Bispecific antibody, drug conjugate thereof, and use thereof

Abstract

Provided are a bispecific antibody, a drug conjugate thereof, and a use thereof. The bispecific antibody comprises an EGFR binding domain and an HER3 binding domain. The provided bispecific antibody and the drug conjugate thereof have excellent endocytosis effects, proliferation inhibition activity and tumor growth inhibition activity, as well as good in vivo safety.

Description

A bispecific antibody, its drug conjugate and its use

[0001] This application claims priority to Chinese Patent Application No. 2023108956544 filed on July 19, 2023, Chinese Patent Application No. 2024102449540 filed on March 4, 2024, and Chinese Patent Application No. 2024109141579 filed on July 9, 2024. The entire contents of the aforementioned Chinese patent applications are incorporated herein by reference. Technical Field

[0002] 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. Background Art

[0003] The epidermal growth factor receptor (EGFR) is a large transmembrane glycoprotein with a molecular weight of approximately 170 kDa and is a member of the ErbB receptor family. The EGFR receptor itself is a tyrosine kinase that forms a dimer upon binding to ligands such as EGF and TNF-a. Through phosphorylation, it activates downstream signaling pathways (such as MAPK, PI3K, and Stat pathways), thereby maintaining cell growth and promoting cell division and proliferation. Due to the conserved nature of ErbB family receptors, EGFR can also form heterodimers with other proteins in the family (such as HER2, HER3, and HER4), thereby regulating cell growth more broadly.

[0004] 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 tremendous clinical benefits in alleviating various cancers, previous efforts to develop anti-HER3 antibodies for cancer treatment have repeatedly failed.

[0005] Antibody drug conjugates (ADCs) consist of three parts: an antibody or its antigen-binding fragment (target), a linker, and a small molecule drug. 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. This fully utilizes the specificity of antibodies or their antigen-binding fragments targeting cells of interest (targeted cells) or binding to highly expressed antigens, as well as the high efficiency of small molecule drugs, thereby reducing or avoiding toxic side effects on non-targeted cells. This means that compared with traditional tumor chemotherapy drugs, antibody drug conjugates for tumors can accurately target tumor cells and reduce the impact on non-tumor cells.

[0006] There is still a need in the art for bispecific antibody-drug conjugates with excellent affinity, specificity, etc.

[0007] Summary of the Invention

[0008] The technical problem to be solved by the present invention is to overcome the drawback of the limited availability of bispecific antibody-drug conjugates targeting EGFR and HER3 in the prior art, thereby providing a bispecific antibody, a drug conjugate thereof, and uses thereof. The bispecific antibody-drug conjugate of the present invention exhibits excellent endocytosis, proliferation inhibition, tumor growth inhibition, and good in vivo safety.

[0009] The present invention mainly solves the above technical problems through the following technical means.

[0010] In order to solve the above technical problems, the first aspect of 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 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 as shown in 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 as shown in 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 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; the amino acid sequences of L2CDR1, L2CDR2 and L2CDR3 contained in the VL2 are as shown in SEQ ID NO:1 NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0011] In some embodiments of the present invention, the amino acid sequence of the H2CDR2 is shown in SEQ ID NO: 77 or 78.

[0012] In some preferred embodiments of the present invention, the amino acid sequences of the framework regions H1FR1, H1FR2, H1FR3, and H1FR4 comprised by the VH1 are as 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;

[0013] The amino acid sequences of the framework regions L1FR1, L1FR2, L1FR3, and L1FR4 comprised by VL1 are as 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;

[0014] the VH2 comprising a framework region H2FR1 having an amino acid sequence as set forth in SEQ ID NO:21, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, for example, comprising an E16D mutation in SEQ ID NO:21; framework regions H2FR2 and H2FR4 having amino acid sequences as set forth in SEQ ID NO:22 and SEQ ID NO:24, respectively, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; and framework region H2FR3 having an amino acid sequence as set forth in SEQ ID NO:23, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, for example, comprising an S18D mutation in SEQ ID NO:23;

[0015] The amino acid sequence of the framework region L2FR1 comprised by 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, having S9D and V15L mutations, or S7E mutations 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.

[0016] In some embodiments of the present invention, the amino acid sequence of the VH1 is shown in SEQ ID NO:28, the amino acid sequence of the VL1 is shown in SEQ ID NO:29, the amino acid sequence of the VH2 is shown in SEQ ID NO:30, SEQ ID NO:79 or SEQ ID NO:80, and the amino acid sequence of the VL2 is shown in SEQ ID NO:31, SEQ ID NO:81 or SEQ ID NO:82.

[0017] In some preferred embodiments of the present invention, the amino acid sequences of VH1, VL1, VH2 and VL2 of the bispecific antibody are 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.

[0018] In some embodiments of the present invention, the EGFR binding domain and the HER3 binding domain further comprise 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 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. NO: 35, or 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; the amino acid sequences of Fc1 and Fc2 are variant sequences of the amino acid sequence as shown in SEQ ID NO: 36, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, for example, having T146W, or S134C and T146W, or T146S, L148A and Y187V, or Y349C, T366S, L368A and Y407V mutations in SEQ ID NO: 36, and the amino acid sequences of Fc1 and Fc2 are not identical.

[0019] In some preferred embodiments of the present invention, the amino acid sequences of CL1 and CL2 are shown in SEQ ID NO:32 or SEQ ID NO:33, respectively, the amino acid sequences of C1H1 and C2H1 are 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 in SEQ ID NO:36.

[0020] In some more preferred embodiments of the present invention, 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.

[0021] In some further more preferred embodiments of the present invention, C1H1 and Fc1, as well as C2H1 and Fc2, are connected by a hinge region, wherein the amino acid sequence of the hinge region is shown in SEQ ID NO: 89.

[0022] In some preferred embodiments of the present invention, 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 heavy chain constant region HC2; wherein the amino acid sequence of CL1 is as set forth in SEQ ID NO:32, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and / or, HC1 comprises C1H1 and Fc1, and HC2 comprises Fc2; wherein the amino acid sequence of C1H1 is as set forth in SEQ ID NO:34, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; and the amino acid sequences of Fc1 and Fc2 are as set forth in SEQ ID NO: A variant sequence of the amino acid sequence shown in NO:36 or a variant sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0023] In some preferred embodiments of the present invention, the amino acid sequence of CL1 is shown in SEQ ID NO:32, the amino acid sequence of C1H1 is shown in SEQ ID NO:34, and the amino acid sequences of Fc1 and Fc2 are shown in SEQ ID NOs:93 and 94, respectively;

[0024] In some preferred embodiments of the present invention, Fc1 and Fc2 are connected via a disulfide bond and a Knob into Hole structure in the hinge region, wherein the Fc1 is a knob-Fc and the Fc2 is a hole-Fc, or the Fc2 is a knob-Fc and the Fc1 is a hole-Fc;

[0025] In some preferred embodiments of the present invention, C1H1 and Fc1 are connected by a hinge region with an amino acid sequence as shown in SEQ ID NO:89; VL2 and VH2 are connected by a hinge region with an amino acid sequence as shown in SEQ ID NO:95; and VH2 and Fc2 are connected by a hinge region with an amino acid sequence as shown in SEQ ID NO:96.

[0026] In some preferred embodiments of the present invention, the bispecific antibody comprises a heavy chain H1, a light chain L1, and a heavy chain H2, wherein the amino acid sequence of H1 is as shown in SEQ ID NO: 37, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or the amino acid sequence of L1 is as shown in SEQ ID NO: 38, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or the 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;

[0027] In some preferred embodiments of the present invention, the bispecific antibody comprises a heavy chain H1, a light chain L1, and a heavy chain H2, wherein 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, and is DBXT005-01. In some preferred embodiments of the present invention, the heavy chain H2 is an Fc+scFv structure.

[0028] In some embodiments of the invention, the bispecific antibody comprises heavy chain H1, light chain L1, heavy chain H2, and light chain L2.

[0029] In some preferred embodiments of the present invention, the 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, and are DBXT001-01; or,

[0030] DBXT001-02 as shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:40, respectively; or

[0031] DBXT001-03 as shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:44 and SEQ ID NO:40, respectively; or

[0032] DBXT001-04 as shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:46 and SEQ ID NO:40, respectively; or

[0033] DBXT001-05 as shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, respectively; or

[0034] DBXT001-06 as shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:50, respectively; or

[0035] DBXT001-07 as shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:54 and SEQ ID NO:50, respectively; or

[0036] DBXT001-08 as shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:56 and SEQ ID NO:50, respectively; or

[0037] DBXT002-01 as shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:57 and SEQ ID NO:58, respectively; or

[0038] DBXT002-02 as shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:59 and SEQ ID NO:58, respectively; or

[0039] DBXT002-03 as shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:60 and SEQ ID NO:58, respectively; or

[0040] DBXT002-04 as shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:61 and SEQ ID NO:58, respectively; or

[0041] DBXT002-05 as shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:62 and SEQ ID NO:63, respectively; or

[0042] DBXT002-06 as shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:64 and SEQ ID NO:63, respectively; or

[0043] DBXT002-07 as shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:65 and SEQ ID NO:63, respectively; or

[0044] DBXT002-08 as shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:66 and SEQ ID NO:63, respectively; or

[0045] DBXT003-01 as shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:57 and SEQ ID NO:40, respectively; or

[0046] DBXT003-02 as shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:59 and SEQ ID NO:40, respectively; or

[0047] DBXT003-03 as shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:60 and SEQ ID NO:40, respectively; or

[0048] DBXT003-04 as shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:61 and SEQ ID NO:40, respectively; or

[0049] DBXT003-05 as shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:62 and SEQ ID NO:50, respectively; or

[0050] DBXT003-06 as shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:64 and SEQ ID NO:50, respectively; or

[0051] DBXT003-07 as shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:65 and SEQ ID NO:50, respectively; or

[0052] DBXT003-08 as shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:66 and SEQ ID NO:50, respectively; or

[0053] DBXT004-01 as shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:67 and SEQ ID NO:68, respectively; or

[0054] DBXT004-02 as shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:69 and SEQ ID NO:68, respectively; or

[0055] DBXT004-03 as shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:70 and SEQ ID NO:68, respectively; or

[0056] DBXT004-04 as shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:71 and SEQ ID NO:68, respectively; or

[0057] DBXT004-05 as shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:72 and SEQ ID NO:73, respectively; or

[0058] DBXT004-06 as shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:74 and SEQ ID NO:73, respectively; or

[0059] DBXT004-07 as shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:75 and SEQ ID NO:73, respectively; or

[0060] 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.

[0061] 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, and the DBXT004 series includes DBXT004-01 to DBXT004-08.

[0062] In order to solve the above technical problems, 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.

[0063] In order to solve the above technical problems, 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.

[0064] In order to solve the above technical problems, 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.

[0065] In some preferred embodiments of the present invention, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a CHO cell.

[0066] In order to solve the above technical problems, 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.

[0067] HC1 is the heavy chain constant region of the first heavy chain, and HC2 is the heavy chain constant region of the second heavy chain. VH1, C1H1, and Fc1 represent the VH, CH1, and Fc regions of the first heavy chain, H1, respectively; CL1 and VL1 represent the CL and VL regions of the first light chain, L1, respectively; VH2, C2H1, and Fc2 represent the VH, CH1, and Fc regions of the second heavy chain, H2, respectively; and CL2 and VL2 represent the CL and VL regions of the second light chain, L2. L1FR1, L1FR2, L1FR3, and L1FR4 represent the framework regions of the light chain variable region of the first light chain; H1FR1, H1FR2, H1FR3, and H1FR4 represent the framework regions of the heavy chain variable region of the first heavy chain; L2FR1, L2FR2, L2FR3, and L2FR4 represent the framework regions of the light chain variable region of the second light chain; and H2FR1, H2FR2, H2FR3, and H2FR4 represent the framework regions of the heavy chain variable region of the second heavy chain.

[0068] Antibody sequences use the Kabat numbering system.

