。
表11
由测试结果可知，10mg/kg和3mg/kg剂量Her3-ADC-07对SW480人结直肠癌移植瘤模型的肿瘤生长有显著抑制作用，并且呈现剂量梯度趋势。
难以预料的是，Her3-ADC-07(DAR8)3mg/kg组的抑瘤效果明显强于Her3-C3-ADC-21(DAR8)3mg/kg组，Her3-ADC-07(DAR8)10mg/kg组的抑瘤效果明显强于Her3-C3-ADC-21(DAR8)10mg/kg组。
另外，动物体重结果显示，整个给药期以及恢复期间，各给药组动物耐受良好。
实施例17.Her3抗体-药物偶联物的血浆稳定性测试
通过在孵育Her3-ADC-07(DAR8)的人血浆中测定生物活性分子A1的释放，评价Her3-ADC-07(DAR8)在血浆中的稳定性。
将Her3-ADC-07(DAR8)分别与过滤除菌后的小鼠、大鼠、犬、猴和人血浆进行三平行孵育。孵育在37℃、5％CO2条件下进行，Her3-ADC-07(DAR8)的终浓度为50μg/mL，孵育总时长为504小时。在孵育过程中分别在时间点0、24、72、168、336和504小时进行取样，加入含有内标(0.5μM甲苯磺丁脲)和0.1％甲酸的甲醇溶液终止反应。使用UPLC-MS/MS分析试验样品,分析条件为:
并计算生物活性分子A1的浓度和释放百分比，计算公式如下：
释放百分比＝所测时间的A1的浓度/理论上最大释放浓度；
理论上最大释放浓度＝ADC孵育浓度/ADC药物分子量×DAR×生物活性分子A1分子量。
在50μg/mL测试浓度下，Her3-ADC-07(DAR8)在小鼠、大鼠、犬、猴、人血浆中的稳定性结果见试验结果表12。
表12.Her3-ADC-07(DAR8)在小鼠、大鼠、犬、猴、人血浆中的A1释 放

*：一个平行中浓度低于定量下限，释放百分比报告为“<最大值”。
试验结果表明，在50μg/mL测试浓度下，Her3-ADC-07(DAR8)在小鼠、大鼠、犬、猴、人血浆中孵育504小时后，生物活性分子A1的释放百分比分别为0.726％、0.497％、0.521％、0.474和0.372％。既Her3-ADC-07(DAR8)在小鼠、大鼠、犬、猴、人血浆中的稳定性好，体外孵育504小时(21天)后，A1释放量小于理论上最大释放浓度的1％。
实施例18.Her3抗体-药物偶联物的安全性评价
治疗窗根据毒理试验中最高非严重毒性剂量(HNSTD)以及药效试验中最小有效剂量(MED)进行评估，Her3-ADC-07(DAR8)其治疗窗根据HNSTD和MED剂量下的体内暴露量比值计算。
18.1 GLP毒理试验
在Her3-ADC-07(DAR8)连续6周及恢复4周的GLP毒理试验中，每剂量 组10只动物，雌雄各半，通过静脉输液重复给予食蟹猴Her3-ADC-07(DAR8)，给药剂量为0、3、10和20mg/kg，每3周给药1次，连续给药3次，给药后恢复4周，评估Her3-ADC-07(DAR8)在食蟹猴中的毒性反应，并伴随进行毒代动力学(TK)研究检测血清样品中的ADC浓度。
