WO2023143365A1 - Her3抗体药物偶联物及其用途 - Google Patents

Her3抗体药物偶联物及其用途 Download PDF

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WO2023143365A1
WO2023143365A1 PCT/CN2023/073130 CN2023073130W WO2023143365A1 WO 2023143365 A1 WO2023143365 A1 WO 2023143365A1 CN 2023073130 W CN2023073130 W CN 2023073130W WO 2023143365 A1 WO2023143365 A1 WO 2023143365A1
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Prior art keywords
drug conjugate
seq
pharmaceutically acceptable
her3 antibody
isomer
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PCT/CN2023/073130
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English (en)
French (fr)
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朱忠远
张禹
李曦
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映恩生物制药（苏州）有限公司
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Publication of WO2023143365A1 publication Critical patent/WO2023143365A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention provides an antibody-drug conjugate specifically binding to Her3 and a composition comprising it. Methods and uses of using the antibody drug conjugates of the invention are also provided.
  • Epidermal growth factor receptor is a huge transmembrane glycoprotein with a molecular weight of about 170KDa and belongs to a member of the ErbB receptor family.
  • the EGFR receptor itself is a tyrosine kinase, which can form dimers when combined with ligands such as EGF, TNF-a, etc., and activate downstream signals (such as MAPK, PI3K, Stat, etc. pathways) by transmitting phosphorylation. Thereby maintaining cell growth and promoting cell division and proliferation.
  • EGFR Due to the conservation of ErbB family receptors, EGFR can also form heterodimers with other family proteins (such as Her2, Her3, Her4), thereby regulating cell growth more extensively.
  • Her3 a member of the ErbB family, plays a key role in cell proliferation, tumor metastasis and drug resistance. While drugs targeting EGFR and Her2 have shown enormous clinical benefit in the mitigation of multiple cancers, previous efforts to develop anti-Her3 antibodies for cancer therapy have repeatedly failed, suggesting that addressing Her3 alone and its pending pathways may not be sufficient Inhibits tumor growth.
  • Antibody drug conjugate consists of three parts: antibody or its antigen-binding fragment (targeting), linker and small molecule drug.
  • Antibodies or antigen-binding fragments thereof are conjugated to small molecule drugs with biological activity such as cytotoxicity such as cytotoxins via cleavable or non-cleavable linkers, making full use of antibodies or antigen-binding fragments thereof to target cells of interest (targeting cells) or the specificity of binding highly expressed antigens and the high efficiency of small molecule drugs, reducing or avoiding the toxic side effects on non-target cells.
  • cytotoxicity such as cytotoxins
  • target cells target cells of interest
  • specificity of binding highly expressed antigens and the high efficiency of small molecule drugs reducing or avoiding the toxic side effects on non-target cells.
  • Anti-Her3 antibody-drug conjugates excellent in affinity, specificity, etc. are still needed in the art.
  • the present application provides an anti-Her3 antibody-drug conjugate, its isomer, a pharmaceutically acceptable salt or a mixture thereof, the structure of the anti-Her3 antibody-drug conjugate is shown in formula (I):
  • D is a cytotoxic drug
  • p represents the average number of connections, and p is selected from integers or decimals of 1 to 10, preferably integers or decimals of 3-8;
  • Ab is an anti-Her3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 2 and SEQ ID NO: 2, respectively.
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR1 shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively LCDR3.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein the structure of the anti-Her3 antibody drug conjugate is shown in formula (I- 1) as shown:
  • n represents the average connection number, and n is selected from integers or decimals of 1 to 10, preferably integers or decimals of 3-8;
  • Ab is an anti-Her3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 2 and SEQ ID NO: 2, respectively.
  • the light chain variable region comprises amino acid sequences such as LCDR1, LCDR2 and LCDR1 shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively LCDR3.
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention comprises:
  • a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:7 or at least 95%, 96%, 97%, 98% or 99% identity thereto, and an amino acid sequence as shown in SEQ ID NO:8
  • a light chain variable region showing or having at least 95%, 96%, 97%, 98% or 99% identity therewith;
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention comprises:
  • the anti-Her3 antibody or antigen-binding fragment of the present invention is a murine antibody or a fragment thereof, a chimeric antibody or an antigen-binding fragment, a humanized antibody or an antigen-binding fragment, or a fully human antibody or an antigen-binding fragment .
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention is a humanized antibody or fragment thereof.
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention is selected from Fab, Fab', Fab'-SH, Fv, scFv, F(ab') 2 , sdAb, diabody or linear antibody.
  • the anti-Her3 antibody of the present invention is a monoclonal antibody.
  • the antibody of the invention is an antibody in IgG1 format, an antibody in IgG2 format, an antibody in IgG3 format, or an antibody in IgG4 format.
  • the antibodies of the invention are antibodies in the IgG1 format.
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention comprises:
  • a heavy chain having an amino acid sequence as shown in SEQ ID NO: 11 or having at least 95%, 96%, 97%, 98% or 99% identity therewith, and an amino acid sequence as shown in SEQ ID NO: 12 or with it A light chain having at least 95%, 96%, 97%, 98% or 99% identity; or
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention comprises:
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein
  • M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O- or -S-;
  • L 3 is -(C(R 1a )(R 1b )) m -, m is selected from 0, 1, 2 or 3, wherein when L 3 contains a methylene unit, 0 or 1 of said L 3
  • the methylene unit can be replaced by -C(O)-, or -C(S)-;
  • L 1 is -(C(R 2a )(R 2b )) n -, n is selected from 1, 2 or 3, wherein when L 1 may contain methylene units, 0 or 1 methylene units of said L 1
  • the methyl unit can be replaced by -C(O)-, or -C(S)-;
  • X is selected from 3 to 6-membered saturated carbocyclyl, 3 to 6-membered saturated heterocyclyl or a single bond, the 3 to 6-membered saturated carbocyclyl and 3 to 6-membered saturated heterocyclyl are optionally 0, 1, 2 or 3 R 3a substitutions;
  • each R 1a , R 1b , R 2a , R 2b , R 3a each independently may be hydrogen, halogen or a C 1-6 aliphatic group which may be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the heteroatoms in the 3-6 membered saturated heterocyclic group of the present invention are selected from N, O and S, and the number of heteroatoms is 1-3.
  • L 3 and X of the present invention are not single bonds at the same time.
  • L2 of the present invention is -O-. In some embodiments, L2 of the present invention is -S-.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein the linker unit M, its L 2 end is connected with the linker unit L Linked, L1 end linked with cytotoxic drug D.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein L 3 is selected from single bond, -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )-;
  • each of R 1a and R 1b independently may be hydrogen, halogen, or a C 1-6 aliphatic group that may be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein L 3 is selected from single bond, -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )-;
  • each R 1a and R 1b independently may be hydrogen, halogen, CH 3 or CH 2 CH 3 .
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 3 is selected from single bond, -CH 2 -, -CH( CH3 )-, -C( CH3 ) 2- , -CH2CH2- , -CH( CH3 ) CH2- or -C( CH3 ) 2CH2- .
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L3 is selected from
  • the anti-Her3 antibody-drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or a mixture thereof, wherein L3 is selected from a single bond.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein L 1 is selected from -C(R 2a )(R 2b )- , -C(R 2a )(R 2b )C(O)- or -C(O)-;
  • each R 2a , R 2b each independently may be hydrogen, halogen or a C 1-6 aliphatic group which may be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein L 1 is selected from -C(R 2a )(R 2b )- , -C(R 2a )(R 2b )C(O)- or -C(O)-;
  • R 2a and R 2b independently can be hydrogen, halogen, CH 3 or CH 2 CH 3 .
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein L 1 is selected from -CH 2 -, -CH 2 C(O )-, -CH(CH 3 )C(O)- or -C(O)-.
  • the anti-Her3 antibody-drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein L 1 is selected from -C(O)-.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is optionally replaced by 0, 1, 2 or 3 R 3a Substituted 3- to 6-membered saturated carbocyclyl or single bond;
  • each R 3a independently can be hydrogen, halogen or a C 1-6 aliphatic group which can be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is optionally replaced by 0, 1, 2 or 3 R 3a Substituted 3- to 6-membered saturated carbocyclyl or single bond;
  • each R 3a independently can be hydrogen, halogen, CH 3 or CH 2 CH 3 .
  • the anti-Her3 antibody drug conjugates of the present invention their isomers, and their pharmaceutically acceptable or a mixture thereof, wherein X is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or a single bond.
  • the anti-Her3 antibody-drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is a single bond.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is cyclopropyl, cyclobutyl, cyclopentyl or Cyclohexyl, preferably cyclobutyl.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein X is Preferably, X is
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • L 2 is -O- or -S-;
  • X is selected from 3 to 6 membered saturated carbocyclyl optionally substituted by 0, 1, 2 or 3 R 3a ;
  • L 1 is selected from -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-;
  • each R 2a , R 2b or R 3a independently can be hydrogen, halogen or a C 1-6 aliphatic group which can be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • L 2 is -O- or -S-;
  • X is selected from 3 to 6 membered saturated carbocyclyl optionally substituted by 0, 1, 2 or 3 R 3a ;
  • each R 3a independently can be hydrogen, halogen or a C 1-6 aliphatic group which can be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • L 2 is -O- or -S-;
  • X is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • L 2 is -O- or -S-;
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • L 2 is -O- or -S-;
  • L 3 is selected from -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )-;
  • L 1 is selected from -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-;
  • each R 1a , R 1b , R 2a or R 2b independently can be hydrogen, halogen or a C 1-6 aliphatic group which can be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • L 2 is -O- or -S-;
  • L 3 is selected from -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )-;
  • each of R 1a and R 1b independently may be hydrogen, halogen, or a C 1-6 aliphatic group that may be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • L 2 is -O- or -S-;
  • L 3 is selected from -CH 2 -, -CH(CH 3 ), -C(CH 3 ) 2 , -CH 2 CH 2 -, -CH(CH 3 )CH 2 - or -C(CH 3 ) 2 CH 2 -.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable A salt or a mixture thereof, wherein said -M- is selected from:
  • L 2 is -O- or -S-;
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein the linker unit M, its L 2 end is connected with the linker unit L connected.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is -L a -L b -L c -,
  • the -L a - is
  • W is -(C(R wa )(R wb )) wn -
  • Y is -(OCH 2 CH 2 ) yn -O yp -
  • Z is -(C(R za )(R zb )) zn ;
  • wn 1, 2, 3 or 6
  • One methylene unit of W is independently replaced by -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )-, or -C(O)-;
  • One methylene unit of Z is independently replaced by -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )-, or -C(O)-;
  • -Cyr- is a 3 to 10 membered saturated carbocyclylene, wherein said -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R cx ;
  • each R wa , R wb , R za , R zb , R wx , R zx , R cx are each independently hydrogen, halogen, -OR r or a C 1-6 aliphatic group optionally substituted by R r ;
  • each R r is independently hydrogen, halogen or C 1-6 aliphatic group
  • R L1 and R L2 are each independently selected from the following group: hydrogen, halogen, -OH and C 1-6 aliphatic group.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is -L a -L b -L c -,
  • the -L a - is
  • W is -(C(R wa )(R wb )) wn -
  • Y is -(OCH 2 CH 2 ) yn -O yp -
  • Z is -(C(R za )(R zb )) zn ;
  • wn 1, 2, 3 or 6
  • One methylene unit of W is independently replaced by -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )-, or -C(O)-;
  • One methylene unit of Z is independently replaced by -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )-, or -C(O)-;
  • -Cyr- is a 3 to 10 membered saturated carbocyclylene, wherein said -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R cx ;
  • each R wa , R wb , R za , R zb , R wx , R zx , R cx are each independently hydrogen, halogen, -OR r or a C 1-6 aliphatic group optionally substituted by R r ;
  • each R r is independently hydrogen, halogen or C 1-6 aliphatic group
  • the -L b - is selected from the following group:
  • R L1 and R L2 are each independently selected from the following group: hydrogen, halogen, -OH and C 1-6 aliphatic group.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L a - is preferably
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L a - is
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L b - is preferably
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -L c - is
  • the anti-Her3 antibody-drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein the linker unit L, its L a end is connected to Ab, The L c- terminus is connected to the linker unit M.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is
  • the anti-Her3 antibody-drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein the structure of the anti-Her3 antibody-drug conjugate is shown in formula (II- 1) or (II-2):
  • the anti-Her3 antibody-drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein the structure of the anti-Her3 antibody-drug conjugate is shown in formula (II- 1) or (II-2):
  • p represents the average number of connections, and p is selected from integers or decimals of 1 to 10, preferably integers or decimals of 3-8;
  • L 2 is -O- or -S-;
  • X is selected from 3 to 6 membered saturated carbocyclyl optionally substituted by 0, 1, 2 or 3 R 3a ;
  • L 3 is selected from -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )-;
  • each R 1a , R 1b or R 3a independently may be hydrogen, halogen or a C 1-6 aliphatic group which may be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody-drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein the structure of the anti-Her3 antibody-drug conjugate is shown in formula (II- 1) or (II-2):
  • p represents the average number of connections, and p is selected from integers or decimals of 1 to 10, preferably integers or decimals of 3-8;
  • L 2 is -O- or -S-;
  • X is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;
  • L 3 is selected from -CH 2 -, -CH(CH 3 )-, -C(CH 3 ) 2 -, -CH 2 CH 2 -, -CH(CH 3 )CH 2 - or -C(CH 3 ) 2 CH2- .
  • p represents the average number of connections, and p is selected from integers or decimals of 1 to 10, preferably integers or decimals of 3-8;
  • the present invention provides an anti-Her3 antibody-drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, wherein the anti-Her3 antibody-drug conjugate is selected from:
  • p represents the average connection number, and p is selected from an integer or a decimal of 1 to 10, preferably an integer or a decimal of 3-8.
  • the hu3F8-2 and hu3F8-3 of the present invention are anti-Her2 antibodies, the heavy chain amino acid sequence of the anti-Her2 antibody hu3F8-2 is shown in SEQ ID NO.:13, and the light chain amino acid sequence is shown in SEQ ID NO.:12 ; The heavy chain amino acid sequence of the anti-Her2 antibody hu3F8-3 is shown in SEQ ID NO.:14, and the light chain amino acid sequence is shown in SEQ ID NO.:12.
  • the average number of connections p in the present invention may be an integer or decimal from 2 to 8.
  • the average number of connections n may be an integer or decimal from 3 to 8.
  • the average connection number n may be an integer or a decimal of 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-Her3 antibody drug conjugate as described herein, its isomer, its pharmaceutically acceptable salt or its mixture, and a pharmaceutically acceptable acceptable carrier or excipient.
  • the present invention provides an anti-Her3 antibody drug conjugate as described herein, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof or a pharmaceutical composition for use in the treatment and/or prevention of Her3 Use in medicine for a disease or condition mediated, preferably, the disease or condition is cancer.
  • the present invention provides a method for treating and/or preventing a Her3-mediated disease or disorder, comprising administering to a subject in need an anti-Her3 antibody drug conjugate as described herein, its Isomers, pharmaceutically acceptable salts thereof or mixtures thereof or pharmaceutical compositions, preferably, the disease or condition is cancer.
  • the present invention provides an anti-Her3 antibody drug conjugate as described herein, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof or a pharmaceutical composition for use in the treatment and/or prevention of A Her3-mediated disease or disorder, preferably, the disease or disorder is cancer.
  • the cancer of the present invention is selected from lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer , bladder cancer, stomach cancer and esophageal cancer.
  • the present invention provides a pharmaceutical combination comprising an antibody drug conjugate as described herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described herein, and one or more additional therapeutic agent.
  • the present invention provides a kit comprising the antibody drug conjugate as described herein, or the pharmaceutical composition as described herein.
  • FIG. 1 Murine 3F8 specifically binds to SP2/0-HER3 cells.
  • Figure 2 Determination of the binding affinity of murine 3F8 to human HER3, HER2 and EGFR by ELISA.
  • Figure 3 The potency of murine 3F8 to recognize human and monkey HER3 was determined by ELISA.
  • FIG. 4 Murine 3F8 blocks NRG1-induced HER3 phosphorylation.
  • FIG. 5 Murine 3F8 is rapidly taken up by cells with different surface levels of HER3.
  • Figure 7 [ 89 Zr]Zr-ch3F8 imaging of gastric PDX model GAS078.
  • Figure 8 Representative [ 89Zr ]Zr-ch3F8 imaging in six PDX phantoms.
  • Figures 9A-9C hu3F8 maintains binding affinity after heat, acid and repeated freeze-thaw stress tests.
  • Figure 9A Three clones of hu3F8 were incubated at pH 3.5 for 0, 2, 4 and 6 hours, followed by ELISA assays to measure binding affinities.
  • Figure 9B Three clones of hu3F8 were incubated at 40°C for different days, followed by ELISA assays to measure binding affinities.
  • Figure 9C Three clones of hu3F8 were freeze-thawed repeatedly for 3 or 5 cycles, followed by ELISA assays to measure binding affinities.
  • FIG. 10 Endocytic activity of antibody drug conjugates.
  • Figure 11A-11C In vitro proliferation inhibition test of antibody-drug conjugates ADC-1-X1 and ADC-1-X2 on HER3-targeted tumor cells;
  • Figure 11A shows HCC1569 tumor cells with high expression of Her3;
  • Figure 11B shows high expression of Her3 MDA-MB-453 tumor cells;
  • Figure 11C shows MDA-MB-231 tumor cells that do not express Her3.
  • FIGS. 12A-12B In vivo anti-tumor efficacy of antibody-drug conjugates ADC-1-X1 and ADC-1-X2 on HCC1569 tumor-bearing mice.
  • Figure 12A shows the change in tumor volume.
  • Figure 12B is the tumor inhibition rate.
  • FIGS. 13A-13B In vivo anti-tumor efficacy of antibody-drug conjugates ADC-1-X1 and ADC-1-X2 on Colo205 tumor-bearing mice.
  • Figure 13A shows the change in tumor volume.
  • Figure 13B is the tumor inhibition rate.
  • Figure 14 In vivo anti-tumor efficacy of antibody-drug conjugate ADC-1-X2 on NCI-H441 lung cancer cell tumor-bearing mice.
  • Figure 15 In vivo anti-tumor efficacy of antibody-drug conjugate ADC-1-X2 on human lung cancer LU1542 subcutaneously xenografted tumor-bearing mice.
  • the term "about” generally means within a range of 0.5% to 10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% above or below the specified value , 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • human epidermal growth factor receptor 3 also known as receptor tyrosine protein kinase ERBB-3 (ERBB3)
  • ERBB3 receptor tyrosine protein kinase ERBB-3
  • Her3 is a member of the EGFR/erBB family. Unlike other erBB family members Her2 and EGFR, Her3 itself does not have kinase activity. Therefore, Her3 must bind to its kinase-active member EGFR or Her2 as a heterodimer to trigger its downstream activity. After binding to its natural ligand NRG1, Her3 undergoes conformational changes, heterodimerization and phosphorylation, and then activates MAPK, PI3K/Akt and PLC ⁇ through signal transduction.
  • antibody refers to any form of antibody that possesses the desired biological activity. Accordingly, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier "monoclonal” characterizes an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • full-length antibody refers to an immunoglobulin molecule that, as it occurs in nature, comprises four peptide chains: two heavy (H) chains (about 50-70 kDa in full length) and two light (L) chains (about 25 kDa) are interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • VH and VL regions can be further subdivided into complementarity determining regions (CDRs) of high variability separated by more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • CDR refers to the complementarity determining regions within the variable sequences of an antibody.
  • the precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al.
  • the boundaries of the CDRs of the variable region of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems are different.
  • the scope of said antibody also covers antibodies whose variable region sequences comprise said particular CDR sequence, but due to the application of a different protocol (e.g. Different assignment systems or combinations) cause the claimed CDR boundaries to be different from the specific CDR boundaries defined in the present invention.
  • antibody-binding fragment of an antibody (“parent antibody”) includes fragments or derivatives of the antibody, typically including at least a fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains the parent antibody. At least some binding specificity of the antibody.
  • antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules such as scFv; specific antibody.
  • the binding fragment or derivative typically retains at least 10% of its antigen binding activity when the antigen binding activity is expressed on a molar concentration basis.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody.
  • antigen-binding fragments of antibodies may include conservative or non-conservative amino acid substitutions that do not appreciably alter their biological activity (referred to as “conservative variants” or “functionally conservative variants” of the antibody).
  • chimeric antibody is an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first and second antibodies are from different species.
  • the variable domains are derived from an antibody of a rodent, etc. (the "parent antibody”), while the constant domain sequences are derived from a human antibody such that the resulting chimeric antibody induces greater The likelihood of an adverse immune response is low.
  • humanized antibody refers to forms of antibodies that contain sequences from both human and non-human (eg, mouse, rat) antibodies.
  • a humanized antibody will comprise substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin.
  • Framework (FR) regions are the framework regions of human immunoglobulin sequences.
  • a humanized antibody optionally can comprise at least a portion of a human immunoglobulin constant region (Fc).
  • Fully human antibody refers to an antibody that comprises only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains as produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Likewise, “mouse antibody” refers to an antibody comprising only mouse immunoglobulin sequences. Alternatively, fully human antibodies may contain rat sugar chains if produced in rats, in rat cells, or in hybridomas derived from rat cells. Likewise, “rat antibody” refers to an antibody comprising only rat immunoglobulin sequences.
  • isotype of an antibody refers to the antibody class (eg, IgM, IgE, IgG such as IgG1, IgG2 or IgG4) that is contributed by the heavy chain constant region genes. Isotypes also include modified forms of one of these classes, wherein modifications have been made to alter Fc function, for example to enhance or decrease effector function or binding to Fc receptors.
  • Fc region is used to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxy-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, as in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed . Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
  • cross-reactivity refers to the binding of antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat). Thus, “cross-reactivity” should be understood as an interspecies reaction with the same molecule X expressed in a different species.
  • the cross-reactive specificity of monoclonal antibodies recognizing human Her3R, monkey, and/or murine Her3R (mouse or rat) can be determined by FACS analysis.
  • affinity refers to an intrinsic binding affinity that reflects the interaction between members of a binding pair.
  • the affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio Kinetic Binding Assay herein.
  • the term "does not bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to it with high affinity, that is, the K D of the binding protein or cell is 1.0 ⁇ 10 ⁇ 6 M or higher, more preferably 1.0 ⁇ 10 -5 M or higher, more preferably 1.0 ⁇ 10 -4 M or higher, 1.0 ⁇ 10 -3 M or higher, more preferably 1.0 ⁇ 10 -2 M or higher.
  • high affinity for an IgG antibody means that the K D for the antigen is 1.0 ⁇ 10 -6 M or lower, preferably 5.0 ⁇ 10 -8 M or lower, more preferably 1.0 ⁇ 10 -8 M or lower, 5.0 ⁇ 10 -9 M or lower, more preferably 1.0 ⁇ 10 -9 M or lower.
  • “High affinity” binding may vary for other antibody subtypes.
  • “high affinity” binding of IgM subtype refers to a KD of 10 -6 M or lower, preferably 10 -7 M or lower, more preferably 10 -8 M or lower.
  • cytotoxic drug generally refers to a toxic drug that can have strong chemical molecules inside tumor cells that disrupt their normal growth. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations.
  • the "cytotoxic drugs” may include toxins, such as small molecule toxins or enzyme active toxins from bacteria, fungi, plants or animals, radioactive isotopes (such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 or radioisotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolysins, for example, can be toxic drugs, including but not limited to camptothecin derivatives, for example, can be Dendritic derivatives Exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H - Benzo[de]pyrano[3
  • linker unit or "linker structure” usually refers to a chemical structural fragment or bond that is connected to a ligand at one end and a cytotoxic drug at the other end, and can also be connected to other linkers before being connected to a cytotoxic drug.
  • the direct or indirect connection to the ligand may mean that the group is directly connected to the ligand through a covalent bond, or may be connected to the ligand through a linker structure.
  • the linker structure may be the structures shown in -Lax-Lb-Lc- and or -La-Lb-Lc- described in this application.
  • chemical fragments comprising acid-labile linker structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linker structures, photolabile linker structures, dimethyl linker structures, or disulfide-containing linker structures can be used or The bond acts as a linker structure.
  • acid-labile linker structures e.g., hydrazones
  • protease-sensitive linker structures e.g., peptidase-sensitive linker structures
  • photolabile linker structures e.g., dimethyl linker structures
  • disulfide-containing linker structures e.g., peptide-sensitive linker structures
  • the bond acts as a linker structure.
  • a structure is "optionally linked to other molecular moieties” generally means that the structure is not linked to any other chemical structure, or that the structure is linked to one or more other chemical structures different from the structure (such as the present application).
  • Ligands described above are connected (for example, connected by a chemical bond, or connected by a linker structure).
  • ligand-drug conjugate generally refers to a ligand linked to a biologically active cytotoxic drug through a stable linker unit.
  • ligand-drug conjugate may be an antibody-drug conjugate (antibody drug conjugate, ADC), and the ADC may refer to a monoclonal antibody or an antibody fragment through a stable linking unit with a biological active cytotoxic drugs.
  • methylene generally refers to a residue derived from a group of 1 carbon atom by removing two hydrogen atoms. Methylene groups can be substituted or unsubstituted, substituted or unsubstituted.
  • alkylene generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived by removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, which It may be a linear or branched chain group containing 1 to 20 carbon atoms, for example an alkylene group containing 1 to 12 carbon atoms, for example an alkylene group containing 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene include, but are not limited to, methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (- CH 2 CH 2 CH 2 CH 2 CH 2 -), etc.