[0069] The present invention also provides a bispecific antibody-drug conjugate, its tautomers, enantiomers, diastereomers, or mixtures of isomers, or pharmaceutically acceptable salts thereof, comprising the following fragments: a bispecific antibody or 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 antigen-binding fragment thereof is as described in any one of the present invention.

[0070] In some embodiments, the cytotoxic drug is camptothecin and its derivatives.

[0071] In some embodiments, the cytotoxic drug is a structure as shown in formula (A-1), a tautomer, an enantiomer, or a diastereomer thereof,

[0072] in,

[0073] M is -L 2 -L 1 -C(O)-;

[0074] L 2 is selected from -NH-, O and S, and L 2 Connected to the connector unit L;

[0075] L 1 -(C(R 1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkylene or 3-6 membered saturated heterocyclylene, said C3-C6 saturated cycloalkylene and said 3-6 membered saturated heterocyclylene are each independently optionally substituted by one or more R 2a replace;

[0076] m is selected from 1, 2, 3 and 4; the heteroatoms in the 3-6 membered saturated heterocyclic group are each independently N, O and S, and the number of heteroatoms is 1, 2 or 3;

[0077] R 1a and R 1b are each independently selected from hydrogen, halogen, hydroxy, amino and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens;

[0078] R 2a is selected from halogen, hydroxy, amino and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens.

[0079] In certain preferred embodiments of the present invention, certain groups in the compounds represented by formula (A-1), (A-2), (A-2a) or (A-2b) are defined as follows, and the unmentioned groups are the same as those described in any scheme of the present invention (referred to as "in some embodiments").

[0080] In some embodiments, L 2 It is preferably -O- or -S-, and more preferably -O-.

[0081] In some embodiments, L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a Selected from: hydrogen, halogen and C1-C6 alkyl; R 1b Selected from: hydrogen, halogen and C1-C6 alkyl.

[0082] In some embodiments, L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a is a C1-C6 alkyl group, preferably a C1-C3 alkyl group; R 1b Selected from: hydrogen and C1-C6 alkyl, preferably selected from: hydrogen and C1-C3 alkyl.

[0083] In some embodiments, L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a -CH3; R 1b Selected from: hydrogen and -CH3.

[0084] In some embodiments, L 1 -(C(R 1a )(R 1b )) m -CH2-; m is 1 or 2, preferably 1.

[0085] In some embodiments, L 1 Selected from:

[0086] In some embodiments, L 1 is a C3-C6 saturated cycloalkylene or a 3-6 membered saturated heterocyclylene, preferably a C3-C6 saturated cycloalkylene, wherein the C3-C6 saturated cycloalkylene and the 3-6 membered saturated heterocyclylene are each independently optionally substituted by one or more R 2a Replacement, R 2aEach is independently selected from: halogen and C1-C6 alkyl.

[0087] In some embodiments, L 1 is optionally replaced by one or more R 2a Substituted: cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; R 2a Each is independently selected from: halogen and C1-C6 alkyl.

[0088] In some embodiments, L 1 Selected from:

[0089] In some embodiments, L 1 In the definitions of and X1, the C3-C6 cycloalkylene group is a cyclobutyl group or a cyclohexyl group, preferably a cyclobutyl group.

[0090] In some embodiments, L 1 In the definition of , 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.

[0091] In some embodiments, R 1a 、R 1b and R 2a In the definition of , the C1-C6 alkyl group is a C1-C3 alkyl group, preferably a methyl group.

[0092] In some embodiments, R, R 1a 、R 1b and R 2a In the definition of , the halogen is F, Cl, Br or I, preferably F, Cl or Br.

[0093] In some embodiments, in the structure shown in formula (A-1),

[0094] M is -L 2 -L 1 -C(O)-;

[0095] L 2 is -O-;

[0096] L 1 -(C(R 1a )(R 1b )) m -CH2- or C3-C6 saturated cycloalkylene, the C3-C6 saturated cycloalkylene is optionally substituted by one or more R 2a replace;

[0097] m is selected from 1 or 2;

[0098] R 1a and R1b are each independently selected from hydrogen, halogen and C1-C6 alkyl, the C1-C6 alkyl being optionally substituted with one or more halogens;

[0099] R 2a is selected from halogen and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens.

[0100] In some embodiments, M is:

[0101] In some embodiments, M is

[0102] In some embodiments, the cytotoxic drug is selected from any of the following structures:

[0103] In some embodiments, the linker unit L is -L a -L b -L c -; where L c connected to the cytotoxic drug;

[0104] -L a -for or -C 1-8 Alkylene-C(O)-, preferably or -C 1-6 Alkylene-C(O)-, more preferably -L a - Each group fragment in the definition is preferably on the right side and the L b connect;

[0105] -L a -More preferably The a end is connected to Ab, and the b end is connected to L b connected

[0106] -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 -L b - Each group fragment in the definition is preferably on the right side and the L c connect;

[0107] -L b - More preferably The C-terminal and La Connected, d end and L c connected;

[0108] -L c - for C 1-6 Alkylene, preferably C 1-3 Alkylene, more preferably

[0109] In some embodiments, the bispecific antibody drug conjugate of the present invention, wherein the linker unit L is Preferably

[0110] In some embodiments, the bispecific antibody drug conjugate of the present invention has a structure as shown in Formula (A-2):

[0111] Wherein, 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;

[0112] Ab and M are as defined in any embodiment of the present invention; L is the linker unit L described in any embodiment of the present invention.

[0113] In some embodiments, the bispecific antibody drug conjugate of the present invention has a structure as shown in Formula (A-2):

[0114] in,

[0115] 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;

[0116] Ab is a bispecific antibody or antigen-binding fragment thereof according to any embodiment of the present invention;

[0117] L is the linker unit L according to any embodiment of the present invention;

[0118] M is -L 2 -L 1 -C(O)-;

[0119] L 2 is -O- or -S-, and L 2 Connect with L;

[0120] L 1 -(C(R1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkylene or 3-6 membered saturated heterocyclylene, wherein the C3-C6 saturated cycloalkylene and 3-6 membered saturated heterocyclylene are each independently optionally substituted by one or more R 2a replace;

[0121] m is 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;

[0122] R 1a and R 1b R 2a Each is independently hydrogen, halogen, hydroxy, amino or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens;

[0123] R 2a is selected from halogen, hydroxy, amino and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens.

[0124] In some embodiments, the bispecific antibody drug conjugate of the present invention has a structure as shown in Formula (A-2a) or (A-2b):

[0125] in,

[0126] p represents the average number of connections, and p is an integer or decimal selected from any one of 1 to 10, preferably any one of 3 to 9; for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98 or 8;

[0127] Ab is a bispecific antibody or antigen-binding fragment thereof according to any embodiment of the present invention;

[0128] L 2 is -NH-, O or S, preferably -O- or -S-; more preferably -O-;

[0129] X1 is selected from the group consisting of 1, 2 or 3 R 2a Substituted C3-C6 cycloalkylene;

[0130] X2 is selected from -(C(R 1a )(R 1b )) m -CH2-;

[0131] m is selected from 1 or 2;

[0132] R 1a and R1b Each is independently hydrogen, halogen or C1-C6 alkyl optionally substituted by 1, 2 or 3 halogens;

[0133] R 2a is selected from halogen, hydroxy, amino and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens.

[0134] In some embodiments, the bispecific antibody drug conjugate of the present invention is selected from the following:

[0135] in,

[0136] 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;

[0137] Ab is a bispecific antibody or an antigen-binding fragment thereof according to any embodiment of the present invention.

[0138] In some embodiments, Ab is selected from the bispecific antibodies DBXT001 series (DBXT001-01-08), DBXT002 series (DBXT002-01-08), DBXT003 series (DBXT003-01-08), and DBXT004 series (DBXT004-01-08) comprising the EGFR binding domain and the HER3 binding domain of the present invention; preferably, Ab is selected from the bispecific antibodies DBXT001 series, DBXT002 series, and DBXT005-01 comprising the EGFR binding domain and the HER3 binding domain of the present invention; more preferably, Ab is selected from the bispecific antibodies DBXT001 series and DBXT005-01 comprising the EGFR binding domain and the HER3 binding domain of the present invention; further preferably, Ab is selected from the bispecific antibodies DBXT001-01 and DBXT005-01 comprising the EGFR binding domain and the HER3 binding domain of the present invention.

[0139] In some embodiments, the Ab is the bispecific antibody DBXT005-01 of the present invention comprising an EGFR binding domain and a HER3 binding domain.

[0140] In some embodiments, the bispecific antibody drug conjugate is selected from any of the following structures:

[0141] Wherein, 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.

[0142] In some embodiments, the bispecific antibody drug conjugate is selected from any of the following structures:

[0143] The amino acid sequences of the bispecific antibodies 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.

[0144] The numbering method of the antibody CDR of the present invention is: Kabat numbering.

[0145] In some embodiments, the bispecific antibody drug conjugate is:

[0146] Wherein, 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;

[0147] 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.

[0148] In some embodiments, the bispecific antibody drug conjugate is selected from the following conjugates:

[0149] Wherein, 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.

[0150] 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.

[0151] In some embodiments, the average connection number p of the present invention can be any integer or decimal from 1 to 10. For example, the average connection number p can be any integer or decimal from 3 to 9. For example, 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, or 9 to 10. Preferably, the average connection number p is 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98 or 8.

[0152] In some embodiments, the connection number p1 of the present invention is any integer from 1 to 10. For example, the connection number p1 is any integer from 3 to 9. For example, the connection number p1 is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Preferably, the connection number p1 is 4, 5 or 6.

[0153] In another aspect, 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 solution 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.

[0154] In some embodiments, the preparation method comprises reacting an anti-EGFR and HER3 bispecific antibody with a compound of formula X2, for example, reacting DBXT005-01 with a compound of formula X2:

[0155] In some embodiments, the reducing agent is a reducing agent conventional in the art for such reactions, such as tris(2-carbonylethyl)phosphine hydrochloride.

[0156] In some embodiments, the buffer is a conventional buffer for such reactions in the art, such as ethylenediaminetetraacetic acid.

[0157] In some embodiments, the solvent is a conventional solvent for such reactions in the art, such as dimethylacetamide.

[0158] In another aspect, the present invention provides a pharmaceutical composition comprising the bispecific antibody according to any one of the present invention, the isolated nucleic acid according to any one of the present invention, the recombinant expression vector according to any one of the present invention, the transformant according to any one of the present invention and / or the bispecific antibody-drug conjugate according to any one of the present invention, and a pharmaceutically acceptable carrier or excipient.

[0159] In another aspect, the present invention provides a method for preparing the pharmaceutical composition of the present invention, comprising combining the bispecific antibody drug conjugate of 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.

[0160] In the present invention, the pharmaceutically acceptable carrier used in the pharmaceutical composition, wherein examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (2005).

[0161] In the present invention, the pharmaceutical composition can be administered in any form, as long as it prevents, alleviates, prevents or cures the symptoms of human or animal patients. For example, it can be prepared into various suitable dosage forms according to the administration route.

[0162] In other embodiments, administration of any of the bispecific antibody drug conjugates or pharmaceutical compositions of the present invention can be combined with another therapeutic approach, which can be selected from, but not limited to, radiation therapy, chemotherapy, immunotherapy, or a combination thereof.

[0163] In another aspect, the present invention provides a pharmaceutical formulation comprising the bispecific antibody drug conjugate of any one of the present invention, or a pharmaceutically acceptable form thereof, or a mixture thereof as an active ingredient, or a pharmaceutical composition of any one of the present invention. In some embodiments, the formulation is in the form of a solid formulation, a semisolid formulation, a liquid formulation, or a gaseous formulation.

[0164] In another aspect, the present invention provides use of the bispecific antibody according to any one of the present invention, the isolated nucleic acid according to any one of the present invention, the recombinant expression vector according to any one of the present invention, the transformant according to any one of the present invention, the bispecific antibody-drug conjugate according to any one of the present invention, and / or the pharmaceutical composition according to any one of the present invention in the preparation of a medicament for treating and / or preventing cancer, preferably, the cancer is EGFR and / or HER3-positive cancer.