GLP毒理试验结果表明，其最高非严重毒性剂量为10mg/kg，该剂量下，动物的临床观察、体重、血生化、凝血等均无明显异常。
对比Her3-ADC-07(DAR8)同类ADC产品，Patritumab Deruxtecan为第一三共的HER3靶点ADC，食蟹猴毒理试验的剂量为0、3、10和30mg/kg，每3周给药1次，连续给药5次。结果表明，在≥3mg/kg的剂量下可见网织红细胞、淋巴细胞和血小板下降；在≥10mg/kg的剂量下，转移酶升高；体重在各剂量下均无明显异常；毒性靶器官为胸腺、骨髓和皮肤(Hashimoto Y,et al.Clin Cancer Res.2019Dec 1；25(23):7151-7161)。
值得关注的是，Patritumab Deruxtecan在≥3mg/kg的剂量下即出现血小板下降，在其临床研究中，血小板下降是剂量爬坡试验中的剂量限制性毒性之一(PA,et al.Cancer Discov.2022Jan；12(1):74-89.)，也是多中心临床研究中最常出现(30％)的治疗期不良事件(TEAE)之一(Yu-H,et al.,Abstract#MA21.06,WCLC 2019)。而对比发现，Her3-ADC-07(DAR8)在最高剂量20mg/kg下未见血小板明显计数异常，且在最高剂量20mg/kg下，未见其在肝、肾、肺等重要脏器中的毒性反应。
18.2裸鼠PK/PD试验
NCI-H358荷瘤裸小鼠单次尾静脉注射给与0.3mg/kg或3mg/kg的Her3-ADC-07(DAR8)，在0h、3h、6h、24h、48h、96h、168h和336h采集血清及肿瘤(每个时间点5只动物)，分析肿瘤体积、重量的同时，检测血清中的ADC浓度。
NCI-H358荷瘤小鼠上单次静脉注射0.3mg/kg或3mg/kg的Her3-ADC-07(DAR8)后，≥0.3mg/kg剂量下可抑制肿瘤生长，说明Her3-ADC-07(DAR8)在该试验中最低起效剂量为0.3mg/kg。
将食蟹猴的最高非严重毒性剂量下的AUC0-336h或者Cmax与CDX小鼠最低起效剂量下的AUC0-336h或者Cmax的比值定义为治疗指数(TI)，各参数的治疗指数如下：
  治疗指数
AUC0-336h(μg/mL*h) 83.2
Cmax(μg/mL) 194.4
综上所述，Her3-ADC-07(DAR8)在食蟹猴10mg/kg剂量(HNSTD)下耐受良好，治疗指数接近100倍，且在该剂量下未发生血小板下降，肺、肝、肾等重要脏器中也未表现出毒性，以上结果均表明Her3-ADC-07(DAR8)在长期用药中具有较高的安全性。
尽管本公开的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本公开的保护范围之内。本公开的保护范围由所附权利要求及其任何等同物给出。