  • Alkylene may be substituted or unsubstituted, substituted or unsubstituted, for example when substituted, substituents may be substituted at any available point of attachment, said substituents are preferably independently optionally selected from alkyl radical, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy
  • substituents in group, heterocycloalkoxy group, cycloalkylthio group, heterocycloalkylthio group and oxo group such as hydrogen, protium, deuterium, tritium, halogen, -NO 2 , -CN, -OH, -SH, -NH2 , -C(O)H, -CO2H , -C(O)C(O)H, -C(O)
  • arylene generally refers to a residue having two hydrogen atoms removed from the same carbon atom or two different carbon atoms of an aromatic ring.
  • aromatic ring may refer to a 6 to 14 membered all-carbon monocyclic or fused polycyclic ring (that is, a ring sharing adjacent pairs of carbon atoms) having a conjugated ⁇ -electron system, which may be 6 to 10 membered, such as benzene and naphthalene.
  • the aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring where the ring bonded to the parent structure is an aryl ring.
  • Aryl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alk Thio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio , and heterocycloalkylthio.
  • heteroarylene generally refers to a residue having two hydrogen atoms removed from the same carbon atom or two different carbon atoms of a heteroaryl ring.
  • heteroaryl ring refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms may be selected from the group consisting of oxygen, sulfur and nitrogen.
  • Heteroaryl can be 5 to 10 membered, can be 5 or 6 membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazole Base etc.
  • the heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring bonded to the parent structure is a heteroaryl ring.
  • Heteroarylene may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkane Oxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, Cycloalkylthio, and heterocycloalkylthio.
  • the substituent is preferably one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkane Oxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloal
  • heterocyclylene generally refers to a stable non-aromatic 3-7 membered monocyclic structure, a fused 7-10-membered bicyclic heterocyclic structure or a bridged 6-10-membered bicyclic heterocyclic structure , these cyclic structures can be saturated or partially saturated, and besides carbon atoms, these cyclic structures also contain one or more heteroatoms, wherein the heteroatoms can be selected from the following groups: oxygen, sulfur and nitrogen. For example, it contains 1-4 heteroatoms as defined above.
  • nitrogen when used to refer to an atom on a heterocyclic ring structure may include substituted nitrogen.
  • a heterocyclylene group may be substituted or unsubstituted.
  • Carbocyclylene generally refers to a residue having two hydrogen atoms removed from the same carbon atom or two different carbon atoms of a carbocyclic ring.
  • Carbocycle generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, the carbocycle contains 3 to 20 carbon atoms, may contain 3 to 12 carbon atoms, may contain 3 to 10 carbon atoms, may Contains 3 to 8 carbon atoms, for example contains 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic carbocycles include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane etc.; polycyclic carbocycles may include spiro, fused and bridged carbocycles.
  • 3- to 6-membered saturated carbocyclyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopropylene, cyclobutylene, cyclopentylene or cyclohexylene.
  • Carbocyclyl and carbocyclylene may be substituted or unsubstituted, for example, may be substituted by one or more halogens, C 1-6 aliphatic groups or C 1-6 aliphatic groups substituted by halogens.
  • partially unsaturated generally refers to a ring structure containing at least one double or triple bond between ring molecules.
  • partially unsaturated encompasses cyclic structures with multiple unsaturations, but is not intended to include aromatic rings or Heteroaromatic ring.
  • unsaturated means that the moiety has one or more degrees of unsaturation.
  • halogen generally refers to fluorine, chlorine, bromine, iodine, for example may be fluorine, chlorine.
  • aliphatic group generally refers to straight-chain, branched-chain or cyclic hydrocarbons of 1 to 12 carbon atoms, which are either fully saturated or carry one or more units of unsaturation , but the unsaturated unit is not an aromatic group.
  • suitable aliphatic groups may include substituted or unsubstituted straight-chain, branched-chain or cyclic alkyl, alkenyl, alkynyl groups and mixtures of these groups; such as (cycloalkyl)alkyl , (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
  • aliphatic groups have 1-12, 1-8, 1-6, 1-4, or 1-3 carbon atoms.
  • C 1-6 aliphatic group refers to an aliphatic group as described above having 1-6 carbon atoms, including but not limited to straight chain, branched chain or Alkyl, alkenyl or alkynyl of ring structure, such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl and the like.
  • a heterocyclic group optionally substituted with an alkyl group means that an alkyl group may but need not be present, and the description may include cases where a heterocyclic group is substituted by an alkyl group and cases where a heterocyclic group is not substituted by an alkyl group. situation.
  • substituted generally refers to one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms are independently substituted by a corresponding number of substituents. Substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (eg, ethylenic) bond.
  • the term 0 or more (for example, 0 or at least 1, 0 or 1, 0) methylene units is "substituted" generally refers to when the structure contains 1 or more
  • One or more hydrogen atoms in a group eg up to 5, eg 1 to 3 hydrogen atoms are independently substituted by a corresponding number of substituents.
  • Substituents are only in their possible chemical positions, and a person skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort.
  • an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated (eg, ethylenic) bond.
  • pharmaceutically acceptable salt or “pharmaceutically acceptable salt” generally refers to the salt of the drug conjugate of the present application, which may have safety and/or effectiveness when used in mammals, and may have Due to the biological activity, the antibody drug conjugate of the present application can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, sulfuric acid Hydrogen salt, citrate, acetate, succinate, ascorbate, oxalate, nitrate, peric acid, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate , maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate.
  • solvate or “solvate” generally means that the drug conjugate of the present application forms a pharmaceutically acceptable solvate with one or more solvent molecules.
  • solvent molecules include water, ethanol, acetonitrile, Isopropanol, DMSO, ethyl acetate.
  • drug loading usually refers to the average amount of cytotoxic drug loaded on each ligand, and can also be expressed as the ratio of cytotoxic drug to antibody amount, and the range of cytotoxic drug loading can be that of each ligand (Ab ) to connect 0-12, for example 1- 10 cytotoxic drugs.
  • the drug loading is expressed as Na, which may be the average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 for example.
  • the drug loading of each ADC molecule after the conjugation reaction can be characterized by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assay and HPLC.
  • “Pharmaceutically acceptable carrier” refers to non-toxic ingredients in pharmaceutical preparations or compositions other than the active ingredient to the subject.
  • Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
  • Subject includes any human or non-human animal.
  • non-human animal includes all vertebrates, eg, mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like.
  • cyno or “cynomolgus monkey” refers to cynomolgus monkeys.
  • Administration "in conjunction with” one or more other therapeutic agents includes both simultaneous (co)administration and sequential administration in any order.
  • “Therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to the Her3 antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic drugs to cells, tissues or subjects, effectively prevent or improve The amount of symptoms of one or more diseases or conditions or the development of such diseases or conditions.
  • a therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an amelioration of symptoms, eg, an amount that treats, cures, prevents or ameliorates an associated medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition.
  • a therapeutically effective dose refers to that ingredient only.
  • a therapeutically effective dose refers to the combined amount of the active ingredients which produces the therapeutic effect, whether in combination, sequentially or simultaneously.
  • An effective amount of a therapeutic agent will result in an increase in diagnostic criteria or parameters of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, most preferably at least 50%.
  • cancer is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancers can be benign (also called benign tumors), pre-malignant, or malignant. The cancer cells may be solid cancer cells or leukemia cancer cells.
  • tumor refers to one or more cells comprising cancer.
  • tumor growth is used herein to refer to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.
  • the present application discloses a new anti-HER3 antibody, which effectively inhibits the growth of tumor cells in vitro and in vivo, and has good safety.
  • the HER3 antibody 3F8 was identified in mouse hybridomas immunized with SP2/0 cells overexpressing human HER3. 3F8 recognizes human and monkey HER3 with nanomolar binding affinity and is highly selective for other ERBB family members. In addition, cells with varying levels of HER3 can rapidly and efficiently uptake HER3, a property considered to be necessary for ideal ADC drugs. PET imaging studies showed that [ 89Zr ]Zr-3F8 was significantly accumulated in tumors of PDX models, suggesting that 3F8 may be a highly efficient carrier to bring cytotoxicity into tumor cells.
  • 3F8 and hu3F8 maintained the aforementioned characteristics of binding affinity, selectivity, and tumor suppression in PDX models.
  • hu3F8 has good developability, and its binding affinity, aggregation, and post-translational modification show little change in the stress tests of repeated freeze-thaw treatments, acid incubation, and 40°C storage.
  • anti-Her3 antibody refers to the ability to bind Her3 protein or fragments thereof with sufficient affinity so that the antibody can be used as a target Her3 protein diagnostic and/or therapeutic agents.
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention includes any anti-Her3 antibody or antigen-binding fragment thereof described in the application number PCT/CN2021/099998, and the entire disclosure of the application is incorporated herein by reference in its entirety.
  • the CDR sequence of the antibody used in the drug conjugate, composition, use or method of the present invention comprises the CDR sequence from the antibody hu3F8-2 described in PCT/CN2021/099998.
  • the variable region sequence of the antibody used in the drug conjugate, composition or use of the present invention comprises the variable region sequence from the antibody hu3F8-1 described in PCT/CN2021/099998.
  • variable region sequence of the antibody used in the drug conjugate, composition, use or method of the present invention comprises the variable region sequence from the antibody hu3F8-2 described in PCT/CN2021/099998. In some aspects, the variable region sequence of the antibody used in the drug conjugate, composition or use of the present invention comprises the variable region sequence from the antibody hu3F8-3 described in PCT/CN2021/099998.
  • the non-limiting and exemplary antibodies used in the examples herein are selected from the humanized antibodies hu3F8-1, hu3F8-2 and hu3F8-3 described in PCT/CN2021/099998, the amino acid sequences of which are shown in the table below.
  • hu3F8-1, hu3F8-2 and hu3F8-3 correspond to clone 1 (clone-1), clone 2 (clone-2) and clone 3 (clone-3) in PCT/CN2021/099998, respectively.
  • Antibodies of the invention can be produced using any suitable method for producing antibodies. Any suitable form of Her3 can be used as an immunogen (antigen) for antibody production. By way of example and not limitation, any Her3 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing murine monoclonal anti-human Her3 antibodies can be produced by methods known in the art. Antibodies derived from rodents (e.g., mice) may cause unwanted antibody immunogenicity when used in vivo as a therapeutic drug, with repeated use leading to an immune response in the body against the therapeutic antibody that results in at least a loss of therapeutic efficacy , and severe, potentially fatal allergic reactions.
  • rodents e.g., mice
  • CDR complementarity-determining region
  • chimeric or humanized antibodies of the invention can be prepared based on the sequence of prepared murine monoclonal hybridoma antibodies.
  • DNA encoding the heavy and light chain immunoglobulins can be obtained from the murine hybridoma of interest and engineered to contain non-murine (eg, human) immunoglobulin sequences using standard molecular biology techniques.
  • the chimeric Her3 antibody of the present invention can use methods known in the art to operably link hybridoma-derived immunoglobulin heavy chain and light chain variable regions with human IgG constant regions (see for example U.S. Patent No. 4,816,567 to Cabilly et al.) was prepared by obtaining a chimeric heavy chain and a chimeric light chain.
  • the constant region comprised by the chimeric antibody of the present invention can be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.
  • the chimeric Her3 antibody of the present invention can be obtained by "mixing and matching" the chimeric light chain and chimeric heavy chain expression plasmids to transfect expression cells, and the Her3 binding of such "mixed and matching" antibodies Testing can be performed using the binding assays described above, as well as other conventional binding assays (eg, ELISA).
  • the murine CDR regions can be inserted into the human germline framework regions using methods known in the art. See US Patent No. 5,225,539 to Winter et al. and US Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.
  • amino acid changes include amino acid deletions, insertions or substitutions.
  • the anti-Her3 antibodies or antigen-binding fragments thereof of the present invention include mutations that have undergone amino acid deletions, insertions, or substitutions, but still have at least one Those antibodies having an amino acid sequence of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity.
  • an antibody of the invention has no more than 1, 2, 3, 4 or 5 amino acid mutations in the CDR region by amino acid deletion, insertion or substitution when compared to the CDR region depicted in the specific sequence.
  • Fc region variants may comprise human Fc region sequences (eg, human IgGl, IgG2, IgG3 or IgG4 Fc regions) comprising amino acid modifications (eg, substitutions) at one or more amino acid positions.
  • cysteine-engineered antibodies such as "thioMAbs,” in which one or more residues of the antibody are replaced with a cysteine residue.
  • the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known and readily available in the art.
  • Moieties suitable for antibody derivatization include, but are not limited to, water soluble polymers.
  • Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl -1,3-dioxane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly(n-ethylene pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyols (such as glycerin), polyvinyl alcohol, and mixture
  • the present application provides an anti-Her3 antibody drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof or a mixture thereof, which may have one or more effects selected from the following groups: (1) has Inhibitory activity on tumor cell proliferation in vitro; (2) targeted inhibition; (3) plasma stability; (4) tumor-inhibiting effect in vivo; (5) bystander effect; (6) ) has anti-transporter transport ability; (7) has in vivo tumor targeting ability; and (8) has good in vivo safety.
  • the present application provides an anti-Her3 antibody drug conjugate, its isomer, its pharmaceutically acceptable salt or a mixture thereof, wherein the structure of the anti-Her3 antibody drug conjugate is shown in formula (I):
  • D is a cytotoxic drug
  • p represents the average number of connections, and p is selected from integers or decimals of 1 to 10, preferably integers or decimals of 3-8;
  • Ab is an anti-Her3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 2 and SEQ ID NO: 2, respectively.
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention comprises: (1) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and an amino acid sequence as shown in SEQ ID NO: 8
  • (III) Amino acid sequence such as the heavy chain variable region shown in SEQ ID NO: 10, and the amino acid sequence such as the light chain variable region shown in SEQ ID NO: 8; more preferably, the anti-Her3 antibody of the present invention or its antigen binding
  • the fragment comprises: (1) the heavy chain with the amino acid sequence
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein the structure of the anti-Her3 antibody drug conjugate is shown in formula (I- 1) as shown:
  • n represents the average connection number, and n is selected from integers or decimals of 1 to 10, preferably integers or decimals of 3-8;
  • Ab is an anti-Her3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 2 and SEQ ID NO: 2, respectively.
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention comprises: (1) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 7, and an amino acid sequence as shown in SEQ ID NO: 8 The light chain variable region of; or (II) the aminoacid sequence such as the heavy chain variable region shown in SEQ ID NO: 9, and the light chain variable region such as the amino acid sequence shown in SEQ ID NO: 8; or (III) The amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 10, and the light chain variable region of the amino acid sequence shown in SEQ ID NO: 8; more preferably, the anti-Her3 antibody of the present invention or its antigen binding Fragment comprises: (1) amino acid sequence such as the heavy chain shown
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein,
  • M is -L 2 -L 3 -XL 1 -;
  • L 2 is -O- or -S-;
  • L 3 is -(C(R 1a )(R 1b )) m -, m is selected from 0, 1, 2 or 3, wherein when L 3 contains a methylene unit, 0 or 1 of said L 3
  • L 1 is -(C(R 2a )(R 2b )) n -, n is selected from 1, 2 or 3, wherein when L 1 may contain methylene units, 0 or 1 methylene units of said L 1
  • X is selected from 3 to 6-membered saturated carbocyclyl, 3 to 6-membered saturated heterocyclyl or a single bond, the 3 to 6-membered saturated carbocyclyl and 3 to 6-membered saturated heterocyclyl are optionally 0, 1, 2 or 3 R 3a are substituted; preferably, X is a 3 to 6 membered saturated carbocyclyl or a single bond optionally substituted by 0, 1, 2 or 3 R 3a ; more preferably, X 3 to 6 membered saturated carbocyclyl or single bond;
  • each R 1a , R 1b , R 2a , R 2b , R 3a each independently may be hydrogen, halogen or a C 1-6 aliphatic group which may be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein, said -M- is selected from:
  • L 2 is -O- or -S-; preferably, L 2 is -O-;
  • X is selected from 3 to 6 membered saturated carbocyclyl optionally substituted by 0, 1, 2 or 3 R; preferably, X is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; more Preferably, X is cyclopropyl; more preferably, X is cyclobutyl; more preferably, X is cyclohexyl;
  • L 1 is selected from -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-; preferably, L 1 is -C(O) -;
  • each R 2a , R 2b or R 3a independently can be hydrogen, halogen or a C 1-6 aliphatic group which can be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • L 2 is -O- or -S-; preferably, L 2 is -O-; preferably, L 2 is -S-;
  • L 3 is selected from -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )-; preferably, L 3 is selected from -CH 2 -, - CH(CH 3 ), -C(CH 3 ) 2 , -CH 2 CH 2 -, -CH(CH 3 )CH 2 - or -C(CH 3 ) 2 CH 2 -; preferably, L 3 is selected from CH 2 -, -CH(CH 3 ), -CH 2 CH 2 -.
  • L 1 is selected from -C(R 2a )(R 2b )-, -C(R 2a )(R 2b )C(O)- or -C(O)-; preferably, preferably, L 1 is -C (O)-;
  • each R 1a , R 1b , R 2a or R 2b independently can be hydrogen, halogen or a C 1-6 aliphatic group which can be optionally substituted by R;
  • each R independently can be hydrogen or halogen.
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said -M- is selected from:
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is -L a -L b -L c -,
  • the -L a - is
  • W is -(C(R wa )(R wb )) wn -, wherein wn is 1, 2, 3 or 6, and one methylene unit of W is independently replaced by -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )-, or -C(O)- instead; preferably, W is -(C(R wa )(R wb )) 2 -or- (C(R wa )(R wb )) 3 -; preferably, W is -CH 2 CH 2 CH 2 - or -CH 2 CH 2 -;
  • Y is -(OCH 2 CH 2 ) yn -O yp -wherein yn is 0, 4 or 8, and yp is 0 or 1; preferably, Y is a single bond;
  • Z is -(C(R za )(R zb )) zn , wherein zn is 1, 2 or 3, and one methylene unit of Z is independently replaced by -Cyr-, -N(R zx )C(O )-, -C(O)N(R zx )-, or -C(O)-substituted, -Cyr- is a 3- to 10-membered saturated carbocyclylene, wherein the -Cyr- is unsubstituted or independently substituted by 1 to 3 substituents R cx ; preferably, Z is -(C(R wa )(R wb )) 2 C(O)- or -(C(R wa )(R wb )) 3 C(O)-; preferably, Z is -CH 2 CH 2 CH 2 C(O)- or CH 2 CH 2 C(O)-;
  • each R wa , R wb , R za , R zb , R wx , R zx , R cx are each independently hydrogen, halogen, -OR r or a C 1-6 aliphatic group optionally substituted by R r ;
  • each R r is independently hydrogen, halogen or C 1-6 aliphatic group
  • said -L a - is preferably
  • the -L b - is selected from the following group: Preferably; the -L b - is Preferably -L b - is
  • the -L c - is wherein R L1 and R L2 are each independently selected from the following group: hydrogen, halogen, -OH and C 1-6 aliphatic groups; preferably, the -L c - is
  • the anti-Her3 antibody drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or its mixture, wherein said L is
  • the anti-Her3 antibody-drug conjugate of the present invention its isomer, its pharmaceutically acceptable salt or the mixture thereof, wherein the structure of the anti-Her3 antibody-drug conjugate is shown in formula (II- 1) or (II-2):
  • L 2 is -O- or -S-; preferably, L 2 is -O-, preferably, L 2 is -S-,
  • X is selected from 3 to 6 membered saturated carbocyclyl optionally substituted by 0, 1, 2 or 3 R; preferably, X is selected from cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl;
  • L 3 is selected from -C(R 1a )(R 1b )- or -C(R 1a )(R 1b )C(R 1a )(R 1b )-, wherein each R 1a , R 1b or R 3a is independently may be hydrogen, halogen, or a C 1-6 aliphatic group optionally substituted by R, wherein each R independently may be hydrogen or halogen; preferably, L 3 is selected from —CH 2 —, —CH( CH 3 )-, -C(CH 3 ) 2 -, -CH 2 CH 2 -, -CH(CH 3 )CH 2 - or -C(CH 3 ) 2 CH 2 -;
  • n represents the average connection number, and n is selected from integers or decimals of 1 to 10, preferably integers or decimals of 3-8;
  • Ab is an anti-Her3 antibody or an antigen-binding fragment thereof, which comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region comprises amino acid sequences such as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 2 and SEQ ID NO: 2, respectively.
  • the anti-Her3 antibody or antigen-binding fragment thereof of the present invention comprises: (1) a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7, and an amino acid sequence as shown in SEQ ID NO: 8
  • (III) Amino acid sequence such as the heavy chain variable region shown in SEQ ID NO: 10, and the amino acid sequence such as the light chain variable region shown in SEQ ID NO: 8; more preferably, the anti-Her3 antibody of the present invention or its antigen binding
  • the fragment comprises: (1) the heavy chain with the amino acid sequence
  • the average number of connections p in the present invention may be an integer or decimal from 2 to 8.
  • the average number of connections n may be an integer or decimal from 3 to 8.
  • the average connection number n may be an integer or a decimal of 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-Her3 antibody drug conjugate as described herein, its isomer, its pharmaceutically acceptable salt or its mixture, and a pharmaceutically acceptable acceptable carrier or excipient.
  • anti-Her3 antibody drug conjugate provided by the present invention or its isomer, its pharmaceutically acceptable salt or its mixture, or its pharmaceutical composition can integrate suitable carriers, excipients and Other agents are administered in combination to provide improved transfer, delivery, tolerance, and the like.
  • composition refers to a formulation that allows the active ingredients contained therein to be present in a biologically effective form and does not contain additional ingredients that would be unacceptably toxic to the subject to which the formulation is administered.
  • compositions or formulations of the invention may also contain one or more other active ingredients as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other .
  • other active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics, or various known anti-tumor or anti-cancer agents, and the active ingredients are suitable in an effective amount for the intended use combination exists.
  • the pharmaceutical composition of the present invention further comprises a composition of a polynucleotide encoding an anti-Her3 antibody.
  • the present invention provides a pharmaceutical combination comprising an antibody drug conjugate as described herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition as described herein, and one or more additional therapeutic agent.
  • the present invention provides a kit comprising the antibody drug conjugate as described herein or its isomer, its pharmaceutically acceptable salt or its mixture or the pharmaceutical combination as described herein object, preferably it further comprises a drug delivery device.
  • the present invention provides an antibody drug conjugate as described herein, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition as described herein for use in the preparation of therapeutic and /or use in medicines for the prevention of Her3-mediated diseases or disorders, preferably, the diseases or disorders are cancers.
  • the present invention provides an antibody drug conjugate as described herein, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition as described herein, for use in the treatment of and /or preventing a Her3-mediated disease or condition, preferably, the disease or condition is cancer.
  • the present invention provides a method for treating and/or preventing a Her3-mediated disease or disorder, comprising administering an antibody drug conjugate as described herein, an isomer thereof, to a subject in need thereof body, a pharmaceutically acceptable salt thereof or a mixture thereof, or a pharmaceutical composition as described herein, preferably, the disease or condition is cancer.
  • the cancer is selected from lung cancer, kidney cancer, urethral cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer cancer, gastric cancer, and esophageal cancer.
  • modes of administration of the present invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (eg, in arthritic joints), via inhalation, aerosol delivery, or intratumoral administration wait.
  • the invention provides for the co-administration of a therapeutically effective amount of one or more therapies (eg, treatment modalities and/or other therapeutic agents) to a subject.
  • the therapy includes surgery and/or radiation therapy.
  • the methods or uses provided herein further comprise administering one or more therapies (eg, treatment modalities and/or other therapeutic agents) to the individual.
  • therapies eg, treatment modalities and/or other therapeutic agents
  • the antibody drug conjugates of the invention, or pharmaceutically acceptable salts thereof, can be used alone or in combination with other therapeutic agents in therapy. For example, it can be co-administered with at least one additional therapeutic agent.
  • the drug conjugates described herein may have inhibitory activity on tumor cell proliferation in vitro.
  • the inhibitory activity may be that when the drug conjugate of the present application is added to the medium of tumor cells, compared with the addition of negative control or control drugs, the cell proliferation ability of the tumor cells is reduced by more than 1%, more than 2%, or more than 4%. , more than 5%, more than 8%, more than 10%, 15% More than, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • the inhibitory activity may have an IC50 value (nM) for tumor cells of 10000 or less, 5000 or less, 4000 or less, 3000 or less, 2000 or less, 1000 or less, 500 or less, 400 or less, 300 or less, 200 or less, 150 or less Below, below 120, below 110, below 100, below 99, below 98, below 97, below 95, below 90, below 80, below 75, below 70, below 65, below 62, below 60, below 50, below 40, 30 or less, 25 or less, 23 or less, 22 or less, 20 or less, 19 or less, 18 or less, 18.5 or less, 17 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8.5 or less, 7 or less, 6.7 or less, 6 or less , 5.9 or less, 5.5 or less, 5.0 or less, 4.8 or less, 4.5 or less, 4.4 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1.0 or less,
  • the drug conjugates described herein may be targeted inhibitory.