[0165] In another aspect, the present invention provides a method for treating and / or preventing cancer, comprising administering to a subject in need thereof the bispecific antibody according to any one of the present invention, the isolated nucleic acid according to any one of the present invention, the recombinant expression vector according to any one of the present invention, the transformant according to any one of the present invention, the bispecific antibody-drug conjugate according to any one of the present invention, and / or the pharmaceutical composition according to any one of the present invention; preferably, the cancer is an EGFR and / or HER3-positive cancer.

[0166] In another aspect, the present invention provides a bispecific antibody according to any one of the present invention, an isolated nucleic acid according to any one of the present invention, a recombinant expression vector according to any one of the present invention, a transformant according to any one of the present invention, a bispecific antibody-drug conjugate according to any one of the present invention, and / or a pharmaceutical composition according to any one of the present invention, for use in treating and / or preventing cancer, preferably, the cancer is an EGFR and / or HER3-positive cancer.

[0167] In some embodiments, the cancer described in the present invention is selected from breast cancer, skin cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract 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.

[0168] In some embodiments, the cancer of the present invention is preferably selected from breast cancer, colorectal cancer, skin cancer, lung cancer, esophageal cancer and oral squamous cell carcinoma.

[0169] In some embodiments, the lung cancer is preferably non-small cell lung cancer, the skin cancer is preferably cutaneous squamous cell carcinoma, and the colorectal cancer is preferably rectal cancer.

[0170] In another aspect, the present invention provides use of the bispecific antibody according to any one of the present invention, the isolated nucleic acid according to any one of the present invention, the recombinant expression vector according to any one of the present invention, the transformant according to any one of the present invention, the bispecific antibody-drug conjugate according to any one of the present invention, and / or the pharmaceutical composition according to any one of the present invention in the preparation of an EGFR and / or HER3 inhibitor.

[0171] In some embodiments, 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.

[0172] In some embodiments, the present invention provides for administering to a subject a therapeutically effective amount of one or more therapies (eg, treatment modalities and / or other therapeutic agents) in combination. In some embodiments, the therapies include surgery and / or radiation therapy.

[0173] In some embodiments, the methods or uses provided herein further comprise administering one or more therapies (e.g., treatment modalities and / or other therapeutic agents) to an individual. The antibody drug conjugates of the present invention or pharmaceutically acceptable salts thereof may be used alone or in combination with other therapeutic agents in the therapy. For example, they may be co-administered with at least one additional therapeutic agent.

[0174] In another aspect, the present invention provides a pharmaceutical combination comprising the bispecific antibody according to any one of the present invention, the isolated nucleic acid according to any one of the present invention, the recombinant expression vector according to any one of the present invention, the transformant according to any one of the present invention, the bispecific antibody-drug conjugate according to any one of the present invention and / or the pharmaceutical composition according to any one of the present invention, and one or more additional therapeutic agents.

[0175] In another aspect, the present invention provides a kit comprising the bispecific antibody according to any one of the present invention, the isolated nucleic acid according to any one of the present invention, the recombinant expression vector according to any one of the present invention, the transformant according to any one of the present invention, the bispecific antibody-drug conjugate according to any one of the present invention, and / or the pharmaceutical composition according to any one of the present invention.

[0176] Definition of terms

[0177] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art.

[0178] Unless otherwise expressly defined elsewhere herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention pertains. For definitions and terminology in this art, those skilled in the art are referred to Current Protocols in Molecular Biology (Ausubel). Amino acid residue abbreviations are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0179] In order to make the present invention more easily understood, certain technical terms are specifically defined below.

[0180] In the present invention, the term "EGFR (Epidermal Growth Factor Receptor)" refers to a receptor for epidermal growth factor (EGF) cell proliferation and signaling. EGFR belongs to the ErbB receptor family, which includes EGFR (ErbB-1), HER2 / c-neu (ErbB-2), HER3 (ErbB-3), and HER 4 (ErbB-4). EGFR, also known as HER1 or ErbB-1, is a glycoprotein that is a tyrosine kinase receptor that penetrates the cell membrane and has a molecular weight of 170 kDa. EGFR is located on the cell membrane surface and is activated by binding to ligands, including EGF and TGFα (transforming growth factor α). Upon activation, EGFR converts 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.

[0181] In the present invention, the term "human epidermal growth factor receptor 3 (HER3)", also known as the receptor tyrosine protein kinase ErbB-3 (ErbB3), is a member of the EGFR / ErbB family. Unlike other ErbB family members HER2 and EGFR, HER3 itself does not have kinase activity. Therefore, HER3 must bind to its kinase-active members EGFR or HER2 as a heterodimer to trigger its downstream activities. After binding to its natural ligand NRG1, HER3 undergoes conformational changes, heterodimerization, and phosphorylation, and then activates MAPK, PI3K / Akt, and PLCγ through signal transduction.

[0182] In the present invention, 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 such variations are appropriate for performing the disclosed methods.

[0183] In the present invention, 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.

[0184] In the present invention, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when explicitly stated to the contrary, such as "only one" or "exactly one" or when used in the claims, "consisting of..." will refer to only one of the listed numbers or one element of the list.

[0185] Unless the context clearly indicates otherwise, the terms "a" and "an" should be understood as meaning "at least one" in the present invention.

[0186] In the present invention, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker. In the present invention, an "antibody-drug conjugate" may be a bispecific antibody-drug conjugate, which may be a bispecific antibody or antigen-binding fragment thereof linked to a biologically active cytotoxic drug fragment via a stable linker.

[0187] In the present invention, the term "cytotoxic drug" generally refers to a toxic drug that has a strong chemical molecule that destroys the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells at a sufficiently high concentration. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 or radioisotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics, nucleolytic enzymes, or derivatives thereof. For example, it can be a toxic drug, including but not limited to camptothecin derivatives; for example, it can be a camptothecin derivative exatecan (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).

[0188] In the present invention, the term "antibody" (Ab) generally refers to an immunoglobulin that is reactive to a specified protein or peptide or a fragment thereof. The antibody can 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 can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. The antibody can also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of the present invention can be derived from any species. The term "antibody" can 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.

[0189] In the present invention, the term "antigen-binding fragment" or "antigen-binding domain" generally refers to a portion of an antibody molecule that comprises the 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. As described herein, an antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily comprise both. An Fd fragment, for example, has two VH regions and generally retains some of the antigen-binding function of an intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) a Fab fragment, a monovalent fragment having a VL, VH, constant light chain (CL) and CH1 domains; (2) a F(ab′)2 fragment, a bivalent fragment having two Fab fragments connected by a disulfide bridge at the hinge region; (3) an 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) single-chain Fv (scFv), for example, derived from an scFV library.Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined using recombinant methods by a synthetic linker, which allows them to be prepared as a single protein chain (called single-chain Fv (scFv)) in which the VL and VH regions pair to form a monovalent molecule (see, for example, 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. USA 85: 5879-5883 (1988)); (8) "VHH" refers to the variable antigen-binding domain of heavy chain antibodies from the Camelidae (camel, dromedary, llama, alpaca, etc.) (see Nguyen VK et al., 2000, The EMBO Journal, 19, 921-930; Muyldermans S., 2001, J. Biotechnol., 74, 277-302 (reviewed in Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407). VHHs are also referred to as nanobodies (Nb).

[0190] In the present invention, the term "variable region" or "variable domain" or "variable antigen-binding domain" generally refers to the domain of the antibody heavy chain or light chain that is involved in the binding of the antibody to the antigen. In the present invention, the term "variable" generally refers to the fact that certain parts of the sequence of the variable domain of an 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 an antibody. It is concentrated in three segments in the light chain variable region and the heavy chain variable region, called complementarity determining regions (CDRs) or hypervariable regions (HVRs), namely LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3. 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 β-pleated sheet 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.

[0191] In the present invention, the variable region of the antibody can be encoded or the CDR of the antibody can be divided by various 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), Lefranc et al. The IMGT numbering scheme and definition rules based on the amino acid sequence alignment of germline V genes, as well as Honneger's numbering scheme (AHo's), Martin numbering scheme, Gelfand numbering scheme, etc., can be seen in Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front. Immunol., 16 October 2018.

[0192] As used herein, the term "monoclonal antibody" or "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of 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 substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0193] In the present invention, the term "multispecific antibody" refers to an antibody comprising two or more antigen binding domains that can bind to two or more different epitopes (e.g., two, three, four or more different epitopes), which may be on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" (abbreviated as bispecific) or "bispecific molecules" that bind to two different antigens or two different epitopes. The 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.

[0194] In the present invention, the term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, as described in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0195] In the present invention, the term "knob-into-hole structure" refers to the mutation of hydrophobic amino acids in the CH3 region of an antibody Fc. The side chain amino acids of one CH3 chain are mutated to form a relatively large hydrophobic amino acid (knob) to enhance hydrophobic interactions; the side chain amino acids of another CH3 chain are mutated to form a smaller amino acid (hole) to reduce steric hindrance. After the mutation, the CH3 with the knob and the CH3 with the hole interact with each other to form a knob-into-hole structure (KiH), which facilitates the formation of heavy chain heterodimers. The KiH mutation primarily occurs in the internal hydrophobic amino acids of the CH3 domain, leaving the exposed amino acids largely unchanged, thus having no impact on the effector function and immunogenicity of the Fc. The term "knob-Fc" refers to a point mutation in the Fc region of an antibody containing T366W to form a knob-like structure. Correspondingly, "hole-Fc" refers to a point mutation in the Fc region of an antibody containing T366S, L368A, and Y407V to form a hole-like structure. To further promote heterodimer formation, point mutations S354C and Y349C can be introduced into the knob-Fc and hole-Fc, respectively, to further promote heterodimer formation through disulfide bonds. Furthermore, to weaken binding to Protein A, point mutations H435R and Y436F can be introduced into the hole-Fc, respectively.

[0196] In the present invention, the term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the variable domains correspond to the variable domains of a non-human immunoglobulin, and all or substantially all of the framework regions (FRs) are framework regions of human immunoglobulin sequences. Optionally, a humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0197] In the present invention, an antibody "isotype" 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 made to alter Fc function, for example to enhance or diminish effector function or binding to an Fc receptor.

[0198] In the present invention, the term "cross-reactivity" refers to binding to antigenic fragments of the same target molecule of human, monkey, and / or murine (mouse or rat) origin. Therefore, "cross-reactivity" should be understood as the interspecies reaction of an antigen-binding molecule (e.g., antibody) with a similar molecule (e.g., BDCA2) expressed in a different species. The cross-reactivity specificity of monoclonal antibodies recognizing human BDCA2, monkey, and / or murine BDCA2 (mouse or rat) can be determined by FACS analysis.

[0199] In the present invention, "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between binding members. The affinity of a molecule X for its partner Y can be generally represented by the equilibrium dissociation constant (KD), which is a product of the dissociation rate constant and the association rate constant (K off (Kd) and K on (Ka)). Affinity can be measured by common methods known in the art. In some embodiments of the present invention, surface plasmon resonance (SPR) technology is used to measure affinity, such as the affinity between the antibody of the present invention and the antigen. In some preferred embodiments of the present invention, a specific method for measuring affinity is the BIAcore method.

[0200] In the present invention, 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 of the binding 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 more, further more preferably 1.0×10 -2 M or higher.

[0201] In the present invention, the term "high affinity" for IgG antibodies refers to a KD of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, further more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype refers to a K D is 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0202] In the present invention, the term "percent (%) amino acid sequence identity" or simply "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 aligning the amino acid sequences (and introducing gaps, if necessary) to obtain the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, 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 alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0203] In the present invention, the term "halogen" generally refers to fluorine, chlorine, bromine, and iodine, for example, it can be fluorine or chlorine.

[0204] In the present invention, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. The term "alkyl" generally refers to a saturated straight or branched aliphatic hydrocarbon group having a residue derived from the same carbon atom or two different carbon atoms of the parent alkane by removing a hydrogen atom, and can be a straight or branched group containing 1 to 20 carbon atoms, for example, a chain alkyl containing 1 to 12 carbon atoms, for example, a chain alkyl containing 1 to 6 carbon atoms, for example, a chain alkyl containing 1 to 3 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. Alkyl can be substituted or non-substituted, substituted or non-substituted, for example, when substituted, the substituent can be substituted on any usable point of attachment.