Claims (28)

  1. 一种结合Her3的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含如下的互补决定区(CDRs)：
    (a)SEQ ID NO:23、25或27所示的重链可变区VH中含有的HCDR1或其序列的变体、HCDR2或其序列的变体、以及HCDR3或其序列的变体；和/或
    (b)SEQ ID NO:24、26或28所示的轻链可变区VL中含有的LCDR1或其序列的变体、LCDR2或其序列的变体、以及LCDR3或其序列的变体；
    优选地，所述序列的变体为与其来源CDR相比为具有一个或几个氨基酸的置换、缺失或添加，例如1个、2个或3个氨基酸的置换、缺失或添加的CDR；更优选地，所述的置换为保守置换。
  2. 如权利要求1所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (1)VH和/或VL，其中按Kabat编号***定义：
    (a)所述VH包含：序列为SEQ ID NO:1的HCDR1，序列为SEQ ID NO:2的HCDR2，序列为SEQ ID NO:3的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:7的LCDR1，序列为SEQ ID NO:8的LCDR2，序列为SEQ ID NO:9的LCDR3；
    (b)所述VH包含：序列为SEQ ID NO:12的HCDR1，序列为SEQ ID NO:13的HCDR2，序列为SEQ ID NO:14的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:18的LCDR1，序列为SEQ ID NO:19的LCDR2，序列为SEQ ID NO:20的LCDR3；
    或，
    (2)VH和/或VL，其中按IMGT编号***定义：
    (a)所述VH包含：序列为SEQ ID NO:4的HCDR1，序列为SEQ ID NO:5的HCDR2，序列为SEQ ID NO:6的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:10的LCDR1，序列为AAS的LCDR2，序列为SEQ ID NO:9的LCDR3；
    (b)所述VH包含：序列为SEQ ID NO:15的HCDR1，序列为SEQ ID NO:16的HCDR2，序列为SEQ ID NO:17的HCDR3；和/或，
    所述VL包含：序列为SEQ ID NO:21的LCDR1，序列为AAS的LCDR2，序列为SEQ ID NO:20的LCDR3；
    可选地，所述的抗体或其抗原结合片段包含下述重链可变区VH和/或轻链可变区VL，其中，与所述Kabat编号***或IMGT编号***定义的CDR相比，所述重链可变区VH和/或轻链可变区VL中至少一个CDR含有突变，所述突变为一个或几个氨基酸的置换、缺失或添加或其任意组合，例如1个、2个或3个氨基酸的置换、缺失或添加或其任意组合；优选地，所述的置换为保守置换；
    更优选地，所述抗体或其抗原结合片段结合人Her3、猴Her3和/或大鼠Her3。
  3. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)SEQ ID NO:23、25或27任一项所示的VH，和/或，SEQ ID NO:24、26或28任一项所示的VL；
    (b)与(a)中的任一VH相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VH；和/或，与(a)中的任一VL相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性的VL；或者
    (c)与(a)中的任一VH相比具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合的VH；和/或，与(a)中的任一VL具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺 失或添加或其任意组合的VL；优选地，所述的置换是保守置换。
  4. 权利要求1-3任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)SEQ ID NO:23所示序列的VH和SEQ ID NO:24所示序列的VL；
    (b)SEQ ID NO:25所示序列的VH和SEQ ID NO:26所示序列的VL；
    (c)SEQ ID NO:27所示序列的VH和SEQ ID NO:28所示序列的VL；
    (d)VH和VL，与(a)至(c)任一组中的VH和VL相比，其VH具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或，其VL具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或者
    (e)VH和VL，与(a)至(c)任一组中的VH和VL相比，其VH具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合；和/或，其VL具有一个或几个氨基酸的置换、缺失或添加或其任意组合例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合；优选地，所述的置换是保守置换。
  5. 一种与权利要求1-4中任一项所述的抗体或其抗原结合片段竞争性结合的，且与权利要求1-4中任一项所述的抗体或其抗原结合片段不同的anti-Her3抗体或其抗原结合片段；优选地，所述anti-Her3抗体或其抗原结合片段与抗体202-2-1竞争性结合，其特征在于不为抗体202-2-1或其抗原结合片段，且与202-2-1或其抗原结合片段具有不同的CDRs；