  • the target inhibition can be that the drug conjugate of the present application is added to the culture medium of tumor cells with high expression of specific targets, compared with the addition of negative control or control drugs, the proliferation ability of tumor cells with high expression of specific targets 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% % or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • the target inhibition can be that the IC50 value (nM) of tumor cells with high expression of a specific target can be less than 10000, less than 5000, less than 4000, less than 3000, less than 2000, less than 1000, less than 500, less than 400, Below 300, below 200, below 185, below 150, below 120, below 110, below 100, below 99, below 98, below 97, below 95, below 91, below 80, below 74, below 70, below 65, below 62 , 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 23 or less, 22 or less, 20 or less, 19 or less, 18 or less, 18.5 or less, 17 or less, 15 or less, 12 or less, 10 or less, 9 or less, 8.5 Less than 7, less than 6.7, less than 6, less than 5.9, less than 5.5, less than 5.0, less than 4.8, less than 4.5, less than 4.4, less than 4, less than 3.5, less than 3, less than 2.5, less than 2, less than 1.5
  • the tumor cells with high expression of the specific target may include but not limited to solid tumor cells, for example, the tumor cells with high expression of the specific target include but not limited to gastric cancer cells, or breast cancer cells, for example, the cells with high expression of the specific target Tumor cells may include, but are not limited to, HCC1569 cells or MDA-MB-453 cells.
  • the specific target may include, but is not limited to, Her3.
  • the drug conjugates described herein can be plasma stable.
  • the plasma stability can be that the drug conjugate of the present application is added to the plasma, and the cytotoxic drug released by the drug conjugate can be stabilized within 1 day, 3 days, 5 days, 7 days, 14 days, 20 days or 30 days.
  • the release rate is not more than 50%, not more than 40%, not more than 30%, not more than 20%, not more than 10%, not more than 7%, not more than 5%, not more than 4%, not more than 3%, not more than 2%, not exceeding 1.9%, not exceeding 1.8%, not exceeding 1.7%, not exceeding 1.6%, not exceeding 1.5%, not exceeding 1.4%, not exceeding 1.3%, not exceeding 1.2%, not exceeding 1.1%, not exceeding 1.0%, not more than 0.9%, not more than 0.8%, not more than 0.7%, not more than 0.6%, not more than 0.5%, not more than 0.4%, not more than 0.3%, not more than 0.2%, or not more than 0.1%.
  • the drug conjugates described in this application can have anti-tumor effect in vivo.
  • the tumor-suppressing effect can be that the drug conjugate of the present application is administered to animals, and compared with adding a negative control or a control drug, the tumors of the animals are reduced in 1 day, 3 days, 5 days, 7 days, 14 days, 20 days Day, 21 days or 30 days volume reduction of 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 55% or more, 60% or more, 70% or more, 73% or more, 75% or more, 80% or more, 90% or more, or 95% or more, or compared to the administration of a negative control or control Drug, the volume of the tumor of the animal is reduced by more than 1.1 times, more than 1.3 times, more than 1.5 times, more than two times in 1 day, 3 days, 5 days, 7 days, 14 days, 20
  • the animals may include but not limited to mammals, for example, the animals may include but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys or humans.
  • the administration may include, but is not limited to, oral administration, intravenous injection, intravenous drip, intraperitoneal injection, or topical administration.
  • the drug conjugates described in this application may have a bystander effect.
  • the bystander killing effect may be that the drug conjugate of the present application has no obvious inhibitory effect on the cell proliferation of tumor cells with low expression of specific targets, and the tumor cells with low expression of specific targets are co-cultured with tumor cells with high expression of specific targets Among them, the drug conjugate of the present application can simultaneously inhibit the cell proliferation of tumor cells with low expression of specific targets and tumor cells with high expression of specific targets.
  • the inhibitory activity may be that the IC50 value (nM) of the tumor cells with low expression of a specific target may be below 10,000, below 5,000, Below 4000, below 3000, below 2000, below 1000, below 500, below 400, below 300, below 200, below 185, below 150, below 120, below 110, below 100, below 99, below 98, below 97, below 95 , 91 or less, 80 or less, 74 or less, 70 or less, 65 or less, 62 or less, 60 or less, 50 or less, 40 or less, 30 or less, 25 or less, 23 or less, 22 or less, 20 or less, 19 or less, 18 or less, 18.5 Below, below 17, below 15, below 12, below 10, below 9, below 8.5, below 7, below 6.7, below 6, below 5.9, below 5.5, below 5.0, below 4.8, below 4.5, below 4.4, below 4, 3.5 or less, 3 or less, 2.5 or
  • the expression of the specific target can be reduced by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, or more than 10% % or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • the tumor cells with high expression of the specific target may include but not limited to solid tumor cells, for example, the tumor cells with high expression of the specific target include but not limited to gastric cancer cells, or breast cancer cells, for example, the cells with high expression of the specific target Tumor cells may include, but are not limited to, HCC1569 cells or MDA-MB-453 cells.
  • the tumor cells with low expression of the specific target may include but not limited to solid tumor cells, for example, the tumor cells with low expression of the specific target include but not limited to breast cancer cells, for example, the tumor cells with low expression of the specific target may include But not limited to HCC1187 cells.
  • the drug conjugates described herein may have anti-transporter transport capabilities.
  • the anti-transport ability can be that compared with the standard product of the transport substrate, the efflux ratio of the drug conjugate described in the present application is reduced by more than 1%, more than 2%, more than 4%, more than 5%, more than 8%, or more than 10% % or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • the test of the efflux ratio can be The methods commonly used by people in the field, or are described in the embodiments of this application.
  • the drug conjugates described in this application may have the ability to target tumors in vivo.
  • the in vivo targeting ability may refer to the administration of the drug conjugate labeled with a signal substance to the animal, and the distribution of the labeled drug conjugate in the tumor tissue of the animal, compared with other tissues and organs, can be Is the distribution increased by 1% or more, 2% or more, 4% or more, 5% or more, 8% or more, 10% or more, 15% or more, 18% or more, 20% or more, 25% or more, 40% or more, 50% or more , more than 60%, more than 70%, more than 80%, more than 90%, or more than 95%, or it can be more than 1.1 times, more than 1.3 times, more than 1.5 times, more than two times, more than three times, more than five times , more than ten times, more than twenty times, more than twenty-two times, more than thirty times, more than fifty times, more than one hundred times, more than five hundred times, more than one thousand times, or more than fifteen hundred
  • the signal substance may be a radioactive substance, for example, the signal substance includes but not limited to 125I.
  • the animals may include but not limited to mammals, for example, the animals may include but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys or humans.
  • the administration may include, but is not limited to, oral administration, intravenous injection, intravenous drip, intraperitoneal injection, or topical administration.
  • the tissue or organ may include, but is not limited to, heart, liver, spleen, lung, kidney, brain, or bone marrow.
  • the drug conjugates described in this application can have good in vivo safety.
  • the in vivo safety can be that after the drug conjugate of the present application is administered to an animal, the release rate of the free toxin in the animal's body is no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than More than 10%, not more than 7%, not more than 5%, not more than 4%, not more than 3%, not more than 2%, not more than 1.9%, not more than 1.8%, not more than 1.7%, not more than 1.6%, not more More than 1.5%, not more than 1.4%, not more than 1.3%, not more than 1.2%, not more than 1.1%, not more than 1.0%, not more than 0.9%, not more than 0.8%, not more than 0.7%, not more than 0.6%, not more More than 0.5%, not more than 0.4%, not more than 0.3%, not more than 0.2%, or not more than 0.1%.
  • the in vivo safety may be that the administration concentration of the drug conjugate described in the present application may be above 0.5 mg/kg, above 1 mg/kg, above 2 mg/kg, or above 3 mg/kg under the condition that animals do not produce toxic manifestations.
  • the animals may include, but are not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, monkeys, or humans.
  • the administration may include, but is not limited to, oral administration, intravenous injection, intravenous drip, intraperitoneal injection, or topical administration.
  • High performance liquid chromatography Waters e2965 high performance liquid chromatography system.
  • Mobile phase B 50mM sodium phosphate (pH 7.0)/isopropanol (75:25V/V);
  • Detection conditions set the mobile phase flow rate to 1ml/min, the detection wavelength to 280nm, and the column temperature to 30°C.
  • Detection conditions set the flow rate of the mobile phase to 0.5ml/min, the detection wavelength to 280nm, and the column temperature to 30°C.
  • Embodiment 1 anti-Her3 antibody
  • NRG1 is from Origene (TP723155).
  • Cell culture medium Roswell Park Memorial Institute (RPMI) 1640, Dulbecco's modified medium (DMEM) and fetal calf serum were from Hyclone.
  • Recombinant Patritumab is prepared in the laboratory.
  • HER3 antibody 3F8 is produced by hybridoma or recombinantly.
  • m3F8 indicates murine 3F8 antibody
  • ch3F8 and hu3F8 indicate chimeric and humanized 3F8 antibody, respectively.
  • SP2/0-EGFR, SP2/0-HER2, SP2/0HER3 cells stably expressing human EGFR, HER2 and HER3 were constructed by the laboratory itself.
  • mice Female, 8-10 weeks old
  • booster immunization was performed with the same number of cells with Freund's incomplete adjuvant.
  • the mice were immunized with the above-mentioned amount of cells every three days, and repeated three times.
  • B lymphocytes were isolated from the spleen and fused with immortal myeloma cells NS-1 cells to generate hybridoma cells.
  • Hybridoma cells were cultured in serial dilutions in 96-well plates. The supernatant was collected, and the antibody recognizing HER3 expressed on the surface of SP2/0 cells was screened by flow cytometry, or the recombinant HER3 antibody was screened by ELISA.
  • RNA extracted from hybridomas was reverse-transcribed into the first cDNA strand with Trizol (ThermoFisher), and following the instructions of the 5' RACE kit (Invitrogen, 18374-058), Rapid amplification of 5' complementary DNA (5'RACE) was used to amplify DNA sequences encoding variable regions using PCR.
  • the PCR product was cloned into pGM-T vector. Positive clones were subjected to DNA sequencing, and protein sequences were deduced accordingly. Amino acids of variable regions are analyzed in the Kabat numbering scheme.
  • Antibody expression DNA encoding antibody heavy and light chains was cloned into expression vector pCDNA3.1(+) (Invitrogen) and expressed in 293T cells. Antibodies were purified using protein A or G columns (GE).
  • Humanization Humanization was done by GenScript Corporation. First, a murine-human chimeric antibody (ch3F8) was generated by replacing the constant region of the heavy chain of the murine antibody with a human IgG1 constant region sequence and the constant region of the light chain of the murine antibody with a human Ig ⁇ constant region sequence. Humanization was then performed on the chimeric antibody following reference (Kuramochi et al). While replacing the mouse framework with a human antibody framework to generate a humanized antibody, the mouse antibody framework residues necessary for affinity and specificity are retained and maintained.
  • ch3F8 murine-human chimeric antibody
  • the codon-optimized DNA sequence encoding the humanized antibody was synthesized by GenScript Company.
  • Antibody expression and purification The plasmids encoding the above antibody DNA sequences were transfected into ExpiCHO-S (Catl. #A2910001, Gibco) cells and then grown in 32°C, 5% CO 2 medium for 12 days. The supernatant was collected after centrifugation at 4000 g for 30 min and filtered through a 0.22 ⁇ m membrane. Protein A-bound antibody (Catl. #17508001, GE) was washed with 20 mM sodium phosphate (pH 7.0) and eluted with 0.1 M glycine (pH 3.0) as recommended by the manufacturer's manual. The eluted fraction was neutralized with 0.1M Tris buffer (pH 9.0), and then exchanged to PBS buffer by ultrafiltration centrifugation. Protein concentration was determined with BCA.
  • SPR Surface Plasmon Resonance
  • FACS detection the cultured cells were digested with 0.25% trypsin-EDTA, and then centrifuged at 1500 rpm for 5 minutes. The cells were resuspended to 5X10 6 cells/mL with PBS FACS solution containing 5% FBS and 0.2% ProClin300. Incubate 50 ⁇ L of the cell suspension with 100 ⁇ L of the primary antibody at a concentration of 1 ⁇ g/ml for 1 h on ice. Wash twice with FACS solution. Resuspend the cells with 100 ⁇ L of FACS solution containing goat anti-mouse IgG-PE (diluted 1:1000), and incubate on ice for 1 hour in the dark. Cells were then washed twice and resuspended in 200 ⁇ L of FACS solution.
  • Western blot detection Transfer the protein separated by SDS-PAGE to a nitrocellulose membrane for Western blot.
  • the primary antibodies used are as follows: anti-HER2 (Cell Signaling, Catl#: 2165S), anti-HER3 (Cell Signaling, Catl#: 12708), anti-p-HER3 (Cell Signaling, Catl#: 4791), anti- ⁇ - Actin (Cell Signaling, Catl No: 4967).
  • Human HER2-ex-huFc, human HER3-huFc and human EGFR-his were diluted to 2 ⁇ g/mL, 50 ⁇ L/well in a 96-well plate, and incubated overnight at 4°C. Wash with 0.5 ⁇ PBST, then incubate with 100 ⁇ L of blocking buffer (PBS+3% BSA) at 37° C. for 2 h, and wash with 0.5 ⁇ PBST.
  • the 3F8 antibody was serially diluted 1:3 with blocking buffer, added at 50 ⁇ L/well, incubated at 37°C for 40-50min, and then washed with 0.5 ⁇ PBST.
  • NRG1 induces HER3 phosphorylation: Cells were cultured on a 6-well plate and used for experiments when the confluence reached 80%. On the day of the experiment, cells were washed twice with PBS, incubated in serum-free medium for 6 hours, and then induced with 10 ⁇ g/mL antibody overnight. To induce HER3 phosphorylation, NRG1 was added to a working concentration of 100 ng/mL 30 minutes before harvesting cells for Western blot.
  • Antibody stability test Store 5 mg/mL purified antibody at 4°C routinely, incubate at 40°C for 7 days and 14 days, transfer to pH 3.5 glycine solution for regeneration and store for 2, 4 and 6 hours for acid Stability assessment, or repeat freeze-thaw treatment for 4 or 6 cycles for freeze-thaw stability assessment. Aggregation was measured by SEC-HPLC and binding affinity was determined by ELISA.
  • Antibody [ 89 Zr]Zr labeling DFO-NCS was conjugated with antibody as described in reference (Zeglis and Lewis, 2015), DFO and antibody were mixed at a molar ratio of 5:1 and incubated at 37°C for 1 hr. DFO-conjugated antibodies were purified by SEC-HPLC.
  • Oxalate 89 Zr solution 0.8 mCi was mixed with DFO-conjugated antibody (0.2 mg/ml) in HEPES/Na 2 CO 2 buffer (pH 7.0-7.5) and incubated at room temperature for 30 minutes. Radiochemical purity was assessed by TLC.
  • the Rf of [ 89 Zr]Zr antibody is 0-0.3, while the Rf of free 89 Zr is 0.6-1.0.
  • PET imaging about 100uCi [ 89 Zr]Zr antibody was injected intravenously into each animal. At indicated times after injection, images were collected and analyzed with a small animal PET imaging machine.
  • Murine 3F8 was co-incubated with SP2/0 wild-type cells or cells overexpressing HER3, HER2 or EGFR. Binding strength was detected with PE-anti-mouse secondary antibody in FACS instrument.
  • the binding affinity of murine 3F8 to human HER3, HER2 and EGFR was determined by ELISA.
  • Murine 3F8 only recognizes HER3 but not HER2 or EGFR.
  • Murine 3F8 exhibits potent binding affinity in the nanomolar range.
  • Table 1 EC50 of murine 3F8 binding to human HER3, HER2 or EGFR
  • the species selectivity of murine 3F8 to human, monkey, rat and mouse HER3 was determined by ELISA.
  • Murine 3F8 recognizes human and monkey HER3 with similar potency, but does not recognize mouse and rat HER3.
  • GraphPad Prism 6.0 was used to analyze the EC50 of murine 3F8 binding to human, monkey, rat and mouse HER3. The results are shown in Table 2.
  • Murine 3F8 shows the same binding affinity for human and cynomolgus HER3 in the nanomolar range.
  • Murine 3F8 blocks NRG1-induced HER3 phosphorylation.
  • NCI-N87, MDA-MB-468 and MDA-MB-453 were treated with the HER3 ligand NRG1 to induce downstream phosphorylation of HER3.
  • the inhibitory effect of murine 3F8 on NRG1-induced p-HER3 was determined by western blot.
  • 3D4 a previously demonstrated anti-HER3 antibody that competes with NRG1 for binding to HER3, was used as a positive control.
  • Murine 3F8 is rapidly taken up by cells with varying surface levels of HER3.
  • Cells with different surface levels of HER3 were incubated with murine 3F8 on ice (control) or at 37°C for 1 or 4 hours.
  • the internalization fraction was determined by subtracting the cell surface signal incubated at 37°C from the ice-incubated control.
  • Anti-HER3 antibody potently inhibits tumor growth in BT474 subcutaneous xenograft model.
  • Anti-HER3 antibody 3F8, 3D4 or 3F8+3D4 combination was injected intravenously at 25 mg/kg once every two weeks for three weeks. Tumor size was monitored every 3-4 days.
  • 3F8 and 3D4 are both anti-HER3 antibodies.
  • 3F8 is a murine antibody.
  • [ 89 Zr]Zr-ch3F8 was used to image the gastric PDX model GAS078.
  • [ 89 Zr]Zr-ch3F8 was intravenously injected into gastric model GAS078. Images were collected at 4, 24, 48, 72, 96 and 168 hours post injection. Radioactive uptake by each organ was analyzed by Olinda and expressed as %ID/g (percent injected dose/gram of tissue).
  • Figure 7 is a representative image of [ 89 Zr]Zr-ch3F8 in the GAS078 model. Ch3F8 indicates chimeric 3F8 antibody. The data showed that the uptake of [ 89 Zr]Zr-ch3F8 in tumors gradually increased over time. Tumor uptake remained stable at 96 hours post-injection, followed by a slight decrease at 168 hours post-injection.
  • [ 89 Zr]Zr-ch3F8 was used to image multiple PDX phantoms.
  • [ 89 Zr]Zr-ch3F8 was injected intravenously. Images were collected 72 hours after injection.
  • Figure 8 shows representative images of [ 89 Zr]Zr-ch3F8 imaging in six animal models.
  • Table 4 lists the expression levels of HER3 in tumor tissues determined by ELISA and the radioactive uptake in major organs and tumor tissues 72 hours after injection. There was considerable tumor uptake in all 6 tested PDX models.
  • Binding affinity of humanized 3F8 (hu3F8) and chimeric 3F8 was determined by Biacore.
  • Three humanized 3F8 clones (clone 1, clone 2 and clone 3) have different heavy chains, and one identical light chain.
  • Clone 1 (clone-1), clone 2 (clone-2) and clone 3 (clone-3) correspond to hu3F8-1, hu3F8-2 and hu3F8-3, respectively.
  • Table 9 Also shown in Table 9 are the EC50s of the three hu3F8 clones subjected to multiple freeze-thaw stress tests.
  • PTM Post-translational modification
  • amino acid sequences of the antibodies of the present application are listed below.
  • MS-ESI m/z 830.3[M+H]+.
  • ultrafiltration tube (MWCO 30KD, manufacturer: Millipore) to replace the sample to the storage buffer, first use 30mM histidine acetate buffer containing 10% dimethyl sulfoxide, pH 5.5 for 3 ultrafiltration times, and then use DMSO-free 30mM histidine acetate buffer solution, pH 5.5, ultrafiltered three times, and then concentrated by ultrafiltration to obtain ADC-1-X1, the concentration was 26.8mg/mL, and the yield was 73%. The purity and DAR value were detected by size exclusion chromatography and hydrophobic chromatography.
  • the reducing agent and protective agent were respectively prepared with ultrapure water as follows: 2 mg/ml TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo), 100 mmol/L ethylenediaminetetraacetic acid sodium salt (manufacturer: Sigma).
  • the purity of the antibody drug conjugate ADC-1-X2 was 99.02%, and the DAR value p was 7.53.
  • linker-payload X2 dimethyl sulfoxide manufactured by Synchronization and aqueous sulfoxide (manufacturer: Sinopharm Group) solution with a concentration of 10 mg/ml, slowly add it according to the molar ratio of drug to antibody 18.0:1, oscillate and mix well, and then place it on a refrigerated constant temperature mixer Reaction, 4°C, 1h.
  • Deruxtecan (CAS 1599440-13-7, purchased from Shanghai Haoyuan Chemical Technology Co., Ltd.) with a concentration of 10 mg/ml in dimethyl sulfoxide (manufacturer: Sinopharm Group), and slowly Add, oscillate and mix well, and then place it on a refrigerated constant temperature mixer for reaction at 4°C for 1h.
  • ultrafiltration tube (MWCO 30KD, manufacturer: Millipore) to replace the sample to the storage buffer, first use 30mM histidine acetate buffer containing 10% dimethyl sulfoxide, pH 5.5 for 3 ultrafiltration times, and then use DMSO-free 30mM histidine acetate buffer, pH 5.5 ultrafiltration 3 times, to obtain reference ADC-1, the concentration is 8.77mg/mL, the yield is 65%.
  • the purity and DAR value were detected by size exclusion chromatography and hydrophobic chromatography.
  • MDA-MB-453 cells were cultured in 10% FBS Leibovitz's L-15 medium.
  • Cell preparation take the MDA-MB-453 cells in the logarithmic growth phase, wash once with PBS, add 2-3ml Versene to digest for 2-3min, after the cells are completely digested, add 10-15ml cell culture medium, and Digested cell wash Take it off, centrifuge at 1000rpm for 5min, discard the supernatant, add cell culture medium to resuspend the cells to make a single cell suspension and adjust the viable cell density to 3x10 5 cells/ml.
  • Cell plating add 50ul/well to a 96-well cell culture plate. Plates were incubated for 18 hours in an incubator (37°C, 5% CO 2 ).
  • Cell culture incubate the culture plate in an incubator for 2, 4, 6, 24 or 48 hours (37° C., 5% CO 2 ).
  • Plate reading operation When the corresponding time point is reached, take out the 96-well cell culture plate, add Hoechst33342 for nuclear staining, place it in operatta CLS to take pictures, and read the number of cells and the number of fluorescent bright spots in each cell. use The imaging and analysis software calculates the average cell fluorescence points
  • Table 12 The number of fluorescent signal bright spots generated by endocytosis of the antibody drug conjugates of this application at different time points in MDA-MB-453 cells
  • the antibody-drug conjugate of the present application has an endocytic effect on MDA-MB-453 cells with high expression of HER3, and the endocytic ability is stronger than that of the reference ADC-1.
  • HCC1569, MDA-MB-453 or MDA-MB-231 cells were cultured in 10% FBS RPMI-1640 medium.
  • Cell preparation Take HCC1569, MDA-MB-453 or MDA-MB-231 cells in the logarithmic growth phase, wash once with PBS, add 2-3ml trypsin to digest for 2-3min, after the cells are completely digested, Add 10-15ml of cell culture medium to elute the digested cells, centrifuge at 1000rpm for 5min, discard the supernatant, then add 10-20ml of cell culture medium to resuspend the cells to make a single cell suspension, trypan blue for cell Stain and count live cells.
  • Cell plating Mix the HCC1569, MDA-MB-453 or MDA-MB-231 single cell suspension, adjust the living cell density to 6x10 4 cells/ml with cell culture medium, and adjust the density of the cell suspension Mix well, add 50ul/well to 96-well cell culture plate. Plates were incubated for 18 hours in an incubator (37°C, 5% CO 2 ).
  • Antibody-drug conjugate concentration 9 concentrations in total, the initial concentration is 100nM, and diluted 3 times until 0.0152nM.
  • Sample addition operation Add the prepared samples to be tested at different concentrations into the culture plate, and each sample has two duplicate holes. The plates were incubated in an incubator (37°C, 5% CO 2 ) for 6 days.
  • Chromogenic operation Take out the 96-well cell culture plate, let it stand for 30 minutes to equilibrate to room temperature, add 50ul CTG reagent to each well, shake for 2 minutes on an orbital shaker, and incubate at room temperature for 10 minutes in the dark.
  • Plate reading operation take out the 96-well cell culture plate, place it in the 2104EnVision microplate reader, and measure the chemiluminescence with the microplate reader.
  • IR Inhibition rate
  • the antibody-drug conjugate of the present application has obvious proliferation inhibitory activity on Her3-positive high-expression cells HCC1569 and MDA-MB-453, and its inhibitory activity is stronger than that of Refer to ADC-1; at the same time, they have no proliferation inhibitory activity on MDA-MB-231 cells that do not express HER3, and have good selectivity.
  • ADC-1-X1 treatment group: 1mg/kg
  • ADC-1-X2 treatment group: 1mg/kg
  • mice were subcutaneously inoculated with 2 ⁇ 10 6 HCC1569 cells on the right front scapula, and the cells were resuspended in 1:1 PBS and Matrigel suspension (0.1 ml/mouse). Observe the growth of the tumor. When the tumor grows to an average volume of 100-150mm 3 The size and body weight of the mice were randomly divided into groups for administration, 6 mice/group. The day of group administration was defined as day 0.
  • TGI% tumor inhibition rate
  • the antibody-drug conjugates of the present application can significantly reduce the tumor volume. Moreover, the antibody-drug conjugate of the present application has better in vivo tumor suppression effect than the reference ADC-1.
  • ADC-III-28 refers to the curative effect of ADC-2 on nude mice with Colo205 transplanted tumor after intraperitoneal injection.
  • ADC-1-X1 treatment group: 1mg/kg
  • ADC-1-X2 treatment group: 1mg/kg
  • mice 8-10 weeks old BALB/c-Nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • Ti mean tumor volume of the treatment group and the positive control group on day i of administration
  • T0 mean tumor volume of the treatment group and positive control group on day 0 of administration
  • Vi mean value of tumor volume of the negative control group on day i of administration The average tumor volume
  • V0 the average tumor volume of the negative control group on the 0th day of administration.
  • Data analysis Data statistics use Excel 2016 statistical software: the average value is calculated by avg; the SD value is calculated by STDEV; the P value of the difference between groups is calculated by TTEST.
  • the antibody-drug conjugates of the present application can significantly reduce the tumor volume. Moreover, the antibody-drug conjugate of the present application has a tumor-inhibiting effect at a lower dosage concentration than that of the reference ADC-1.
  • mice Male NU/NU nude mice were used as test animals to evaluate the anti-tumor effect of the antibody-drug conjugate of the present application on NCI-H441 tumor-bearing mice.
  • Blank control normal saline
  • Reference ADC-1 (positive control group): 0.3mg/kg or 1mg/kg
  • ADC-1-X2 treatment group: 0.3mg/kg or 1mg/kg
  • Experimental animals 6-8 week old NU/NU nude mice, purchased from Weitong Lihua Experimental Animal Technology Co., Ltd.
  • mice were subcutaneously inoculated with 5 ⁇ 10 6 NCI-H441 cells on the right back, and the cells were resuspended in 0.1 ml of PBS and Matrigel (1:1), and the tumor growth was observed regularly until the tumor grew to an average volume of 115.24 mm 3 According to the size of the tumor and the body weight of the mice, the mice were randomly divided into groups for administration, with 6 animals in each group. The day of group administration was defined as day 0. The administration was administered by tail vein injection, administered once, and the experiment was ended 28 days after the administration. Tumor volume and body weight were measured twice a week, and the data were recorded.
  • TGI% tumor inhibition rate
  • Data analysis Data statistics use Excel 2016 statistical software: the average value is calculated by avg; the SD value is calculated by STDEV; the P value of the difference between groups is calculated by TTEST.
  • the antibody drug conjugate of the present application can significantly reduce the tumor volume, and the antibody drug conjugate of the present application has better tumor inhibition than the reference ADC-1 at the same concentration.
  • mice Female BALB/c nude mice were used as test animals to evaluate the anti-tumor effect of the antibody-drug conjugate of the present application on mice with subcutaneous xenograft tumors of human lung cancer LU1542.
  • Reference ADC-1 (positive control group): 3mg/kg or 10mg/kg
  • ADC-1-X2 treatment group: 3mg/kg or 10mg/kg
  • mice BALB/c nude mice at 6-8 weeks, purchased from Beijing Ankai Yibo Biotechnology Co., Ltd.
  • Tumor tissues were harvested from lung cancer xenograft model LU1542 tumor-bearing mice, cut into 2-3mm diameter tumor masses and inoculated subcutaneously at the right anterior shoulder blade of Balb/c nude mice.
  • the day of group administration was defined as day 0.
  • the administration was administered by tail vein injection, administered once, and the experiment was ended 28 days after the administration. Tumor volume and body weight were measured twice a week, and the data were recorded.
  • TGI% tumor inhibition rate
  • Data analysis Data statistics use Excel 2016 statistical software: the average value is calculated by avg; the SD value is calculated by STDEV; the P value of the difference between groups is calculated by TTEST.
  • the antibody-drug conjugates of the present application can significantly reduce the tumor volume. Moreover, the antibody-drug conjugate of the present application has a tumor-inhibiting effect at a lower dosage concentration than the reference ADC, and has better tumor-inhibiting effect at the same concentration.