[0205] In the present invention, the term "alkylene" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of an alkane group. It can be a straight-chain or branched group containing 1 to 20 carbon atoms. For example, the term "methylene" can refer to a residue derived from a group containing 1 carbon atom by removing two hydrogen atoms. The methylene group can be substituted or unsubstituted, substituted or unsubstituted; for example, an alkylene group containing 1 to 12 carbon atoms, for example, an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-), 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-), and the like.

[0206] In the present invention, the term "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, cyclooctane, and the like; polycyclic cycloalkyls include spirocyclic, fused, and bridged cycloalkyls.

[0207] The term "cycloalkylene" is a divalent group that is attached to the rest of the molecule by two single bonds, and the rest of the definition is the same as the term "cycloalkyl".

[0208] As used herein, the term "partially unsaturated" generally refers to a cyclic structure containing at least one double or triple bond between the ring molecules. The term "partially unsaturated" encompasses cyclic structures with multiple unsaturations, but is not intended to include aromatic or heteroaromatic rings as defined herein. The term "unsaturated" refers to a moiety having one or more degrees of unsaturation.

[0209] In the present invention, the term "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 is a heteroatom selected from nitrogen, oxygen, or sulfur, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; even more preferably, it contains 3 to 6 ring atoms, of which 1 to 3 are heteroatoms; and most preferably, it contains 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl. Polycyclic heterocyclyls include spirocyclic, fused, and bridged heterocyclyls. The heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring; the ring connected to the parent structure is the heterocyclyl.

[0210] The term "heteroalkylene" is a divalent group that is attached to the rest of the molecule by two single bonds, and otherwise has the same definition as the term "cycloalkylene".

[0211] In the present invention, the term "each independently" generally refers to a variable being applicable in any instance, regardless of whether other variables with the same or different definitions exist in the same compound. For example, the variable may refer to the type or number of substituents in the compound, or the type of atoms in the compound. For example, if R occurs twice in a compound and R is defined as "independently carbon or nitrogen," both R may be carbon, both R may be nitrogen, or one R may be carbon and the other R may be nitrogen.

[0212] In the present invention, the term "optional" or "optionally" generally means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that alkyl substitution may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0213] In the present invention, the term "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 independently replaced 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) whether substitution is possible or not without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0214] In the present invention, unless otherwise specified, the "connection" of a group to a group can generally be in any orientation; for example, the "connection" of a group X to a group Y can generally be in any orientation, and any orientation generally means that when a group X is used to connect a group Y and a group Z, the two or more connection sites of the group X can be arbitrarily connected to the group Y or the group Z.

[0215] In the present invention, as known to those skilled in the art, terms such as "alkyl" and "cycloalkyl" may be preceded by an identifier to indicate the number of atoms present in the group in a particular case, for example, C1-C4 alkyl, C3-C7 cycloalkyloxy, C1-C4 alkylcarbonylamino, etc. The subscript number after "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group with three carbon atoms (e.g., n-propyl, isopropyl); C 1-10 In the examples, the members of the group may have any number of carbon atoms falling within the range of 1-10.

[0216] In the present invention, the compounds or antibody drug conjugates of the present invention include tautomers, mesomorphs, racemates, enantiomers, and / or diastereomers thereof. In the present invention, 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, for example, melting points, boiling points, spectral properties and reactivity. In the present invention, the terms "tautomers" or "tautomeric forms" are used interchangeably and generally refer to structural isomers of different energies that can be converted into each other through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via reorganization of some of the bonding electrons. In the present invention, the term "mesomer" generally refers to a molecule containing an asymmetric atom, but having factors of symmetry such that the total intramolecular optical rotation is zero. The term "racemate" or "racemic mixture" refers to a composition composed of equimolar amounts of two enantiomeric species.

[0217] In the present invention, the term "linker unit" or "linker structure" generally refers to a chemical structure fragment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end, and can also be connected to a cytotoxic drug after being connected to other linkers. The directly or indirectly connected ligand can refer to the group being directly connected to the ligand through a covalent bond, or it can be connected to the ligand through a linker structure. For example, a chemical structure fragment or bond comprising an acid-labile linker structure (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure can be used as the linker structure.

[0218] In the present invention, the term "optionally linked to other molecular moieties" generally means that the structure is not linked to any other chemical structure, or that the structure is linked to one or more other chemical structures (such as the ligands described in the present invention) that are different from the structure (for example, linked by a chemical bond or linked by a linker structure).

[0219] In the present invention, the term "drug loading" generally refers to the average amount of cytotoxic drug (payload) loaded per ligand, and can also be expressed as the ratio of the amount of cytotoxic drug to the amount of antibody (drug / antibody ratio, DAR). The cytotoxic drug loading can range from 0-12, for example, 1-10, cytotoxic drugs per ligand (Ab). In embodiments of the present invention, the drug loading is expressed as p or p1, and can illustratively be the average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The drug loading per ADC molecule after the conjugation reaction can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0220] In the present invention, certain atoms in the compounds or antibody drug conjugates of the present invention may appear in more than one isotopic form. For example, hydrogen may appear in the form of protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), carbon can exist in three different isotopes ( 12 C. 13 C and 14 C) naturally occurring. Examples of isotopes that can be incorporated into the compounds of the present invention also include, but are not limited to 15 N. 18 O. 17 O. 18 F. 32 P. 33 P. 129 I. 131 I. 123 I. 124 I. 125I, or similar isotopes. Thus, the compounds or antibody drug conjugates of the present invention may be enriched in one or more of these isotopes relative to the natural abundance of these isotopes. As is known to those skilled in the art, such isotopically enriched compounds may be used in a variety of applications. For example, heavy isotopes such as deuterium ( 2 Deuterium (H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability. 2 The natural abundance of deuterium (H) is approximately 0.015%. Therefore, there is one deuterium atom for approximately every 6,500 hydrogen atoms in nature. Therefore, the deuterium-containing compounds or antibody drug conjugates of the present invention have a deuterium abundance greater than 0.015% at one or more positions (as appropriate). Unless otherwise indicated, the structures described herein may also include compounds or antibody drug conjugates that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds or antibody drug conjugates that have the same structure as the present invention except for the replacement of hydrogen atoms by deuterium or tritium, or the replacement of carbon atoms by carbon-13 or carbon-14, are within the scope of the present invention.

[0221] In 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, with other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity. Conventional pharmaceutical composition preparations can be found in various pharmacopoeias. Pharmaceutical compositions can be in the form of sterile injectable aqueous or oily suspensions for intramuscular and subcutaneous administration. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable preparations can also be sterile injectable solutions or suspensions prepared in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Sterile fixed oils can also be conveniently used as solvents or suspending media. For example, any blended fixed oil, including synthetic mono- or diglycerides, can be used. Fatty acids, such as oleic acid, can also be used to prepare injectable formulations.

[0222] In the present invention, 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.

[0223] In the present invention, 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 pharmaceutical carrier that does not itself induce the production of antibodies 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 acids, polyglycolic acids, polyamino 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 buffering substances, etc., may also be present in these carriers.

[0224] As used herein, the terms "treatment" and "treating" generally refer to an approach for obtaining a beneficial or desired result, including, but not limited to, a therapeutic benefit. A therapeutic benefit includes, but is not limited to, eradication, inhibition, reduction, or amelioration of the underlying disorder being treated. Additionally, a therapeutic benefit is achieved by eradication, inhibition, reduction, or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, but the patient may still suffer from the underlying disorder.

[0225] As used herein, the terms "prevention" and "preventing" generally refer to an approach for obtaining a beneficial or desired result, including, but not limited to, a prophylactic benefit. For prophylactic benefit, a pharmaceutical composition can be administered to a patient at risk for 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.

[0226] In the present invention, 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.

[0227] In the present invention, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of a compound or antibody drug conjugate of the present invention, alone or in combination with other therapeutic agents, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions or the progression of the disease or condition when administered to a cell, tissue or subject. A therapeutically effective dose also refers to a dose sufficient to cause amelioration of symptoms, such as an amount that treats, cures, prevents or ameliorate a related medical condition or increases the rate of treatment, cure, prevention or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in 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 by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0228] In the present invention, the term "cancer" refers to a group of cells that show an abnormally high level of proliferation and growth. Cancer may be benign (also known as a benign tumor), pre-malignant or malignant. Cancer cells may be solid cancer cells or leukemia cancer cells. In the present invention, the term "tumor" refers to one or more cells comprising cancer. In the present invention, the term "tumor growth" refers to the proliferation or growth of one or more cells comprising cancer, which results in a corresponding increase in the size or extent of the cancer. The terms "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.

[0229] The amino acid numbers of the antibodies of the present invention are numbered according to the natural sequence from N-terminus to C-terminus of the antibody sequence.

[0230] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0231] The reagents and raw materials used in the present invention are commercially available.

[0232] The positive progress effect of the present invention is:

[0233] The bispecific antibodies of the present invention have the following advantages:

[0234] 1. It can simultaneously bind to EGFR and HER3 antigens and EGFR / HER3 cell lines, and exhibits anti-tumor activity superior to that of monoclonal antibodies. Compared with the parental monoclonal antibody and the control antibody SI-1X6.4, 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.

[0235] 2. Compared with related monoclonal antibody combination therapies, the bispecific antibodies of the present invention have the advantages of good compliance and controllable quality.

[0236] 3. The stability characterization of the bispecific antibodies in the present invention is mainly reflected in the research on monomer purity and thermal stability. After affinity purification and molecular sieve separation, the bispecific antibody monomer content can reach 95%. The results of the structural and activity analysis of the antibody after heat treatment show that the antibody can still maintain a good molecular conformation and complete biological activity under harsh environments, which is conducive to the industrial production and packaging and storage of the antibody. In general, 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) that is significantly better than Cetuximab and EGFR. In vivo data show that in EGFR+HER3 tumor cells, the anti-tumor activity of the bispecific antibodies is better than the EGFR and HER3 monoclonal antibody combination group. Therefore, the bispecific antibodies of the present invention have broad application prospects due to their excellent developability and activity.

[0237] The bispecific antibody drug conjugates described in any one of the present invention have an in vivo tumor inhibition effect, which is specifically manifested as follows:

[0238] (1) It has a better tumor inhibitory effect on the human esophageal cancer cell line OE-19 with low EGFR expression and medium HER3 expression;

[0239] (2) It has a better tumor inhibitory effect on the human non-small cell lung cancer cell line NCI-H441 that expresses EGFR and HER3;

[0240] (3) It has a better tumor inhibitory effect on the human oral squamous cell carcinoma cell line CAL-27 with high EGFR expression and low HER3 expression;

[0241] (4) It has a better tumor inhibitory effect in the human esophageal cancer OE-19 subcutaneous xenograft model that is resistant to EGFR ADC;

[0242] (5) It has a better tumor inhibitory effect in the subcutaneous xenograft model of human skin cancer A431 with high EGFR expression and low HER3 expression;

[0243] (6) It has a better tumor inhibitory effect in the subcutaneous xenograft model of human colon cancer SW620 that does not express EGFR but only expresses HER3;

[0244] (7) It has a better tumor inhibitory effect in the SW48 subcutaneous xenograft model of human colon cancer with low EGFR expression and HER3 low expression;

[0245] (8) It has a better tumor inhibitory effect in the subcutaneous xenograft model of non-small cell lung cancer NCI-H1975 (EGFR L858R / T790M / C797S triple mutation) that is resistant to third-generation TKI. BRIEF DESCRIPTION OF THE DRAWINGS

[0246] Figure 1 shows the binding of the bispecific antibody and its corresponding parental monoclonal antibody to cell lines expressing different levels of EGFR / HER3.

[0247] Figure 2. Binding of bispecific antibodies and their corresponding parental monoclonal antibodies to cell lines expressing different levels of EGFR / HER3.

[0248] FIG3 shows the endocytic activity of the bispecific antibody and its corresponding parental monoclonal antibody (pHrodo assay).