    可选地，所述与抗体202-2-1竞争性结合的anti-Her3抗体或其抗原结合片段结合人Her3的domain 3，优选地，结合SEQ ID No.31所示人Her3的第328至499位氨基酸；优选地，所述与抗体202-2-1竞争性结合的anti- Her3抗体或其抗原结合片段结合如下空间表位：SEQ ID No.31所示的人Her3的第466位组氨酸，470位色氨酸，471位苏氨酸，478位苏氨酸，483位至487位(天冬氨酸-异亮氨酸-赖氨酸-组氨酸-天冬酰胺)，484位异亮氨酸，490位精氨酸和491位精氨酸；
    优选地，所述anti-Her3抗体或其抗原结合片段阻断Her3配体依赖性和Her3配体非依赖性信号转导；
    优选地，所述anti-Her3抗体或其抗原结合片段抑制Her3配体依赖性和Her3配体非依赖性AKT磷酸化。
  6. 权利要求1-5任一项所述抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段是嵌合抗体、人源化抗体或全人源抗体；
    可选地，所述抗体或其抗原结合片段选自Fab、Fab’、(Fab’)2、Fv片段例如scFv或二硫键连接的Fv(dsFv)、双抗体(diabody)和多特异性抗体。
  7. 权利要求1-6任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含：
    (a)人免疫球蛋白的重链恒定区CH或其变体；和/或
    (b)人免疫球蛋白的轻链恒定区CL或其变体，
    其中，所述变体与其所源自的野生型序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；或者，所述变体与其所源自的野生型序列相比具有一个或多个氨基酸的置换、缺失或添加或其任意组合例如至多50个、至多45个、至多40个、至多35个、至多30个、至多25个、至多20个、至多15个、至多10个或至多5个氨基酸的置换、缺失或添加或其任意组合；例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的置换、缺失或添加或其任意组合；优选地，所述的置换是保守置换；
    优选地，所述重链恒定区是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区；和/或，所述轻链恒定区是κ或λ轻链恒定区；
    更优选地，所述抗体或其抗原结合片段包含人IgG1重链恒定区；和/或，所述抗体或其抗原结合片段包含人κ轻链恒定区。
  8. 权利要求7所述的抗体或其抗原结合片段，其中，
    所述重链恒定区包括SEQ ID NO:29所示的CH或其变体，所述变体与SEQ ID NO:29相比具有至多20个氨基酸的保守置换例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换，例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的保守置换；或者与SEQ ID NO:29相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；和/或
    所述轻链恒定区包括SEQ ID NO:30所示的CL或其变体，所述变体与SEQ ID NO:30相比具有至多20个氨基酸的保守置换例如至多20个、至多15个、至多10个或至多5个氨基酸的保守置换，例如1个、2个、3个、4个、5个、6个、7个、8个、9个或10个氨基酸的保守置换，或者与SEQ ID NO:30相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％的序列同一性；
    优选地，所述抗体或其抗原结合片段包含SEQ ID NO:29所示的重链恒定区CH和SEQ ID NO:30所示的轻链恒定区CL。
  9. 权利要求1-8任一项所述的抗体或其抗原结合片段，其中，所述抗体选自以下组：
    (a)包括SEQ ID NO:23所示的VH和SEQ ID NO:29所示的CH的重链，和，包括SEQ ID NO:24所示的VL和SEQ ID NO:30所示的CL的轻链；优选包括SEQ ID NO:11所示的重链和SEQ ID NO:22所示的轻链；
    (b)包括SEQ ID NO:25所示的VH和SEQ ID NO:29所示的CH的重链，和，包括SEQ ID NO:26所示的VL和SEQ ID NO:30所示的CL的轻链；或
    (c)包括SEQ ID NO:27所示的VH和SEQ ID NO:29所示的CH的重链，和，包括SEQ ID NO:28所示的VL和SEQ ID NO:30所示的CL的轻链。
  10. 权利要求1-9中任一项所述的抗体，其中所述抗体是多特异性抗体；
    优选地，所述多特异性抗体是双特异性抗体或三特异性抗体或四特异性抗体。
  11. 分离的核酸分子，其编码权利要求1-10任一项所述的抗体或其抗原结合片段。
  12. 载体，其包含权利要求11所述的分离的核酸分子；优选地，所述载体为克隆载体或表达载体。
  13. 宿主细胞，其包含权利要求11所述的分离的核酸分子或权利要求12所述的载体。
  14. 制备权利要求1-10任一项所述的抗体或其抗原结合片段，其包括，在允许所述抗体或其抗原结合片段的条件下，培养权利要求13所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
  15. 一种抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其中抗体为权利要求1-10任一项所述的抗体或其抗原结合片段，其通过接头与偶联部分连接；
    所述偶联部分可选自：可检测的标记、放射性同位素、荧光物质、发光物质、有色物质、酶、聚乙二醇、核素、核酸、小分子毒素、具有结合活性多肽、蛋白、受体、配体，以及其它抑制肿瘤细胞生长、促进肿瘤细胞凋亡或坏死的活性物质。
