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WO2025016453A1 - 一种双特异性抗体、其药物偶联物及其用途 - Google Patents

一种双特异性抗体、其药物偶联物及其用途 Download PDF

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Publication number
WO2025016453A1
WO2025016453A1 PCT/CN2024/106435 CN2024106435W WO2025016453A1 WO 2025016453 A1 WO2025016453 A1 WO 2025016453A1 CN 2024106435 W CN2024106435 W CN 2024106435W WO 2025016453 A1 WO2025016453 A1 WO 2025016453A1
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seq
cancer
bispecific antibody
amino acid
drug conjugate
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PCT/CN2024/106435
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English (en)
French (fr)
Inventor
周蕴华
花海清
杨俊杰
朱忠远
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映恩生物制药（苏州）有限公司
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Publication of WO2025016453A1 publication Critical patent/WO2025016453A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present invention relates to the field of biotechnology, and specifically to a bispecific antibody comprising an EGFR binding domain and a HER3 binding domain, a drug conjugate thereof, and uses thereof.
  • Epidermal growth factor receptor is a huge transmembrane glycoprotein with a molecular weight of about 170KDa, and is a member of the ErbB receptor family.
  • the EGFR receptor itself is a tyrosine kinase that can form dimers when bound to ligands such as EGF, TNF-a, etc., and activate downstream signals (such as MAPK, PI3K, Stat, etc.) by transmitting phosphorylation, thereby maintaining cell growth and promoting cell division and proliferation.
  • ligands such as EGF, TNF-a, etc.
  • downstream signals such as MAPK, PI3K, Stat, etc.
  • EGFR Due to the conservatism of the ErbB family receptors, EGFR can also form heterodimers with other family proteins (such as HER2, HER3, HER4), thereby more widely regulating cell growth.
  • HER3 is a member of the ErbB family and plays a key role in cell proliferation, tumor metastasis and drug resistance. Although drugs targeting EGFR and HER3 have shown great clinical benefits in alleviating a variety of cancers, previous efforts to develop anti-HER3 antibodies for cancer treatment have repeatedly failed.
  • Antibody drug conjugates consist of three parts: antibodies or their antigen-binding fragments (targets), linkers, and small molecule drugs.
  • Antibodies or their antigen-binding fragments are conjugated to small molecule drugs with biological activity, such as cytotoxicity, such as cytotoxins, via cleavable or non-cleavable linkers, making full use of the specificity of antibodies or their antigen-binding fragments targeting cells of interest (targeted cells) or binding to highly expressed antigens and the high efficiency of small molecule drugs, reducing or avoiding toxic side effects on non-targeted cells.
  • cytotoxicity such as cytotoxins
  • the technical problem to be solved by the present invention is to overcome the defect that there are few bispecific antibody drug conjugates targeting EGFR and HER3 in the prior art, and to provide a bispecific antibody, its drug conjugate and its use.
  • the bispecific antibody drug conjugate of the present invention has good endocytosis effect, proliferation inhibition activity, tumor growth inhibition activity and good in vivo safety.
  • the present invention mainly solves the above technical problems through the following technical means.
  • the present invention provides a bispecific antibody comprising an EGFR binding domain and a HER3 binding domain, wherein the EGFR binding domain comprises a heavy chain variable region VH1 and a light chain variable region VL1, and the HER3 binding domain comprises a
  • the structural domain comprises a heavy chain variable region VH2 and a light chain variable region VL2; wherein the amino acid sequences of H1CDR1, H1CDR2 and H1CDR3 contained in the VH1 are shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; the amino acid sequences of L1CDR1, L1CDR2 and L1CDR3 contained in the VL1 are shown as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; the amino acid sequences of H2CDR1, H2CDR2 and H2CDR3 contained in the VH2 are shown as SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9,
  • the amino acid sequence of the H2CDR2 is shown in SEQ ID NO:77 or 78.
  • the amino acid sequences of the framework regions H1FR1, H1FR2, H1FR3 and H1FR4 comprised in the VH1 are shown in SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, respectively, or are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • amino acid sequences of the framework regions L1FR1, L1FR2, L1FR3 and L1FR4 comprised in the VL1 are shown in SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20, respectively, or are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the amino acid sequence of the framework region H2FR1 contained in the VH2 is as shown in SEQ ID NO:21 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, for example, having an E16D mutation in SEQ ID NO:21;
  • the amino acid sequences of the framework regions H2FR2 and H2FR4 are as shown in SEQ ID NO:22 and SEQ ID NO: 24 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the amino acid sequence of the framework region H2FR3 is as shown in SEQ ID NO:23 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, for example having an S18D mutation in SEQ ID NO:23;
  • the amino acid sequence of the framework region L2FR1 comprised in the VL2 is as shown in SEQ ID NO:25 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, for example, it has S9D and V15L mutations, or S7E mutation on SEQ ID NO:25; the amino acid sequences of the framework regions L2FR2, L2FR3 and L2FR4 are as shown in SEQ ID NO:18, SEQ ID NO:26 and SEQ ID NO:27, respectively, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
  • the amino acid sequence of VH1 is shown as SEQ ID NO:28
  • the amino acid sequence of VL1 is shown as SEQ ID NO:29
  • the amino acid sequence of VH2 is shown as SEQ ID NO:30, SEQ ID NO:79 or SEQ ID NO:80
  • the amino acid sequence of VL2 is shown as SEQ ID NO:31, SEQ ID NO:81 or SEQ ID NO:82.
  • the amino acid sequences of VH1, VL1, VH2 and VL2 of the bispecific antibody are as shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, respectively; or, as shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:79 and SEQ ID NO:81, respectively; or, as shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:79 and SEQ ID NO:31, respectively; or, as shown in SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:80 and SEQ ID NO:82, respectively.
  • the EGFR binding domain and the HER3 binding domain further include a light chain constant region and a heavy chain constant region, respectively, the EGFR binding domain comprises a light chain constant region CL1 and a heavy chain constant region HC1, and the HER3 binding domain comprises a light chain constant region CL2 and a heavy chain constant region HC3.
  • the domain comprises a light chain constant region CL2 and a heavy chain constant region HC2; wherein the amino acid sequences of CL1 and CL2 are as shown in SEQ ID NO:32 or SEQ ID NO:33, respectively, or are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the amino acid sequences of CL1 and CL2 are not identical sequences; and/or, HC1 comprises C1H1 and Fc1, and HC2 comprises C2H1 and Fc2; wherein the amino acid sequences of C1H1 and C2H1 are as shown in SEQ ID NO:34 or SEQ ID NO:35, or are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the amino acid sequences of C1H1 and C2H1 are not identical sequences; the amino acid sequences of Fc1 and Fc2 are as shown in SEQ ID NO:
  • the amino acid sequences of CL1 and CL2 are as shown in SEQ ID NO:32 or SEQ ID NO:33, respectively, the amino acid sequences of C1H1 and C2H1 are as shown in SEQ ID NO:34 or SEQ ID NO:35, and the amino acid sequences of Fc1 and Fc2 are variant sequences of the amino acid sequence shown in SEQ ID NO:36, for example, having T146W, or S134C and T146W, or T146S, L148A and Y187V, or Y349C, T366S, L368A and Y407V mutations on SEQ ID NO:36.
  • Fc1 and Fc2 are connected via a disulfide bond and a Knob into Hole structure in the hinge region; the Fc1 is knob-Fc and the Fc2 is hole-Fc, or Fc2 is knob-Fc and Fc1 is hole-Fc.
  • C1H1 and Fc1, C2H1 and Fc2 are connected by a hinge region, wherein the amino acid sequence of the hinge region is as shown in SEQ ID NO:89.
  • the EGFR binding domain comprises a light chain constant region CL1 and a heavy chain constant region HC1
  • the HER3 binding domain comprises a heavy chain constant region HC2
  • the amino acid sequence of CL1 is as shown in SEQ ID NO:32 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto
  • HC1 comprises C1H1 and Fc1
  • HC2 comprises F c2
  • the amino acid sequence of C1H1 is as shown in SEQ ID NO:34 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto
  • the amino acid sequences of Fc1 and Fc2 are variant sequences of the amino acid sequence shown in SEQ ID NO:36 or are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical there
  • amino acid sequence of CL1 is shown in SEQ ID NO:32
  • amino acid sequence of C1H1 is shown in SEQ ID NO:34
  • amino acid sequences of Fc1 and Fc2 are shown in SEQ ID NO:93 and 94, respectively;
  • Fc1 and Fc2 are connected via a disulfide bond and a Knob into Hole structure in the hinge region, and the Fc1 is knob-Fc and the Fc2 is hole-Fc, or Fc2 is knob-Fc and Fc1 is hole-Fc;
  • C1H1 and Fc1 are connected by a hinge region as shown in an amino acid sequence such as SEQ ID NO:89; VL2 and VH2 are connected by a hinge region as shown in an amino acid sequence such as SEQ ID NO:95; and VH2 and Fc2 are connected by a hinge region as shown in an amino acid sequence such as SEQ ID NO:96.
  • the bispecific antibody comprises a heavy chain H1, a light chain L1 and a heavy chain H2, the amino acid sequence of H1 being as shown in SEQ ID NO: 37 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and/or the amino acid sequence of L1 being as shown in SEQ ID NO: 38 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.
  • amino acid sequence of H2 is as shown in SEQ ID NO:90 or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;
  • the bispecific antibody comprises a heavy chain H1, a light chain L1 and a heavy chain H2, and the amino acid sequences of the heavy chain H1, the light chain L1 and the heavy chain H2 are shown in SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 90, respectively, which is DBXT005-01.
  • the heavy chain H2 is an Fc+scFv structure.
  • the bispecific antibody comprises heavy chain H1, light chain L1, heavy chain H2 and light chain L2.
  • amino acid sequences of the heavy chain H1, light chain L1, heavy chain H2 and light chain L2 of the bispecific antibody are shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively, which are DBXT001-01; or,
  • SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:40, respectively, are DBXT001-02; or,
  • SEQ ID NO:43 As shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:44 and SEQ ID NO:40, respectively, which are DBXT001-03; or,
  • SEQ ID NO:45 As shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:46 and SEQ ID NO:40, respectively, which is DBXT001-04; or,
  • SEQ ID NO:47 As shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, respectively, are DBXT001-05; or,
  • SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:50, respectively, are DBXT001-06; or,
  • SEQ ID NO:53 As shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:54 and SEQ ID NO:50, respectively, are DBXT001-07; or,
  • SEQ ID NO:55 As shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:56 and SEQ ID NO:50, respectively, are DBXT001-08; or,
  • SEQ ID NO:37 As shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:57 and SEQ ID NO:58, respectively, are DBXT002-01; or,
  • SEQ ID NO:41 As shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:59 and SEQ ID NO:58, respectively, DBXT002-02; or,
  • SEQ ID NO:43 As shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:60 and SEQ ID NO:58, respectively, DBXT002-03; or,
  • SEQ ID NO:45 As shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:61 and SEQ ID NO:58, respectively, which is DBXT002-04; or,
  • SEQ ID NO:47 As shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:62 and SEQ ID NO:63, respectively, DBXT002-05; or,
  • SEQ ID NO:51 As shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:64 and SEQ ID NO:63 respectively, which is DBXT002-06; or,
  • SEQ ID NO: 53 As shown in SEQ ID NO: 53, SEQ ID NO: 48, SEQ ID NO: 65 and SEQ ID NO: 63, respectively, DBXT002-07; or,
  • SEQ ID NO: 55 As shown in SEQ ID NO: 55, SEQ ID NO: 48, SEQ ID NO: 66 and SEQ ID NO: 63, respectively, which is DBXT002-08; or,
  • SEQ ID NO:37 As shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:57 and SEQ ID NO:40, respectively, which are DBXT003-01; or,
  • SEQ ID NO:41 As shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:59 and SEQ ID NO:40, respectively, DBXT003-02; or,
  • SEQ ID NO:43 As shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:60 and SEQ ID NO:40, respectively, DBXT003-03; or,
  • SEQ ID NO:45 As shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:61 and SEQ ID NO:40, respectively, which are DBXT003-04; or,
  • SEQ ID NO:47 As shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:62 and SEQ ID NO:50, respectively, are DBXT003-05; or,
  • SEQ ID NO:51 As shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:64 and SEQ ID NO:50, respectively, DBXT003-06; or,
  • SEQ ID NO:53 As shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:65 and SEQ ID NO:50, respectively, are DBXT003-07; or,
  • SEQ ID NO:55 As shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:66 and SEQ ID NO:50, respectively, are DBXT003-08; or,
  • SEQ ID NO: 37 As shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 67 and SEQ ID NO: 68, respectively, which are DBXT004-01; or,
  • SEQ ID NO:41 As shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:69 and SEQ ID NO:68, respectively, DBXT004-02; or,
  • SEQ ID NO:43 As shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:70 and SEQ ID NO:68, respectively, which is DBXT004-03; or,
  • SEQ ID NO:45 As shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:71 and SEQ ID NO:68, respectively, which is DBXT004-04; or,
  • SEQ ID NO:47 As shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:72 and SEQ ID NO:73, respectively, are DBXT004-05; or,
  • SEQ ID NO:51 As shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:74 and SEQ ID NO:73, respectively, DBXT004-06; or,
  • SEQ ID NO:55 As shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:76 and SEQ ID NO:73 respectively, they are DBXT004-08.
  • the DBXT001 series includes DBXT001-01 to DBXT001-08
  • the DBXT002 series includes DBXT002-01 to DBXT002-08
  • the DBXT003 series includes DBXT003-01 to DBXT003-0108
  • the DBXT004 series includes DBXT004-01 to DBXT004-08.
  • the second aspect of the present invention provides an isolated nucleic acid, which encodes the bispecific antibody as described in the first aspect of the present invention.
  • the third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid as described in the second aspect of the present invention.
  • the fourth aspect of the present invention provides a transformant, which contains the nucleic acid as described in the second aspect of the present invention or the recombinant expression vector as described in the third aspect of the present invention in a host cell.
  • the host cell is a eukaryotic cell, preferably a mammalian cell, such as a CHO cell.
  • the fifth aspect of the present invention provides a method for preparing the bispecific antibody as described in the first aspect of the present invention, the method comprising culturing the transformant as described in the fourth aspect of the present invention to obtain the bispecific antibody.
  • HC1 is the heavy chain constant region of the first heavy chain
  • HC2 is the heavy chain constant region of the second heavy chain.
  • VH1, C1H1 and Fc1 are the VH, CH1 and Fc regions of the first heavy chain H1, respectively
  • CL1 and VL1 are the CL and VL regions of the first light chain L1, respectively
  • VH2, C2H1 and Fc2 are the VH, CH1 and Fc regions of the second heavy chain H2, respectively
  • CL2 and VL2 are the CL and VL regions of the second light chain L2, respectively.
  • L1FR1, L1FR2, L1FR3 and L1FR4 are the framework regions of the light chain variable region of the first light chain, respectively, H1FR1, H1FR2, H1FR3 and H1FR4 are the framework regions of the heavy chain variable region of the first heavy chain, respectively, L2FR1, L2FR2, L2FR3 and L2FR4 are the framework regions of the light chain variable region of the second light chain, respectively, and H2FR1, H2FR2, H2FR3 and H2FR4 are the framework regions of the heavy chain variable region of the second heavy chain, respectively.
  • Antibody sequences use the Kabat numbering system.
  • the present invention also provides a bispecific antibody-drug conjugate, its tautomer, enantiomer, diastereomer, or isomer mixture, or a pharmaceutically acceptable salt thereof, which comprises the following fragments: a bispecific antibody or an antigen-binding fragment thereof comprising an EGFR binding domain and a HER3 binding domain, a linker unit L, and a cytotoxic drug; wherein the bispecific antibody or the antigen-binding fragment thereof is as described in any one of the present invention.
  • the cytotoxic drug is camptothecin and its derivatives.
  • the cytotoxic drug is a structure as shown in formula (A-1), its tautomers, enantiomers, or diastereomers,
  • M is -L 2 -L 1 -C(O)-;
  • L 2 is selected from -NH-, O and S, and L 2 is connected to the linker unit L;
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -, C 3 -C 6 saturated cycloalkylene or 3-6 membered saturated heterocyclylene, and the C 3 -C 6 saturated cycloalkylene and the 3-6 membered saturated heterocyclylene are each independently optionally substituted by one or more R 2a ;
  • n is selected from 1, 2, 3 and 4; the heteroatoms in the 3-6 membered saturated heterocyclic group are independently N, O and S, and the number of heteroatoms is 1, 2 or 3;
  • R 1a and R 1b are each independently selected from hydrogen, halogen, hydroxy, amino and C 1 -C 6 alkyl, wherein the C 1 -C 6 alkyl is optionally substituted with one or more halogens;
  • R 2a is selected from halogen, hydroxy, amino and C 1 -C 6 alkyl, wherein the C 1 -C 6 alkyl is optionally substituted with one or more halogens.
  • L2 is preferably -O- or -S-, more preferably -O-.
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -;
  • R 1a is selected from the group consisting of hydrogen, halogen, and C 1 -C 6 alkyl;
  • R 1b is selected from the group consisting of hydrogen, halogen, and C 1 -C 6 alkyl.
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -;
  • R 1a is C 1 -C 6 alkyl, preferably C 1 -C 3 alkyl;
  • R 1b is selected from hydrogen and C 1 -C 6 alkyl, preferably selected from hydrogen and C 1 -C 3 alkyl.
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -; R 1a is -CH 3 ; R 1b is selected from: hydrogen and -CH 3 .
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -; m is 1 or 2, preferably 1.
  • L1 is selected from:
  • L 1 is C 3 -C 6 saturated cycloalkylene or 3-6 membered saturated heterocyclylene, preferably C 3 -C 6 saturated cycloalkylene, wherein the C 3 -C 6 saturated cycloalkylene and 3-6 membered saturated heterocyclylene are each independently optionally substituted by one or more R 2a , and R 2a is each independently selected from: halogen and C 1 -C 6 alkyl.
  • L 1 is optionally substituted with one or more R 2a : cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; each R 2a is independently selected from: halogen and C 1 -C 6 alkyl.
  • L1 is selected from:
  • the C 3 -C 6 cycloalkylene group is cyclobutyl or cyclohexyl, preferably cyclobutyl.
  • the heteroatoms of the 3-6 membered heterocyclylene group are each independently N or O, and the number of heteroatoms is preferably 1 or 2.
  • the C 1 -C 6 alkyl group is a C 1 -C 3 alkyl group, preferably a methyl group.
  • the halogen is F, Cl, Br or I, preferably F, Cl or Br.
  • M is -L 2 -L 1 -C(O)-;
  • L2 is -O-
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 - or C 3 -C 6 saturated cycloalkylene, wherein the C 3 -C 6 saturated cycloalkylene is optionally substituted by one or more R 2a ;
  • n 1 or 2;
  • R 2a is selected from halogen and C 1 -C 6 alkyl, wherein the C 1 -C 6 alkyl is optionally substituted with one or more halogens.
  • M is:
  • M is N
  • the cytotoxic drug is selected from any of the following structures:
  • the linker unit L is -L a -L b -L c -; wherein L c is connected to the cytotoxic drug;
  • Each group fragment in the definition of -L a - is preferably connected to the L b at the right end;
  • -L a - is further preferably The a end is connected to Ab, and the b end is connected to L b
  • -L b - is a polypeptide-NH- of -1 to 6 natural amino acids, preferably a polypeptide-NH- of -2 to 4 natural amino acids, more preferably selected from any of the following structures: More preferably Each group fragment in the definition of -L b - is preferably connected to the L c at the right end;
  • -L c - is C 1-6 alkylene, preferably C 1-3 alkylene, more preferably
  • the bispecific antibody drug conjugate of the present invention wherein the linker unit L is Preferably
  • the bispecific antibody drug conjugate of the present invention has a structure as shown in formula (A-2):
  • p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 3 to 9; For example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98 or 8;
  • the bispecific antibody drug conjugate of the present invention has a structure as shown in formula (A-2):
  • p represents the average number of connections, and p is any integer or decimal from 1 to 10; preferably any integer or decimal from 3 to 9; for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98 or 8;
  • Ab is a bispecific antibody or an antigen-binding fragment thereof according to any embodiment of the present invention.
  • L is the linker unit L described in any embodiment of the present invention.
  • M is -L 2 -L 1 -C(O)-;
  • L2 is -O- or -S-, and L2 is connected to L;
  • L 1 is -(C(R 1a )(R 1b )) m -CH 2 -, C 3 -C 6 saturated cycloalkylene or 3-6 membered saturated heterocyclylene, and the C 3 -C 6 saturated cycloalkylene and 3-6 membered saturated heterocyclylene are each independently optionally substituted by one or more R 2a ;
  • n 1, 2, 3 or 4;
  • the heteroatoms in the 3-6 membered saturated heterocyclic group are each independently N, O or S, and the number of heteroatoms is 1, 2 or 3;
  • R 1a , R 1b and R 2a are each independently hydrogen, halogen, hydroxy, amino or C 1 -C 6 alkyl, wherein the C 1 -C 6 alkyl is optionally substituted with one or more halogens;
  • R 2a is selected from halogen, hydroxy, amino and C 1 -C 6 alkyl, wherein the C 1 -C 6 alkyl is optionally substituted with one or more halogens.
  • the bispecific antibody drug conjugate of the present invention has a structure as shown in formula (A-2a) or (A-2b):
  • p represents the average connection number, and p is selected from any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98 or 8;
  • Ab is a bispecific antibody or an antigen-binding fragment thereof according to any embodiment of the present invention.
  • L2 is -NH-, O or S, preferably -O- or -S-; more preferably -O-;
  • X 1 is selected from C 3 -C 6 cycloalkylene optionally substituted by 1, 2 or 3 R 2a ;
  • X2 is selected from -(C( R1a )( R1b )) m - CH2- ;
  • n 1 or 2;
  • R 1a and R 1b are each independently hydrogen, halogen or C 1 -C 6 alkyl optionally substituted by 1, 2 or 3 halogens;
  • R 2a is selected from halogen, hydroxy, amino and C 1 -C 6 alkyl, wherein the C 1 -C 6 alkyl is optionally substituted with one or more halogens.
  • the bispecific antibody drug conjugate of the present invention wherein the bispecific antibody drug conjugate is selected from the following:
  • p represents the average connection number, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98 or 8;
  • Ab is a bispecific antibody or an antigen-binding fragment thereof according to any embodiment of the present invention.
  • Ab is selected from the bispecific antibodies DBXT001 series (DBXT001-01-08), DBXT002 series (DBXT002-01-08), DBXT003 series (DBXT003-01-08) of the present invention comprising an EGFR binding domain and a HER3 binding domain.
  • Ab is selected from the bispecific antibodies DBXT001 series, DBXT002 series and DBXT005-01 comprising an EGFR binding domain and a HER3 binding domain of the present invention; more preferably, Ab is selected from the bispecific antibodies DBXT001 series and DBXT005-01 comprising an EGFR binding domain and a HER3 binding domain of the present invention; further preferably, Ab is selected from the bispecific antibodies DBXT001-01 and DBXT005-01 comprising an EGFR binding domain and a HER3 binding domain of the present invention.
  • Ab is the bispecific antibody DBXT005-01 of the present invention comprising an EGFR binding domain and a HER3 binding domain.
  • the bispecific antibody drug conjugate is selected from any of the following structures:
  • p represents the average connection number, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9; for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98 or 8.
  • DBXT001 (DBXT001-01-08), DBXT002 (DBXT002-01-08), DBXT003 (DBXT003-01-08) and DBXT004 (DBXT004-01-08) comprising the EGFR binding domain and the HER3 binding domain of the present invention are shown in the sequence listing of the present invention.
  • the numbering method of the antibody CDR of the present invention is: Kabat numbering.
  • the bispecific antibody drug conjugate is:
  • p represents the average number of connections, and p is any integer or decimal from 1 to 10, preferably any integer or decimal from 3 to 9, and more preferably any integer or decimal from 4 to 6; for example, 5.99;
  • DBXT005-01 is a bispecific antibody against EGFR and HER3, the amino acid sequence of its heavy chain H1 is shown in SEQ ID NO:37, the amino acid sequence of its light chain L1 is shown in SEQ ID NO:38, and the amino acid sequence of its heavy chain H2 is shown in SEQ ID NO:90.
  • the bispecific antibody drug conjugate is selected from the following conjugates:
  • p1 represents the number of connections, and p1 is any integer from 1 to 10, preferably any integer from 3 to 9, and more preferably any integer from 4 to 6; for example, 4, 5 or 6.
  • DBXT005-01 is a bispecific antibody against EGFR and HER3, the amino acid sequence of its heavy chain H1 is shown in SEQ ID NO:37, the amino acid sequence of its light chain L1 is shown in SEQ ID NO:38, and the amino acid sequence of its heavy chain H2 is shown in SEQ ID NO:90.
  • the average connection number p of the present invention can be any integer or decimal from 1 to 10.
  • the average connection number p can be any integer or decimal from 3 to 9.
  • the average connection number p can be any integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10.
  • the average connection number p is 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98 or 8.
  • connection number p1 of the present invention is any integer from 1 to 10.
  • the connection number p1 is any integer from 3 to 9.
  • the connection number p1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the connection number p1 is 4, 5 or 6.
  • the present invention provides a method for preparing the bispecific antibody-drug conjugate according to any one of the present invention, comprising the following steps: mixing the bispecific antibody dissolved in a buffer with the linker unit L-cytotoxic drug dissolved in a solvent under the action of a reducing agent to obtain the bispecific antibody-drug conjugate.
  • the preparation method comprises reacting the anti-EGFR and HER3 bispecific antibody with a compound of formula X2, for example, reacting DBXT005-01 with a compound of formula X2:
  • the reducing agent is a reducing agent conventional in the art for such reactions, such as tris(2-carbonylethyl)phosphine hydrochloride.
  • the buffer is a conventional buffer for such reactions in the art, such as ethylenediaminetetraacetic acid.
  • the solvent is a conventional solvent for such reactions in the art, such as dimethylacetamide.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bispecific antibody as described in any one of the present invention, the isolated nucleic acid as described in any one of the present invention, the recombinant expression vector as described in any one of the present invention, the transformant as described in any one of the present invention and/or the bispecific antibody-drug conjugate as described in any one of the present invention, and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a method for preparing the pharmaceutical composition of the present invention, comprising combining the bispecific antibody drug conjugate according to any one of the present invention, or a pharmaceutically acceptable form thereof, or a mixture thereof, with one or more pharmaceutically acceptable carriers or excipients.
  • the pharmaceutically acceptable carrier used in the pharmaceutical composition examples of suitable pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (2005).
  • the pharmaceutical composition can be administered in any form as long as it can prevent, alleviate, prevent or cure the symptoms of human or animal patients.
  • it can be prepared into various suitable dosage forms according to the administration route.
  • the administration of the bispecific antibody drug conjugate or the pharmaceutical composition described in any one of the present invention can be combined with another treatment method.
  • the other treatment method can be selected from, but not limited to: radiotherapy, chemotherapy, immunotherapy, or a combination thereof.
  • the present invention provides a pharmaceutical preparation comprising the bispecific antibody drug conjugate according to any one of the present invention.
  • the preparation is in the form of a solid preparation, a semi-solid preparation, a liquid preparation or a gaseous preparation.
  • the present invention provides use of the bispecific antibody as described in any one of the present invention, the isolated nucleic acid as described in any one of the present invention, the recombinant expression vector as described in any one of the present invention, the transformant as described in any one of the present invention, the bispecific antibody-drug conjugate as described in any one of the present invention, and/or the pharmaceutical composition as described in any one of the present invention in the preparation of a medicament for treating and/or preventing cancer, preferably, the cancer is a cancer that positively expresses EGFR and/or HER3.
  • the present invention provides a method for treating and/or preventing cancer, comprising administering to a subject in need thereof a bispecific antibody as described in any one of the present invention, an isolated nucleic acid as described in any one of the present invention, a recombinant expression vector as described in any one of the present invention, a transformant as described in any one of the present invention, a bispecific antibody-drug conjugate as described in any one of the present invention, and/or a pharmaceutical composition as described in any one of the present invention; preferably, the cancer is a cancer that positively expresses EGFR and/or HER3.
  • the present invention provides a bispecific antibody as described in any one of the present invention, an isolated nucleic acid as described in any one of the present invention, a recombinant expression vector as described in any one of the present invention, a transformant as described in any one of the present invention, a bispecific antibody-drug conjugate as described in any one of the present invention, and/or a pharmaceutical composition as described in any one of the present invention, which is used for treating and/or preventing cancer, preferably, the cancer is a cancer that positively expresses EGFR and/or HER3.
  • the cancer described in the present invention is selected from breast cancer, skin cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, bile duct cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, digestive tract cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, thyroid cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma and melanoma.
  • the cancer described in the present invention is preferably selected from breast cancer, colorectal cancer, skin cancer, lung cancer, esophageal cancer and oral squamous cell carcinoma.
  • the lung cancer is preferably non-small cell lung cancer
  • the skin cancer is preferably cutaneous squamous cell carcinoma
  • the colorectal cancer is preferably rectal cancer.
  • the present invention provides use of the bispecific antibody as described in any one of the present invention, the isolated nucleic acid as described in any one of the present invention, the recombinant expression vector as described in any one of the present invention, the transformant as described in any one of the present invention, the bispecific antibody-drug conjugate as described in any one of the present invention and/or the pharmaceutical composition as described in any one of the present invention in the preparation of EGFR and/or HER3 inhibitors.
  • the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration, etc.
  • the invention provides for co-administering to a subject a therapeutically effective amount of one or more therapies (eg, treatment modalities and/or other therapeutic agents).
  • the therapy comprises surgery and/or radiation therapy.
  • the methods or uses provided by the present invention further include administering one or more therapies (e.g., treatment modalities and/or other therapeutic agents) to an individual.
  • therapies e.g., treatment modalities and/or other therapeutic agents
  • the antibody drug conjugate of the present invention or a pharmaceutically acceptable salt thereof may be used alone or in combination with other therapeutic agents in the therapy. For example, it may be co-administered with at least one additional therapeutic agent.
  • the present invention provides a pharmaceutical combination comprising the bispecific antibody according to any one of the present invention,
  • the present invention provides a kit comprising the bispecific antibody as described in any one of the present invention, the isolated nucleic acid as described in any one of the present invention, the recombinant expression vector as described in any one of the present invention, the transformant as described in any one of the present invention, the bispecific antibody-drug conjugate as described in any one of the present invention and/or the pharmaceutical composition as described in any one of the present invention.
  • EGFR Epidermal growth factor Receptor
  • Epidermal Growth Factor Receptor is a receptor for epidermal growth factor (EGF) cell proliferation and signal transduction.
  • EGFR belongs to a member of the ErbB receptor family, which includes EGFR (ErbB-1), HER2/c-neu (ErbB-2), HER3 (ErbB-3) and HER 4 (ErbB-4).
  • EGFR is also known as HER1 and ErbB-1, and mutations or overexpression generally cause tumors.
  • EGFR is a glycoprotein, a tyrosine kinase receptor, which penetrates the cell membrane and has a molecular weight of 170KDa.
  • EGFR is located on the surface of the cell membrane and is activated by binding to ligands, including EGF and TGF ⁇ (transforming growth factor ⁇ ). After activation, EGFR is converted from a monomer to a dimer, although there is also evidence that dimers exist before activation. EGFR may also be activated by polymerization with other members of the ErbB receptor family, such as ErbB-2/HER2/neu.
  • HER3 human epidermal growth factor receptor 3
  • ErbB3 receptor tyrosine protein kinase ErbB-3
  • HER3 is a member of the EGFR/ErbB family.
  • HER2 and EGFR Unlike other ErbB family members HER2 and EGFR, HER3 itself does not have kinase activity. Therefore, HER3 must bind to its kinase active members EGFR or HER2 as a heterodimer to trigger its downstream activity. After binding to its natural ligand NRG1, HER3 undergoes conformational changes, heterodimerization and phosphorylation, and then activates MAPK, PI3K/Akt and PLC ⁇ through signal transduction.
  • the term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ⁇ 5%, ⁇ 2%, ⁇ 1% and ⁇ 0.1%, as these variations are suitable for performing the disclosed methods.
  • the term "and/or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.