[0249] FIG4 shows the endocytic activity (incucyte assay) of the bispecific antibody and its corresponding parental monoclonal antibody and a control antibody.

[0250] Figure 5 shows the endocytic activity (incucyte assay) of the bispecific antibody and its corresponding parental monoclonal antibody and control antibody.

[0251] Figure 6 is the in vivo efficacy evaluation of the antibody drug conjugate on human esophageal cancer cell line OE-19 tumor-bearing mice expressing EGFR low expression and HER3.

[0252] FIG7 is an in vivo evaluation of the efficacy of the antibody drug conjugate on human non-small cell lung cancer cell line NCI-H441 tumor-bearing mice expressing EGFR and HER3.

[0253] FIG8 is an in vivo evaluation of the efficacy of the antibody-drug conjugate in mice bearing the human oral squamous cell carcinoma cell line CAL-27 with high EGFR expression and low HER3 expression.

[0254] Figure 9 shows the in vivo efficacy evaluation of bispecific antibody drug conjugates with different DAR values ​​in NCI-H1975 tumor-bearing mice.

[0255] Figure 10 shows the in vivo efficacy evaluation of bispecific antibody drug conjugates with different DAR values ​​in OE-19 tumor-bearing mice.

[0256] FIG11 is a comparison of the in vivo efficacy of the bispecific antibody drug conjugate of the present invention and a control antibody drug conjugate in NCI-H1975 tumor-bearing mice.

[0257] FIG12 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.

[0258] FIG13 shows the in vivo efficacy of the bispecific antibody-drug conjugate linker-cytotoxin X1 of the present invention in NCI-H1975 tumor-bearing mice.

[0259] FIG14 shows the in vivo efficacy of the bispecific antibody-drug conjugate of the present invention in human esophageal cancer OE-19 tumor-bearing mice.

[0260] FIG15 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.

[0261] FIG16 shows the in vivo efficacy of the bispecific antibody-drug conjugate of the present invention in mice bearing xenografts of the human colon cancer cell line SW620 that does not express EGFR but only expresses HER3.

[0262] FIG17 shows the in vivo efficacy of the bispecific antibody-drug conjugate of the present invention in mice bearing xenografts of the human colon cancer cell line SW48 that expresses EGFR and low expression of HER3.

[0263] FIG18 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. DETAILED DESCRIPTION

[0264] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0265] Sample testing

[0266] The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0267] Example

[0268] The following examples are provided to demonstrate and further explain some preferred embodiments and aspects of the present invention and should not be construed as limiting the scope thereof.

[0269] Example 1 Preparation of bispecific antibodies

[0270] In the anti-EGFR × HER3 bispecific antibody of the present invention, the anti-HER3 antibody or antigen-binding fragment thereof was prepared with reference to clone 2 of hu3F8 (PCT / CN2022 / 098929), and the anti-EGFR antibody or antigen-binding fragment thereof was prepared with reference to zalutumumab (WO2002100348). Furthermore, based on these antibodies, some amino acid mutations were made in the CDR / FR / Fc regions. The CDR regions of the bispecific antibody are numbered according to the Kabat numbering convention.

[0271] Based on the amino acid sequences of the antibody variable regions, primers were designed for PCR to construct VH / VK gene segments, yielding the variable regions. The variable regions were then combined with the constant region gene segments to construct the complete bispecific antibody sequence. After transfection into CHO cells, the monoclonal antibodies anti-EGFR and anti-HER3, as well as the bispecific antibodies DBXT001-01-08, DBXT002-01-08, DBXT003-01-08, DBXT004-01-08, and DBXT005-01, were expressed and purified using conventional methods.

[0272] Cetuximab monoclonal antibody: prepared by conventional methods; control antibody SI-1X6.4: prepared according to WO2023083381A1.

[0273] Antibody names and sequences are shown in Table 1.

[0274] Table 1 Amino acid sequences of antibodies

[0275] Antibody expression and purification

[0276] 1. CHO-K1 cells from ECACC were revived and inoculated into culture medium (Shanghai Aupuma Biotech Co., Ltd., Cat. No. C673017). They were subcultured at 8% CO2 and 37°C. The density of cells reached 6.0×10 6 / mL, take 1.45×10 8 cells, centrifuge and remove the supernatant;

[0277] 2. Add 0.5 mL of electroporation buffer to the cell pellet and mix well;

[0278] 3. Add four plasmids in a ratio of H1:L1:H2:L2 = 2:3:2:3. Mix the cell-plasmid suspension thoroughly and add it to the electroporation tube. Place it in the electroporator for electroporation.

[0279] 4. After the electroporation is completed, transfer the cells in the electroporated tube into a shake flask containing 20 mL of culture medium and incubate at room temperature for 40 minutes;

[0280] 5. After incubation, place the shake flask in a shaker at 37°C, 110 rpm, and 8% CO2. After 24 hours, add feed / sodium butyrate / double antibiotic (penicillin + streptomycin) and continue incubation for 4 days.

[0281] 6. On the 4th day after transfection, the supernatant was collected by centrifugation and purified by Protein A affinity chromatography (Borgron), eluted with sodium acetate buffer (pH 3.4), collected in separate tubes, and sent for SEC analysis;

[0282] 7. The eluate that meets the requirements is purified by gel filtration chromatography (Borgron 200, size: 16mm×700mm), eluted with 1×PBS, collected in different tubes, and sent for SEC. The eluate that meets the requirements is exchanged into buffer (25mM His-HCl, 6% Sucrose, pH 6.0) and stored at -20℃.

[0283] Example 2 Antibody Affinity Detection (SPR)

[0284] 2.1 Objective: The affinity of anti-EGFR and HER3 bispecific antibodies and the parental monoclonal antibody was tested using Biacore T200 (Cytiva).

[0285] Experimental methods:

[0286] 1. The ProA chip captured EGFR-HER3 dual antibody (1 μg / mL). The analytes were human EGFR (human EGFR, His-Avi Tag, Kactus Biosystem, EGF-HM401), with a concentration range of 3.125-400 nM in serial dilutions, for a total of 8 concentrations; human HER3 (human HER3, His-Avi Tag, Kactus Biosystem, HER-HM403), with a concentration range of 6.25-200 nM in serial dilutions, for a total of 8 concentrations; monkey EGFR (rhesus EGFR, C-His Tag, Kactus Biosystem, EGF-CM101), with a concentration range of 6.25-400 nM in serial dilutions, for a total of 8 concentrations; monkey HER3 (rhesus HER3, C-His Tag, Kactus Biosystem, EGF-CM101), with a concentration range of 6.25-400 nM in serial dilutions, for a total of 8 concentrations. Biosystem, HER-CM403), with serial dilutions ranging from 3.125 to 200 nM, for a total of 8 concentrations;

[0287] 2. The ProA chip captured the EGFR parental mAb (1 μg / mL) and the analyte was human EGFR (human EGFR, His-Avi Tag, Kactus Biosystem, EGF-HM401). The concentration was serially diluted from 6.25 to 400 nM, for a total of 8 concentrations. The ProA chip captured the HER3 parental mAb (3 μg / mL) and the analyte was human HER3 (human HER3, His-Avi Tag, Kactus Biosystem, HER-HM403). The concentration was serially diluted from 3.125 to 100 nM, for a total of 8 concentrations.

[0288] 3. Affinity data were analyzed and fitted using a 1:1 Langmuir binding model. Affinity (KD) is the ratio of the dissociation constant (Kd) to the association constant (Ka). Affinity results are shown in Table 2.

[0289] Table 2 Affinity of bispecific antibodies and monoclonal antibodies to human and monkey antigens

[0290] Experimental Conclusions: The affinity of the DBXT001-01 bispecific antibody for human EGFR is approximately 16-fold weaker than its affinity for HER3. The affinity of the DBXT001-01 bispecific antibody for human EGFR is approximately 3-fold weaker than that of the parental EGFR antibody, while its affinity for HER3 is comparable to that of the parental HER3 antibody. Furthermore, the affinity of the DBXT001-01 bispecific antibody for human EGFR or HER3 is comparable to that for monkey EGFR or HER3. This affinity design of the bispecific antibody can reduce on-target toxicity of the bispecific antibody in widely distributed normal tissues expressing EGFR.

[0291] 2.2 Antibody affinity detection (SPR)

[0292] Objective: To detect the affinity of anti-EGFR and HER3 dual antibodies using Biacore T200 (Cytiva).

[0293] Experimental methods:

[0294] 1. The ProA chip captured SI-1X6.4 (7.5 μg / mL). The analytes were human EGFR (His-Avi Tag, Kactus Biosystem, EGF-HM401) at five serial dilutions ranging from 1.17 to 18.75 nM; and human HER3 (His-Avi Tag, Kactus Biosystem, HER-HM403) at five serial dilutions ranging from 4.69 to 75 nM. Similarly, the ProA chip captured DBXT005-01 (6 μg / mL). The analytes were human EGFR at five serial dilutions ranging from 9.38 to 150 nM; and human HER3 at five serial dilutions ranging from 4.69 to 75 nM.

[0295] 2. Affinity data were analyzed and fitted using a 1:1 Langmuir binding model. Affinity (KD) is the ratio of the dissociation constant (Kd) to the association constant (Ka). Affinity results are shown in Table 3.

[0296] Table 3 Affinity of bispecific antibodies and human antigens

[0297] Experimental Conclusions: The affinity of the bispecific antibody DBXT005-01 for human EGFR is approximately 15-fold weaker than that of the control antibody SI-1X6.4, while its affinity for HER3 is 25-fold stronger. This affinity design of the bispecific antibody can reduce on-target toxicity in normal tissues that widely express EGFR and enhance its affinity for tumors that overexpress EGFR and underexpress HER3.

[0298] Example 3 Binding of bispecific antibodies and their corresponding parental monoclonal antibodies to cell lines expressing different levels of EGFR / HER3

[0299] 3.1 Test Purpose:

[0300] Flow cytometry was used to compare the affinity of DBXT001-01 and its parent monoclonal antibody for cell lines with different EGFR / HER3 expression levels.

[0301] 1. Establishment of a mouse monoclonal cell line CT26 stably expressing human EGFR and HER3 (CoBio). Tumor cell lines NCI-H441, NCI-H1975, and T47D were obtained from ATCC.

[0302] 2. All cell lines were cultured in complete medium at 37°C, 5% CO2;

[0303] 3. Harvest cells in the logarithmic growth phase and detect cell viability using the trypan blue exclusion method to ensure that the cell viability is above 90%. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Wash the cells once with PBS, resuspend them in FACS buffer to prepare a single cell suspension and adjust the cell density to 5×10 6 cells / mL;

[0304] 4. Add 50 μL of cell suspension to each well of a 96-well plate to make the starting concentration of the test solution 100 nM, and dilute 5-fold for a total of 6 concentrations; after mixing, place at 4°C and incubate for 40 minutes;

[0305] 5. Wash the cells three times with 400 μL of FACS Buffer each time, centrifuge at 1000 rpm for 5 min, and resuspend the cells in 100 μL of FACS Buffer.

[0306] 6. Add 2 μL of PE-labeled secondary antibody (PE anti-human IgG Fc Antibody), mix well, and incubate at 4°C in the dark for 40 min.

[0307] 7. Wash the cells three times with 400 μL of FACS Buffer each time, centrifuge at 1000 rpm for 5 minutes, and resuspend the cells in 250 μL of FACS Buffer.

[0308] 8. Detect fluorescence value by flow cytometry.

[0309] The experimental results are shown in Figure 1 and Table 4.

[0310] Table 4 Affinity of parental monoclonal antibodies and bispecific antibodies to cell lines

[0311] Experimental conclusion:

[0312] The affinity of DBXT001-01 for cell lines overexpressing either EGFR or HER3 alone is lower than that of the parent bivalent monoclonal antibody and lower than that of cetuximab for cell lines overexpressing EGFR. However, the affinity of DBXT001-01 for tumor cell lines co-expressing EGFR and HER3 is comparable to that of the parent bivalent monoclonal antibody. This may facilitate the bivalent antibody's reduced binding to normal tissues and enhanced binding to tumor tissues co-expressing EGFR and HER3.

