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WO2022078425A1 - 抗her3抗体和抗her3抗体药物偶联物及其医药用途 - Google Patents

抗her3抗体和抗her3抗体药物偶联物及其医药用途 Download PDF

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WO2022078425A1
WO2022078425A1 PCT/CN2021/123733 CN2021123733W WO2022078425A1 WO 2022078425 A1 WO2022078425 A1 WO 2022078425A1 CN 2021123733 W CN2021123733 W CN 2021123733W WO 2022078425 A1 WO2022078425 A1 WO 2022078425A1
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seq
antibody
her3
variable region
her3 antibody
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PCT/CN2021/123733
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English (en)
French (fr)
Inventor
杨阳
于佳
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to AU2021360625A priority Critical patent/AU2021360625A1/en
Priority to CA3196940A priority patent/CA3196940A1/en
Priority to JP2023518435A priority patent/JP2023545925A/ja
Priority to EP21879468.3A priority patent/EP4230653A4/en
Priority to US18/032,086 priority patent/US20240026028A1/en
Priority to CN202180054438.6A priority patent/CN116133694A/zh
Priority to KR1020237014336A priority patent/KR20230086702A/ko
Priority to MX2023004189A priority patent/MX2023004189A/es
Publication of WO2022078425A1 publication Critical patent/WO2022078425A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • 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
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    • 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
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    • A61K47/6855—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
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    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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    • C07K—PEPTIDES
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    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to anti-HER3 antibodies, anti-HER3 antibody-ixitecan analog conjugates, methods for their preparation, pharmaceutical compositions comprising the same, and uses thereof for the preparation of medicaments for the treatment of HER3-mediated diseases or disorders ; Especially in the preparation of anticancer drugs.
  • HER3 epidermal growth factor receptor3 (epidermal growth factor receptor3, ErbB-3 or HER3) is one of the epidermal growth factor receptor (EGFR) family members. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3) and HER4 (erbB4). These receptors all contain 3 parts: extracellular region, transmembrane region and intracellular region, of which the extracellular region contains 4 domains, the intracellular region contains 1 intracellular tyrosine kinase domain for signal transduction and 1 cytoplasmic tail with tyrosine phosphorylated residues. Cell signaling is initiated when ligands bind to extracellular domains I and III.
  • EGFR epidermal growth factor receptor
  • HER3 receptor activation and physiological effects are similar to other family members, the difference is that its ligands include neuregulin 1 (NRG-1) and neuregulin 2 (NRG-2), and HER3 cannot be formed after activation Homomers, can only form heterodimers with EGFR or HER2. In the process of forming heterodimers of HER3, the intracellular domain of HER3 exhibits high tyrosine phosphorylase activity.
  • HER3 has six P85 (PI-3K subunits) binding This specific structure determines that when HER3 interacts with the p85 regulatory subunit, it is able to recruit up to six PI-3K to the regulatory subunit site, thereby strongly activating the PI-3K signaling pathway.
  • the HER3/HER2 dimer is the most active of the HER dimers.
  • EGFR is widely distributed on the surface of mammalian epithelial cells, fibroblasts, glial cells, and keratinocytes. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation.
  • HER3 is highly expressed in various common malignant tumors, such as breast cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma and melanoma.
  • EGFR gene mutation which leads to its high level expression or overactivation
  • HER3 gene mutation rate is low, and its high expression is mainly due to the increase of mRNA transcription, thereby increasing protein translation, and it is often co-expressed with HER2.
  • the occurrence, progression and survival of subjects are closely related to the occurrence of various tumors, so the research on anti-tumor drugs targeting HER3 is of great significance.
  • the present disclosure relates to anti-HER3 antibodies, anti-HER3 antibody-ixitecan analog conjugates, and uses thereof.
  • the present disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody has one or more of the following characteristics:
  • the anti-HER3 antibody binds to HER3 protein with an apparent affinity EC 50 of less than 0.5 nM, and the apparent affinity EC 50 is determined by an ELISA method;
  • the anti-HER3 antibody binds to the HER3 protein expressed by MCF7 cells with an apparent affinity EC 50 of less than 0.2 nM, and the apparent affinity EC 50 is determined by the FACS method;
  • the anti-HER3 antibody can be endocytosed by cells expressing human HER3.
  • the present disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody has one or more of the following characteristics:
  • the anti-HER3 antibody binds to HER3 protein with an apparent affinity EC 50 of less than 0.5 nM, and the apparent affinity EC 50 is determined by an ELISA method;
  • the anti-HER3 antibody binds to the HER3 protein expressed by MCF7 cells with an apparent affinity EC 50 of less than 0.2 nM, and the apparent affinity EC 50 is determined by the FACS method;
  • the anti-HER3 antibody can be endocytosed by cells expressing human HER3, and when the anti-HER3 antibody is determined by the method of Test Example 3, its IC 50 is less than 2nM;
  • the anti-HER3 antibody can be endocytosed by cells expressing human HER3, and when the anti-HER3 antibody is measured by the method of Test Example 4, its FITC signal is greater than 300.
  • the anti-HER3 antibody of any of the above comprises (1) HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region set forth in SEQ ID NO:7; and (2) SEQ ID NO : LCDR1, LCDR2 and LCDR3 contained in the light chain variable region shown in :8.
  • the anti-HER3 antibody of any of the above comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively; the light chain variable region comprises SEQ ID NO: 11, respectively; NO:12, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:13 and SEQ ID NO:14;
  • the CDR regions described therein are determined according to the Chothia numbering rules.
  • the anti-HER3 antibody of any of the above comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; the light chain variable region comprises respectively as SEQ ID NO: 17 NO:18, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:19 and SEQ ID NO:20;
  • the CDR regions described therein are determined according to the IMGT numbering rules.
  • the anti-HER3 antibody of any of the above comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, respectively; the light chain variable region comprises HCDR1 as shown in SEQ ID NO:23, respectively; NO:24, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:25 and SEQ ID NO:26;
  • the CDR regions described therein are determined according to the Kabat numbering rules.
  • the present disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively; the light chain variable region comprises SEQ ID NO: 11, respectively; NO:12, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:13 and SEQ ID NO:14;
  • the CDR regions described therein are determined according to the Chothia numbering rules.
  • the present disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively; the light chain variable region comprises respectively as SEQ ID NO: 17 NO:18, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:19 and SEQ ID NO:20;
  • the CDR regions described therein are determined according to the IMGT numbering rules.
  • the present disclosure provides an isolated anti-HER3 antibody, wherein the anti-HER3 antibody comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, respectively; the light chain variable region comprises HCDR1 as shown in SEQ ID NO:23, respectively; NO:24, LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:25 and SEQ ID NO:26;
  • the CDR regions described therein are determined according to the Kabat numbering rules.
  • the anti-HER3 antibody of any of the above is a human antibody or antigen-binding fragment.
  • the anti-HER3 antibody of any of the above comprises a heavy chain variable region and a light chain variable region, wherein:
  • the amino acid sequence of the heavy chain variable region is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of SEQ ID NO:7 and/or the amino acid sequence of the light chain variable region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity;
  • the anti-HER3 antibody of any of the above comprises a heavy chain variable region and a light chain variable region, wherein:
  • amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:7; and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:8; or
  • the anti-HER3 antibody of any of the above further comprises an antibody heavy chain constant region and a light chain constant region; preferably, the heavy chain constant region is selected from the group consisting of human IgGl, IgG2, IgG3 and IgG4 constant regions and conventional variants thereof, the light chain constant region is selected from the group consisting of human antibody kappa and lambda chain constant regions and conventional variants thereof; more preferably, the antibody comprises a heavy chain constant region as shown in SEQ ID NO: 5 and The light chain constant region as set forth in SEQ ID NO:6.
  • the anti-HER3 antibody of any of the above comprises:
  • a heavy chain having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:27, and/or having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:28 light chain;
  • the anti-HER3 antibody of any of the above comprises:
  • the anti-HER3 antibody of any of the above has one or more of the following characteristics:
  • the anti-HER3 antibody binds to HER3 protein with an apparent affinity EC50 of less than 0.5 nM, and the apparent affinity EC50 is determined by ELISA;
  • the anti-HER3 antibody binds to the HER3 protein expressed by MCF7 cells with an apparent affinity EC50 of less than 0.2 nM, and the apparent affinity EC50 is determined by the FACS method;
  • the anti-HER3 antibody can be endocytosed by cells expressing human HER3.
  • the anti-HER3 antibody of any of the above has one or more of the following characteristics:
  • the anti-HER3 antibody binds to HER3 protein with an apparent affinity EC50 of less than 0.5 nM, and the apparent affinity EC50 is determined by ELISA;
  • the anti-HER3 antibody binds to the HER3 protein expressed by MCF7 cells with an apparent affinity EC50 of less than 0.2 nM, and the apparent affinity EC50 is determined by the FACS method;
  • the anti-HER3 antibody can be endocytosed by cells expressing human HER3, and when the anti-HER3 antibody is determined by the method of Test Example 3, its IC50 is less than 2nM;
  • the anti-HER3 antibody can be endocytosed by cells expressing human HER3, and when the anti-HER3 antibody is measured by the method of Test Example 4, its FITC signal is greater than 300.
  • the present disclosure also provides an isolated anti-HER3 antibody, wherein the antibody competes with the anti-HER3 antibody of any preceding item for binding to human HER3.
  • the present disclosure also provides a nucleic acid molecule encoding the anti-HER3 antibody of any preceding item.
  • the present disclosure also provides a host cell comprising the nucleic acid molecule of any preceding item.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of an anti-HER3 antibody as described in any preceding item, or a nucleic acid molecule as previously described, and one or more pharmaceutical agents acceptable carrier, diluent or excipient.
  • the present disclosure also provides an immunoconjugate comprising the anti-HER3 antibody of any preceding item and an effector molecule, wherein the effector molecule is conjugated to the anti-HER3 antibody; preferably , the effector molecule is selected from the group consisting of radioisotopes, antineoplastic agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof .
  • the present disclosure also provides an in vivo or in vitro method for immunodetection or determination of HER3, the method comprising contacting an anti-HER3 antibody as described in any preceding item with a subject or a sample from the subject A step of.
  • the present disclosure also provides an antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof:
  • Y is selected from -O-(CR a R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 - , -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR a R b ) m -CR 1 R 2 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from hydrogen atoms, deuterium atoms, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkane cycloalkyl, and heterocyclyl; alternatively, R and R together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl
  • R 2 is selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl ; alternatively, R1 and R2 are formed together with the carbon atom to which they are attached cycloalkyl or heterocyclyl;
  • R and R together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl ;
  • n is an integer from 0 to 4.
  • n 1 to 10, n is a decimal or an integer
  • Pc is an anti-HER3 antibody as described in any preceding item.
  • the antibody-drug conjugate of general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any preceding item wherein n is 1 to 8, and n is a decimal or an integer . In some embodiments, n is from 3 to 8, and n is a decimal or an integer.
  • Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-;
  • R a and R b are the same or different, and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, and a C 1-6 alkyl group;
  • R 1 is halogenated C 1-6 alkyl or C 3-6 cycloalkyl
  • R 2 is selected from hydrogen atom, halogenated C 1-6 alkyl and C 3-6 cycloalkyl;
  • R 1 and R 2 together with the carbon atom to which they are attached form a C 3-6 cycloalkyl
  • m 0 or 1.
  • L 1 is selected from -(succinimid-3-yl-N)-WC(O)-, -CH 2 -C(O)-NR 3 -WC(O)- and -C(O)-WC ( O)-, wherein W is selected from C 1-8 alkyl, C 1-8 alkyl-C 3-6 cycloalkyl and straight chain heteroalkyl of 1 to 8 chain atoms, the 1 to 8 chain Atomic straight chain heteroalkyl contains 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 alkyl, C 1-8 alkyl-C 3-6 cycloalkyl and 1 Straight chain heteroalkyl groups of up to 8 chain atoms are each independently optionally further selected from halogen, hydroxy, cyano, amino, C 1-6 alkyl, haloC 1-6 alkyl, deuterated C 1- Substituted by one or more substituents in 6 alkyl, C 1-6 alkoxy and C 3-6 cycloalkyl
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and a chemical bond, wherein p 1 is an integer from 1 to 20;
  • L3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( An amino acid residue formed from the amino acids of K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further selected from halogen, hydroxy, cyano, amino , substituted with one or more substituents in alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( An amino acid residue formed from the amino acids of K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further selected from halogen, hydroxy, cyano, amino , substituted with one or more substituents in alkyl, chlor
  • L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 , -C(O)NR 5 (CH 2 ) t - and a chemical bond, wherein t is an integer from 1 to 6;
  • R 3 , R 4 and R 5 are the same or different, and are each independently selected from a hydrogen atom, a C 1-6 alkyl group, a halogenated C 1-6 alkyl group, a deuterated C 1-6 alkyl group, and a C 1-6 alkyl group Hydroxyalkyl;
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, halogen, C 1-6 alkyl, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl and C 1-6 hydroxy alkyl.
  • L1 is s 1 is an integer from 2 to 8;
  • L 2 is a chemical bond
  • L 3 is a tetrapeptide residue
  • L 4 is -NR 5 (CR 6 R 7 )t-, R 5 , R 6 or R 7 are the same or different, and each is independently a hydrogen atom or a C 1-6 alkyl group, and t is 1 or 2;
  • the L 1 end is connected with Pc, and the L 4 end is connected with Y.
  • L 3 is a tetrapeptide residue of GGFG.
  • the antibody-drug conjugate of general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding items is of general formula (Pc-L a -YD)
  • Pc is the anti-HER3 antibody as previously described
  • n is an integer from 0 to 4; for example, m is selected from 0, 1, 2, 3 and 4;
  • n is 1 to 10, and n is a decimal or an integer; specifically, n is a decimal or an integer between 2-8, inclusive; more specifically, n is a decimal or an integer between 2-7, inclusive; Optionally, n is a decimal or integer between 2-3, 3-4, 4-5, 5-6, 6-7 or 7-8, inclusive;
  • R 1 is selected from halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl
  • R 2 is selected from hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl ; alternatively, R1 and R2 are formed together with the carbon atom to which they are attached cycloalkyl or heterocyclyl;
  • W is selected from C 1-8 alkyl, C 1-8 alkyl-C 3-6 cycloalkyl and straight-chain heteroalkyl of 1 to 8 chain atoms, the straight-chain heteroalkyl of 1 to 8 chain atoms
  • the alkyl group contains 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 alkyl, C 1-8 alkyl-C 3-6 cycloalkyl and 1 to 8 chain atoms
  • the straight chain heteroalkyl groups are each independently optionally further selected from halogen, hydroxy, cyano, amino, C 1-8 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 alkyl, C substituted by one or more substituents of 1-6 alkoxy and C 3-6 cycloalkyl;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and a chemical bond, wherein p 1 is an integer from 1 to 20;
  • L3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( An amino acid residue formed from the amino acids of K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further selected from halogen, hydroxy, cyano, amino , substituted with one or more substituents in alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;
  • the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( An amino acid residue formed from the amino acids of K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further selected from halogen, hydroxy, cyano, amino , substituted with one or more substituents in alkyl, chlor
  • R 5 is selected from hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, and a hydroxyalkyl group.
  • the antibody-drug conjugate of general formula (Pc-LYD) or a pharmaceutically acceptable salt thereof according to any one of the preceding items is of general formula (Pc-L a -YD)
  • Pc is the anti-HER3 antibody according to any one of the preceding items
  • n is an integer from 0 to 4; for example, m is selected from 0, 1, 2, 3 and 4;
  • n is 1 to 10, and n is a decimal or an integer; specifically, n is a decimal or an integer between 2-8, inclusive; more specifically, n is a decimal or an integer between 2-7, inclusive; Optionally, n is a decimal or integer between 2-3, 3-4, 4-5, 5-6, 6-7 or 7-8, inclusive;
  • R 1 is selected from halogen, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl, C 3-6 cycloalkyl, C 3-6 cycloalkyl C 1-6 alkyl, C 1-6 Alkoxy C 1-6 alkyl, heterocyclyl, aryl and heteroaryl;
  • R 2 is selected from hydrogen atom, halogen, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl, C 3 -6 cycloalkyl, C 3-6 cycloalkyl C 1-6 alkyl, C 1-6 alkoxy C 1-6 alkyl, heterocyclyl, aryl and heteroaryl; or, R 1 and R 2 together with the carbon atom to which it is attached forms C 3-6 cycloalkyl or heterocyclyl;
  • W is selected from C 1-8 alkyl, C 1-8 alkyl-C 3-6 cycloalkyl and straight-chain heteroalkyl of 1 to 8 chain atoms, the straight-chain heteroalkyl of 1 to 8 chain atoms
  • the alkyl group contains 1 to 3 heteroatoms selected from N, O and S, wherein said C 1-8 alkyl, C 1-8 alkyl-C 3-6 cycloalkyl and 1 to 8 chain atoms
  • the straight chain heteroalkyl groups are each independently optionally further selected from halogen, hydroxy, cyano, amino, C 1-6 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 alkyl, C substituted by one or more substituents of 1-6 alkoxy and C 3-6 cycloalkyl;
  • L 2 is selected from -NR 4 (CH 2 CH 2 O)p 1 CH 2 CH 2 C(O)-, -NR 4 (CH 2 CH 2 O)p 1 CH 2 C(O)-, -S(CH 2 ) p 1 C(O)- and a chemical bond, wherein p 1 is an integer from 1 to 20;
  • L3 is a peptide residue composed of 2 to 7 amino acid residues, wherein the amino acid residue is selected from phenylalanine (F), glycine (G), valine (V), lysine ( An amino acid residue formed from the amino acids of K), citrulline, serine (S), glutamic acid (Q) and aspartic acid (D), and optionally further selected from halogen, hydroxy, cyano, amino , C 1-6 alkyl, chloro C 1-6 alkyl, deuterated C 1-6 alkyl, C 1-6 alkoxy and C 3-6 cycloalkyl in one or more substituents replace;
  • R 5 is selected from hydrogen atom, C 1-6 alkyl, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl and C 1-6 hydroxyalkyl;
  • R 6 and R 7 are the same or different, and are each independently selected from a hydrogen atom, halogen, C 1-6 alkyl, halogenated C 1-6 alkyl, deuterated C 1-6 alkyl and C 1-6 hydroxy alkyl;
  • the heterocyclyl group contains 3 to 6 ring atoms, of which 1-3 are heteroatoms selected from nitrogen, oxygen and sulfur.
  • the antibody-drug conjugate or a pharmaceutically acceptable salt thereof represented by the general formula (Pc-L-Y-D) of any preceding item is:
  • n is 1 to 8, and n is a decimal or an integer; specifically, n is a decimal or an integer between 2 and 8, inclusive; more specifically, n is a decimal or an integer between 2 and 7, inclusive; Optionally, n is a decimal or integer between 2-3, 3-4, 4-5, 5-6, 6-7 or 7-8, inclusive;
  • HER3-29 is an anti-HER3 antibody comprising a heavy chain as set forth in SEQ ID NO:27 and a light chain as set forth in SEQ ID NO:28.
  • the antibody-drug conjugate represented by the general formula (Pc-L-Y-D) or a pharmaceutically acceptable salt thereof according to any one of the preceding items is preferably 3 to 8, and n is a decimal or an integer.
  • the present disclosure also provides a method for preparing an antibody-drug conjugate of the general formula (Pc-L a -YD) or a pharmaceutically acceptable salt thereof as described in any preceding item, which Include the following steps:
  • Pc' is coupled with the compound represented by the general formula (L a -YD) to obtain the compound represented by the general formula (Pc-L a -YD);
  • Pc' is the Pc obtained after reduction
  • n, m, W, L 2 , L 3 , R 1 , R 2 , R 5 , R 6 and R 7 are as defined in any of the preceding items.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-HER3 antibody as described in any preceding item, or a nucleic acid molecule as described in any preceding item, or an antibody as described in any preceding item- A drug conjugate or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, diluents or carriers.