  • the term "or” should be understood to have the same meaning as “and/or” as defined above.
  • “or” or “and/or” should be interpreted as inclusive, that is, including at least one of the number or elements in the list, but More than one, and optionally, additional unlisted items are also included. Only when explicitly stated to the contrary, such as “only one” or “exactly one” or when used in a claim, “consisting of" will refer to only one number listed or one element of a list.
  • the term "antibody drug conjugate” generally refers to an antibody connected to a biologically active cytotoxic drug via a stable linker.
  • the "antibody drug conjugate” may be a bispecific antibody drug conjugate, which may refer to a bispecific antibody or an antigen-binding fragment thereof connected to a biologically active cytotoxic drug fragment via a stable linker.
  • cytotoxic drug generally refers to a toxic drug, which may have a strong chemical molecule in tumor cells that destroys its normal growth. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations.
  • the "cytotoxic drug” may include toxins, such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals, radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 or radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics, nucleolytic enzymes, or their derivatives.
  • toxins such as small molecule toxins or enzyme-active toxins from bacteria, fungi, plants or animals, radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi
  • camptothecin derivatives can be a toxic drug, including but not limited to camptothecin derivatives; for example, it can be a camptothecin derivative exatecan (chemical name: chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione).
  • camptothecin derivative exatecan chemical name: chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione
  • the term "antibody” generally refers to an immunoglobulin reactive to a specified protein or peptide or a fragment thereof.
  • the antibody may be an antibody from any class, including but not limited to IgG, IgA, IgM, IgD and IgE, and an antibody from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4).
  • the antibody may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4.
  • the antibody may also have a light chain selected from, for example, kappa ( ⁇ ) or lambda ( ⁇ ).
  • the antibodies of the present invention may be derived from any species.
  • antibody may include complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules, as long as these antibodies exhibit the desired biological activity.
  • an antigen binding domain generally refers to a portion of an antibody molecule that contains amino acids responsible for the specific binding between the antibody and the antigen.
  • the portion of an antigen that is specifically recognized and bound by an antibody is referred to as an "epitope" as described herein.
  • an antigen binding domain may typically contain an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily contain both.
  • VL antibody light chain variable region
  • VH antibody heavy chain variable region
  • a Fd fragment for example, has two VH regions and generally retains some antigen binding functions of a complete antigen binding domain.
  • antigen-binding fragments of antibodies include (1) a Fab fragment, a monovalent fragment having VL, VH, a constant light chain (CL) and a CH1 domain; (2) a F(ab′) 2 fragment, a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; (3) a Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli,” Nature 341:544-546 (1989), which is incorporated herein by reference in its entirety), which has a VH domain; (6) isolated complementarity determining regions (CDRs); and (7) a single-chain Fv (scFv), for example derived from an scFV library.
  • a Fab fragment
  • Fv fragment VL and VH
  • VL and VH the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that enables them to be prepared as a single protein chain (called a single-chain Fv) in which the VL and VH regions pair to form a monovalent molecule.
  • Fv scFv
  • Huston et al. "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci.
  • VHH refers to the variable antigen binding domain of heavy chain antibodies from Camelidae (camels, dromedaries, llamas, alpacas, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J Biotechnol., 74, 277-302 and review Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHH can also be called nanobody (Nanobody, Nb).
  • variable region or “variable domain” or “variable antigen binding domain” generally refers to the domain of the antibody heavy chain or light chain that participates in the binding of the antibody to the antigen.
  • variable generally refers to that some parts of the sequence of the variable domain of the antibody vary strongly, forming the binding and specificity of various specific antibodies to their specific antigens. Variability is not evenly distributed throughout the variable region of the antibody. It is concentrated in three segments in the light chain variable region and the heavy chain variable region, which are called complementary determining regions (CDRs) or hypervariable regions (HVRs), which are LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, respectively.
  • CDRs complementary determining regions
  • HVRs hypervariable regions
  • variable region The more highly conserved parts of the variable region are called framework regions (FRs).
  • the variable domains of natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), most of which adopt a ⁇ -folded configuration and are connected by three CDR structural loop regions.
  • the CDRs in each chain are held together by the FR regions and, together with the CDRs from the other chain, form the antibody antigen-binding site.
  • variable region of an antibody can be encoded or the CDR of an antibody can be divided by a variety of methods, such as the Kabat numbering scheme and definition rules based on sequence variability (see, Kabat et al., Protein Sequences in Immunology, Fifth Edition, National Institutes of Health, Bethesda, Maryland (1991)), the Chothia numbering scheme and definition rules based on the position of the structural loop region (see, Al-Lazikani et al., J Mol Biol 273:927-48, 1997), the IMGT numbering scheme and definition rules based on the amino acid sequence alignment of germline V genes of Lefranc et al., There are also Honneger’s numbering scheme (AHo’s), Martin’s numbering scheme, Gelfand’s numbering scheme, etc., see Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition, Front. Immuno
  • the term "monoclonal antibody” or “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • multispecific antibody refers to an antibody comprising two or more antigen binding domains, capable of binding to two or more different epitopes (e.g., two, three, four or more different epitopes), the epitopes of which may be on the same or different antigens.
  • multispecific antibodies include "bispecific antibodies” (abbreviated as bispecific) or "bispecific molecules” that bind to two different antigens or two different epitopes.
  • bispecific antibodies targeting EGFR and HER3 herein may be referred to as, for example, "anti-EGFR and HER3" or “anti-EGFR/HER3” or “EGFR ⁇ HER3” bispecific molecules, or bispecific antibodies comprising an EGFR binding domain and a HER3 binding domain, or other similar terms.
  • the term "Fc region” is used herein to define the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain.
  • the C-terminal lysine (Lys447) in the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, such as Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).
  • the term "Knob into Hole structure” refers to mutating the hydrophobic amino acids of CH3 of antibody Fc.
  • the side chain amino acids of one CH3 chain are mutated to form a relatively large hydrophobic amino acid (knob) to strengthen the hydrophobic force; the side chain amino acids of another CH3 are mutated to form a small amino acid (hole) to reduce steric hindrance; after the mutation, the CH3 with Knob and the CH3 with Hole form a Knob into Hole structure (KiH) in the form of hydrophobic interaction, which is conducive to the formation of heavy chain heterodimers; the KiH mutation mainly occurs in the internal hydrophobic amino acids of the spatial structure of the CH3 domain, and the amino acids exposed to the outside after the mutation are almost unchanged, so it does not affect the effector function of Fc and the immunogenicity caused.
  • knob-Fc refers to a point mutation containing T366W in the Fc region of an antibody to form a knob-like spatial structure.
  • hole-Fc refers to point mutations T366S, L368A, and Y407V in the Fc region of an antibody to form a hole-like spatial structure.
  • point mutations S354C and Y349C can be introduced into knob-Fc and hole-Fc, respectively, to further promote the formation of heterodimers through disulfide bonds.
  • point mutations H435R and Y436F can be introduced into hole-Fc, respectively.
  • humanized antibody refers to an antibody form containing sequences from human and non-human (e.g., mouse, rat) antibodies.
  • a humanized antibody comprises substantially all of at least one, usually two variable domains, wherein all or substantially all of the variable domains are equivalent to the variable domains of non-human immunoglobulins, and all or substantially all of the framework regions (FR) are framework regions of human immunoglobulin sequences.
  • a humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).
  • an “isotype” antibody refers to the class of antibody provided by the heavy chain constant region gene (e.g., IgM, IgE, IgG such as IgG1, IgG2 or IgG4).
  • Isotypes also include modified forms of one of these classes, where the modification has been generated to alter Fc function, such as to enhance or reduce effector function or binding to Fc receptors.
  • cross-reaction refers to the binding of antigen fragments of the same target molecule of human, monkey, and/or murine (mouse or rat) origin. Therefore, “cross-reaction” should be understood as the interspecies reaction of an antigen binding molecule (e.g., antibody) with a similar molecule (e.g., BDCA2) expressed in different species.
  • an antigen binding molecule e.g., antibody
  • BDCA2 a similar molecule
  • the cross-reaction specificity of monoclonal antibodies recognizing human BDCA2, monkey, and/or murine BDCA2 (mouse or rat) can be determined by FACS analysis.
  • affinity refers to the intrinsic binding affinity that reflects the interaction between the members of the binding.
  • the affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (K off (Kd) and K on (Ka), respectively).
  • KD equilibrium dissociation constant
  • Kd dissociation rate constant
  • Ka association rate constant
  • affinity is measured using surface plasmon resonance (SPR) technology.
  • SPR surface plasmon resonance
  • a specific method for measuring affinity is the BIAcore method.
  • the term "not binding" to a protein or cell means not binding to the protein or cell, or not binding to the protein or cell with high affinity, i.e., the KD for binding to the protein or cell is 1.0 ⁇ 10-6 M or higher, preferably 1.0 ⁇ 10-5 M or higher, more preferably 1.0 ⁇ 10-4 M or higher, 1.0 ⁇ 10-3 M or higher, and even more preferably 1.0 ⁇ 10-2 M or higher.
  • the term "high affinity" means that the KD for the antigen is 1.0 ⁇ 10 -6 M or less for IgG antibodies. Preferably, 5.0 ⁇ 10 -8 M or less, more preferably 1.0 ⁇ 10 -8 M or less, 5.0 ⁇ 10 -9 M or less, and even more preferably 1.0 ⁇ 10 -9 M or less.
  • "high affinity” binding may vary.
  • "high affinity” binding for the IgM subtype means a KD of 10 -6 M or less, preferably 10 -7 M or less, and more preferably 10 -8 M or less.
  • percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after the amino acid sequences are aligned (and gaps are introduced if necessary) to obtain the maximum percentage sequence identity, and any conservative substitutions are not considered as part of the sequence identity.
  • Sequence alignments can be performed using various methods in the art to determine the percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software.
  • One skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to obtain maximum alignment over the entire length of the compared sequences.
  • halogen generally refers to fluorine, chlorine, bromine, and iodine, for example, it can be fluorine or chlorine.
  • alkyl generally refers to an alkane residue derived from a hydrogen atom.
  • alkyl generally refers to a saturated straight or branched aliphatic hydrocarbon radical having a residue derived from the same carbon atom or two different carbon atoms of the parent alkane, which can be a straight or branched group containing 1 to 20 carbon atoms, such as a chain alkyl containing 1 to 12 carbon atoms, such as a chain alkyl containing 1 to 6 carbon atoms, such as a chain alkyl containing 1 to 3 carbon atoms.
  • alkyl examples include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc.
  • Alkyl can be substituted or non-substituted, alternative or non-substituted, such as when substituted, the substituent can be substituted on any usable point of attachment.
  • alkylene generally refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of the parent alkane by removing two hydrogen atoms, which can be a straight or branched group containing 1 to 20 carbon atoms, for example, the term “methylene” can refer to a residue derived from a group of 1 carbon atom by removing two hydrogen atoms. Methylene can be substituted or unsubstituted, substituted or unsubstituted; for example, an alkylene containing 1 to 12 carbon atoms, for example, 1 to 6 carbon atoms.
  • Non-limiting examples of alkylene groups include, but are not limited to, methylene (—CH 2 —), 1,1-ethylene (—CH(CH 3 )—), 1,2-ethylene (—CH 2 CH 2 )—), 1,1-propylene (—CH(CH 2 CH 3 )—), 1,2-propylene (—CH 2 CH(CH 3 )—), 1,3-propylene (—CH 2 CH 2 CH 2 —), 1,4-butylene (—CH 2 CH 2 CH 2 CH 2 —), and 1,5-butylene (—CH 2 CH 2 CH 2 CH 2 CH 2 —), and the like.
  • cycloalkyl refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, and more preferably 3 to 6 carbon atoms.
  • Non-limiting examples of monocyclic cycloalkyls include cyclopropane, cyclobutane, cyclopentane, cyclopentenyl, cyclohexane, cyclohexenyl, cyclohexadienyl, cycloheptane, cycloheptatrienyl, cyclooctanyl, etc.; polycyclic cycloalkyls include cycloalkyls of spiro rings, fused rings, and bridged rings.
  • cycloalkylene is a divalent group which is attached to the rest of the molecule by two single bonds, and the rest is the same as the definition of the term “cycloalkyl”.
  • partially unsaturated generally refers to a ring structure containing at least one double bond or triple bond between ring molecules.
  • partially unsaturated encompasses ring structures with multiple unsaturations, but is not intended to include aromatic or heteroaromatic rings as defined in the present invention.
  • unsaturated means that the moiety has one or more degrees of unsaturation.
  • heterocyclyl refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic light substituent containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen or sulfur, and the remaining ring atoms are carbon.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl and homopiperazinyl, etc.
  • Polycyclic heterocyclic groups include spirocyclic, fused and bridged heterocyclic groups.
  • the heterocyclic ring can be fused to an aryl, heteroaryl or cycloalkyl ring, and the ring connected to the parent structure is a heterocyclic group.
  • heteroalkylene is a divalent group which is attached to the rest of the molecule by two single bonds and otherwise has the same definition as the term “cycloalkylene”.
  • each independently generally means that the variable applies to any case, regardless of whether the variable with the same or different definition exists in the same compound.
  • the variable may refer to the type, number of substituents of the compound, or the type of atoms in the compound.
  • R appears twice in the compound and R is defined as "independently carbon or nitrogen” both R can be carbon, both R can be nitrogen, or one R can be carbon and the other R can be nitrogen.
  • the term “optional” or “optionally” generally means that the subsequently described event or circumstance may but need not occur, and the description includes occasions where the event or circumstance occurs or does not occur.
  • a heterocyclic group optionally substituted with an alkyl group means that alkyl substitution may but need not exist, and the description may include situations where the heterocyclic group is substituted with an alkyl group and situations where the heterocyclic group is not substituted with an alkyl group.
  • substituted generally refers to one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, which are replaced independently of each other by a corresponding number of substituents.
  • the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort.
  • an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g. olefinic) bond.
  • connection between groups can generally be in any orientation; the “connection” between group X and group Y can generally be in any orientation, and any orientation generally means that when group X is used to connect group Y and group Z, two or more connection sites of the group X can be arbitrarily connected to group Y or group Z.
  • alkyl alkyl
  • cycloalkyl alkyl
  • an identifier to indicate the number of atoms present in the group in a particular case, for example, C 1 -C 4 alkyl, C 3 -C 7 cycloalkyloxy, C 1 -C 4 alkylcarbonylamino, etc., and the subscript number following "C” indicates the number of carbon atoms present in the group.
  • C 3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl); in C 1-10 , the members of the group may have any number of carbon atoms falling within the range of 1-10.
  • the compounds or antibody drug conjugates of the present invention include tautomers, mesomers, racemates, enantiomers, and/or diastereomers thereof.
  • the term “diastereomer” generally refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as, melting points, boiling points, spectral properties and reactivity.
  • tautomers or “tautomeric forms” are used interchangeably and generally refer to structural isomers of different energies that can be mutually converted by low energy barriers.
  • proton tautomers also known as prototropic tautomers
  • proton migration such as keto-enol isomerization and imine-enamine isomerization
  • Valence tautomers include interconversions via reorganization of some of the bonding electrons.
  • the term "mesomorph” generally refers to a molecule containing an asymmetric atom, but having a symmetry factor such that the total intramolecular optical rotation is zero.
  • racemate or “racemic mixture” refers to a mixture composed of equimolar amounts of a compound. A composition consisting of two enantiomeric substances.
  • linker unit or “linker structure” generally refers to a chemical structure fragment or bond connected to a ligand at one end and to a cytotoxic drug at the other end, and may also be connected to a cytotoxic drug after being connected to other linkers.
  • the directly or indirectly connected ligand may refer to the group being directly connected to the ligand through a covalent bond, or may be connected to the ligand through a linker structure.
  • a chemical structure fragment or bond comprising an acid-labile linker structure (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure may be used as a linker structure.
  • an acid-labile linker structure e.g., hydrazone
  • a protease-sensitive linker structure e.g., peptidase-sensitive linker structure
  • photolabile linker structure e.g., a photolabile linker structure
  • dimethyl linker structure e.g., dimethyl linker structure
  • disulfide-containing linker structure e.g., a disulfide-containing linker structure
  • the term "optionally linked to other molecular parts" of a certain structure generally means that the structure is not linked to any other chemical structure, or that the structure is linked to one or more other chemical structures (such as the ligands described in the present invention) different from the structure (for example, linked by a chemical bond, or linked by a linker structure).
  • the term "drug loading” generally refers to the average amount of cytotoxic drugs (payload) loaded on each ligand, and can also be expressed as the ratio of the amount of cytotoxic drugs to antibodies (drug/antibody ratio, DAR), and the range of cytotoxic drug loading can be 0-12, for example, 1-10 cytotoxic drugs connected to each ligand (Ab).
  • the drug loading is expressed as p or p1, which can be illustratively an average of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.
  • the drug loading of each ADC molecule after the coupling reaction can be identified by conventional methods such as UV/visible light spectroscopy, mass spectrometry, ELISA test and HPLC characteristics.
  • certain atoms of the compounds of the present invention or antibody drug conjugates may appear in more than one isotopic form.
  • hydrogen may exist in the form of protium ( 1H ), deuterium ( 2H ) and tritium ( 3H ), and carbon may exist naturally in three different isotopes ( 12C , 13C and 14C ).
  • isotopes that can be incorporated into the compounds of the present invention also include, but are not limited to, 15N , 18O , 17O , 18F , 32P, 33P , 129I , 131I , 123I , 124I , 125I , or similar isotopes.
  • the compounds of the present invention or antibody drug conjugates may be enriched in one or more of these isotopes.
  • isotope-enriched compounds can be used for a variety of purposes.
  • substitution with heavy isotopes such as deuterium ( 2H ) may provide certain therapeutic advantages, which may be due to higher metabolic stability.
  • the natural abundance of deuterium ( 2H ) is about 0.015%. Therefore, there is one deuterium atom for approximately every 6500 hydrogen atoms in nature. Therefore, the deuterium abundance of the deuterium-containing compound or antibody drug conjugate of the present invention at one or more positions (as the case may be) is greater than 0.015%.
  • the structures described in the present invention may also include compounds or antibody drug conjugates that differ only in the presence or absence of one or more isotopically enriched atoms.
  • compounds or antibody drug conjugates that are consistent with the structure of the present invention except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon 13 or carbon 14, are within the scope of the present invention.
  • the term "pharmaceutical composition” generally refers to a mixture containing one or more compounds of the present invention or their physiologically/pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients.
  • the pharmaceutical composition can be used to promote administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.
  • the preparation of conventional pharmaceutical compositions can be found in the pharmacopoeias of various countries.
  • the pharmaceutical composition can be in the form of a sterile injection water or oil suspension for intramuscular and subcutaneous administration.
  • the suspension can be prepared with a suitable dispersant or wetting agent and suspending agent according to known techniques.
  • the sterile injection preparation can also be a sterile injection solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, such as a solution prepared in 1,3-butanediol.
  • sterile fixed oils can be conveniently used as solvents or suspension media.
  • any blended fixed oils including synthetic mono- or diglycerides can be used.
  • fatty acids such as oleic acid can also be used to prepare injections.
  • the term "pharmaceutically acceptable salt” or “pharmaceutically usable salt” generally refers to a salt of the compound or antibody-drug conjugate of the present invention. Such salts may be safe and/or effective when used in mammals and may have the desired biological activity.
  • the compound or antibody-drug conjugate of the present invention may form a salt with an acid.
  • the term "pharmaceutically acceptable carrier” generally refers to a carrier or vehicle for administering therapeutic agents, such as antibodies or polypeptides, genes and other therapeutic agents.
  • the term refers to any drug carrier that does not itself induce the production of antibodies that are harmful to the individual receiving the composition and can be administered without excessive toxicity.
  • Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates and inactivated viral particles. These carriers are well known to those skilled in the art.
  • Pharmaceutically acceptable carriers in therapeutic compositions may include liquids, such as water, saline, glycerol and ethanol. Auxiliary substances, such as wetting agents or emulsifiers, pH buffer substances, etc., may also be present in these carriers.
  • treatment and “treating” generally refer to methods of obtaining beneficial or desired results, including but not limited to therapeutic benefits.
  • Therapeutic benefits include but are not limited to eradication, inhibition, reduction or improvement of the underlying disorder being treated.
  • therapeutic benefits are achieved by eradication, inhibition, reduction or improvement of one or more physiological symptoms associated with the underlying disorder, so that improvement is observed in the patient, but the patient may still suffer from the underlying disorder.
  • prevention and preventing generally refer to methods of obtaining beneficial or desired results, including but not limited to preventive benefits.
  • a pharmaceutical composition can be administered to a patient at risk of developing a particular disease or to a patient reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.
  • the term "subject” or “patient” generally refers to humans (i.e., males or females of any age group, for example, pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults or elderly people)) and/or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats and/or dogs; and/or birds, including commercially relevant birds such as chickens, ducks, geese, quail and/or turkeys.
  • humans i.e., males or females of any age group, for example, pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults or elderly people)) and/or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercial
  • the terms "therapeutically effective amount”, “therapeutically effective dose” and “effective amount” refer to the amount of the compound or antibody drug conjugate of the present invention, when administered to a cell, tissue or subject alone or in combination with other therapeutic drugs, that effectively prevents or improves the symptoms of one or more diseases or conditions or the development of the disease or condition.
  • a therapeutically effective dose also refers to a dose sufficient to cause symptomatic improvement, such as an amount that treats, cures, prevents or improves a related medical condition or increases the rate of treatment, cure, prevention or improvement of such a condition.
  • a therapeutically effective dose refers only to that ingredient.
  • a therapeutically effective dose refers to the combined amount of active ingredients that cause a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • An effective amount of a therapeutic agent will result in an increase in a diagnostic criterion or parameter by at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50%.
  • cancer refers to a group of cells that show abnormally high levels of proliferation and growth.
  • Cancer may be benign (also known as benign tumors), pre-malignant or malignant.
  • Cancer cells may be solid cancer cells or leukemia cancer cells.
  • tumor refers to one or more cells comprising cancer.
  • tumor growth refers to the proliferation or growth of one or more cells comprising cancer, which leads to a corresponding increase in the size or degree of cancer.
  • EGFR and/or HER3 positively expressed cancer and "EGFR and/or HER3 expressed cancer” have the same definition, and are cancers in which cancer cells express EGFR and/or HER3, preferably expressed on the surface of cancer cells.
  • amino acid numbers of the antibodies of the present invention are numbered according to the natural sequence from the N-terminus to the C-terminus of the antibody sequence.
  • the reagents and raw materials used in the present invention are commercially available.
  • the bispecific antibody of the present invention has a better endocytosis effect on the human tumor cell line T47D, the human tumor cell line NCI-H1975 and the mouse CT26 cell line that simultaneously express EGFR and HER3.
  • the bispecific antibodies of the present invention have the advantages of good compliance and controllable quality.
  • the stability characterization of the bispecific antibody in the present invention is mainly reflected in the study of monomer purity and thermal stability. After affinity purification and molecular sieve separation, the monomer content of the bispecific antibody can reach 95%. The results of the structural and activity analysis of the antibody after heat treatment prove that the antibody can still maintain a good molecular conformation and complete biological activity under harsh conditions, which is conducive to the industrial production and packaging and storage of the antibody.
  • the EGFR and HER3 bispecific antibodies of the present invention show good molecular stability and have in vitro activity (binding molecular level and cellular level) significantly better than Cetuximab and EGFR.
  • the bispecific antibody of the present invention has broad application prospects due to its excellent developability and activity.
  • the bispecific antibody drug conjugate of any one of the present invention has an in vivo tumor inhibition effect, which is specifically manifested as follows:
  • Figure 1 shows the binding of the bispecific antibody and its corresponding parental monoclonal antibody to cell lines expressing different levels of EGFR/HER3.
  • FIG3 shows the endocytic activity of the bispecific antibody and its corresponding parental monoclonal antibody (pHrodo method).
  • FIG. 4 shows the endocytic activity (incucyte method) of the bispecific antibody and its corresponding parental monoclonal antibody and control antibody.
  • FIG. 5 shows the endocytic activity (incucyte method) of the bispecific antibody and its corresponding parental monoclonal antibody and control antibody.
  • Figure 6 shows the in vivo drug effects of antibody-drug conjugates on human esophageal cancer cell line OE-19 bearing mice with low EGFR expression and HER3 expression Effectiveness evaluation.
  • FIG. 7 is an in vivo efficacy evaluation of the antibody-drug conjugate on human non-small cell lung cancer cell line NCI-H441 tumor-bearing mice expressing EGFR and HER3.
  • FIG8 is an in vivo evaluation of the efficacy of the antibody-drug conjugate on human oral squamous cell carcinoma cell line CAL-27 tumor-bearing mice with high EGFR expression and low HER3 expression.
  • FIG9 is an in vivo efficacy evaluation of bispecific antibody-drug conjugates with different DAR values in NCI-H1975 tumor-bearing mice.
  • Figure 10 is the in vivo efficacy evaluation of bispecific antibody drug conjugates with different DAR values in OE-19 tumor-bearing mice.
  • FIG. 11 is a comparison of the in vivo efficacy of the bispecific antibody drug conjugate of the present invention and the control antibody drug conjugate in NCI-H1975 tumor-bearing mice.
  • FIG. 12 is a comparison of the in vivo efficacy of the bispecific antibody drug conjugate of the present invention and the control antibody drug conjugate in OE-19 tumor-bearing mice.
  • FIG. 13 shows the in vivo efficacy of the bispecific antibody-drug conjugate linker-cytotoxin X1 of the present invention in NCI-H1975 tumor-bearing mice.
  • FIG. 14 shows the in vivo efficacy of the bispecific antibody-drug conjugate of the present invention in human esophageal cancer OE-19 tumor-bearing mice.
  • FIG. 15 shows the in vivo efficacy of the bispecific antibody-drug conjugate of the present invention in mice bearing human skin cancer A431 xenograft tumors with high EGFR expression and low HER3 expression.
  • FIG. 16 shows the in vivo efficacy of the bispecific antibody-drug conjugate of the present invention in mice bearing xenograft tumors of the human colon cancer cell line SW620 that does not express EGFR but only expresses HER3.
  • FIG. 17 shows the in vivo efficacy of the bispecific antibody drug conjugate of the present invention in mice bearing xenograft tumors of the human colon cancer cell line SW48 that lowly expresses HER3 in EGFR.
  • Figure 18 shows the in vivo efficacy of the bispecific antibody-drug conjugate of the present invention in mice bearing non-small cell lung cancer NCI-H1975 (EGFR L858R T790M C797S triple mutation) xenograft tumors.
  • the anti-HER3 antibody or its antigen-binding fragment is prepared with reference to clone 2 of hu3F8 (PCT/CN2022/098929), and the anti-EGFR antibody or its antigen-binding fragment is prepared with reference to Zalutumumab (WO2002100348).
  • some amino acid mutations are performed in the CDR/FR/Fc region.
  • the CDR region of the bispecific antibody is determined according to the Kabat numbering rule.
  • Cetuximab monoclonal antibody prepared by conventional methods; control antibody SI-1X6.4: prepared with reference to WO2023083381A1.
  • CHO-K1 cells from ECACC, inoculate in culture medium (Shanghai Aupumai Biotech Co., Ltd., Catalog No. C673017), subculture at 8% CO 2 , 37°C, and make the density reach 6.0 ⁇ 10 6 cells/mL during transfection. Take 1.45 ⁇ 10 8 cells, centrifuge and remove the supernatant;
  • the ProA chip captured the EGFR parent monoclonal antibody (1 ⁇ g/mL), and the analyte was human EGFR (human EGFR, His-Avi Tag, Kactus Biosystem, EGF-HM401), and the concentration was diluted in the range of 6.25-400 nM, with a total of 8 concentrations;
  • the ProA chip captured the HER3 parent monoclonal antibody (3 ⁇ g/mL), and the analyte was human HER3 (human HER3, His-Avi Tag, Kactus Biosystem, HER-HM403), and the concentration was diluted in the range of 3.125-100 nM, with a total of 8 concentrations;
  • the affinity of the bispecific antibody DBXT001-01 to human EGFR is about 16 times weaker than that to HER3; the affinity of the bispecific antibody DBXT001-01 to human EGFR is about 3 times weaker than that of the parent EGFR antibody, while the affinity of the bispecific antibody DBXT001-01 to HER3 is comparable to that of the parent HER3 antibody.
  • the affinity of the bispecific antibody DBXT001-01 to human EGFR or HER3 is comparable to that of monkey EGFR or HER3.
  • Such a bispecific antibody affinity design can reduce the on-target toxicity of the bispecific antibody to widely distributed normal tissues expressing EGFR.
  • ProA chip captured SI-1X6.4 (7.5 ⁇ g/mL), and the analytes were human EGFR (human EGFR, His-Avi Tag, Kactus Biosystem, EGF-HM401), with concentrations in the range of 1.17-18.75nM, and 5 concentrations in total; human HER3 (human HER3, His-Avi Tag, Kactus Biosystem, HER-HM403), with concentrations in the range of 4.69-75nM, and 5 concentrations in total; similarly, ProA chip captured DBXT005-01 (6 ⁇ g/mL), and the analyte human EGFR concentration was diluted in the range of 9.38-150nM, and 5 concentrations in total; when the analyte was human HER3, the concentration was diluted in the range of 4.69-75nM, and 5 concentrations in total;
  • the affinity of the bispecific antibody DBXT005-01 to human EGFR is about 15 times weaker than that of the control antibody SI-1X6.4, while its affinity to HER3 is 25 times stronger than that of the control antibody SI-1X6.4.
  • Such a bispecific antibody affinity design can reduce the affinity of the bispecific antibody to widely distributed expression EGFR has on-target toxicity in normal tissues and enhances affinity for tumors with high EGFR expression and low HER3 expression.
  • Tumor cell lines NCI-H441, NCI-H1975 and T47D are from ATCC;
  • PE-labeled secondary antibody PE anti-human IgG Fc Antibody
  • the affinity of DBXT001-01 for cell lines that overexpress EGFR or HER3 alone is lower than that of the bivalent parent monoclonal antibody, and is also lower than that of Cetuximab for cell lines that overexpress EGFR; however, the affinity of DBXT001-01 for tumor cell lines that express both EGFR and HER3 is not weaker than that of the bivalent parent monoclonal antibody. This may be beneficial for the dual antibody to reduce binding to normal tissues and enhance simultaneous expression. Binding of EGFR and HER3 in tumor tissue.