  16. 如权利要求15所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    所述的抗体-药物偶联物如下式所示：
    Tb-(L-D)q,
    其中：
    Tb是如权利要求1-10中任一项所述的抗体或其抗原结合片段，并且；
    D是小分子毒素药物部分；
    L是键或连接分子，其共价连接Tb和D；
    q是1-16之间的整数，且表示共价连接至Tb的L-D的数目。
  17. 如权利要求16所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，
    所述的抗体-药物偶联物具有式I所示结构：
    其中，
    L1选自 各Z独立地选自直接键、碳碳三键、碳碳双键和酰胺基；Rx、Ry独立地选自H和C1-4烷基；各m独立地选自0、1、2、3、4、5和6；y1选自1-6之间任意整数；各y2独立地选自0-15之间任意整数；各y3独立地选自1、2和3；各y4独立地选自0和1；1位通过S原子与Tb相连，2位与L2或L3相连；
    L2不存在或存在，L2存在时，L2选自： y1选自1-6之间任意整数，各y2独立地选自0-10之间任意整数，各y3独立地选自1或2，各y4独立地选自0或1，1位与L1相连，2位与L3相连；
    L3选自氨基酸残基或由2-10个氨基酸残基组成的短肽；所述的氨基酸残基选自天然氨基酸残基、非天然氨基酸残基、或选自AA1所示氨基酸残基或其立体异构体；
    AA1所示的氨基酸残基中，Ra和Rb中任一个为H，另一个为H、或者，Ra与Rb和与它们共同相连的碳原子一起形成5-6元杂环，所述的5-6元杂环为哌啶环或哌嗪环，
    r、r1、r1a和r1b各自独立地为0、1、2、3、4或5；
    Rm1、Rn1、Rm1a、Rn1a、Rm1b和Rn1b各自独立地为H、C1-6烷基或-COORx1，其中，Rx1为C1-6烷基；
    或者，Rm1和Rn1、Rm1a和Rn1a、以及Rm1b和Rn1b和与它们共同相连的氮原子一起，形成5-6元杂环，所述的5-6元杂环中，杂原子选自1个或2个N原子；所述的5-6元杂环任选地被一个或多个R0’所取代；
    Rz选自C1-6烷基；
    R0、R0’各自独立地选自C1-6烷基、-NRm2Rn2或任选被C1-6烷基取代的5-6元杂环基；所述的5-6元杂环中，杂原子选自1个或2个N原子；
    Rm2和Rn2各自独立地选自H和C1-6烷基；
    L4不存在或存在，L4存在时，L4选自 1位与L3相连，2位与D相连；
    R1、R2各自独立地选自H、卤素和C1-4烷基；或者，R1和R2和与其相连的碳原子一起形成5-6元杂环，所述杂环含有1个、2个或3个O，S或N或其任意组合；
    R3选自H和C1-4烷基；或者，R3和X和与其相连的碳原子一起形成5-6元碳环；
    W不存在或存在，W存在时，W选自-O-、-S-、-NR4-、 1位与X相连，2位与L4或L3相连；
    X选自任选取代的-(CH2)n1-、1位和母环相连，2位和W或L4相连；所述取代基选自1个或2个C1-4烷基；
    R4、R5、R7各自独立地选自H和C1-4烷基；
    n、n1、n2、n3各自独立地选自0到6之间的任意整数。
  18. 如权利要求17所述的抗体-药物偶联物、其立体异构体、其前药、 其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一种或多种：
    (1)L1选自 1位通过S原子与Tb相连，2位与L2或L3相连；
    (2)L2不存在或存在，L2存在时，L2选自 1位与L1相连，2位与L3相连；
    (3)L3选自AA1、AA1-Gly、Val-Cit、Val-AA1-Gly、AA1-Ala-Asn和Gly-Gly-Phe-Gly；
    (4)AA1所示氨基酸残基选自
    (5)L4不存在或存在，L4存在时，L4选自 1位与L3相连，2位与D相连；
    (6)R1选自H和卤素，R2选自H和C1-4烷基；或者，R1和R2和与其相连的碳原子形成虚线表示所述杂环与苯环稠合的位置；
    (7)R3为H或R3和X和与其相连的碳原子一起形成虚线表示所述碳环与苯环和吡啶环稠合的位置；
    (8)各R4独立地选自H、甲基、乙基、正丙基、异丙基和叔丁基，R5为H；
    (9)各R7独立地选自H和C1-4烷基；
    (10)n选自1、2和3；
    (11)n1选自1、2、3和4；
    (12)n2为1；
    (13)n3为0；
    (14)W选自-O-、-NR4-和1位X相连，2位与L4或L3相连；
    (15)X选自任选取代的 1位和母环相连，2位和W或L4相连；所述取代基选自1个或2个 C1-4烷基，或者2个C1-4烷基。
  19. 如权利要求18所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一种或多种：
    (1)L1选自1位通过S原子与Tb相连，2位与L2或L3相连；
    (2)L2不存在；
    (3)L3选自 1位与L1或L2相连，2位与L4或D相连；
    (4)L4选自1位与L3相连，2位与D相连；
    (5)R1为H或F，R2为H或甲基；或者，R1和R2和与其相连的碳原子一起形成
    (6)R3为H；
    (7)W选自-O-、-NR4-和1位X相连，2位与L4或L3相连；
    (8)X选自1位和母环相连，2位和W相连。
  20. 如权利要求17或18所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一种或多种：
    (1)的结构选自以下结构片段：
    其中，1位与Tb相连，2位与D相连；
    例如，的结构选自以下结构片段：