  • the present disclosure also provides an anti-HER3 antibody as described in any preceding item, or a nucleic acid molecule as described in any preceding item, or an antibody-drug conjugate as described in any preceding item, or a pharmaceutically acceptable Use of an acceptable salt, or a pharmaceutical composition as described in any preceding item, in the manufacture of a medicament for the treatment of a HER3 mediated disease or disorder.
  • the present disclosure also provides an anti-HER3 antibody as described in any preceding item, or a nucleic acid molecule as described in any preceding item, or an antibody-drug conjugate as described in any preceding item, or a pharmaceutically acceptable An acceptable salt, or the use of a pharmaceutical composition as described in any preceding item in the manufacture of a medicament for the treatment and/or prevention of tumors and cancers, wherein the tumors and cancers are selected from breast cancer, non-small cell lung cancer, Stomach cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, and melanoma.
  • the present disclosure also provides a kit comprising an anti-HER3 antibody as described in any preceding item, or a nucleic acid molecule as described in any preceding item, or an antibody-drug as described in any preceding item A conjugate or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described in any preceding item.
  • the present disclosure also provides a method of preventing or treating a disease or disorder, the method comprising administering to a subject a therapeutically effective amount of an anti-HER3 antibody as described in any preceding item, or as in any preceding item
  • the disease or disorder is preferably a tumor, an autoimmune disease, or an infectious disease; in some embodiments, the disease or disorder is a disease or disorder associated with HER3.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-HER3 antibody, antibody-drug conjugate or pharmaceutically acceptable salt thereof of any preceding item, and one or more pharmaceutically acceptable salts thereof. acceptable excipients, diluents or carriers.
  • the unit dose of the pharmaceutical composition contains 0.1-3000 mg or 1-1000 mg of an anti-HER3 antibody or an antibody-drug conjugate as described above.
  • the present disclosure provides the use of the antibody-drug conjugate of any preceding item, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the same, as a medicament.
  • the present disclosure provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the antibody-drug conjugate as described in any preceding item in the manufacture of a medicament for the treatment of a HER3-mediated disease or condition
  • the HER3-mediated disease or disorder is a HER3 high-expressing cancer, a medium-expressing cancer, or a low-expressing cancer.
  • the present disclosure provides the use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same in the manufacture of a medicament for the treatment or prevention of cancer as described in any preceding item,
  • the tumors and cancers are selected from breast cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, and melanoma.
  • the present disclosure further relates to a method for treating and/or preventing a tumor, the method comprising administering to a subject in need thereof a therapeutically effective dose of an antibody-drug conjugate as described in any preceding item or A pharmaceutically acceptable salt thereof or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same; in some embodiments, the tumor is a cancer associated with high HER3 expression, a medium-expressing cancer or a low-expressing cancer .
  • the present disclosure further relates to a method for treating or preventing a tumor or cancer, the method comprising administering to a subject in need thereof a therapeutically effective dose of the antibody drug conjugate of any preceding item, or A pharmaceutically acceptable salt or a pharmaceutical composition comprising the same; wherein the tumor and cancer
  • the tumor and cancer are selected from breast cancer, non-small cell lung cancer, gastric cancer, ovarian cancer, prostate cancer, Bladder cancer, colorectal cancer, head and neck squamous cell carcinoma, and melanoma.
  • the present disclosure further provides an anti-HER3 antibody or antibody-drug conjugate thereof as described in any preceding item as a medicament, in some embodiments, as a medicament for the treatment of cancer or tumor, more preferably as a treatment for HER3-mediated of cancer drugs.
  • Active compounds can be formulated to be suitable for use by any suitable method.
  • the active compounds may be presented in unit doses or in such a manner that a subject can self-administer a single dose.
  • Unit doses of active compounds or compositions described in this disclosure may be expressed as tablets, capsules, cachets, vials, powders, granules, lozenges, suppositories, reconstituted powders, or liquids.
  • the administered dose of active compound or composition used in the methods of treatment of the present disclosure will generally vary with the severity of the disease, the subject's weight, and the relative efficacy of the active compound.
  • a suitable unit dose may range from 0.1 mg to 1000 mg.
  • the pharmaceutical composition of the present disclosure may contain one or more excipients selected from the following ingredients: fillers, diluents, binders, wetting agents, disintegrants or excipients, etc. .
  • the composition may contain from 0.1 to 99% by weight of active compound.
  • the HER3 antibody and antibody-drug conjugate provided by the present disclosure have good affinity with cell surface antigens, good endocytosis efficiency and strong tumor inhibition efficiency, and have a wider drug application window, which is suitable for clinical drug application .
  • Figure 1 Binding activity of the disclosed antibodies and positive antibodies to HER3 protein.
  • Figure 2 Binding activity of the disclosed antibodies and positive antibodies to MCF7 cells.
  • Figure 3 Antibodies of the present disclosure and positive antibodies were tested for endocytic activity with DT3C.
  • Figure 4 Antibodies of the present disclosure and positive antibodies were tested for endocytic activity with pHrodo.
  • Figure 5 Efficacy of ADC samples of the present disclosure on SW620 xenografts in tumor-bearing nude mice.
  • a trade name When a trade name is used in this disclosure, it is intended to include the formulation of the trade name product, the drug and the active drug portion of the trade name product.
  • antibody drug conjugate refers to the linking of an antibody to a biologically active drug. Among them, the antibody can be conjugated to the drug directly or via a linking unit.
  • drug loading refers to the average amount of drug carried by each antibody-drug conjugate molecule in a population of antibody-drug conjugates, and can also be expressed as the ratio of the amount of drug to the amount of antibody.
  • the range of drug loading can be 0-12 drugs linked to each antibody, exemplified by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 drugs, the value can be decimal, also Can be an integer.
  • each antibody is loaded with 1 to about 10 drugs; in certain embodiments, each antibody is loaded with about 1 to about 9, 1 to about 8, about 3 drugs to about 7, about 3 to about 6, about 3 to about 5, about 2, about 3, about 4, about 5, about 6, about 7, about 8 drugs,
  • the value can be a decimal or an integer.
  • Drug loading can be identified by conventional methods, such as UV/visible spectroscopy, mass spectrometry, ELISA assays and HPLC characterization.
  • the cytotoxic drug is coupled to the sulfhydryl group of the antibody through a linking unit.
  • Ligand cytotoxic drug conjugate loading can be controlled by the following non-limiting methods, including:
  • antibody in this disclosure is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (eg, bispecific antibodies), full length antibodies or antigen binding thereof Fragments (also referred to as "antigen-binding portions”) so long as they exhibit the desired antigen-binding activity.
  • Full-length antibodies are immunoglobulins (Ig) comprising at least two heavy chains and two light chains interconnected by disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains, and epsilon chains.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH).
  • the heavy chain constant region contains three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL).
  • the heavy and light chain variable regions include hypervariable regions (also called complementarity determining regions, abbreviated as CDRs or HVRs) and framework regions (also called framework regions, abbreviated as FRs) with relatively conserved sequences.
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VL and VH consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • inventions of the human antibody heavy chain constant region and the human antibody light chain constant region mentioned in the present disclosure refer to the human-derived heavy chain constant regions disclosed in the prior art that do not alter the structure and function of the antibody variable region or variants of the light chain constant region
  • exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed reengineering and amino acid substitutions of the heavy chain constant region, specifically replacing YTE as known in the art Mutations, L234A and/or L235A mutations, S228P mutations, 265A (eg D265A) and/or 297A (eg N297A), and/or mutations to obtain a knob-into-hole structure (so that the antibody heavy chain has knob-Fc and hole- Fc combination), these mutations have been shown to confer novel properties of the antibody without altering the function of the variable region of the antibody.
  • human antibody (HuMAb), “human antibody”, “fully human antibody”, “fully human antibody” are used interchangeably, the amino acid sequence of which corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from amino acid sequences of non-human origin using human antibody repertoires or other human antibody coding sequences.
  • This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • antigen-binding fragment or “functional fragment” or “antigen-binding portion” refers to one or more fragments of an intact antibody that retain the ability to specifically bind an antigen. Fragments of full-length antibodies can be used for the antigen-binding function of antibodies.
  • binding fragments encompassed by the term "antigen-binding fragment” include: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) a VH and Fv fragment composed of VL domains; (v) dsFv, a stable antigen-binding fragment formed by VH and VL via interchain disulfide bonds; (vi) diabodies and bispecific antibodies comprising scFv, dsFv, Fab and other fragments and multispecific antibodies.
  • Antigen binding moieties can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • amino acid difference or “amino acid mutation” refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to the original protein or polypeptide, including 1, 2, 3, or Insertion, deletion or substitution of more amino acids.
  • antibody framework region refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • the amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); light
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and amino acid residues in VL are numbered 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acid residue numbers in VH are approximately 27-38 (HCDR1), 56-65 (HCDR2), and 105-117 (HCDR3)
  • CDR amino acid residues in VL are approximately 27-38 (LCDR1 ), 56-65 (LCDR2) and 105-117 (LCDR3)
  • CDR amino acids in VH are numbered 26-35 (HCDR1), 50-58 (HCDR2), and 95-102 (HCDR3)
  • amino acid residues in VL are numbered 24-34 (LCDR1), 50- 56 (LCDR2) and 89-97 (LCDR3).
  • epitopes refers to a site on an antigen that is bound by an antibody (eg, a specific site on a HER3 molecule).
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • an antibody or antigen-binding fragment binds with an affinity (KD) of less than about 10-8 M, eg, less than about 10-9 M, 10-10 M, 10-11 M, 10-12 M, or less.
  • KD affinity
  • the term “compete” in the context of antigen-binding proteins competing for the same epitope (eg, neutralizing antigen-binding proteins or neutralizing antibodies), it means competition between antigen-binding proteins, as determined by the following assay:
  • the antigen-binding protein eg, antibody or immunologically functional fragment thereof
  • the antigen-binding protein prevents or inhibits (eg, reduces) the interaction of a reference antigen-binding protein (eg, ligand or reference antibody) with a common antigen (eg, HER3 antigen or specific binding of its fragments).
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, eg, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, eg, Kirkland et al., 1986, J. Immunol. 137:3614-3619
  • solid-phase Phase Direct Labeling Assay Solid Phase Direct Labeling Sandwich Assay (see e.g.
  • Solid Phase Direct Labeling with I-125 Label RIA see, eg, Morel et al., 1988, Molec. Immunol. 25:7-15
  • solid-phase direct biotin-avidin EIA see, eg, Cheung, et al., 1990, Virology 176:546-552
  • directly labeled RIA Methyl et al., 1990, Scand. J. Immunol. 32:77-82
  • the assay involves the use of purified antigen bound to a solid surface or cell bearing either an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Antigen-binding proteins identified by competitive assays include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to adjacent epitopes sufficiently close to the binding epitope of the reference antigen-binding protein protein, the two epitopes sterically prevent each other from binding. Additional details regarding methods for determining competitive binding are provided in the Examples herein.
  • a competing antigen binding protein when present in excess, it will inhibit (eg decrease) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or 75% or more specific binding of the reference antigen binding protein to a common antigen. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • nucleic acid molecule refers to a DNA molecule or an RNA molecule. Nucleic acid molecules may be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Amino acid sequence identity refers to the alignment of amino acid sequences and, where necessary, the introduction of gaps to achieve a maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity, in the first sequence to the second sequence.
  • the percentage of amino acid residues that are identical For purposes of determining percent amino acid sequence identity, alignment can be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software.
  • One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcR-expressing non-specific cytotoxic cells eg, natural killer (NK) cells, neutrophils, and macrophages
  • NK cells Primary cells that regulate ADCC, NK cells express only FcyRIII, while monocytes express FcyRI, FcyRII and FCYRIII.
  • FcR-expressing non-specific cytotoxic cells eg, natural killer (NK) cells, neutrophils, and macrophages
  • NK cells express only FcyRIII
  • monocytes express FcyRI, FcyRII and FCYRIII.
  • in vitro and in vivo ADCC assays can be performed, such as those described by Clynes et al. (PNASUSA 95:652-656 (1998)), US Pat.
  • ADCP antibody-dependent cellular phagocytosis
  • phagocytic cells eg, macrophages, neutrophils, and dendritic cells
  • Internalized antibody-coated target cells or virions are contained in vesicles called phagosomes, which then fuse with one or more lysosomes to form phagolysosomes.
  • ADCP can be assessed by using in vitro cytotoxicity assays with macrophages as effector cells and videomicroscopy (eg van Bij et al. Journal of Hepatology Vol. 53, No. 4, October 2010, No. 677– 685).
  • CDC complement-dependent cytotoxicity
  • in vitro assays eg, the CDC assay, in which normal human serum is used as a source of complement
  • a reduction in CDC activity can be achieved by comparing the CDC activity of the polypeptide or antibody to its parent polypeptide or antibody without the second mutation within the same assay CDC activity was measured.
  • the assay can be performed as described by Romeuf et al. (Romeuf et al., Br J Haematol. 2008 Mar; 140(6):635-43).
  • antibodies or antibody fragments described in this disclosure can be coupled to effector molecules by any means.
  • antibodies or antibody fragments can be chemically or recombinantly attached to cytotoxic drugs.
  • Chemical means for preparing fusions or conjugates are known in the art and can be used to prepare immunoconjugates.
  • the method for conjugating the antibody or antibody fragment and the drug must be able to link the antibody to the cytotoxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.
  • both the antibody and the cytotoxic drug are proteins and can be conjugated using techniques well known in the art.
  • crosslinkers are generally selected based on reactive functional groups available or inserted on the antibody or cytotoxic drug. Additionally, if no reactive groups are present, a photoactivatable crosslinker can be used. In some cases, it may be desirable to include a spacer between the antibody and the cytotoxic drug.
  • Crosslinking agents known in the art include homobifunctional agents: glutaraldehyde, dimethyl adipimide, and bis(diazobenzidine), and heterobifunctional agents: m-maleimide Benzoyl-N-hydroxysuccinimide and sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide.
  • Crosslinkers that can be used to couple effector molecules to antibody fragments include, for example, TPCH (S-(2-thiopyridyl)-L-cysteine hydrazide) and TPMPH (S-(2-thiopyridyl) ) mercapto-propionic hydrazide).
  • TPCH and TPMPH react on the carbohydrate portion of the glycoprotein that has previously been oxidized by mild periodate treatment, thereby forming a hydrazone bond between the hydrazide portion of the crosslinker and the periodate-generated aldehyde.
  • Heterobifunctional crosslinkers GMBS N-( ⁇ -maleimidobutyryloxy)-succinimide
  • SMCC succinimidyl 4-(N-maleimido- Methyl)cyclohexane
  • GMBS N-( ⁇ -maleimidobutyryloxy)-succinimide
  • SMCC succinimidyl 4-(N-maleimido- Methyl)cyclohexane
  • a cross-linking agent can be used to introduce a long spacer arm between the components, such as 3-(2-pyridyldithio)propionic acid n - Succinimidyl ester (SPDP).
  • SPDP 3-(2-pyridyldithio)propionic acid n - Succinimidyl ester
  • expression vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cell into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors having a bacterial origin of replication) or may integrate into the host cell's genome upon introduction into the host cell, thereby following The host genome replicates together (eg, a non-episomal mammalian vector).
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), 293 cells and NSO cells.
  • the host cells in the present disclosure do not comprise cells derived from human embryos.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind human HER3. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody was eluted by pH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are as follows:
  • Exogenous refers to a substance produced outside an organism, cell, or human body, as the case may be.
  • Endogenous refers to a substance produced in a cell, organism, or human body as the case may be.
  • Homology refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. Two DNA molecules are homologous when a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine, then the molecules are homologous at that position . The percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100.
  • sequences when sequences are optimally aligned, two sequences are 60% homologous if 6 matches or homology at 10 positions in the two sequences; if 95 matches at 100 positions in the two sequences or homologous, then the two sequences are 95% homologous.
  • comparisons are made when aligning two sequences to give the greatest percent homology.
  • the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are chosen to give the maximum match between the respective sequences over the entire length of the respective reference sequences.
  • the following references refer to BLAST algorithms frequently used in sequence analysis: BLAST ALGORITHMS: Altschul, S.F. et al., (1990) J. Mol. Biol. 215:403-410; Gish, W.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included. Where a different name is meant, it is clear from the context.
  • Polymerase chain reaction or "PCR” as used herein refers to a procedure or technique in which a specified portion of nucleic acid, RNA and/or DNA is amplified in small amounts as described in, eg, US Pat. No. 4,683,195. Generally, sequence information from the end of the target region or beyond is required to allow the design of oligonucleotide primers; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified. The 5'-terminal nucleotides of the two primers can be identical to the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR as used herein is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample that involves the use of known nucleic acids and nucleic acid polymerases as primers to amplify or Generate a specific portion of nucleic acid.
  • isolated refers to a purified state, and in this case means that the named molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term “isolated” is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.
  • drug refers to chemical substances that can change or identify the physiological functions and pathological states of the body, and can be used to prevent, diagnose and treat diseases. Drugs include cytotoxic drugs. There is no strict boundary between drugs and poisons. Poisons refer to chemical substances that have a toxic effect on the body in small doses and damage human health. Any drug dose that is too large can produce toxic reactions.
  • Cytotoxic drugs substances that inhibit or prevent the function of cells and/or cause cell death or destruction.
  • cytotoxic drugs can kill tumor cells at a sufficiently high concentration, but due to the lack of specificity, when killing tumor cells, they can also lead to apoptosis of normal cells, resulting in serious side effects.
  • Cytotoxic drugs include toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (eg At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P32 and Lu radioisotopes), chemotherapeutic drugs, antibiotics and nucleolysin.
  • the toxin may be a small molecule toxin from bacteria, fungi, plants or animals and derivatives thereof, including camptothecin derivatives such as ixatecan, maytansinoids and derivatives thereof ( CN101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivatives, such as MMAF, MMAE, 3024 (WO 2016/127790 A1, compound 7), diphtheria toxin, exotoxin, ricin (ricin) A Chain, abrin A chain, modeccin, ⁇ -succinyl toxin (sarcin), Aleutites fordii toxin, dianthin toxin, Phytolaca americana toxin (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin , Selinomycin (mitogellin) limited to asper
  • chemotherapeutic drug is a chemical compound that can be used to treat tumors. This definition also includes antihormonal agents that act to modulate, reduce, block or inhibit the effects of hormones that promote cancer growth, and are often in the form of systemic or systemic therapy. They can themselves be hormones.
  • chemotherapy drugs include alkylating agents such as thiotepa, cyclosphamide (CYTOXAN TM ), alkyl sulfonates such as busulfan, improsulfan and piper piposulfan, aziridines such as benaodopa, carboquone, meturedopa and uredopa, aziridine and methylamelamine include Altretamine, triethylenemelamine, triethylene phosphoramide, triethylene phosphoramide and trimethylolomelamine, nitrogen mustards such as chlorambucil , naphthalene mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, oxazolam hydrochloride; melphalan, new Nitrogen mustard (novembichin), cholesteryl phenylacetate mustard, prednimustine, trofosfamide, uracil mustard; nitrosureas such as nitrosour
  • anti-hormonal agents that modulate or inhibit the effect of hormones on tumors, such as anti-estrogen agents including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxytamoxifen, trioxifene (trioxifene), keoxifene, LY117018, onapristone, and toremifene (Fareston), and antiandrogens such as flutamide (flutamide), nilutamide ( nilutamide), bicalutamide, leuprolide and goserelin, and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing.
  • anti-estrogen agents including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole , 4-hydroxytamoxifen, trioxifene (trioxifene), keoxifene, LY117018, onapristone, and torem
  • alkyl refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably 1 to 12 (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, more preferably alkyl groups containing 1 to 6 carbon atoms.
  • Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1 ,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2- Methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3 -Dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2 -Methylhexyl, 3-methylhexyl, 4-methylhe
  • lower alkyl groups containing 1 to 6 carbon atoms include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl base, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-Methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylpropyl butyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl base and 2,3-dimethylbutyl, etc.
  • Alkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from H atoms, D atoms, halogens, alkanes alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and One or more substituents in a heteroaryl group.
  • heteroalkyl refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, wherein alkyl is as defined above.
  • alkylene refers to a saturated straight or branched chain aliphatic hydrocarbon group having 2 residues derived by removing two hydrogen atoms from the same or two different carbon atoms of the parent alkane, which are Straight or branched chain groups containing 1 to 20 carbon atoms, preferably 1 to 12 (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbons atom, more preferably an alkylene group containing 1 to 8 carbon atoms, most preferably an alkylene group containing 1 to 6 carbon atoms.
  • 1 to 12 eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12
  • Non-limiting examples of alkylene groups include, but are not limited to, methylene ( -CH2- ), 1,1-ethylene (-CH( CH3 )-), 1,2-ethylene ( -CH2) CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -), and the like.
  • Alkylene may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from alkyl, alkenyl, alkynyl , alkoxy, haloalkoxy, cycloalkyloxy, heterocyclyloxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclyl, aryl One or more of the substituents of oxo, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.
  • alkenyl refers to an alkyl compound having a carbon-carbon double bond in the molecule, wherein alkyl is as defined above.
  • Alkenyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from hydrogen atoms, alkyl groups, alkoxy groups, halogens, haloalkyl groups, haloalkoxy groups one or more of the substituents in a radical, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl .
  • alkynyl refers to an alkyl compound having a carbon-carbon triple bond in the molecule, wherein alkyl is as defined above.
  • Alkynyl groups may be substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from hydrogen atoms, alkyl groups, alkoxy groups, halogens, haloalkyl groups, haloalkoxy groups one or more of the substituents in a radical, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl .
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to 8 carbon atoms Carbon atoms (eg 3, 4, 5, 6, 7 and 8) carbon atoms, more preferably 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatriene and cyclooctyl, etc.; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.
  • spirocycloalkyl refers to a 5- to 20-membered polycyclic group with one carbon atom (called a spiro atom) shared between the single rings, which may contain one or more double bonds, but none of the rings are fully conjugated ⁇ electron system.
  • a spiro atom a 5- to 20-membered polycyclic group with one carbon atom (called a spiro atom) shared between the single rings, which may contain one or more double bonds, but none of the rings are fully conjugated ⁇ electron system.
  • it is 6 to 14 yuan, more preferably 7 to 10 yuan (eg 7, 8, 9 or 10 yuan).
  • spirocycloalkyl groups are classified into mono-spirocycloalkyl groups, double-spirocycloalkyl groups or poly-spirocycloalkyl groups, preferably mono-spirocycloalkyl groups and double-spirocycloalkyl groups. More preferably 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospirocycloalkyl.
  • spirocycloalkyl include:
  • fused cycloalkyl refers to an all-carbon polycyclic group of 5 to 20 members in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more of the rings may contain one or more rings. Multiple double bonds, but none of the rings have a fully conjugated pi electron system.
  • it is 6 to 14 yuan, more preferably 7 to 10 yuan (eg 7, 8, 9 or 10 yuan).
  • fused cycloalkyl preferably bicyclic or tricyclic, more preferably 5-membered/5-membered or 5-membered/6-membered bicycloalkyl.
  • fused cycloalkyl groups include:
  • bridged cycloalkyl refers to an all-carbon polycyclic group of 5 to 20 members, any two rings sharing two non-directly connected carbon atoms, which may contain one or more double bonds, but none of the rings have complete Conjugated pi electron system.
  • it is 6 to 14 yuan, more preferably 7 to 10 yuan (eg 7, 8, 9 or 10 yuan).
  • it can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic.
  • bridged cycloalkyl groups include:
  • the cycloalkyl ring includes a cycloalkyl (including monocyclic, spiro, fused and bridged) as described above fused to an aryl, heteroaryl or heterocycloalkyl ring where it is attached to the parent structure
  • Rings taken together are cycloalkyl groups, non-limiting examples including indanyl, tetrahydronaphthyl, benzocycloheptanyl, and the like; preferably phenylcyclopentyl or tetrahydronaphthyl.
  • Cycloalkyl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from hydrogen atoms, halogens, alkyls, Alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl one or more substituents in the group.
  • alkoxy refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl, cycloalkyl are as defined above.
  • alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy.
  • Alkoxy can be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more of the following groups independently selected from H atom, D atom, halogen, alkyl, alkoxy , haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, sulfur, S ( O) or S(O) 2 heteroatoms, excluding ring moieties of -OO-, -OS- or -SS-, the remaining ring atoms being carbon.
  • It preferably contains 3 to 12 (eg 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) ring atoms, of which 1 to 4 (eg 1, 2, 3 and 4) are heterocyclic atom; more preferably contains 3 to 8 ring atoms (eg 3, 4, 5, 6, 7 and 8) of which 1-3 are heteroatoms (eg 1, 2 and 3); more preferably contains 3 to 6 ring atoms, of which 1-3 are heteroatoms; most preferably 5 or 6 ring atoms, of which 1-3 are heteroatoms.
  • Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, tetrahydropyranyl, 1,2,3,6-tetrahydropyridyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholine base and homopiperazinyl, etc.
  • Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls.
  • spiroheterocyclyl refers to a 5- to 20-membered polycyclic heterocyclic group with one atom (called a spiro atom) shared between the single rings, wherein one or more ring atoms are selected from nitrogen, oxygen, sulfur, A heteroatom of S(O) or S(O) 2 , and the remaining ring atoms are carbon. It may contain one or more double bonds, but none of the rings have a fully conjugated pi electron system. Preferably it is 6 to 14 yuan, more preferably 7 to 10 yuan (eg 7, 8, 9 or 10 yuan).
  • spiroheterocyclyls are classified into mono-spiroheterocyclyl, bis-spiroheterocyclyl or poly-spiroheterocyclyl, preferably mono-spiroheterocyclyl and bis-spiroheterocyclyl. More preferably, it is a 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiroheterocyclyl group.
  • Non-limiting examples of spiroheterocyclyl include:
  • fused heterocyclyl refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, and one or more of the rings may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O) or S(O) 2 , and the remaining ring atoms are carbon.
  • it is 6 to 14 yuan, more preferably 7 to 10 yuan (eg 7, 8, 9 or 10 yuan).
  • fused heterocyclyl groups include:
  • bridged heterocyclyl refers to a 5- to 14-membered, polycyclic heterocyclyl group in which any two rings share two atoms that are not directly connected, which may contain one or more double bonds, but none of the rings have a complete common A pi-electron system of a yoke in which one or more ring atoms are heteroatoms selected from nitrogen, oxygen, sulfur, S(O) or S(O) 2 and the remaining ring atoms are carbon.
  • it is 6 to 14 yuan, more preferably 7 to 10 yuan (eg 7, 8, 9 or 10 yuan).
  • bridged heterocyclyl groups include:
  • the heterocyclyl ring includes a heterocyclyl group (including monocyclic, spiroheterocycle, fused heterocycle and bridged heterocycle) as described above fused to an aryl, heteroaryl or cycloalkyl ring, wherein the rings that are structurally linked together are heterocyclyl groups, non-limiting examples of which include:
  • Heterocyclyl groups may be substituted or unsubstituted, and when substituted, the substituents may be substituted at any available point of attachment, preferably independently optionally selected from hydrogen atoms, halogens, alkyl groups, Alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl one or more substituents in the group.
  • aryl refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (fused polycyclic are rings that share adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, preferably 6 to 10 membered , such as phenyl and naphthyl.
  • the aryl ring includes an aryl ring fused to a heteroaryl, heterocyclyl or cycloalkyl ring as described above, wherein the ring linked to the parent structure is an aryl ring, non-limiting examples of which include :
  • Aryl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from hydrogen atoms, halogens, alkyl groups, alkanes Oxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl one or more substituents in .
  • heteroaryl refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen.
  • Heteroaryl is preferably 5 to 10 membered (eg 5, 6, 7, 8, 9 or 10 membered), more preferably 5 or 6 membered, eg furyl, thienyl, pyridyl, pyrrolyl, N-alkane pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl and the like.
  • the heteroaryl ring includes a heteroaryl group as described above fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, non-limiting examples of which include :
  • Heteroaryl groups may be substituted or unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably independently optionally selected from hydrogen atoms, halogens, alkyls, Alkoxy, haloalkyl, haloalkoxy, cycloalkyloxy, heterocyclyloxy, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl one or more substituents in the group.
  • amino protecting group is used to protect the amino group with a group that is easy to remove in order to keep the amino group unchanged when the other part of the molecule is reacted.
  • Non-limiting examples include (trimethylsilyl)ethoxymethyl, tetrahydropyranyl, t-butoxycarbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may be optionally substituted with 1-3 substituents selected from halogen, alkoxy or nitro.
  • hydroxyl protecting group is a suitable group for hydroxyl protection known in the art, see the literature ("Protective Groups in Organic Synthesis", 5 Th Ed. TW Greene & P. GMWuts) for hydroxyl protecting groups.
  • the hydroxyl protecting group can be a (C 1-10 alkyl or aryl) 3 silyl group, such as: triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl Silyl, tert-butyldiphenylsilyl, etc.; can be C 1-10 alkyl or substituted alkyl, preferably alkoxy or aryl substituted alkyl, more preferably C 1-6 alkoxy substituted C 1-6 alkyl or phenyl substituted C 1-6 alkyl, most preferably C 1-4 alkoxy substituted C 1-4 alkyl, for example: methyl, tert-butyl, allyl, benzyl
  • cycloalkyloxy refers to cycloalkyl-O-, wherein cycloalkyl is as defined above.
  • heterocyclyloxy refers to heterocyclyl-O-, wherein heterocyclyl is as defined above.
  • alkylthio refers to alkyl-S-, wherein alkyl is as defined above.
  • haloalkyl refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above.
  • haloalkoxy refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.
  • deuterated alkyl refers to an alkyl group substituted with one or more deuterium atoms, wherein alkyl is as defined above.
  • hydroxyalkyl refers to an alkyl group substituted with hydroxy, wherein alkyl is as defined above.
  • halogen refers to fluorine, chlorine, bromine or iodine.
  • hydroxy refers to -OH.
  • thiol refers to -SH.
  • amino refers to -NH2 .
  • cyano refers to -CN.
  • nitro refers to -NO2 .
  • carboxylate refers to -C(O)O(alkyl), -C(O)O(cycloalkyl), (alkyl)C(O)O- or (cycloalkyl)C(O )O-, wherein alkyl, cycloalkyl are as defined above.
  • the compounds of the present disclosure include isotopic derivatives thereof.
  • isotopic derivatives refers to compounds that differ in structure only by the presence of one or more isotopically enriched atoms.
  • “deuterium” or “tritium” is used in place of hydrogen
  • 18F -fluorine label 18F isotope
  • 18F isotope is used in place of fluorine, or11C- , 13C- , or14C -enriched
  • Compounds in which carbon ( 11 C-, 13 C-, or 14 C-carbon labels; 11 C-, 13 C-, or 14 C-isotopes) in place of carbon atoms are within the scope of this disclosure.
  • Such compounds can be used, for example, as analytical tools or probes in biological assays, or as tracers for in vivo diagnostic imaging of disease, or as tracers for pharmacodynamic, pharmacokinetic or receptor studies.
  • Various deuterated forms of the compounds of the present disclosure mean that each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can refer to the relevant literature to synthesize deuterated forms of the compounds.
  • deuterated starting materials can be used in preparing deuterated forms of the compounds, or they can be synthesized using conventional techniques using deuterated reagents including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran , Deuterated lithium aluminum hydride, deuterated iodoethane and deuterated iodomethane, etc.
  • Deuterated compounds generally retain comparable activity to undeuterated compounds, and when deuterated at certain specific sites can achieve better metabolic stability, resulting in certain therapeutic advantages.
  • Optional or “optionally” means that the subsequently described event or circumstance can but need not occur, and that the description includes instances where the event or circumstance occurs or instances where it does not.
  • a heterocyclic group optionally substituted with an alkyl group means that an alkyl group may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl group and the case where the heterocyclic group is not substituted with an alkyl group .
  • Substituted means that one or more hydrogen atoms in a group, preferably 1 to 5, more preferably 1 to 3 hydrogen atoms, independently of one another, are substituted by the corresponding number of substituents.
  • a person skilled in the art can determine possible or impossible substitutions (either experimentally or theoretically) without undue effort.
  • amino or hydroxyl groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (eg, olefinic) bonds.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, and other components such as a physiological/pharmaceutically acceptable carrier and excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that, within the scope of sound medical judgment, are suitable for use in contact with subject tissue without undue toxicity, irritation, allergy reactions or other problems or complications, have a reasonable benefit/risk ratio, and are effective for the intended use.
  • linker unit refers to a chemical structural fragment or bond that is linked to a ligand (in this disclosure, an antibody) at one end and a drug at the other end. Drugs are linked.
  • a linker may comprise one or more linker members.
  • exemplary linker members include 6-maleimidocaproyl (“MC”), maleimidopropionyl (“MP”), valine-citrulline (“val-cit” or “vc”).
  • MC 6-maleimidocaproyl
  • MP maleimidopropionyl
  • val-cit valine-citrulline
  • PAB p-aminobenzyloxycarbonyl
  • linker reagents N-succinimidyl 4- (2-Pyridylthio)valerate (“SPP”), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 carboxylate (“SMCC”, in Also referred to herein as "MCC”) and N-succinimidyl(4-iodo-acetyl)aminobenzoate (“SIAB”).
  • Linkers can include stretchers, spacers and amino acid units, and can be synthesized by methods known in the art, such as those described in US2005-0238649A1.
  • the linker can be a "cleavable linker" that facilitates the release of the drug in the cell.
  • acid-labile linkers eg, hydrazones
  • protease-sensitive linkers eg, peptidase-sensitive linkers
  • photolabile linkers eg, dimethyl linkers
  • disulfide-containing linkers can be used (Chari et al. Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).
  • Connector components include but are not limited to:
  • MC 6-maleimidocaproyl
  • Val-Cit or "vc” valine-citrulline (exemplary dipeptide in a protease cleavable linker)
  • PAB p-aminobenzyloxycarbonyl (an example of a "self-sacrificing" linker assembly)
  • Me-Val-Cit N-methyl-valine-citrulline (where the linker peptide bond has been modified to prevent cleavage by cathepsin B)
  • MC(PEG)6-OH maleimidocaproyl-polyethylene glycol (can be attached to antibody cysteine)
  • SPDP N-succinimidyl 3-(2-pyridyldithio)propionate
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
  • carrier is used for the drugs of the present disclosure, and refers to a system capable of changing the way the drug enters the human body and its distribution in the body, controlling the release rate of the drug, and delivering the drug to the targeted organ.
  • Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability.
  • polymer surfactants that can be used as carriers can self-assemble to form aggregates in various forms due to their unique amphiphilic structure.
  • Preferred examples are micelles, microemulsions, gels, liquid crystals, vesicles, etc. . These aggregates have the ability to encapsulate drug molecules, and at the same time have good permeability to the membrane, and can be used as excellent drug carriers.
  • excipient refers to an addition other than the main drug in a pharmaceutical preparation, and may also be called an adjuvant.
  • adjuvant such as binders, fillers, disintegrating agents and lubricants in tablets; matrix parts in semi-solid preparations ointments and creams; preservatives, antioxidants, flavorings, fragrances in liquid preparations, Solubilizers, emulsifiers, solubilizers, osmotic pressure regulators, and colorants can all be called excipients.
  • the term "diluent” is also known as filler, the main purpose of which is to increase the weight and volume of the tablet.
  • the addition of the diluent not only ensures a certain volume, but also reduces the dosage deviation of the main components and improves the compression molding of the drug.
  • an absorbent to absorb the oily substances, so as to keep the "dry” state, so as to facilitate the tableting.
  • pharmaceutical composition means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, as well as other components such as physiological/pharmaceutically acceptable Carriers and Excipients.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • the pharmaceutical compositions may be in the form of sterile injectable aqueous solutions.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • the sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in an oily phase.
  • the active ingredient is dissolved in a mixture of soybean oil and lecithin.
  • the oil solution is then processed into a mixture of water and glycerol to form a microemulsion.
  • the injectable solution or microemulsion can be injected into the bloodstream of a subject by local bolus injection.
  • solutions and microemulsions are preferably administered in a manner that maintains a constant circulating concentration of the compounds of the present disclosure.
  • a continuous intravenous drug delivery device can be used.
  • An example of such a device is the Deltec CADD-PLUS.TM.5400 intravenous pump.
  • the pharmaceutical compositions may be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration.
  • This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • sterile fixed oils are conveniently employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can also be used in the preparation of injectables.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions Contact with animals, humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment as it applies to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • pharmaceutically acceptable salt or “pharmaceutically acceptable salt” refers to salts of the antibody-drug conjugates of the present disclosure, or salts of the compounds described in the present disclosure, which when used in mammals have It is safe and effective, and has due biological activity.
  • the antibody drug conjugate of the present disclosure contains at least one amino group, so it can form a salt with an acid.
  • Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide acid salt, hydroiodate, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pearate, hydrogen phosphate, dihydrogen phosphate, Salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, ethanesulfonate, benzenesulfonate and p-toluenesulfonate acid salt.
  • Treatment means administering an internal or external therapeutic agent, eg, a composition comprising any one of the binding compounds of the present disclosure, to a subject having one or more disease symptoms for which the therapeutic agent is known Treat these symptoms.
  • the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a subject or population to be treated, to induce regression of such symptoms or to inhibit progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to alleviate symptoms of any particular disease may vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the level of the drug that produces the desired effect in the subject. ability.
  • Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of each target disease, the method of The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce target disease symptoms in a statistically significant number of subjects.
  • H test Kruskal-Wallis test
  • Jonckheere-Terpstra test Jonckheere-Terpstra test
  • Wilcoxon test determine that it should reduce target disease symptoms in a statistically significant number of subjects.
  • the pCDH-Her3 lentiviral expression vector plasmid and pVSV-G, pCMV-dR8.91 lentiviral system packaging vector were transfected into virus packaging cells 293T with Lipofectamine 3000 transfection reagent, and the supernatant of the medium containing the virus was collected, filtered and filtered. Ultracentrifugation was performed, and the concentrated virus was used to infect Chinese hamster ovary cells CHO-K1, screened by puromycin for two to three weeks, and then subjected to FACS single-cell sorting.
  • HER3 expression on the surface of lentivirus-infected CHO-K1 cells was detected by FACS, and monoclonal cell lines with high HER3 expression were selected.
  • the selected monoclonal cell lines were expanded and stored for subsequent experiments.
  • HuMan ErbB3 amino acid sequence (UniProtKB-P21860-1, AA Ser 20-Thr 643)
  • the positive control antibody U3 was prepared with reference to WO2007077028A2 (page 118, U1-59). Wherein the heavy chain and light chain amino acid sequences of U3 are as follows:
  • sequences of the heavy chain variable region and light chain variable region of the fully human antibody molecule HER3-29 are as follows:
  • HER3-29 heavy chain variable region
  • the CDR sequences obtained by different numbering rules are as follows:
  • heavy chain CDR1 DYAMH SEQ ID NO: 21 heavy chain CDR2 GISWNSGSIGYADSVKG SEQ ID NO: 22 heavy chain CDR3 EGLPGLDY SEQ ID NO: 23 light chain CDR1 RASQHVGTYLN SEQ ID NO: 24 light chain CDR2 GAANLQS SEQ ID NO: 25 light chain CDR3 QQSYNTPFS SEQ ID NO: 26
  • the heavy and light chain sequences of the fully human antibody molecule HER3-29 are as follows:
  • the DAR value calculation method of the ADC of the present disclosure adopts RP-HPLC (reverse-phase high performance liquid chromatography), which is as follows:
  • Naked antibody and ADC samples for testing were added to 4 ⁇ L of DDT (sigma) to reduce, and water bathed at 37 °C for 1 hour, and then taken out into the inner cannula.