  • Tumor cell lines BT-474 and MDA-MB-468 were obtained from ATCC; mouse CT26 monoclonal cell lines stably expressing human EGFR or human HER3 were obtained from Kebai Biotechnology;
  • PE-labeled secondary antibody PE anti-human IgG Fc Antibody
  • test product codes are shown in Table 5
  • DBXT005-01 has stronger target antigen saturation ability than the control antibody SI-1X6.4; and its antigen saturation ability is also stronger than the corresponding parental monoclonal antibody.
  • the endocytic effect of the antibody drug targeting EGFR and HER3 of the present invention on T47D cells expressing both EGFR and HER3 was detected, and the endocytic activity was compared with that of the parent monoclonal antibody.
  • the cells were co-incubated with a fixed concentration of antibody drug and endocytosis indicator reagent pHrodo, and the endocytic ability of the antibody drug was evaluated by observing the fluorescent signal generated by pHrodo accompanying the antibody drug entering the cell at different time points.
  • T47D cells were cultured in RPMI-1640 medium containing 0.2 units/mL bovine insulin and 10% FBS.
  • Cell preparation Take T47D cells in the logarithmic growth phase, wash once with PBS, digest for 2-3 minutes, add 10-15mL cell culture medium after the cells are completely digested, wash out the digested cells, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells into a single cell suspension and adjust the live cell density to 3 ⁇ 10 5 cells/mL.
  • Cell plating Add 50 ⁇ L/well to a 96-well cell culture plate and culture the plate in an incubator for 48 hours (37° C., 5% CO 2 ).
  • the culture plate was incubated in an incubator for 48 hours (37° C., 5% CO 2 ).
  • Plate reading operation When the corresponding time point is reached, take out the 96-well cell culture plate, digest the cells, and read the cell number and fluorescence value through FACS.
  • the antibody drug targeting EGFR and HER3 of the present invention has a better endocytosis effect on T47D cells that simultaneously express EGFR and HER3.
  • the endocytosis efficiency of the antibody drug targeting EGFR and HER3 of the present invention was detected in the cell line NCI-H1975 expressing both EGFR and HER3 and the cell line CT26 overexpressing both EGFR and HER3 compared with other antibody drugs targeting both EGFR and HER3.
  • Fabfluor-pH was co-incubated, and the internalization ability of the antibody-drug conjugate was evaluated by continuously observing the changes in the fluorescence signal of living cells for 48 hours.
  • NCI-H1975 cells were cultured in RPMI1640 medium containing 10% FBS; EGFR and The culture medium of HER3 CT26 cell line is 1640+10% FBS+10 ⁇ g/mL puromycin+20 ⁇ g/mL blasticidin;
  • Cell preparation Take cells in the logarithmic growth phase, wash once with PBS, and digest for 2-3 minutes. After the cells are completely digested, add 10-15mL of cell culture medium to wash out the digested cells, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells into a single cell suspension and adjust the live cell density to 1x105 cells/mL.
  • Cell plating Add 50 ⁇ L/well of a 96-well cell culture plate and place the culture plate in an incubator for overnight culture (37° C., 5% CO 2 ).
  • Labeling of the antibody-drug conjugate to be tested For high-concentration antibody labeling, dilute the test sample stock solution to 240 nM 4 ⁇ working solution (final concentration 60 nM). The Fabfluor-pH stock solution was diluted to 720 nM 4 ⁇ working solution (final concentration 180 nM), the two were mixed thoroughly, and incubated at 37°C in the dark for 15 minutes. The low concentration antibody labeling was to dilute the test sample stock solution to 40 nM 4 ⁇ working solution (final concentration 10 nM), The Fabfluor-pH stock solution was diluted to a 120 nM 4 ⁇ working solution (final concentration 30 nM), the two were mixed thoroughly, and incubated at 37°C in the dark for 15 minutes.
  • Capture and analyze images transfer the labeled test sample working solution to the corresponding wells of the experimental plate, transfer the experimental plate to the Incucyte live cell analysis device, set the scanning and photography program, and use the Incucyte live cell analysis system to obtain images. Quantification is performed at intervals of 2 hours for 48 hours. The analysis results are expressed as: total fluorescence integrated intensity (RCU X ⁇ m 2 /image).
  • the antibody drug targeting EGFR and HER3 of the present invention has a better endocytosis effect on cells expressing both EGFR and HER3 at both high and low concentrations compared to the parent monoclonal antibody.
  • the antibody drug targeting EGFR and HER3 of the present invention has a better endocytosis effect than the control antibody SI-1X6.4 at both high and low concentrations.
  • the endocytosis efficiency of the antibody drug targeting EGFR and HER3 of the present invention was detected in cell lines A431 and NCI-H1975 expressing different levels of EGFR and HER3 and CT26 cell line overexpressing EGFR and HER3 at the same time.
  • Fabfluor-pH was co-incubated, and the internalization ability of the antibody-drug conjugate was evaluated by continuously observing the changes in the fluorescence signal of living cells for 24 hours.
  • A431 cells were cultured in DMEM medium containing 10% FBS; NCI-H1975 cells were cultured in RPMI1640 medium containing 10% FBS; the culture medium of CT26 cell lines stably transfected with EGFR and HER3 was 1640+10% FBS+10 ⁇ g/mL puromycin+20 ⁇ g/mL blasticidin;
  • Cell preparation Take cells in the logarithmic growth phase, wash once with PBS, and digest for 2-3 minutes. After the cells are completely digested, add 10-15mL of cell culture medium to wash out the digested cells, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells into a single cell suspension and adjust the live cell density to 1x105 cells/mL.
  • Cell plating Add 50 ⁇ L/well of a 96-well cell culture plate and place the culture plate in an incubator for overnight culture (37° C., 5% CO 2 ).
  • Label the antibody-drug conjugate to be tested Mix the test sample/reference sample working solution and the labeling reagent working solution in a 1:3 molar ratio and a 1:1 volume ratio. The mixture was mixed at a ratio of 1:1 and incubated in a cell culture incubator (37°C, 5% CO 2 ) in the dark for 15 minutes to allow the two to be fully coupled.
  • Capture and analyze images transfer the labeled test sample working solution to the corresponding wells of the experimental plate, transfer the experimental plate to the Incucyte live cell analysis device, set the scanning and photography program, and use the Incucyte live cell analysis system to obtain images. Quantification is performed at intervals of 2 hours for 24 hours. The analysis results are expressed as: total fluorescence integrated intensity (RCU X ⁇ m 2 /image).
  • the antibody DBXT005-01 targeting EGFR and HER3 of the present invention has a better endocytic effect on tumor cells expressing different levels of EGFR or HER3.
  • benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a solution of 27a (5.00 g, 43.0 mmol) and NaHCO 3 (10.9 g, 129 mmol) in DMF (50 mL), and the mixture was reacted at 25° C. for 17 hours.
  • HATU 74 mg, 0.19 mmol
  • DMF 2.5 mL
  • 27f 90 mg, 0.13 mmol
  • KI1 92 mg, 0.19 mmol
  • DIEA 50 mg, 0.39 mmol
  • LCMS showed that the basic reaction was complete.
  • MS-ESI m/z 830.3[M+H]+.
  • Source of Isotype IgG1 purchased from Bio-Tech (Taicang).
  • ADC-7 (DBXT001-01-X1-DAR4)
  • X1 (2.61 mg, 2.43 ⁇ mol) was dissolved in 1.75 mL DMA, added to the above solution, placed in a thermostatic stirrer, stirred at 60 rpm, and oscillated at 22 ° C for 2 hours to stop the reaction.
  • reaction solution was desalted and purified on an AKTA G-25 gel column (desalting column: HiPrep 26/10 Desalting column, 53 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-7 (30 mM histidine-hydrochloric acid pH 5.5; 68 mg, 22 mg/mL, yield: 97%), which was stored at -80 ° C.
  • X1 (4.30 mg, 4.00 ⁇ mol) was dissolved in 1.75 mL DMA, added to the above solution, placed in a thermostatic stirrer, stirred at 60 rpm, and oscillated at 22 ° C for 2 hours to stop the reaction.
  • reaction solution was desalted and purified on an AKTA G-25 gel column (desalting column: HiPrep 26/10 Desalting column, 53 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-8 (30 mM histidine-hydrochloric acid pH 5.5; 69 mg, 23 mg/mL, yield: 99%), which was stored at -80 ° C.
  • X1 (5.05 mg, 4.70 ⁇ mol) was dissolved in 1.75 mL DMA, added to the above solution, placed in a thermostatic stirrer, stirred at 60 rpm, and oscillated at 22 ° C for 2 hours to stop the reaction.
  • reaction solution was desalted and purified on an AKTA G-25 gel column (desalting column: HiPrep 26/10 Desalting column, 53 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-9 (30 mM histidine-hydrochloric acid pH 5.5; 68 mg, 22.4 mg/mL, yield: 97%), which was stored at -80 ° C.
  • ADC-4 (DBXT001-01-X2-DAR4)
  • reaction solution was desalted and purified on an AKTA G-25 gel column (desalting column: HiPrep 26/10 Desalting column, 53 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-4 (30 mM histidine-hydrochloric acid pH 5.5; 67.2 mg, 22 mg/mL, yield: 96%), which was stored at -80 ° C.
  • X2 (4.58 mg, 4.31 ⁇ mol) was dissolved in 1.75 mL DMA, added to the above solution, placed in a thermostatic stirrer, stirred at 60 rpm, and oscillated at 22 ° C for 2 hours to stop the reaction.
  • reaction solution was desalted and purified on an AKTA G-25 gel column (desalting column: HiPrep 26/10 Desalting column, 3 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration and concentration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-5 (30 mM histidine-hydrochloric acid pH 5.5; 66.5 mg, 25 mg/mL, yield: 95%), which was stored at -80 ° C.
  • X2 (5.49 mg, 5.17 ⁇ mol) was dissolved in 1.75 mL DMA, added to the above solution, placed in a constant temperature stirrer, stirred at 60 rpm, and reacted at 22 ° C for 2 hours to stop the reaction.
  • reaction solution was precipitated on an AKTA plate with G-
  • the product was purified by desalting with a 25 gel column (desalting column: HiPrep 26/10 Desalting column, 53 mL; elution phase: 30 mM histidine-hydrochloric acid pH 5.5), and concentrated by ultrafiltration with a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-6 (30 mM histidine-hydrochloric acid pH 5.5; 67.9 mg, 29.2 mg/mL, yield: 97%), which was stored at -80°C.
  • TCEP tri(2-carboxyethyl)phosphine
  • the antibody was slowly added with 200mM histidine-hydrochloric acid, pH 6.0 (so that the final concentration of histidine-hydrochloric acid in the coupling reaction solution is 20mM), dimethyl sulfoxide containing 0.1% v/v acetic acid, and linker-cytotoxin (Linker-payload) solution (10 equivalents, 10mM X2 solution (dimethyl sulfoxide containing 0.1% v/v acetic acid)) at room temperature.
  • the reaction system contained 3% v/v dimethyl sulfoxide (DMSO) containing 0.1% v/v acetic acid.
  • the linker-payload (2 equivalents, 10 mM X2 solution (dimethyl sulfoxide containing 0.1% v/v acetic acid) was added. After thorough mixing, the coupling reaction solution was shaken on a shaker at 22°C for 2 hours (shaker speed 60 rpm).
  • the ADC solution is replaced with a storage buffer (20 mM histidine-hydrochloric acid, pH 6.0) using a desalting column (40K). Samples are taken for inspection to obtain the ADC product.
  • ADC-14 (BL-B01D1, DAR8) refers to patent WO2023083381A1, as follows:
  • Antibody SI-1X6.4 was reduced with 15 equivalents of tris(2-carboxyethyl)phosphine (TCEP) in PBS 7.4 buffer (PBS 7.4, 2 mM EDTA, pH 7.4) and incubated on a shaker at 22°C for 17.5 hours (shaker speed 60 rpm). The reaction solution was used directly in the next coupling reaction without removing excess TCEP.
  • TCEP tris(2-carboxyethyl)phosphine
  • DMSO dimethyl sulfoxide
  • linker-payload 10 equivalents, 10 mM Ed-04 in DMSO
  • the reaction system contained 10% v/v dimethyl sulfoxide (DMSO).
  • the linker-payload (2 equivalents, 10 mM Ed-04 in DMSO) was added. After thorough mixing, the coupling reaction solution was shaken on a shaker at 22°C for 1.5 hours (shaker speed 60 rpm).
  • the corresponding bispecific antibody drug conjugates can be prepared by replacing the antibody clone DBXT001-01 in Table 8 with DBXT001-02-08, DBXT003-01-08 and DBXT004-01-08.
  • Example 7 In vivo efficacy evaluation of antibody drug conjugates on human esophageal cancer cell line OE-19 tumor-bearing mice expressing EGFR low expression HER3
  • ADC-6 antitumor effect of ADC-6 was evaluated after subcutaneous ectopic inoculation of OE-19 in mice to form transplanted tumors, and the antitumor effect was compared with that of the parental monoclonal antibody ADC.
  • Blank control group (control group): normal saline
  • ADC-1 treatment group: Day 0: 1 mg/kg; Day 12: 3 mg/kg
  • ADC-2 treatment group: Day 0: 1 mg/kg; Day 12: 3 mg/kg
  • mice 6-7 week old female BALB/c Nude mice, purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2mM L-Glutamine. OE-19 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • OE-19 cells 5 ⁇ 10 6 OE-19 cells were subcutaneously inoculated on the right back of the experimental mice. The cells were resuspended in 1:1 PBS and Matrigel (0.1 mL/mouse), and the tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups using StudyDirectorTM, and the test article was injected intravenously (iv) on the same day (day 0), once on the same day and once on the 12th day, for a total of 2 injections.
  • the dose of the treatment group on the same day was 1 mg/kg
  • the dose of the treatment group on the 12th day was 3 mg/kg.
  • the end point of the experiment was the 25th day after grouping, and the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • the anti-tumor efficacy of the compound is evaluated by T/C (%).
  • the percentage value of T/C (%) is an indicator reflecting the inhibition of tumor growth, and T and C represent the average tumor volume of the drug-treated group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a dosing group on a certain day, and T0 is the average tumor volume of this dosing group at the beginning of dosing; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V0 is the average tumor volume of the vehicle control group at the beginning of dosing.
  • Tumor volume is expressed as mean ⁇ standard error
  • bispecific ADC has a better tumor inhibition effect than monospecific ADC.
  • Example 8 In vivo efficacy evaluation of antibody drug conjugates on human non-small cell lung cancer cell line NCI-H441 tumor-bearing mice expressing EGFR and HER3
  • ADC-6 antitumor effect of ADC-6 was evaluated after subcutaneous ectopic inoculation of NCI-H441 in mice to form xenograft tumors, and compared with the antitumor effect of the parental monoclonal antibody ADC.
  • ADC-2 treatment group: 0.5 mg/kg
  • ADC-6 treatment group: 0.5 mg/kg
  • NCI-H441 (CL-00759) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. NCI-H441 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • mice were subcutaneously inoculated with 5 ⁇ 10 6 NCI-H441 cells on the right back.
  • the cells were resuspended in 1:1 PBS and Matrigel (0.1 mL/mouse), and the tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups using StudyDirectorTM, and the test article was injected intravenously (iv) on the same day (day 0), for a total of 1 injection.
  • the dose for the treatment group on that day was 0.5 mg/kg.
  • the end point of the experiment was the 35th day after grouping.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • the anti-tumor efficacy of the compound is evaluated by T/C (%).
  • the percentage value of T/C (%) is an index reflecting the inhibition of tumor growth, and T and C represent the average tumor volume of the drug-treated group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a drug-treated group on a certain day, T 0 is the average tumor volume of this drug-treated group at the beginning of drug administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration.
  • the two groups of samples were compared using the independent sample T-test (T-Test), and the data were analyzed using SPSS. P ⁇ 0.05 was considered to be significantly different.
  • the graphics software was GraphPad Prism.
  • Tumor volume is expressed as mean ⁇ standard error
  • the p value was calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents not significant).
  • dual-antibody ADC has better tumor inhibition effect than monoclonal antibody ADC.
  • Example 9 In vivo efficacy evaluation of antibody drug conjugates on human oral squamous cell carcinoma cell line CAL-27 bearing mice with high EGFR expression and low HER3 expression
  • ADC-6 antitumor effect of ADC-6 was evaluated after subcutaneous ectopic inoculation of CAL-27 cells into xenografts in mice, and compared with the antitumor effects of the parental monoclonal antibody ADC and EGFR monoclonal antibody.
  • Blank control group (control group): normal saline
  • ADC-2 treatment group: 3 mg/kg
  • ADC-6 treatment group: 3 mg/kg
  • mice Female NOD/Scid mice aged 6-7 weeks were purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • CAL-27 (CL-00599) cells were cultured in DMEM medium containing 10% fetal bovine serum. CAL-27 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • mice were subcutaneously inoculated with 1 ⁇ 10 7 CAL-27 cells on the right back.
  • the cells were resuspended in 1:1 PBS and Matrigel (0.1 mL/mouse). The tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups using StudyDirectorTM, and the test article was injected intravenously (iv) on the same day (day 0), once on the same day and on the 14th day, for a total of 2 injections, with a treatment group dose of 3 mg/kg.
  • the end point of the experiment was the 27th day after grouping, and the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • the anti-tumor efficacy of the compound is evaluated by T/C (%).
  • the percentage value of T/C (%) is an index reflecting the inhibition of tumor growth, and T and C represent the average tumor volume of the drug-treated group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a drug-treated group on a certain day, T 0 is the average tumor volume of this drug-treated group at the beginning of drug administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration.
  • the two groups of samples were compared using the independent sample T-test (T-Test), and the data were analyzed using SPSS. P ⁇ 0.05 was considered to be significantly different.
  • the graphics software was GraphPad Prism.
  • Tumor volume is expressed as mean ⁇ standard error
  • the p value was calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents not significant).
  • the dual-antibody ADC has a better tumor inhibition effect than EGFR monoclonal antibody or HER3 ADC, and is not inferior to EGFR ADC.
  • Example 10 Evaluation of the efficacy of antibody drug conjugates with different DAR values in NCI-H1975 and OE-19 tumor-bearing mice
  • mice 6-8 week old female Balb/c nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • NCI-H1975 (CL-00650) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, and OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2mM L-Glutamine.
  • Cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in nude mice.
  • mice were subcutaneously inoculated with 5 ⁇ 10 6 NCI-H1975 cells on the right back, and the cells were resuspended in PBS (0.1 mL/mouse). The tumor growth was observed regularly.
  • OE-19 cells 5 ⁇ 10 6 OE-19 cells were subcutaneously inoculated on the right back of the experimental mice. The cells were resuspended in 1:1 PBS and Matrigel (0.1 mL/mouse), and the tumor growth was observed regularly.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • the anti-tumor efficacy of the compound is evaluated by T/C (%).
  • the percentage value of T/C (%) is an index reflecting the inhibition of tumor growth, and T and C represent the average tumor volume of the drug-treated group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a drug-treated group on a certain day, T 0 is the average tumor volume of this drug-treated group at the beginning of drug administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration.
  • the two groups of samples were compared using the independent sample T-test (T-Test), and the data were analyzed using SPSS. P ⁇ 0.05 was considered to be significantly different.
  • the graphics software was GraphPad Prism.
  • mice in the NCI-H1975 blank control group reached the euthanasia criteria after 18 days, the statistical analysis data was incomplete, so the statistical analysis was performed on the 18th day when the number of animals was complete; similarly, some mice in the OE-19 blank control group reached the euthanasia criteria after 21 days, and the statistical analysis data was incomplete, so the statistical analysis was performed on the 21st day when the number of animals was complete;
  • Tumor volume is expressed as mean ⁇ standard error
  • T/C (%) TD / VD ⁇ 100
  • TGI (TGI (%) [1-( TD - T0 )/( VD - V0 )] ⁇ 100);
  • the p value was calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents not significant).
  • the dual-antibody coupling linker-cytotoxin X2 of the present invention targeting EGFR and HER3 has an anti-tumor effect, and the tumor inhibition increases with the increase of DAR value. The effect is enhanced.
  • Example 11 Comparison of the efficacy of the present antibody-drug conjugate and the control antibody conjugate in NCI-H1975 tumor-bearing mice
  • mice 6-8 week old female Balb/c nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • NCI-H1975 (CL-00650) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in nude mice.
  • mice were subcutaneously inoculated with 5 ⁇ 10 6 NCI-H1975 cells on the right back, and the cells were resuspended in 1:1 DPBS:Matrigel (0.2 mL/mouse). The tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups using StudyDirectorTM, and the day of grouping was designated as Day 0.
  • the specific dosing regimen is shown in Table 16.
  • the end point of the experiment was Day 26 after grouping.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • the anti-tumor efficacy of the compound is evaluated by T/C (%).
  • the percentage value of T/C (%) is an index reflecting the inhibition of tumor growth, and T and C represent the average tumor volume of the drug-treated group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a drug-treated group on a certain day, T 0 is the average tumor volume of this drug-treated group at the beginning of drug administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration.
  • the two groups of samples were compared using the independent sample T-test (T-Test), and the data were analyzed using SPSS. P ⁇ 0.05 was considered to be significantly different.
  • the graphics software was GraphPad Prism.
  • mice in the NCI-H1975 blank control group reached the euthanasia criteria after 17 days, the statistical analysis data was incomplete, so the statistical analysis was performed on the 17th day when the number of animals was complete;
  • Tumor volume is expressed as mean ⁇ standard error
  • p value is calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference)
  • the dual-antibody coupled linker-cytotoxin X2 (DAR8) of the present invention targeting EGFR and HER3 has a stronger anti-tumor effect than the control antibody SI-1X6.4 coupled linker-cytotoxin X2 (DAR8).
  • Example 12 Comparison of the efficacy of the present antibody-drug conjugate and the control antibody conjugate in OE-19 tumor-bearing mice
  • mice 6-8 week old female Balb/c nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2 mM L-Glutamine.
  • OE-19 cells 5 ⁇ 10 6 OE-19 cells were subcutaneously inoculated on the right back of the experimental mice. The cells were resuspended in 1:1 PBS and Matrigel (0.1 mL/mouse), and the tumor growth was observed regularly.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • the anti-tumor efficacy of the compound is evaluated by T/C (%).
  • the percentage value of T/C (%) is an index reflecting the inhibition of tumor growth, and T and C represent the average tumor volume of the drug-treated group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a drug-treated group on a certain day, T 0 is the average tumor volume of this drug-treated group at the beginning of drug administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration.
  • the two groups of samples were compared using the independent sample T-test (T-Test), and the data were analyzed using SPSS. P ⁇ 0.05 was considered to be significantly different.
  • the graphics software was GraphPad Prism.
  • Tumor volume is expressed as mean ⁇ standard error
  • the p value is calculated based on the tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference)
  • the dual-antibody coupled linker-cytotoxin X2 (DAR8) of the present invention targeting EGFR and HER3 can overcome EGFR resistance and has a stronger tumor inhibition effect than the control antibody SI-1X6.4 coupled linker-cytotoxin X2 (DAR8).
  • Example 13 Evaluation of the efficacy of antibody-drug conjugate linker-cytotoxin X1 in NCI-H1975 tumor-bearing mice
  • the tested drugs and dosing schedules are shown in Table 20.
  • NCI-H1975 (Cell Bank of Chinese Academy of Sciences) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, and cells in the exponential growth phase were collected and resuspended in DPBS to a suitable concentration for subcutaneous tumor inoculation in nude mice.
  • mice were subcutaneously inoculated with 5 ⁇ 10 6 NCI-H1975 cells on the right back (the DPBS used to resuspend the cells was mixed with BD Matrigel matrix gel in a ratio of 1:1, Catalogue: 356234, 0.2 mL/mouse), and the tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups using StudyDirectorTM.
  • the day of grouping was designated as Day 0.
  • the specific dosing regimen is shown in Table 20.
  • the end point of the experiment was the 20th day after grouping.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • the anti-tumor efficacy of the compound is evaluated by T/C (%).
  • the percentage value of T/C (%) is an index reflecting the inhibition of tumor growth, and T and C represent the average tumor volume of the drug-treated group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a drug-treated group on a certain day, T 0 is the average tumor volume of this drug-treated group at the beginning of drug administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration.
  • the two groups of samples were compared using the independent sample T-test (T-Test), and the data were analyzed using SPSS. P ⁇ 0.05 was considered to be significantly different.
  • the graphics software was GraphPad Prism.
  • mice in the NCI-H1975 blank control group reached the euthanasia criteria after 18 days, the statistical analysis data was incomplete, so the statistical analysis was performed on the 18th day when the number of animals was complete;
  • Tumor volume is expressed as mean ⁇ standard error
  • the p value was calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents not significant).
  • the antibody-drug conjugate of the dual-antibody coupling linker-cytotoxin X1 of the present invention targeting EGFR and HER3 has a significant effect of inhibiting tumor growth.
  • Example 14 Comparison of the efficacy of the dual-antibody ADC of the present invention and the control ADC in a mouse model resistant to EGFR ADC
  • test drugs and dosing regimens are shown in Table 22.
  • mice 6-7 week old female BALB/c Nude mice, purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2mM L-Glutamine. OE-19 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • OE-19 cells 5 ⁇ 10 6 OE-19 cells were subcutaneously inoculated on the right back of the experimental mice. The cells were resuspended in 1:1 PBS and Matrigel (0.1 mL/mouse), and the tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups using StudyDirectorTM and injected with the test article intravenously (iv) starting on the same day (day 1) for a total of one injection.
  • the end point of the experiment was the 23rd day after grouping.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • the anti-tumor efficacy of the compound is evaluated by T/C (%).
  • the percentage value of T/C (%) is an index reflecting the inhibition of tumor growth, and T and C represent the average tumor volume of the drug-treated group and the control group on a certain day, respectively.
  • TGI (%) [1-(T i -T 0 )/(V i -V 0 )] ⁇ 100, where Ti is the average tumor volume of a drug-treated group on a certain day, T 0 is the average tumor volume of this drug-treated group at the beginning of drug administration; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of drug administration.
  • Tumor volume is expressed as mean ⁇ standard error
  • the p value was calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents not significant).
  • Example 15 Comparison of the efficacy of the bispecific ADC of the present invention and the control ADC in a mouse model with high EGFR expression and low HER3 expression
  • test drugs and dosing regimens are shown in Table 244.
  • mice Female NCG mice aged 6-8 weeks were purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • A431 (CRL-1555) cells were cultured in DMEM containing 10% fetal bovine serum and 2 mM L-Glutamine. A431 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • mice were subcutaneously inoculated with 5 ⁇ 10 6 A431 cells on the right back, and the tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups and injected with the test article once intravenously (iv) starting on the same day (day 0).
  • the end point of the experiment was the 21st day after grouping.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • TGI (%) [1-(T i /T 0 )/(V i /V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain dosing group on a certain day, T 0 is the average tumor volume of this dosing group at the beginning of dosing; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of dosing.
  • Tumor volume is expressed as mean ⁇ standard error
  • p value is calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference). Comparison of tumor volume between each experimental group and the blank control group.
  • the p value is calculated based on the tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference). Comparison of tumor volume between the dual-antibody ADC and control ADC test groups in this example.
  • the antibody conjugate of the present invention has a better tumor inhibition effect than the control ADC.
  • the control ADC cannot inhibit tumor growth at this dosage concentration.
  • the mice in each administration group tolerated well and there was no significant decrease in body weight.
  • Example 16 Comparison of the efficacy of the dual-antibody ADC of this example and the control ADC in a xenograft mouse model of a human colon cancer cell line that does not express EGFR but only expresses HER3
  • test drugs and dosing regimens are shown in Table 26.
  • mice Female NCG mice aged 6-8 weeks were purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • SW620 (CCL-227) cells were cultured in DMEM containing 10% fetal bovine serum and 2 mM L-Glutamine. SW620 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • the experimental mice were subcutaneously inoculated with 5 ⁇ 10 6 SW620 cells on the right back, and the tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups and injected with the test article intravenously (iv) starting on the same day (day 0).
  • the end point of the experiment was the 59th day after grouping.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • TGI (%) [1-(T i /T 0 )/(V i /V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain dosing group on a certain day, T 0 is the average tumor volume of this dosing group at the beginning of dosing; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of dosing.
  • Tumor volume is expressed as mean ⁇ standard error
  • TGI (%) [1-(T 31 /T 0 )/(V 31 /V 0 )] ⁇ 100);
  • p value is calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference). Comparison of tumor volume between each experimental group and the blank control group.
  • the p value is calculated based on the tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference). Comparison of tumor volume between the dual-antibody ADC and control ADC test groups in this example.
  • N/A means that the mice in the control group were euthanized on day 31 according to animal welfare principles.
  • Example 17 Comparison of the efficacy of the dual-antibody ADC of the present invention and the control ADC in a xenograft tumor mouse model of a human colon cancer cell line expressing low expression of HER3 in EGFR
  • test drugs and dosing regimens are shown in Table 28.
  • mice Female NCG mice aged 6-8 weeks were purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • SW48 (CCL-231) cells were cultured in DMEM containing 10% fetal bovine serum and 2 mM L-Glutamine.
  • SW48 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • mice were subcutaneously inoculated with 1 ⁇ 10 7 SW48 cells on the right back, and the tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups and injected with the test article intravenously (iv) starting on the same day (day 0).
  • the end point of the experiment was the 45th day after grouping.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 5 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • TGI (%) [1-(T i /T 0 )/(V i /V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain dosing group on a certain day, T 0 is the average tumor volume of this dosing group at the beginning of dosing; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of dosing.
  • Tumor volume is expressed as mean ⁇ standard error
  • TGI (%) [1-(T 21 -T 0 )/(V 21 -V 0 )] ⁇ 100);
  • p value is calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference). Comparison of tumor volume between each experimental group and the blank control group.
  • the p value is calculated based on the tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference). Comparison of tumor volume between the dual-antibody ADC and control ADC test groups in this example.
  • N/A means that the mice in the control group were euthanized on the 21st day according to animal welfare principles.
  • Example 18 Comparison of the efficacy of the bispecific ADC of the present invention and the control ADC on osimertinib-resistant human non-small cell lung cancer cell line xenograft tumor mouse model
  • test drugs and dosing regimens are shown in Table 30.
  • mice Female NOD/SCID mice aged 6-7 weeks were purchased from Jicui Yaokang Biotechnology Co., Ltd.
  • NCI-H1975 EGFR L858R/T790M/C797S (CL-01195) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 100 ⁇ g/mL Hygromycin.
  • NCI-H1975 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in NOD/SCID mice.
  • 1 ⁇ 10 7 NCI-H1975 EGFR L858R/T790M/C797S cells were subcutaneously inoculated on the right back of the experimental mice, and the cells were resuspended in 1:1 PBS and Matrigel (0.2mL/mouse), and the tumor growth was observed regularly.
  • the tumor-bearing mice were randomly divided into groups and injected with the test article intravenously (iv) starting on the same day (day 0).
  • the end point of the experiment was the 35th day after grouping.
  • the tumor volume and body weight were measured twice a week and the data were recorded.
  • mice There were 6 mice in each control group or treatment group.
  • the tumor inhibition rate was calculated by measuring the tumor volume.
  • TGI (%) [1-(T i /T 0 )/(V i /V 0 )] ⁇ 100, where Ti is the average tumor volume of a certain dosing group on a certain day, T 0 is the average tumor volume of this dosing group at the beginning of dosing; Vi is the average tumor volume of the vehicle control group on a certain day (the same day as Ti ), and V 0 is the average tumor volume of the vehicle control group at the beginning of dosing.
  • Tumor volume is expressed as mean ⁇ standard error
  • TGI (%) [1-(T 35 /T 0 )/(V 35 /V 0 )] ⁇ 100);
  • p value is calculated based on tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference). Comparison of tumor volume between each experimental group and the blank control group.
  • the p value is calculated based on the tumor volume (* represents p ⁇ 0.05, ** represents p ⁇ 0.01, *** represents p ⁇ 0.001, ns represents no significant difference). Comparison of tumor volume between the dual-antibody ADC and control ADC test groups in this example.