    其中，1位与Tb相连，2位与W相连；
    (2)所示的结构片段为 1位与L4连接；
    例如，的结构选自以下结构片段：


    (3)的结构选自以下结构片段：
    其中，1位通过S原子与Tb相连，2位与W相连；
    (4).的结构选自以下结构片段：
    其中，1位与L4连接。
  21. 如权利要求17或18所述的抗体-药物偶联物、其立体异构体、其前 药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，其满足如下条件中的一种或多种：
    (1)所述的抗体-药物偶联物具有式I-1、I-2、I-3所示结构：
    (2)所述的抗体-药物偶联物具有式I-1A、I-1B、式I-2A、I-2B、式I-3A或I-3B所示结构：

    (3)所述的抗体-药物偶联物具有式I-A、I-B所示结构：

    其中，Tb、L1、L2、L3、L4、X、R1、R2、R3、R4、R5、Ra、Rb、n和q定义如权利要求16至20中任一项所述。
  22. 如权利要求15至21中任一项所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，所述的抗体-药物偶联物选自：




    其中，
    Tb为如上任一权利要求所述的抗Her3抗体，
    q为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16；q优选1、2、3、4、5、6、7、8；
    例如，

    其中，Her3mAb为202-2-1抗体。
  23. 如权利要求15至21中任一项所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物，其特征在于，所述的抗体-药物偶联物选自：
    其中，Tb为如上任一权利要求所述的抗Her3抗体，q为1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16；q优选1、2、3、4、5、6、7、8；
    例如，

    其中，
    Her3mAb为202-2-1抗体。
  24. 一种抗体-药物偶联物，其包含如权利要求16至23中任一项所述的 抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物中的两种或多种，所述抗体-药物偶联物中的抗体-药物偶联物具有一种、两种或多种q值。
  25. 如权利要求24所述的抗体-药物偶联物，其特征在于，
    所述抗体-药物偶联物中的药物与抗体的比例(DAR)选自1-10中的整数或小数；
    例如选自：2、2.5、3、3.5、4、4.5,5、5.5、6、6.5、7、7.2、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4、8.5、8.7、8.9和9。
  26. 药物组合物，其包含权利要求1-10任一项所述的抗体或其抗原结合片段、权利要求11所述的核酸分子、权利要求12所述的载体、权利要求13所述的宿主细胞，权利要求15至23所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物、权利要求24至25所述的抗体-药物偶联物以及药学上可接受的载体和/或赋形剂。
  27. 权利要求1-10任一项所述的抗体或其抗原结合片段、权利要求11所述的核酸分子、权利要求12所述的载体、权利要求13所述的宿主细胞、权利要求15至23所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物、权利要求24至25所述的抗体-药物偶联物和/或权利要求26所述的药物组合物，在制备用于预防和/或***和/或传染病的药物中的用途；
    可选地，所述肿瘤为实体肿瘤；
    可选地，所述肿瘤选自食管癌(例如食管腺癌和食管鳞状细胞癌)、脑瘤、肺癌(例如小细胞性肺癌和非小细胞性肺癌)、鳞状上皮细胞癌、膀胱癌、胃癌、卵巢癌、腹膜癌、胰腺癌、乳腺癌、头颈癌、子***、子宫内膜癌、结直肠癌、肝癌、肾癌、尿路上皮癌、实体瘤、非霍奇金淋巴瘤、中枢神经***肿瘤(例如神经胶质瘤、多形性胶质母细胞瘤、胶质瘤或肉瘤)、***癌或甲状腺癌。
    可选地，所述肿瘤选自：结直肠癌(如结肠癌)，肺癌(如非小细胞 肺癌)，乳腺癌，***癌。
  28. 一种用于在受试者中预防和/或***和/或传染病的方法，所述方法包括向有此需要的受试者施用有效量的权利要求1-10任一项所述的抗体或其抗原结合片段、权利要求11所述的核酸分子、权利要求12所述的载体，权利要求13所述的宿主细胞、权利要求15至23所述的抗体-药物偶联物、其立体异构体、其前药、其药学上可接受的盐或其药学上可接受的溶剂合物、权利要求24至25所述的抗体-药物偶联物和/或权利要求26所述的药物组合物，其中所述受试者是哺乳动物；
    优选地，所述受试者是人。
PCT/CN2023/072657 2022-01-25 2023-01-17 一种针对Her3的抗体，偶联物及其用途 WO2023143263A1 (zh)

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CN106163559A (zh) * 2014-04-10 2016-11-23 第三共株式会社 抗her3抗体‑药物偶联物
CN105367657A (zh) * 2014-08-14 2016-03-02 上海生物制品研究所有限责任公司 抗her3抗体、其制法及其应用
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