Abstract

一种抗Her3抗体药物偶联物具有Ab-(L-M-D) p (I)结构，其中，L和M是接头单元，D是细胞毒性药物，P表示平均连接数，Ab为抗Her3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ IDNO:6所示的LCDR1、LCDR2和LCDR3。一种药物组合物，包含式(I)结构抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。上述抗体药物偶联物或组合物可用于制备治疗和/或预防Her3介导的疾病或病症的药物。

Description

Her3抗体药物偶联物及其用途 技术领域
本发明提供了与Her3特异性结合的抗体药物偶联物和包含其的组合物。还提供了使用本发明的抗体药物偶联物的方法和用途。
背景技术
表皮生长因子受体(EGFR)是一个巨大的跨膜糖蛋白，分子量约为170KDa，属于ErbB受体家族的一个成员。EGFR受体本身是一种酪氨酸激酶，当与配体如EGF、TNF-a等结合后可形成二聚体，通过传递磷酸化作用激活下游信号(如MAPK,PI3K,Stat等通路)，从而维持细胞生长，促进细胞分裂增殖。由于ErbB家族受体的保守性，EGFR还能与家族其他蛋白(如Her2,Her3,Her4)形成异源二聚体，从而更广泛的调控细胞的生长。
Her3是ErbB家族的成员，在细胞增殖、肿瘤转移和耐药中起关键作用。虽然靶向EGFR和Her2的药物在缓解多种癌症方面表现出巨大的临床益处，但之前开发用于癌症治疗的抗Her3抗体的努力屡屡失败，这表明仅处理Her3及其悬而未决的途径可能不足以抑制肿瘤生长。
抗体药物偶联体(antibody drug conjugate，ADC)由抗体或其抗原结合片段(靶向)、接头和小分子药物三部分组成。抗体或其抗原结合片段经由可裂解或不可裂解的接头与具有生物活性例如细胞毒性的小分子药物如细胞毒素缀合，充分利用了抗体或其抗原结合片段靶向于感兴趣的细胞(靶向细胞)的特异性或结合高表达抗原的特异性以及小分子药物的高效性，降低或避免了对非靶向细胞的毒副作用。这意味着，与传统肿瘤化疗药物相比，用于肿瘤的抗体药物偶联物能精准地靶向肿瘤细胞并降低对非肿瘤细胞的影响。
本领域仍需要在亲和力、特异性等方面优异的抗Her3抗体药物偶联物。
发明内容
一方面，本申请提供一种抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，所述抗Her3抗体药物偶联物结构如式(I)所示：
Ab-(L-M-D)p      (Ⅰ)
其中，
L和M是接头单元；
D是细胞毒性药物；
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab为抗Her3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3,和所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗Her3抗体药物偶联物结构如式(I-1)所示：
其中，
L、M是接头单元；
p表示平均连接数，且n选自1到10的整数或小数，优选3-8的整数或小数；
Ab为抗Her3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3,和所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在一些实施方式中，本发明所述抗Her3抗体或其抗原结合片段包含：
(I)氨基酸序列如SEQ ID NO:7所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID NO:8所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链可变区；或
(II)氨基酸序列如SEQ ID NO:9所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID NO:8所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链可变区；或
(III)氨基酸序列如SEQ ID NO:10所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID NO:8所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链可变区。
在一些实施方式中，本发明所述抗Her3抗体或其抗原结合片段包含：
(I)氨基酸序列如SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；或
(II)氨基酸序列如SEQ ID NO:9所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；或
(III)氨基酸序列如SEQ ID NO:10所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区。
在一些实施方式中，本发明所述抗Her3抗体或抗原结合片段为鼠源抗体或其片段、嵌合抗体或抗原结合片段、人源化抗体或抗原结合片段、或全人抗体或抗原结合片段。
在一些实施方式中，本发明所述抗Her3抗体或其抗原结合片段为人源化抗体或其片段。
在一些实施方式中，本发明所述抗Her3抗体或其抗原结合片段选自Fab、Fab'、Fab'-SH、 Fv、scFv、F(ab')2、sdAb、双抗体或线性抗体。
在一些实施方式中，本发明所述抗Her3抗体为单克隆抗体。
在一些实施方式中，本发明所述抗体为IgG1形式的抗体、IgG2形式的抗体、IgG3形式的抗体或IgG4形式的抗体。
在一些实施方式中，本发明所述抗体为IgG1形式的抗体。
在一些实施方式中，本发明所述抗Her3抗体或其抗原结合片段包含：
(1)氨基酸序列如SEQ ID NO:11所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO:12所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链；或
(2)氨基酸序列如SEQ ID NO:13所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO:12所示或与其具有至少95％、96％、97％、98％或99％同一性所示的轻链；或
(3)氨基酸序列如SEQ ID NO:14所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO:12所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链。
在一些实施方式中，本发明所述抗Her3抗体或其抗原结合片段包含：
(1)氨基酸序列如SEQ ID NO:11所示的重链，和氨基酸序列如SEQ ID NO:12所示的轻链；或
(2)氨基酸序列如SEQ ID NO:13所示的重链，和氨基酸序列如SEQ ID NO:12所示或的轻链；或
(3)氨基酸序列如SEQ ID NO:14所示的重链，和氨基酸序列如SEQ ID NO:12所示的轻链。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中
M为-L2-L3-X-L1-；
L2为-O-或-S-；
L3为-(C(R1a)(R1b))m-，m选自0、1、2或3，其中当L3包含亚甲基单元时，所述L3的0个或1个亚甲基单元可以被-C(O)-、或-C(S)-替代；
L1为-(C(R2a)(R2b))n-，n选自1、2或3,其中当L1可以包含亚甲基单元时，所述L1的0个或1个亚甲基单元可以被-C(O)-、或-C(S)-替代；
X选自3到6元饱和的碳环基、3到6元饱和的杂环基或单键，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被0、1、2或3个R3a取代；
其中每个R1a，R1b，R2a，R2b，R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述3-6元饱和的杂环基中的杂原子选自N、O和S，杂原子数为1-3个。
在一些实施方式中，本发明L3和X不同时为单键。
在一些实施方式中，本发明L2为-O-。在一些实施方式中，本发明L2为-S-。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述的接头单元M，其L2端与接头单元L相连，L1端与细胞毒性药物D相连。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L3选自单键、-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；
其中每个R1a和R1b各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L3选自单键、-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；
其中每个R1a和R1b各自独立可以为氢、卤素、CH3或CH2CH3。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L3选自单键、-CH2-、-CH(CH3)-、-C(CH3)2-、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L3选自
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L3选自单键。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；
其中每个R2a，R2b各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；
其中每个R2a，R2b各自独立可以为氢、卤素、CH3或CH2CH3。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L1选自-CH2-、-CH2C(O)-、-CH(CH3)C(O)-或-C(O)-。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中L1选自-C(O)-。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中X为任选被0、1、2或3个R3a取代的3到6元饱和的碳环基或单键；
其中每个R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中X为任选被0、1、2或3个R3a取代的3到6元饱和的碳环基或单键；
其中每个R3a各自独立可以为氢、卤素、CH3或CH2CH3。
在一些实施方式中，本发明所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受 的盐或其混合物,其中X为环丙基、环丁基、环戊基、环己基或单键。
在一些实施方式中，本发明所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中X为单键。
在一些实施方式中，本发明所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中X为环丙基、环丁基、环戊基或环己基，优选为环丁基。
在一些实施方式中，本发明所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中X为优选地，X为
在一些实施方式中，本发明所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
L2为-O-或-S-；
X选自任选被0、1、2或3个R3a取代的3到6元饱和的碳环基；
L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；
其中每个R2a，R2b或R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
L2为-O-或-S-；
X选自任选被0、1、2或3个R3a取代的3到6元饱和的碳环基；
其中每个R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
L2为-O-或-S-；
X选自环丙基、环丁基、环戊基或环己基。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
L2为-O-或-S-；
X选自
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
L2为-O-或-S-；
L3选自-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；
L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；
其中每个R1a，R1b，R2a或R2b各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
L2为-O-或-S-；
L3选自-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；
其中每个R1a和R1b各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
L2为-O-或-S-；
L3选自-CH2-、-CH(CH3)、-C(CH3)2、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的 盐或其混合物,其中所述-M-选自：
L2为-O-或-S-；
L3选自
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述的接头单元M，其L2端与接头单元L相连。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述L为-La-Lb-Lc-，
所述-La-为
其中，W为-(C(Rwa)(Rwb))wn-，Y为-(OCH2CH2)yn-Oyp-，Z为-(C(Rza)(Rzb))zn；
其中wn为1、2、3或6，
W的1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；
其中yn为0、4或8，yp为0或1；
其中zn为1、2或3，
Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代；
-Cyr-为3到10元饱和的亚碳环基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；
其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx,Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1-6脂肪族基团；
其中每个Rr各自独立地为氢、卤素或C1-6脂肪族基团；
所述-Lb-由2到7个氨基酸构成的肽残基，所述-Lb-的肽残基为由选自以下组中的氨基酸形成的肽残基：苯丙氨酸、甘氨酸、丙氨酸、缬氨酸、瓜氨酸、赖氨酸、丝氨酸、谷氨酸、和天冬氨酸；
所述-Lc-为
其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6脂肪族基团。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述L为-La-Lb-Lc-，
所述-La-为
其中，W为-(C(Rwa)(Rwb))wn-，Y为-(OCH2CH2)yn-Oyp-，Z为-(C(Rza)(Rzb))zn；
其中wn为1、2、3或6，
W的1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；
其中yn为0、4或8，yp为0或1；
其中zn为1、2或3，
Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代；
-Cyr-为3到10元饱和的亚碳环基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；
其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx,Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1-6脂肪族基团；
其中每个Rr各自独立地为氢、卤素或C1-6脂肪族基团；
所述-Lb-选自以下组：
所述-Lc-为
其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6脂肪族基团。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-La-为优选为
在一些实施方式中，本发明所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-La-为
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-Lb-为优选为
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-Lc-为
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述的接头单元L，其La端与Ab相连，Lc端与接头单元M相连。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述L为
在一些实施方式中，本发明所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述L为
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体药物偶联物结构如式(Ⅱ-1)或(Ⅱ-2)所示：

其中，
Ab、L2、X、p和L3分别如本文任一方案所定义。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体药物偶联物结构如式(Ⅱ-1)或(Ⅱ-2)所示：
其中，
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab如本文任一方案所定义；
L2为-O-或-S-；
X选自任选被0、1、2或3个R3a取代的3到6元饱和的碳环基；
L3选自-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；
其中每个R1a、R1b或R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体药物偶联物结构如式(Ⅱ-1)或(Ⅱ-2)所示：
其中，
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab如本文任一方案所定义；
L2为-O-或-S-；
X选自环丙基、环丁基、环戊基或环己基；
L3选自-CH2-、-CH(CH3)-、-C(CH3)2-、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体药物偶联物选自以下结构式：



其中，
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab如本文任一方案所定义。
在又一个方面，本发明提供了一种抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体药物偶联物选自：