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Abstract

提供一种双特异性抗体、其药物偶联物及其用途。所述双特异性抗体包含EGFR结合结构域和HER3结合结构域。提供的双特异性抗体及其药物偶联物具有很好的内吞效应、增殖抑制活性、肿瘤生长抑制活性及良好的体内安全性。

Description

一种双特异性抗体、其药物偶联物及其用途
本申请要求申请日为2023/7/19的中国专利申请2023108956544、申请日为2024/3/4的中国专利申请2024102449540和申请日为2024/7/9的中国专利申请2024109141579的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明生物技术领域，具体涉及一种包含EGFR结合结构域和HER3结合结构域的双特异性抗体、其药物偶联物及其用途。
背景技术
表皮生长因子受体(EGFR)是一个巨大的跨膜糖蛋白，分子量约为170KDa，属于ErbB受体家族的一个成员。EGFR受体本身是一种酪氨酸激酶，当与配体如EGF、TNF-a等结合后可形成二聚体，通过传递磷酸化作用激活下游信号(如MAPK，PI3K，Stat等通路)，从而维持细胞生长，促进细胞分裂增殖。由于ErbB家族受体的保守性，EGFR还能与家族其他蛋白(如HER2，HER3，HER4)形成异源二聚体，从而更广泛地调控细胞的生长。
HER3是ErbB家族的成员，在细胞增殖、肿瘤转移和耐药中起关键作用。虽然靶向EGFR和HER3的药物在缓解多种癌症方面表现出巨大的临床益处，但之前开发用于癌症治疗的抗HER3抗体的努力屡屡失败。
抗体药物偶联物(antibody drug conjugate，ADC)由抗体或其抗原结合片段(靶向)、接头和小分子药物三部分组成。抗体或其抗原结合片段经由可裂解或不可裂解的接头与具有生物活性例如细胞毒性的小分子药物如细胞毒素缀合，充分利用了抗体或其抗原结合片段靶向于感兴趣的细胞(靶向细胞)的特异性或结合高表达抗原的特异性以及小分子药物的高效性，降低或避免了对非靶向细胞的毒副作用。这意味着，与传统肿瘤化疗药物相比，用于肿瘤的抗体药物偶联物能精准地靶向肿瘤细胞并降低对非肿瘤细胞的影响。
本领域仍需要在亲和力、特异性等方面优异的双特异性抗体药物偶联物。
发明内容
本发明所要解决的技术问题是为了克服现有技术中靶向EGFR和HER3的双特异性抗体药物偶联物较少的缺陷，而提供了一种双特异性抗体、其药物偶联物及其用途。本发明的双特异性抗体药物偶联物具有很好的内吞效应、增殖抑制活性、肿瘤生长抑制活性及良好的体内安全性。
本发明主要是通过以下技术手段解决上述技术问题的。
为了解决上述技术问题，本发明第一方面提供了一种包含EGFR结合结构域和HER3结合结构域的双特异性抗体，所述EGFR结合结构域包括重链可变区VH1和轻链可变区VL1，所述HER3结合 结构域包含重链可变区VH2和轻链可变区VL2；其中，所述VH1包含的H1CDR1、H1CDR2和H1CDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示；所述VL1包含的L1CDR1、L1CDR2和L1CDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；所述VH2包含的H2CDR1、H2CDR2和H2CDR3的氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示；所述VL2包含的L2CDR1、L2CDR2和L2CDR3氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示。
在本发明的一些实施方案中，所述H2CDR2的氨基酸序列如SEQ ID NO:77或78所示。
在本发明的一些优选实施方案中，所述VH1包含的框架区H1FR1、H1FR2、H1FR3和H1FR4的氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；
所述VL1包含的框架区L1FR1、L1FR2、L1FR3和L1FR4的氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；
所述VH2包含的框架区H2FR1的氨基酸序列如SEQ ID NO:21所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，例如在SEQ ID NO:21上具有E16D突变；框架区H2FR2和H2FR4的氨基酸序列分别如SEQ ID NO:22和SEQ ID NO:24所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；框架区H2FR3的氨基酸序列如SEQ ID NO:23所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，例如在SEQ ID NO:23上具有S18D突变；
所述VL2包含的框架区L2FR1的氨基酸序列如SEQ ID NO:25所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，例如在SEQ ID NO:25上具有S9D和V15L突变，或S7E突变；框架区L2FR2、L2FR3和L2FR4的氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:26和SEQ ID NO:27所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性。
在本发明的一些实施方案中，所述VH1的氨基酸序列如SEQ ID NO:28所示，所述VL1的氨基酸序列如SEQ ID NO:29所示，所述VH2的氨基酸序列如SEQ ID NO:30、SEQ ID NO:79或SEQ ID NO:80所示，所述VL2的氨基酸序列如SEQ ID NO:31、SEQ ID NO:81或SEQ ID NO:82所示。
在本发明的一些优选实施方案中，所述双特异性抗体的VH1、VL1、VH2和VL2的氨基酸序列分别如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:31所示；或，分别如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:79和SEQ ID NO:81所示；或，分别如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:79和SEQ ID NO:31所示；或，分别如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:80和SEQ ID NO:82所示。
在本发明的一些实施方案中，所述EGFR结合结构域和HER3结合结构域还分别包括轻链恒定区和重链恒定区，所述EGFR结合结构域包含轻链恒定区CL1和重链恒定区HC1，所述HER3结合结 构域包含轻链恒定区CL2和重链恒定区HC2；其中，CL1和CL2的氨基酸序列分别如SEQ ID NO:32或SEQ ID NO:33所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，且CL1和CL2的氨基酸序列不为同一序列；和/或，HC1包含C1H1和Fc1，HC2包含C2H1和Fc2；其中，C1H1和C2H1的氨基酸序列如SEQ ID NO:34或SEQ ID NO:35所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，且C1H1和C2H1的氨基酸序列不为同一序列；Fc1和Fc2的氨基酸序列为如SEQ ID NO:36所示的氨基酸序列的变体序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，例如在SEQ ID NO:36上具有T146W，或S134C和T146W，或T146S、L148A和Y187V，或Y349C、T366S、L368A和Y407V突变，且Fc1和Fc2的氨基酸序列不为同一序列。
在本发明的一些优选实施方案中，CL1和CL2的氨基酸序列分别如SEQ ID NO:32或SEQ ID NO:33所示，C1H1和C2H1的氨基酸序列如SEQ ID NO:34或SEQ ID NO:35所示，和，Fc1和Fc2的氨基酸序列为如SEQ ID NO:36所示的氨基酸序列的变体序列，例如在SEQ ID NO:36上具有T146W，或S134C和T146W，或T146S、L148A和Y187V，或Y349C、T366S、L368A和Y407V突变。
在本发明的一些更优选实施方案中，Fc1和Fc2之间通过铰链区的二硫键和Knob into Hole结构连接；所述Fc1为knob-Fc，所述Fc2为hole-Fc，或Fc2为knob-Fc，Fc1为hole-Fc。
在本发明的一些进一步更优选实施方案中，C1H1和Fc1、C2H1和Fc2之间由铰链区相连，其中，所述铰链区的氨基酸序列如SEQ ID NO:89所示。
在本发明的一些优选实施方案中，所述EGFR结合结构域包含轻链恒定区CL1和重链恒定区HC1，所述HER3结合结构域包含重链恒定区HC2；其中，CL1的氨基酸序列如SEQ ID NO:32所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；和/或，HC1包含C1H1和Fc1，HC2包含Fc2；其中，C1H1的氨基酸序列如SEQ ID NO:34所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；Fc1和Fc2的氨基酸序列为如SEQ ID NO:36所示氨基酸序列的变体序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性。
在本发明的一些优选实施方案中，CL1的氨基酸序列如SEQ ID NO:32所示，C1H1的氨基酸序列如SEQ ID NO:34所示，和，Fc1和Fc2的氨基酸序列分别为如SEQ ID NO:93和94所示；
在本发明的一些优选实施方案中，Fc1和Fc2之间通过铰链区的二硫键和Knob into Hole结构连接，所述Fc1为knob-Fc，所述Fc2为hole-Fc，或Fc2为knob-Fc，Fc1为hole-Fc；
在本发明的一些优选实施方案中，C1H1和Fc1之间由氨基酸序列如SEQ ID NO:89所示的铰链区相连；VL2和VH2之间由氨基酸序列如SEQ ID NO:95所示的铰链区相连；VH2和Fc2之间由氨基酸序列如SEQ ID NO:96所示的铰链区相连。
在本发明的一些优选实施方案中，所述双特异性抗体包含重链H1、轻链L1和重链H2，所述H1的氨基酸序列如SEQ ID NO:37所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，和/或，所述L1的氨基酸序列如SEQ ID NO:38所示或与其具有至少90％、 91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，和/或，所述H2的氨基酸序列如SEQ ID NO:90所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；
在本发明的一些优选实施方案中，所述双特异性抗体包含重链H1、轻链L1和重链H2，所述重链H1、轻链L1和重链H2的氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:90所示，为DBXT005-01。在本发明的一些优选实施方案中，重链H2为Fc+scFv结构。
在本发明的一些实施方案中，所述双特异性抗体包含重链H1、轻链L1、重链H2和轻链L2。
在本发明的一些优选实施方案中，所述双特异性抗体的重链H1、轻链L1、重链H2和轻链L2的氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示，为DBXT001-01；或，
分别如SEQ ID NO:41、SEQ ID NO:38、SEQ ID NO:42和SEQ ID NO:40所示，为DBXT001-02；或，
分别如SEQ ID NO:43、SEQ ID NO:38、SEQ ID NO:44和SEQ ID NO:40所示，为DBXT001-03；或，
分别如SEQ ID NO:45、SEQ ID NO:38、SEQ ID NO:46和SEQ ID NO:40所示，为DBXT001-04；或，
分别如SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49和SEQ ID NO:50所示，为DBXT001-05；或，
分别如SEQ ID NO:51、SEQ ID NO:48、SEQ ID NO:52和SEQ ID NO:50所示，为DBXT001-06；或，
分别如SEQ ID NO:53、SEQ ID NO:48、SEQ ID NO:54和SEQ ID NO:50所示，为DBXT001-07；或，
分别如SEQ ID NO:55、SEQ ID NO:48、SEQ ID NO:56和SEQ ID NO:50所示，为DBXT001-08；或，
分别如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:57和SEQ ID NO:58所示，为DBXT002-01；或，
分别如SEQ ID NO:41、SEQ ID NO:38、SEQ ID NO:59和SEQ ID NO:58所示，为DBXT002-02；或，
分别如SEQ ID NO:43、SEQ ID NO:38、SEQ ID NO:60和SEQ ID NO:58所示，为DBXT002-03；或，
分别如SEQ ID NO:45、SEQ ID NO:38、SEQ ID NO:61和SEQ ID NO:58所示，为DBXT002-04；或，
分别如SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:62和SEQ ID NO:63所示，为DBXT002-05；或，
分别如SEQ ID NO:51、SEQ ID NO:48、SEQ ID NO:64和SEQ ID NO:63所示，为DBXT002-06；或，
分别如SEQ ID NO:53、SEQ ID NO:48、SEQ ID NO:65和SEQ ID NO:63所示，为DBXT002-07；或，
分别如SEQ ID NO:55、SEQ ID NO:48、SEQ ID NO:66和SEQ ID NO:63所示，为DBXT002-08；或，
分别如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:57和SEQ ID NO:40所示，为DBXT003-01；或，
分别如SEQ ID NO:41、SEQ ID NO:38、SEQ ID NO:59和SEQ ID NO:40所示，为DBXT003-02；或，
分别如SEQ ID NO:43、SEQ ID NO:38、SEQ ID NO:60和SEQ ID NO:40所示，为DBXT003-03；或，
分别如SEQ ID NO:45、SEQ ID NO:38、SEQ ID NO:61和SEQ ID NO:40所示，为DBXT003-04；或，
分别如SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:62和SEQ ID NO:50所示，为DBXT003-05；或，
分别如SEQ ID NO:51、SEQ ID NO:48、SEQ ID NO:64和SEQ ID NO:50所示，为DBXT003-06；或，
分别如SEQ ID NO:53、SEQ ID NO:48、SEQ ID NO:65和SEQ ID NO:50所示，为DBXT003-07；或，
分别如SEQ ID NO:55、SEQ ID NO:48、SEQ ID NO:66和SEQ ID NO:50所示，为DBXT003-08；或，
分别如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:67和SEQ ID NO:68所示，为DBXT004-01；或，
分别如SEQ ID NO:41、SEQ ID NO:38、SEQ ID NO:69和SEQ ID NO:68所示，为DBXT004-02；或，
分别如SEQ ID NO:43、SEQ ID NO:38、SEQ ID NO:70和SEQ ID NO:68所示，为DBXT004-03；或，
分别如SEQ ID NO:45、SEQ ID NO:38、SEQ ID NO:71和SEQ ID NO:68所示，为DBXT004-04；或，
分别如SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:72和SEQ ID NO:73所示，为DBXT004-05；或，
分别如SEQ ID NO:51、SEQ ID NO:48、SEQ ID NO:74和SEQ ID NO:73所示，为DBXT004-06；或，
分别如SEQ ID NO:53、SEQ ID NO:48、SEQ ID NO:75和SEQ ID NO:73所示，为DBXT004-07；或，
分别如SEQ ID NO:55、SEQ ID NO:48、SEQ ID NO:76和SEQ ID NO:73所示，为DBXT004-08。
DBXT001系包括DBXT001-01～DBXT001-08，DBXT002系包括DBXT002-01～DBXT002-08，DBXT003系包括DBXT003-01～DBXT003-0108，DBXT004系包括DBXT004-01～DBXT004-08。
为了解决上述技术问题，本发明第二方面提供了一种分离的核酸，所述核酸编码如本发明第一方面所述的双特异性抗体。
为了解决上述技术问题，本发明第三方面提供了一种重组表达载体，所述重组表达载体包含如本发明第二方面所述的核酸。
为了解决上述技术问题，本发明第四方面提供了一种转化体，所述转化体在宿主细胞中包含如本发明第二方面所述的核酸或如本发明第三方面所述的重组表达载体。
在本发明一些较佳实施方案中，所述宿主细胞为真核细胞，优选为哺乳动物细胞，例如CHO细胞。
为了解决上述技术问题，本发明第五方面提供了一种制备如本发明第一方面所述的双特异性抗体的方法，所述方法包括培养如本发明第四方面所述的转化体，获得所述双特异性抗体。
HC1为第一重链的重链恒定区，HC2为第二重链的重链恒定区。VH1、C1H1和Fc1分别为第一重链H1的VH、CH1和Fc区域，CL1和VL1分别为第一轻链L1的CL和VL区域，VH2、C2H1和Fc2分别为第二重链H2的VH、CH1和Fc区域，CL2和VL2分别为第二轻链L2的CL和VL区域。L1FR1、L1FR2、L1FR3和L1FR4分别为第一轻链的轻链可变区的框架区，H1FR1、H1FR2、H1FR3和H1FR4分别为第一重链的重链可变区的框架区，L2FR1、L2FR2、L2FR3和L2FR4分别为第二轻链的轻链可变区的框架区，H2FR1、H2FR2、H2FR3和H2FR4分别为第二重链的重链可变区的框架区。
抗体序列使用Kabat编号系统。
本发明还提供了一种双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其包含以下片段：包含EGFR结合结构域和HER3结合结构域的双特异性抗体或其抗原结合片段、接头单元L和细胞毒性药物；其中，所述双特异性抗体或其抗原结合片段如本发明任一项所述。
在一些实施方式中，所述细胞毒性药物为喜树碱及其衍生物。
在一些实施方式中，所述细胞毒性药物为如式(A-1)所示结构、其互变异构体、对映异构体、或非对映异构体，
其中，
M为-L2-L1-C(O)-；
L2选自-NH-、O和S，且L2与所述的接头单元L连接；
L1为-(C(R1a)(R1b))m-CH2-、C3-C6饱和的亚环烷基或3-6元饱和的亚杂环基，所述C3-C6饱和的亚环烷基和所述3-6元饱和的亚杂环基各自独立地任选被一个或多个R2a取代；
m选自1、2、3和4；所述的3-6元饱和的亚杂环基中的杂原子各自独立地为N、O和S，杂原子数为1、2或3个；
R1a和R1b各自独立地选自氢、卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代；
R2a选自卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代。
在本发明某些优选实施方案中，所述的如式(A-1)、(A-2)、(A-2a)或(A-2b)所示的化合物中的某些基团如下定义，未提及的基团同本发明任一方案所述(简称“在一些实施方案中”)。
在一些实施方案中，L2优选为-O-或-S-，更优选为-O-。
在一些实施方案中，L1为-(C(R1a)(R1b))m-CH2-；R1a选自：氢、卤素和C1-C6烷基；R1b选自：氢、卤素和C1-C6烷基。
在一些实施方案中，L1为-(C(R1a)(R1b))m-CH2-；R1a为C1-C6烷基，优选为C1-C3烷基；R1b选自：氢和C1-C6烷基，优选选自：氢和C1-C3烷基。
在一些实施方案中，L1为-(C(R1a)(R1b))m-CH2-；R1a为-CH3；R1b选自：氢和-CH3。
在一些实施方案中，L1为-(C(R1a)(R1b))m-CH2-；m为1或2，优选为1。
在一些实施方案中，L1选自：
在一些实施方案中，L1为C3-C6饱和的亚环烷基或3-6元饱和的亚杂环基，优选为C3-C6饱和的亚环烷基，其中所述C3-C6饱和的亚环烷基和3-6元饱和的亚杂环基各自独立地任选被一个或多个R2a取代，R2a各自独立地选自：卤素和C1-C6烷基。
在一些实施方案中，L1为任选被一个或多个R2a取代的：环丙基、环丁基、环戊基或环己基；R2a各自独立地选自：卤素和C1-C6烷基。
在一些实施方案中，L1选自：
在一些实施方案中，L1和X1的定义中，所述C3-C6亚环烷基为环丁基或环己基，优选为环丁基。
在一些实施方案中，L1的定义中，所述3-6元亚杂环基的杂原子各自独立地为N或O，杂原子数优选为1或2个。
在一些实施方案中，R1a、R1b和R2a的定义中，所述C1-C6烷基为C1-C3烷基，优选为甲基。
在一些实施方案中，R、R1a、R1b和R2a的定义中，所述卤素为F、Cl、Br或I，优选为F、Cl或Br。
在一些实施方式中，所述如式(A-1)所示结构中，
M为-L2-L1-C(O)-；
L2为-O-；
L1为-(C(R1a)(R1b))m-CH2-或C3-C6饱和的亚环烷基，所述C3-C6饱和的亚环烷基任选被一个或多个R2a取代；
m选自1或2；
R1a和R1b各自独立地选自氢、卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代；
R2a选自卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代。
在一些实施方式中，M为：
在一些实施方案中，M为
在一些实施方案中，所述细胞毒性药物选自以下任一结构：
在一些实施方案中，所述接头单元L为-La-Lb-Lc-；其中Lc与所述的细胞毒性药物连接；
-La-为或-C1-8亚烷基-C(O)-，优选为或-C1-6亚烷基-C(O)-，更优选为-La-定义中的各基团片段优选右端与所述Lb连接；
-La-进一步优选为其中a端和Ab相连，b端与Lb相连
-Lb-为-1至6个天然氨基酸的多肽-NH-，优选为-2至4个天然氨基酸的多肽-NH-，更优选选自以下任一结构：
进一步优选为 -Lb-定义中的各基团片段优选右端与所述Lc连接；
-Lb-更优选为其中c端和La相连，d端与Lc相连；
-Lc-为C1-6亚烷基，优选为C1-3亚烷基，更优选为
在一些实施方案中，本发明所述的双特异性抗体药物偶联物，其中所述接头单元L为 优选为
在一些实施方案中，本发明所述的双特异性抗体药物偶联物，其结构如式(A-2)所示：
其中，p表示平均连接数，且p为1到10中任一整数或小数；优选为3到9中任一整数或小数； 例如为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8；
Ab和M分别如本发明任一实施方案所定义；L为本发明任一实施方案所述的接头单元L。
在一些实施方案中，本发明所述的双特异性抗体药物偶联物，其结构如式(A-2)所示：
其中，
p表示平均连接数，且p为1到10中任一整数或小数；优选为3到9中任一整数或小数；例如为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8；
Ab为如本发明任一实施方案所述的双特异性抗体或其抗原结合片段；
L为本发明任一实施方案所述的接头单元L；
M为-L2-L1-C(O)-；
L2为-O-或-S-，且L2与L连接；
L1为-(C(R1a)(R1b))m-CH2-、C3-C6饱和的亚环烷基或3-6元饱和的亚杂环基，所述C3-C6饱和的亚环烷基和3-6元饱和的亚杂环基各自独立地任选被一个或多个R2a取代；
m为1、2、3或4；所述的3-6元饱和的杂环基中的杂原子各自独立地为N、O或S，杂原子数为1、2或3个；
R1a和R1bR2a各自独立地为氢、卤素、羟基、氨基或C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代；
R2a选自卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代。
在一些实施方案中，本发明所述的双特异性抗体药物偶联物，其结构如式(A-2a)或(A-2b)所示：

其中，
p表示平均连接数，且p选自1到10中任一的整数或小数，优选3到9中任一整数或小数；例如为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8；
Ab为如本发明任一实施方案所述的双特异性抗体或其抗原结合片段；
L2为-NH-、O或S，优选为-O-或-S-；更优选为-O-；
X1选自任选被1、2或3个R2a取代的C3-C6亚环烷基；
X2选自-(C(R1a)(R1b))m-CH2-；
m选自1或2；
R1a和R1b各自独立为氢、卤素或被1、2或3个卤素任选取代的C1-C6烷基；
R2a选自卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代。
在一些实施方案中，本发明所述的双特异性抗体药物偶联物，其中所述双特异性抗体药物偶联物选自以下：



其中，
p表示平均连接数，且p为1到10中任一整数或小数，优选3到9中任一整数或小数；例如为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8；
Ab为如本发明任一实施方案所述的双特异性抗体或其抗原结合片段。
在一些实施方案中，Ab选自本发明的包含EGFR结合结构域和HER3结合结构域的双特异性抗体DBXT001系(DBXT001-01～08)、DBXT002系(DBXT002-01～08)、DBXT003系(DBXT003-01～08) 和DBXT004系(DBXT004-01～08)；优选地，Ab选自本发明的包含EGFR结合结构域和HER3结合结构域的双特异性抗体DBXT001系、DBXT002系和DBXT005-01；更优选地，Ab选自本发明的包含EGFR结合结构域和HER3结合结构域的双特异性抗体DBXT001系和DBXT005-01；进一步优选地，Ab选自本发明的包含EGFR结合结构域和HER3结合结构域的双特异性抗体DBXT001-01和DBXT005-01。
在一些实施方案中，Ab为本发明的包含EGFR结合结构域和HER3结合结构域的双特异性抗体DBXT005-01。
在一些实施方案中，所述双特异性抗体药物偶联物选自以下任一结构：
其中，p表示平均连接数，且p为1到10中任一整数或小数，优选3到9中任一整数或小数；例如为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8。
在一些实施方案中，所述双特异性抗体药物偶联物选自以下任一结构：