[0313] 3.2 Affinity of different bispecific antibodies and cell lines expressing different EGFR / HER3 levels

[0314] Experimental purpose: To compare the affinity of different bispecific antibodies and the parental monoclonal antibody to EGFR / HER3 cell lines with different expression levels by flow cytometry

[0315] Experimental methods:

[0316] 1. Tumor cell lines BT-474 and MDA-MB-468 were obtained from ATCC; mouse monoclonal cell lines stably expressing human EGFR or human HER3 were obtained from Kebai Biotechnology.

[0317] 2. All cell lines were cultured in complete medium at 37°C, 5% CO2;

[0318] 3. Harvest cells in the logarithmic growth phase and detect cell viability using the trypan blue exclusion method to ensure that the cell viability is above 90%. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant. Wash the cells once with PBS, resuspend them in FACS buffer to prepare a single cell suspension and adjust the cell density to 5×10 6 cells / mL;

[0319] 4. Add 50 μL of cell suspension to each well of a 96-well plate to make the starting concentration of the test solution 150 nM, and dilute 4-fold for a total of 8 concentrations; after mixing, place at 4°C and incubate for 40 minutes;

[0320] 5. Wash the cells three times with 400 μL of FACS Buffer each time, centrifuge at 1000 rpm for 5 min, and resuspend the cells in 100 μL of FACS Buffer.

[0321] 6. Add 2 μL of PE-labeled secondary antibody (PE anti-human IgG Fc Antibody), mix well, and incubate at 4°C in the dark for 40 min.

[0322] 7. Wash the cells three times with 400 μL of FACS Buffer each time, centrifuge at 1000 rpm for 5 minutes, and resuspend the cells in 250 μL of FACS Buffer.

[0323] 8. Detect fluorescence value by flow cytometry.

[0324] The test product codes are shown in Table 5

[0325] The experimental results are shown in Figure 2 and Table 6.

[0326] Table 5 Different compositions of bispecific antibodies or monospecific antibodies

[0327] Table 6 Affinity of different bispecific antibodies and cell lines with different EGFR / HER3 expression levels

[0328] Experimental conclusion: For cell lines with different EGFR and HER3 expression levels, DBXT005-01 has a stronger ability to saturate target antigens than the control antibody SI-1X6.4; and its saturation antigen capacity is also stronger than the corresponding parental monoclonal antibody.

[0329] Example 4 Endocytic activity of antibodies (pHrodo method)

[0330] Purpose of the test

[0331] The endocytic activity of the antibody drug targeting EGFR and HER3 was tested in T47D cells expressing both EGFR and HER3, and compared with the endocytic activity of the parental monoclonal antibody. The cells were incubated with a fixed concentration of the antibody drug and the endocytosis indicator reagent pHrodo. The endocytic activity of the antibody drug was evaluated by observing the fluorescence signal generated by pHrodo at different time points as the antibody drug entered the cells.

[0332] Experimental methods

[0333] 1. Cell culture: T47D cells were cultured in RPMI-1640 medium containing 0.2 units / mL bovine insulin and 10% FBS.

[0334] 2. Cell preparation: Take T47D cells in the logarithmic growth phase, wash them once with PBS, and digest them for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to elute the digested cells. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells into a single cell suspension and adjust the viable cell density to 3×10 5 cells / mL.

[0335] 3. Cell plating: Add 50 μL / well of the cell culture plate to a 96-well cell culture plate and incubate the plate in an incubator (37° C., 5% CO 2 ) for 48 hours.

[0336] 4. Sample addition: After incubating the antibody drug to be tested with Fab-pHrodo to form a complex, adjust the concentration to 6nM or 60nM and add 50μL / well to the cells.

[0337] 5. Cell culture: Incubate the culture plate in an incubator for 48 hours (37° C., 5% CO 2 ).

[0338] 6. Plate reading: At the corresponding time point, remove the 96-well cell culture plate, digest the cells, and read the cell number and fluorescence value by FACS.

[0339] Experimental results Figure 3.

[0340] Experimental conclusion:

[0341] Compared with the parent monoclonal antibody, the antibody drug targeting EGFR and HER3 of the present invention has a better endocytic effect on T47D cells that express both EGFR and HER3.

[0342] Example 5 Endocytic activity of antibodies (incucyte method)

[0343] 5.1 Test Purpose:

[0344] The endocytosis efficiency of the antibody drug targeting EGFR and HER3 of the present invention was tested 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 endocytic ability of the antibody drug conjugate was evaluated by continuously observing the changes in the fluorescence signal of living cells for 48 hours.

[0345] Experimental methods:

[0346] 1. Cell culture: NCI-H1975 cells were cultured in RPMI1640 medium supplemented with 10% FBS. CT26 cells stably transfected with both EGFR and HER3 were cultured in RPMI1640 medium supplemented with 10% FBS, 10 μg / mL puromycin, and 20 μg / mL blasticidin.

[0347] 2. Cell preparation: Take cells in the logarithmic growth phase, wash them once with PBS, and digest them for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to elute the digested cells. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells into a single cell suspension and adjust the viable cell density to 1x10 5 cells / mL.

[0348] 3. Cell plating: Add 50 μL / well of the cell culture plate to a 96-well cell culture plate and culture the plate in an incubator overnight (37°C, 5% CO2).

[0349] 4. Label the antibody-drug conjugate to be tested: For high concentration antibody labeling, dilute the test sample stock solution to 240nM 4× working solution (final concentration 60nM). Fabfluor-pH stock solution was diluted to 720nM 4× working solution (final concentration 180nM), the two were mixed thoroughly, and incubated at 37℃ in the dark for 15 minutes. Low concentration antibody labeling was done by diluting the test sample stock solution to 40nM 4× working solution (final concentration 10nM). The Fabfluor-pH stock solution was diluted to a 4× working solution of 120 nM (final concentration 30 nM), the two were thoroughly mixed, and incubated at 37°C in the dark for 15 minutes.

[0350] 5. 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 acquire images. Quantification is performed at 2-hour intervals for 48 hours. The analysis results are expressed as: total fluorescence integrated intensity (RCU X μm 2 / image).

[0351] The experimental results are shown in Figures 4A and 4B.

[0352] Experimental conclusion:

[0353] 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. Furthermore, 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.

[0354] 5.2 Internalization of Different BisAbs in Cell Lines Expressing Different EGFR / HER3 Levels (Incucyte Method)

[0355] Purpose of the test:

[0356] The endocytosis efficiency of the antibody drug targeting EGFR and HER3 of the present invention was tested in comparison with other antibody drugs targeting EGFR and HER3 in cell lines A431 and NCI-H1975 expressing different levels of EGFR and HER3 and in CT26 cell line overexpressing both EGFR and HER3. Fabfluor-pH was co-incubated, and the endocytic ability of the antibody drug conjugate was evaluated by continuously observing the changes in the fluorescence signal of living cells for 24 hours.

[0357] Experimental methods:

[0358] 1. Cell Culture: A431 cells were cultured in DMEM medium supplemented with 10% FBS; NCI-H1975 cells were cultured in RPMI1640 medium supplemented with 10% FBS; CT26 cells stably transfected with both EGFR and HER3 were cultured in RPMI1640 medium supplemented with 10% FBS, 10 μg / mL puromycin, and 20 μg / mL blasticidin.

[0359] 2. Cell preparation: Take cells in the logarithmic growth phase, wash them once with PBS, and digest them for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to elute the digested cells. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells into a single cell suspension and adjust the viable cell density to 1x10 5 cells / mL.

[0360] 3. Cell plating: Add 50 μL / well of the cell culture plate to a 96-well cell culture plate and culture the plate in an incubator overnight (37°C, 5% CO2).

[0361] 4. Label the antibody-drug conjugate to be tested: Mix the test sample / control sample working solution and the labeling reagent working solution at a 1:3 molar ratio and a 1:1 volume ratio, and incubate in a cell culture incubator (37°C, 5% CO2) in the dark for 15 minutes to allow for full coupling between the two.

[0362] 5. 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 acquire images. Quantification is performed at 2-hour intervals for 24 hours. The analysis results are expressed as: total fluorescence integrated intensity (RCU X μm 2 / image).

[0363] The experimental results are shown in Figures 5A, 5B, 5C, 5D and Table 7.

[0364] Table 7 24-hour endocytosis of 1 nM dual antibody in cell lines with different EGFR / HER3 expression levels

[0365] Experimental conclusion:

[0366] Compared with the control antibody, the antibody DBXT005-01 targeting EGFR and HER3 of the present invention has a better endocytic effect on tumor cells with different expression levels of EGFR or HER3.

[0367] Example 6 Preparation of Bispecific Antibody Drug Conjugates (ADCs)

[0368] 6.1 Preparation of Linker-Payload

[0369] Preparation of Linker-payload X1

[0370] first step

[0371] To a solution of 27a (5.00 g, 43.0 mmol) and NaHCO₃ (10.9 g, 129 mmol) in DMF (50 mL) was added benzyl bromide (11.0 g, 64.6 mmol) dropwise under nitrogen. The mixture was allowed to react at 25°C for 17 hours. TLC (PE / EA = 2 / 1) indicated completion of the reaction. The reaction solution was added to 500 mL of water and extracted twice with EA (250 mL). The mixture was separated, washed with saturated aqueous sodium chloride (500 mL), dried over anhydrous Na₂SO₄, and concentrated through a column chromatography (PE:EA = 3:2) to afford 5.1 g of a colorless liquid (yield: 57.1%).

[0372] Step 2

[0373] Under nitrogen, a solution of 27b (4.50 g, 21.8 mmol) in THF (10 mL) was added dropwise to a solution of KI2 (4.00 g, 10.9 mmol) and TsOH (800 mg, 4.65 mmol) in THF (30 mL) at 0°C. The mixture was allowed to react at 25°C for 2 hours. TLC (PE / EA = 1 / 2) indicated the reaction was complete. The reaction solution was added to 200 mL of water and extracted twice with EA (200 mL). The mixture was dried over anhydrous Na2SO4 and concentrated through a column chromatography (PE / EA = 3 / 2) to afford 1.56 g of a white solid (yield: 26%).

[0374] Step 3

[0375] To a solution of 27c (800 mg, 1.55 mmol) in EtOH (8 mL) and EA (8 mL) was added Pd / C (80 mg) under hydrogen at 0°C, and the mixture was stirred at 0°C for 2.5 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered through celite, washed with EA (200 mL), and the filter cake was concentrated and dissolved in THF (20 mL). The product was then dried to afford 600 mg of a white solid (91% yield).

[0376] Step 4

[0377] Under nitrogen, DIEA (152 mg, 1.18 mmol) was added to a solution of 27d (220 mg, 0.515 mmol), HY-13631A (250 mg, 0.47 mmol), and HATU (214 mg, 0.56 mmol) in DMF (6 mL) at 0°C. The mixture was allowed to react for 2 hours at 0°C. LCMS indicated the reaction was complete. The reaction solution was added to 150 mL of aqueous citric acid (pH = 4), filtered, and the filter cake was washed with 175 mL of water, filtered, and pumped dry to afford 260 mg of a brown solid (yield: 66%).

[0378] Step 5

[0379] Under nitrogen, diethylamine (8 mL) was added dropwise to a solution of 27e (260 mg, 0.309 mmol) in DCM (30 mL) at 0°C. The mixture was allowed to react for 3 hours at 0°C. LCMS indicated the reaction was complete. The reaction solution was added to 600 mL of petroleum ether at 0°C, resulting in the precipitation of a solid. After the solid was allowed to adsorb to the bottom of the flask, the solution was decanted and pumped dry with an oil pump to afford 90 mg of a brown solid (yield: 47.1%).

[0380] Step 6

[0381] Under nitrogen, HATU (74 mg, 0.19 mmol) was added to a solution of 27f (90 mg, 0.13 mmol), KI1 (92 mg, 0.19 mmol), and DIEA (50 mg, 0.39 mmol) in DMF (2.5 mL) at 0°C and allowed to react for 2 hours. LCMS indicated the reaction was complete. The reaction solution was added to a pH 4 aqueous citric acid solution (30 mL) at 0°C. A flocculent solid precipitated, which was filtered and purified by preparative chromatography (DCM / MecOH = 10 / 1) to afford 9.2 mg of a light yellow solid, X1, in a 6% yield.