其中，
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数。
本发明hu3F8-2和hu3F8-3为抗Her2抗体，所述抗Her2抗体hu3F8-2的重链氨基酸序列如SEQ ID NO.:13所示，轻链氨基酸序列如SEQ ID NO.:12所示；所述抗Her2抗体hu3F8-3的重链氨基酸序列如SEQ ID NO.:14所示，轻链氨基酸序列如SEQ ID NO.:12所示。
在一些实施方式中，本发明所述平均连接数p可以为2到8的整数或小数。例如，所述平均连接数n可以为3到8的整数或小数。例如，所述平均连接数n可以为1到2、2到3、3到4、4到5、5到6、6到7、7到8、8到9、9到10的整数或小数。
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了如本文所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或药物组合物在制备用于治疗和/或预防Her3介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了一种治疗和/或预防Her3介导的疾病或病症的方法，其包括向有需要的受试者施用如本文所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或药物组合物，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了如本文所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或药物组合物，其用于治疗和/或预防Her3介导的疾病或病症，优选地，所述疾病或病症为癌症。
在一些实施方式中，本发明所述癌症选自肺癌、肾癌、尿道癌、结肠直肠癌、前列腺癌、多形性成胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、黑色素瘤、肝癌、膀胱癌、胃癌和食道癌。
在又一个方面，本发明提供了一种药物组合，其包含如本文所述的抗体药物偶联物或其药学上可接受的盐或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的抗体药物偶联物、或本文所述的药物组合物。
附图说明
图1：鼠源3F8特异性结合SP2/0-HER3细胞。
图2：通过ELISA测定鼠源3F8与人HER3、HER2和EGFR的结合亲和力。
图3：通过ELISA测定鼠源3F8识别人和猴HER3的效力。
图4：鼠源3F8阻断NRG1诱导的HER3磷酸化。
图5：鼠源3F8被不同HER3表面水平的细胞迅速摄取。
图6：抗HER3抗体在BT474皮下异种移植模型中抑制肿瘤生长。
图7：胃PDX模型GAS078的[89Zr]Zr-ch3F8成像。
图8：在6个PDX模型中具有代表性的[89Zr]Zr-ch3F8成像。
图9A-9C：hu3F8在热、酸和重复冻融的应激测试后保持结合亲和力。图9A，将hu3F8的三个克隆在pH3.5下孵育0、2、4和6小时，然后进行ELISA测定以测量结合亲和力。图9B，将hu3F8的三个克隆在40℃下孵育不同天，然后进行ELISA测定以测量结合亲和力。图9C，将hu3F8的三个克隆重复冻融3或5个周期，然后进行ELISA测定以测量结合亲和力。
图10：抗体药物偶联物的细胞内吞活性。
图11A-11C：抗体药物偶联物ADC-1-X1和ADC-1-X2对HER3靶标的肿瘤细胞的体外增殖抑制测试；图11A为Her3高表达的HCC1569肿瘤细胞；图11B为Her3高表达的MDA-MB-453肿瘤细胞；图11C为Her3不表达的MDA-MB-231肿瘤细胞。
图12A-12B：抗体药物偶联物ADC-1-X1和ADC-1-X2对HCC1569荷瘤小鼠的抗肿瘤体内药效。图12A为肿瘤体积变化。图12B为肿瘤抑制率。
图13A-13B：抗体药物偶联物ADC-1-X1和ADC-1-X2对Colo205荷瘤小鼠的抗肿瘤体内药效。图13A为肿瘤体积变化。图13B为肿瘤抑制率。
图14：抗体药物偶联物ADC-1-X2对NCI-H441肺癌细胞荷瘤小鼠的抗肿瘤体内药效。
图15：抗体药物偶联物ADC-1-X2对人源肺癌LU1542皮下异种移植荷瘤小鼠的抗肿瘤体内药效。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
为了可以更容易地理解本发明，某些科技术语具体定义如下。除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocolsin Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。本文(包括权利要求书)所用单数形式包括其相应的复数形式，除非文中另有明确规定。
术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
术语“人表皮生长因子受体3(Her3)”，也称为受体酪氨酸蛋白激酶ERBB-3(ERBB3)，是EGFR/erBB家族的成员。与其他erBB家族成员Her2和EGFR不同，Her3本身不具有激酶活性。因此，Her3必须与其激酶活性成员EGFR或Her2结合，作为异二聚体来触发其下游活性。在与其天然配体NRG1结合后，Her3经历构象变化、异二聚化和磷酸化，然后通过信号转导激活MAPK、PI3K/Akt和PLCγ。
术语“抗体”是指具有所需生物活性的任何形式的抗体。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“全长抗体”，是指在天然存在时包含四条肽链的免疫球蛋白分子：两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
术语“CDR”是指抗体可变序列内的互补决定区。在重链和轻链的各个可变区中存在3个CDR，其对于各个重链和轻链可变区被命名为HCDR1、HCDR2和HCDR3或LCDR1、LCDR2和LCDR3。本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342:877-883；Al-Lazikani等人,“Standard conformations for the canonical structures of immunoglobulins”,Journal of Molecular Biology,273,927-948(1997))基于抗体序列可变性的Kabat(Kabat等人,Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services,National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999Nucleic Acids Research,27,209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
应该注意，基于不同的指派系统获得的同一抗体的可变区的CDR的边界可能有所差异。即不同指派系统下定义的同一抗体可变区的CDR序列有所不同。因此，在涉及用本发明定义的具体CDR序列限定抗体时，所述抗体的范围还涵盖了这样的抗体，其可变区序列包含所述的具体CDR序列，但是由于应用了不同的方案(例如不同的指派系统或组合)而导致其所声称的CDR边界与本发明所定义的具体CDR边界不同。
术语抗体(“亲代抗体”)的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗体结合片段的实例包括但不限于Fab，Fab'，F(ab')2和Fv片段；双抗体；线性抗体；单链抗体分子，例如scFv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
术语“全人抗体”是指只包含人免疫球蛋白蛋白质序列的抗体。如在小鼠中、在小鼠细胞中或在来源于小鼠细胞的杂交瘤中产生，则全人抗体可含有鼠糖链。同样，“小鼠抗体”是指仅包含小鼠免疫球蛋白序列的抗体。或者，如果在大鼠中、在大鼠细胞中或在来源于大鼠细胞的杂交瘤中产生，则全人抗体可含有大鼠糖链。同样，“大鼠抗体”是指仅包含大鼠免疫球蛋白序列的抗体。
术语“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
术语“Fc区”用于定义包含至少一部分恒定区的免疫球蛋白重链的C端区域。该术语包括天然序列Fc区和变异Fc区。在一些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羧基末端。但是，Fc区的C端赖氨酸(Lys447)可能存在或不存在(此段中的编号是根据EU编号系统，也称为EU索引，如Rabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991)。
术语“交叉反应”指的是对人类、猴、和/或鼠源(小鼠或大鼠)相同靶分子的抗原片段的结合。因此，“交叉反应”应被理解为与在不同物种中表达的相同分子X的种属间反应。识别人Her3R、猴、和/或鼠Her3R(小鼠或大鼠)的单克隆抗体的交叉反应特异性可通过FACS分析确定。
术语“亲和力”或“结合亲和力”指反映结合对子的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是kdis和kon)的比值。亲和力可以由本领域已知的常见方法测量。用于测量亲和力的一个具体方法是本文中的ForteBio动力学结合测定法。
术语“不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的KD为1.0×10-6M或更高，更优选1.0×10-5M或更高，更优选1.0×10-4M或更高、1.0×10-3M或更高，更优选1.0×10-2M或更高。
术语“高亲和性”对于IgG抗体而言，是指对于抗原的KD为1.0×10-6M或更低，优选5.0×10-8M或更低，更优选1.0×10-8M或更低、5.0×10-9M或更低，更优选1.0×10-9M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指KD为10-6M或更低，优选10-7M或更低，更优选10-8M或更低。
术语“细胞毒性药物”通常指毒性药物，所述细胞毒性药物可以在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物可以在足够高的浓度下杀死肿瘤细胞。所述“细胞毒性药物”可以包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32或Lu的放射性同位素)，毒性药物，化疗药物，抗生素和核溶酶，例如，可以是毒性药物，包括但不限于喜树碱衍生物，例如，可以是喜树碱衍生物依沙替康(化学名：(1S,9S)-1-氨基-9-乙基-5-氟-2,3-二氢-9-羟基-4-甲基-1H,12H-苯并[de]吡喃并[3’,4’:6,7]咪唑并[1,2-b]喹啉-10,13(9H,15H)-二酮)。
术语“接头单元”或“接头结构”通常指指一端与配体连接而另一端与细胞毒性药物相连的化学结构片段或键，也可以连接其他接头后再与细胞毒性药物相连。所述直接或间接连接配体可以是指所述基团通过共价键直接连接配体，也可以是通过接头结构连接配体。例如，接头结构可以是本申请所述的-Lax-Lb-Lc-和或-La-Lb-Lc-所示的结构。例如，可以使用包含酸不稳定接头结构(例如腙)、蛋白酶敏感(例如肽酶敏感)接头结构、光不稳定接头结构、二甲基接头结构、或含二硫化物接头结构的化学结构片段或键作为接头结构。
术语某个结构“任选地与其它分子部分相连接”通常是指该结构不与任何其它化学结构相连接，或者该结构与一个或多个不同于该结构的其它化学结构(例如本申请所述的配体)相连接(例如，通过化学键连接、或通过接头结构连接)。
术语“配体-药物偶联物”通常是指配体通过稳定的连接单元与具有生物活性的细胞毒性药物相连。在本申请中“配体-药物偶联物”可以为抗体-药物偶联物(antibody drug conjugate，ADC)，所述ADC可以是指把单克隆抗体或者抗体片段通过稳定的连接单元与具有生物活性的细胞毒性药物相连。
在本申请中，术语“亚甲基”通常是指1个碳原子的基团除去两个氢原子所衍生的残基。亚甲基可以是取代的或非取代的，替代或者非替代的。术语“亚烷基”通常指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其可以为包含1至20个碳原子的直链或支链基团，例如含有1至12个碳原子，例如含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH2-)、1,1-亚乙基(-CH(CH3)-)、1,2-亚乙基(-CH2CH2)-、1,1-亚丙基(-CH(CH2CH3)-)、1,2-亚丙基(-CH2CH(CH3)-)、1,3-亚丙基(-CH2CH2CH2-)、1,4-亚丁基(-CH2CH2CH2CH2-)和1,5-亚丁基(- CH2CH2CH2CH2CH2-)等。亚烷基可以是取代的或非取代的，替代或者非替代的，例如当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、杂环烷硫基和氧代基中的一个或多个取代基所取代，例如可以是氢、氕、氘、氚、卤素、-NO2、-CN、-OH、-SH、-NH2、-C(O)H、-CO2H、-C(O)C(O)H、-C(O)CH2C(O)H、-S(O)H、-S(O)2H、-C(O)NH2、-SO2NH2、-OC(O)H、-N(H)SO2H、或C1-6脂肪族基团。
术语“亚芳基”通常是指具有两个从芳环的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基。术语“芳环”可以指具有共轭的π电子体系的6至14元全碳单环或稠合多环(也就是共享毗邻碳原子对的环)，可以为6至10元，例如苯和萘。所述芳环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环。芳基可以是取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自以下组：烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、和杂环烷硫基。
在本申请中，术语“亚杂芳基”通常是指具有两个从杂芳环的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基。术语“杂芳环”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子可以选自以下组：氧、硫和氮。杂芳基可以为5至10元，可以为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环。亚杂芳基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自以下组：烷基、烯基、炔基、烷氧基、烷硫基、烷基氨基、卤素、巯基、羟基、硝基、氰基、环烷基、杂环烷基、芳基、杂芳基、环烷氧基、杂环烷氧基、环烷硫基、和杂环烷硫基。
术语“亚杂环基”通常是指稳定的不具有芳香性的3元-7元单环结构，融合的7元-10元双环杂环结构或桥联的6元-10元双环杂环结构，这些环状结构即可以是饱和的，也可以是部分饱和的，除碳原子外，这些环状结构中还含有一个或多个杂原子，其中杂原子可以选自以下组：氧、硫和氮。例如是含有1-4个上述定义的杂原子。当用来表示杂环环状结构上的原子时，术语“氮”可以包括发生过取代反应的氮。亚杂环基可以是取代的或非取代的。
术语“亚碳环基”通常是指具有两个从碳环的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基。术语“碳环”通常指饱和或部分不饱和单环或多环环状烃，碳环包含3至20个碳原子，可以包含3至12个碳原子，可以包含3至10个碳原子，可以包含3至8个碳原子，例如包含3至6个碳原子。单环碳环的非限制性实例包括环丙烷、环丁烷、环戊烷、环戊烯、环己烷、环己烯、环己二烯、环庚烷、环庚三烯、环辛烷等；多环碳环可以包括螺环、稠环和桥环的碳环。例如，“3到6元饱和的碳环基”包括环丙基、环丁基、环戊基、环己基、亚环丙基、亚环丁基、亚环戊基或亚环己基。碳环基和亚碳环基可以是取代的或非取代的，例如可以被一个或多个卤素、C1-6脂肪族基团或被卤素取代的C1-6脂肪族基团所取代。
在本申请中，术语“部分不饱和的”通常是指环状结构中环分子间至少含一个双键或三键。术语“部分不饱和”涵盖带有多处不饱和的环状结构，但并非意在包括本申请所定义的芳环或 杂芳环。术语"不饱和的"表示部分具有一个或多个不饱和度。
术语“卤素”通常是指氟、氯、溴、碘，例如可以是氟、氯。
术语“脂肪族基团”通常是指具有1-12个碳原子的直链烃、支链烃或环状结构的烃，这些烃或者是完全饱和烃；或者带有一个或多个不饱和单元，但不饱和单元不是芳香类基团。例如，适用的脂肪族基团可以包括取代的或未取代的直链、支链或环状结构的烷基、烯基、炔基以及这些基团的混合物；比如是(环烷基)烷基、(环烯基)烷基或(环烷基)烯基。例如，脂肪族基团具有1-12、1-8、1-6、1-4或1-3个碳原子。例如，“C1-6脂肪族基团”指的是具有1-6个碳原子的如上所述的脂肪族基团，包括但不限于具有1-6个碳原子的直链、支链或环状结构的烷基、烯基或炔基，例如甲基、乙基、丙基、异丙基、正丁基、异丁基、叔丁基、戊基、己基等。
在本申请中，术语“任选”或“任选地”通常意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明可以包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
术语“取代的”通常指基团中的一个或多个氢原子，例如为最多5个，例如为1～3个氢原子彼此独立地被相应数目的取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
在本申请中，术语0个或多个(例如，0个或至少1个、0个或1个、0个)亚甲基单元被“替代”通常指当所述结构包含1个或多个亚甲基单元时，所述一个或多个亚甲基单元可以不被替代，或被一个或多个不是亚甲基的基团(例如-NHC(O)-、-C(O)NH-、-C(O)-、-OC(O)-、-C(O)O-、-NH-、-O-、-S-、-SO-、-SO2-、-PH-、-P(＝O)H-、-NHSO2-、-SO2NH-、-C(＝S)-、-C(＝NH)-、-N＝N-、-C＝N-、-N＝C-或-C(＝N2)-)所替代。
基团中的一个或多个氢原子，例如为最多5个，例如为1～3个氢原子彼此独立地被相应数目的取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
术语“药学上可接受的盐”或“可药用盐”通常是指本申请药物偶联物的盐，这类盐用于哺乳动物体内时可以具有安全性和/或有效性，且可以具有应有的生物活性，本申请抗体药物偶联物可以与酸形成盐，药学上可接受的盐的非限制性实例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐、对甲苯磺酸盐。
术语“溶剂化物”或“溶剂化合物”通常是指本申请的药物偶联物与一种或多种溶剂分子形成可药用的溶剂化物，溶剂分子的非限制性实例包括水、乙醇、乙腈、异丙醇、DMSO、乙酸乙酯。
术语“载药量”通常是指每个配体上加载的细胞毒性药物平均数量，也可以表示为细胞毒性药物和抗体量的比值，细胞毒性药物载量的范围可以是每个配体(Ab)连接0-12个，例如1- 10个细胞毒性药物。在本申请的实施方式中，载药量表示为Na，示例性的可以为1，2，3，4，5，6，7，8，9，10的均值。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的载药量。
“药学上可接受的载体”是指药物制剂或组合物中除活性成分以外的对受试者无毒的成分。药学上可接受的载体包括但不限于缓冲剂，赋形剂，稳定剂或防腐剂。
术语“包含”通常是指包括明确指定的特征，但不排除其他要素。术语“以上”、“以下”通常是指包含本数的情况。
“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。如本文中所用，术语“cyno”或“食蟹猴”是指食蟹猴。
“联合”一种或多种其它治疗剂的施用包括同时(共同)施用和任意次序的连续施用。
“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的Her3抗体或其抗原结合片段当单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的抗体或其抗原结合片段的量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。
术语“癌症”在本文中用于指表现出异常高水平的增殖和生长的一组细胞。癌症可能是良性的(也称为良性肿瘤)，恶性前或恶性。癌细胞可以是实体癌细胞或白血病癌细胞。本文使用的术语“肿瘤”是指包含癌症的一个或多个细胞。术语“肿瘤生长”在本文中用于指代包含癌症的一种或多种细胞的增殖或生长，其导致癌症的大小或程度的相应增加。
抗Her3抗体
本申请公开了一种新的抗HER3抗体，其在体外和体内有效抑制肿瘤细胞生长，具有很好的安全性。用过表达人HER3的SP2/0细胞免疫的小鼠杂交瘤中鉴定出HER3抗体3F8。3F8以纳摩尔结合亲和力识别人和猴HER3，并对其他ERBB家族成员具有高选择性。此外，具有不同HER3水平的细胞可以快速有效地摄取HER3，HER3被认为是理想的ADC药物所必需的特性。PET成像研究表明，[89Zr]Zr-3F8在PDX模型的肿瘤中显著积累，表明3F8可能是一种高效载体，将细胞毒性带入肿瘤细胞。3F8和hu3F8在PDX模型中保持了上述结合亲和力、选择性和肿瘤抑制的特征。hu3F8具有良好的可开发性，在反复冻融处理、酸温育和40℃储存的应激试验中，其结合亲和力、聚集和翻译后修饰几乎没有变化。
本发明术语“抗Her3抗体”、“抗Her3”、“Her3抗体”或“结合Her3的抗体”是指能够以足够的亲合力结合Her3蛋白或其片段以致所述抗体可以用作靶向Her3中的诊断剂和/或治疗剂。
本发明所述的抗Her3抗体或其抗原结合片段包括申请号为PCT/CN2021/099998中描述的任意一个抗Her3抗体或其抗原结合片段，该申请公开的全部内容通过引入全文并入本文。 在一些方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体的CDR序列包括来自于PCT/CN2021/099998中描述的抗体hu3F8-2的CDR序列。在一些方案中，在本发明的药物偶联物、组合物或用途中使用的抗体的可变区序列包括来自于PCT/CN2021/099998中描述的抗体hu3F8-1的可变区序列。在一些方案中，在本发明的药物偶联物、组合物、用途或方法中使用的抗体的可变区序列包括来自于PCT/CN2021/099998中描述的抗体hu3F8-2的可变区序列。在一些方案中，在本发明的药物偶联物、组合物或用途中使用的抗体的可变区序列包括来自于PCT/CN2021/099998中描述的抗体hu3F8-3的可变区序列。
在本文实施例中所用的非限制性、示范性抗体选自PCT/CN2021/099998中描述的人源化抗体hu3F8-1，hu3F8-2和hu3F8-3，其氨基酸序列见下表。
在本文中，hu3F8-1、hu3F8-2和hu3F8-3分别对应PCT/CN2021/099998中的克隆1(clone-1)、克隆2(clone-2)和克隆3(clone-3)。
人源化抗Her3抗体氨基酸序列(KABAT方案)
可采用用于产生抗体的任何合适方法来产生本发明的抗体。任何合适形式的Her3都可用作产生抗体的免疫原(抗原)。通过举例而非限制，任何Her3变体或其片段都可用作免疫原。在一些实施方式中，产生鼠源的单克隆抗人Her3抗体的杂交瘤细胞可通过本领域公知的方法产生。来源于啮齿动物(如小鼠)的抗体在体内用作治疗药物时可能引起不需要的抗体免疫原性，重复使用导致人体产生针对治疗性抗体的免疫应答，这类免疫应答至少导致丧失治疗功效，而严重的则导致潜在致死过敏反应。降低啮齿动物抗体的免疫原性的一种方法包括嵌合抗体的产生，其中将小鼠可变区与人恒定区融合(Liu等(1987)Proc.Natl.Acad.Sci.USA 84:3439-43)。然而，嵌合抗体中的完整啮齿动物可变区的保留仍可能在患者中引起有害的免疫原性。将啮齿动物可变结构域的互补决定区(CDR)环移植到人构架上(即人源化)已被用于进一步将啮齿动物序列减至最低(Jones等(1986)Nature 321:522；Verhoeyen等(1988)Science 239:1534)。
在一些实施方式中，本发明的嵌合或人源化抗体可基于所制备的鼠单克隆杂交瘤抗体的序列来制备。编码重链和轻链免疫球蛋白的DNA可以从目标鼠杂交瘤中获得，并且使用标准分子生物学技术进行工程改造以包含非鼠(例如人)免疫球蛋白序列。
在一些实施方式中，本发明所述的嵌合Her3抗体，可使用本领域已知的方法将杂交瘤来源的免疫球蛋白重链和轻链可变区与人IgG恒定区有效连接(参见例如属于Cabilly等人的美国专利No.4,816,567)，获得嵌合型重链和嵌合型轻链来制备。在一些实施方式中，本发明的嵌合抗体包含的恒定区可选自任何人IgG亚型，如IgG1、IgG2、IgG3、IgG4，优选IgG4。
在一些实施方式中，本发明的嵌合Her3抗体可由嵌合型轻链与嵌合型重链表达质粒“混合和匹配”转染表达细胞获得，此类“混合和匹配”的抗体的Her3结合可使用上述结合测定和其它常规结合测定(例如，ELISA)来进行测试。
本发明所述的人源化抗体，可以使用本领域已知的方法将鼠源CDR区插入人种系框架区。参见Winter等人的美国专利No.5,225,539及Queen等人的美国专利No.5,530,101；5,585,089；5,693,762和6,180,370。
在一些实施方式中，氨基酸变化包括氨基酸缺失、插入或置换。在一些实施方式中，本发明的抗Her3抗体或其抗原结合片段包括具有已通过氨基酸缺失、插入或置换突变的，但仍与上述抗体(特别地在上述序列中描绘的CDR区中)有至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性的氨基酸序列的那些抗体。在一些实施方式中，本发明的抗体与具体序列中描绘的CDR区相比较时，在CDR区中已通过氨基酸缺失、插入或置换的氨基酸突变不超过1、2、3、4或5个。
在一些实施方式中，可在本文中所提供抗体的Fc区中引入一个或多个氨基酸修饰，以此产生Fc区变体。Fc区变体可包含在一或多个氨基酸位置处包含氨基酸修饰(例如置换)的人Fc区序列(例如人IgG1、IgG2、IgG3或IgG4Fc区)。
在一些实施方式中，可能需要产生经半胱氨酸工程改造的抗体，例如“硫代MAb”，其中抗体的一或多个残基经半胱氨酸残基置换。
在一些实施方式中，本文中所提供的抗体可进一步经修饰为含有本领域中已知且轻易获得的其他非蛋白质部分。适合抗体衍生作用的部分包括，但不限于，水溶性聚合物。水溶性聚合物的非限制性实例包括，但不限于，聚乙二醇(PEG)、乙二醇/丙二醇共聚物、羧甲基纤维素、葡聚糖、聚乙烯醇、聚乙烯吡咯烷酮、聚-1，3-二烷、聚-1，3，6-三烷、乙烯/马来酸酐共聚物、聚氨基酸(均聚物或无规共聚物)、及葡聚糖或聚(n-乙烯基吡咯烷酮)聚乙二醇、丙二醇均聚物、聚环氧丙烷/氧化乙烯共聚物、聚氧乙基化多元醇(例如甘油)、聚乙烯醇、及其混合物。
药物偶联物
本申请提供了一种抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物形式，其可以具有选自以下组的一种或多种效果：(1)具有对肿瘤细胞的体外增殖的抑制活性；(2)具有靶向抑制性；(3)具有血浆稳定性；(4)具有体内抑瘤效果；(5)具有旁观杀伤效应(Bystander Effect)；(6)具有抗转运体转运能力；(7)具有体内肿瘤靶向能力；和(8)具有良好的体内安全性。
本申请提供一种抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗Her3抗体药物偶联物结构如式(I)所示：
Ab-(L-M-D)p    (I)
其中，
L和M是接头单元；
D是细胞毒性药物；
p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
Ab为抗Her3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3,和所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；优选地，本发明所述抗Her3抗体或其抗原结合片段包含：(I)氨基酸序列如SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；或(II)氨基酸序列如SEQ ID NO:9所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；或(III)氨基酸序列如SEQ ID NO:10所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；更优选地，本发明所述抗Her3抗体或其抗原结合片段包含：(1)氨基酸序列如SEQ ID NO:11所示的重链，和氨基酸序列如SEQ ID NO:12所示的轻链；或(2)氨基酸序列如SEQ ID NO:13所示的重链，和氨基酸序列如SEQ ID NO:12所示或的轻链；或(3)氨基酸序列如SEQ ID NO:14所示的重链，和氨基酸序列如SEQ ID NO:12所示的轻链。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗Her3抗体药物偶联物结构如式(I-1)所示：
其中，
L和M是接头单元；
p表示平均连接数，且n选自1到10的整数或小数，优选3-8的整数或小数；
Ab为抗Her3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3,和所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；优选地，本发明所述抗Her3抗体或其抗原结合片段包含：(I)氨基酸序列如SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；或(II)氨基酸序列如SEQ ID NO:9所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；或(III)氨基酸序列如SEQ ID NO:10所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；更优选地，本发明所述抗Her3抗体或其抗原结合片段包含：(1)氨基酸序列如SEQ ID NO:11所示的重链，和氨基酸序列如SEQ  ID NO:12所示的轻链；或(2)氨基酸序列如SEQ ID NO:13所示的重链，和氨基酸序列如SEQ ID NO:12所示或的轻链；或(3)氨基酸序列如SEQ ID NO:14所示的重链，和氨基酸序列如SEQ ID NO:12所示的轻链。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中，
M为-L2-L3-X-L1-；
L2为-O-或-S-；
L3为-(C(R1a)(R1b))m-，m选自0、1、2或3，其中当L3包含亚甲基单元时，所述L3的0个或1个亚甲基单元可以被-C(O)-、或-C(＝S)-替代；优选地，L3选自单键、-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；更优选地，L3选自单键、-CH2-、-CH(CH3)、-C(CH3)2、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-；
L1为-(C(R2a)(R2b))n-，n选自1、2或3,其中当L1可以包含亚甲基单元时，所述L1的0个或1个亚甲基单元可以被-C(O)-、或-C(＝S)-替代；优选地，L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；更优选地，L1选自-CH2-、-CH2C(O)-、-CH(CH3)C(O)-或-C(O)-；
X选自3到6元饱和的碳环基、3到6元饱和的杂环基或单键，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被0、1、2或3个R3a取代；优选地，X为任选被0、1、2或3个R3a取代的3到6元饱和的碳环基或单键；更优选地，X为3到6元饱和的碳环基或单键；
其中每个R1a，R1b，R2a，R2b，R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中，所述-M-选自：
L2为-O-或-S-；优选地，L2为-O-；
X选自任选被0、1、2或3个R3a取代的3到6元饱和的碳环基；优选地，X选自环丙基、环丁基、环戊基或环己基；更优选地，X为环丙基；更优选地，X为环丁基；更优选地，X为环己基；
L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；优选地，L1为-C(O)-；
其中每个R2a，R2b或R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
L2为-O-或-S-；优选地，L2为-O-；优选地，L2为-S-；
L3选自-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；优选地，L3选自-CH2-、-CH(CH3)、-C(CH3)2、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-；优选地，L3选自CH2-、-CH(CH3)、-CH2CH2-。
L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；优选地，优选地，L1为-C(O)-；
其中每个R1a，R1b，R2a或R2b各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
其中每个R各自独立地可以为氢或卤素。
在一些优选地实施例中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述L为-La-Lb-Lc-，
所述-La-为
其中，W为-(C(Rwa)(Rwb))wn-，其中wn为1、2、3或6，W的1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；优选地，W为-(C(Rwa)(Rwb))2-或-(C(Rwa)(Rwb))3-；优选地，W为-CH2CH2CH2-或-CH2CH2-；
Y为-(OCH2CH2)yn-Oyp-其中yn为0、4或8，yp为0或1；优选地，Y为单键；
Z为-(C(Rza)(Rzb))zn，其中zn为1、2或3，Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代，-Cyr-为3到10元饱和的亚碳环基，其中所述-Cyr-是未取代的或独立地被1到3个取代基Rcx取代；优选地，Z为-(C(Rwa)(Rwb))2C(O)-或-(C(Rwa)(Rwb))3C(O)-；优选地，Z为-CH2CH2CH2C(O)-或CH2CH2C(O)-；
其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx,Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1-6脂肪族基团；
其中每个Rr各自独立地为氢、卤素或C1-6脂肪族基团；
优选地，所述-La-为优选为
所述-Lb-选自以下组：
优选地；所述-Lb-为优选-Lb-为
所述-Lc-为其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6脂肪族基团；优选地，所述-Lc-为
在一些特别优选地实施例中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述L为
在一些实施方式中，本发明所述抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体药物偶联物结构如式(Ⅱ-1)或(Ⅱ-2)所示：