本发明的包含EGFR结合结构域和HER3结合结构域的双特异性抗体DBXT001(DBXT001-01～08)、DBXT002(DBXT002-01～08)、DBXT003(DBXT003-01～08)和DBXT004(DBXT004-01～08)的氨基酸序列如本发明序列表所示。
本发明抗体CDR的编号方法为：Kabat编号。
在一些实施方案中，所述双特异性抗体药物偶联物为：
其中，p表示平均连接数，且p为1到10中任一整数或小数，优选3到9中任一整数或小数，更优选为4到6中任一整数或小数；例如为5.99；
DBXT005-01为抗EGFR和HER3的双特异性抗体，其重链H1氨基酸序列如SEQ ID NO:37所示，轻链L1的氨基酸序列如SEQ ID NO:38所示，重链H2的氨基酸序列如SEQ ID NO:90所示。
在一些实施方案中，所述双特异性抗体药物偶联物选自如下偶联物：
其中，p1表示连接数，且p1为1到10中任一整数，优选3到9中任一整数，更优选为4到6中任一整数；例如为4、5或6。
DBXT005-01为抗EGFR和HER3的双特异性抗体，其重链H1氨基酸序列如SEQ ID NO:37所示，轻链L1的氨基酸序列如SEQ ID NO:38所示，重链H2的氨基酸序列如SEQ ID NO:90所示。
在一些实施方案中，本发明所述平均连接数p可以为1到10中任一整数或小数。例如，所述平均连接数p可以为3到9中任一整数或小数。例如，所述平均连接数p可以为1到2、2到3、3到4、4到5、5到6、6到7、7到8、8到9、9到10中任一整数或小数。优选地，所述平均连接数p为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8。
在一些实施方案中，本发明所述连接数p1为1到10中任一整数。例如，所述连接数p1为3到9中任一整数。例如，所述连接数p1为1、2、3、4、5、6、7、8、9或10。优选地，所述连接数p1为4、5或6。
在又一个方面，本发明提供了一种如制备本发明任一项所述的双特异性抗体药物偶联物的方法，其包括以下步骤：在还原剂的作用下，将溶于缓冲液的所述双特异性抗体与溶于溶剂的接头单元L-细胞毒性药物混合，即得所述双特异性抗体药物偶联物。
在一些实施方案中，所述制备方法包括将抗EGFR和HER3的双特异性抗体与式X2的化合物反应，例如，将DBXT005-01与式X2的化合物反应：
在一些实施方案中，所述还原剂为本领域此类反应常规的还原剂，例如三(2-羰基乙基)磷盐酸盐。
在一些实施方案中，所述缓冲液为本领域此类反应常规的缓冲液，例如为乙二胺四乙酸。
在一些实施方案中，所述溶剂为本领域此类反应常规的溶剂，例如二甲基乙酰胺。
在又一个方面，本发明提供了一种药物组合物，其包含如本发明任一项所述的双特异性抗体、如本发明任一项所述的分离的核酸、如本发明任一项所述的重组表达载体、如本发明任一项所述的转化体和/或如本发明任一项所述的双特异性抗体药物偶联物，和药学上可接受的载体或赋形剂。
在又一个方面，本发明提供了一种制备本发明的药物组合物的方法，所述方法包括将本发明任一项所述的双特异性抗体药物偶联物，或其药学上可接受的形式，或者它们的混合物，与一种或多种药学上可接受的载体或赋形剂组合。
本发明中，所述的药物组合物中使用的药学上可接受的载体，其中适合的药学上可接受的载体的实例如在Remington’s Pharmaceutical Sciences(2005)中所述。
本发明中，所述的药物组合物可以以任意形式施用，只要其实现预防、减轻、防止或者治愈人类或动物患者的症状即可。例如，可根据给药途径制成各种适宜的剂型。
在另一些实施方案中，本发明任一项所述的双特异性抗体药物偶联物，或所述的药物组合物的施用可以与另外的治疗方法组合。所述另外的治疗方法可以选自，但不限于：放射疗法、化疗疗法、免疫疗法，或其组合。
在又一个方面，本发明提供了一种药物制剂，其包含本发明任一项所述的双特异性抗体药物偶联 物，或其药学上可接受的形式，或它们的混合物作为活性成分，或者本发明任一项所述的药物组合物。在一些实施方案中，所述制剂的形式为固体制剂、半固体制剂、液体制剂或气态制剂。
在又一个方面，本发明提供了如本发明任一项所述的双特异性抗体、如本发明任一项所述的分离的核酸、如本发明任一项所述的重组表达载体、如本发明任一项所述的转化体、如本发明任一项所述的双特异性抗体药物偶联物，和/或如本发明任一项所述的药物组合物在制备用于治疗和/或预防癌症的药物中的用途，优选地，所述癌症为EGFR和/或HER3阳性表达的癌症。
在又一个方面，本发明提供了一种治疗和/或预防癌症的方法，其包括向有需要的受试者施用如本发明任一项所述的双特异性抗体、如本发明任一项所述的分离的核酸、如本发明任一项所述的重组表达载体、如本发明任一项所述的转化体、如本发明任一项所述的双特异性抗体药物偶联物，和/或如本发明任一项所述的药物组合物；优选地，所述癌症为EGFR和/或HER3阳性表达的癌症。
在又一个方面，本发明提供了如本发明任一项所述的双特异性抗体、如本发明任一项所述的分离的核酸、如本发明任一项所述的重组表达载体、如本发明任一项所述的转化体、如本发明任一项所述的双特异性抗体药物偶联物，和/或如本发明任一项所述的药物组合物，其用于治疗和/或预防癌症，优选地，所述癌症为EGFR和/或HER3阳性表达的癌症。
在一些实施方式中，本发明所述癌症选自乳腺癌、皮肤癌、胃癌、结直肠癌、肺癌、食管癌、胆道癌、头颈癌、甲状腺癌、卵巢癌、子宫内膜癌、胰腺癌、前列腺癌、膀胱癌、胃肠道癌、消化道癌、子宫颈癌、鳞状细胞癌、腹膜癌、肝癌、肾癌、甲状腺癌、白血病、恶性淋巴瘤、血浆瘤、骨髓瘤、胶质瘤、骨肉瘤、肉瘤、口腔鳞癌和黑色素瘤。
在一些实施方式中，本发明所述癌症优选选自乳腺癌、结直肠癌、皮肤癌、肺癌、食管癌和口腔鳞癌。
在一些实施方式中，所述的肺癌优选为非小细胞肺癌，所述皮肤癌优选为皮肤鳞癌，所述的结直肠癌优选为直肠癌。
在又一个方面，本发明提供了如本发明任一项所述的双特异性抗体、如本发明任一项所述的分离的核酸、如本发明任一项所述的重组表达载体、如本发明任一项所述的转化体、如本发明任一项所述的双特异性抗体药物偶联物和/或如本发明任一项所述的药物组合物在制备EGFR和/或HER3抑制剂中的应用。
在一些实施方式中，本发明给药方式包括但不限于口服、静脉内、皮下、肌内、动脉内、关节内(例如在关节炎关节中)、通过吸入、气雾剂递送或肿瘤内给予等。
在一些实施方式中，本发明提供了向受试者联合施用治疗有效量的一种或多种疗法(例如治疗方式和/或其它治疗剂)。在一些实施方式中，所述疗法包括手术治疗和/或放射疗法。
在一些实施方式中，本发明提供的方法或用途还包括向个体施用一种或多种疗法(例如治疗方式和/或其它治疗剂)。可以单独或与疗法中的其它治疗剂组合使用本发明的抗体药物偶联物或其药学上可接受的盐。例如，可以与至少一种另外的治疗剂共施用。
在又一个方面，本发明提供了一种药物组合，其包含如本发明任一项所述的双特异性抗体、如本 发明任一项所述的分离的核酸、如本发明任一项所述的重组表达载体、如本发明任一项所述的转化体、如本发明任一项所述的双特异性抗体药物偶联物和/或如本发明任一项所述的药物组合物，以及一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包括如本发明任一项所述的双特异性抗体、如本发明任一项所述的分离的核酸、如本发明任一项所述的重组表达载体、如本发明任一项所述的转化体、如本发明任一项所述的双特异性抗体药物偶联物和/或如本发明任一项所述的药物组合物。
术语定义
除非另有说明，本发明的实施将采用分子生物学(包括重组技术)、微生物学、细胞生物学、生物化学和免疫学的常规技术，这些都在本领域的技术范围内。
除非本文其它部分另有明确定义，否则本文所用的科技术语都具有本发明所属领域普通技术人员通常理解的含义。关于本领域的定义及术语，专业人员具体可参考Current Protocolsin Molecular Biology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
为了更容易地理解本发明，某些科技术语具体定义如下。
在本发明中，术语“EGFR(Epidermal Growth Factor Receptor)”是上皮生长因子(EGF)细胞增殖和信号传导的受体。EGFR属于ErbB受体家族的一种，该家族包括EGFR(ErbB-1)、HER2/c-neu(ErbB-2)、HER3(ErbB-3)和HER 4(ErbB-4)。EGFR也被称作HER1、ErbB-1，突变或过表达一般会引发肿瘤。EGFR是一种糖蛋白，属于酪氨酸激酶型受体，细胞膜贯通，分子量170KDa。EGFR位于细胞膜表面，靠与配体结合来激活，包括EGF和TGFα(transforming growth factorα)。激活后，EGFR由单体转化为二聚体，尽管也有证据表明，激活前也存在二聚体。EGFR还可能和ErbB受体家族的其他成员聚合来激活，例如ErbB-2/HER2/neu。
在本发明中，术语“人表皮生长因子受体3(HER3)”，也称为受体酪氨酸蛋白激酶ErbB-3(ErbB3)，是EGFR/ErbB家族的成员。与其他ErbB家族成员HER2和EGFR不同，HER3本身不具有激酶活性。因此，HER3必须与其激酶活性成员EGFR或HER2结合，作为异二聚体来触发其下游活性。在与其天然配体NRG1结合后，HER3经历构象变化、异二聚化和磷酸化，然后通过信号转导激活MAPK、PI3K/Akt和PLCγ。
在本发明中，术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值，包括但不限于±5％、±2％、±1％和±0.1％，因为这些变化适于进行所公开的方法。
在本发明中，术语“和/或”应理解为意指可选项中的任一项或可选项中的任意两项或更多项的组合。
在本发明中，术语“或”应被理解为与如上定义的“和/或”具有相同的含义。例如，当分离列表中的项目时，“或”或“和/或”应被解释为包容性的，即，包括数量或元素列表中的至少一个，但 也包括多于一个，以及任选地，额外的未列出的项目。只有明确指出相反的术语下，例如“只有一个”或“的确一个”或者在权利要求中使用“由...组成”时，将指的是仅列出的一个数字或列表的一个元素。
除非上下文明确指出相反的情况，否则本发明中，词“一”和“一个”应理解为“至少一个”。
在本发明中，术语“抗体药物偶联物”通常是指抗体通过稳定的连接单元与具有生物活性的细胞毒性药物相连。在本发明中“抗体药物偶联物”可以为双特异性抗体药物偶联物，所述双特异性抗体药物偶联物可以是指把双特异性抗体或其抗原结合片段通过稳定的连接单元与具有生物活性的细胞毒性药物片段相连。
在本发明中，术语“细胞毒性药物”通常指毒性药物，所述细胞毒性药物可以在肿瘤细胞内具有较强破坏其正常生长的化学分子。细胞毒性药物可以在足够高的浓度下杀死肿瘤细胞。所述“细胞毒性药物”可以包括毒素，如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素，放射性同位素(例如At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32或Lu的放射性同位素)，毒性药物，化疗药物，抗生素，核溶酶，或它们的衍生物。例如，可以是毒性药物，包括但不限于喜树碱衍生物；例如，可以是喜树碱衍生物依沙替康(化学名：化学名：(1S,9S)-1-氨基-9-乙基-5-氟-2,3-二氢-9-羟基-4-甲基-1H,12H-苯并[de]吡喃并[3’,4’:6,7]咪唑并[1,2-b]喹啉-10,13(9H,15H)-二酮)。
在本发明中，术语“抗体”(antibody，Ab)通常是指对指定蛋白质或肽或其片段有反应性的免疫球蛋白。抗体可以是来自任何类的抗体，包括但不限于IgG、IgA、IgM、IgD和IgE，及来自任何亚类(例如IgG1、IgG2、IgG3、和IgG4)的抗体。抗体可具有选自例如IgG1、IgG2、IgG3、或IgG4的重链恒定区。抗体还可具有选自例如kappa(κ)或lambda(λ)的轻链。本发明的抗体可衍生自任何物种。术语“抗体”可包括完整的多克隆抗体、完整的单克隆抗体、多特异性抗体(例如双特异性抗体)、嵌合抗体、人源化抗体、人抗体、包含抗体的融合蛋白和任何其他经修饰的免疫球蛋白分子，只要这些抗体展示出所需的生物活性即可。
在本发明中，术语“抗原结合片段”或“抗原结合结构域”通常是指抗体分子的一部分，其包含负责抗体与抗原之间的特异性结合的氨基酸。抗原中由抗体特异性地识别和结合的部分是称作如本文所述的“表位”。如本文所述，抗原结合结构域可典型地包含抗体轻链可变区(VL)和抗体重链可变区(VH)；然而，其并非必须包含两者。Fd片段例如具有两个VH区并且通常保留完整抗原结合结构域的一些抗原结合功能。抗体的抗原结合片段的实例包括(1)Fab片段，具有VL、VH、恒定轻链(CL)和CH1结构域的单价片段；(2)F(ab′)2片段，具有由铰链区的二硫桥连接的两个Fab片段的二价片段；(3)具有两个VH和CH1结构域的Fd片段；(4)具有抗体单臂的VL和VH结构域的Fv片段，(5)dAb片段(Ward等人，“Binding Activities of a Repertoire of Single Immunoglobulin Variable DomainsSecreted From Escherichia coli”，Nature 341:544-546(1989)，其以引用的方式整体并入本发明)，其具有VH结构域；(6)分离的互补决定区(CDR)；(7)单链Fv(scFv)，例如源于scFV-文库。尽管Fv片段的两个结构域VL和VH是由独立基因编码，但其可通过合成连接子使用重组方法接合，合成连接子使得其被制备为其中VL和VH区配对以形成单价分子的单一蛋白链(称为单链 Fv(scFv))(可参见例如Huston等人，“Protein Engineering of AntibodyBinding Sites:Recovery of Specific Activity in an Anti-Digoxin Single-ChainFv Analogue Produced in Escherichia coli,”Proc.Natl.Acad.Sci.USA 85:5879-5883(1988))；(8)“VHH”涉及来自骆驼科(骆驼、单峰骆驼、美洲驼、羊驼等)重链抗体的可变抗原结合结构域(参见Nguyen V.K.等人，2000，The EMBO Journal，19，921-930；Muyldermans S.，2001，J Biotechnol.，74，277-302以及综述Vanlandschoot P.等人，2011，Antiviral Research 92，389-407)。VHH也可称为纳米抗体(Nanobody，Nb)。
在本发明中，术语“可变区”或“可变结构域”或“可变抗原结合结构域”通常是指参与抗体与抗原的结合的抗体重链或轻链的结构域。在本发明中，术语“可变”通常是指，抗体的可变结构域的序列的某些部分变化强烈，形成各种特定抗体对其特定抗原的结合和特异性。变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链可变区和重链可变区中的三个区段，被称为互补决定区(CDR)或高变区(HVR)，分别为LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3。可变区中更高度保守的部分被称为框架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区(H-FR1，H-FR2，H-FR3，H-FR4，L-FR1，L-FR2，L-FR3，L-FR4)，大部分采用β-折叠构型，通过三个CDR结构环区连接。每条链中的CDR通过FR区紧密靠近在一起，并与来自另一条链的CDR一起形成抗体的抗原结合位点。
在本发明中，可以通过多种方法来编码抗体的可变区或划分抗体的CDR，例如基于序列可变性的Kabat编号方案和定义规则(参见，Kabat等人，免疫学的蛋白质序列，第五版，美国国立卫生研究院，贝塞斯达，马里兰州(1991))，基于结构环区域位置的Chothia编号方案和定义规则(参见，A1-Lazikani等人，JMol Biol 273:927-48,1997)，Lefranc等人的基于种系V基因的氨基酸序列比对的IMGT编号方案和定义规则，还有Honneger’s编号方案(AHo’s)，Martin编号方案，Gelfand编号方案等，可参见Mathieu Dondelinger等人，Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface/Residue Definition,Front.Immunol.,16 October 2018。
在本发明中，术语“单克隆抗体”或“单抗”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
在本发明中，术语“多特异性抗体”是指包含两个或更多个抗原结合结构域，能够结合两个或更多个不同的表位(例如，两个、三个、四个或更多个不同的表位)，表位可以在相同或不同的抗原上的抗体。多特异性抗体的示例包括结合两个不同抗原或两个不同表位的“双特异性抗体”(简称双抗)或“双特异性分子”。本文靶向EGFR和HER3的双特异性抗体可被称为例如“抗EGFR和HER3”或“抗EGFR/HER3”或“EGFR×HER3”双特异性分子，或包含EGFR结合结构域和HER3结合结构域的双特异性抗体，或其他类似术语。
在本发明中，本文术语“Fc区”用于定义包含至少一部分恒定区的免疫球蛋白重链的C端区域。 该术语包括天然序列Fc区和变异Fc区。在一些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羧基末端。但是，Fc区的C端赖氨酸(Lys447)可能存在或不存在(此段中的编号是根据EU编号系统，也称为EU索引，如Rabat等人，Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,MD,1991)。
在本发明中，术语“Knob into Hole结构”，是将抗体Fc的CH3的疏水氨基酸进行突变。将一条链CH3的侧链氨基酸突变形成分子比较大的疏水氨基酸(knob)，以加强疏水作用力；另一个CH3侧链氨基酸突变形成小的氨基酸(hole)，用于减少空间位阻；突变后带有Knob的和带有Hole的CH3以疏水作用形式形成Knob into Hole结构(KiH)，有利于重链异源二聚体的形成；KiH突变主要是发生在CH3结构域的空间结构的内部疏水氨基酸，突变后暴露在外面的氨基酸几乎没有变化，所以不影响Fc的效应功能和引起的免疫原性。术语“knob-Fc”是指在抗体Fc区包含T366W的点突变，以形成类似knob的空间结构。相对应地，“hole-Fc”是指在抗体Fc区包含T366S、L368A、Y407V的点突变，以形成类似hole的空间结构。为进一步地促进异二聚体的形成，还可在knob-Fc和hole-Fc分别引入S354C和Y349C的点突变，通过二硫键进一步促进异二聚体的形成。同时，为了减弱和蛋白A的结合，还可在hole-Fc分别引入H435R和Y436F的点突变。
在本发明中，术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的可变结构域相当于非人免疫球蛋白的可变结构域，而所有或基本所有的框架区(FR)是人免疫球蛋白序列的框架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
在本发明中，“同种型”抗体是指由重链恒定区基因提供的抗体种类(例如，IgM、IgE、IgG诸如IgGl、IgG2或IgG4)。同种型还包括这些种类之一的修饰形式，其中修饰已被产生来改变Fc功能，例如以增强或减弱效应子功能或对Fc受体的结合。
在本发明中，术语“交叉反应”指的是对人类、猴、和/或鼠源(小鼠或大鼠)相同靶分子的抗原片段的结合。因此，“交叉反应”应被理解为抗原结合分子(例如，抗体)与在不同物种中表达的同类分子(例如，BDCA2)的种属间反应。识别人BDCA2、猴、和/或鼠BDCA2(小鼠或大鼠)的单克隆抗体的交叉反应特异性可通过FACS分析确定。
在本发明中，“亲和力”或“结合亲和力”指反映结合的成员之间相互作用的固有结合亲和力。分子X对其配偶物Y的亲和力可以通常由平衡解离常数(KD)代表，平衡解离常数是解离速率常数和结合速率常数(分别是Koff(Kd)和Kon(Ka))的比值。亲和力可以由本领域已知的常见方法测量。在本发明的一些实施方案中，利用表面等离子共振(SPR)技术测量亲和力，例如本发明的抗体与抗原之间的亲和力。在本发明的一些优选的实施方案中，用于测量亲和力的一个具体方法是BIAcore法。
在本发明中，术语“不结合”蛋白或细胞是指，不与蛋白或细胞结合，或者不以高亲和力与其结合，即结合蛋白或细胞的KD为1.0×10-6M或更高，优选1.0×10-5M或更高，更优选1.0×10-4M或更高、1.0×10-3M或更高，进一步更优选1.0×10-2M或更高。
在本发明中，术语“高亲和性”对于IgG抗体而言，是指对于抗原的KD为1.0×10-6M或更低， 优选5.0×10-8M或更低，更优选1.0×10-8M或更低、5.0×10-9M或更低，进一步更优选1.0×10-9M或更低。对于其他抗体亚型，“高亲和性”结合可能会变化。例如，IgM亚型的“高亲和性”结合是指KD为10-6M或更低，优选10-7M或更低，更优选10-8M或更低。
在本发明中，术语“百分比(％)氨基酸序列同一性”或简称“同一性”定义为在将氨基酸序列进行比对(并在必要时导入空位)以获取最大百分比序列同一性，且不将任何保守取代视为序列同一性的部分之后，候选氨基酸序列中的氨基酸残基与参比氨基酸序列中的相同氨基酸残基的百分比。可使用本领域各种方法进行序列比对以便测定百分比氨基酸序列同一性，例如，使用公众可得到的计算机软件如BLAST、BLAST-2、ALIGN或MEGALIGN(DNASTAR)软件。本领域技术人员可以决定测量比对的适宜参数，包括对所比较的序列全长获得最大比对所需的任何算法。
在本发明中，术语“卤素”通常是指氟、氯、溴、碘，例如，可以是氟、氯。
在本发明中，术语“烷基”通常是指烷除去氢原子所衍生的残基。术语“烷基”通常指饱和的直链或支链脂肪族烃基，其具有从母体烷的相同碳原子或两个不同的碳原子上除去氢原子所衍生的残基，其可以为包含1至20个碳原子的直链或支链基团，例如含有1至12个碳原子，例如含有1至6个碳原子的链烷基，例如含有1至3个碳原子的链烷基。烷基的非限制性实例包括但不限于甲基、乙基、丙基、丙基、丁基等。烷基可以是取代的或非取代的，替代或者非替代的，例如当被取代时，取代基可以在任何可使用的连接点上被取代。
在本发明中，术语“亚烷基”通常指饱和的直链或支链脂肪族烃基，其具有2个从母体烷的相同碳原子或两个不同的碳原子上除去两个氢原子所衍生的残基，其可以为包含1至20个碳原子的直链或支链基团，例如，术语“亚甲基”可以是指1个碳原子的基团除去两个氢原子所衍生的残基。亚甲基可以是取代的或非取代的，替代或者非替代的；例如含有1至12个碳原子，例如含有1至6个碳原子的亚烷基。亚烷基的非限制性实例包括但不限于亚甲基(-CH2-)、1,1-亚乙基(-CH(CH3)-)、1,2-亚乙基(-CH2CH2)-)、1,1-亚丙基(-CH(CH2CH3)-)、1,2-亚丙基(-CH2CH(CH3)-)、1,3-亚丙基(-CH2CH2CH2-)、1,4-亚丁基(-CH2CH2CH2CH2-)和1,5-亚丁基(-CH2CH2CH2CH2CH2-)等。
在本发明中，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10个碳原子，优选包含3至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烷基、环丁烷基、环戊烷基、环戊烯基、环己烷基、环己烯基、环己二烯基、环庚烷基、环庚三烯基、环辛烷基等；多环环烷基包括螺环、稠环和桥环的环烷基。
术语“亚环烷基”为二价基团，其通过两个单键与分子其余部分相连，其余定义同术语“环烷基”。
在本发明中，术语“部分不饱和的”通常是指环状结构中环分子间至少含一个双键或三键。术语“部分不饱和”涵盖带有多处不饱和的环状结构，但并非意在包括本发明所定义的芳环或杂芳环。术语"不饱和的"表示部分具有一个或多个不饱和度。
在本发明中，术语“杂环基”指饱和或部分不饱和单环或多环环状轻取代基，其包含3至20个环原子，其中一个或多个环原子为选自氮、氧或硫的杂原子，其余环原子为碳。优选包含3至12个 环原子，其中1～4个是杂原子；更优选包含3至8个环原子，其中1-3是杂原子；进一步更优选包含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、哌啶基、吗啉基、硫代吗啉基和高哌嗪基的等。多环杂环基包括螺环、稠环和桥环的杂环基。所述杂环基环可以稠合于芳基、杂芳基或环烷基环上，其与母体结构连接在一起的环为杂环基。
术语“亚杂烷基”为二价基团，其通过两个单键与分子其余部分相连，其余定义同术语“亚环烷基”。
在本发明中，术语“各自独立地”通常是指变量适用于任何一种情况，而不考虑在相同化合物中具有相同或不同定义的变量存在与否。例如其中的变量可以是指化合物的取代基种类、数量或化合物中原子的种类等。例如，在化合物中出现2次R并且R被定义为“独立地碳或氮”时，两个R可以均为碳，两个R可以均为氮，或一个R可以为碳而另一个R为氮。
在本发明中，术语“任选”或“任选地”通常意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选被烷基取代的杂环基团”意味着烷基取代可以但不必须存在，该说明可以包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
在本发明中，术语“取代的”通常指基团中的一个或多个氢原子，例如为最多5个，例如为1～3个氢原子彼此独立地被相应数目的取代基取代。取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。
在本发明中，除非特别说明，基团与基团的“连接”通常可以处于任一定向；以基团X与基团Y的“连接”通常可以处于任一定向，任一定向通常是指在基团X用于连接体Y和基团Z时，所述基团X的两个或更多个连接位点可以任意地与基团Y或基团Z连接。
在本发明中，如本领域技术人员可知的，“烷基”、“环烷基”等之类的术语可以在名称前加一个标识表示在特定情况下基团中存在的原子数，例如，C1-C4烷基，C3-C7环烷氧基，C1-C4烷基羰基氨基等，“C”后所跟下标数字表示在基团中存在的碳原子数。例如，C3烷基是指具有三个碳原子的烷基(例如，正丙基，异丙基)；C1-10中，基团的成员可具有落入1-10范围内的任何数目的碳原子。
在本发明中，本发明的化合物或抗体药物偶联物包含其互变异构体、内消旋体、外消旋体、对映异构体、和/或非对映异构体。在本发明中，术语“非对映异构体”通常是指具有两个或更多个手性中心并且其分子不是彼此的镜像的立体异构体。非对映异构体可以具有不同的物理性质，例如、熔点、沸点、波谱性质和反应性。在本发明中，术语“互变异构体”或“互变异构形式”可互换使用，通常是指可通过低能垒(low energy barrier)互相转化的不同能量的结构异构体。例如，质子互变异构体(protontautomer)(也称为质子移变互变异构体(prototropic tautomer)包括通过质子迁移进行的互相转化，诸如酮-烯醇异构化和亚胺-烯胺异构化。价键互变异构体(valence tautomer)包括通过一些成键电子的重组进行的互相转化。在本发明中，术语“内消旋体”通常是指分子内含有不对称性的原子，但具有对称因素而使分子内总旋光度为零。术语“外消旋体”或“外消旋混合物”是指由等摩尔量的 两种对映异构体物质构成的组合物。
在本发明中，术语“接头单元”或“接头结构”(linker)通常指指一端与配体连接而另一端与细胞毒性药物相连的化学结构片段或键，也可以连接其他接头后再与细胞毒性药物相连。所述直接或间接连接配体可以是指所述基团通过共价键直接连接配体，也可以是通过接头结构连接配体。例如，可以使用包含酸不稳定接头结构(例如腙)、蛋白酶敏感(例如肽酶敏感)接头结构、光不稳定接头结构、二甲基接头结构、或含二硫化物接头结构的化学结构片段或键作为接头结构。
在本发明中，术语某个结构“任选地与其它分子部分相连接”通常是指该结构不与任何其它化学结构相连接，或者该结构与一个或多个不同于该结构的其它化学结构(例如本发明所述的配体)相连接(例如，通过化学键连接、或通过接头结构连接)。
在本发明中，术语“载药量”通常是指每个配体上加载的细胞毒性药物(payload)平均数量，也可以表示为细胞毒性药物和抗体量的比值(drug/antibody ratio,DAR)，细胞毒性药物载量的范围可以是每个配体(Ab)连接0-12个，例如1-10个细胞毒性药物。在本发明的实施方式中，载药量表示为p或p1，示例性地可以为1、2、3、4、5、6、7、8、9和10的均值。可用常规方法如UV/可见光光谱法，质谱，ELISA试验和HPLC特征鉴定偶联反应后每个ADC分子的载药量。
在本发明中，本发明的化合物或抗体药物偶联物的某些原子可能以一种以上的同位素形式出现。例如，氢可能以氕(1H)、氘(2H)和氚(3H)的形式存在，碳可能以三种不同的同位素(12C、13C和14C)自然存在。可并入本发明化合物中的同位素示例还包括但不限于15N、18O、17O、18F、32P、33P、129I、131I、123I、124I、125I，或者类似的同位素。因此，相对于这些同位素的自然丰度，本发明的化合物或抗体药物偶联物可富集在一种或多种这些同位素中。如本领域技术人员所知，此类同位素富集化合物可用于多种用途。例如，用重同位素如氘(2H)替代可能会提供某些治疗优势，这可以是由于更高的代谢稳定性。例如，氘(2H)的自然丰度约为0.015％。因此，自然界中大约每6500个氢原子，就有一个氘原子。因此，本发明的含氘化合物或抗体药物偶联物在一个或多个位置(视情况而定)的氘丰度大于0.015％。除非另有指明，否则本发明所述的结构还可以包括仅在是否存在一个或多个同位素富集原子方面存在差别的化合物或抗体药物偶联物。举例而言，除了氢原子被氘或氚所取代，或碳原子被碳13或碳14所取代之外，其余部分均与本发明结构一致的化合物或抗体药物偶联物均在本发明的范围之内。
在本发明中，术语“药物组合物”通常是指含有一种或多种本发明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。药物组合物可以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。常规的药物组合物的制备可以见各国药典。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。例如，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。
在本发明中，术语“药学上可接受的盐”或“可药用的盐”通常是指本发明化合物或抗体药物偶联物的盐，这类盐用于哺乳动物体内时可以具有安全性和/或有效性，且可以具有应有的生物活性，本发明的化合物或抗体药物偶联物可以与酸形成盐。
在本发明中，术语“药学上可接受的载体”通常是指给予治疗剂，例如抗体或多肽、基因和其它治疗剂的载体或载剂。该术语指本身不诱导对接受组合物的个体有害的抗体产生并且可以给予而不产生过度毒性的任何药物载体。合适的载体可以是大的、代谢缓慢的大分子，例如蛋白质、多糖、聚乳酸、聚乙醇酸、多聚氨基酸、氨基酸共聚物、脂质聚集物和灭活的病毒颗粒。本领域技术人员熟知这些载体。治疗组合物中药学上可接受的载体可包括液体，例如水、盐水、甘油和乙醇。这些载体中也可存在辅助物质，例如润湿剂或乳化剂、pH缓冲物质等。
在本发明中，术语“治疗(treatment)”和“治疗(treating)”通常是指获得有益或希望的结果的方法，所述有益或希望的结果包括但不限于治疗益处。治疗益处包括但不限于根除、抑制、减少或改善所治疗的潜在障碍。另外，治疗益处是通过根除抑制、减少或改善与潜在的障碍相关的一种或多种生理症状实现的，从而在患者中观察到改善，但是患者仍然可能患有潜在障碍。
在本发明中，术语“预防(prevention)”和“预防(preventing)”通常是指获得有益或希望的结果的方法，所述有益或希望的结果包括但不限于预防益处。为了预防益处，可以向处于患上特定疾病的风险的患者或向报告具有疾病的一种或多种生理症状的患者施用药物组合物，即使尚未诊断出该疾病。
在本发明中，术语“受试者”或“患者”通常是指人类(即，任何年龄组的男性或女性，例如，小儿对象(例如，婴儿、儿童、青少年)或成人对象(例如，年轻人、中年人或老年人))和/或其他灵长类动物(例如，食蟹猴、恒河猴)；哺乳动物，包括商业上相关的哺乳动物，如牛、猪、马、绵羊、山羊、猫和/或犬；和/或鸟类，包括商业上相关的鸟类，如鸡、鸭、鹅、鹌鹑和/或火鸡。
在本发明中，术语“治疗有效量”、“治疗有效剂量”和“有效量”是指本发明的化合物或抗体药物偶联物单独或与其它治疗药物组合给予细胞、组织或受试者时，有效预防或改善一种或多种疾病或病况的症状或该疾病或病况的发展的量。治疗有效剂量还指足以导致症状改善的剂量，例如治疗、治愈、预防或改善相关医学病况或者提高这类病况的治疗、治愈、预防或改善的速度的量。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。
在本发明中，术语“癌症”指表现出异常高水平的增殖和生长的一组细胞。癌症可能是良性的(也称为良性肿瘤)，恶性前或恶性。癌细胞可以是实体癌细胞或白血病癌细胞。在本发明中，术语“肿瘤”是指包含癌症的一个或多个细胞。在本发明中，术语“肿瘤生长”指代包含癌症的一种或多种细胞的增殖或生长，其导致癌症的大小或程度的相应增加。术语“EGFR和/或HER3阳性表达的癌症”“EGFR和/或HER3表达的癌症”具有相同的定义，是癌细胞表达EGFR和/或HER3的癌症，优选是在癌细胞表面表达。
本发明抗体的氨基酸编号为分别根据抗体序列从N端至C端的自然顺序编号。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
本发明的双特异性抗体有以下优势：
1.可以同时结合EGFR和HER3抗原和EGFR/HER3细胞株，更表现出了优于单克隆抗体用药的抗肿瘤活性，相较母本单抗和对照抗体SI-1X6.4，本发明的双特异性抗体对同时表达EGFR和HER3的人肿瘤细胞株T47D、人肿瘤细胞株NCI-H1975和小鼠CT26细胞株具有更好的内吞效应。
2.与相关的单克隆抗体联合治疗相比，本发明的双特异性抗体具有依从性好、质量可控的优点。
3.本发明中双特异性抗体的稳定性表征主要体现在为单体纯度、热稳定性的研究上。亲和纯化和分子筛分离后，双特异性抗体单体含量可达95％。抗体经热处理后的结构和活性分析结果证明，该抗体在恶劣环境下仍能保持良好的分子构象和完整的生物活性，有利于抗体的工业化生产及包装贮存。总体来说，本发明的EGFR和HER3双特异性抗体显示出良好的分子稳定性，并具有显著优于Cetuximab和EGFR的体外活性(结合分子水平和细胞水平)。体内数据证明，在EGFR+HER3肿瘤细胞中，双特异性抗体的抗肿瘤活性优于EGFR和HER3单克隆抗体联合用药组。因此，本发明的双特异性抗体由于其优异的可开发性和活性，具有广阔的应用前景。
本发明任一项所述的双特异性抗体药物偶联物具有体内抑瘤效果，具体表现为：
(1)对EGFR低表达HER3中表达的人食管癌细胞株OE-19具有更好的抑瘤效果；
(2)对EGFR中表达HER3中表达的人非小细胞肺癌细胞株NCI-H441具有更好的抑瘤效果；
(3)对EGFR高表达HER3低表达的人口腔鳞癌细胞株CAL-27具有更好的抑瘤效果；
(4)在对EGFR ADC耐药的人食管癌OE-19皮下异种移植模型中具有更好的抑瘤效果；
(5)在EGFR高表达HER3低表达的人皮肤癌A431皮下异种移植模型中具有更好的抑瘤效果；
(6)在不表达EGFR仅表达HER3的人结肠癌SW620皮下异种移植模型中具有更好的抑瘤效果；
(7)在EGFR中表达HER3低表达的人结肠癌SW48皮下异种移植模型中具有更好的抑瘤效果；
(8)在对第三代TKI耐药的非小细胞肺癌NCI-H1975(EGFR L858R/T790M/C797S三突变)皮下异种移植模型中具有更好的抑瘤效果。
附图说明
图1为双特异性抗体及其对应母本单抗和表达不同水平的EGFR/HER3细胞株结合。
图2双特异性抗体及其对应母本单抗和表达不同水平的EGFR/HER3细胞株结合。
图3为双特异性抗体及其对应母本单抗的内吞活性(pHrodo法)。
图4为双特异性抗体及其对应母本单抗和对照抗体的内吞活性(incucyte法)。
图5双特异性抗体及其对应母本单抗和对照抗体的内吞活性(incucyte法)。
图6为抗体药物偶联物对EGFR低表达HER3中表达的人食管癌细胞株OE-19荷瘤小鼠体内药 效评价。
图7为抗体药物偶联物对EGFR中表达HER3中表达的人非小细胞肺癌细胞株NCI-H441荷瘤小鼠体内药效评价。
图8为抗体药物偶联物对EGFR高表达HER3低表达的人口腔鳞癌细胞株CAL-27荷瘤小鼠体内药效评价。
图9为双特异性抗体药物偶联物不同DAR值在NCI-H1975荷瘤小鼠体内药效评价。
图10为双特异性抗体药物偶联物不同DAR值在OE-19荷瘤小鼠体内药效评价。
图11为本发明双特异性抗体药物偶联物与对照抗体药物偶联物在NCI-H1975荷瘤小鼠体内药效的比较。
图12为本发明双特异性抗体药物偶联物与对照抗体药物偶联物在OE-19荷瘤小鼠体内药效的比较。
图13为本发明双特异性抗体药物偶联接头-细胞毒素X1在NCI-H1975荷瘤小鼠体内药效。
图14为本发明双特异性抗体药物偶联物在人食管癌OE-19荷瘤小鼠体内药效。
图15为本发明双特异性抗体药物偶联物在EGFR高表达HER3低表达的人皮肤癌A431异种移植瘤小鼠体内药效。
图16为本发明双特异性抗体药物偶联物在不表达EGFR仅表达HER3的人结肠癌细胞株SW620异种移植瘤小鼠体内药效。
图17为本发明双特异性抗体药物偶联物在EGFR中表达HER3低表达的人结肠癌细胞株SW48异种移植瘤小鼠体内药效。
图18为本发明双特异性抗体药物偶联物在非小细胞肺癌NCI-H1975(EGFR L858R T790M C797S三突变)异种移植瘤小鼠体内药效。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
样品检测
本发明包括所叙述特定实施方式的所有组合。本发明的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得可以实现。然而，应理解，尽管详细描述及特定实施例指示本发明的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本发明的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得可以实现。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。
实施例
提供以下实施例以证明并进一步解释本发明的一些优选的实施方式和方面，不应被解释为限制其范围。
实施例1双特异性抗体的制备
本发明的抗EGFR×HER3双特异性抗体中，抗HER3抗体或其抗原结合片段参考hu3F8的克隆2(PCT/CN2022/098929)制备，抗EGFR抗体或其抗原结合片段参考Zalutumumab(WO2002100348)制备，同时，在上述抗体的基础上，对其CDR/FR/Fc区进行了部分氨基酸突变。其中，双特异性抗体的CDR区依照Kabat编号规则确定。
根据抗体可变区的氨基酸序列，设计引物PCR搭建得到VH/VK基因片段，获得可变区。抗体可变区再与恒定区基因片段进行组合，构建完整双特异性抗体序列。经过转染CHO细胞后，按照常规表达纯化方法得到单抗anti-EGFR和单抗anti-HER3，及双特异性抗体DBXT001-01～08、DBXT002-01～08、DBXT003-01～08、DBXT004-01～08和DBXT005-01。
Cetuximab单抗：采用常规方法制备；对照抗体SI-1X6.4：参照WO2023083381A1制备。
抗体名称和序列见表1。
表1抗体的氨基酸序列