  • High performance liquid chromatograph Agilent 1200 was used for detection, the chromatographic column was Agilent PLRP-S 1000A 8 ⁇ m 4.6*250mm, column temperature: 80°C; DAD detector wavelength 280nm; flow rate: 1mL/min; injection volume: 40 ⁇ L; By comparing the spectrum of the sample with the naked antibody, the positions of the light and heavy chains are distinguished, and then the spectrum of the detected sample is integrated to calculate the DAR value.
  • Preparation example Take 5.78mg of DTT, add 150 ⁇ L of purified water to dissolve it fully, prepare a 0.25M DTT solution, and store it at -20°C.
  • Preparation example measure 1000mL of purified water in a graduated cylinder, add 1mL of TFA (sigma), mix thoroughly before use, and store at 2-8°C for 14 days.
  • Preparation example measure 1000 mL of acetonitrile in a graduated cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.
  • the positions of the light and heavy chains are distinguished, and then the spectrum of the detected sample is integrated to calculate the DAR value.
  • HC DAR ⁇ (number of connected drugs * percentage of peak area)/sum of HC peak areas
  • the drug moiety of the conjugates of the present disclosure can be any suitable drug. Particularly suitable drugs are described, for example, in PCT Publication No. WO2020063676A1 (hereby incorporated by reference in its entirety).
  • Compound 9A of the present disclosure is N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9- Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran [3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxo Hetero-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo
  • the present disclosure uses the following method to prepare an antibody-drug conjugate represented by the general formula of ADC (HER3-29-9A) by adjusting the reaction parameters.
  • TCEP in an aqueous solution (10 mM, 128 ⁇ L, 1.281 ⁇ mol), placed on a water bath shaker, and shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • aqueous solution (10 mM, 111 ⁇ L, 1.111 ⁇ mol), placed in a water bath shaker, and shaken at 37° C. for 3 hours to stop the reaction.
  • the reaction solution was cooled to 25°C with a water bath.
  • U3-1402 was prepared as a positive control.
  • the HER3 protein was diluted to 1 ⁇ g/mL with pH 7.4 PBS (Original Bio, B320) buffer, added to a 96-well microtiter plate at a volume of 100 ⁇ L/well, and incubated at 4°C overnight. After discarding the liquid, 300 ⁇ L of 5% nonfat milk (BD, 232100) diluted with PBS was added to each well for blocking, and incubated at 37° C. for 2 hours. After blocking, the blocking solution was discarded, and the plate was washed 3 times with PBST buffer (pH 7.4 PBS containing 0.1% tween-20), and then 100 ⁇ L of serially diluted antibody solution was added to each well and incubated at 37°C for 1 hour.
  • pH 7.4 PBS Olinal Bio, B320
  • MCF7 cells (ATCC, HTB-22) were prepared into a cell suspension of 1 ⁇ 10 6 cells/mL with FACS buffer (2% fetal bovine serum (Gibco, 10099141) pH 7.4 PBS (Sigma, P4417-100TAB)), 100 ⁇ L/well was added to a 96-well round bottom plate. After removing the supernatant by centrifugation, add 50 ⁇ L/well of different concentrations of the antibody to be tested diluted with FACS buffer, and incubate in a refrigerator at 4°C for 1 hour in the dark.
  • FACS buffer 2% fetal bovine serum (Gibco, 10099141) pH 7.4 PBS (Sigma, P4417-100TAB)
  • the purpose of this experiment is to kill cells by activated diphtheria toxin (DT) after DT3C protein enters cells, which indirectly reflects the endocytosis of HER3 antibody.
  • DT diphtheria toxin
  • the in vitro endocytic activity of the antibodies was evaluated according to IC50 and Imax.
  • DT3C is a recombinantly expressed fusion protein composed of Fragment A (toxin part only) of diphtheria toxin and 3C fragment (IgG binding part) of group G Streptococcus.
  • the protein has a high affinity for the Fc structure of the antibody and enters the cell together when the antibody undergoes endocytosis. Under the action of intracellular furin, the toxic DT is released.
  • DT can inhibit the activity of EF2-ADP ribosylation, block the protein translation process, and eventually lead to cell death.
  • DT3C that did not enter the cells did not have the activity of killing cells. The endocytic activity of the antibody was evaluated based on cell killing.
  • DT3C was prepared at a concentration of 4 ⁇ in serum-free medium and filtered into a sterile solution through a small 0.22 ⁇ m filter. Prepare 4 ⁇ concentration of antibody in serum-free medium, mix 80 ⁇ L DT3C (400 nM) and 80 ⁇ L antibody (66 nM) according to the volume of 1:1, and incubate at room temperature for 30 minutes. Add 50 ⁇ L of diluted antibody to 50 ⁇ L of cells and incubate for three days in an incubator. 50 ⁇ L of CTG (CellTiter-Glo TM reagent, G7573) was added to each well, incubated in the dark at room temperature for 10 minutes, and the chemiluminescence was read on Victor3. The results are shown in Figure 3 and Table 6.
  • the purpose of this experiment is to reflect the endocytosis of HER3 antibody according to the change of fluorescence signal after the dye is internalized.
  • the in vitro endocytic activity of the antibody was evaluated according to the intensity of the fluorescent signal.
  • the pH-sensitive pHrodo iFL dye-conjugated Fab fragment can directly bind to the Fc region of the HER3 antibody without affecting the antibody's antigen recognition.
  • the pHrodo iFL dye is almost non-fluorescent at neutral pH, and the dye is simultaneously internalized when the HER3 antibody undergoes endocytosis, and the fluorescence signal increases gradually as the pH decreases.
  • the endocytic activity of the antibody was evaluated based on the enhancement of the fluorescent signal.
  • HER3/CHOK1 cells were cultured with DMEM/F12+10%FBS+10 ⁇ g/mL puromycin, and the cell suspension was prepared with fresh cell culture medium on the first day of the experiment, the density was 2 ⁇ 10 5 cells/mL, with 100 ⁇ L/mL The wells were added to a 96-well cell culture plate, and incubated with 5% carbon dioxide at 37°C for 24 hours.
  • the purpose of this experiment was to detect the cell-killing effect of ADC samples, and to evaluate the in vitro activity of Her3-ADC according to IC 50 and Imax.
  • MCF7 cells human breast cancer cells
  • SW620 cells human colon cancer cells, Nanjing Kebai, CBP60036
  • WiDr cells human colorectal cancer cells
  • ADC samples were diluted with PBS to 15 ⁇ M (10 ⁇ concentration). With this first concentration, it was diluted five times with PBS, a total of 8 concentrations. Add 15 ⁇ L of 10 ⁇ strength solution to each well. Incubate at 37°C with 5% carbon dioxide for 6 days.
  • ADC samples ADC-1, ADC-2, ADC-3 have better killing activity on cells than positive controls ADC-4 and ADC-5.
  • SW620 cells (5 ⁇ 10 6 cells/cell) were inoculated subcutaneously in the right flank of Balb/c nude mice, and 7 days later, they were divided into 9 groups, 8 cells/group, for a total of 9 groups.
  • the average packet volume is 134.75mm 3 .
  • the ADC was injected intraperitoneally for 3 doses, once every 5 days. Each animal was injected with 0.1mL/10g according to body weight. Tumor volume and body weight were measured twice a week and the data were recorded.
  • V 1/2 ⁇ L length ⁇ L short 2
  • T/C(%) (TT 0 )/(CC 0 ) ⁇ 100%, where T and C are the tumor volumes of the treatment group and control group at the end of the experiment; T 0 and C 0 are the beginning of the experiment tumor volume.
  • ADC-1 and ADC-2 of the present disclosure are more effective than positive control ADC-5 on SW620 xenografts in tumor-bearing nude mice.

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Abstract

提供了抗HER3抗体和抗HER3抗体药物偶联物及其医药用途。具体而言，该抗HER3抗体，如通式(Pc-L-Y-D)所示的抗HER3抗体-药物偶联物，其中Pc为抗HER3抗体，L、Y和n如说明书中所定义。

Description

抗HER3抗体和抗HER3抗体药物偶联物及其医药用途 技术领域
本披露涉及抗HER3抗体、抗HER3抗体-依喜替康类似物偶联物，其制备方法，包含其的药物组合物，以及其用于制备治疗HER3介导的疾病或病症的药物中的用途；尤其在用于制备抗癌药物中的用途。
背景技术
这里的陈述仅提供与本披露有关的背景信息，而不必然地构成现有技术。
HER3(epidermal growth factor receptor3，ErbB-3或HER3)是表皮生长因子受体(EGFR)家族成员之一。该家族包括HER1(erbB1，EGFR)、HER2(erbB2，NEU)、HER3(erbB3)及HER4(erbB4)。这些受体均包含细胞外区、跨膜区和胞内区3个部分，其中细胞外区包含4个结构域，胞内区包含1个用来信号传导的细胞内酪氨酸激酶结构域和1个位于胞浆内的带有酪氨酸磷酸化残基的尾部。当配体与细胞外结构域I和III结合时，细胞信号传导启动。在正常情况下，这些受体介导细胞分裂、迁移、存活和器官发育。EGFR家族成员发生突变时，其产生的异常信号传导刺激细胞存活，与癌症进展相关。HER3受体被激活并产生生理作用的基本原理与其他家族成员相似，区别在于其配体包括神经调节蛋白1(NRG-1)和神经调节蛋白2(NRG-2)，并且活化后HER3不能形成同源聚体，只能与EGFR或HER2形成异源二聚体。HER3在形成异源二聚体过程中，其胞内结构域部分表现较高的酪氨酸磷酸化酶活性，通过结构解析发现HER3胞内结构域有6个P85(PI-3K亚基)结合位点，该特定结构决定HER3与P85调节亚基相互作用时，能够募集多达6个PI-3K至调节亚基位点，从而强烈激活PI-3K信号通路。事实上，HER3/HER2二聚体是HER二聚体中活性最强的。EGFR广泛分布于哺乳动物上皮细胞、成纤维细胞、胶质细胞、角质细胞等细胞表面。EGFR信号通路对细胞的生长、增殖和分化等生理过程发挥重要的作用。
HER3高表达于各种常见恶性肿瘤，例如乳腺癌、胃癌、卵巢癌、前列腺癌、膀胱癌、结肠直肠癌、头颈部鳞状细胞癌和黑色素瘤等。与EGFR基因突变导致其高水平表达或过度活化不同的是，HER3基因突变率低，其高表达原因主要是mRNA转录增加，从而蛋白质翻译增多，并且常常与HER2共同高表达HER3的高表达与多种肿瘤的发生、进展及受试者的生存期都有密切联系，因此以HER3为靶点的抗肿瘤药物研究具有重要意义。
发明内容
本披露涉及抗HER3抗体，抗HER3抗体-依喜替康类似物偶联物以及其用途。
本披露提供了一种分离的抗HER3抗体，其中所述抗HER3抗体具有以下特征中的一种或多种：
a.所述的抗HER3抗体以小于0.5nM的表观亲和力EC 50与HER3蛋白结合，所述的表观亲和力EC 50是通过ELISA方法测定的；
b.所述的抗HER3抗体以小于0.2nM的表观亲和力EC 50与MCF7细胞表达的HER3蛋白结合，所述的表观亲和力EC 50是通过FACS方法测定的；
c.所述的抗HER3抗体能被表达人HER3的细胞内吞。
本披露提供了一种分离的抗HER3抗体，其中所述抗HER3抗体具有以下特征中的一种或多种：
a.所述的抗HER3抗体以小于0.5nM的表观亲和力EC 50与HER3蛋白结合，所述的表观亲和力EC 50是通过ELISA方法测定的；
b.所述的抗HER3抗体以小于0.2nM的表观亲和力EC 50与MCF7细胞表达的HER3蛋白结合，所述的表观亲和力EC 50是通过FACS方法测定的；
c.所述的抗HER3抗体能被表达人HER3的细胞内吞，当采用测试例3的方法测定所述的抗HER3抗体时，其IC 50小于2nM；
d.所述的抗HER3抗体能被表达人HER3的细胞内吞，当采用测试例4的方法测定所述的抗HER3抗体时，其FITC信号大于300。
在一些实施方案中，如上任一项所述的抗HER3抗体包含(1)SEQ ID NO:7所示的重链可变区中所包含的HCDR1，HCDR2和HCDR3；和(2)SEQ ID NO:8所示的轻链可变区中所包含的LCDR1，LCDR2和LCDR3。
在一些实施方案中，如上任一项所述的抗HER3抗体包含重链可变区和轻链可变区，其中：
a.所述重链可变区包含分别如SEQ ID NO:9，SEQ ID NO:10和SEQ ID NO:11所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:12，SEQ ID NO:13和SEQ ID NO:14所示的LCDR1，LCDR2和LCDR3；
其中所述的CDR区按Chothia编号规则确定。
在一些实施方案中，如上任一项所述的抗HER3抗体包含重链可变区和轻链可变区，其中：
b.所述重链可变区包含分别如SEQ ID NO:15，SEQ ID NO:16和SEQ ID NO:17所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:18，SEQ ID NO:19和SEQ ID NO:20所示的LCDR1，LCDR2和LCDR3；
其中所述的CDR区按IMGT编号规则确定。
在一些实施方案中，如上任一项所述的抗HER3抗体包含重链可变区和轻链可变区，其中：
c.所述重链可变区包含分别如SEQ ID NO:21，SEQ ID NO:22和SEQ ID NO:23所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:24，SEQ ID NO:25和SEQ ID NO:26所示的LCDR1，LCDR2和LCDR3；
其中所述的CDR区按Kabat编号规则确定。本披露提供了一种分离的抗HER3抗体，其中所述抗HER3抗体包含重链可变区和轻链可变区，其中：
a.所述重链可变区包含分别如SEQ ID NO:9，SEQ ID NO:10和SEQ ID NO:11所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:12，SEQ ID NO:13和SEQ ID NO:14所示的LCDR1，LCDR2和LCDR3；
其中所述的CDR区按Chothia编号规则确定。
本披露提供了一种分离的抗HER3抗体，其中所述抗HER3抗体包含重链可变区和轻链可变区，其中：
b.所述重链可变区包含分别如SEQ ID NO:15，SEQ ID NO:16和SEQ ID NO:17所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:18，SEQ ID NO:19和SEQ ID NO:20所示的LCDR1，LCDR2和LCDR3；
其中所述的CDR区按IMGT编号规则确定。
本披露提供了一种分离的抗HER3抗体，其中所述抗HER3抗体包含重链可变区和轻链可变区，其中：