其中，
L2为-O-或-S-；优选地，L2为-O-，优选地，L2为-S-，
X选自任选被0、1、2或3个R3a取代的3到6元饱和的碳环基；优选地，X选自环丙基、环丁基、环戊基或环己基；
L3选自-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-，其中每个R1a、R1b或R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团，其中每个R各自独立地可以为氢或卤素；优选地，L3选自-CH2-、-CH(CH3)-、-C(CH3)2-、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-；
p表示平均连接数，且n选自1到10的整数或小数，优选3-8的整数或小数；
Ab为抗Her3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3,和所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；优选地，本发明所述抗Her3抗体或其抗原结合片段包含：(I)氨基酸序列如SEQ ID NO:7所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；或(II)氨基酸序列如SEQ ID NO:9所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；或(III)氨基酸序列如SEQ ID NO:10所示的重链可变区，和氨基酸序列如SEQ ID NO:8所示的轻链可变区；更优选地，本发明所述抗Her3抗体或其抗原结合片段包含：(1)氨基酸序列如SEQ ID NO:11所示的重链，和氨基酸序列如SEQ ID NO:12所示的轻链；或(2)氨基酸序列如SEQ ID NO:13所示的重链，和氨基酸序列如SEQ ID NO:12所示或的轻链；或(3)氨基酸序列如SEQ ID NO:14所示的重链，和氨基酸序列如SEQ ID NO:12所示的轻链。
在一些实施方式中，本发明所述平均连接数p可以为2到8的整数或小数。例如，所述平均连接数n可以为3到8的整数或小数。例如，所述平均连接数n可以为1到2、2到3、3到4、4到5、5到6、6到7、7到8、8到9、9到10的整数或小数。
药物组合物和药物制剂
在又一个方面，本发明提供了一种药物组合物，其包含如本文所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。
应理解，本发明提供的抗Her3抗体药物偶联物或、其异构体、其药学上可接受的盐或其混合物，或其药物组合物可以整合制剂中合适的运载体、赋形剂和其他试剂以联合给药，从而提供改善的转移、递送、耐受等。
术语“药物组合物”指这样的制剂，其允许包含在其中的活性成分的生物学活性有效的形式存在，并且不包含对施用所述制剂的受试者具有不可接受的毒性的另外的成分。
可以通过将具有所需纯度的本发明的抗Her3抗体药物偶联物或其药学上可接受的盐与一种或多种任选的药用辅料(Remington's Pharmaceutical Sciences，第16版，Osol,A.编辑(1980))混合来制备包含本文所述的抗Her3抗体的药物制剂，优选地以水溶液或冻干制剂的形式。
本发明的药物组合物或制剂还可以包含一种或多种其它活性成分，所述活性成分是被治疗的特定适应证所需的，优选具有不会不利地影响彼此的互补活性的那些活性成分。在一些实施方式中，其它的活性成分为化疗剂、免疫检查点抑制剂、生长抑制剂、抗生素或已知的各种抗肿瘤或抗癌剂，所述活性成分以对于目的用途有效的量合适地组合存在。在一些实施方式中，本发明的药物组合物还包含编码抗Her3抗体的多核苷酸的组合物。
在又一个方面，本发明提供了一种药物组合，其包含如本文所述的抗体药物偶联物或其药学上可接受的盐或本文所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本文所述的抗体药物偶联物或、其异构体、其药学上可接受的盐或其混合物或本文所述的药物组合物，优选其进一步包括给药装置。
医药用途
在又一个方面，本发明提供了如本文所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，或本文所述的药物组合物在制备用于治疗和/或预防Her3介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了如本文所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，或本文所述的药物组合物，其用于治疗和/或预防Her3介导的疾病或病症，优选地，所述疾病或病症为癌症。
在又一个方面，本发明提供了一种治疗和/或预防Her3介导的疾病或病症的方法，其包括向有需要的受试者施用如本文所述的抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，或本文所述的药物组合物，优选地，所述疾病或病症为癌症。
在一些实施方式中，所述癌症选自肺癌、肾癌、尿道癌、结肠直肠癌、前列腺癌、多形性成胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、黑色素瘤、肝癌、膀胱癌、胃癌、和食道癌。
在一些实施方式中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
在一些实施方式中，本发明提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。
在一些实施方式中，本发明提供的方法或用途还包括向个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体药物偶联物或其药学上可接受的盐。例如，可以与至少一种另外的治疗剂共施用。
本申请所述的药物偶联物可以具有对肿瘤细胞的体外增殖的抑制活性。所述抑制活性可以是本申请的药物偶联物加入肿瘤细胞的培养基中，相比于加入阴性对照或者对照药物，所述肿瘤细胞细胞增殖能力下降1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％ 以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上。例如，所述抑制活性可以是对于肿瘤细胞的IC50值(nM)可以为10000以下、5000以下、4000以下、3000以下、2000以下、1000以下、500以下、400以下、300以下、200以下、150以下、120以下、110以下、100以下、99以下、98以下、97以下、95以下、90以下、80以下、75以下、70以下、65以下、62以下、60以下、50以下、40以下、30以下、25以下、23以下、22以下、20以下、19以下、18以下、18.5以下、17以下、15以下、12以下、10以下、9以下、8.5以下、7以下、6.7以下、6以下、5.9以下、5.5以下、5.0以下、4.8以下、4.5以下、4.4以下、4以下、3.5以下、3以下、2.5以下、2以下、1.5以下、1.0以下、0.5以下、0.3以下、0.29以下、0.25以下、0.21以下、0.20以下、0.18以下、0.17以下、0.15以下、0.12以下、0.10以下、0.09以下、0.08以下、0.07以下、0.06以下、0.05以下、0.04以下、0.03以下、0.02以下或0.01以下。例如，所述肿瘤细胞可以包括但不限于实体瘤细胞，例如所述肿瘤细胞包括但不限于胃癌细胞、或乳腺癌细胞，例如所述肿瘤细胞可以包括但不限于HCC1569细胞或MDA-MB-453细胞。
本申请所述的药物偶联物可以具有靶向抑制性。所述靶向抑制性可以是本申请的药物偶联物加入特定靶标高表达的肿瘤细胞的培养基中，相比于加入阴性对照或者对照药物，所述特定靶标高表达的肿瘤细胞细胞增殖能力下降1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上。例如，所述靶向抑制性可以是对于特定靶标高表达的肿瘤细胞的IC50值(nM)可以为10000以下、5000以下、4000以下、3000以下、2000以下、1000以下、500以下、400以下、300以下、200以下、185以下、150以下、120以下、110以下、100以下、99以下、98以下、97以下、95以下、91以下、80以下、74以下、70以下、65以下、62以下、60以下、50以下、40以下、30以下、25以下、23以下、22以下、20以下、19以下、18以下、18.5以下、17以下、15以下、12以下、10以下、9以下、8.5以下、7以下、6.7以下、6以下、5.9以下、5.5以下、5.0以下、4.8以下、4.5以下、4.4以下、4以下、3.5以下、3以下、2.5以下、2以下、1.5以下、1.0以下、0.5以下、0.3以下、0.29以下、0.25以下、0.21以下、0.20以下、0.18以下、0.17以下、0.15以下、0.12以下、0.10以下、0.09以下、0.08以下、0.07以下、0.06以下、0.05以下、0.04以下、0.03以下、0.02以下或0.01以下。例如，所述特定靶标高表达的肿瘤细胞可以包括但不限于实体瘤细胞，例如所述特定靶标高表达的肿瘤细胞包括但不限于胃癌细胞、或乳腺癌细胞，例如所述特定靶标高表达的肿瘤细胞可以包括但不限于HCC1569细胞或MDA-MB-453细胞。所述特定靶标可以包括但不限于Her3。
本申请所述的药物偶联物可以具有血浆稳定性。所述血浆稳定性可以是本申请的药物偶联物加入血浆中，所述药物偶联物释放的细胞毒性药物在1天、3天、5天、7天、14天、20天或30天的释放率不超过50％、不超过40％、不超过30％、不超过20％、不超过10％、不超过7％、不超过5％、不超过4％、不超过3％、不超过2％、不超过1.9％、不超过1.8％、不超过1.7％、不超过1.6％、不超过1.5％、不超过1.4％、不超过1.3％、不超过1.2％、不超过1.1％、不超过1.0％、不超过0.9％、不超过0.8％、不超过0.7％、不超过0.6％、不超过0.5％、不超过0.4％、不超过0.3％、不超过0.2％或不超过0.1％。
本申请所述的药物偶联物可以具有体内抑瘤效果。所述抑瘤效果可以是本申请的药物偶联物施用于动物，相比于加入阴性对照或者对照药物，所述动物的肿瘤在1天、3天、5天、7天、14天、20天、21天或30天的体积减少1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、55％以上、60％以上、70％以上、73％以上、75％以上、80％以上、90％以上、或95％以上，或者相比于施用阴性对照或者对照药物，所述动物的肿瘤在1天、3天、5天、7天、14天、20天、21天或30天的体积减少1.1倍以上，1.3倍以上，1.5倍以上，两倍以上，三倍以上，五倍以上，十倍以上，二十倍以上，二十二倍以上，三十倍以上，五十倍以上，一百倍以上，五百倍以上，一千倍以上，或一千五百倍以上。所述动物可以包括但不限于哺乳动物，例如所述动物可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠、猴或人。所述施用可以包括但不限于口服、静脉注射、静脉滴注、腹腔注射、或局部给药。
本申请所述的药物偶联物可以具有旁观杀伤效应(Bystander Effect)。所述旁观杀伤效应可以是本申请的药物偶联物对特定靶标低表达的肿瘤细胞细胞的细胞增殖无明显抑制作用，而在特定靶标低表达的肿瘤细胞与特定靶标高表达的肿瘤细胞共培养中，本申请的药物偶联物可以同时抑制特定靶标低表达的肿瘤细胞与特定靶标高表达的肿瘤细胞的细胞增殖。例如，在特定靶标低表达的肿瘤细胞与特定靶标高表达的肿瘤细胞共培养中，所述抑制活性可以是对于特定靶标低表达的肿瘤细胞的IC50值(nM)可以为10000以下、5000以下、4000以下、3000以下、2000以下、1000以下、500以下、400以下、300以下、200以下、185以下、150以下、120以下、110以下、100以下、99以下、98以下、97以下、95以下、91以下、80以下、74以下、70以下、65以下、62以下、60以下、50以下、40以下、30以下、25以下、23以下、22以下、20以下、19以下、18以下、18.5以下、17以下、15以下、12以下、10以下、9以下、8.5以下、7以下、6.7以下、6以下、5.9以下、5.5以下、5.0以下、4.8以下、4.5以下、4.4以下、4以下、3.5以下、3以下、2.5以下、2以下、1.5以下、1.0以下、0.5以下、0.3以下、0.29以下、0.25以下、0.21以下、0.20以下、0.18以下、0.17以下、0.15以下、0.12以下、0.10以下、0.09以下、0.08以下、0.07以下、0.06以下、0.05以下、0.04以下、0.03以下、0.02以下或0.01以下。所述特定靶标低表达的肿瘤细胞与特定靶标高表达的肿瘤细胞相比，所述特定靶标的表达可以是降低1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上。例如，所述特定靶标高表达的肿瘤细胞可以包括但不限于实体瘤细胞，例如所述特定靶标高表达的肿瘤细胞包括但不限于胃癌细胞、或乳腺癌细胞，例如所述特定靶标高表达的肿瘤细胞可以包括但不限于HCC1569细胞或MDA-MB-453细胞。例如，所述特定靶标低表达的肿瘤细胞可以包括但不限于实体瘤细胞，例如所述特定靶标低表达的肿瘤细胞包括但不限于乳腺癌细胞，例如所述特定靶标低表达的肿瘤细胞可以包括但不限于HCC1187细胞。
本申请所述的药物偶联物可以具有抗转运体转运能力。所述抗转运能力可以是相比于转运底物的标准品，本申请所述药物偶联物的外排比降低1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上。例如，所述外排比的测试可以可以为本领 域人员常用方法，或记载在本申请的实施例之中。
本申请所述的药物偶联物可以具有体内肿瘤靶向能力。所述体内靶向能力可以是指将用信号物质标记的所述药物偶联物施用于动物，所述标记的药物偶联物在动物的肿瘤组织的分布，相较于其他组织和器官，可以是分布增加1％以上、2％以上、4％以上、5％以上、8％以上、10％以上、15％以上、18％以上、20％以上、25％以上、40％以上、50％以上、60％以上、70％以上、80％以上、90％以上、或95％以上，或者可以是分布增加1.1倍以上，1.3倍以上，1.5倍以上，两倍以上，三倍以上，五倍以上，十倍以上，二十倍以上，二十二倍以上，三十倍以上，五十倍以上，一百倍以上，五百倍以上，一千倍以上，或一千五百倍以上。所述信号物质可以是放射性物质，例如所述信号物质包括但不限于125I。所述动物可以包括但不限于哺乳动物，例如所述动物可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠、猴或人。所述施用可以包括但不限于口服、静脉注射、静脉滴注、腹腔注射、或局部给药。所述组织或器官可以可以包含但不限于心、肝、脾、肺、肾、脑、或骨髓。
本申请所述的药物偶联物可以具有良好的体内安全性。所述体内安全性可以是本申请的药物偶联物在施用于动物后，所述动物的体内游离毒素释放率不超过50％、不超过40％、不超过30％、不超过20％、不超过10％、不超过7％、不超过5％、不超过4％、不超过3％、不超过2％、不超过1.9％、不超过1.8％、不超过1.7％、不超过1.6％、不超过1.5％、不超过1.4％、不超过1.3％、不超过1.2％、不超过1.1％、不超过1.0％、不超过0.9％、不超过0.8％、不超过0.7％、不超过0.6％、不超过0.5％、不超过0.4％、不超过0.3％、不超过0.2％或不超过0.1％。例如，所述体内安全性可以是在动物不产生毒性表现的情况下本申请所述药物偶联物的施用浓度可以是0.5mg/kg以上、1mg/kg以上、2mg/kg以上、3mg/kg以上、4mg/kg以上、5mg/kg以上、10mg/kg以上、20mg/kg以上、30mg/kg以上、50mg/kg以上、70mg/kg以上、100mg/kg以上、200mg/kg以上、500mg/kg以上、或1000mg/kg以上。例如所述动物可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠、猴或人。所述施用可以包括但不限于口服、静脉注射、静脉滴注、腹腔注射、或局部给药。
样品检测
1.ADC DAR值分析方法–HIC-HPLC(疏水色谱法)
高效液相色谱仪：沃特世e2965高效液相色谱系统。
色谱柱：MabPacTM HIC-Butyl 5μm 4.6×100mm(厂家：Thermo)；
流动相A：1.5M(NH4)2SO4+50mM磷酸钾盐(pH 7.0)；
流动相B：50mM磷酸钠盐(pH 7.0)/异丙醇(75:25V/V)；
按照以下洗脱程序进行洗脱，
检测条件：设置流动相流速为1ml/min，检测波长为280nm，柱温30℃。
2.SEC纯度分析–SEC-HPLC(分子排阻色谱法)
高效液相色谱仪：1260安捷伦液相色谱仪。
色谱柱：Waters Xbridge BEH200 SEC(7.8×300mm,3.5μm)
流动相：50mM磷酸钾盐+200mM精氨酸(pH 6.80)+10％异丙醇
检测条件：设置流动相流速为0.5ml/min，检测波长为280nm，柱温30℃。
本发明包括所叙述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
实施例
提供以下实施例以证明并进一步解释本发明的一些优选的实施方式和方面，不应被解释为限制其范围。
实施例1、抗Her3抗体
材料和方法：以下试剂购自Southern Biotech，按规定稀释使用：山羊抗鼠IgG-HRP(1030-05,1:5000稀释)、山羊抗人IgG-PE(2040-09,1:1000稀释)、羊抗人Kappa IgG-HRP，2061-05，1:20000稀释)，山羊抗兔IgG-HRP(4030-05,1:5000 dilution)、FITC标记链酶亲和素(7100-02，1:500稀释)，小鼠抗人Kappa-APC(9230-11,1:500稀释)，小鼠IgG APC(0107-11,0.1mg/ml)。NRG1来自Origene(TP723155)。细胞培养基Roswell Park Memorial Institute(RPMI)1640、Dulbecco改良培养基(DMEM)和胎牛血清来自Hyclone。重组Patritumab为实验室制备所得。HER3抗体3F8是由杂交瘤或重组产生。m3F8表示鼠源3F8抗体，而ch3F8和hu3F8分别表示嵌合和人源化3F8抗体。
细胞培养：研究中使用的SP2/0、SP2/0-HER3、SP2/0-HER2、SP2/O-EGFR、NCI-N87、MDA-MB-468、MDA-MB-453、7901、HT29、MCF-7、SK-BR-3部分从ATCC购买并使用其推荐的适当培养基培养。稳定表达人EGFR、HER2和HER3的SP2/0-EGFR、SP2/0-HER2、SP2/0HER3细胞为实验室自己构建所得。
抗HER3杂交瘤的制备：在第一天，BALB/c小鼠(雌性，8-10周龄)腹腔注射1-2x106表达人HER3的SP2/0-HER3细胞，该细胞与Freund’s完全佐剂一起注射。第8天，用相同数量的带有Freund’s不完全佐剂的细胞进行增强免疫。从第14天开始，每三天用上述量的细胞免疫小鼠，并重复三次。最后一次免疫后三天，从脾脏中分离B淋巴细胞，并与永生骨髓瘤细胞NS-1细胞融合，以产生杂交瘤细胞。
杂交瘤细胞在96孔板中以梯度稀释培养。收集上清液，通过流式细胞术筛选识别SP2/0细胞表面表达HER3的抗体，或通过ELISA筛选重组HER3抗体。
抗体可变区的DNA克隆和测序：用Trizol(ThermoFisher)从杂交瘤中提取的总RNA，逆转录到第一条cDNA链后，按照5'RACE试剂盒(Invitrogen，18374-058)的说明，采用PCR快速扩增5'互补DNA(5'RACE)，以扩增编码可变区的DNA序列。将PCR产物克隆到pGM-T载体中。阳性克隆进行DNA测序，据此推断蛋白质序列。在Kabat编号方案分析可变区的氨基酸。
抗体表达：将编码抗体重链和轻链的DNA克隆到表达载体pCDNA3.1(+)(Invitrogen)中，并在293T细胞中表达。用蛋白A或G柱(GE)纯化抗体。
人源化：人源化由GenScript公司完成。首先，通过用人IgG1恒定区序列替换鼠源抗体重链的恒定区并用人Igκ恒定区序列替换鼠源抗体轻链的恒定区域，产生鼠源-人嵌合抗体(ch3F8)。然后按照参考文献(Kuramochi et al)，在嵌合抗体上进行人源化。在用人抗体框架代替小鼠框架以产生人源化抗体的同时，保留并维持亲和力和特异性所必需的小鼠抗体框架残基。
由GenScript公司合成编码人源化抗体的优化密码子的DNA序列。
抗体表达和纯化：编码上述抗体DNA序列的质粒转染ExpiCHO-S(Catl.#A2910001，Gibco)细胞后在32℃，5％CO2的培养基中生长并保持12天。在4000g离心30分钟后收集上清液，并通过0.22μm膜过滤。按照制造商手册所推荐，用20mM磷酸钠(pH7.0)洗涤与蛋白A结合的抗体(Catl.#17508001，GE)，并用0.1M甘氨酸(pH3.0)洗脱。用0.1M Tris缓冲液(pH9.0)中和洗脱的部分，然后通过超滤离心将其换至PBS缓冲液。用BCA测定蛋白浓度。
表面等离子体共振(SPR)亲和力测定：抗体动力学和亲和力用Biacore T200测定。简言之，将重组人HER3抗体固定在蛋白A芯片(GE，Cat.29-1275-5)上。抗原浓度从50nM到最终0.78125nM，通过2倍连续稀释产生，通过芯片以确定亲和力和动力学。
FACS检测：用0.25％胰蛋白酶-EDTA消化培养的细胞，然后以1500rpm离心5分钟。用含有5％FBS和0.2％ProClin300的PBS FACS溶液将细胞重悬到5X106cells/mL。将50μL的细胞悬浮液与100μL浓度为1μg/ml的一抗冰上孵育1小时。用FACS溶液洗涤两次。用100μL含有山羊抗小鼠IgG-PE(1:1000稀释)的FACS溶液再细胞重悬，并在避光条件下冰孵育1小时。然后将细胞洗涤两次并重新悬浮在200μL FACS溶液中。
Western blot检测：将SDS-PAGE分离的蛋白质转移到硝化纤维膜上进行Western blot。使用一抗如下所示：抗HER2(细胞信号，Catl#：2165S)、抗HER3(细胞信号传导，Catl#:12708)、抗p-HER3(胞信号传导，Catl#：4791)、抗β-肌动蛋白(细胞信号转导，Catl号：4967)。
ELISA检测：将人HER2-ex-huFc、人HER3-huFc和人EGFR-his在96孔板中稀释至2μg/mL、50μL/孔，在4℃下孵育过夜。用0.5×PBST洗涤，然后在37℃下与100μL封闭缓冲液(PBS+3％BSA)孵育2h，并用0.5×PBST洗涤。3F8抗体用封闭缓冲液以1:3连续梯度稀释，以50μL/孔加入，在37℃孵育40-50min，然后用0.5×PBST洗涤。用封闭缓冲溶液以1:20000稀释山羊抗小鼠IgG-HRP(Southern Biotech，1030-05)，50μL//孔加入，在避光条件中孵育30分钟，然后用0.5×PBST冲洗。在检测之前，将50μL鲁米诺(Luminol)缓冲 液A+B以1:1混合加入每个孔中。
NRG1诱导HER3磷酸化：细胞在6孔板上培养，当融合度达到80％时用于实验。实验当天，用PBS洗涤细胞两次，在无血清培养基中孵育6小时，然后用10μg/mL的抗体诱导过夜。为了诱导HER3磷酸化，在收集细胞进行Western blot前30分钟，添加NRG1至100ng/mL的工作浓度。
抗体稳定性试验：按常规将5mg/mL的纯化抗体储存在4℃下，在40℃下孵育7天和14天后移至pH3.5的甘氨酸溶液中再生并保存2、4和6小时进行酸稳定性评估，或重复冻融处理4或6个循环进行冻融稳定性评估。通过SEC-HPLC测量聚集，并通过ELISA测定结合亲和力。
抗体[89Zr]Zr标记：DFO-NCS与抗体偶联，如参考文献所示(Zeglis和Lewis，2015)，DFO和抗体以5:1的摩尔比混合，并在37℃孵育1小时。DFO偶联抗体用SEC-HPLC纯化。
将草酸89Zr溶液0.8mCi与DFO偶联抗体(0.2mg/ml)在HEPES/Na2CO2缓冲液(pH 7.0-7.5)中混合，并在室温下孵育30分钟。用TLC评估放射化学纯度。[89Zr]Zr抗体Rf为0-0.3，而游离89Zr的Rf为0.6-1.0。
PET成像：每只动物静脉注射约100uCi[89Zr]Zr抗体。在注射后的指定时间，用小型动物PET成像机收集和分析图像。
体内药效研究：根据IACUC的指导原则对动物进行维护和使用。分别从Charles River和SPF Biotech购买的BALC/b裸小鼠和NPG小鼠，在25℃下，在12小时的黑暗/光明循环中，自由获取食物和饮料。患者来源的异种移植(PDX)模型是通过皮下植入冷冻保存的组织碎片而产生的。当肿瘤大小达到100-200mm3时，动物开始接受药物治疗，并在1000mm3时实施安乐死。每天观察健康状况。每3天监测并记录肿瘤大小和体重。
软件：使用Olinda、GraphPad Prism 6.0或EXCEL分析数据。
实施例1.1
将鼠源3F8与SP2/0野生型细胞或过度表达HER3、HER2或EGFR的细胞共孵育。在FACS仪中用PE-抗鼠二抗检测结合强度。
结果如图1所示。3F8特异性结合SP2/0-HER3细胞，但不结合其他细胞。
实施例1.2
用ELISA测定鼠源3F8与人HER3、HER2和EGFR的结合亲和力。
结果如图2所示。鼠源3F8只识别HER3而不识别HER2或EGFR。
实施例1.3
用GraphPad Prism 6.0分析结合人HER3、HER2和EGFR的鼠源3F8的EC50。结果如表1所示。鼠源3F8在纳摩尔范围内表现出强效的结合亲和力。
表1鼠源3F8与人HER3、HER2或EGFR结合的EC50