抗体表达和纯化
1.将来源于ECACC的CHO-K1细胞复苏，接种于培养基中(上海奥浦迈生物科技股份有限公司，货号C673017)，于8％ CO2，37℃传代培养，转染时使其密度达到6.0×106个/mL，取1.45×108个细胞，离心去上清；
2.在细胞沉淀中加入0.5mL电转液，混匀；
3.按照H1:L1:H2:L2＝2:3:2:3的比例加入4个质粒，细胞质粒混悬液充分混匀后加入电击管，放入电转仪进行电击；
4.电击完成后，将电击管内细胞转入含有20mL培养液摇瓶内，室温静止孵育40min；
5.孵育结束后将摇瓶放入摇床37℃，110rpm，8％ CO2培养，24小时后添加补料/丁酸钠/双抗(青霉素+链霉素)，继续培养4天；
6.转染后第4天离心收集上清，上清经过Protein A亲和层析柱纯化(博格隆)，用醋酸钠缓冲液(pH 3.4)洗脱，分管收集，送测SEC；
7.将符合要求的分段洗脱液经过凝胶过滤层析精纯(博格隆200，规格：16mm×700mm)，1×PBS洗脱，分管收集，送测SEC，符合要求的分管洗脱液换液到缓冲液(25mM His-HCl，6％ Sucrose，pH 6.0)中-20℃保存。
实施例2抗体亲和力检测(SPR)
2.1目的：抗EGFR和HER3双抗和母本单抗用Biacore T200(Cytiva)进行亲和力检测。
实验方法：
1.ProA芯片俘获EGFR-HER3双抗(1μg/mL)，分析物分别为人的EGFR(human EGFR，His-Avi Tag，Kactus Biosystem，EGF-HM401)，浓度在3.125-400nM范围内倍比稀释，一共8个浓度；人的HER3(human HER3，His-Avi Tag，Kactus Biosystem，HER-HM403)，浓度在6.25-200nM范围内倍比稀释，一共8个浓度；猴的EGFR(rehsus EGFR，C-His Tag，Kactus Biosystem，EGF-CM101)，浓度在6.25-400nM范围内倍比稀释，一共8个浓度；猴的HER3(rhesus HER3，C-His Tag，Kactus Biosystem，HER-CM403)，浓度在3.125-200nM范围内倍比稀释，一共8个浓度；
2.ProA芯片俘获EGFR母本单抗(1μg/mL)，分析物为人的EGFR(human EGFR，His-Avi Tag，Kactus Biosystem，EGF-HM401)，浓度在6.25-400nM范围内倍比稀释，一共8个浓度；ProA芯片俘获HER3母本单抗(3μg/mL)，分析物为人的HER3(human HER3，His-Avi Tag，Kactus Biosystem，HER-HM403)，浓度在3.125-100nM范围内倍比稀释，一共8个浓度；
3.亲和力数据用1:1的Langmuir结合模式进行分析和拟合，亲和力(KD)为解离常数(Kd)与结合常数(Ka)的比值。亲和力结果见表2。
表2双抗和单抗与人猴抗原的亲和力
实验结论：双抗DBXT001-01与人EGFR亲和力比与HER3亲和力弱大约16倍；双抗DBXT001-01与人EGFR亲和力比母本EGFR抗体亲和力弱大约3倍，而双抗DBXT001-01与HER3的亲和力与母本HER3抗体相当。并且双抗DBXT001-01与人的EGFR或者HER3亲和力与猴的EGFR或者HER3亲和力相当。这样的双抗亲和力设计可以减少双抗对广泛分布的表达EGFR正常组织的在靶毒性。
2.2抗体亲和力检测(SPR)
目的：抗EGFR和HER3双抗用Biacore T200(Cytiva)进行亲和力检测。
实验方法：
1.ProA芯片俘获SI-1X6.4(7.5μg/mL)，分析物分别为人的EGFR(human EGFR，His-Avi Tag，Kactus Biosystem，EGF-HM401)，浓度在1.17-18.75nM范围内倍比稀释，一共5个浓度；人的HER3(human HER3，His-Avi Tag，Kactus Biosystem，HER-HM403)，浓度在4.69-75nM范围内倍比稀释，一共5个浓度；同样，ProA芯片俘获DBXT005-01(6μg/mL)，分析物人的EGFR浓度在9.38-150nM范围内倍比稀释，一共5个浓度；分析物为人的HER3时，浓度在4.69-75nM范围内倍比稀释，一共5个浓度；
2.亲和力数据用1:1的Langmuir结合模式进行分析和拟合，亲和力(KD)为解离常数(Kd)与结合常数(Ka)的比值。亲和力结果见表3。
表3双抗和人抗原的亲和力
实验结论：本发明双抗DBXT005-01与人EGFR亲和力比对照抗体SI-1X6.4弱约15倍，而与HER3的亲和力比对照抗体SI-1X6.4强25倍。这样的双抗亲和力设计可以减少双抗对广泛分布表达 EGFR正常组织的在靶毒性，增强对高表达EGFR低表达HER3肿瘤的亲和力。
实施例3双抗及其对应母本单抗和表达不同水平的EGFR/HER3细胞株结合
3.1测试目的：
通过流式检测对比DBXT001-01和其母本单抗对不同表达水平的EGFR/HER3细胞株的亲和力实验方法：
1.建立稳定表达人EGFR和HER3的小鼠CT26单克隆细胞株(科佰生物)，肿瘤细胞株NCI-H441、NCI-H1975和T47D来源于ATCC；
2.所有细胞株于37℃，5％ CO2条件下培养于完全培养基；
3.收获处于对数生长期的细胞并用台盼蓝排斥法检测细胞活力，确保细胞活力在90％以上，1000r/min离心5min后，弃上清。使用PBS洗涤细胞一次，用FACS Buffer重悬制成单细胞悬液并调整细胞密度至5×106cells/mL；
4.向96孔板中分别加入50μL的细胞悬液，使受试品工作液起始浓度为100nM，5倍稀释，共6个浓度；混匀后，放置于4℃，孵育40min；
5.使用FACS Buffer洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用100μL FACS Buffer重悬细胞；
6加入2μL的PE标记的二抗(PE anti-human IgG Fc Antibody)，混匀后，放于4℃避光孵育40min；
7.使用FACS Buffer洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用250μL FACS Buffer重悬细胞；
8.流式细胞仪检测荧光值。
实验结果如图1和表4所示。
表4母本单抗和双抗与细胞株的亲和力
实验结论：
DBXT001-01对单独过表达EGFR或者HER3细胞株的亲和力低于二价的母本单抗，也低于Cetuximab对过表达EGFR细胞株的亲和力；但是DBXT001-01对同时表达EGFR和HER3肿瘤细胞株的亲和力不弱于二价的母本单抗。这可能有利于双抗减少和正常组织的结合而增强和同时表达 EGFR和HER3的肿瘤组织的结合。
3.2不同双抗和不同表达水平EGFR/HER3的细胞株的亲和力
实验目的：通过流式检测对比不同双抗和母本单抗对不同表达水平的EGFR/HER3细胞株的亲和力
实验方法：
1.肿瘤细胞株BT-474和MDA-MB-468来源于ATCC；稳定表达人EGFR或者人HER3的小鼠CT26单克隆细胞株来自科佰生物；
2.所有细胞株于37℃，5％ CO2条件下培养于完全培养基；
3.收获处于对数生长期的细胞并用台盼蓝排斥法检测细胞活力，确保细胞活力在90％以上，1000r/min离心5min后，弃上清。使用PBS洗涤细胞一次，用FACS Buffer重悬制成单细胞悬液并调整细胞密度至5×106cells/mL；
4.向96孔板中分别加入50μL的细胞悬液，使受试品工作液起始浓度为150nM，4倍稀释，共8个浓度；混匀后，放置于4℃，孵育40min；
5.使用FACS Buffer洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用100μL FACS Buffer重悬细胞；
6加入2μL的PE标记的二抗(PE anti-human IgG Fc Antibody)，混匀后，放于4℃避光孵育40min；
7.使用FACS Buffer洗涤细胞3次，每次400μL，1000r/min速度下，离心5min，最后使用250μL FACS Buffer重悬细胞；
8.流式细胞仪检测荧光值。
受试品代号如表5所示
实验结果如图2和表6所示。
表5双抗或者单抗不同组成
表6不同双抗和不同EGFR/HER3表达水平细胞株的亲和力
实验结论：对不同EGFR和HER3表达水平细胞株，DBXT005-01饱和靶抗原能力比对照抗体SI-1X6.4强；并且饱和抗原能力也比对应母本单抗强。
实施例4抗体的内吞活性(pHrodo法)
测试目的
检测本发明针对EGFR和HER3靶标的抗体药物，在同时表达EGFR和HER3的T47D细胞上的内吞效应，以及与母本单抗的内吞活性的比较。将细胞和固定浓度的抗体药物和内吞指示试剂pHrodo进行共孵育，通过观察不同时间点细胞内伴随抗体药物进入细胞的pHrodo所产生的荧光信号来评价抗体药物内吞能力。
实验方法
1.细胞培养：T47D细胞使用含0.2Unites/mL牛胰岛素和10％ FBS的RPMI-1640培养基培养。
2.细胞准备：取对数生长期的T47D细胞，用PBS洗涤1次之后，消化2-3min，待细胞消化完全后，加入10-15mL细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，加入细胞培养液将细胞重悬制成单细胞悬液并调整活细胞密度至3×105cells/mL。
3.细胞铺板：以50μL/孔加入96孔细胞培养板。将培养板在培养箱培养48小时(37℃，5％CO2)。
4.加样操作：将待测抗体药物与Fab-pHrodo共同孵育形成复合物后调整浓度至6nM或60nM，以50μL/孔加到细胞。
5.细胞培养：将培养板在培养箱孵育48小时(37℃，5％ CO2)。
6.读板操作：到达对应时间点，取出96孔细胞培养板，将细胞消化后通过FACS读取细胞数量及荧光数值。
实验结果图3。
实验结论：
本发明针对EGFR和HER3靶标的抗体药物相较母本单抗对同时表达EGFR和HER3的T47D细胞具有更好的内吞效应。
实施例5抗体的内吞活性(incucyte法)
5.1测试目的：
检测本发明针对EGFR和HER3靶标的抗体药物与其他针对EGFR和HER3靶标的抗体药物相比，在同时表达EGFR和HER3的细胞株NCI-H1975和同时过表达EGFR和HER3的CT26细胞株的内吞效率。将细胞和固定浓度的抗体药物偶联物和内吞指示试剂Fabfluor-pH进行共孵育，通过连续观察48小时活细胞的荧光信号变化来评价抗体药物偶联物的内吞能力。
实验方法：
1.细胞培养：NCI-H1975细胞使用含10％ FBS的RPMI1640培养基培养；同时稳转EGFR和 HER3的CT26细胞株的培养基为1640+10％FBS+10μg/mL puromycin+20μg/mL blasticidin；
2.细胞准备：取对数生长期的细胞，用PBS洗涤1次之后，消化2-3min，待细胞消化完全后，加入10-15mL细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，加入细胞培养液将细胞重悬制成单细胞悬液并调整活细胞密度至1x105cells/mL。
3细胞铺板：以50μL/孔加入96孔细胞培养板。将培养板放在培养箱培养过夜(37℃，5％ CO2)。
4.标记待测抗体药物偶联物：高浓度的抗体标记为将供试品储液稀释为240nM的4×工作液(终浓度60nM)，将Fabfluor-pH储液稀释为720nM的4×工作液(终浓度180nM)，将两者充分混合，37℃避光孵育15分钟。低浓度的抗体标记为将供试品储液稀释为40nM的4×工作液(终浓度10nM)，将Fabfluor-pH储液稀释为120nM的4×工作液(终浓度30nM)，将两者充分混合，37℃避光孵育15分钟。
5.俘获分析图像：转移标记后的供试品工作液至实验板的相应孔中，将实验板转移至Incucyte活细胞分析设备中，设置扫描拍照程序，使用Incucyte活细胞分析系统获取图像。以2小时的间隔进行定量，持续48小时。分析结果表示为：总荧光综合强度(RCU Xμm2/图像)。
实验结果如图4A，4B所示。
实验结论：
本发明针对EGFR和HER3靶标的抗体药物在高浓度和低浓度相较母本单抗都对同时表达EGFR和HER3的细胞具有更好的内吞效应。同时本发明针对EGFR和HER3靶标的抗体药物在高浓度和低浓度都比对照抗体SI-1X6.4有更好的内吞效应。
5.2不同双抗在不同表达水平EGFR/HER3的细胞株上的内吞(incucyte法)
测试目的：
检测本发明针对EGFR和HER3靶标的抗体药物与其他针对EGFR和HER3靶标的抗体药物相比，在表达不同水平EGFR和HER3的细胞株A431和NCI-H1975以及同时过表达EGFR和HER3的CT26细胞株的内吞效率。将细胞和固定浓度的抗体药物偶联物和内吞指示试剂Fabfluor-pH进行共孵育，通过连续观察24小时活细胞的荧光信号变化来评价抗体药物偶联物的内吞能力。
实验方法：
1.细胞培养：A431细胞使用含10％ FBS的DMEM培养基培养；NCI-H1975细胞使用含10％FBS的RPMI1640培养基培养；同时稳转EGFR和HER3的CT26细胞株的培养基为1640+10％FBS+10μg/mL puromycin+20μg/mL blasticidin；
2.细胞准备：取对数生长期的细胞，用PBS洗涤1次之后，消化2-3min，待细胞消化完全后，加入10-15mL细胞培养液，将经过消化的细胞洗脱下来，1000rpm离心5min，弃上清，加入细胞培养液将细胞重悬制成单细胞悬液并调整活细胞密度至1x105cells/mL。
3细胞铺板：以50μL/孔加入96孔细胞培养板。将培养板放在培养箱培养过夜(37℃，5％ CO2)。
4.标记待测抗体药物偶联物：将供试品/对照品工作液及标记试剂工作液以1:3摩尔比、1:1体积 比进行混合，于细胞培养箱(37℃，5％ CO2)中避光孵育15分钟使二者充分偶联。
5.俘获分析图像：转移标记后的供试品工作液至实验板的相应孔中，将实验板转移至Incucyte活细胞分析设备中，设置扫描拍照程序，使用Incucyte活细胞分析系统获取图像。以2小时的间隔进行定量，持续24小时。分析结果表示为：总荧光综合强度(RCU Xμm2/图像)。
实验结果如图5A，5B、5C、5D和表7所示。
表7双抗1nM在不同表达水平EGFR/HER3的细胞株上24小时的内吞
实验结论：
本发明针对EGFR和HER3靶标的抗体DBXT005-01相较对照抗体对不同表达EGFR或者HER3水平的肿瘤细胞具有更好的内吞效应。
实施例6双特异性抗体药物偶联物(ADC)的制备
6.1接头-细胞毒素(Linker-payload)的制备
Linker-payload X1的制备

第一步
氮气保护下，向27a(5.00g，43.0mmol)，NaHCO3(10.9g，129mmol)的DMF(50mL)溶液中滴加苄溴(11.0g，64.6mmol)，并在25℃反应17小时。TLC(PE/EA＝2/1)显示反应完全，将反应液加入到500mL水中，用EA(250mL)萃取两遍，分液后经饱和氯化钠水溶液(500mL)洗涤，无水Na2SO4干燥，浓缩过柱(PE:EA＝3:2)得5.1g无色液体，收率：57.1％。
第二步
氮气保护下，向KI2(4.00g，10.9mmol)，TsOH(800mg，4.65mmol)的THF(30mL)溶液中，在0℃下，滴加27b(4.50g，21.8mmol)的THF(10mL)溶液，并在25℃反应2小时。TLC(PE/EA＝1/2)显示反应完全，将反应液加入到200mL水中，用EA(200mL)萃取两次分液，无水Na2SO4干燥，浓缩过柱(PE/EA＝3/2)得白色固体1.56g，收率：26％。
第三步
氢气环境下，在0℃下，向27c(800mg，1.55mmol)的EtOH(8mL)和EA(8mL)混合溶液中加入Pd/C(80mg)，在0℃搅拌2.5小时。LCMS显示反应完全。反应液经硅藻土过滤，用EA(200mL)洗涤滤饼浓缩后用THF(20mL)溶解旋干得白色固体600mg，收率：91％。
第四步
氮气保护下，在0℃下，向27d(220mg，0.515mmol)，HY-13631A(250mg，0.47mmol)和HATU(214mg，0.56mmol)的DMF(6mL)溶液中加入DIEA(152mg，1.18mmol)，并在0℃反应2小时。LCMS显示反应完全。将反应液加入柠檬酸水溶液(pH＝4)(150mL)中，过滤，并用175mL水洗涤滤饼，滤干，用油泵拉干得棕色固体260mg，收率：66％。
第五步
氮气保护下，在0℃下，向27e(260mg，0.309mmol)的DCM(30mL)溶液中滴加二乙胺(8mL)，并在0℃反应3小时。LCMS显示反应完全。将反应液加入0℃的石油醚溶液(600mL)中，有固体析出，静置待固体吸附于瓶底后，倒出溶液，用油泵拉干，得棕色固体90mg，收率：47.1％。
第六步
氮气保护下，在0℃下，向27f(90mg，0.13mmol)，KI1(92mg，0.19mmol)和DIEA(50mg，0.39mmol)的DMF(2.5mL)溶液中加入HATU(74mg，0.19mmol)并在0℃反应2小时。LCMS显示基本反应结束。在0℃下，将反应液加入PH＝4的柠檬酸水溶液(30mL)中，有絮状固体析出，过滤，经制备板(DCM/MecOH＝10/1)得9.2mg淡黄色固体X1，收率：6％。
MS m/z(ESI)：1074[M+1]
H-NMR(400MHz，MeOD)：7.65(d,1H),7.62(s,1H),7.30-7.21(m,5H),6.79(s,2H),5.69-5.65(m,1H),5.57(d,1H),5.43-5.10(m,3H),4.70(d,2H),4.48-4.39(m,2H),4.10-4.05(m,1H),4.01-3.75(m,5H),3.46(t,2H),3.22-3.15(m,2H),3.07-3.00(m,1H),2.75(m,1H),2.62(m,1H),2.45(s,3H),2.37-2.20(m,6H),2.10-2.02(m,2H),2.00-1.92(m,2H)1.68-1.57(m,6H),1.01(t,3H)
Linker-payload X2的制备

第一步
将34a(5g，48.0mmol)、K2CO3(19.9g，144.0mmol)溶于DMF(20mL)中，滴加苄溴(12.3g，72.0mmol)，25℃下反应17小时。TLC(PE/EA＝3/1)检测原料反应完全。将反应液加入水(200mL)中、用EA(250mL)萃取分液，用饱和NaCl洗涤，无水Na2SO4干燥后浓缩过柱(PE:EA＝2:1)得8.7g无色液体34b，收率93％。MS-ESI:m/z 195.1[M+H]+。
第二步
取34c(7.3g，19.8mmol)、TsOH(1.46g，8.5mmol)溶于THF(20mL)中，氮气保护并降温至0℃，滴加43b(7.7g，39.6mmol)的THF(10mL)溶液，加完后0℃反应2小时。TLC(PE/EA＝2/1)显示原料大部分反应。将反应液倒入100mL水中，DCM(100mL)萃取，分液并用饱和NaCl洗涤，无水Na2SO4干燥后过柱(PE/EA＝1/1)，得3.9g无色稠状物34d，收率：39％。MS-ESI:m/z503.3[M+H]+。
第三步
氢气环境下，在0℃下，向34d(1.9g，3.78mmol)的EtOH(100mL)和EA(100mL)混合溶液中加入Pd/C(1g，10wt.％)，在0℃反应3小时。TLC(PE/EA＝2/1)显示反应完全。反应液经硅藻土过滤，用EA/EtOH(1:1，100mL×3)洗涤滤饼，滤液浓缩，并用THF(50mL×3)溶解旋干并重复三次得1g灰色固体34e，收率：64％。MS-ESI:m/z 435.2[M+Na]+。
第四步
在氮气保护下，0℃下向34e(426mg，1.03mmol)，KI4(500mg，0.94mmol)和HATU(429mg，1.13mmol)的DMF(20mL)溶液中滴加DIEA(303mg，2.35mmol)，加完后在0℃反应2小时。LCMS显示反应结束。将反应液滴到300mL水中，搅拌后静置5分钟，过滤，滤饼用DCM/MeOH(10:1，100mL)的溶液溶解后，干燥旋干拌样，柱层析(EA:MeOH＝30:1)得600mg黄色固体34f，收率：77％。MS-ESI:m/z 830.3[M+H]+。
第五步
氮气保护下，在0℃下，向34f(150mg，0.18mmol)的DCM(5mL)溶液中滴加二乙胺(5mL)，并在0℃反应2小时。LCMS显示反应完全。将石油醚溶液(100mL×6)加入反应液中，有固体析出，静置待固体沉淀后，倒出溶液，再用油泵拉干，得120mg白色粉末34g，LCMS显示产物含量为70％，收率：76％。MS-ESI:m/z 608.3[M+H]+。
第六步
在氮气保护下，向34g(60mg，0.099mmol)、43h(51mg，0.108mmol)、和DIEA(32mg，0.25mmol)的DMF(1mL)溶液中在0℃下加入HATU(45mg，0.118mmol)的DMF(1mL)溶液，并在0℃下反应2小时。LCMS显示原料反应完全。将反应液直接过反相柱，洗脱剂((MeCN/MeOH＝1/1):H2O＝60％:40％)，纯化得14.8mg黄色固体X2，收率14％。
MS-ESI:m/z 1062.4[M+H]+。
1H NMR(400MHz,Methanol-d4)δ7.69–7.61(m,2H),7.22–7.16(m,2H),7.16–7.09(m,3H),6.76(s,2H),5.70–5.64(m,1H),5.60(d,J＝16.4Hz,1H),5.40–5.31(m,2H),5.26(d,J＝19.0Hz,1H),4.65–4.50(m,7H),4.25–4.16(m,1H),3.87(d,J＝16.7Hz,1H),3.83–3.76(m,3H),3.72(d,J＝17.0Hz,2H),3.44(t,J＝7.1Hz,2H),3.25–3.17(m,2H),3.10–3.02(m,1H),2.92–2.83(m,1H),2.45–2.39(m,5H),2.32–2.20(m,5H),1.97–1.89(m,2H),1.63–1.50(m,4H),1.34–1.20(m,6H),0.99(t,J＝7.3Hz,3H).
Linker-payload X3的制备