c.所述重链可变区包含分别如SEQ ID NO:21，SEQ ID NO:22和SEQ ID NO:23所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:24，SEQ ID NO:25和SEQ ID NO:26所示的LCDR1，LCDR2和LCDR3；
其中所述的CDR区按Kabat编号规则确定。
在一些实施方案中，如上任一项所述的抗HER3抗体是人抗体或抗原结合片段。
在一些实施方案中，如上任一项所述的抗HER3抗体包含重链可变区和轻链可变区，其中：
所述重链可变区的氨基酸序列与SEQ ID NO:7具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性，和/或所述轻链可变区的氨基酸序列与SEQ ID NO:8具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性；
在一些实施方案中，如上任一项所述的抗HER3抗体包含重链可变区和轻链可变区，其中：
所述重链可变区的氨基酸序列如SEQ ID NO:7所示；和所述轻链可变区的氨基酸序列如SEQ ID NO:8所示；或
在一些实施方案中，如上任一项所述的抗HER3抗体进一步包含抗体重链恒定区和轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常 规变体；更优选地，所述抗体包含如SEQ ID NO:5所示的重链恒定区和如SEQ ID NO:6所示的轻链恒定区。
在一些实施方案中，如上任一项所述的抗HER3抗体包含：
与SEQ ID NO:27具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的重链，和/或与SEQ ID NO:28具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的轻链；
在一些实施方案中，如上任一项所述的抗HER3抗体包含：
如SEQ ID NO:27所示的重链和如SEQ ID NO:28所示的轻链。
在一些实施方案中，如上任一项所述的抗HER3抗体具有以下特征中的一种或多种：
a.所述的抗HER3抗体以小于0.5nM的表观亲和力EC50与HER3蛋白结合，所述的表观亲和力EC50是通过ELISA方法测定的；
b.所述的抗HER3抗体以小于0.2nM的表观亲和力EC50与MCF7细胞表达的HER3蛋白结合，所述的表观亲和力EC50是通过FACS方法测定的；
c.所述的抗HER3抗体能被表达人HER3的细胞内吞。
在一些实施方案中，如上任一项所述的抗HER3抗体具有以下特征中的一种或多种：
a.所述的抗HER3抗体以小于0.5nM的表观亲和力EC50与HER3蛋白结合，所述的表观亲和力EC50是通过ELISA方法测定的；
b.所述的抗HER3抗体以小于0.2nM的表观亲和力EC50与MCF7细胞表达的HER3蛋白结合，所述的表观亲和力EC50是通过FACS方法测定的；
c.所述的抗HER3抗体能被表达人HER3的细胞内吞，当采用测试例3的方法测定所述的抗HER3抗体时，其IC50小于2nM；
d.所述的抗HER3抗体能被表达人HER3的细胞内吞，当采用测试例4的方法测定所述的抗HER3抗体时，其FITC信号大于300。
在一些实施方案中，本披露还提供一种分离的抗HER3抗体，其中所述抗体与如前任一项所述的抗HER3抗体竞争性结合人HER3。
在一些实施方案中，本披露还提供一种核酸分子，其编码如前任一项所述的抗HER3抗体。
在一些实施方案中，本披露还提供一种宿主细胞，其包含如前任一项所述的核酸分子。
在一些实施方案中，本披露还提供一种药物组合物，其含有治疗有效量的如前任一项所述的抗HER3抗体，或如前所述的核酸分子，以及一种或更多种药学上可接受的载体、稀释剂或赋形剂。
在一些实施方案中，本披露还提供一种免疫偶联物，其包含如前任一项所述 的抗HER3抗体和效应分子，其中，所述效应分子偶联至所述抗HER3抗体；优选地，所述效应分子选自放射性同位素、抗肿瘤剂、免疫调节剂、生物反应修饰剂、凝集素、细胞毒性药物、发色团、荧光团、化学发光化合物、酶、金属离子，以及其任何组合。
在一些实施方案中，本披露还提供一种用于免疫检测或测定HER3的体内或体外方法，所述方法包括如前任一项所述的抗HER3抗体接触受试者或来自受试者的样品的步骤。
在一些实施方案中，本披露还提供一种通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2021123733-appb-000001
其中：
Y选自-O-(CR aR b) m-CR 1R 2-C(O)-、-O-CR 1R 2-(CR aR b) m-、-O-CR 1R 2-、-NH-(CR aR b) m-CR 1R 2-C(O)-和-S-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素、烷基、卤代烷基、氘代烷基、烷氧基、羟基、氨基、氰基、硝基、羟烷基、环烷基和杂环基；或者，R a和R b与其相连接的碳原子一起形成环烷基或杂环基；
R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
或者，R a和R 2与其相连的碳原子一起形成环烷基或杂环基；
m为0至4的整数；
n为1至10，n是小数或整数；
L为接头单元；
Pc为如前任一项所述的抗HER3抗体。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中n为1至8，n是小数或整数。在一些实施方案中，n为3至8，n是小数或整数。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，
其中：
Y为-O-(CR aR b) m-CR 1R 2-C(O)-；
R a和R b相同或不同，且各自独立地选自氢原子、氘原子、卤素和C 1-6烷基；
R 1为卤代C 1-6烷基或C 3-6环烷基；
R 2选自氢原子、卤代C 1-6烷基和C 3-6环烷基；
或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基；
m为0或1。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中Y选自：
Figure PCTCN2021123733-appb-000002
其中Y的O端与接头单元L相连。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1选自-(琥珀酰亚胺-3-基-N)-W-C(O)-、-CH 2-C(O)-NR 3-W-C(O)-和-C(O)-W-C(O)-，其中W选自C 1-8烷基、C 1-8烷基-C 3-6环烷基和1至8个链原子的直链杂烷基，所述1至8个链原子的直链杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、C 1-8烷基-C 3-6环烷基和1至8个链原子的直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-6环烷基中的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(Q)和天冬氨酸(D)中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
L 4选自-NR 5(CR 6R 7) t-、-C(O)NR 5、-C(O)NR 5(CH 2) t-和化学键，其中t为1至6的整数；
R 3、R 4和R 5相同或不同，且各自独立地选自氢原子、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和C 1-6羟烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和C 1-6羟烷基。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中接头单元-L-为-L 1-L 2-L 3-L 4-，
L 1为
Figure PCTCN2021123733-appb-000003
s 1为2至8的整数；
L 2为化学键；
L 3为四肽残基；
L 4为-NR 5(CR 6R 7)t-，R 5、R 6或R 7相同或不同，且各自独立地为氢原子或C 1-6烷基，t为1或2；
其中所述的L 1端与Pc相连，L 4端与Y相连。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，L 3为GGFG的四肽残基。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中-L-为：
Figure PCTCN2021123733-appb-000004
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其中-L-Y-任选自：
Figure PCTCN2021123733-appb-000005
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物 偶联物或其药学上可接受的盐，其为通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
Figure PCTCN2021123733-appb-000006
其中，
Pc为如前所述的抗HER3抗体；
m为0至4的整数；例如，m选自0、1、2、3和4；
n为1至10，n是小数或整数；具体地，n为2-8之间的小数或整数，包括两个端点值；更具体地n为2-7的小数或整数，包括端点值；可选地，n为2-3、3-4、4-5、5-6、6-7或7-8之间的小数或整数，包括端点值；
R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
W选自C 1-8烷基、C 1-8烷基-C 3-6环烷基和1至8个链原子的直链杂烷基，所述1至8个链原子的直链杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、C 1-8烷基-C 3-6环烷基和1至8个链原子的直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、C 1-8烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-6环烷基的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(Q)和天冬氨酸(D)中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
R 5选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、 氘代烷基和羟烷基。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，其为通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：其中，
Pc为如前任一项所述的抗HER3抗体；
m为0至4的整数；例如，m选自0、1、2、3和4；
n为1至10，n是小数或整数；具体地，n为2-8之间的小数或整数，包括两个端点值；更具体地n为2-7的小数或整数，包括端点值；可选地，n为2-3、3-4、4-5、5-6、6-7或7-8之间的小数或整数，包括端点值；
R 1选自卤素、卤代C 1-6烷基、氘代C 1-6烷基、C 3-6环烷基、C 3-6环烷基C 1-6烷基、C 1-6烷氧基C 1-6烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代C 1-6烷基、氘代C 1-6烷基、C 3-6环烷基、C 3-6环烷基C 1-6烷基、C 1-6烷氧基C 1-6烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成C 3-6环烷基或杂环基；
W选自C 1-8烷基、C 1-8烷基-C 3-6环烷基和1至8个链原子的直链杂烷基，所述1至8个链原子的直链杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、C 1-8烷基-C 3-6环烷基和1至8个链原子的直链杂烷基各自独立地任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-6环烷基的一个或多个取代基所取代；
L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸(F)、甘氨酸(G)、缬氨酸(V)、赖氨酸(K)、瓜氨酸、丝氨酸(S)、谷氨酸(Q)和天冬氨酸(D)中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、C 1-6烷基、氯代C 1-6烷基、氘代C 1-6烷基、C 1-6烷氧基和C 3-6环烷基中的一个或多个取代基所取代；
R 5选自氢原子、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和C 1-6羟烷基；
R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、C 1-6烷基、卤代C 1-6烷基、氘代C 1-6烷基和C 1-6羟烷基；
所述的杂环基包含3至6个环原子，其中1-3个是选自氮、氧和硫的杂原子。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物为：
Figure PCTCN2021123733-appb-000007
其中：
n为1至8，n是小数或整数；具体地，n为2-8之间的小数或整数，包括两个端点值；更具体地n为2-7的小数或整数，包括端点值；可选地，n为2-3、3-4、4-5、5-6、6-7或7-8之间的小数或整数，包括端点值；
HER3-29为抗HER3抗体，其包含如SEQ ID NO：27所示的重链和如SEQ ID NO：28所示的轻链。
在一些实施方案中，如前任一项所述的通式(Pc-L-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，优选为3至8，n是小数或整数。
在一些实施方案中，本披露还提供一种制备如前任一项所述通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐的方法，其包括以下步骤：
Figure PCTCN2021123733-appb-000008
Pc’与通式(L a-Y-D)所示的化合物进行偶联反应，得到通式(Pc-L a-Y-D)所示的化合物；
其中：
Pc’为Pc经还原后所得；
n、m、W、L 2、L 3、R 1、R 2、R 5、R 6和R 7如前任一项中所定义。
在一些实施方案中，本披露还提供一种药物组合物，其包含如前任一项所述的抗HER3抗体，或如前任一项所述的核酸分子，或如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，以及一种或多种药学上可接受的赋形剂、稀释剂或载体。
在一些实施方案中，本披露还提供如前任一项所述的抗HER3抗体，或如前任一项所述的核酸分子，或如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或如前任一项所述的药物组合物在制备用于治疗HER3介导的疾病或病症的药物中的用途。
在一些实施方案中，本披露还提供如前任一项所述的抗HER3抗体，或如前任一项所述的核酸分子，或如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或如前任一项所述的药物组合物在制备用于治疗和/或预防肿瘤和癌症的药物中的用途，其中所述肿瘤和癌症选自乳腺癌、非小细胞肺癌、胃癌、卵巢癌、前列腺癌、膀胱癌、结肠直肠癌、头颈部鳞状细胞癌和黑色素瘤。
在一些实施方案中，本披露还提供一种试剂盒，其包含如前任一项所述的抗HER3抗体，或如前任一项所述的核酸分子，或如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或如前任一项所述的药物组合物。
在一些实施方案中，本披露还提供一种预防或治疗疾病或病症的方法，所述方法包括向受试者施用治疗有效量的如前任一项所述的抗HER3抗体，或如前任一项所述的核酸分子，或如前任一项所述的抗体-药物偶联物或其药学上可接受的盐，或如前任一项所述的药物组合物。在一些实施方案中，所述疾病或病症优选为肿瘤、自身免疫性疾病或感染性疾病；在一些实施方案中，所述疾病或病症为与HER3有关的疾病或病症。
另一方面，本披露提供了一种药物组合物，其包含如前任一项所述的抗HER3抗体、抗体-药物偶联物或其药学上可接受的盐，以及一种或多种药学上可接受的赋形剂、稀释剂或载体。在一些实施方案中，所述单位剂量的药物组合物中含有0.1-3000mg或1-1000mg如前所述的抗HER3抗体或如前所述的抗体药物偶联物。
另一方面，本披露提供了如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或包含其的药物组合物作为药物的用途。
另一方面，本披露提供了如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或包含其的药物组合物在制备用于治疗HER3介导的疾病或病症的药 物中的用途，在一些实施方案中，所述HER3介导的疾病或病症为HER3高表达癌症，中表达癌症或低表达癌症。
另一方面，本披露提供了如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或包含其的药物组合物在制备用于治疗或预防癌症的药物中的用途，在一些实施方案中，所述肿瘤和癌症选自乳腺癌、非小细胞肺癌、胃癌、卵巢癌、前列腺癌、膀胱癌、结肠直肠癌、头颈部鳞状细胞癌和黑色素瘤。
另一方面，本披露进一步涉及一种用于治疗和/或预防肿瘤的方法，该方法包括向需要其的受试者施用治疗有效剂量的如前任一项所述的抗体-药物偶联物或其药学上可接受的盐或其药学上可接受的盐或包含其的药物组合物；在一些实施方案中，其中所述的肿瘤为与HER3高表达相关的癌症，中表达癌症或低表达癌症。
另一方面，本披露进一步涉及一种用于治疗或预防肿瘤或癌症的方法，该方法包括向需要其的受试者施用治疗有效剂量的如前任一项所述的抗体药物偶联物或其药学上可接受的盐或包含其的药物组合物；其中所述肿瘤和癌症在一些实施方案中，其中所述肿瘤和癌症选自乳腺癌、非小细胞肺癌、胃癌、卵巢癌、前列腺癌、膀胱癌、结肠直肠癌、头颈部鳞状细胞癌和黑色素瘤。
另一方面，本披露进一步提供如前任一项所述的抗HER3抗体或其抗体-药物偶联物作为药物，在一些实施方案中，作为治疗癌症或肿瘤的药物，更优选作为治疗HER3介导的癌症的药物。
可将活性化合物(例如根据本公开所述的化合物或其药学上可接受的盐、本公开所述的配体-药物偶联物或其药学上可接受的盐)制成适合于通过任何适当途径给药的形式，活性化合物可以以单位剂量的方式，或者是以受试者能够以单剂进行自我给药的方式。本披露所述的活性化合物或组合物的单位剂量的表达方式可以是片剂、胶囊、扁囊剂、瓶装药水、药粉、颗粒剂、锭剂、栓剂、再生药粉或液体制剂。
本披露治疗方法中所用活性化合物或组合物的施用剂量通常将随疾病的严重性、受试者的体重和活性化合物的相对功效而改变。不过，作为一般性指导，合适的单位剂量可以是0.1mg～1000mg。
本披露的药物组合物除活性化合物外，可含有一种或多种辅料，所述辅料选自以下成分：填充剂、稀释剂、粘合剂、润湿剂、崩解剂或赋形剂等。根据给药方法的不同，组合物可含有0.1至99重量％的活性化合物。
本披露提供的HER3抗体及抗体药物偶联物具有与细胞表面抗原良好的亲和力，良好的细胞内吞效率和很强的肿瘤抑制效率，并且具有更宽的药物应用窗口，适于临床的药物应用。
附图说明
图1：本披露的抗体和阳性抗体与HER3蛋白的结合活性。
图2：本披露的抗体和阳性抗体与MCF7细胞的结合活性。
图3：用DT3C测试本披露的抗体和阳性抗体的细胞内吞活性。
图4：用pHrodo测试本披露的抗体和阳性抗体的细胞内吞活性。
图5：本披露ADC样品对荷瘤裸鼠SW620移植瘤的疗效。
具体实施方式
术语
除非另有限定，本文所用的所有技术和科学术语均与本披露所属领域普通技术人员的通常理解一致。虽然也可采用与本文所述相似或等同的任何方法和材料实施或测试本披露，但本文描述了优选的方法和材料。描述和要求保护本披露时，依据以下定义，使用下列术语。
当本披露中使用商品名时，旨在包括该商品名产品的制剂、该商品名产品的药物和活性药物部分。
术语“抗体-药物偶联物”(antibody drug conjugate，ADC)指将抗体与具有生物活性的药物相连。其中，抗体可以直接地或经连接单元地与药物偶联。
术语“载药量”是指抗体-药物偶联物群体中，每个抗体-药物偶联物分子载有的药物平均数量，也可以表示为药物量和抗体量的比值。载药量的范围可以是每个抗体连接0-12个药物，示例性的为1、2、3、4、5、6、7、8、9、10个药物，该数值可以是小数，也可以是整数。在某些实施方案中，每个抗体载有1个到约10个药物；在某些实施方案中，每个抗体载有约1个至约9个，1个到约8个，约3个至约7个，约3个至约6个，约3个至约5个，约2个、约3个、约4个、约5个、约6个、约7个、约8个药物，该数值可以是小数，也可以是整数。载药量可用常规方法鉴定，例如UV/可见光光谱法、质谱、ELISA试验和HPLC特征。
本披露的一个实施方式中，细胞毒性药物通过连接单元偶联在抗体的巯基上。
可以用以下非限制性方法控制配体细胞毒性药物偶联物的载量，包括：
(1)控制连接试剂和单抗的摩尔比，
(2)控制反应时间和温度，
(3)选择不同的反应试剂。
本披露所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
本披露的术语“抗体”以最广义使用，其涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体，多特异性抗体(例如双特异性抗体)，全长抗体或其抗原结合片段(也称“抗原结合部分”)，只要它们展现出期望的抗原结合活性。 全长抗体是包含由二硫键互相连接的至少两条重链和两条轻链的免疫球蛋白(Ig)。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
全长抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(缩写为Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。每条重链由重链可变区(缩写为VH)和重链恒定区(缩写为CH)组成。重链恒定区包含CH1、CH2和CH3三个结构域。每条轻链由轻链可变区(缩写为VL)和轻链恒定区(缩写为CL)组成。重链可变区和轻链可变区包括高变区(也称为互补性决定区，缩写为CDR或HVR)和序列相对保守的骨架区(也称框架区，缩写为FR)。每个VL和VH由从氨基末端排到羧基末端按以下顺序排列的3个CDR 4个FR组成：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。
本披露中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A突变，S228P突变，265A(例如D265A)和/或297A(例如N297A)，和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
本披露中“人抗体”(HuMAb)、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，其氨基酸序列对应于由人或人细胞产生的抗体的氨基酸序列、或衍生自利用人抗体组库或其它人抗体编码序列的非人来源的氨基酸序列。该人抗体的定义明确排除了包含非人抗原结合残基的人源化抗体。
术语“抗原结合片段”或“功能片段”或“抗原结合部分”是指保持特异性结合抗原的能力的完整抗体的一个或更多个片段。可利用全长抗体的片段来进行抗体的抗原结合功能。示例性的，涵盖在术语“抗原结合片段”的结合片段的示例包括：(i)Fab片段，一种由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，一种包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和 VL结构域组成的Fv片段；(v)dsFv，由VH和VL经链间二硫键形成的稳定的抗原结合片段；(vi)包含scFv、dsFv、Fab等片段的双抗体、双特异性抗体和多特异性抗体。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法将这两个结构域通过能够使它们形成为单条蛋白质链的人工肽接头来接合，其中VL和VH配对形成单价分子，称为单链Fv(scFv)(参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883))。此类单链抗体也包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个、2个、3个或更多个氨基酸的插入、缺失或替换。