实施例1.4
用ELISA测定鼠源3F8对人、猴、大鼠和小鼠HER3的种属选择性。
结果如图3所示。鼠源3F8识别人和猴HER3，具有相似的效力，但不识别小鼠和大鼠HER3。
实施例1.5
用GraphPad Prism 6.0分析鼠源3F8与人、猴、大鼠和小鼠HER3结合的EC50。结果如表2所示。鼠源3F8显示在纳摩尔范围内对人和食蟹猴HER3具有相同的结合亲和力。
表2鼠源3F8与人、食蟹猴、大鼠或小鼠HER3结合的EC50
实施例1.6
鼠源3F8阻断NRG1诱导的HER3磷酸化。NCI-N87、MDA-MB-468和MDA-MB-453用HER3配体NRG1处理，以诱导HER3的下游磷酸化。通过western blot测定鼠源3F8抑制NRG1诱导的p-HER3的作用。3D4是之前证实的抗HER3抗体，其与NRG1竞争结合HER3，用作阳性对照。
结果如图4所示。数据显示，鼠源3F8降低了HER3蛋白磷酸化水平，但对总HER3蛋白水平没有影响。HER2的Western blot也显示3F8对HER2蛋白水平没有影响。
实施例1.7
鼠源3F8被不同HER3表面水平的细胞迅速吸收。将不同HER3表面水平的细胞与鼠源3F8冰孵育(对照)或在37℃下孵育1或4小时。通过从冰孵育的对照中减去37℃孵育的细胞表面信号来确定内化分数。
结果如图5所示。数据表明，鼠源3F8被迅速内化到细胞中。大部分在1小时内被吸收，并随着孵育时间延长至4小时，细胞内片段量略有增加，这表明3F8内吞是一个快速和连续的过程。
实施例1.8
抗HER3抗体在BT474皮下异种移植模型中有效地抑制肿瘤生长。抗HER3抗体3F8、3D4或3F8+3D4组合以25mg/kg静脉注射，每两周一次，持续三周。每3-4天监测一次肿瘤大小。3F8和3D4都是抗HER3抗体。3F8是鼠源抗体。
结果如图6所示。3F8和3D4均显著抑制肿瘤生长(单因素方差分析，p<0.05)，而3F8显示出更好的疗效。3F8和3D4的组合与单独使用3F8疗效相当。
实施例1.9
[89Zr]Zr-ch3F8用于对胃PDX模型GAS078成像。将[89Zr]Zr-ch3F8静脉注射到胃模型GAS078中。在注射后4、24、48、72、96和168小时收集图像。用Olinda分析每个器官的放射性摄取，并以％ID/g(注射剂量百分比/克组织)表示。
图7是GAS078模型中[89Zr]Zr-ch3F8的代表性成像。Ch3F8表示嵌合3F8抗体。数据显示，随着时间的推移，肿瘤中[89Zr]Zr-ch3F8的摄取逐渐增加。肿瘤摄取在注射后96小时保持稳定，随后在注射后168小时略有下降。
数据也显示在表3中。肿瘤，心脏、肝脏、肾脏和脾脏的主要器官中[89Zr]Zr-ch3F8的摄取量以％ID/g(％注射剂量/克组织)进行评估。
表3肿瘤和主要器官的放射性摄取
实施例1.10
[89Zr]Zr-ch3F8用于对多个PDX模型成像。静脉注射[89Zr]Zr-ch3F8。在注射后72小时收集图像。图8显示了6个动物模型中[89Zr]Zr-ch3F8成像的代表性图像。表4列出了通过ELISA测定的肿瘤组织中HER3表达水平以及注射后72小时主要器官和肿瘤组织中的放射性摄取。在所有6个测试的PDX模型中都有相当数量的肿瘤摄取。
表4肿瘤组织中HER3表达水平(ng/mg)和主要器官和肿瘤组织中的放射性摄取(％ID/g)

实施例1.11
人源化3F8(hu3F8)和嵌合3F8的结合亲和力。通过Biacore测定3个人源化3F8克隆和嵌合3F8的结合动力学。三个人源化3F8克隆(clone 1、clone 2和clone 3)具有不同的重链，和一条相同的轻链。克隆1(clone-1)、克隆2(clone-2)和克隆3(clone-3)分别对应hu3F8-1，hu3F8-2和hu3F8-3。
使用Biacore T200评估软件3.1版处理表6中列出的所有数据。
表6人源化3F8(hu3F8)与嵌合3F8的结合亲和力
实施例1.12
将hu3F8的三个克隆在pH3.5下孵育0、2、4和6小时，然后进行ELISA以测量结合亲和力。结果(图9A)表明，酸处理对结合亲和力几乎没有影响。
三个hu3F8克隆在pH3.5下孵育0、2、4和6小时后的EC50测定。EC50通过ELISA测定。结果(表7)表明，酸处理对结合亲和力几乎没有影响。
表7在pH3.5下孵育0、2、4和6小时后，hu3F8的三个克隆的EC50
将三个hu3F8克隆在40℃孵育不同时间，然后进行ELISA以测定结合亲和力。平行测定嵌合抗体。结果如图9B所示。
三个hu3F8克隆在40℃下孵育不同时间的EC50测定。将三个人源化3F8抗体克隆在40℃的生理盐水中孵育7或14天，并通过ELISA测定热应激试验后的结合亲和力。平行测量嵌合体3F8。结果(表8)表明热应力对结合亲和力几乎没有影响。
表8 hu3F8的三个克隆在40℃下孵育不同天的EC50
将hu3F8的三个克隆冻融3或5个周期，然后进行ELISA以测量结合亲和力。平行测定嵌合抗体。结果(图9C)表明，重复冻融对结合亲和力几乎没有影响。
表9中还显示了经过多次冻融应激试验的三个hu3F8克隆的EC50。
表9 hu3F8的三个克隆经过多次冻融应激试验的EC50
综上，这些数据表明，hu3F8在热、酸和重复冻融的测试后保持结合亲和力，表明hu3F8具有良好的稳定性。
实施例1.13
3个hu3F8克隆在稳定性试验后的聚集性及其在4℃下储存的聚集性评估。用SEC-HPLC测定了三种hu3F8抗体在酸处理、反复冻融和40℃温育后的聚集情况，并与4℃保存的每种抗体进行了比较。结果列于表10中。所有克隆在应激测试后都保持了95％以上的单体，表明几乎没有聚集的趋势。
表10应激测试后hu3F8三个克隆的聚集性评估结果

实施例1.14
hu3F8的翻译后修饰(PTM)分析。P1:hu3F8无应激；P2:hu3F8在40℃下应激2周。用胰蛋白酶消化无应激或40℃应激2周的hu3F8，然后用质谱分析翻译后修饰。结果显示，在HC:372-393时脱酰胺略有增加。总的来说，应激前后的PTM没有太大的差异，表明具有良好的可开发性。
表11 hu3F8的PTM分析结果
下面列出了本申请抗体的氨基酸序列。
鼠源3F8轻链可变区氨基酸序列：
鼠源3F8重链可变区氨基酸序列：
人源化3F8重链氨基酸序列(Clone 1/hu3F8-1)
人源化3F8重链氨基酸序列(Clone 2/hu3F8-2)
人源化3F8重链氨基酸序列(Clone 3/hu3F8-3)
人源化3F8轻链氨基酸序列(Clones 1-3/hu3F8-1～3)
实施例2、抗Her3抗体药物偶联物(ADC)的制备
1.1.接头-细胞毒素(linker-payload)的制备
linker-payload X1

第一步
氮气保护下，向27a(5.00g，43.0mmol)，NaHCO3(10.9g，129mmol)的DMF(50mL)溶液中滴加苄溴(11.0g，64.6mmol)，并在25℃反应17小时。TLC(PE/EA＝2/1)显示反应完全，将反应液加入到500mL水中，用EA(250mL)萃取两遍，分液后经饱和氯化钠水溶液(500mL)洗涤，无水Na2SO4干燥，浓缩过柱(PE:EA＝3:2)得5.1g无色液体，收率：57.1％。
第二步
氮气保护下，向KI2(4.00g，10.9mmol)，TsOH(800mg，4.65mmol)的THF(30mL)溶液中，在0℃下，滴加27b(4.50g，21.8mmol)的THF(10mL)溶液，并在25℃反应2小时。TLC(PE/EA＝1/2)显示反应完全，将反应液加入到200mL水中，用EA(200mL)萃取两次分液，无水Na2SO4干燥，浓缩过柱(PE/EA＝3/2)得白色固体1.56g，收率：26％。
第三步
氢气环境下，在0℃下，向27c(800mg，1.55mmol)的EtOH(8mL)和EA(8mL)混合溶液中加入Pd/C(80mg)，在0℃搅拌2.5小时。LCMS显示反应完全。反应液经硅藻土过滤，用 EA(200mL)洗涤滤饼浓缩后用THF(20mL)溶解旋干得白色固体600mg，收率：91％
第四步
氮气保护下，在0℃下，向27d(220mg，0.515mmol)，HY-13631A(250mg，0.47mmol)和HATU(214mg，0.56mmol)的DMF(6mL)溶液中加入DIEA(152mg，1.18mmol)，并在0℃反应2小时。LCMS显示反应完全。将反应液加入柠檬酸水溶液(pH＝4)(150mL)中，过滤，并用175mL水洗涤滤饼，滤干，用油泵拉干得棕色固体260mg，收率：66％。
第五步
氮气保护下，在0℃下，向27e(260mg，0.309mmol)的DCM(30mL)溶液中滴加二乙胺(8mL)，并在0℃反应3小时。LCMS显示反应完全。将反应液加入0℃的石油醚溶液(600mL)中，有固体析出，静置待固体吸附于瓶底后，倒出溶液，用油泵拉干，得棕色固体90mg，收率：47.1％。
第六步
氮气保护下，在0℃下，向27f(90mg，0.13mmol)，KI-1(92mg，0.19mmol)和DIEA(50mg，0.39mmol)的DMF(2.5mL)溶液中加入HATU(74mg，0.19mmol)并在0℃反应2小时。LCMS显示基本反应结束。在0℃下，将反应液加入PH＝4的柠檬酸水溶液(30mL)中，有絮状固体析出，过滤，经制备板(DCM/MecOH＝10/1)得9.2mg淡黄色固体X1，收率：6％。
MS m/z(ESI)：1074[M+1]
H-NMR(400MHz，MeOD)：7.65(d,1H),7.62(s,1H),7.30-7.21(m,5H),6.79(s,2H),5.69-5.65(m,1H),5.57(d,1H),5.43-5.10(m,3H),4.70(d,2H),4.48-4.39(m,2H),4.10-4.05(m,1H),4.01-3.75(m,5H),3.46(t,2H),3.22-3.15(m,2H),3.07-3.00(m,1H),2.75(m,1H),2.62(m,1H),2.45(s,3H),2.37-2.20(m,6H),2.10-2.02(m,2H),2.00-1.92(m,2H)1.68-1.57(m,6H),1.01(t,3H)
linker-payload X2

第一步
将34a(5g，48.0mmol)、K2CO3(19.9g，144.0mmol)溶于DMF(20mL)中，滴加苄溴(12.3g，72.0mmol)，25℃下反应17小时。TLC(PE/EA＝3/1)检测原料反应完全。将反应液加入水(200mL)中、用EA(250mL)萃取分液，用饱和NaCl洗涤，无水Na2SO4干燥后浓缩过柱(PE:EA＝2:1)得8.7g无色液体34b，收率93％。MS-ESI:m/z 195.1[M+H]+。
第二步
取34c(7.3g，19.8mmol)、TsOH(1.46g，8.5mmol)溶于THF(20mL)中，氮气保护并降温至0℃，滴加43b(7.7g，39.6mmol)的THF(10mL)溶液，加完后0℃反应2小时。TLC(PE/EA＝2/1)显示原料大部分反应。将反应液倒入100mL水中，DCM(100mL)萃取，分液并用饱和NaCl洗涤，无水Na2SO4干燥后过柱(PE/EA＝1/1)，得3.9g无色稠状物34d，收率：39％。MS-ESI:m/z 503.3[M+H]+。
第三步
氢气环境下，在0℃下，向34d(1.9g，3.78mmol)的EtOH(100mL)和EA(100mL)混合溶液中加入Pd/C(1g，10wt.％)，在0℃反应3小时。TLC(PE/EA＝2/1)显示反应完全。反应液经硅藻土过滤，用EA/EtOH(1:1，100mL×3)洗涤滤饼，滤液浓缩，并用THF(50mL×3)溶解旋干并重复三次得1g灰色固体34e，收率：64％。MS-ESI:m/z 435.2[M+Na]+。
第四步
在氮气保护下，0℃下向34e(426mg，1.03mmol)，KI4(500mg，0.94mmol)和HATU(429mg，1.13mmol)的DMF(20mL)溶液中滴加DIEA(303mg，2.35mmol)，加完后在0℃反应2小时。LCMS显示反应结束。将反应液滴到300mL水中，搅拌后静置5分钟，过滤，滤饼用DCM/MeOH(10:1，100mL)的溶液溶解后，干燥旋干拌样，柱层析(EA:MeOH＝30:1)得600mg黄色固体34f，收率：77％。MS-ESI:m/z 830.3[M+H]+。
第五步
氮气保护下，在0℃下，向34f(150mg，0.18mmol)的DCM(5mL)溶液中滴加二乙胺(5mL)，并在0℃反应2小时。LCMS显示反应完全。将石油醚溶液(100mL×6)加入反应液中，有固体析出，静置待固体沉淀后，倒出溶液，再用油泵拉干，得120mg白色粉末34g，LCMS显示产物含量为70％，收率：76％。MS-ESI:m/z 608.3[M+H]+。
第六步
在氮气保护下，向34g(60mg，0.099mmol)、43h(51mg，0.108mmol)、和DIEA(32mg，0.25mmol)的DMF(1mL)溶液中在0℃下加入HATU(45mg，0.118mmol)的DMF(1mL)溶液，并在0℃下反应2小时。LCMS显示原料反应完全。将反应液直接过反相柱，洗脱剂((MeCN/MeOH＝1/1)：H2O＝60％：40％)，纯化得14.8mg黄色固体X2，收率14％。
MS-ESI:m/z 1062.4[M+H]+。
1H NMR(400MHz,Methanol-d4)δ7.69–7.61(m,2H),7.22–7.16(m,2H),7.16–7.09(m,3H),6.76(s,2H),5.70–5.64(m,1H),5.60(d,J＝16.4Hz,1H),5.40–5.31(m,2H),5.26(d,J＝19.0Hz,1H),4.65–4.50(m,7H),4.25–4.16(m,1H),3.87(d,J＝16.7Hz,1H),3.83–3.76(m,3H),3.72(d,J＝17.0Hz,2H),3.44(t,J＝7.1Hz,2H),3.25–3.17(m,2H),3.10–3.02(m,1H),2.92–2.83(m,1H),2.45–2.39(m,5H),2.32–2.20(m,5H),1.97–1.89(m,2H),1.63–1.50(m,4H),1.34–1.20(m,6H),0.99(t,J＝7.3Hz,3H).
linker-payload X3

第一步
向32a(2.00g，6.6mmol)，K2CO3(1.82g，13.2mmol)的MeCN(20mL)中加入溴丙烯(960mg，7.92mmol)，在20℃下搅拌5小时。TLC(PE/EA＝1/2)显示反应结束。将反应液倒入水100mL中，将pH值调至5，用EA(100mL)萃取三次，无水硫酸钠干燥，旋干过柱纯化(PE/EA＝2/1)得1.83g白色固体32b，收率：81％。
第二步
向32b(1.38g，4.02mmol)的DCM(10mL)中加入TFA(10mL)，在25℃下搅拌17小时。TLC(PE/EA＝1/3)显示反应结束。将反应液旋干得0.91g黄色粘状物32c，收率不计。
第三步
向32c(910mg，4.87mmol)，NaHCO3(613mg，7.3mmol)的DME/H2O(20mL/10mL)中加入41d(1.92g，4.87mmol)，在25℃下搅拌3小时。TLC(DCM/MeOH＝1/1)显示反应结束。将反应液倒入水100mL中，用aq.HCl(1N)将pH值调至5，用EA(150mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(DCM/MeOH＝20/1)得1.53g白色固体32e，收率：67％。MS-ESI:m/z 467.4[M+H]+。
第四步
向32f(3g，5.83mmol)的MeOH(50mL)中加入Pd/C(600mg)，在25℃在氢气球下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.9g白色固体32g，收率：77％。
第五步
向32g(789mg，1.86mmol)，KI4(900mg，1.69mmol)，三乙胺(342mg，3.38mmol) 的DMF(10mL)中加入HATU(707mg，1.86mmol)，在0℃下搅拌3.5小时。TLC(EA)显示反应结束。将反应液倒入H2O(80mL)，用EA(100mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(EA)得1.186g白色固体32h，收率：83％。MS-ESI:m/z 842.3[M+H]+。
第六步
将32h(1.186g，1.41mmol)的DCM/二乙胺(20mL，20/1)在25℃下搅拌17小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液倒入石油醚(200mL)中过滤得768mg白色固体32i，收率：88％。MS-ESI:m/z 620.3[M+H]+。
第七步
向32i(676mg，1.09mmol)，32e(508mg，1.09mmol)，DIEA(423mg，3.27mmol)的DMF(10mL)中加入HATU(414mg，1.09mmol)，在20℃下搅拌17小时。TLC(PE/EA＝1/5)显示反应结束。将反应液倒入水中(30mL)过滤，滤饼过柱纯化(DCM/MeOH＝50/1)511mg白色固体32j，收率：44％。MS-ESI:m/z 1068.3[M+H]+。
第八步
将32j(482mg，0.451mmol)的二乙胺/DCM(10mL，1/5)的溶液在10℃下搅拌17小时。TLC(EA)显示反应结束。将反应液倒入PE(300mL)中过滤得301mg白色固体32k，收率不计。
第九步
向32k(301mg，0.356mmol)，Pd(PPh3)4(82mg，0.071mmol)的THF(5mL)中加入吗啡啉(93mg，1.07mmol)，在25℃下搅拌5小时。LCMS显示反应结束。将反应液制备得108mg白色固体32l，收率：38％。MS-ESI:m/z 806.3[M+H]+。
第十步
向32l(108mg，0.134mmol)，三乙胺(41mg，0.402mmol)的THF(2mL)和DMF(2mL)中加入溴乙酰溴(27mg，0.134mmol)，在0℃下搅拌1小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液直接制备得15mg白色固体X3，收率：12％。
MS-ESI:m/z 926.3[M+H]+。
1H NMR(400MHz,DMSO-d6)δ12.11(s,1H),8.54–8.42(m,3H),8.27–8.16(m,2H),7.78(d,J＝11.0Hz,1H),7.30(s,1H),6.53(s,1H),5.61–5.51(m,1H),5.42(s,2H),5.20–5.05(m,2H),4.56–4.42(m,2H),4.32–4.22(m,1H),3.96–3.87(m,3H),3.79(d,J＝5.6Hz,2H),3.70(d,J＝5.9Hz,2H),3.25–3.08(m,2H),2.61–2.53(m,2H),2.45–2.36(m,4H),2.36–2.22(m,3H),2.20–2.03(m,4H),1.99–1.68(m,4H),0.87(t,J＝7.3Hz,3H).
linker-payload X4