[0382] MS m / z(ESI):1074[M+1]

[0383] 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)

[0384] Preparation of Linker-payload X2

[0385] first step

[0386] 34a (5 g, 48.0 mmol) and K2CO3 (19.9 g, 144.0 mmol) were dissolved in DMF (20 mL), and benzyl bromide (12.3 g, 72.0 mmol) was added dropwise. The mixture was allowed to react at 25°C for 17 hours. TLC (PE / EA = 3 / 1) confirmed the complete reaction. The reaction mixture was added to water (200 mL) and extracted with EA (250 mL). The mixture was washed with saturated NaCl, dried over anhydrous Na2SO4, and concentrated to obtain 8.7 g of 34b as a colorless liquid (93% yield). MS-ESI: m / z 195.1 [M+H]+.

[0387] Step 2

[0388] 34c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) were dissolved in THF (20 mL) under nitrogen atmosphere and cooled to 0°C. A solution of 43b (7.7 g, 39.6 mmol) in THF (10 mL) was added dropwise. The mixture was allowed to react at 0°C for 2 hours. TLC (PE / EA = 2 / 1) showed that most of the starting material had reacted. The reaction solution was poured into 100 mL of water and extracted with DCM (100 mL). The mixture was separated and washed with saturated NaCl, dried over anhydrous Na2SO4, and filtered through a column (PE / EA = 1 / 1) to give 3.9 g of a colorless viscous product 34d (yield: 39%). MS-ESI: m / z 503.3 [M+H]+.

[0389] Step 3

[0390] To a solution of 34d (1.9 g, 3.78 mmol) in EtOH (100 mL) and EA (100 mL) was added Pd / C (1 g, 10 wt.%) under hydrogen at 0°C for 3 hours. TLC (PE / EA = 2 / 1) indicated the reaction was complete. The reaction mixture was filtered through celite, and the filter cake was washed with EA / EtOH (1:1, 100 mL × 3). The filtrate was concentrated and dissolved in THF (50 mL × 3) and the mixture was dried by spin drying. This was repeated three times to afford 1 g of a gray solid 34e in a 64% yield. MS-ESI: m / z 435.2 [M+Na]+.

[0391] Step 4

[0392] Under nitrogen, DIEA (303 mg, 2.35 mmol) was added dropwise to a solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol), and HATU (429 mg, 1.13 mmol) in DMF (20 mL) at 0°C. The mixture was allowed to react at 0°C for 2 hours. LCMS indicated the reaction was complete. The reaction solution was added dropwise to 300 mL of water, stirred, allowed to stand for 5 minutes, and filtered. The filter cake was dissolved in DCM / MeOH (10:1, 100 mL), dried, and then spin-dried. The mixture was purified by column chromatography (EA:MeOH = 30:1) to afford 600 mg of 34f as a yellow solid in a 77% yield. MS-ESI: m / z 830.3 [M+H]+.

[0393] Step 5

[0394] Under nitrogen, diethylamine (5 mL) was added dropwise to a solution of 34f (150 mg, 0.18 mmol) in DCM (5 mL) at 0°C. The mixture was allowed to react for 2 hours at 0°C. LCMS indicated the reaction was complete. Petroleum ether solution (100 mL x 6) was added to the reaction mixture, causing solid precipitation. After standing for solid precipitation, the solution was decanted and pumped dry using an oil pump to yield 120 mg of a white powder (34 g). LCMS indicated a product content of 70%, yielding 76%. MS-ESI: m / z 608.3 [M+H]+.

[0395] Step 6

[0396] Under nitrogen, a solution of HATU (45 mg, 0.118 mmol) in DMF (1 mL) was added to a solution of 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) at 0°C. The mixture was allowed to react at 0°C for 2 hours. LCMS indicated complete reaction of the starting materials. The reaction solution was directly purified by a reverse phase column using an eluent (MeCN / MeOH = 1 / 1: HO = 60%:40%) to afford 14.8 mg of a yellow solid, X2, in a 14% yield.

[0397] MS-ESI: m / z 1062.4[M+H]+.

[0398] 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),5.6 0(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,1 H),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).

[0399] Preparation of Linker-payload X3

[0400] first step

[0401] To a solution of 32a (2.00 g, 6.6 mmol) and KCO (1.82 g, 13.2 mmol) in MeCN (20 mL) was added allyl bromide (960 mg, 7.92 mmol), and the mixture was stirred at 20°C for 5 hours. TLC (PE / EA = 1 / 2) indicated the reaction was complete. The reaction solution was poured into 100 mL of water, the pH was adjusted to 5, and the mixture was extracted three times with EA (100 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (PE / EA = 2 / 1) to afford 1.83 g of 32b as a white solid (yield: 81%).

[0402] Step 2

[0403] To a solution of 32b (1.38 g, 4.02 mmol) in DCM (10 mL) was added TFA (10 mL) and stirred at 25°C for 17 hours. TLC (PE / EA = 1 / 3) indicated the reaction was complete. The reaction solution was dried to afford 0.91 g of a yellow viscous product 32c. The yield was negligible.

[0404] Step 3

[0405] To a solution of 32c (910 mg, 4.87 mmol) and NaHCO₃ (613 mg, 7.3 mmol) in DME / H₂O (20 mL / 10 mL) was added 41d (1.92 g, 4.87 mmol) and stirred at 25°C for 3 hours. TLC (DCM / MeOH = 1 / 1) indicated the reaction was complete. The reaction solution was poured into 100 mL of water, the pH was adjusted to 5 with aq. HCl (1 N), and the mixture was extracted twice with EA (150 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (DCM / MeOH = 20 / 1) to afford 1.53 g of 32e as a white solid in a 67% yield. MS-ESI: m / z 467.4 [M+H]⁺.

[0406] Step 4

[0407] To a solution of 32f (3 g, 5.83 mmol) in MeOH (50 mL) was added Pd / C (600 mg) and stirred at 25°C under a hydrogen balloon for 5 hours. TLC (EA) indicated the reaction was complete. The reaction solution was filtered and dried to afford 1.9 g of a white solid (32g, yield: 77%).

[0408] Step 5

[0409] HATU (707 mg, 1.86 mmol) was added to 32 g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol), and triethylamine (342 mg, 3.38 mmol) in DMF (10 mL). The mixture was stirred at 0°C for 3.5 hours. TLC (EA) indicated the reaction was complete. The reaction solution was poured into H2O (80 mL), extracted twice with EA (100 mL), dried over anhydrous sodium sulfate, and purified by column chromatography (EA) to afford 1.186 g of a white solid (32h, yield: 83%). MS-ESI: m / z 842.3 [M+H]+.

[0410] Step 6

[0411] A solution of 32h (1.186 g, 1.41 mmol) in DCM / diethylamine (20 mL, 20 / 1) was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 10 / 1) indicated the reaction was complete. The reaction solution was poured into petroleum ether (200 mL) and filtered to afford 768 mg of 32i as a white solid in an 88% yield. MS-ESI: m / z 620.3 [M+H]+.

[0412] Step 7

[0413] To a solution of 32i (676 mg, 1.09 mmol), 32e (508 mg, 1.09 mmol), and DIEA (423 mg, 3.27 mmol) in DMF (10 mL) was added HATU (414 mg, 1.09 mmol), and the mixture was stirred at 20°C for 17 hours. TLC (PE / EA = 1 / 5) indicated the reaction was complete. The reaction solution was poured into water (30 mL) and filtered. The filter cake was purified by column chromatography (DCM / MeOH = 50 / 1) to afford 511 mg of 32j as a white solid in a 44% yield. MS-ESI: m / z 1068.3 [M+H]+.

[0414] Step 8

[0415] A solution of 32j (482 mg, 0.451 mmol) in diethylamine / DCM (10 mL, 1 / 5) was stirred at 10°C for 17 hours. TLC (EA) indicated the reaction was complete. The reaction solution was poured into PE (300 mL) and filtered to afford 32k as a white solid (301 mg). The yield was negligible.

[0416] Step 9

[0417] To a solution of 32k (301 mg, 0.356 mmol) and Pd(PPh3)4 (82 mg, 0.071 mmol) in THF (5 mL) was added morpholine (93 mg, 1.07 mmol) and stirred at 25°C for 5 hours. LCMS indicated the reaction was complete. The reaction mixture afforded 108 mg of 32l as a white solid, yield: 38%. MS-ESI: m / z 806.3 [M+H]+.

[0418] Step 10

[0419] To a solution of 321 (108 mg, 0.134 mmol) and triethylamine (41 mg, 0.402 mmol) in THF (2 mL) and DMF (2 mL) was added bromoacetyl bromide (27 mg, 0.134 mmol), and the mixture was stirred at 0°C for 1 hour. TLC (DCM / MeOH = 10 / 1) indicated the reaction was complete. The reaction solution was directly used to obtain 15 mg of a white solid, X3, in a 12% yield.

[0420] MS-ESI: m / z 926.3[M+H]+.

[0421] 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),7.3 0(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).

[0422] Preparation of Linker-payload X4

[0423] first step

[0424] To a solution of 33a (2.00 g, 2.58 mmol) in MeOH (20 mL) was added Pd / C (400 mg, 10 wt.%) and stirred at 20°C for 5 h. TLC (EA) indicated the reaction was complete. The reaction solution was filtered and dried to afford 1.3 g of 33b as a white solid (yield: 74%).

[0425] Step 2

[0426] To a solution of 33b (0.55 g, 0.802 mmol), KI4 (427 mg, 0.802 mmol), and DIPEA (310 mg, 2.40 mmol) in DMF (5 mL) was added HATU (305 mg, 0.802 mmol), and the mixture was stirred at 0°C for 2 hours. TLC (DCM / MeOH = 1 / 10) indicated the reaction was complete. The reaction solution was poured into water (40 mL) and filtered to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 20 / 1) to afford 33c as a yellow solid (360 mg, 41% yield).

[0427] Step 3

[0428] To a solution of 33c (360 mg, 0.326 mmol) in DCM (10 mL) was added diethylamine (2 mL). The mixture was stirred at 25°C for 17 hours. TLC (DCM / MeOH = 5 / 1) indicated the reaction was complete. The reaction solution was poured into PE (100 mL) and filtered to afford 205 mg of 33d as a white solid in a 71% yield. MS-ESI: m / z 881.3 [M+H]+.

[0429] Step 4

[0430] To a solution of 33d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) and water (1 mL) was added a solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL), and the mixture was stirred at 0°C for 1 hour. The reaction solution directly produced 15 mg of a white solid X4, with a yield of 6%.

[0431] MS-ESI: m / z 1001.2[M+H]+.

[0432] 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),5.20–5.0 7(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),2.83–2.7 4(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).

[0433] 6.2 Preparation of Antibody-Drug Conjugates

[0434] Table 8

[0435] Source of Isotype IgG1: purchased from Bio-Tech (Taicang).

[0436] Preparation of DBXT001-01-X1

[0437] ADC-7(DBXT001-01-X1-DAR4)

[0438] To the buffer solution of antibody DBXT001-01 (PBS pH 7.2; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM EDTA solution (0.875 mL) and prepared tris(2-carbonylethyl)phosphine hydrochloride solution (3.48 mM, 0.379 mL, 1.32 μmol) were added. The mixture was placed in a thermostatic stirrer at 60 rpm and reacted at 22°C for 2 hours, and the reaction was stopped.

[0439] X1 (2.61 mg, 2.43 μmol) was dissolved in 1.75 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer at 60 rpm and shaken at 22°C for 2 hours to stop the reaction. After filtration with activated carbon, the reaction solution was desalted and purified using an AKTA G-25 gel column (HiPrep 26 / 10 Desalting column, 53 mL; elution phase: 30 mM histidine-HCl, pH 5.5). The solution was then concentrated by ultrafiltration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-7 (30 mM histidine-HCl, pH 5.5; 68 mg, 22 mg / mL, yield: 97%), which was stored at -80°C.