术语“抗体框架区”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia”编号规则、“ABM”编号规则、“contact”编号规则(参见Martin,ACR.Protein Sequence and Structure Analysis of Antibody Variable Domains[J].2001)和ImMunoGenTics(IMGT)编号规则(参见Lefranc M.P.,Dev.Comp.Immunol.，27，55-77(2003))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为27-38(HCDR1)、56-65(HCDR2)和105-117(HCDR3)，VL中的CDR氨基酸残基编号大致为27-38(LCDR1)、 56-65(LCDR2)和105-117(LCDR3)。遵循AbM规则，VH中的CDR氨基酸编号为26-35(HCDR1)、50-58(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。
术语“表位”或“抗原决定簇”是指抗原上被抗体结合的部位(例如，HER3分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体或抗原结合片段对预先确定的抗原上的表位的结合。通常，抗体或抗原结合片段以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M、10 -12M或更小的亲和力(KD)结合。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如HER3抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用结合荷有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白任一种的固态表面或细胞的纯化的抗原。通过测量在所测抗原结合蛋白存在下结合固态表面或细胞的标记的量来测量竞争性抑制。通常所测抗原结合蛋白过量存在。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、 90-95％、95-97％或97％或更多。
本文中使用的术语“核酸分子”是指DNA分子或RNA分子。核酸分子可以是单链或双链的，优选是双链DNA或单链mRNA或修饰的mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
氨基酸序列“同一性”指在比对氨基酸序列及必要时引入间隙，以达成最大序列同一性百分比，且不将任何保守性取代视为序列同一性的一部分，第一序列中与第二序列中的氨基酸残基同一的氨基酸残基的百分比。为测定氨基酸序列同一性百分比的目的，比对可以通过属于本领域技术的范围内的多种方式来实现，例如使用公开可得到的计算机软件，诸如BLAST、BLAST-2、ALIGN、ALIGN-2或Megalign(DNASTAR)软件。本领域技术人员可确定适用于测量比对的参数，包括在所比较的序列全长上达成最大比对所需的任何算法。
“抗体依赖性的细胞介导的细胞毒性”或“ADCC”指的是细胞介导的反应，其中表达FcR的非特异性细胞毒性细胞(例如天然杀伤(NK)细胞、嗜中性粒细胞、和巨噬细胞)识别靶细胞上结合的抗体，并且随后引起靶细胞的裂解。调节ADCC的原代细胞、NK细胞仅表达FcyRIII，而单核细胞表达FcyRI、FcyRII和FCYRIII。为了评估目的分子的ADCC活性，可进行体内外ADCC测定，诸如Clynes等人(PNASUSA 95:652-656(1998))、美国专利号US5500362、US5821337等所述。
“抗体依赖性细胞吞噬作用”或“ADCP”指通过吞噬细胞(例如，巨噬细胞、嗜中性粒细胞和树突细胞)内化来消除经抗体包被的靶细胞或病毒体的机制。内化的抗体包被的靶细胞或病毒体被包含在称为吞噬体的囊泡中，所述囊泡随后与一个或多个溶酶体融合，以形成吞噬溶酶体。ADCP可以通过使用用巨噬细胞作为效应细胞的体外细胞毒性测定和电视显微术(videomicroscopy)来评价(如van Bij等于Journal of Hepatology第53卷，第4期，2010年10月，第677–685)。“补体依赖性细胞毒性”或“CDC”是指补体参与的细胞毒作用，即通过抗体与细胞或病毒体上的相应抗原结合，形成复合物而激活补体经典途径，所形成的攻膜复合物对靶细胞发挥裂解效应。CDC可以通过体外测定(如CDC测定，其中使用正常人血清作为补体源)或在C1q浓度系列中评价。CDC活性的降低(例如由于在多肽或抗体中引入第二突变而导致的CDC活性的降低)可以通过在相同的测定法内比较多肽或抗体的CDC活性与其不具有第二突变的亲本多肽或抗体的CDC活性来测量。为了评估抗体诱导CDC的能力，可如Romeuf等人所述的测定方法(Romeuf等人，Br J Haematol.2008Mar；140(6):635-43)进行测定。
本披露所描述的抗体或抗体片段可以通过任何方式偶联至效应分子。举例来说，抗体或抗体片段可以通过化学或重组方式附接至细胞毒性药物。制备融合物或偶联物的化学方式在本领域中是已知的并且可以用于制备免疫偶联物。用于偶联抗体或抗体片段和药物的方法必须能够连接抗体与细胞毒性药物而不会干扰抗体或抗体片段结合至标靶分子的能力。
在一个实施方案中，抗体和细胞毒性药物都是蛋白质并且可以使用本领域中熟知的技术偶联。存在本领域中所公开的数百种交联剂，其可以偶联两种蛋白质。交联剂一般基于在抗体或细胞毒性药物上可用或插入的反应官能团来选择。另外，如果不存在反应基团，那么可以使用光可活化交联剂。在某些情况下，可能需要在抗体与细胞毒性药物之间包括间隔子。本领域中已知的交联剂包括同型双功能剂：戊二醛、二甲基己二亚酰胺化物和双(重氮基联苯胺)，以及异型双功能剂：间马来酰亚胺基苯甲酰基-N-羟基琥珀酰亚胺和磺基-间马来酰亚胺基苯甲酰基-N-羟基琥珀酰亚胺。
可以用于使效应分子偶合至抗体片段的交联剂包括例如TPCH(S-(2-硫代吡啶基)-L-半胱氨酸酰肼)和TPMPH(S-(2-硫代吡啶基)巯基-丙酰肼)。TPCH和TPMPH在先前已经通过温和高碘酸盐处理而氧化的糖蛋白的碳水化合物部分反应，由此在交联剂的酰肼部分与高碘酸盐产生的醛之间形成腙键。异型双功能交联剂GMBS(N-(γ-马来酰亚胺基丁酰氧基)-琥珀酰亚胺)和SMCC(琥珀酰亚胺基4-(N-马来酰亚胺基-甲基)环己烷)与伯胺反应，由此将马来酰亚胺基引入组分上。这个马来酰亚胺基随后可以与另一种组分上的可以由交联剂引入的硫氢基反应，由此在组分之间形成稳定的硫醚键。如果组分之间的位阻干扰任一种组分的活性，那么可以使用交联剂，将长的间隔臂引入组分之间，诸如3-(2-吡啶基二硫基)丙酸n-琥珀酰亚胺酯(SPDP)。因此，存在许多适合的交联剂，其可以被使用并且各自取决于其对最佳免疫偶联物产量的影响来选择。
术语“表达载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。公开所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或更多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics (IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、293细胞和NS0细胞。在一些实施方案中，本披露中的宿主细胞不包含来自人胚胎的细胞。
本披露工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人HER3特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene，The Benjamin/Cummings Pub.Co.，第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。示例性保守取代如下：
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同源性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS):Altschul，S.F.等人，(1990)J.Mol.Biol.215:403-410；Gish，W.等人，(1993)Nature Genet.3:266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266:131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25:3389-3402；Zhang，J.等人，(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初 转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4,683,195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press，N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个示例，但不是唯一的示例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“分离的”指纯化状态，并且在这种情况下意味着在指定的分子基本上不含其他生物分子，例如核酸、蛋白质、脂质、碳水化合物或其他材料，例如细胞碎片和生长培养基。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
术语“药物”是指可以改变或查明机体的生理功能及病理状态，可用以预防，诊断和治疗疾病的化学物质。药物包括细胞毒性药物。药物和毒物之间并无严格界限，毒物是指在较小剂量即对机体产生毒害作用，损害人体健康的化学物质，任何药物剂量过大都可产生毒性反应。
细胞毒性药物，即抑制或防止细胞的功能和/或引起细胞死亡或破坏的物质。细胞毒性药物原则上在足够高的浓度下都可以杀死肿瘤细胞，但是由于缺乏特异性，在杀伤肿瘤细胞的同时，也会导致正常细胞的凋亡，导致严重的副作用。细胞毒性药物包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At 211、I 131、I 125、Y 90、Re 186、Re 188、Sm 153、Bi 212、P 32和Lu的放射性同位素)，化疗药物，抗生素和核溶酶。
在一些实施方式中，毒素可以是来自细菌、真菌、植物或动物的小分子毒素及其衍生物，包括喜树碱类衍生物如伊沙替康，美登木素生物碱及其衍生物(CN101573384)如DM1、DM3、DM4，auristatin F(AF)及其衍生物，如MMAF、MMAE、3024(WO 2016/127790 A1，化合物7)，白喉毒素、外毒素、蓖麻毒蛋白(ricin)A链、相思豆毒蛋白(abrin)A链、modeccin、α-帚曲霉素(sarcin)、油桐(Aleutites fordii)毒蛋白、香石竹(dianthin)毒蛋白、美洲商陆(Phytolaca americana)毒蛋白(PAPI、PAPII和PAP-S)、苦瓜(Momordica charantia)抑制 物、麻疯树毒蛋白(curcin)、巴豆毒蛋白(crotin)、肥皂草(sapaonaria officinalis)抑制物、白树毒蛋白(gelonin)、丝林霉素(mitogellin)局限曲霉素(restrictocin)、酚霉素(phenomycin)、依诺霉素(enomycin)和单端孢菌素(trichothecenes)。
术语“化疗药物”是可用于治疗肿瘤的化学化合物。该定义还包括起调节、降低、阻断或抑制可促进癌生长的激素效果作用的抗激素剂，且常常是系统或全身治疗的形式。它们自身可以是激素。化疗药物示例包括烷化剂，如噻替哌(thiotepa)，环磷酰胺(cyclosphamide)(CYTOXAN TM)，烷基磺酸脂如白消安(busulfan)，英丙舒凡(improsulfan)和哌泊舒凡(piposulfan)，氮丙啶(aziridine)如苯并多巴(benaodopa)，卡波醌(carboquone)，美妥替哌(meturedopa)和尿烷亚胺(uredopa)，氮丙啶和methylamelamine包括六甲蜜胺(altretamine)，三亚胺嗪(triethylenemelamine)，三亚乙基磷酰胺，三亚乙基硫代磷酰胺和三羟甲基蜜胺(trimethylolomelamine)，氮芥(nitrogen mustards)如苯丁酸氮芥，萘氮芥，胆磷酰胺(cholophosphamide)，雌氮芥(estramustine)，异环磷酰胺(ifosfamide)，氮芥(mechlorethamine)，盐酸氧氮芥；左旋苯丙氨酸氮芥(melphalan)，新氮芥(novembichin)，胆甾醇苯乙酸氮芥，松龙苯芥(prednimustine)，曲磷胺(trofosfamide)，尿嘧啶氮芥；亚硝基脲(nitrosureas)如亚硝基脲氮芥(carmustine)，氯脲菌素(chlorozotocin)，福莫司汀(fotemustine)，洛莫司汀(lomustine)，尼莫司汀(nimustine)，雷莫司汀(ranimustine)，抗生素如阿克拉霉素，放线菌素，authramycin，重氮丝氨酸，博来霉素，放线菌素C(cactinomycin)，加利车霉素(calicheamicin)，carabicin，洋红霉素(chromomycin)，嗜癌素(carzinophilin)，色霉素，放线菌素D，柔红菌素(daunorubicin)，地托比星(detorubicin)，6-重氮-5-氧-L-正亮氨酸，阿霉素(doxorubicin)，表阿霉素(epirubicin)，依索比星(esorubicin)，伊达比星(idarubicin)，发波霉素(marcellomycin)，丝裂霉素，霉酚酸，诺加霉素(nogalamycin)，橄榄霉素(olivomycin)，培洛霉素(peplomycin)，potfiromycin，嘌呤霉素，三铁阿霉素(quelamycin)，罗多比星(rodorubicin)，链黑菌素，链脲霉素(streptozocin)，杀结核菌素，乌苯美司(ubenimex)，净司他丁(zinostatin)，佐柔比星(zorubicin)，抗代谢药如氨甲蝶吟，5-氟尿嘧啶(5-FU)，叶酸类似物如二甲叶酸(denopterin)，氨甲蝶呤，蝶罗呤，三甲曲沙(trimetrexate)，喋吟类似物氟达拉滨(f1udarabine)，6-巯基蝶呤，硫咪蝶呤，硫鸟蝶呤，嘧啶类似物如安西他滨(ancitabine)，阿扎胞苷(azacitidine)，6-氮尿苷，卡莫氟(carmofur)，阿糖胞苷，双脱氧尿苷，去氟氧尿苷(doxitluridine)，依诺他滨(enocitabine)，氟尿苷，5-FU，雄激素类如二甲睾酮(calusterone)，丙酸甲雄烷酮(dromostanolong propionate)，环硫雄醇(epitiostanol)，美雄氨(mepitiostane)，睾内酯(testolactone)，抗肾上腺类如氨鲁米特(aminoglutethimide)，米托坦(mitotane)，曲洛司坦(trilostane)，叶酸补充剂如frolinic acid，醋葡内脂，醛磷酰胺糖苷(aldophosphamideglycoside)，氨基乙酰丙酸(aminolevulinic acid)，安吖啶(amsacrine)，bestrabucil，比生群(biasntrene)， 依达曲沙(edatraxate)，defofamine，秋水仙胺，地吖醌(diaziquone)，elfomithine，依利醋铵(elliptiniumacetate)，依托格鲁(etoglucid)，硝酸镓，羟基脲，香菇多糖(lentinan)，氯尼达明(lonidamine)，米托胍腙(mitoguazone)，米托蒽醌(mitoxantrone)，莫哌达醇(mopidamol)，硝呋旦(nitracrine)，喷司他丁(pintostatin)，phenamet，吡柔比星(pirarubicin)，鬼臼树酸(podophyllinic acid)，2-乙基酰肼，丙卡巴肼(procarbazine)，
Figure PCTCN2021123733-appb-000009
雷佐生(razoxane)，西索菲兰(sizofiran)，锗螺胺(spirogermanium)，细交链孢菌酮酸，三亚胺醌；2，2'，2"-三氯二乙胺(trichlorrotriethylamine)，乌拉坦(urethan)，长春碱酰胺，达卡巴嗪(dacarbazine)，甘露醇氮芥，二溴甘露醇(mitobronitol)，二溴卫矛醇，哌溴烷坑(pipobroman)，gacytosine，阿拉伯糖苷("Ara-C")，环磷酰胺，三胺硫磷(thiotepa)，紫杉烷，如紫杉醇(
Figure PCTCN2021123733-appb-000010
Bristol-Myers Squibb Oncology，Princeton，NJ)和docetaxel(
Figure PCTCN2021123733-appb-000011
Rhone-Poulenc Rorer，Antony，France)，苯丁酸氮芥，吉西他滨(gemcitabine)，6-硫代鸟嘌呤，巯基嘌呤，氨甲蝶呤，铂类似物如顺铂和卡铂，长春花碱，铂，依托泊甙(etoposide)(VP-16)，异环磷航胶，丝裂霉素C，米托蒽醌，长春新碱，长春瑞宾(vinorelbine)，新霉酰胺(navelbine)，novantrone，替尼泊甙(teniposide)，柔红霉素，氨基蝶呤；xeloda，伊拜磷酸盐(ibandronate)，CPT-11，拓扑异构酶抑制剂RFS2000，二氟甲基鸟氨酸(DMFO)，维甲酸esperamicins，capecitabine，以及上述任何物质的可药用盐，酸或衍生物。此定义还包括能调节或抑制激素对肿瘤的作用的抗激素制剂，如抗雌激素制剂包括他莫昔芬(tamoxifen)，雷洛昔芬(raloxifene)，芳香酶抑制剂4(5)-咪唑，4-羟基他莫昔芬，曲沃昔芬(trioxifene)，keoxifene，LY117018，onapristone，和托瑞米芬(Fareston)，和抗雄激素制剂如氟他氨(flutamide)，尼鲁米特(nilutamide)，bicalutamide，亮丙瑞林(leuprolide)和戈舍瑞林(goserelin)，和上述任何物质的可药用盐，酸或衍生物。
术语“烷基”指饱和脂肪族烃基团，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个(例如1、2、3、4、5、6、7、8、9、10、11和12个)碳原子的烷基，更优选为含有1至6个碳原子的烷基。非限制性示例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基、2,3-二甲基丁基、正庚基、2-甲基己基、3-甲基己基、4-甲基己基、5-甲基己基、2,3-二甲基戊基、2,4-二甲基戊基、2,2-二甲基戊基、3,3-二甲基戊基、2-乙基戊基、3-乙基戊基、正辛基、2,3-二甲基己基、2,4-二甲基己基、2,5-二甲基己基、2,2-二甲基己基、3,3-二甲基己基、4,4-二甲基己基、2-乙基己基、3-乙基己基、4-乙基己基、2-甲基-2-乙基戊基、2-甲基-3-乙基戊基、正 壬基、2-甲基-2-乙基己基、2-甲基-3-乙基己基、2,2-二乙基戊基、正癸基、3,3-二乙基己基、2,2-二乙基己基，及其各种支链异构体等。更优选的是含有1至6个碳原子的低级烷基，非限制性实施例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基、2-甲基戊基、3-甲基戊基、4-甲基戊基和2,3-二甲基丁基等。烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自H原子、D原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基。
术语“杂烷基”指含有一个或多个选自N、O或S的杂原子的烷基，其中烷基如上所定义。
术语“亚烷基”指饱和的直链或支链脂肪族烃基，其具有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，其为包含1至20个碳原子的直链或支链基团，优选含有1至12个(例如1、2、3、4、5、6、7、8、9、10、11和12个)碳原子，更优选含有1至8个碳原子的亚烷基，最优选含有1至6个碳原子的亚烷基。亚烷基的非限制性示例包括但不限于亚甲基(-CH 2-)、1,1-亚乙基(-CH(CH 3)-)、1,2-亚乙基(-CH 2CH 2)-、1,1-亚丙基(-CH(CH 2CH 3)-)、1,2-亚丙基(-CH 2CH(CH 3)-)、1,3-亚丙基(-CH 2CH 2CH 2-)、1,4-亚丁基(-CH 2CH 2CH 2CH 2-)等。亚烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自烷基、烯基、炔基、烷氧基、卤代烷氧基、环烷基氧基、杂环基氧基、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。
术语“烯基”指分子中含有碳碳双键的烷基化合物，其中烷基的定义如上所述。烯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，其独立地选自氢原子、烷基、烷氧基、卤素、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基。
术语“炔基”指分子中含有碳碳三键的烷基化合物，其中烷基的定义如上所述。炔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，其独立地选自氢原子、烷基、烷氧基、卤素、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基。
术语“环烷基”指饱和或部分不饱和单环或多环环状烃取代基，环烷基环包含3至20个碳原子，优选包含3至12个碳原子，优选包含3至8个碳原子(例如3、4、5、6、7和8个)碳原子，更优选包含3至6个碳原子。单环环烷基的非限制性示例包括环丙基、环丁基、环戊基、环戊烯基、环己基、环己烯基、环己二烯基、环庚基、环庚三烯基和环辛基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“螺环烷基”指5至20元，单环之间共用一个碳原子(称螺原子)的多环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元(例如7、8、9或10元)。根据环与环之间共用螺原子的数目将螺环烷基分为单螺环烷基、双螺环烷基或多螺环烷基，优选为单螺环烷基和双螺环烷基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺环烷基。螺环烷基的非限制性示例包括：
Figure PCTCN2021123733-appb-000012
术语“稠环烷基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对碳原子的全碳多环基团，其中一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元(例如7、8、9或10元)。根据组成环的数目可以分为双环、三环、四环或多环稠环烷基，优选为双环或三环，更优选为5元/5元或5元/6元双环烷基。稠环烷基的非限制性示例包括：
Figure PCTCN2021123733-appb-000013
术语“桥环烷基”指5至20元，任意两个环共用两个不直接连接的碳原子的全碳多环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元(例如7、8、9或10元)。根据组成环的数目可以分为双环、三环、四环或多环桥环烷基，优选为双环、三环或四环，更优选为双环或三环。桥环烷基的非限制性示例包括：
Figure PCTCN2021123733-appb-000014