第一步
向32a(2.00g，6.6mmol)，K2CO3(1.82g，13.2mmol)的MeCN(20mL)中加入溴丙烯(960mg，7.92mmol)，在20℃下搅拌5小时。TLC(PE/EA＝1/2)显示反应结束。将反应液倒入水100mL中，将pH值调至5，用EA(100mL)萃取三次，无水硫酸钠干燥，旋干过柱纯化(PE/EA＝2/1)得1.83g白色固体32b，收率：81％。
第二步
向32b(1.38g，4.02mmol)的DCM(10mL)中加入TFA(10mL)，在25℃下搅拌17小时。TLC(PE/EA＝1/3)显示反应结束。将反应液旋干得0.91g黄色粘状物32c，收率不计。
第三步
向32c(910mg，4.87mmol)，NaHCO3(613mg，7.3mmol)的DME/H2O(20mL/10mL)中加入41d(1.92g，4.87mmol)，在25℃下搅拌3小时。TLC(DCM/MeOH＝1/1)显示反应结束。将反应液倒入水100mL中，用aq.HCl(1N)将pH值调至5，用EA(150mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(DCM/MeOH＝20/1)得1.53g白色固体32e，收率：67％。MS-ESI:m/z 467.4[M+H]+。
第四步
向32f(3g，5.83mmol)的MeOH(50mL)中加入Pd/C(600mg)，在25℃在氢气球下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.9g白色固体32g，收率：77％。
第五步
向32g(789mg，1.86mmol)，KI4(900mg，1.69mmol)，三乙胺(342mg，3.38mmol)的DMF(10mL)中加入HATU(707mg，1.86mmol)，在0℃下搅拌3.5小时。TLC(EA)显示反应结束。将反应液倒入H2O(80mL)，用EA(100mL)萃取两次，无水硫酸钠干燥，旋干过柱纯化(EA)得1.186g白色固体32h，收率：83％。MS-ESI:m/z 842.3[M+H]+。
第六步
将32h(1.186g，1.41mmol)的DCM/二乙胺(20mL，20/1)在25℃下搅拌17小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液倒入石油醚(200mL)中过滤得768mg白色固体32i，收率：88％。MS-ESI:m/z 620.3[M+H]+。
第七步
向32i(676mg，1.09mmol)，32e(508mg，1.09mmol)，DIEA(423mg，3.27mmol)的DMF(10mL)中加入HATU(414mg，1.09mmol)，在20℃下搅拌17小时。TLC(PE/EA＝1/5)显示反应结束。将反应液倒入水中(30mL)过滤，滤饼过柱纯化(DCM/MeOH＝50/1)511mg白色固体32j，收率：44％。MS-ESI:m/z 1068.3[M+H]+。
第八步
将32j(482mg，0.451mmol)的二乙胺/DCM(10mL，1/5)的溶液在10℃下搅拌17小时。TLC(EA)显示反应结束。将反应液倒入PE(300mL)中过滤得301mg白色固体32k，收率不计。
第九步
向32k(301mg，0.356mmol)，Pd(PPh3)4(82mg，0.071mmol)的THF(5mL)中加入吗啡啉(93mg，1.07mmol)，在25℃下搅拌5小时。LCMS显示反应结束。将反应液制备得108mg白色固体32l，收率：38％。MS-ESI:m/z 806.3[M+H]+。
第十步
向32l(108mg，0.134mmol)，三乙胺(41mg，0.402mmol)的THF(2mL)和DMF(2mL)中加入溴乙酰溴(27mg，0.134mmol)，在0℃下搅拌1小时。TLC(DCM/MeOH＝10/1)显示反应结束。将反应液直接制备得15mg白色固体X3，收率：12％。
MS-ESI:m/z 926.3[M+H]+。
1H NMR(400MHz,DMSO-d6)δ12.11(s,1H),8.54–8.42(m,3H),8.27–8.16(m,2H),7.78(d,J＝11.0Hz,1H),7.30(s,1H),6.53(s,1H),5.61–5.51(m,1H),5.42(s,2H),5.20–5.05(m,2H),4.56–4.42(m,2H),4.32–4.22(m,1H),3.96–3.87(m,3H),3.79(d,J＝5.6Hz,2H),3.70(d,J＝5.9Hz,2H),3.25–3.08(m,2H),2.61–2.53(m,2H),2.45–2.36(m,4H),2.36–2.22(m,3H),2.20–2.03(m,4H),1.99–1.68(m,4H),0.87(t,J＝7.3Hz,3H).
Linker-payload X4的制备
第一步
向33a(2.00g，2.58mmol)的MeOH(20mL)中加入Pd/C(400mg，10wt.％)，在20℃下搅拌5小时。TLC(EA)显示反应结束。将反应液过滤旋干得1.3g白色固体33b，收率：74％。
第二步
向33b(0.55g，0.802mmol)，KI4(427mg，0.802mmol)和DIPEA(310mg，2.40mmol)的DMF(5mL)中加入HATU(305mg，0.802mmol)，在0℃下搅拌2小时。TLC(DCM/MeOH＝1/10)显示反应结束。将反应液倒入水(40mL)中，过滤得粗品，经柱纯化(DCM/MeOH＝20/1)得360mg黄色固体33c，收率41％。
第三步
向33c(360mg，0.326mmol)的DCM(10mL)中加入二乙胺(2mL)。在25℃下搅拌17小时。TLC(DCM/MeOH＝5/1)显示反应结束。将反应液倒入PE(100mL)中，过滤得205mg白色固体33d，收率：71％。MS-ESI:m/z 881.3[M+H]+。
第四步
向33d(205mg，0.233mmol)和三乙胺(118mg，1.17mmol)的DMF(1mL)和水(1mL)中加入溴乙酰溴(94mg，0.446mmol)的THF(2mL)溶液，并在0度搅拌1小时，反应液直接制备得15mg白色固体X4，收率：6％。
MS-ESI:m/z 1001.2[M+H]+。
1H NMR(400MHz,DMSO-d6)δ8.57–8.50(m,1H),8.50–8.43(m,2H),8.35–8.29(m,1H),8.19–8.12(m,2H),7.80(d,J＝10.8Hz,1H),7.27–7.14(m,7H),6.53(s,1H),5.59–5.51(m,1H),5.44–5.39(m,2H),5.20–5.07(m,2H),4.56–4.44(m,3H),3.92(s,3H),3.80–3.68(m,5H),3.41(s,1H),3.21–3.12(m,2H),2.83–2.74(m,1H),2.58–2.55(m,3H),2.39(s,4H),2.18–2.03(m,4H),1.93–1.78(m,2H),0.87(t,J＝7.3Hz,3H).
6.2抗体药物偶联物的制备
表8
Isotype IgG1来源：购于百英生物(太仓)。
DBXT001-01-X1的制备
ADC-7(DBXT001-01-X1-DAR4)
向抗体DBXT001-01的缓冲液(PBS pH 7.2；70mg，8.52mg/mL，0.47μmol)加入10mM EDTA溶液(0.875mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(3.48mM，0.379mL，1.32μmol)，置于 恒温搅拌器中，搅拌转速为60rpm，于22℃反应2小时，并停止反应；
将X1(2.61mg，2.43μmol)溶解于1.75mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。活性炭过滤后，在AKTA上将反应液用G-25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10 Desalting column，53mL；洗脱相：30mM组氨酸-盐酸pH 5.5)，并用30KD超滤管超滤浓缩后得到示例性产物ADC-7的溶液(30mM组氨酸-盐酸pH 5.5；68mg，22mg/mL，收率：97％)，于-80℃储存。
HIC DAR分析检测并计算得DAR值n＝4.06；SEC主峰纯度为98.2％。
ADC-8(DBXT001-01-X1-DAR6)
向抗体DBXT001-01的缓冲液(PBS pH 7.2；70mg，8.52mg/mL，0.47μmol)加入10mM EDTA溶液(0.875mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(3.48mM，0.598mL，2.08μmol)，置于恒温搅拌器中，搅拌转速为60rpm，于22℃反应2小时，并停止反应；
将X1(4.30mg，4.00μmol)溶解于1.75mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。活性炭过滤后，在AKTA上将反应液用G-25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10 Desalting column，53mL；洗脱相：30mM组氨酸-盐酸pH 5.5)，并用30KD超滤管超滤浓缩后得到示例性产物ADC-8的溶液(30mM组氨酸-盐酸pH 5.5；69mg，23mg/mL，收率：99％)，-80℃储存。
HIC DAR分析检测并计算得DAR值n＝6.11；SEC主峰纯度为99.2％。
ADC-9(DBXT001-01-X1-DAR8)
向抗体DBXT001-01的缓冲液(PBS pH 7.2；70mg，8.52mg/mL，0.47μmol)加入10mM EDTA溶液(0.875mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(3.48mM，1.12mL，3.89μmol)，置于恒温搅拌器中，搅拌转速为60rpm，于22℃反应2小时，并停止反应；
将X1(5.05mg，4.70μmol)溶解于1.75mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。活性炭过滤后，在AKTA上将反应液用G-25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10 Desalting column，53mL；洗脱相：30mM组氨酸-盐酸pH5.5)，并用30KD超滤管超滤浓缩后得到示例性产物ADC-9的溶液(30mM组氨酸-盐酸pH 5.5；68mg，22.4mg/mL，收率：97％)，于-80℃储存。
HIC DAR分析检测并计算得DAR值n＝7.99；SEC主峰纯度为99.0％。
DBXT001-01-X2的制备
ADC-4(DBXT001-01-X2-DAR4)
向抗体DBXT001-01的缓冲液(PBS pH 7.4；70mg，8.52mg/mL，0.47μmol)加入10mM EDTA溶液(0.875mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(3.48mM，0.379mL，1.32μmol)，置于恒温搅拌器中，搅拌转速为60rpm，于37℃反应2小时，并停止反应；
将X2(2.75mg，2.59μmol)溶解于1.75mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。活性炭过滤后，在AKTA上将反应液用G-25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10 Desalting column，53mL；洗脱相：30mM组氨酸-盐酸pH 5.5)，并用30KD超滤管超滤浓缩后得到示例性产物ADC-4的溶液(30mM组氨酸-盐酸pH 5.5；67.2mg，22mg/mL，收率：96％)，于-80℃储存。
HIC DAR分析检测并计算得DAR值n＝4.10；SEC主峰纯度为98.4％。
ADC-5(DBXT001-01-X2-DAR6)
向抗体DBXT001-01的缓冲液(PBS pH 7.4；70mg，8.52mg/mL，0.47μmol)加入10mM EDTA溶液(0.875mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(3.48mM，0.598mL，2.08μmol)，置于恒温搅拌器中，搅拌转速为60rpm，于37℃反应2小时，并停止反应；
将X2(4.58mg，4.31μmol)溶解于1.75mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。活性炭过滤后，在AKTA上将反应液用G-25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10 Desalting column，3mL；洗脱相：30mM组氨酸-盐酸pH 5.5)，并用30KD超滤管超滤浓缩后得到示例性产物ADC-5的溶液(30mM组氨酸-盐酸pH 5.5；66.5mg，25mg/mL，收率：95％)，于-80℃储存。
HIC DAR分析检测并计算得DAR值n＝6.05；SEC主峰纯度为98.9％。
ADC-6(DBXT001-01-X2-DAR8)
向抗体DBXT001-01的缓冲液(PBS pH 7.2；70mg，8.52mg/mL，0.47μmol)加入10mM EDTA溶液(0.875mL)和配制好的三(2-羰基乙基)磷盐酸盐溶液(3.48mM，1.12mL，3.89μmol)，置于恒温搅拌器中，搅拌转速为60rpm，于22℃反应2小时，并停止反应；
将X2(5.49mg，5.17μmol)溶解于1.75mL DMA中，加入到上述溶液中，置于恒温搅拌器中，搅拌转速为60rpm，于22℃振荡反应2小时，停止反应。活性炭过滤后，在AKTA上将反应液用G- 25凝胶柱脱盐纯化(脱盐柱：HiPrep 26/10 Desalting column，53mL；洗脱相：30mM组氨酸-盐酸pH 5.5)，并用30KD超滤管超滤浓缩后得到示例性产物ADC-6的溶液(30mM组氨酸-盐酸pH 5.5；67.9mg，29.2mg/mL，收率：97％)，于-80℃储存。
HIC DAR分析检测并计算得DAR值n＝7.98；SEC主峰纯度为99.0％。
ADC-13(DBXT005-01-X2,DAR6)
在PBS 7.4缓冲液中(PBS 7.4，2mM EDTA，pH 7.4)，用20当量的三(2-羧乙基)膦(TCEP)还原抗体DBXT005-01，在摇床上22℃振荡反应3小时(摇床转速60rpm)。
中间监测后，补加5当量的三(2-羧乙基)膦(TCEP)在摇床上22℃继续振荡反应1小时。不除去多余TCEP，将反应液直接用于下一步偶联反应。
还原后的抗体在常温下缓慢加入200mM组氨酸-盐酸，pH 6.0(使偶联反应液中组氨酸-盐酸终浓度为20mM)，含0.1％v/v醋酸的二甲基亚砜和接头-细胞毒素(Linker-payload)溶液(10当量，10mM X2溶液(含0.1％v/v醋酸的二甲基亚砜))。反应体系中有3％v/v的含0.1％v/v醋酸的二甲基亚砜(DMSO)。充分混匀后，偶联反应液在摇床上22℃振荡反应1.5小时(摇床转速60rpm)。
中间监测后，补加接头-细胞毒素(Linker-payload)(2当量，10mM X2溶液(含0.1％v/v醋酸的二甲基亚砜))。充分混匀后，偶联反应液在摇床上22℃继续振荡反应2小时(摇床转速60rpm)。
反应结束后，将ADC溶液用脱盐柱(40K)换液到储存缓冲液中(20mM组氨酸-盐酸，pH 6.0)。取样送检，得到ADC产品。
检测结果如下：
ADC-14(BL-B01D1，DAR8)的制备参照专利WO2023083381A1，具体如下：
在PBS 7.4缓冲液中(PBS 7.4，2mM EDTA，pH 7.4)，用15当量的三(2-羧乙基)膦(TCEP)还原抗体SI-1X6.4，在摇床上22℃振荡反应17.5小时(摇床转速60rpm)。不除去多余TCEP，将反应液直接用于下一步偶联反应。
还原后的抗体在常温下缓慢加入二甲基亚砜(DMSO)和接头-细胞毒素(Linker-payload)(10当量，10mM Ed-04的DMSO溶液)。反应体系中含有10％v/v的二甲基亚砜(DMSO)。充分混匀后，偶联反应液在摇床上22℃振荡反应2小时(摇床转速60rpm)。
中间监测后，补加接头-细胞毒素(Linker-payload)(2当量，10mM Ed-04的DMSO溶液)。充分混匀后，偶联反应液在摇床上22℃继续振荡反应1.5小时(摇床转速60rpm)。
反应结束后，将200mM组氨酸-盐酸，pH 5.5加入反应液中，使得反应液中组氨酸-盐酸浓度为20mM，然后将反应液用0.22μm PES针孔式滤器过滤，随后将ADC溶液用AKTA通过脱盐柱(HiPrepTM 26/10 Desalting)换液到储存缓冲液中(20mM组氨酸-盐酸，pH 5.5)。用Amicon(30K MWCO)将反应液进行浓缩，最后用0.22μm PES针孔式滤器过滤得到ADC产品，取样送检。
检测结果如下：
参考DBXT001-01-X2的制备过程，仅替换表8中对应的抗体克隆来制备ADC-1～3、ADC-10和ADC-11。
参考上述实验过程，经将表8中抗体克隆DBXT001-01替换为DBXT001-02～08、DBXT003-01～08和DBXT004-01～08，可以制备的相应的双特异性抗体药物偶联物。
实施例7：抗体药物偶联物对EGFR低表达HER3中表达的人食管癌细胞株OE-19荷瘤小鼠体内药效评价
为研究ADC-6对人食管癌细胞株体内形成肿瘤的抑制作用，在小鼠皮下异位接种OE-19形成移植瘤后，评估ADC-6的抗肿瘤效果，并且和母本单抗ADC的抗肿瘤效果进行比较。
1.受试药物及材料
空白对照组(对照组)：生理盐水
ADC-1(治疗组)：Day 0:1mg/kg；Day 12:3mg/kg
ADC-2(治疗组)：Day 0:1mg/kg；Day 12:3mg/kg
ADC-6(治疗组)：Day 0:1mg/kg；Day 12:3mg/kg
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：6-7周龄的雌性BALB/c Nude小鼠，购自集萃药康生物科技有限公司。
4.试验方法：
OE-19(CL-00806)细胞培养在含10％胎牛血清和2mM L-Glutamine的RPMI1640培养液中。收集指数生长期的OE-19细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种5×106OE-19细胞，细胞重悬在1:1的PBS与基质胶中(0.1mL/只)，定期观察肿瘤生长情况。
当肿瘤长至约140.15mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射受试品，当天和第12天各一次，共注射2次，当天治疗组剂量为1mg/kg，第12天治疗组剂量为3mg/kg。试验终点时间为分组后第25天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]× 100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P<0.05为具有显著性差异。作图软件为GraphPad Prism。
表9.受试物对OE-19细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后25天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T25/V25×100)和TGI(TGI(％)＝[1-(T25-T0)/(V25-V0)]×100)反映；
实验结果如图6及表9所示，在OE-19模型，单抗ADC-1(EGFR-ADC)不能抑制肿瘤生长，双抗ADC-6(EGFR-HER3 ADC)比单抗ADC-2(HER3-ADC)有更好的抑瘤效果。
实验结论：
对EGFR-ADC耐药的模型，双抗ADC有比单抗ADC更好的抑瘤效果。
实施例8：抗体药物偶联物对EGFR中表达HER3中表达的人非小细胞肺癌细胞株NCI-H441荷瘤小鼠体内药效评价
为研究ADC-6对人非小细胞肺癌细胞株体内形成肿瘤的抑制作用，在小鼠皮下异位接种NCI-H441形成移植瘤后，评估ADC-6的抗肿瘤效果，并且和母本单抗ADC的抗肿瘤效果进行比较。
1.受试药物及材料
空白对照组(对照组)：生理盐水
ADC-1(治疗组)：0.5mg/kg
ADC-2(治疗组)：0.5mg/kg
ADC-6(治疗组)：0.5mg/kg
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：6-7周龄的雄性NU/NU小鼠，购自北京维通利华实验动物技术有限公司。
4.试验方法：
NCI-H441(CL-00759)细胞培养在含10％胎牛血清的RPMI1640培养液中。收集指数生长期的NCI-H441细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种5×106NCI-H441细胞，细胞重悬在1:1的PBS与基质胶中(0.1mL/只)，定期观察肿瘤生长情况。
当肿瘤长至约139.12mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射受试品，共注射1次，当天治疗组剂量为0.5mg/kg。试验终点时间为分组后第35天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P<0.05为具有显著性差异。作图软件为GraphPad Prism。
表10.受试物对NCI-H441细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后35天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T35/V35×100)和TGI(TGI(％)＝[1-(T35-T0)/(V35-V0)]×100)反映；
3.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。
实验结果如图7及表10所示，在NCI-H441模型中，ADC-6比ADC-2有更好的抑瘤效果。
实验结论：
在EGFR和HER3表达量相当的模型中，双抗ADC比单抗ADC有更好的抑瘤效果。
实施例9：抗体药物偶联物对EGFR高表达HER3低表达的人口腔鳞癌细胞株CAL-27荷瘤小鼠体内药效评价
为研究ADC-6对人口腔鳞癌细胞株体内形成肿瘤的抑制作用，在小鼠皮下异位接种CAL-27形成移植瘤后，评估ADC-6的抗肿瘤效果，并且和母本单抗ADC及EGFR单抗的抗肿瘤效果进行比较。
1.受试药物及材料
空白对照组(对照组)：生理盐水
ADC-1(治疗组)：3mg/kg
ADC-2(治疗组)：3mg/kg
ADC-6(治疗组)：3mg/kg
EGFR单抗Zalutumumab(治疗组)：3mg/kg
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：6-7周龄的雌性NOD/Scid小鼠，购自集萃药康生物科技有限公司。
4.试验方法：
CAL-27(CL-00599)细胞培养在含10％胎牛血清的DMEM培养液中。收集指数生长期的CAL-27细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种1×107CAL-27细胞，细胞重悬在1:1的PBS与基质胶中(0.1mL/只)，定期观察肿瘤生长情况。
当肿瘤长至约144.75mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第0天)开始通过静脉(i.v.)注射受试品，当天和第14天各一次，共注射2次，治疗组剂量为3mg/kg。试验终点时间为分组后第27天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P<0.05为具有显著性差异。作图软件为GraphPad Prism。
表11.受试物对CAL-27细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后27天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T27/V27×100)和TGI(TGI(％)＝[1-(T27-T0)/(V27-V0)]×100)反映；
3.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。
实验结果如图8及表11所示，在CAL-27模型中，ADC-1和ADC-6都有显著的抑瘤效果，且优于ADC-2和EGFR单抗。
实验结论：
在对靶向EGFR敏感的EGFR高表达HER3低表达模型中，双抗ADC比EGFR单抗或HER3ADC有更优的抑瘤效果，且不劣于EGFR ADC。
实施例10：抗体药物偶联物不同DAR值在NCI-H1975和OE-19荷瘤小鼠体内药效评价
为研究DBXT001-01偶联接头-细胞毒素X2的不同DAR值在人非小细胞肺癌细胞株NCI-H1975和人食管癌细胞株OE-19体内形成肿瘤的抑制作用，在小鼠右侧背部皮下分别异位接种NCI-H1975或者OE-19形成移植瘤后，评估双抗偶联毒素不同DAR值后的抗肿瘤效果。
受试药物及给药方案见表12和表13。
表12.人肺癌NCI-H1975皮下异种移植模型中的给药途径、剂量及方案
表13.人食管癌OE-19细胞株皮下异种移植模型中的给药途径、剂量及方案
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：6-8周龄的雌性Balb/c裸鼠，购自集萃药康生物科技有限公司。
4.试验方法：
NCI-H1975(CL-00650)细胞培养在含10％胎牛血清的RPMI1640培养液中，OE-19(CL-00806)细胞培养在含10％胎牛血清和2mM L-Glutamine的RPMI1640培养液中。收集指数生长期的细胞，PBS重悬至适合浓度用于裸鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种5×106NCI-H1975细胞，细胞重悬在PBS中(0.1mL/只)。定期观察肿瘤生长情况。
实验小鼠于右侧背部皮下接种5×106OE-19细胞，细胞重悬在1:1的PBS与基质胶中(0.1mL/只)，定期观察肿瘤生长情况。
当肿瘤长至约121.71mm3(NCI-H1975)或者139.93mm3(OE-19)，对荷瘤小鼠进行StudyDirectorTM随机分组，分组当天定为Day0，具体给药方案见表12和表13。试验终点时间为分 组后第31天(NCI-H1975)，或第35天(OE-19)。每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P<0.05为具有显著性差异。作图软件为GraphPad Prism。
表14.受试物对NCI-H1975细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后18天1肿瘤体积计算得出)
表15.受试物对OE-19细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后21天1肿瘤体积计算得出)
1.因为NCI-H1975空白对照组有小鼠在18天后达到安乐死标准，统计学分析数据不完整，因此采取动物数量完整的第18天进行统计分析；同样，OE-19空白对照组有小鼠在21天后达到安乐死标准，统计学分析数据不完整，因此采取动物数量完整的第21天进行统计分析；
2.肿瘤体积用平均值±标准误表示；
3.肿瘤生长抑制由T/C(T/C(％)＝TD/VD×100)和TGI(TGI(％)＝[1-(TD-T0)/(VD-V0)]×100)反映；
4.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。
实验结果如图9和图10及表14和表15所示，在NCI-H1975模型的给药方案中，ADC-2，ADC-4，ADC-5，ADC-6都有抑制肿瘤作用。
实验结论：
靶向EGFR和HER3的本发明双抗偶联接头-细胞毒素X2有抑瘤效果，且随着DAR值升高抑瘤 效果增强。
实施例11：本抗体药物偶联物与对照抗体偶联物在NCI-H1975荷瘤小鼠体内药效的比较
为比较DBXT001-01偶联接头-细胞毒素X2与对照抗体SI-1X6.4偶联接头-细胞毒素X2对人非小细胞肺癌细胞株NCI-H1975体内形成肿瘤的抑制作用，在小鼠右侧背部皮下异位接种NCI-H1975形成移植瘤后，评估两者的抗肿瘤效果。
受试药物及给药方案见表16。
表16.人肺癌NCI-H1975皮下异种移植模型中的给药途径、剂量及方案
a.按照分子量进行了摩尔换算，保证各治疗组之间摩尔浓度一致。
1.配制方法：所有样品均用生理盐水稀释配制。
2.试验动物：6-8周龄的雌性Balb/c裸鼠，购自集萃药康生物科技有限公司。
3.试验方法：
NCI-H1975(CL-00650)细胞培养在含10％胎牛血清的RPMI1640培养液中。收集指数生长期的细胞，PBS重悬至适合浓度用于裸鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种5×106NCI-H1975细胞，细胞重悬在1:1的DPBS:Matrigel中(0.2mL/只)。定期观察肿瘤生长情况。
当肿瘤长至约164mm3(NCI-H1975)，对荷瘤小鼠进行StudyDirectorTM随机分组，分组当天定为Day0，具体给药方案见表16。试验终点时间为分组后第26天。每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P<0.05为具有显著性差异。作图软件为GraphPad Prism。
表17.受试物对NCI-H1975细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后17天1肿瘤体积计算得出)

1.因为NCI-H1975空白对照组有小鼠在17天后达到安乐死标准，统计学分析数据不完整，因此采取动物数量完整的第17天进行统计分析；
2.肿瘤体积用平均值±标准误表示；
3.肿瘤生长抑制由T/C(T/C(％)＝T17/V17×100)和TGI(TGI(％)＝[1-(T17-T0)/(V17-V0)]×100)反映；
4.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)
实验结果如图11及表17所示，在NCI-H1975模型的给药方案中，ADC-3，ADC-6，ADC-10都有显著的抑制肿瘤作用，其中ADC-6显示出了最强的抑制肿瘤作用，ADC-3和ADC-10药效相当。
实验结论：
靶向EGFR和HER3的本发明双抗偶联接头-细胞毒素X2(DAR8)比对照抗体SI-1X6.4偶联接头-细胞毒素X2(DAR8)有更强的抑瘤效果。
实施例12：本抗体药物偶联物与对照抗体偶联物在OE-19荷瘤小鼠体内药效的比较
为比较DBXT001-01偶联接头-细胞毒素X2或者接头-细胞毒素X1与对照抗体SI-1X6.4偶联接头-细胞毒素X2对人食管癌细胞株OE-19体内形成肿瘤的抑制作用，在小鼠右侧背部皮下异位接种OE-19形成移植瘤后，评估体内的抗肿瘤效果。
受试药物及给药方案见表18。
表18.人食管癌OE-19皮下异种移植模型中的给药途径、剂量及方案
a.按照分子量进行了摩尔换算，保证各治疗组之间摩尔浓度一致。
1.配制方法：所有样品均用生理盐水稀释配制。
2.试验动物：6-8周龄的雌性Balb/c裸鼠，购自集萃药康生物科技有限公司。
3.试验方法：
OE-19(CL-00806)细胞培养在含10％胎牛血清和2mM L-Glutamine的RPMI1640培养液中。
实验小鼠于右侧背部皮下接种5×106OE-19细胞，细胞重悬在1:1的PBS与基质胶中(0.1mL/只)，定期观察肿瘤生长情况。
当肿瘤长至约138mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，分组当天定为Day0，具体给药方案见表18。试验终点时间为分组后第21天。每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P<0.05为具有显著性差异。作图软件为GraphPad Prism。
表19.受试物对OE-19细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后21天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T21/V21×100)和TGI(TGI(％)＝[1-(T21-T0)/(V21-V0)]×100)反映。
3.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)
实验结果如图12及表19所示，在OE-19模型的给药方案中，ADC-3，ADC-6，ADC-9，ADC-10都有抑制肿瘤作用，其中ADC-6显示出了最强的抑制肿瘤作用，ADC-3，ADC-9和ADC-10药效相当。
实验结论：
在靶向EGFR治疗耐药的模型中，靶向EGFR和HER3的本发明双抗偶联接头-细胞毒素X2(DAR8)能克服EGFR耐药，并且比对照抗体SI-1X6.4偶联接头-细胞毒素X2(DAR8)有更强的抑瘤效果。
实施例13：抗体药物偶联接头-细胞毒素X1在NCI-H1975荷瘤小鼠体内药效评价
为研究DBXT001-01偶联接头-细胞毒素X1(DAR8)在人非小细胞肺癌细胞株NCI-H1975体内形成肿瘤的抑制作用，在小鼠右侧背部皮下异位接种NCI-H1975形成移植瘤后，评估本抗体偶联物的抗肿瘤效果。
受试药物及给药方案见表20。
表20.人肺癌NCI-H1975皮下异种移植模型中的给药途径、剂量及方案
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：NCI-H1975：6-8周龄的雌性CB-17SCID，购自北京维通利华实验动物技术有限公司；
4.试验方法：
NCI-H1975(中国科学院细胞库)细胞培养在含10％胎牛血清的RPMI1640培养液中，收集指数生长期的细胞，DPBS重悬至适合浓度用于裸鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种5×106NCI-H1975细胞(重悬细胞的DPBS1:1与BD Matrigel基质胶混匀，Catalogue:356234，0.2mL/只)，定期观察肿瘤生长情况。
当肿瘤长至约201.31mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，分组当天定为Day0，具体给药方案见表20。试验终点时间为分组后第20天。每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
两组样本之间比较采用独立样本T检验(T-Test)，数据使用SPSS进行分析，P<0.05为具有显著性差异。作图软件为GraphPad Prism。
表21.受试物对NCI-H1975细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后18天1肿瘤体积计算得出)
1.因为NCI-H1975空白对照组有小鼠在18天后达到安乐死标准，统计学分析数据不完整，因此采取动物数量完整的第18天进行统计分析；
2.肿瘤体积用平均值±标准误表示；
3.肿瘤生长抑制由T/C(T/C(％)＝T18/V18×100)和TGI(TGI(％)＝[1-(T18-T0)/(V18-V0)]×100)反映；
4.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。
实验结果如图13及表21所示，在NCI-H1975模型的给药方案中，ADC-9有显著抑制肿瘤作用，而空白对照组和ADC-12没有抑制肿瘤作用。
实验结论：
靶向EGFR和HER3的本发明双抗偶联接头-细胞毒素X1的抗体药物偶联物有显著的抑制肿瘤生长作用。
实施例14本发明双抗ADC与对照ADC在对EGFR ADC耐药的小鼠模型体内药效比较
为比较DBXT005-01 ADC(ADC-13)和对照ADC(BL-B01D1，ADC-14)对EGFR ADC耐药模型体内药效，在小鼠皮下异位接种OE-19形成移植瘤后，比较两者的抗肿瘤效果。
1.受试药物及及给药方案见表22。
表22.人食管癌OE-19皮下异种移植模型中的给药途径、剂量及方案
2.配制方法：所有样品均用生理盐水稀释配制。
3.试验动物：6-7周龄的雌性BALB/c Nude小鼠，购自集萃药康生物科技有限公司。
4.试验方法：
OE-19(CL-00806)细胞培养在含10％胎牛血清和2mM L-Glutamine的RPMI1640培养液中。收集指数生长期的OE-19细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种5×106OE-19细胞，细胞重悬在1:1的PBS与基质胶中(0.1mL/只)，定期观察肿瘤生长情况。
当肿瘤长至约151mm3，对荷瘤小鼠进行StudyDirectorTM随机分组，并于当天(第1天)开始通过静脉(i.v.)注射受试品，共注射1次，试验终点时间为分组后第23天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用T/C(％)评价。T/C(％)的百分比值是一项反映肿瘤生长抑制的指标，T和C分别表示给药组和对照组在某一天的平均肿瘤体积。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
样本组之间比较采用ONE-WAY ANOVA，P<0.05为具有显著性差异。作图软件和统计分析为GraphPad Prism。
表23.受试物对OE-19细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后23天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T23/V23×100)和TGI(TGI(％)＝[1-(T23-T0)/(V23-V0)]×100)反映；
3.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。
实验结果如图14及表23所示，在EGFR ADC耐药模型中，本发明抗体偶联物比对照ADC-14有更好的抑瘤效果。
实施例15本发明双抗ADC与对照ADC在EGFR高表达HER3低表达的小鼠模型体内药效比较
为比较DBXT005-01 ADC(ADC-13)和对照ADC(BL-B01D1，ADC-14)对EGFR高表达HER3低表达细胞株体内形成肿瘤的抑制作用，在小鼠皮下异位接种A431形成移植瘤后，比较两者的抗肿瘤效果。
1.受试药物及给药方案见表244。
表24.人皮肤癌A431皮下异种移植模型中的给药途径、剂量及方案
2.配制方法：所有样品均用辅料溶剂稀释配制。
3.试验动物：6-8周龄的雌性NCG小鼠，购自集萃药康生物科技有限公司。
4.试验方法：
A431(CRL-1555)细胞培养在含10％胎牛血清和2mM L-Glutamine的DMEM培养液中。收集指数生长期的A431细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种5×106A431细胞，定期观察肿瘤生长情况。
当肿瘤长至约137mm3，对荷瘤小鼠进行随机分组，并于当天(第0天)开始通过静脉(i.v.)注射受试品共注射1次，试验终点时间为分组后第21天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用TGI(％)评价。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti/T0)/(Vi/V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
样本组之间比较采用ONE-WAY ANOVA，P<0.05为具有显著性差异。作图软件和统计分析为GraphPad Prism。
表25.受试物对A431细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后21天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由T/C(T/C(％)＝T21/V21×100)和TGI(TGI(％)＝[1-(T21/T0)/(V21/V0)]×100)反映；
3.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。各试验组和空白对照组之间的肿瘤体积比较。
4.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。本实施例双抗ADC和对照ADC试验组的肿瘤体积比较。
实验结果如图15及表25所示，在EGFR高表达，HER3低表达的肿瘤模型中，在相同的给药剂量下，本发明抗体偶联物比对照ADC具有更好的抑瘤作用。对照ADC在该给药浓度下不能抑制肿瘤生长。各给药组小鼠耐受良好，体重无明显下降。
实施例16本实施例双抗ADC与对照ADC在不表达EGFR仅表达HER3的人结肠癌细胞株异种移植瘤小鼠模型的药效比较
为比较DBXT005-01 ADC(ADC-13)和对照ADC(BL-B01D1，ADC-14)对不表达EGFR仅表达HER3的人结肠癌细胞株体内形成肿瘤的抑制作用，在小鼠皮下异位接种SW620形成移植瘤后，比较两者的抗肿瘤效果。
1.受试药物及给药方案见表26。
表26.人结肠癌SW620皮下异种移植模型中的给药途径、剂量及方案
2.配制方法：所有样品均用辅料溶剂稀释配制。
3.试验动物：6-8周龄的雌性NCG小鼠，购自集萃药康生物科技有限公司。
4.试验方法：
SW620(CCL-227)细胞培养在含10％胎牛血清和2mM L-Glutamine的DMEM培养液中。收集指数生长期的SW620细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种5×106SW620细胞，定期观察肿瘤生长情况。
当肿瘤长至约131mm3，对荷瘤小鼠进行随机分组，并于当天(第0天)开始通过静脉(i.v.)注射受试品，试验终点时间为分组后第59天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用TGI(％)评价。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti/T0)/(Vi/V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
样本组之间比较采用ONE-WAY ANOVA，P<0.05为具有显著性差异。作图软件和统计分析为GraphPad Prism。
表27.受试物对SW620细胞皮下异种移植肿瘤模型的抑瘤药效评价
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由TGI(TGI(％)＝[1-(T31/T0)/(V31/V0)]×100)反映；
3.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。各试验组和空白对照组之间的肿瘤体积比较。
4.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。本实施例双抗ADC和对照ADC试验组的肿瘤体积比较。
5.N/A代表对照组小鼠按照动物福利原则在第31天已经安乐死。
实验结果如图16及表27所示，在EGFR不表达，HER3中表达的肿瘤模型中，所有试验组对比空白对照组都有显著的抑制肿瘤生长作用；在相同的给药剂量下，本发明抗体偶联物比对照ADC具有更好的抑瘤作用，能让肿瘤完全消退并持续到第59天；对照组ADC不能让肿瘤完全消退，不能抑制肿瘤重新生长。各给药组小鼠耐受良好，体重无明显下降。
实施例17本发明双抗ADC与对照ADC在EGFR中表达HER3低表达的人结肠癌细胞株异种移植瘤小鼠模型的药效比较
为比较DBXT005-01 ADC(ADC-13)和对照ADC(BL-B01D1，ADC-14)对EGFR中表达HER3低表达的人结肠癌细胞株体内形成肿瘤的抑制作用，在小鼠皮下异位接种SW48形成移植瘤后，比较两者的抗肿瘤效果。
1.受试药物及给药方案见表28。
表28.人结肠癌SW48皮下异种移植模型中的给药途径、剂量及方案
2.配制方法：所有样品均用辅料溶剂稀释配制。
3.试验动物：6-8周龄的雌性NCG小鼠，购自集萃药康生物科技有限公司。
4.试验方法：
SW48(CCL-231)细胞培养在含10％胎牛血清和2mM L-Glutamine的DMEM培养液中。收集指数生长期的SW48细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
实验小鼠于右侧背部皮下接种1×107SW48细胞，定期观察肿瘤生长情况。
当肿瘤长至约131mm3，对荷瘤小鼠进行随机分组，并于当天(第0天)开始通过静脉(i.v.)注射受试品，试验终点时间为分组后第45天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组5只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用TGI(％)评价。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti/T0)/(Vi/V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
样本组之间比较采用ONE-WAY ANOVA，P<0.05为具有显著性差异。作图软件和统计分析为GraphPad Prism。
表29.受试物对SW48细胞皮下异种移植肿瘤模型的抑瘤药效评价
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由TGI(TGI(％)＝[1-(T21-T0)/(V21-V0)]×100)反映；
3.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。各试验组和空白对照组之间的肿瘤体积比较。
4.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。本实施例双抗ADC和对照ADC试验组的肿瘤体积比较。
5.N/A代表对照组小鼠按照动物福利原则在第21天已经安乐死。
实验结果如图17及表29所示，在EGFR中表达，HER3低表达的肿瘤模型中，所有试验组对比空白对照组都有显著的抑制肿瘤生长作用；在相同的给药剂量下，本发明抗体偶联物比对照ADC具有更好的抑瘤作用，能让部分小鼠肿瘤完全消退并持续抑制肿瘤生长到第45天；对照组ADC不能让肿瘤完全消退，且不能抑制肿瘤复发。各给药组小鼠耐受良好，体重无明显下降。
实施例18：本发明双抗ADC与对照ADC对奥西替尼耐药的人非小细胞肺癌细胞株异种移植瘤小鼠模型的药效比较
为比较DBXT005-01 ADC(ADC-13)和对照ADC(BL-B01D1，ADC-14)对奥西替尼耐药的非小细胞肺癌细胞株体内形成肿瘤的抑制作用，在小鼠皮下异位接种NCI-H1975(EGFR三突变L858R/T790M/C797S)形成移植瘤后，比较两者的抗肿瘤效果。
1.受试药物及给药方案见表30。
表30.人非小细胞肺癌NCI-H1975(EGFR三突变L858R/T790M/C797S)皮下异种移植模型中的给药途径、剂量及方案
2.配制方法：所有样品均用辅料溶剂稀释配制。
3.试验动物：6-7周龄的雌性NOD/SCID小鼠，购自集萃药康生物科技有限公司。
4.试验方法：
NCI-H1975 EGFR L858R/T790M/C797S(CL-01195)细胞培养在含10％胎牛血清和100μg/mL Hygromycin的RPMI1640培养液中。收集指数生长期的NCI-H1975细胞，PBS重悬至适合浓度用于NOD/SCID小鼠皮下肿瘤接种。实验小鼠于右侧背部皮下接种1×107NCI-H1975 EGFR L858R/T790M/C797S细胞，细胞重悬在1:1的PBS与基质胶中(0.2mL/只)，定期观察肿瘤生长情况。
当肿瘤长至约169.46mm3，对荷瘤小鼠进行随机分组，并于当天(第0天)开始通过静脉(i.v.)注射受试品，试验终点时间为分组后第35天，每周测量2次瘤体积和体重，记录数据。
对照组或治疗组每组6只小鼠。通过测量肿瘤体积计算抑瘤率。
肿瘤体积的计算公式为：V＝0.5a×b2，a和b分别表示肿瘤的长径和短径。化合物的抑瘤疗效用TGI(％)评价。肿瘤生长抑制率用下列公式计算：TGI(％)＝[1-(Ti/T0)/(Vi/V0)]×100，其中Ti为某一天某给药组的平均肿瘤体积，T0为此给药组在开始给药时的平均肿瘤体积；Vi为某一天(与Ti同一天)溶媒对照组的平均肿瘤体积，V0为溶媒对照组在开始给药时的平均肿瘤体积。
样本组之间比较采用ONE-WAY ANOVA，P<0.05为具有显著性差异。作图软件和统计分析为GraphPad Prism。
表31.受试物对NCI-H1975 EGFR L858R/T790M/C797S细胞皮下异种移植肿瘤模型的抑瘤药效评价
(基于给药后35天肿瘤体积计算得出)
1.肿瘤体积用平均值±标准误表示；
2.肿瘤生长抑制由TGI(TGI(％)＝[1-(T35/T0)/(V35/V0)]×100)反映；
3.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。各试验组和空白对照组之间的肿瘤体积比较。
4.p值根据肿瘤体积计算(*代表p<0.05,**代表p<0.01，***代表p<0.001,ns代表无显著意义)。本实施例双抗ADC和对照ADC试验组的肿瘤体积比较。
实验结果如图18及表31所示，在对第三代TKI奥西替尼耐受的非小细胞肺癌肿瘤模型中，所有试验组对比空白对照组都有显著的抑制肿瘤生长作用；在相同的给药剂量下，本发明抗体偶联物比对照ADC具有更好的抑瘤作用，各给药组小鼠耐受良好，体重无明显下降。
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虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (32)