第一步
向33a(2.00g，2.58mmol)的MeOH(20mL)中加入Pd/C(400mg，10wt.％)，在20℃下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.3g白色固体33b，收率：74％。
第二步
向33b(0.55g，0.802mmol)，KI4(427mg，0.802mmol)和DIPEA(310mg，2.40mmol)的DMF(5mL)中加入HATU(305mg，0.802mmol)，在0℃下搅拌2小时。TLC(DCM/MeOH＝1/10)显示反应结束。将反应液倒入水(40mL)中，过滤得粗品，经柱纯化(DCM/MeOH＝20/1)得360mg黄色固体33c，收率41％。
第三步
向33c(360mg，0.326mmol)的DCM(10mL)中加入二乙胺(2mL)。在25℃下搅拌17小时。TLC(DCM/MeOH＝5/1)显示反应结束。将反应液倒入PE(100Ml)中，过滤得205mg白色固体33d，收率：71％。MS-ESI:m/z 881.3[M+H]+。
第四步
向33d(205mg，0.233mmol)和三乙胺(118mg，1.17mmol)的DMF(1mL)和水(1mL)中加入溴乙酰溴(94mg，0.446mmol)的THF(2mL)溶液，并在0度搅拌1小时，反应液直接制备得15mg白色固体X4，收率：6％。
MS-ESI:m/z 1001.2[M+H]+。
1H NMR(400MHz,DMSO-d6)δ8.57–8.50(m,1H),8.50–8.43(m,2H),8.35–8.29(m,1H),8.19–8.12(m,2H),7.80(d,J＝10.8Hz,1H),7.27–7.14(m,7H),6.53(s,1H),5.59–5.51(m,1H),5.44–5.39(m,2H),5.20–5.07(m,2H),4.56–4.44(m,3H),3.92(s,3H),3.80–3.68(m,5H),3.41(s,1H),3.21–3.12(m,2H),2.83–2.74(m,1H),2.58–2.55(m,3H),2.39(s,4H),2.18–2.03(m,4H),1.93–1.78(m,2H),0.87(t,J＝7.3Hz,3H).
2.2抗Her3抗体药物偶联物的制备
抗体药物偶联物ADC-1-X1的制备(hu3F8-2-X1)
用超纯水分别配制还原剂和保护剂如下：2mg/ml TCEP(Tris-2-carboxyethyl-phosphine，厂家：Thermo)、100mmol/L乙二胺四乙酸钠盐(厂家：Sigma)。取150mg 7.4mg/ml的抗Her3抗体(hu3F8-2)置于50ml离心管，加入50mM磷酸钠盐缓冲液,pH7.5稀释抗体反应浓度至5mg/ml，按照反应液总体积5％加入100mmol/L乙二胺四乙酸钠盐震荡混匀后加入2mg/ml TCEP进行抗体还原，TCEP与抗体的摩尔比为6.0∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。配制浓度为10mg/ml的linker-payload X1的二甲基亚砜(厂家：国药集团)溶液，按照药物与抗体摩尔比18.0∶1缓慢加入，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO 30KD，厂家：密理博)置换样品至储存缓冲液，先用含有10％二甲基亚砜的30mM组氨酸醋酸缓冲液，pH 5.5超滤3次，再用无DMSO的30mM组氨酸醋酸缓冲液，pH 5.5超滤3次，再经超滤浓缩得到ADC-1-X1，浓度为26.8mg/mL，收率73％。用分子排阻色谱法和疏水层析色谱法检测纯度和DAR值。
经检测，抗体药物偶联物ADC-1-X1的纯度为98.68％，DAR值p为7.12。
抗体药物偶联物ADC-2-X1的制备(hu3F8-3-X1)
用超纯水分别配制还原剂和保护剂如下：2mg/ml TCEP(Tris-2-carboxyethyl-phosphine，厂家：Thermo)、100mmol/L乙二胺四乙酸钠盐(厂家：Sigma)。取150mg 7.5mg/ml的抗Her3抗体(hu3F8-3)置于50ml离心管，加入50mM磷酸钠盐缓冲液,pH7.5稀释抗体反应浓度至5mg/ml，按照反应液总体积5％加入100mmol/L乙二胺四乙酸钠盐震荡混匀后加入2mg/ml TCEP进行抗体还原，TCEP与抗体的摩尔比为6.0∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。配制浓度为10mg/ml的linker-payload X1的二甲基亚砜(厂家：国药集团)溶液，按照药物与抗体摩尔比18.0∶1缓慢加入，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO 30KD，厂家：密理博)置换样品至储存缓冲液，先用含有10％二甲基亚砜的30mM组氨酸醋酸缓冲液，pH 5.5超滤3次，再用无DMSO的30mM组氨 酸醋酸缓冲液，pH 5.5超滤3次，再经超滤浓缩得到抗体药物偶联物ADC-2-X1，浓度为21.15mg/mL，收率67.3％。用分子排阻色谱法和疏水层析色谱法检测纯度和DAR值。
经检测，抗体药物偶联物ADC-2-X1的纯度为99.08％，DAR值p为7.01。
抗体药物偶联物ADC-1-X2的制备(hu3F8-2-X2)
用超纯水分别配制还原剂和保护剂如下：2mg/ml TCEP(Tris-2-carboxyethyl-phosphine，厂家：Thermo)、100mmol/L乙二胺四乙酸钠盐(厂家：Sigma)。取150mg 7.4mg/ml的抗Her3抗体(hu3F8-2)置于50ml离心管，加入50mM磷酸钠盐缓冲液,pH7.5稀释抗体反应浓度至5mg/ml，按照反应液总体积5％加入100mmol/L乙二胺四乙酸钠盐震荡混匀后加入2mg/ml TCEP进行抗体还原，TCEP与抗体的摩尔比为6.0∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。配制浓度为10mg/ml的linker-payload X2的二甲基亚砜(厂家：国药集团)溶液，按照药物与抗体摩尔比21∶1缓慢加入，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO 30KD，厂家：密理博)置换样品至储存缓冲液，先用含有10％二甲基亚砜的30mM组氨酸醋酸缓冲液，pH 5.5超滤3次，再用无DMSO的30mM组氨酸醋酸缓冲液，pH 5.5超滤3次，再经超滤浓缩得到抗体药物偶联物ADC-1-X2，浓度为20.56mg/mL，收率64％。用分子排阻色谱法和疏水层析色谱法检测纯度和DAR值。
经检测，抗体药物偶联物ADC-1-X2的纯度为99.02％，DAR值p为7.53。
抗体药物偶联物ADC-2-X2的制备(hu3F8-3-X2)
用超纯水分别配制还原剂和保护剂如下：2mg/ml TCEP(Tris-2-carboxyethyl-phosphine，厂家：Thermo)、100mmol/L乙二胺四乙酸钠盐(厂家：Sigma)。取150mg 7.5mg/ml的抗Her3抗体(hu3F8-3)置于50ml离心管，加入50mM磷酸钠盐缓冲液,pH7.5稀释抗体反应浓度至5mg/ml，按照反应液总体积5％加入100mmol/L乙二胺四乙酸钠盐震荡混匀后加入2mg/ml TCEP进行抗体还原，TCEP与抗体的摩尔比为6.0∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。配制浓度为10mg/ml的linker-payload X2的二甲基亚砜(厂家：国药集团)溶液，按照药物与抗体摩尔比18.0∶1缓慢加入，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO 30KD，厂家：密理博)置换样品至储存缓冲液，先用含有10％二甲基亚砜的30mM组氨酸醋酸缓冲液，pH 5.5超滤3次，再用无DMSO的30mM组氨酸醋酸缓冲液，pH 5.5超滤3次，再经超滤浓缩得到抗体药物偶联物ADC-2-X2，浓度为21.63mg/mL，收率67.3％。用分子排阻色谱法和疏水层析色谱法检测纯度和DAR值。
经检测，抗体药物偶联物ADC-2-X2的纯度为99.24％，DAR值p为7.11。
参照ADC-1(U3-1402)的制备
用超纯水分别配制还原剂和保护剂如下：2mg/ml TCEP(Tris-2-carboxyethyl-phosphine，厂家：Thermo)、100mmol/L乙二胺四乙酸钠盐(厂家：Sigma)。取40mg 6.9mg/ml的Patritumab单抗(抗体序列使用专利“WO2007077028”中序列号70重链，序列号72轻链，转染CHO细胞后按照常规抗体表达纯化得到，抗体纯度>95％)置于50ml离心管，加入50mM磷酸钠盐缓冲液,pH7.5稀释抗体反应浓度至5mg/ml，按照反应液总体积5％加入100mmol/L乙二胺四乙酸钠盐震荡混匀后加入2mg/ml TCEP进行抗体还原，TCEP与抗体的摩尔比为6.0∶1，震荡混匀后置于制冷型恒温混匀仪上反应，37℃，2h。配制浓度为10mg/ml的Deruxtecan(CAS 1599440-13-7，购自上海皓元化学科技有限公司)的二甲基亚砜(厂家：国药集团)溶液，按照药物与抗体摩尔比18.0∶1缓慢加入，震荡混匀后置于制冷型恒温混匀仪上反应，4℃，1h。用超滤管(MWCO 30KD，厂家：密理博)置换样品至储存缓冲液，先用含有10％二甲基亚砜的30mM组氨酸醋酸缓冲液，pH 5.5超滤3次，再用无DMSO的30mM组氨酸醋酸缓冲液，pH 5.5超滤3次，得到参照ADC-1，浓度为8.77mg/mL，收率65％。用分子排阻色谱法和疏水层析色谱法检测纯度和DAR值。
经检测，参照ADC-1的纯度为99.42％，DAR值p为7.58。
实施例3.抗体药物偶联物的细胞内吞活性
测试目的
检测本申请针对HER3靶标的抗体药物偶联物，在HER3表达的MDA-MB-453细胞上的内吞效应。将细胞和固定浓度的抗体药物偶联物(ADC)和内吞指示试剂pHrodo进行共孵育，通过观察不同时间点细胞内伴随抗体药物偶联物进入细胞的pHrodo所产生的荧光信号亮点数来评价抗体药物偶联物内吞能力。
实验方法
1、细胞培养：MDA-MB-453细胞使用10％FBS Leibovitz's L-15培养基培养。
2、细胞准备：取对数生长期的MDA-MB-453细胞，用PBS洗涤1次之后，加入2-3ml Versene消化2-3min，待细胞消化完全后，加入10-15ml细胞培养液，将经过消化的细胞洗 脱下来，1000rpm离心5min,弃上清，加入细胞培养液将细胞重悬制成单细胞悬液并调整活细胞密度至3x105cells/ml。
3、细胞铺板：以50ul/孔加入96孔细胞培养板。将培养板在培养箱培养18小时(37℃，5％CO2)。
4、加样操作：将待测抗体药物偶联物与Fab-pHrodo共同孵育形成复合物后调整浓度至10nM，以50ul/孔加到细胞。
5、细胞培养：将培养板在培养箱孵育2、4、6、24或48小时(37℃，5％CO2)。
6、读板操作：到达对应时间点，取出96孔细胞培养板，加入Hoechst33342进行细胞核染色，置于operatta CLS中拍照，读取细胞数量及每个细胞内荧光亮点数。使用imaging and analysis软件计算平均细胞荧光点数
表12本申请抗体药物偶联物在MDA-MB-453细胞不同时间点内吞产生荧光信号亮点数
结论：根据图10和表12的结果，本申请针抗体药物偶联物对HER3高表达MDA-MB-453细胞具有内吞效应，且内吞能力强于参照ADC-1。
实施例4抗体药物偶联物对HER3靶标的肿瘤细胞的体外增殖抑制测试
测试目的
为了检测本申请针对HER3靶标的抗体药物偶联物，对Her3高表达的HCC1569和MDA-MB-453以及Her3不表达的MDA-MB-231肿瘤细胞体外增殖的抑制活性。以不同浓度的抗体药物偶联物体外处理细胞，经6天培养后，采用CTG试剂对细胞的增值进行检测根据50％生长抑制浓度(IC50)值评价该化合物的体外活性。
实验方法
1、细胞培养：HCC1569、MDA-MB-453或MDA-MB-231细胞用10％FBS RPMI-1640培养基培养。
2、细胞准备：取对数生长期的HCC1569、MDA-MB-453或MDA-MB-231细胞，用PBS洗涤1次之后，加入2-3ml胰蛋白酶消化2-3min，待细胞消化完全后，加入10-15ml细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min,弃上清，接着加入10-20ml细胞培养液将细胞重悬，制成单细胞悬液，台盼兰进行细胞染色并计数活细胞。
3、细胞铺板：将HCC1569、MDA-MB-453或MDA-MB-231单细胞悬液混匀，用细胞培养液分别调整活细胞密度至6x104cells/ml,将密度调整过后的细胞悬液混匀，以50ul/孔加入96孔细胞培养板。将培养板在培养箱培养18小时(37℃，5％CO2)。
4、抗体药物偶联物浓度：共9个浓度，起始浓度为100nM，3倍稀释直到0.0152nM。
5、加样操作：向培养板中加入配置的不同浓度的待测样品，每个样品两复孔。将培养板在培养箱孵育6天(37℃，5％CO2)。
6、显色操作：取出96孔细胞培养板，放置30分钟使其平衡至室温，向每孔加入50ul CTG试剂，轨道摇床上振摇2分钟，室温避光孵育10分钟。
7、读板操作：取出96孔细胞培养板，置于2104EnVision酶标仪中，用酶标仪测定化学发光。
数据分析
用下列公式来计算检测化合物的抑制率(Inhibition rate，IR)：IR(％)＝(1–(RLU化合物–RLU空白对照)/(RLU溶媒对照–RLU空白对照))*100％。用Microsoft Excel计算不同浓度化合物的抑制率，Graphpad Prism 9对数据进行处理分析。
表13本申请抗体药物偶联物对HCC1569和MDA-MB-453细胞体外增殖抑制的IC50值
注:“-”表示无增殖抑制活性。
结论：根据表13和图11(11A/11B/11C)的结果，本申请抗体药物偶联物对Her3阳性高表达细胞HCC1569和MDA-MB-453具有明显的增殖抑制活性且其抑制活性强于参照ADC-1；同时，它们对HER3不表达的MDA-MB-231细胞无增殖抑制活性，具有良好的选择性。
实施例5抗体药物偶联物对HCC1569荷瘤小鼠药效评价
测试目的
评价本申请的抗体药物偶联物在HCC1569乳腺癌细胞皮下移植雌性NOD/SCID小鼠模型中的抗肿瘤作用。
1.受试药物及材料
空白对照组(阴性对照组)：PBS
参照ADC-1(阳性对照组)：1mg/kg
ADC-1-X1(治疗组)：1mg/kg
ADC-1-X2(治疗组)：1mg/kg
2.配制方法：所有样品均用PBS稀释配制。
3.试验动物
8-10周NOD/SCID小鼠，购自江苏集萃药康生物技术有限公司。
4.试验方法
实验小鼠于右前肩胛处皮下接种2×106HCC1569细胞，细胞重悬在1:1PBS和Matrigel混悬液(0.1ml/只)中。观察肿瘤生长情况，待肿瘤生长至平均体积100-150mm3时根据肿瘤 大小和小鼠体重随机分组给药，6只/组。分组给药当天定义为第0天。
采用尾静脉注射给药，每周给药1次，给药21天后结束实验。每周测量2次瘤体积和体重，记录数据。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI％(TGI＝(1-(Ti-T0)/(Vi-V0)))。Ti：治疗组和阳性对照组在给药第i天的肿瘤体积均值；T0：治疗组和阳性对照组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值；V0：阴性对照组在给药第0天的肿瘤体积均值。
数据分析
数据统计使用Excel 2016统计软件：平均值以avg计算；SD值以STDEV计算；组间差异P值以TTEST计算。
实验结果如表14和图12，在实验结束时，空白对照组平均肿瘤体积为1078.32mm3。
表14本申请抗体药物偶联物对HCC1569荷瘤小鼠的体内肿瘤抑制效果
结论：本申请的抗体药物偶联物均能显著缩小肿瘤体积。且本申请的抗体药物偶联物比参照ADC-1有更好的体内肿瘤抑制效果。
实施例6抗体药物偶联物对Colo205荷瘤小鼠药效评价
试验目的
以BALB/c Nude裸鼠为受试动物，评价本申请的抗体药物偶联物的抗肿瘤作用。
ADC-III-28，参照ADC-2腹腔注射给药后，对Colo205移植瘤裸小鼠的疗效。
1.受试药物及材料
空白对照(阴性对照组)：PBS
参照ADC-1(阳性对照组)：1mg/kg
ADC-1-X1(治疗组)：1mg/kg
ADC-1-X2(治疗组)：1mg/kg
2.配制方法：均用PBS稀释配制。
3.试验动物
8-10周BALB/c-Nude小鼠，购自江苏集萃药康生物技术有限公司。
4.试验方法
在小鼠右肋部皮下接种Colo205细胞,1×107cells(100μL/只)。肿瘤生长达80-120mm3时将动物随机分组，5只/组。分组给药当天定义为第0天。采用尾静脉注射给药，每两周给药1次，给药22天后结束实验。每周测量2次瘤体积和体重，记录数据。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI％(TGI＝(1-(Ti-T0)/(Vi-V0)))。Ti：治疗组和阳性对照组在给药第i天的肿瘤体积均值；T0：治疗组和阳性对照组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值；V0：阴性对照组在给药第0天的肿瘤体积均值。
数据分析数据统计使用Excel 2016统计软件：平均值以avg计算；SD值以STDEV计算；；组间差异P值以TTEST计算。
实验结果如表15和图13，在实验结束时，空白对照组平均肿瘤体积为515.92mm3。
表15本申请抗体药物偶联物对Colo205荷瘤小鼠的体内肿瘤抑制效果
结论：本申请的抗体药物偶联物均能显著缩小肿瘤体积。且本申请的抗体药物偶联物比参照ADC-1在更低的给药浓度观察到肿瘤抑制效果。
实施例7抗体药物偶联物对NCI-H441肺癌细胞荷瘤小鼠药效评价
试验目的
以雄性NU/NU裸鼠为受试动物，评价本申请的抗体药物偶联物对NCI-H441荷瘤小鼠的抗肿瘤作用。
1.受试药物及材料
空白对照(阴性对照组)：生理盐水
参照ADC-1(阳性对照组)：0.3mg/kg或1mg/kg
ADC-1-X2(治疗组)：0.3mg/kg或1mg/kg
2.配制方法：均用生理盐水稀释配制。
3.试验动物：6-8周NU/NU裸鼠，购自维通利华实验动物技术有限公司。
4.试验方法
实验小鼠于右侧背部皮下接种5×106NCI-H441细胞，细胞重悬在0.1ml的PBS和Matrigel(1:1)，定期观察肿瘤生长情况，待肿瘤生长至平均体积115.24mm3时根据肿瘤大小和小鼠体重随机分组给药，每组6只动物。分组给药当天定义为第0天。采用尾静脉注射给药，给药1次，给药28天后结束实验。每周测量2次瘤体积和体重，记录数据。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI％(TGI＝(1-(Ti-T0)/(Vi-V0)))。Ti：治疗组和阳性对照组在给药第i天的肿瘤体积均值；T0：治疗组和阳性对照组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值；V0：阴性对照组在给药第0天的肿瘤体积均值。
数据分析数据统计使用Excel 2016统计软件：平均值以avg计算；SD值以STDEV计算；；组间差异P值以TTEST计算。
实验结果如表16和图14，在实验结束时，空白对照组平均肿瘤体积为1138.85mm3。
表16本申请抗体药物偶联物对NCI-H441荷瘤小鼠的体内肿瘤抑制效果
结论：本申请的抗体药物偶联物能显著缩小肿瘤体积，且本申请的抗体药物偶联物比参照ADC-1在同等浓度下有更好的肿瘤抑制。
实施例8抗体药物偶联物对人源肺癌LU1542皮下异种移植荷瘤小鼠药效评价
试验目的
以雌性BALB/c nude鼠为受试动物，评价本申请的抗体药物偶联物对人源肺癌LU1542皮下异种移植瘤小鼠的抗肿瘤作用。
1.受试药物及材料
空白对照(阴性对照组)：PBS
参照ADC-1(阳性对照组)：3mg/kg或10mg/kg
ADC-1-X2(治疗组)：3mg/kg或10mg/kg
2.配制方法：均用PBS稀释配制。
3.试验动物：6-8周BALB/c nude鼠，购自北京安凯毅博生物技术有限公司。
4.试验方法
从肺癌异种移植模型LU1542荷瘤小鼠收取肿瘤组织，切成直径为2-3mm的瘤块接种于Balb/c裸小鼠右前肩胛处皮下。当荷瘤鼠平均肿瘤体积到达136.8mm3时，将小鼠随机分组，每组6只动物。分组给药当天定义为第0天。采用尾静脉注射给药，给药1次，给药28天后结束实验。每周测量2次瘤体积和体重，记录数据。实验结束时，将小鼠安乐死，计算肿瘤抑制率TGI％(TGI＝(1-(Ti-T0)/(Vi-V0)))。Ti：治疗组和阳性对照组在给药第i天的肿瘤体积均值；T0：治疗组和阳性对照组在给药第0天的肿瘤体积均值；Vi：阴性对照组在给药第i天的肿瘤体积均值；V0：阴性对照组在给药第0天的肿瘤体积均值。
数据分析数据统计使用Excel 2016统计软件：平均值以avg计算；SD值以STDEV计算；；组间差异P值以TTEST计算。
实验结果如表17和图15，在实验结束时，空白对照组平均肿瘤体积为1580.09mm3。
表17本申请抗体药物偶联物对NCI-H441荷瘤小鼠的体内肿瘤抑制效果
结论：本申请的抗体药物偶联物均能显著缩小肿瘤体积。且本申请的抗体药物偶联物比参照ADC在更低的给药浓度观察到肿瘤抑制效果，且同等浓度下有更好的肿瘤抑制。
本申请的序列(SEQ ID NO.)：

Claims (29)

  1. 一种抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，所述抗Her3抗体药物偶联物结构如式(I)所示：
    Ab-(L-M-D)p  (Ⅰ)
    其中，
    L和M是接头单元；
    D是细胞毒性药物；
    p表示平均连接数，且p选自1到10的整数或小数，优选3到8的整数或小数；
    Ab为抗Her3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3,和所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ IDNO:6所示的LCDR1、LCDR2和LCDR3。
  2. 如权利要求1所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，所述抗Her3抗体药物偶联物结构如式(I-1)所示：

    其中，
    L和M是接头单元；
    p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数；
    Ab为抗Her3抗体或其抗原结合片段，其包含重链可变区和轻链可变区，所述重链可变区包含氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3,和所述轻链可变区包含氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ IDNO:6所示的LCDR1、LCDR2和LCDR3。
  3. 如权利要求1或2所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体或其抗原结合片段包含：
    (I)氨基酸序列如SEQ ID NO:7所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID NO:8所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链可变区；或
    (II)氨基酸序列如SEQ ID NO:9所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID NO:8所示或与其具有至少95％、96％、97％、98％ 或99％同一性的轻链可变区；或
    (III)氨基酸序列如SEQ ID NO:10所示或与其具有至少95％、96％、97％、98％或99％同一性的重链可变区，和氨基酸序列如SEQ ID NO:8所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链可变区。
  4. 如权利要求1或2所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体或其抗原结合片段为鼠源抗体或其片段、嵌合抗体或其片段、人源化抗体或其片段。
  5. 如权利要求1或2所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体或其抗原结合片段选自Fab、Fab'、Fab'-SH、Fv、scFv、F(ab')2、sdAb、双抗体或线性抗体。
  6. 如权利要求1或2所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，其中所述抗体为IgG1形式的抗体、IgG2形式的抗体、IgG3形式的抗体或IgG4形式的抗体。
  7. 如权利要求1-6中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体或其抗原结合片段包含：
    (1)氨基酸序列如SEQ ID NO:11所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO:12所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链；或
    (2)氨基酸序列如SEQ ID NO:13所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO:12所示或与其具有至少95％、96％、97％、98％或99％同一性所示的轻链；或
    (3)氨基酸序列如SEQ ID NO:14所示或与其具有至少95％、96％、97％、98％或99％同一性的重链，和氨基酸序列如SEQ ID NO:12所示或与其具有至少95％、96％、97％、98％或99％同一性的轻链。
  8. 如权利要求1-7中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中
    M为-L2-L3-X-L1-；
    L2为-O-或-S-；
    L3为-(C(R1a)(R1b))m-，m选自0、1、2或3，其中当L3包含亚甲基单元时，所述L3的0个或1个亚甲基单元可以被-C(O)-、或-C(S)-替代；
    L1为-(C(R2a)(R2b))n-，n选自1、2或3,其中当L1可以包含亚甲基单元时，所述L1的0个或1个亚甲基单元可以被-C(O)-、或-C(S)-替代；
    X选自3到6元饱和的碳环基、3到6元饱和的杂环基或单键，所述3到6元饱和的碳环基和3到6元饱和的杂环基任选被0、1、2或3个R3a取代；
    其中每个R1a，R1b，R2a，R2b，R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地可以为氢或卤素。
  9. 如权利要求8所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其 混合物，其中L3选自单键、-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；
    其中每个R1a和R1b各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地可以为氢或卤素。
  10. 如权利要求9所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L3选自单键、-CH2-、-CH(CH3)-、-C(CH3)2-、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-。
  11. 如权利要求8所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；
    其中每个R2a，R2b各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地可以为氢或卤素。
  12. 如权利要求11所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中L1选自-CH2-、-CH2C(O)-、-CH(CH3)C(O)-或-C(O)-。
  13. 如权利要求8所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中X为任选被0、1、2或3个R3a取代的3到6元饱和的碳环基或单键；
    其中每个R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地可以为氢或卤素。
  14. 如权利要求13所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中X为3到6元饱和的碳环基或单键。
  15. 如权利要求1-14中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：

    L2为-O-或-S-；
    X选自任选被0、1、2或3个R3a取代的3到6元饱和的碳环基；
    L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；
    其中每个R2a，R2b或R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地可以为氢或卤素。
  16. 如权利要求15所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：

    L2为-O-或-S-；
    X选自3到6元饱和的碳环基。
  17. 如权利要求1-14中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接 受的盐或其混合物,其中所述-M-选自：

    L2为-O-或-S-；
    L3选自-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；
    L1选自-C(R2a)(R2b)-、-C(R2a)(R2b)C(O)-或-C(O)-；
    其中每个R1a，R1b，R2a或R2b各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地可以为氢或卤素。
  18. 如权利要求17所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：

    L2为-O-或-S-；
    L3选自-CH2-、-CH(CH3)、-C(CH3)2-、-CH2CH2-、-CH(CH3)CH2-或-C(CH3)2CH2-。
  19. 如权利要求1-18中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-M-选自：
  20. 如权利要求1-19中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述L为-La-Lb-Lc-，
    所述-La-为
    其中，W为-(C(Rwa)(Rwb))wn-，Y为-(OCH2CH2)yn-Oyp-，Z为-(C(Rza)(Rzb))zn；
    其中wn为1、2、3或6，
    W的1个亚甲基单元各自独立地被-Cyr-、-N(Rwx)C(O)-、-C(O)N(Rwx)-、或-C(O)-替代；
    其中yn为0、4或8，yp为0或1；
    其中zn为1、2或3，
    Z的1个亚甲基单元各自独立地被-Cyr-、-N(Rzx)C(O)-、-C(O)N(Rzx)-、或-C(O)-替代；
    -Cyr-为3到10元饱和的亚碳环基，其中所述-Cyr-是未取代的或独立地被1到3个取代 基Rcx取代；
    其中每个Rwa，Rwb，Rza，Rzb，Rwx，Rzx,Rcx各自独立地为氢、卤素、-ORr或被Rr任选取代的C1-6脂肪族基团；
    其中每个Rr各自独立地为氢、卤素或C1-6脂肪族基团；
    所述-Lb-选自以下组：
    所述-Lc-为

    其中RL1、RL2各自独立地选自以下组：氢、卤素、-OH和C1-6脂肪族基团。
  21. 如权利要求20所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述-La-为
    所述-Lb-为
    所述-Lc-为
  22. 如权利要求1-21中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述L为
  23. 如权利要求1-21中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述L为
  24. 如权利要求1-23中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体药物偶联物结构如式(Ⅱ-1)或(Ⅱ-2)所示：

    其中，
    p如权利要求1所述；
    Ab如权利要求1-7中任一项所述；
    L2为-O-或-S-；
    X选自任选被0、1、2或3个R3a取代的3到6元饱和的碳环基；
    L3选自-C(R1a)(R1b)-或-C(R1a)(R1b)C(R1a)(R1b)-；
    其中每个R1a、R1b或R3a各自独立可以为氢、卤素或可以被R任选取代的C1-6脂肪族基团；
    其中每个R各自独立地可以为氢或卤素。
  25. 如权利要求1-24中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体药物偶联物选自以下结构式：




    其中，
    p如权利要求1所述；
    Ab如权利要求1-7中任一项所述。
  26. 一种抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物,其中所述抗Her3抗体药物偶联物选自：





    其中，
    p表示平均连接数，且p选自1到10的整数或小数，优选3-8的整数或小数。
  27. 一种药物组合物，其包含如权利要求1-26中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物，和药学上可接受的载体或赋形剂。
  28. 如权利要求1-26中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或权利要求27所述的药物组合物在制备用于治疗和/或预防Her3介导的疾病或病症的药物中的用途，优选地，所述疾病或病症为癌症；更优选地，所述癌症选自肺癌、肾癌、尿道癌、结肠直肠癌、前列腺癌、多形性成胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、黑色素瘤、肝癌、膀胱癌、胃癌和食道癌。
  29. 一种治疗和/或预防Her3介导的疾病或病症的方法，其包括向有需要的受试者施用如权利要求1-26中任一项所述的抗Her3抗体药物偶联物、其异构体、其药学上可接受的盐或其混合物或权利要求27所述的药物组合物，优选地，所述疾病或病症为癌症；更优选地，所述癌症选自肺癌、肾癌、尿道癌、结肠直肠癌、前列腺癌、多形性成胶质细胞瘤、卵巢癌、胰腺癌、乳腺癌、黑色素瘤、肝癌、膀胱癌、胃癌和食道癌。
PCT/CN2023/073130 2022-01-28 2023-01-19 Her3抗体药物偶联物及其用途 WO2023143365A1 (zh)

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