所述环烷基环包括如上所述的环烷基(包括单环、螺环、稠环和桥环)稠合于芳基、杂芳基或杂环烷基环上，其中与母体结构连接在一起的环为环烷基，非限制性示例包括茚满基、四氢萘基和苯并环庚烷基等；优选苯基并环戊基或四氢萘基。
环烷基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自氢原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基。
术语“烷氧基”指-O-(烷基)和-O-(非取代的环烷基)，其中烷基、环烷基的定义如上所述。烷氧基的非限制性示例包括：甲氧基、乙氧基、丙氧基、丁氧基、环丙氧基、环丁氧基、环戊氧基和环己氧基。烷氧基可以是任选取代的或非取代的，当被取代时，取代基优选为一个或多个以下基团，其独立地选自H原子、D原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基。
术语“杂环基”指饱和或部分不饱和单环或多环环状烃取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧、硫、S(O)或S(O) 2的杂原子，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包含3至12个(例如3、4、5、6、7、8、9、10、11和12个)环原子，其中1～4个(例如1、2、3和4个)是杂原子；更优选包含3至8个环原子(例如3、4、5、6、7和8个)，其中1-3是杂原子(例如1、2和3个)；更优选包含3至6个环原子，其中1-3个是杂原子；最优选包含5或6个环原子，其中1-3个是杂原子。单环杂环基的非限制性示例包括吡咯烷基、四氢吡喃基、1,2,3,6-四氢吡啶基、哌啶基、哌嗪基、吗啉基、硫代吗啉基和高哌嗪基等。多环杂环基包括螺环、稠环和桥环的杂环基。
术语“螺杂环基”指5至20元的，单环之间共用一个原子(称螺原子)的多环杂环基团，其中一个或多个环原子为选自氮、氧、硫、S(O)或S(O) 2的杂原子，其余环原子为碳。其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统。优选为6至14元，更优选为7至10元(例如7、8、9或10元)。根据环与环之间共用螺原子的数目将螺杂环基分为单螺杂环基、双螺杂环基或多 螺杂环基，优选为单螺杂环基和双螺杂环基。更优选为4元/4元、4元/5元、4元/6元、5元/5元或5元/6元单螺杂环基。螺杂环基的非限制性示例包括：
Figure PCTCN2021123733-appb-000015
术语“稠杂环基”指5至20元，系统中的每个环与体系中的其他环共享毗邻的一对原子的多环杂环基团，一个或多个环可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧、硫、S(O)或S(O) 2的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(例如7、8、9或10元)。根据组成环的数目可以分为双环、三环、四环或多环稠杂环基，优选为双环或三环，更优选为5元/5元或5元/6元双环稠杂环基。稠杂环基的非限制性示例包括：
Figure PCTCN2021123733-appb-000016
术语“桥杂环基”指5至14元，任意两个环共用两个不直接连接的原子的多环杂环基团，其可以含有一个或多个双键，但没有一个环具有完全共轭的π电子系统，其中一个或多个环原子为选自氮、氧、硫、S(O)或S(O) 2的杂原子，其余环原子为碳。优选为6至14元，更优选为7至10元(例如7、8、9或10元)。根据组成环的数目可以分为双环、三环、四环或多环桥杂环基，优选为双环、三环或四环，更优选为双环或三环。桥杂环基的非限制性示例包括：
Figure PCTCN2021123733-appb-000017
所述杂环基环包括如上所述的杂环基(包括单环、螺杂环、稠杂环和桥杂环)稠合于芳基、杂芳基或环烷基环上，其中与母体结构连接在一起的环为杂环基，其非限制性示例包括：
Figure PCTCN2021123733-appb-000018
等。
杂环基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自氢原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基。
术语“芳基”指具有共轭的π电子体系的6至14元全碳单环或稠合多环(稠合多环是共享毗邻碳原子对的环)基团，优选为6至10元，例如苯基和萘基。所述芳基环包括如上所述的芳基环稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，其非限制性示例包括：
Figure PCTCN2021123733-appb-000019
芳基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自氢原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基。
术语“杂芳基”指包含1至4个杂原子、5至14个环原子的杂芳族体系，其中杂原子选自氧、硫和氮。杂芳基优选为5至10元(例如5、6、7、8、9或10元)，更优选为5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、哒嗪基、咪唑基、吡唑基、三唑基、四唑基等。所述杂芳基环包括如上述的杂芳基稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，其非限制性示例包括：
Figure PCTCN2021123733-appb-000020
Figure PCTCN2021123733-appb-000021
杂芳基可以是取代的或非取代的，当被取代时，取代基可以在任何可使用的连接点上被取代，所述取代基优选独立地任选选自氢原子、卤素、烷基、烷氧基、卤代烷基、卤代烷氧基、环烷基氧基、杂环基氧基、羟基、羟烷基、氰基、氨基、硝基、环烷基、杂环基、芳基和杂芳基中的一个或多个取代基。
术语“氨基保护基”是为了使分子其它部位进行反应时氨基保持不变，用易于脱去的基团对氨基进行保护。非限制性实施例包含(三甲基硅)乙氧基甲基、四氢吡喃基、叔丁氧羰基、乙酰基、苄基、烯丙基和对甲氧苄基等。这些基团可任选地被选自卤素、烷氧基或硝基中的1-3个取代基所取代。
术语“羟基保护基”是本领域已知的适当的用于羟基保护的基团，参见文献(“Protective Groups in Organic Synthesis”，5 Th Ed.T.W.Greene&P.G.M.Wuts)中的羟基保护基团。作为示例，优选地，所述的羟基保护基可以是(C 1-10烷基或芳基) 3硅烷基，例如：三乙基硅基，三异丙基硅基，叔丁基二甲基硅基，叔丁基二苯基硅基等；可以是C 1-10烷基或取代烷基，优选烷氧基或芳基取代的烷基，更优选C 1-6烷氧基取代的C 1-6烷基或苯基取代的C 1-6烷基，最优选C 1-4烷氧基取代的C 1-4烷基，例如：甲基，叔丁基，烯丙基，苄基，甲氧基甲基(MOM)，乙氧基乙基，2-四氢吡喃基(THP)等；可以是(C 1-10烷基或芳香基)酰基，例如：甲酰基，乙酰基，苯甲酰基、对硝基苯甲酰基等；可以是(C 1-6烷基或C 6-10芳基)磺酰基；也可以是(C 1-6烷氧基或C 6-10芳基氧基)羰基。
术语“环烷基氧基”指环烷基-O-，其中环烷基如上所定义。
术语“杂环基氧基”指杂环基-O-，其中杂环基如上所定义。
术语“烷硫基”指烷基-S-，其中烷基如上所定义。
术语“卤代烷基”指烷基被一个或多个卤素取代，其中烷基如上所定义。
术语“卤代烷氧基”指烷氧基被一个或多个卤素取代，其中烷氧基如上所定义。
术语“氘代烷基”指烷基被一个或多个氘原子取代，其中烷基如上所定义。
术语“羟烷基”指被羟基取代的烷基，其中烷基如上所定义。
术语“卤素”指氟、氯、溴或碘。
术语“羟基”指-OH。
术语“巯基”指-SH。
术语“氨基”指-NH 2。
术语“氰基”指-CN。
术语“硝基”指-NO 2。
术语“氧代基”指“＝O”。
术语“羰基”指C＝O。
术语“羧基”指-C(O)OH。
术语“羧酸酯基”指-C(O)O(烷基)、-C(O)O(环烷基)、(烷基)C(O)O-或(环烷基)C(O)O-，其中烷基、环烷基如上所定义。
本披露的化合物包含其同位素衍生物。术语“同位素衍生物”指结构不同仅在于存在一种或多种同位素富集原子的化合物。例如，具有本披露的结构，用“氘”或“氚”代替氢，或者用 18F-氟标记( 18F同位素)代替氟，或者用 11C-， 13C-，或者 14C-富集的碳( 11C-， 13C-，或者 14C-碳标记； 11C-， 13C-，或者 14C-同位素)代替碳原子的化合物处于本披露的范围内。这样的化合物可用作例如生物学测定中的分析工具或探针，或者可以用作疾病的体内诊断成像示踪剂，或者作为药效学、药动学或受体研究的示踪剂。本披露的各种氘化形式的化合物是指与碳原子连接的各个可用的氢原子可独立地被氘原子替换。本领域技术人员能够参考相关文献合成氘化形式的化合物。在制备氘代形式的化合物时可使用市售的氘代起始物质，或它们可使用常规技术采用氘代试剂合成，氘代试剂包括但不限于氘代硼烷、三氘代硼烷四氢呋喃溶液、氘代氢化锂铝、氘代碘乙烷和氘代碘甲烷等。氘代物通常可以保留与未氘代的化合物相当的活性，并且当氘代在某些特定位点时可以取得更好的代谢稳定性，从而获得某些治疗优势。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该说明包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代的”指基团中的一个或多个氢原子，优选为1～5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。本领域技术人员能够在不付出过多努力的情况下(通过实验或理论)确定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。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的 载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本文所用的术语“药学上可接受的”是指这些化合物、材料、组合物和/或剂型，在合理的医学判断范围内，适用于与受试者组织接触而没有过度毒性、刺激性、过敏反应或其他问题或并发症，具有合理的获益/风险比，并且对预期的用途是有效。
本文所使用的，单数形式的“一个”、“一种”和“该”包括复数引用，反之亦然，除非上下文另外明确指出。
当将术语“约”应用于诸如pH、浓度、温度等的参数时，表明该参数可以变化±10％，并且有时更优选地在±5％之内。如本领域技术人员将理解的，当参数不是关键的时，通常仅出于说明目的给出数字，而不是限制。
术语“接头单元”、“连接单元”或“连接片段”是指一端与配体(本披露中为抗体)连接而另一端与药物相连的化学结构片段或键，也可以连接其他接头后再与药物相连。
接头可以包含一种或多种接头构件。例示性的接头构件包括6-马来酰亚氨基己酰基(“MC”)、马来酰亚氨基丙酰基(“MP”)、缬氨酸-瓜氨酸(“val-cit”或“vc”)、丙氨酸-苯丙氨酸(“ala-phe”)、对氨基苄氧羰基(“PAB”)、及那样源自与接头试剂的偶联的：N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯(“SPP”)、N-琥珀酰亚氨基4-(N-马来酰亚氨基甲基)环己烷-1羧酸酯(“SMCC”，在本文中也称作“MCC”)和N-琥珀酰亚氨基(4-碘-乙酰基)氨基苯甲酸酯(“SIAB”)。接头可以包括延伸物、间隔物和氨基酸单元，可以通过本领域已知方法合成，诸如US2005-0238649A1中所记载的。接头可以是便于在细胞中释放药物的“可切割接头”。例如，可使用酸不稳定接头(例如腙)、蛋白酶敏感(例如肽酶敏感)接头、光不稳定接头、二甲基接头、或含二硫化物接头(Chari等，Cancer Research 52:127-131(1992)；美国专利No.5，208，020)。
接头组件包括但不限于：
MC＝6-马来酰亚氨基己酰基，结构如下：
Figure PCTCN2021123733-appb-000022
Val-Cit或“vc”＝缬氨酸-瓜氨酸(蛋白酶可切割接头中的例示二肽)
瓜氨酸＝2-氨基-5-脲基戊酸
PAB＝对氨基苄氧羰基(“自我牺牲”接头组件的例示)
Me-Val-Cit＝N-甲基-缬氨酸-瓜氨酸(其中接头肽键已经修饰以防止其受到组织蛋白酶B的切割)
MC(PEG)6-OH＝马来酰亚氨基己酰基-聚乙二醇(可附着于抗体半胱氨酸)
SPP＝N-琥珀酰亚氨基4-(2-吡啶基硫代)戊酸酯
SPDP＝N-琥珀酰亚氨基3-(2-吡啶基二硫代)丙酸酯
SMCC＝琥珀酰亚氨基-4-(N-马来酰亚氨基甲基)环己烷-1-羧酸酯
IT＝亚氨基硫烷
常规的药物组合物的制备见中国药典。
术语“载体”用于本披露的药物，是指能改变药物进入人体的方式和在体内的分布、控制药物的释放速度并将药物输送到靶向器官的体系。药物载体释放和靶向系统能够减少药物降解及损失，降低副作用，提高生物利用度。如可作为载体的高分子表面活性剂由于其独特的两亲性结构，可以进行自组装，形成各种形式的聚集体，优选的示例如胶束、微乳液、凝胶、液晶、囊泡等。这些聚集体具有包载药物分子的能力，同时又对膜有良好的渗透性，可以作为优良的药物载体。
术语“赋形剂”是在药物制剂中除主药以外的附加物，也可称为辅料。如片剂中的粘合剂、填充剂、崩解剂和润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂和着色剂等均可称为赋形剂。
术语“稀释剂”又称填充剂，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐和微晶纤维素等。
术语“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
药物组合物可以是无菌注射水溶液形式。可在使用的可接受的溶媒和溶剂中有水、林格氏液和等渗氯化钠溶液。无菌注射制剂可以是其中活性成分溶于油相的无菌注射水包油微乳。例如将活性成分溶于大豆油和卵磷脂的混合物中。然后将油溶液加入水和甘油的混合物中处理形成微乳。可通过局部大量注射，将注射液或微乳注入受试者的血流中。或者，最好按可保持本披露化合物恒定循环浓度的方式给予溶液和微乳。为保持这种恒定浓度，可使用连续静脉内递药装置。这种装置的示例是Deltec CADD-PLUS.TM.5400型静脉注射泵。
药物组合物可以是用于肌内和皮下给药的无菌注射水或油混悬液的形式。可按已知技术，用上述那些适宜的分散剂或湿润剂和悬浮剂配制该混悬液。无菌注射制剂也可以是在无毒肠胃外可接受的稀释剂或溶剂中制备的无菌注射溶液或混悬液，例如1，3-丁二醇中制备的溶液。此外，可方便地用无菌固定油作为溶 剂或悬浮介质。为此目的，可使用包括合成甘油单或二酯在内的任何调和固定油。此外，脂肪酸例如油酸也可以制备注射剂。
“施用”、“给予”和“处理”，当其应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当其应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
术语“药学上可接受的盐”或“可药用盐”是指本披露抗体-药物偶联物的盐，或本披露中所述的化合物的盐，这类盐用于哺乳动物体内时具有安全性和有效性，且具有应有的生物活性，本披露抗体药物偶联物至少含有一个氨基，因此可以与酸形成盐，可药用盐的非限制性示例包括：盐酸盐、氢溴酸盐、氢碘酸盐、硫酸盐、硫酸氢盐、柠檬酸盐、乙酸盐、琥珀酸盐、抗坏血酸盐、草酸盐、硝酸盐、梨酸盐、磷酸氢盐、磷酸二氢盐、水杨酸盐、柠檬酸氢盐、酒石酸盐、马来酸盐、富马酸盐、甲酸盐、苯甲酸盐、甲磺酸盐、乙磺酸盐、苯磺酸盐和对甲苯磺酸盐。
“治疗”意指给予受试者内用或外用治疗剂，例如包含本披露的任一种结合化合物的组合物，所述受试者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本披露的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
在以上说明书中提出了本披露一种或多种实施方案的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本披露，但是以下描述优选的方法和材料。通过说明书和权利要求书，本披露的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形 式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本披露所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入。提出以下实施例是为了更全面地说明本披露的优选实施方案。这些实施例不应以任何方式理解为限制本披露的范围，本披露的范围由权利要求书限定。
以下结合实施例用于进一步描述本发明，但这些实施例并非限制本发明的范围。
本披露实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
实施例1：HER3高表达细胞株构建
将pCDH-Her3慢病毒表达载体质粒与pVSV-G，pCMV-dR8.91慢病毒系统包装载体用Lipofectamine 3000转染试剂转染至病毒包装细胞293T中，收集含有病毒的培养基上清，过滤并进行超高速离心，使用浓缩后的病毒感染中国仓鼠卵巢细胞CHO-K1，经嘌呤霉素筛选两至三周，再进行FACS单细胞分选。
通过FACS检测慢病毒感染的CHO-K1细胞表面的HER3表达，挑选出HER3表达量高的单克隆细胞株。
将挑选出的单克隆细胞株扩大培养，储存以便后续实验。
HuMan ErbB3氨基酸序列(UniProtKB-P21860-1，AA Ser 20-Thr 643)
Figure PCTCN2021123733-appb-000023
HuMan ErbB3核苷酸序列
Figure PCTCN2021123733-appb-000024
实施例2：抗人HER3单克隆抗体产生
2.1阳性对照抗体制备
阳性对照抗体U3参照WO2007077028A2(第118页，U1-59)制备。其中U3的重链和轻链氨基酸序列如下：
U3重链：
Figure PCTCN2021123733-appb-000025
U3轻链：
Figure PCTCN2021123733-appb-000026
2.2本披露的抗体制备
利用全人源天然噬菌体抗体库和抗原Biotinylated HuMan ErbB3(购买于北京百普赛斯生物科技股份有限公司，货号：ER3-H82E6)，经过淘选后，采用ELISA方法进行噬菌体检测，获得阳性克隆。将阳性克隆测序，获得序列后，将阳性克隆序列插入蛋白表达载体Phr-IgG中，并通过HEK293和Expi-CHO-S细胞进行表达。纯化后进行FACS和内吞活性验证实验，得到全人源抗体分子HER3-29。全人源抗体分子HER3-29的抗体的恒定区：
人IgG1的重链恒定区：
Figure PCTCN2021123733-appb-000027
人源κ轻链恒定区：
Figure PCTCN2021123733-appb-000028
全人源抗体分子HER3-29的重链可变区和轻链可变区序列如下：
HER3-29重链可变区：
Figure PCTCN2021123733-appb-000029
HER3-29轻链可变区：
Figure PCTCN2021123733-appb-000030
不同编号规则获得的CDR序列如下：
表1、Chothia编号规则获得的CDR序列
抗体 HER3-29 序列号
重链CDR1 GFTFDDY SEQ ID NO:9
重链CDR2 SWNSGS SEQ ID NO:10
重链CDR3 EGLPGLDY SEQ ID NO:11
轻链CDR1 RASQHVGTYLN SEQ ID NO:12
轻链CDR2 GAANLQS SEQ ID NO:13
轻链CDR3 QQSYNTPPFS SEQ ID NO:14
表2、IMGT编号规则获得的CDR序列
抗体 HER3-29 序列号
重链CDR1 GFTFDDYA SEQ ID NO:15
重链CDR2 ISWNSGSI SEQ ID NO:16
重链CDR3 AKEGLPGLDY SEQ ID NO:17
轻链CDR1 QHVGTY SEQ ID NO:18
轻链CDR2 GAA SEQ ID NO:19
轻链CDR3 QQSYNTPPFS SEQ ID NO:20
表3、Kabat编号规则获得的CDR序列
抗体 HER3-29 序列号
重链CDR1 DYAMH SEQ ID NO:21
重链CDR2 GISWNSGSIGYADSVKG SEQ ID NO:22
重链CDR3 EGLPGLDY SEQ ID NO:23
轻链CDR1 RASQHVGTYLN SEQ ID NO:24
轻链CDR2 GAANLQS SEQ ID NO:25
轻链CDR3 QQSYNTPPFS SEQ ID NO:26
全人源抗体分子HER3-29的重链序列和轻链序列如下：
HER3-29重链：
Figure PCTCN2021123733-appb-000031
HER3-29轻链：
Figure PCTCN2021123733-appb-000032
实施例3：ADC的制备
ADC的DAR值测定
本披露的ADC的DAR值计算方法采用了RP-HPLC(反相高效液相色谱)，具体如下：
1、测定方法：
裸抗和供测试ADC样品(浓度为1mg/mL)，加入4μL DDT(sigma)还原，37℃水浴1小时，结束后取出到内插管中。使用高效液相色谱仪Agilent 1200进行检测，色谱柱选用Agilent PLRP-S 1000A 8μm 4.6*250mm，柱温：80℃；DAD检测器波长280nm；流速：1mL/min；进样量为：40μL；之后通过样品与裸抗的谱图比对，区分出轻重链的位置，然后对检测样品的谱图进行积分，计算出 DAR值。
2、溶液配制
1)0.25M DTT溶液：
配制示例：取DTT 5.78mg，加入150μL纯化水充分溶解后，配得0.25M DTT溶液，-20℃保存。
2)流动相A(0.1％TFA水溶液)：
配制示例：量筒量取1000mL纯化水，加入1mL TFA(sigma)，充分混匀后使用，2-8℃保存14天。
3)流动相B(0.1％TFA乙腈溶液)：
配制示例：量筒量取1000mL乙腈，加入1mLTFA，充分混匀后使用，2-8℃保存14天。
3、数据分析
通过样品与裸抗的谱图比对，区分出轻重链的位置，然后对检测样品的谱图进行积分，计算出DAR值。
计算公式如下：
名称 连接药物数
LC 0
LC+1 2
HC 0
HC+1 2
HC+2 4
HC+3 6
LC峰面积总和＝LC峰面积+LC+1峰面积
HC峰面积总和＝HC峰面积+HC+1峰面积+HC+2峰面积+HC+3峰面积
LC DAR＝Σ(连接药物数*峰面积百分比)/LC峰面积总和
HC DAR＝Σ(连接药物数*峰面积百分比)/HC峰面积总和
DAR＝LC DAR+HC DAR
药物
本披露偶联物的药物部分可以是任意适宜的药物。特别适宜的药物描述于例如PCT公开号WO2020063676A1(通过援引完整收入本文)。本披露的化合物9A(即WO2020063676A1实施例9的化合物9-A)是N-((2R,10S)-10-苄基-2-环丙基-1-(((1S,9S)-9-乙基-5-氟-9-羟基-4-甲基-10,13-二氧代-2,3,9,10,13,15-六氢-1H,12H-苯并[de]吡喃并[3',4':6,7]吲哚嗪并[1,2-b]喹啉-1-基)氨基)-1,6,9,12,15-五氧代-3-氧杂-5,8,11,14-四氮杂十六-16-基)-6-(2,5-二氧代-2,5-二氢-1H-吡咯-1-基) 己酰胺，其具有如下的结构：
Figure PCTCN2021123733-appb-000033
本披露用以下方法，通过调节反应参数，制备如ADC通式(HER3-29-9A)所示的抗体-药物偶联物。
Figure PCTCN2021123733-appb-000034
实施例3-1 ADC-1
在37℃条件下，向抗体HER3-29的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，11.8mL，797nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，208.2μL，2.082μMol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9A(参考WO2020063676，实施例9的化合物9-A制备所得，该申请的全部内容引入本披露)(8.6mg，8.006μmol)溶解于500μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到偶联物HER3-29-9A的示例性产物ADC-1的PBS缓冲液(4.02mg/mL，27.9mL)，于4℃储存。RP-HPLC计算平均值：DAR＝4.19。