  1. 一种包含EGFR结合结构域和HER3结合结构域的双特异性抗体，其特征在于，所述EGFR结合结构域包括重链可变区VH1和轻链可变区VL1，所述HER3结合结构域包含重链可变区VH2和轻链可变区VL2；其中，所述VH1包含的H1CDR1、H1CDR2和H1CDR3的氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示；所述VL1包含的L1CDR1、L1CDR2和L1CDR3的氨基酸序列分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示；所述VH2包含的H2CDR1、H2CDR2和H2CDR3的氨基酸序列分别如SEQ ID NO:7、SEQ ID NO:8和SEQ ID NO:9所示；所述VL2包含的L2CDR1、L2CDR2和L2CDR3氨基酸序列分别如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示。
  2. 如权利要求1所述的双特异性抗体，其特征在于，所述H2CDR2的氨基酸序列如SEQ ID NO:77或78所示；
    优选地，所述VH1包含的框架区H1FR1、H1FR2、H1FR3和H1FR4的氨基酸序列分别如SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；
    所述VL1包含的框架区L1FR1、L1FR2、L1FR3和H1FR4的氨基酸序列分别如SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19和SEQ ID NO:20所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；
    所述VH2包含的框架区H2FR1的氨基酸序列如SEQ ID NO:21所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，例如在SEQ ID NO:21上具有E16D突变；框架区H2FR2和H2FR4的氨基酸序列分别如SEQ ID NO:22和SEQ ID NO:24所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；框架区H2FR3的氨基酸序列如SEQ ID NO:23所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，例如在SEQ ID NO:23上具有S18D突变；
    所述VL2包含的框架区L2FR1的氨基酸序列如SEQ ID NO:25所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，例如在SEQ ID NO:25上具有S9D和V15L突变，或S7E突变；框架区L2FR2、L2FR3和L2FR4的氨基酸序列分别如SEQ ID NO:18、SEQ ID NO:26和SEQ ID NO:27所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性。
  3. 如权利要求1或2所述的双特异性抗体，其特征在于，所述VH1的氨基酸序列如SEQ ID NO:28所示，所述VL1的氨基酸序列如SEQ ID NO:29所示，所述VH2的氨基酸序列如SEQ ID NO:30、SEQ ID NO:79或SEQ ID NO:80所示，所述VL2的氨基酸序列如SEQ ID NO:31、SEQ ID NO:81或SEQ ID NO:82所示；
    优选地，所述双特异性抗体的VH1、VL1、VH2和VL2的氨基酸序列分别如SEQ ID NO:28、 SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:31所示；或，分别如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:79和SEQ ID NO:81所示；或，分别如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:79和SEQ ID NO:31所示；或，分别如SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:80和SEQ ID NO:82所示。
  4. 如权利要求1-3任一项所述的双特异性抗体，其特征在于，所述EGFR结合结构域和HER3结合结构域还分别包括轻链恒定区和重链恒定区，所述EGFR结合结构域包含轻链恒定区CL1和重链恒定区HC1，所述HER3结合结构域包含轻链恒定区CL2和重链恒定区HC2；其中，CL1和CL2的氨基酸序列分别如SEQ ID NO:32或SEQ ID NO:33所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，且CL1和CL2的氨基酸序列不为同一序列；和/或，HC1包含C1H1和Fc1，HC2包含C2H1和Fc2；其中，C1H1和C2H1的氨基酸序列如SEQ ID NO:34或SEQ ID NO:35所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，且C1H1和C2H1的氨基酸序列不为同一序列；Fc1和Fc2的氨基酸序列为如SEQ ID NO:36所示的氨基酸序列的变体序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，例如在SEQ ID NO:36上具有T146W，或S134C和T146W，或T146S、L148A和Y187V，或Y349C、T366S、L368A和Y407V突变，且Fc1和Fc2的氨基酸序列不为同一序列；
    优选地，CL1和CL2的氨基酸序列分别如SEQ ID NO:32或SEQ ID NO:33所示，C1H1和C2H1的氨基酸序列分别如SEQ ID NO:34或SEQ ID NO:35所示，和，Fc1和Fc2的氨基酸序列为如SEQ ID NO:36所示的氨基酸序列的变体序列，例如在SEQ ID NO:36上具有T146W，或S134C和T146W，或T146S、L148A和Y187V，或Y349C、T366S、L368A和Y407V突变；
    更优选地，Fc1和Fc2之间通过铰链区的二硫键和Knob into Hole结构连接，所述Fc1为knob-Fc，所述Fc2为hole-Fc，或Fc2为knob-Fc，Fc1为hole-Fc；
    进一步更优选地，C1H1和Fc1、C2H1和Fc2之间由铰链区相连，其中，所述铰链区的氨基酸序列如SEQ ID NO:89所示。
  5. 如权利要求1-3任一项所述的双特异性抗体，其特征在于，所述EGFR结合结构域包含轻链恒定区CL1和重链恒定区HC1，所述HER3结合结构域包含重链恒定区HC2；其中，CL1的氨基酸序列如SEQ ID NO:32所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；和/或，HC1包含C1H1和Fc1，HC2包含Fc2；其中，C1H1的氨基酸序列如SEQ ID NO:34所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性的；Fc1和Fc2的氨基酸序列为如SEQ ID NO:36所示的氨基酸序列的变体序列或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；
    优选地，CL1的氨基酸序列如SEQ ID NO:32所示，C1H1的氨基酸序列如SEQ ID NO:34所示，和，Fc1和Fc2的氨基酸序列分别为如SEQ ID NO:93和94所示；
    更优选地，Fc1和Fc2之间通过铰链区的二硫键和Knob into Hole结构连接，所述Fc1为knob- Fc，所述Fc2为hole-Fc，或Fc2为knob-Fc，Fc1为hole-Fc；
    进一步更优选地，C1H1和Fc1之间由氨基酸序列如SEQ ID NO:89所示的铰链区相连；VL2和VH2之间由氨基酸序列如SEQ ID NO:95所示的铰链区相连；VH2和Fc2之间由氨基酸序列如SEQ ID NO:96所示的铰链区相连。
  6. 如权利要求1-3或5中任一项所述的双特异性抗体，其特征在于，所述双特异性抗体包含重链H1、轻链L1和重链H2，所述H1的氨基酸序列如SEQ ID NO:37所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，和/或，所述L1的氨基酸序列如SEQ ID NO:38所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性，和/或，所述H2的氨基酸序列如SEQ ID NO:90所示或与其具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％同一性；
    优选地，所述重链H1、轻链L1和重链H2的氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38和SEQ ID NO:90所示。
  7. 如权利要求1-4任一项所述的双特异性抗体，其特征在于，所述双特异性抗体的重链H1、轻链L1、重链H2和轻链L2的氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示；或，
    分别如SEQ ID NO:41、SEQ ID NO:38、SEQ ID NO:42和SEQ ID NO:40所示；或，
    分别如SEQ ID NO:43、SEQ ID NO:38、SEQ ID NO:44和SEQ ID NO:40所示；或，
    分别如SEQ ID NO:45、SEQ ID NO:38、SEQ ID NO:46和SEQ ID NO:40所示；或，
    分别如SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49和SEQ ID NO:50所示；或，
    分别如SEQ ID NO:51、SEQ ID NO:48、SEQ ID NO:52和SEQ ID NO:50所示；或，
    分别如SEQ ID NO:53、SEQ ID NO:48、SEQ ID NO:54和SEQ ID NO:50所示；或，
    分别如SEQ ID NO:55、SEQ ID NO:48、SEQ ID NO:56和SEQ ID NO:50所示；或，
    分别如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:57和SEQ ID NO:58所示；或，
    分别如SEQ ID NO:41、SEQ ID NO:38、SEQ ID NO:59和SEQ ID NO:58所示；或，
    分别如SEQ ID NO:43、SEQ ID NO:38、SEQ ID NO:60和SEQ ID NO:58所示；或，
    分别如SEQ ID NO:45、SEQ ID NO:38、SEQ ID NO:61和SEQ ID NO:58所示；或，
    分别如SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:62和SEQ ID NO:63所示；或，
    分别如SEQ ID NO:51、SEQ ID NO:48、SEQ ID NO:64和SEQ ID NO:63所示；或，
    分别如SEQ ID NO:53、SEQ ID NO:48、SEQ ID NO:65和SEQ ID NO:63所示；或，
    分别如SEQ ID NO:55、SEQ ID NO:48、SEQ ID NO:66和SEQ ID NO:63所示；或，
    分别如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:57和SEQ ID NO:40所示；或，
    分别如SEQ ID NO:41、SEQ ID NO:38、SEQ ID NO:59和SEQ ID NO:40所示；或，
    分别如SEQ ID NO:43、SEQ ID NO:38、SEQ ID NO:60和SEQ ID NO:40所示；或，
    分别如SEQ ID NO:45、SEQ ID NO:38、SEQ ID NO:61和SEQ ID NO:40所示；或，
    分别如SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:62和SEQ ID NO:50所示；或，
    分别如SEQ ID NO:51、SEQ ID NO:48、SEQ ID NO:64和SEQ ID NO:50所示；或，
    分别如SEQ ID NO:53、SEQ ID NO:48、SEQ ID NO:65和SEQ ID NO:50所示；或，
    分别如SEQ ID NO:55、SEQ ID NO:48、SEQ ID NO:66和SEQ ID NO:50所示；或，
    分别如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:67和SEQ ID NO:68所示；或，
    分别如SEQ ID NO:41、SEQ ID NO:38、SEQ ID NO:69和SEQ ID NO:68所示；或，
    分别如SEQ ID NO:43、SEQ ID NO:38、SEQ ID NO:70和SEQ ID NO:68所示；或，
    分别如SEQ ID NO:45、SEQ ID NO:38、SEQ ID NO:71和SEQ ID NO:68所示；或，
    分别如SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:72和SEQ ID NO:73所示；或，
    分别如SEQ ID NO:51、SEQ ID NO:48、SEQ ID NO:74和SEQ ID NO:73所示；或，
    分别如SEQ ID NO:53、SEQ ID NO:48、SEQ ID NO:75和SEQ ID NO:73所示；或，
    分别如SEQ ID NO:55、SEQ ID NO:48、SEQ ID NO:76和SEQ ID NO:73所示。
  8. 一种分离的核酸，其特征在于，所述核酸编码如权利要求1～7任一项所述的双特异性抗体。
  9. 一种重组表达载体，其特征在于，所述重组表达载体包含如权利要求8所述的核酸。
  10. 一种转化体，其特征在于，所述转化体在宿主细胞中包含如权利要求8所述的核酸或如权利要求9所述的重组表达载体；
    较佳地，所述宿主细胞为真核细胞，优选为哺乳动物细胞，例如CHO细胞。
  11. 一种制备如权利要求1～7任一项所述的双特异性抗体的方法，所述方法包括培养如权利要求10所述的转化体，获得所述双特异性抗体。
  12. 一种双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，所述双特异性抗体药物偶联物包含以下片段：如权利要求1～7中任一项所述的双特异性抗体或其抗原结合片段、接头单元L和细胞毒性药物。
  13. 如权利要求12所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，所述细胞毒性药物为喜树碱及其衍生物；
    优选地，所述细胞毒性药物为如式(A-1)所示结构、其互变异构体、对映异构体、或非对映异构体，
    其中，
    M为-L2-L1-C(O)-；
    L2为-NH-、O或S，优选为-O-或-S-，更优选为-O-，且L2与所述的接头单元L连接；
    L1为-(C(R1a)(R1b))m-CH2-、C3-C6饱和的亚环烷基或3-6元饱和的亚杂环基，所述C3-C6饱和的亚环烷基和3-6元饱和的亚杂环基各自独立地任选被一个或多个R2a取代；
    m选自1、2、3和4；所述的3-6元饱和的亚杂环基中的杂原子各自独立地为N、O和S，杂原子数为1、2或3个；
    各个R1a和R1b独立地为氢、卤素、羟基、氨基或C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代；
    R2a选自卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代。
  14. 如权利要求13所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，L1为-(C(R1a)(R1b))m-CH2-；各个R1a选自：氢、卤素和C1-C6烷基；各个R1b选自：氢、卤素和C1-C6烷基；
    或，L1为-(C(R1a)(R1b))m-CH2-；R1a为C1-C6烷基，优选为C1-C3烷基；R1b选自：氢和C1-C6烷基，优选选自：氢和C1-C3烷基；或，L1为-(C(R1a)(R1b))m-CH2-；R1a为-CH3；R1b选自：氢和-CH3；
    或，L1为C3-C6饱和的亚环烷基或3-6元饱和的亚杂环基，优选为C3-C6饱和的亚环烷基，其中所述C3-C6饱和的亚环烷基和3-6元饱和的亚杂环基各自独立地任选被一个或多个R2a取代，各个R2a独立地为卤素或C1-C6烷基；
    或，L1为任选被一个或多个R2a取代的：环丙基、环丁基、环戊基或环己基；R2a各自独立地选自：卤素和C1-C6烷基；
    或，L1为
    或，L1为
  15. 如权利要求14所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，所述如式(A-1)所示结构中，
    M为-L2-L1-C(O)-；
    L2为-O-；
    L1为-(C(R1a)(R1b))m-CH2-或C3-C6饱和的亚环烷基，所述C3-C6饱和的亚环烷基任选被一个或多个R2a取代；
    m选自1或2；
    R1a和R1b各自独立地选自氢、卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代；
    R2a选自卤素和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代。
  16. 如权利要求13-15中任一项所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，M为：
    优选为
  17. 如权利要求12-16中任一项所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其中所述细胞毒性药物选自如下任一结构：
  18. 如权利要求12-17中任一项所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，所述接头单元L为-La-Lb-Lc-；其中Lc与所述的细胞毒性药物连接；
    -La-为或-C1-8亚烷基-C(O)-，优选为或-C1-6 亚烷基-C(O)-，更优选为进一步优选 其中a端和Ab相连，b端与Lb相连；
    -Lb-为-1至6个天然氨基酸的多肽-NH-，优选为-2至4个天然氨基酸的多肽-NH-，更优选选自以下任一结构：
    进一步优选为 更优选为其中c端和La相连，d端与Lc相连；
    -Lc-为C1-6亚烷基，优选为C1-3亚烷基，更优选为
  19. 如权利要求18所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其中所述接头单元L为 优选为
  20. 如权利要求12-19中任一项所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，所述双特异性抗体药物偶联物的结构如式(A-2)所示：
    其中，p表示平均连接数，且p为1到10中任一整数或小数；优选为3到9中任一整数或小数；例如为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8；
    Ab为如权利要求1-7中任一项所述的双特异性抗体或其抗原结合片段；
    M为如权利要求13-17中任一项所述的抗体药物偶联物所定义；
    L为如权利要求18或19所述的接头单元L。
  21. 如权利要求12-20中任一项所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，所述双特异性抗体药物偶联物的结构如式(A-2a)或(A-2b)所示：

    其中，
    p表示平均连接数，且p选自1到10中任一的整数或小数，优选3到9中任一整数或小数；例如为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8；
    Ab为如权利要求1-7中任一项所述的双特异性抗体或其抗原结合片段；
    L2为-NH-、O或S，优选为-O-或-S-；更优选为-O-；
    X1选自任选被1、2或3个R2a取代的C3-C6亚环烷基；
    X2选自-(C(R1a)(R1b))m-CH2-；
    m选自1或2；
    R1a和R1b各自独立为氢、卤素或被1、2或3个卤素任选取代的C1-C6烷基；
    R2a选自卤素、羟基、氨基和C1-C6烷基，所述C1-C6烷基任选被一个或多个卤素取代；
    较佳地，所述双特异性抗体药物偶联物的结构选自以下：



    其中，
    p表示平均连接数，且p为1到10中任一整数或小数，优选3到9中任一整数或小数；例如为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8；
    Ab为如权利要求1-7中任一项所述的双特异性抗体或其抗原结合片段；
    较佳地，Ab选自双特异性抗体DBXT001系、DBXT002系、DBXT003系和DBXT004系；优选选自双特异性抗体DBXT001系、DBXT002系和DBXT005-01；更优选选自双特异性抗体DBXT001系和DBXT005-01；进一步优选选自双特异性抗体DBXT001-01和DBXT005-01。
  22. 如权利要求21所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，所述双特异性抗体药物偶联物选自如下任一：
    其中，p表示平均连接数，且p为1到10中任一整数或小数，优选3到9中任一整数或小数；例如为4、4.06、4.10、6、6.11、6.05、7.99、7.98或8；
    较佳地，所述双特异性抗体药物偶联物选自如下任一：




    其中，DBXT001-01为抗EGFR和HER3的双特异性抗体，其重链H1、轻链L1、重链H2和轻链L2的氨基酸序列分别如SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39和SEQ ID NO:40所示。
  23. 如权利要求12-21中任一项所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，所述双特异性抗体药物偶联物为：
    其中，p表示平均连接数，且p为1到10中任一整数或小数，优选3到9中任一整数或小数，更优选为4到6中任一整数或小数；例如为5.99；
    DBXT005-01为抗EGFR和HER3的双特异性抗体，其重链H1氨基酸序列如SEQ ID NO:37所示，轻链L1的氨基酸序列如SEQ ID NO:38所示，重链H2的氨基酸序列如SEQ ID NO:90所示。
  24. 如权利要求12-19中任一项所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐，其特征在于，所述双特异性抗体药物偶联物为：
    其中，p1表示连接数，且p1为1到10中任一整数，优选3到9中任一整数，更优选为4到6中任一整数；例如为4、5或6；
    DBXT005-01为抗EGFR和HER3的双特异性抗体，其重链H1氨基酸序列如SEQ ID NO:37所示，轻链L1的氨基酸序列如SEQ ID NO:38所示，重链H2的氨基酸序列如SEQ ID NO:90所示。
  25. 一种制备如权利要求12～24中任一项所述的双特异性抗体药物偶联物、其互变异构体、对映异构体、非对映异构体、或异构体的混合物、或其药学上可用的盐的方法，其包括以下步骤：在还原剂的作用下，将溶于缓冲液的所述双特异性抗体与溶于溶剂的接头单元L-细胞毒性药物混合，即得所述双特异性抗体药物偶联物；其中，所述的还原剂优选为三(2-羰基乙基)磷盐酸盐，所述的缓冲液优选为乙二胺四乙酸，所述的溶剂优选为二甲基乙酰胺。
  26. 如权利要求25所述的方法，包括将抗EGFR和HER3的双特异性抗体与式X2的化合物反

    应，例如，将DBXT005-01与式X2的化合物反应。
  27. 一种药物组合物，其包含如权利要求1～7中任一项所述的双特异性抗体、如权利要求8所述的分离的核酸、如权利要求9所述的重组表达载体、如权利要求10所述的转化体和/或如权利要求12～22中任一项所述的双特异性抗体药物偶联物，以及药学上可接受的载体或赋形剂。
  28. 如权利要求1～7中任一项所述的双特异性抗体、如权利要求8所述的分离的核酸、如权利要求9所述的重组表达载体、如权利要求10所述的转化体、如权利要求12～24任一项所述的双特异性抗体药物偶联物和/或权利要求27所述的药物组合物在制备用于治疗和/或预防癌症的药物中的用途，优选地，所述癌症为EGFR和/或Her3阳性表达的癌症，例如所述癌症选自乳腺癌、皮肤癌、胃癌、结直肠癌、肺癌、食管癌、胆道癌、头颈癌、甲状腺癌、卵巢癌、子宫内膜癌、胰腺癌、前列腺癌、膀胱癌、胃肠道癌、消化道癌、子宫颈癌、鳞状细胞癌、腹膜癌、肝癌、肾癌、甲状腺癌、白血病、恶性淋巴瘤、血浆瘤、骨髓瘤、胶质瘤、骨肉瘤、肉瘤、口腔鳞癌和黑色素瘤；优选选自乳腺癌、结直肠癌、皮肤癌、肺癌、食管癌和口腔鳞癌；所述的肺癌优选为非小细胞肺癌，所述皮肤癌优选为皮肤鳞癌，所述的结直肠癌优选为直肠癌。
  29. 一种治疗和/或预防癌症的方法，其包括向有需要的受试者施用如权利要求1～7中任一项所述的双特异性抗体、如权利要求12～24任一项所述的双特异性抗体药物偶联物和/或权利要求27所述的药物组合物；优选地，所述癌症为EGFR和/或Her3阳性表达的癌症，例如所述癌症选自乳腺癌、皮肤癌、胃癌、结直肠癌、肺癌、食管癌、胆道癌、头颈癌、甲状腺癌、卵巢癌、子宫内膜癌、胰腺癌、前列腺癌、膀胱癌、胃肠道癌、消化道癌、子宫颈癌、鳞状细胞癌、腹膜癌、肝癌、肾癌、甲状腺癌、白血病、恶性淋巴瘤、血浆瘤、骨髓瘤、胶质瘤、骨肉瘤、肉瘤、口腔鳞癌和黑色素瘤；优选选自乳腺癌、结直肠癌、皮肤癌、肺癌、食管癌和口腔鳞癌；所述的肺癌优选为非小细胞肺癌，所述皮肤癌优选为皮肤鳞癌，所述的结直肠癌优选为直肠癌。
  30. 一种用于预防和/或治疗癌症的如权利要求1～7中任一项所述的双特异性抗体、如权利要求12～24任一项所述的双特异性抗体药物偶联物和/或权利要求27所述的药物组合物；优选地，所述癌症为EGFR和/或Her3阳性表达的癌症，例如所述癌症选自乳腺癌、皮肤癌、胃癌、结直肠癌、肺癌、食管癌、胆道癌、头颈癌、甲状腺癌、卵巢癌、子宫内膜癌、胰腺癌、前列腺癌、膀胱癌、胃肠道癌、消化道癌、子宫颈癌、鳞状细胞癌、腹膜癌、肝癌、肾癌、甲状腺癌、白血病、恶性淋巴瘤、血浆瘤、骨髓瘤、胶质瘤、骨肉瘤、肉瘤、口腔鳞癌和黑色素瘤；优选选自乳腺癌、结直肠癌、皮肤癌、肺癌、食管癌和口腔鳞癌；所述的肺癌优选为非小细胞肺癌，所述皮肤癌优选为皮肤鳞癌，所述的结直肠癌优选为直肠癌。
  31. 一种联合疗法，其特征在于，其包括分别向有需要的受试者施用如权利要求1～7中任一项所述的双特异性抗体、如权利要求12～24中任一项所述的双特异性抗体药物偶联物和/或权利要求27所述的药物组合物，以及第二治疗剂；
    较佳地，所述癌症为EGFR和/或Her3阳性表达的癌症，例如所述癌症选自乳腺癌、皮肤癌、胃癌、结直肠癌、肺癌、食管癌、胆道癌、头颈癌、甲状腺癌、卵巢癌、子宫内膜癌、胰腺癌、前列腺 癌、膀胱癌、胃肠道癌、消化道癌、子宫颈癌、鳞状细胞癌、腹膜癌、肝癌、肾癌、甲状腺癌、白血病、恶性淋巴瘤、血浆瘤、骨髓瘤、胶质瘤、骨肉瘤、肉瘤、口腔鳞癌和黑色素瘤；优选选自乳腺癌、结直肠癌、皮肤癌、肺癌、食管癌和口腔鳞癌；所述的肺癌优选为非小细胞肺癌，所述皮肤癌优选为皮肤鳞癌，所述的结直肠癌优选为直肠癌。
  32. 如权利要求1～7中任一项所述的双特异性抗体、如权利要求8所述的分离的核酸、如权利要求9所述的重组表达载体、如权利要求10所述的转化体、如权利要求12～24任一项所述的双特异性抗体药物偶联物和/或权利要求27所述的药物组合物在制备EGFR和/或HER3抑制剂中的应用。
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