实施例3-2 ADC-2
在37℃条件下，向抗体HER3-29的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，11.8mL，797nmol)加入配置好的三(2-羧乙 基)膦(TCEP)的水溶液(10mM，128μL，1.281μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9A(6.88mg，6.405μmol)溶解于400μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到偶联物HER3-29-9A的示例性产物ADC-2的PBS缓冲液(4.24mg/mL，27.2mL)，于4℃储存。RP-HPLC计算平均值：DAR＝2.91。
实施例3-3 ADC-3
在37℃条件下，向抗体HER3-29的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，3.1mL，209nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，111μL，1.111μMol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9A(3.37mg，3.137μmol)溶解于120μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到偶联物HER3-29-9A的示例性产物ADC-3的PBS缓冲液(1.48mg/mL，12.8mL)，于4℃储存。RP-HPLC计算平均值：DAR＝7.27。
实施例3-4 ADC-4
Figure PCTCN2021123733-appb-000035
在37℃条件下，向抗体U3的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，3.1mL，209nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，111μL，1.111μmol)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物9A(3.37mg，3.137μmol)溶解于120μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到偶联物U3-9A的示例性产物ADC-4的PBS缓冲液(1.48mg/mL，12.8mL)，于4℃储存。RP-HPLC计算平均值：DAR＝6.76。
实施例3-5 ADC-5
Figure PCTCN2021123733-appb-000036
参考WO2015155998A1说明书第156页的实施例12，制备U3-1402，做为阳性对照。在37℃条件下，向抗体U3的PBS缓冲水溶液(pH＝6.5的0.05M的PBS缓冲水溶液；10.0mg/mL，3.1mL，236nmol)加入配置好的三(2-羧乙基)膦(TCEP)的水溶液(10mM，130μL)，置于水浴振荡器，于37℃下振荡反应3小时，停止反应。将反应液用水浴降温至25℃。
将化合物1402(3.67mg，3.54μmol)溶解于180μL DMSO中，加入到上述反应液中，置于水浴振荡器，于25℃下振荡反应3小时，停止反应。将反应液用Sephadex G25凝胶柱脱盐纯化(洗脱相：pH为6.5的0.05M的PBS缓冲水溶液，含0.001M的EDTA)，得到偶联物U3-1402的示例性产物ADC-5的PBS缓冲液(1.53mg/mL，15.4mL)，于4℃储存。RP-HPLC计算平均值：DAR＝6.97。
测试例
测试例1：抗体与游离HER3蛋白的结合实验
用pH 7.4的PBS(源培生物，B320)缓冲液将HER3蛋白稀释至1μg/mL，以100μL/孔的体积加入96孔酶标板中，4℃过夜孵育。弃去液体后，每孔加入300μL用PBS稀释的5％脱脂牛奶(BD，232100)进行封闭，37℃孵育2小时。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.1％tween-20)洗板3次后，每孔加入100μL梯度稀释的抗体溶液，于37℃孵育1小时。孵育结束后用PBST洗板3次，每孔加入100μL小鼠抗人(Mouse Anti-HuMan)IgG(H+L)(Jackson ImmunoResearch，209-035-088，1:8000稀释)，37℃孵育1小时。用PBST洗板3次后，每孔加入100μLTMB显色底物(KPL，5120-0077)，室温孵育10-15分钟，每孔加入50μL 1M H 2SO 4终止反应，用酶标仪读取在450nm处的吸收值，用软件拟合出抗体与抗原的结合曲线，见图1。计算出EC 50值，结果见表4。
表4、抗体与HER3蛋白的结合活性
抗体 HER3-29 U3
EC 50(nM) 0.14 0.56
结论：本披露抗体HER3-29与HER3蛋白的结合活性优于对照抗体U3。
测试例2：抗体与表达HER3的细胞的结合实验
将MCF7细胞(ATCC，HTB-22)用FACS缓冲液(2％胎牛血清(Gibco，10099141)pH7.4PBS(Sigma，P4417-100TAB))制备成1×10 6细胞/mL的细胞悬液，100μL/孔加入96孔圆底板中。离心去除上清后加入50μL/孔用FACS缓冲液稀释的不同浓度待测抗体，放于4℃冰箱中避光孵育1小时。以FACS缓冲液300g离心洗涤3次后，加入工作浓度的Alexa Fluor 488山羊抗人(Goat anti-HuMan)IgG(H+L)(invitrogen，A-11013)，放于4℃冰箱中避光孵育40分钟。以FACS缓冲液300g离心洗涤3次后，在BD FACSCantoII流式细胞仪上检测几何平均数荧光强度，见图2。EC 50值见表5。
表5、抗体细胞水平结合活性
抗体 HER3-29 U3
EC 50(nM) 0.057 0.249
结论：本披露抗体HER3-29与表达HER3蛋白的细胞的结合活性优于对照抗体U3。
测试例3：DT3C抗体内吞实验
本实验的目的是根据DT3C蛋白进入细胞后，活化的白喉毒素(DT)对细胞进行杀伤，间接反映了HER3抗体的内吞情况。根据IC 50和Imax来评价抗体的体外内吞活性。
DT3C是重组表达的融合蛋白，由白喉毒素的Fragment A(仅毒素部分)和G群链球菌的3C片段(IgG结合部分)融合而成。该蛋白能够与抗体的Fc结构高度亲和，在抗体发生内吞时一同进入细胞。在胞内弗林蛋白酶的作用下，释放出具有毒性的DT。DT能够抑制EF2-ADP核糖基化的活性，阻断蛋白翻译过程，最终导致细胞死亡。而未进入细胞的DT3C则不具有杀伤细胞的活性。根据细胞杀伤情况来评价抗体的内吞活性。
用含20％low IgG FBS的新鲜细胞培养基，制取重组表达HER3的CHOK1细胞悬液，细胞密度为2×10 4细胞/mL，以50μL/孔加入细胞培养板中，5％二氧化碳37℃16小时培养。
用无血清培养基配制成4×浓度的DT3C，0.22μm小滤器过滤成无菌溶液。用无血清培养基配制4×浓度的抗体，将80μL DT3C(400nM)和80μL抗体(66nM)按照1:1的体积混匀，室温下静置孵育30分钟。取50μL稀释好的抗体加入50μL的细胞中，培养箱中孵育三天。每孔加入50μL CTG(CellTiter-Glo TM试剂,G7573)，室温下避光孵育10分钟，Victor3上读取化学发光，结果见图3和表6。
表6、抗体的内吞活性
抗体 HER3-29 U3
Imax 48％ 19％
IC 50(nM) 0.51 2.81
结论：本披露抗体HER3-29的细胞内吞活性优于对照抗体U3。
测试例4：pHrodo抗体内吞实验
本实验的目的是根据染料被内化后荧光信号变化来反映HER3抗体的内吞情况。根据荧光信号的强弱来评价抗体的体外内吞活性。
pH敏感的pHrodo iFL染料偶联结合的Fab片段，可直接结合至HER3抗体的Fc区，且不影响抗体的抗原识别。pHrodo iFL染料在中性pH值时几乎不发荧光，在HER3抗体发生内吞时，染料同时被内化，随着pH降低，荧光信号会逐渐增强。根据荧光信号的增强情况来评价抗体的内吞活性。
HER3/CHOK1细胞用DMEM/F12+10％FBS+10μg/mL嘌呤霉素培养，实验第一天用含新鲜细胞培养基制取细胞悬液，密度为2×10 5细胞/mL，以100μL/孔加入96孔细胞培养板中，5％二氧化碳37℃培养24小时。
将板中的细胞液吸出50μL，每孔加入50μL的抗体和pHrodo染料混合液，每个抗体样品都设置两个复孔。并设置单加染料组和同型IgG1对照组。
培养箱培养24小时后，吸出培养基，每孔加入50μL胰酶，消化2分钟，50μL新鲜培养基终止消化。用排枪将同一样品复孔的细胞转到圆底板同一个孔中。1500rpm，离心2分钟，弃去培养液，用FACS Buffer(PBS+2.5％FBS)洗一次细胞，1500rpm，离心2分钟。加入200μL的FACS Buffer(PBS+2.5％FBS)，重悬细胞，用流式细胞仪检测FITC信号。用Flowjo 7.6分析数据，结果见图4和表7。
表7、抗体内吞活性
抗体 HER3-29 U3
FITC信号 373 267
结论：本披露抗体HER3-29的细胞内吞活性优于对照抗体U3。
测试例5：ADC分子细胞活性实验
本实验的目的是检测ADC样品对细胞杀伤作用，根据IC 50和Imax来评价Her3-ADC的体外活性。
MCF7细胞(人乳腺癌细胞)，SW620细胞(人结肠癌细胞，南京科佰，CBP60036)，WiDr细胞(人大肠癌细胞)用胰酶消化，新鲜培养基中和，1000rpm离心后用培养液重悬，计数后将细胞悬液密度调整为500细胞/孔，加入到96孔细胞培养板中，第11列不铺细胞只加入135μL的培养基，5％二氧化碳37℃16 小时培养。
ADC样品用PBS稀释成15μM(10×浓度)。以此为首浓度，用PBS五倍稀释，共8个浓度。每孔加入15μL的10×浓度溶液。5％二氧化碳37℃培养6天。
每孔加入70μL CTG，室温下避光孵育10分钟，将白色底膜贴在细胞培养板底部，置于Victor3上读取化学发。本实验的数据使用了数据处理软件GraphPad prism5.0，见表8。
表8、HER3-29-9A不同DAR值体外杀伤实验
Figure PCTCN2021123733-appb-000037
结论：本披露ADC样品ADC-1，ADC-2，ADC-3对细胞的杀伤活性优于阳性对照ADC-4和ADC-5。
体内活性生物学评价
测试例6：HER3高表达CDX模型体内药效评价
将SW620细胞(5×10 6个/只)接种于Balb/c裸鼠右肋部皮下，7天后分9组，8只/组，共9组。平均分组体积134.75mm 3。ADC腹腔注射，共给药3次，每隔5天给药一次。每只按体重注射0.1mL/10g。每周测量2次瘤体积和体重，记录数据。使用Excel统计软件记录数据：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT(每组动物数)计算；采用GraphPad Prism软件作图，采用Two-way ANOVA或One-way ANOVA对数据进行统计学分析。
肿瘤体积(V)计算公式为：V＝1/2×L 长×L 短 2
相对肿瘤增殖率T/C(％)＝(T-T 0)/(C-C 0)×100％，其中T、C为实验结束时治疗组和对照组的肿瘤体积；T 0、C 0为实验开始时的肿瘤体积。
抑瘤率TGI(％)＝1-T/C(％)。结果见图5及表9。
表9、ADC对荷瘤裸鼠SW620移植瘤的疗效
Figure PCTCN2021123733-appb-000038
Figure PCTCN2021123733-appb-000039
结论：本披露ADC-1，ADC-2对荷瘤裸鼠SW620移植瘤的疗效优于阳性对照ADC-5。

Claims (19)

  1. 一种分离的抗HER3抗体，其中所述抗HER3抗体具有以下特征中的一种或多种：
    a.所述的抗HER3抗体以小于0.5nM的表观亲和力EC 50与HER3蛋白结合，所述的表观亲和力EC 50是通过ELISA方法测定的；
    b.所述的抗HER3抗体以小于0.2nM的表观亲和力EC 50与MCF7细胞表达的HER3蛋白结合，所述的表观亲和力EC 50是通过FACS方法测定的；
    c.所述的抗HER3抗体能被表达人HER3的细胞内吞；优选的，当采用DT3C抗体内吞实验测定所述的抗HER3抗体时，其IC 50小于2nM；
    d.所述的抗HER3抗体能被表达人HER3的细胞内吞；优选的，当采用pHrodo抗体内吞实验测定所述的抗HER3抗体时，其FITC信号大于300。
  2. 根据权利要求1所述的分离的抗HER3抗体，其中所述抗HER3抗体包含1)SEQ ID NO:7所示的重链可变区中所包含的HCDR1，HCDR2和HCDR3；和2)SEQ ID NO:8所示的轻链可变区中所包含的LCDR1，LCDR2和LCDR3；
    优选的，其中所述抗HER3抗体包含重链可变区和轻链可变区，其中：
    a.所述重链可变区包含分别如SEQ ID NO:9，SEQ ID NO:10和SEQ ID NO:11所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:12，SEQ ID NO:13和SEQ ID NO:14所示的LCDR1，LCDR2和LCDR3；
    其中所述的CDR区按Chothia编号规则确定；或
    b.所述重链可变区包含分别如SEQ ID NO:15，SEQ ID NO:16和SEQ ID NO:17所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:18，SEQ ID NO:19和SEQ ID NO:20所示的LCDR1，LCDR2和LCDR3；
    其中所述的CDR区按IMGT编号规则确定；或
    c.所述重链可变区包含分别如SEQ ID NO:21，SEQ ID NO:22和SEQ ID NO:23所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:24，SEQ ID NO:25和SEQ ID NO:26所示的LCDR1，LCDR2和LCDR3；
    其中所述的CDR区按Kabat编号规则确定。
  3. 一种分离的抗HER3抗体，其中所述抗HER3抗体包含重链可变区和轻链可变区，其中：
    a.所述重链可变区包含分别如SEQ ID NO:9，SEQ ID NO:10和SEQ ID NO:11所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:12，SEQ ID NO:13和SEQ ID NO:14所示的LCDR1，LCDR2和LCDR3；
    如上所述的CDR区按Chothia编号规则确定；或
    b.所述重链可变区包含分别如SEQ ID NO:15，SEQ ID NO:16和SEQ ID NO: 17所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:18，SEQ ID NO:19和SEQ ID NO:20所示的LCDR1，LCDR2和LCDR3；
    如上所述的CDR区按IMGT编号规则确定；或
    c.所述重链可变区包含分别如SEQ ID NO:21，SEQ ID NO:22和SEQ ID NO:23所示的HCDR1，HCDR2和HCDR3；所述轻链可变区包含分别如SEQ ID NO:24，SEQ ID NO:25和SEQ ID NO:26所示的LCDR1，LCDR2和LCDR3；
    如上所述的CDR区按Kabat编号规则确定。
  4. 根据权利要求1至3任一项所述的分离的抗HER3抗体，其中所述抗HER3抗体是人抗体或抗原结合片段。
  5. 根据权利要求1至4任一项所述的分离的抗HER3抗体，其包含重链可变区和轻链可变区，其中：
    所述重链可变区的氨基酸序列与SEQ ID NO:7具有至少90％的序列同一性，和/或所述轻链可变区的氨基酸序列与SEQ ID NO:8具有至少90％的序列同一性；
    优选地，所述的抗HER3抗体包含重链可变区和轻链可变区，其中：
    所述重链可变区的氨基酸序列如SEQ ID NO:7所示；和所述轻链可变区的氨基酸序列如SEQ ID NO:8所示。
  6. 根据权利要求1至5中任一项所述的分离的抗HER3抗体，其包含：
    与SEQ ID NO:27具有至少85％序列同一性的重链，和/或与SEQ ID NO:28具有至少85％序列同一性的轻链；
    优选地，所述的抗HER3抗体包含：
    如SEQ ID NO:27所示的重链和如SEQ ID NO:28所示的轻链。
  7. 根据权利要求1至6中任一项所述的分离的抗HER3抗体，其中所述抗HER3抗体具有以下特征中的一种或多种：
    a.所述的抗HER3抗体以小于0.5nM的表观亲和力EC 50与HER3蛋白结合，所述的表观亲和力EC 50是通过ELISA方法测定的；
    b.所述的抗HER3抗体以小于0.2nM的表观亲和力EC 50与MCF7细胞表达的HER3蛋白结合，所述的表观亲和力EC 50是通过FACS方法测定的；
    c.所述的抗HER3抗体能被表达人HER3的细胞内吞；优选的，当采用DT3C抗体内吞实验测定所述的抗HER3抗体时，其IC 50小于2nM；
    d.所述的抗HER3抗体能被表达人HER3的细胞内吞；优选的，当采用pHrodo抗体内吞实验测定所述的抗HER3抗体时，其FITC信号大于300。
  8. 一种分离的抗HER3抗体，其中所述抗体与权利要求1至7中任一项所述 的抗HER3抗体竞争性结合人HER3。
  9. 一种核酸分子，其编码权利要求1至8中任一项所述的分离的抗HER3抗体。
  10. 一种宿主细胞，其包含如权利要求9所述的核酸分子。
  11. 一种免疫偶联物，其包含：权利要求1至8中任一项所述的分离的抗HER3抗体和效应分子，其中，所述效应分子偶联至所述抗HER3抗体；
    优选地，所述效应分子选自抗肿瘤剂、免疫调节剂、生物反应修饰剂、凝集素、细胞毒性药物、发色团、荧光团、化学发光化合物、酶、金属离子，以及其任何组合。
  12. 一种用于免疫检测或测定HER3的方法，所述方法包括使权利要求1至8中任一项所述的分离的抗HER3抗体接触受试者或来自受试者的样品的步骤。
  13. 一种通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐：
    Figure PCTCN2021123733-appb-100001
    其中，
    Pc为如权利要求1至8中任一项所述的分离的抗HER3抗体；
    m为0至4的整数；
    n为1至10，n是小数或整数；
    R 1选自卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；R 2选自氢原子、卤素、卤代烷基、氘代烷基、环烷基、环烷基烷基、烷氧基烷基、杂环基、芳基和杂芳基；或者，R 1和R 2与其相连接的碳原子一起形成环烷基或杂环基；
    W选自C 1-8烷基、C 1-8烷基-C 3-6环烷基和1至8个链原子的直链杂烷基，所述1至8个链原子的直链杂烷基包含1至3个选自N、O和S的杂原子，其中所述的C 1-8烷基、C 1-8烷基-C 3-6环烷基和1至8个链原子的直链杂烷基各自独立地任 选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基的一个或多个取代基所取代；
    L 2选自-NR 4(CH 2CH 2O)p 1CH 2CH 2C(O)-、-NR 4(CH 2CH 2O)p 1CH 2C(O)-、-S(CH 2)p 1C(O)-和化学键，其中p 1为1至20的整数；
    L 3为由2至7个氨基酸残基构成的肽残基，其中所述的氨基酸残基选自苯丙氨酸、甘氨酸、缬氨酸、赖氨酸、瓜氨酸、丝氨酸、谷氨酸和天冬氨酸中的氨基酸形成的氨基酸残基，并任选进一步被选自卤素、羟基、氰基、氨基、烷基、氯代烷基、氘代烷基、烷氧基和环烷基中的一个或多个取代基所取代；
    R 5选自氢原子、烷基、卤代烷基、氘代烷基和羟烷基；
    R 6和R 7相同或不同，且各自独立地选自氢原子、卤素、烷基、卤代烷基、氘代烷基和羟烷基。
  14. 根据权利要求13所述的通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐，所述抗体-药物偶联物为：
    Figure PCTCN2021123733-appb-100002
    其中：
    n为1至8，n是小数或整数；优选的n为3至8，n是小数或整数；
    HER3-29为抗HER3抗体，其包含如SEQ ID NO：27所示的重链和如SEQ ID NO：28所示的轻链。
  15. 一种制备如权利要求13所述的通式(Pc-L a-Y-D)所示的抗体-药物偶联物或其药学上可接受的盐的方法，其包括以下步骤：
    Figure PCTCN2021123733-appb-100003
    Pc’与通式(L a-Y-D)所示的化合物进行偶联反应，得到通式(Pc-L a-Y-D)所示的化合物；
    其中：
    Pc’为Pc经还原后所得，Pc、n、m、W、L 2、L 3、R 1、R 2、R 5、R 6和R 7如权利要求13中所定义。
  16. 一种药物组合物，其包含根据权利要求1至8中任一项所述的分离的抗HER3抗体，或权利要求9所述的核酸分子，或权利要求13或14所述的抗体-药物偶联物或其药学上可接受的盐；以及一种或多种药学上可接受的赋形剂、稀释剂或载体。
  17. 根据权利要求1至8中任一项所述的分离的抗HER3抗体，或权利要求9所述的核酸分子，或权利要求13或14所述的抗体-药物偶联物或其药学上可接受的盐，或根据权利要求16所述的药物组合物在制备用于治疗HER3介导的疾病或病症的药物中的用途。
  18. 根据权利要求1至8中任一项所述的分离的抗HER3抗体，或权利要求9所述的核酸分子，或权利要求13或14所述的抗体-药物偶联物或其药学上可接受的盐，或根据权利要求16所述的药物组合物在制备用于治疗和/或预防肿瘤的药物 中的用途；
    优选的，所述肿瘤选自乳腺癌、非小细胞肺癌、胃癌、卵巢癌、前列腺癌、膀胱癌、结肠直肠癌、头颈部鳞状细胞癌和黑色素瘤。
  19. 一种试剂盒，其包含根据权利要求1至8中任一项所述的分离的抗HER3抗体，或权利要求9所述的核酸分子，或权利要求13或14所述的抗体-药物偶联物或其药学上可接受的盐，或根据权利要求16所述的药物组合物。
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WO2023208216A1 (en) * 2022-04-29 2023-11-02 Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. Antibody-drug conjugates and preparation methods and use thereof
WO2023237050A1 (en) * 2022-06-09 2023-12-14 Beigene, Ltd. Antibody drug conjugates
WO2024083166A1 (zh) * 2022-10-19 2024-04-25 普众发现医药科技(上海)有限公司 抗体-药物偶联物及其制备方法和在抗肿瘤中的用途
WO2024088388A1 (en) * 2022-10-28 2024-05-02 Hansoh Bio Llc Ligand-cytotoxicity drug conjugates and pharmaceutical uses thereof
WO2024109840A1 (zh) * 2022-11-22 2024-05-30 康诺亚生物医药科技（成都）有限公司 稠环类化合物及其偶联物和用途
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WO2024131835A1 (en) * 2022-12-20 2024-06-27 Hansoh Bio Llc Ligand-cytotoxicity drug conjugates and pharmaceutical uses thereof

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