[0440] The DAR value detected and calculated by HIC DAR analysis was n=4.06; the purity of the main peak by SEC was 98.2%.

[0441] ADC-8(DBXT001-01-X1-DAR6)

[0442] To the buffer solution of antibody DBXT001-01 (PBS pH 7.2; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM EDTA solution (0.875 mL) and prepared tris(2-carbonylethyl)phosphine hydrochloride solution (3.48 mM, 0.598 mL, 2.08 μmol) were added. The mixture was placed in a thermostatic stirrer at 60 rpm and reacted at 22°C for 2 hours, and the reaction was stopped.

[0443] X1 (4.30 mg, 4.00 μmol) was dissolved in 1.75 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer at 60 rpm and shaken at 22°C for 2 hours to stop the reaction. After filtration with activated carbon, the reaction solution was desalted and purified using an AKTA G-25 gel column (HiPrep 26 / 10 Desalting column, 53 mL; elution phase: 30 mM histidine-HCl pH 5.5). The solution was then concentrated by ultrafiltration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-8 (30 mM histidine-HCl pH 5.5; 69 mg, 23 mg / mL, yield: 99%), which was then stored at -80°C.

[0444] The DAR value detected and calculated by HIC DAR analysis was n=6.11; the purity of the main peak by SEC was 99.2%.

[0445] ADC-9(DBXT001-01-X1-DAR8)

[0446] To the buffer solution of antibody DBXT001-01 (PBS pH 7.2; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM EDTA solution (0.875 mL) and prepared tris(2-carbonylethyl)phosphine hydrochloride solution (3.48 mM, 1.12 mL, 3.89 μmol) were added. The mixture was placed in a thermostatic stirrer at 60 rpm and reacted at 22°C for 2 hours, and the reaction was stopped.

[0447] X1 (5.05 mg, 4.70 μmol) was dissolved in 1.75 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer at 60 rpm and shaken at 22°C for 2 hours to stop the reaction. After filtration with activated carbon, the reaction solution was desalted and purified using an AKTA G-25 gel column (HiPrep 26 / 10 Desalting column, 53 mL; elution phase: 30 mM histidine-HCl, pH 5.5). The solution was then concentrated by ultrafiltration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-9 (30 mM histidine-HCl, pH 5.5; 68 mg, 22.4 mg / mL, yield: 97%), which was stored at -80°C.

[0448] The DAR value detected and calculated by HIC DAR analysis was n=7.99; the purity of the main peak by SEC was 99.0%.

[0449] Preparation of DBXT001-01-X2

[0450] ADC-4 (DBXT001-01-X2-DAR4)

[0451] To the buffer solution of antibody DBXT001-01 (PBS pH 7.4; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM EDTA solution (0.875 mL) and prepared tris(2-carbonylethyl)phosphine hydrochloride solution (3.48 mM, 0.379 mL, 1.32 μmol) were added. The mixture was placed in a thermostatic stirrer at 60 rpm and reacted at 37°C for 2 hours, and the reaction was stopped.

[0452] X2 (2.75 mg, 2.59 μmol) was dissolved in 1.75 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer at 60 rpm and shaken at 22°C for 2 hours to stop the reaction. After filtration with activated carbon, the reaction solution was desalted and purified using an AKTA G-25 gel column (HiPrep 26 / 10 Desalting column, 53 mL; elution phase: 30 mM histidine-HCl, pH 5.5). The solution was then concentrated by ultrafiltration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-4 (30 mM histidine-HCl, pH 5.5; 67.2 mg, 22 mg / mL, yield: 96%), which was stored at -80°C.

[0453] The DAR value detected and calculated by HIC DAR analysis was n=4.10; the purity of the main peak of SEC was 98.4%.

[0454] ADC-5(DBXT001-01-X2-DAR6)

[0455] To the buffer solution of antibody DBXT001-01 (PBS pH 7.4; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM EDTA solution (0.875 mL) and prepared tris(2-carbonylethyl)phosphine hydrochloride solution (3.48 mM, 0.598 mL, 2.08 μmol) were added. The mixture was placed in a thermostatic stirrer at 60 rpm and reacted at 37°C for 2 hours, and the reaction was stopped.

[0456] X2 (4.58 mg, 4.31 μmol) was dissolved in 1.75 mL of DMA and added to the above solution. The mixture was stirred at 60 rpm in a thermostatic stirrer at 22°C for 2 hours, and the reaction was stopped. After filtration with activated carbon, the reaction solution was desalted and purified using an AKTA G-25 gel column (HiPrep 26 / 10 Desalting column, 3 mL; elution phase: 30 mM histidine-HCl, pH 5.5). The solution was then concentrated by ultrafiltration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-5 (30 mM histidine-HCl, pH 5.5; 66.5 mg, 25 mg / mL, yield: 95%), which was stored at -80°C.

[0457] The DAR value detected and calculated by HIC DAR analysis was n=6.05; the purity of the main peak by SEC was 98.9%.

[0458] ADC-6(DBXT001-01-X2-DAR8)

[0459] To the buffer solution of antibody DBXT001-01 (PBS pH 7.2; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM EDTA solution (0.875 mL) and prepared tris(2-carbonylethyl)phosphine hydrochloride solution (3.48 mM, 1.12 mL, 3.89 μmol) were added. The mixture was placed in a thermostatic stirrer at 60 rpm and reacted at 22°C for 2 hours, and the reaction was stopped.

[0460] X2 (5.49 mg, 5.17 μmol) was dissolved in 1.75 mL of DMA and added to the above solution. The mixture was placed in a thermostatic stirrer at 60 rpm and shaken at 22°C for 2 hours to stop the reaction. After filtration with activated carbon, the reaction solution was desalted and purified using an AKTA G-25 gel column (HiPrep 26 / 10 Desalting column, 53 mL; elution phase: 30 mM histidine-HCl, pH 5.5). The solution was then concentrated by ultrafiltration using a 30KD ultrafiltration tube to obtain a solution of the exemplary product ADC-6 (30 mM histidine-HCl, pH 5.5; 67.9 mg, 29.2 mg / mL, yield: 97%), which was stored at -80°C.

[0461] The DAR value detected and calculated by HIC DAR analysis was n=7.98; the purity of the main peak by SEC was 99.0%.

[0462] ADC-13(DBXT005-01-X2,DAR6)

[0463] Antibody DBXT005-01 was reduced with 20 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 3 hours (shaker speed: 60 rpm).

[0464] After the intermediate monitoring, 5 equivalents of tris(2-carboxyethyl)phosphine (TCEP) were added and the reaction was continued on a shaker at 22° C. for 1 hour. The reaction solution was directly used for the next coupling reaction without removing the excess TCEP.

[0465] After reducing the antibody, slowly add 200 mM histidine-HCl, pH 6.0 (to a final histidine-HCl concentration of 20 mM in the coupling reaction solution), 0.1% v / v acetic acid in dimethyl sulfoxide, and a linker-cytotoxin solution (10 equivalents, 10 mM X2 solution in dimethyl sulfoxide containing 0.1% v / v acetic acid) at room temperature. The reaction system contains 3% v / v DMSO containing 0.1% v / v acetic acid. After thorough mixing, the coupling reaction solution is shaken at 22°C (60 rpm) for 1.5 hours.

[0466] After the intermediate monitoring, 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).

[0467] After the reaction is completed, the ADC solution is exchanged into a storage buffer (20 mM histidine-hydrochloric acid, pH 6.0) using a desalting column (40K). Samples are taken and sent for testing to obtain the ADC product.

[0468] The test results are as follows:

[0469] The preparation of ADC-14 (BL-B01D1, DAR8) refers to patent WO2023083381A1, as follows:

[0470] 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 the reaction was shaken at 22°C for 17.5 hours (60 rpm). The reaction solution was used directly in the next coupling reaction without removing excess TCEP.

[0471] After reducing the antibody, slowly add dimethyl sulfoxide (DMSO) and the linker-payload (10 equivalents, 10 mM Ed-04 in DMSO) at room temperature. The reaction system contains 10% v / v dimethyl sulfoxide (DMSO). After thorough mixing, the coupling reaction solution is shaken at 22°C (60 rpm) for 2 hours.

[0472] After the intermediate monitoring, linker-payload (2 equivalents, 10 mM Ed-04 in DMSO) was added and thoroughly mixed. The coupling reaction solution was shaken on a shaker at 22° C. for 1.5 hours (shaker speed: 60 rpm).

[0473] After the reaction, 200 mM histidine-hydrochloric acid, pH 5.5, was added to the reaction solution to make the histidine-hydrochloric acid concentration in the reaction solution 20 mM. The reaction solution was then filtered with a 0.22 μm PES pinhole filter. The ADC solution was then passed through a desalting column (HiPrep TM The reaction solution was exchanged with storage buffer (20 mM histidine-hydrochloric acid, pH 5.5) using a 26 / 10 desalting method. The reaction solution was concentrated using an Amicon (30K MWCO) and finally filtered through a 0.22 μm PES pinhole filter to obtain the ADC product, which was then sampled and sent for testing.

[0474] The test results are as follows:

[0475] Referring to the preparation process of DBXT001-01-X2, only the corresponding antibody clones in Table 8 were replaced to prepare ADC-1 to 3, ADC-10 and ADC-11.

[0476] With reference to the above experimental process, 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.

[0477] Example 7: In vivo efficacy evaluation of antibody drug conjugates on human esophageal cancer cell line OE-19 bearing mice with low EGFR expression and HER3 expression

[0478] To investigate the inhibitory effect of ADC-6 on tumor formation in human esophageal cancer cell lines in vivo, the antitumor efficacy of ADC-6 was evaluated after subcutaneous ectopic inoculation of OE-19 cells into xenografts in mice, and compared with that of the parental monoclonal antibody ADC.

[0479] 1. Test drugs and materials

[0480] Blank control group (control group): normal saline

[0481] ADC-1 (treatment group): Day 0: 1 mg / kg; Day 12: 3 mg / kg

[0482] ADC-2 (treatment group): Day 0: 1 mg / kg; Day 12: 3 mg / kg

[0483] ADC-6 (treatment group): Day 0: 1 mg / kg; Day 12: 3 mg / kg

[0484] 2. Preparation method: All samples were diluted with physiological saline.

[0485] 3. Experimental animals: 6-7 week old female BALB / c nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0486] 4. Test method:

[0487] OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2 mM 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.

[0488] The experimental mice were subcutaneously inoculated with 5×10 6 OE-19 cells were resuspended in 1:1 PBS and Matrigel (0.1 mL / cell), and tumor growth was observed regularly.

[0489] When the tumor grows to about 140.15 mm 3 Tumor-bearing mice were randomly divided into groups using StudyDirect™. The test article was administered intravenously (IV) starting on day 0, once on day 0 and once on day 12, for a total of two injections. The dose for the treatment group on day 0 was 1 mg / kg, and the dose for the treatment group on day 12 was 3 mg / kg. The endpoint of the study was day 25 after grouping. Tumor volume and body weight were measured twice weekly, and the data were recorded.

[0490] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0491] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the major and minor diameters of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated using T / C (%). The T / C (%) percentage value is an indicator of tumor growth inhibition, where T and C represent the average tumor volume of the treatment group and the control group, respectively, on a given day. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]× 100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0492] 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 a significant difference. The graphics software was GraphPad Prism.

[0493] Table 9. Evaluation of the antitumor efficacy of the test substances in the OE-19 cell subcutaneous xenograft tumor model

[0494] (Calculated based on tumor volume 25 days after administration)

[0495] 1. Tumor volume is expressed as mean ± standard error;

[0496] 2. Tumor growth inhibition was determined by T / C (T / C (%) = T 25 / V 25 × 100) and TGI (TGI (%) = [1-(T 25 -T0) / (V 25 -V0)]×100) reflection;

[0497] The experimental results are shown in Figure 6 and Table 9. In the OE-19 model, monoclonal antibody ADC-1 (EGFR-ADC) cannot inhibit tumor growth, while dual antibody ADC-6 (EGFR-HER3 ADC) has a better tumor inhibition effect than monoclonal antibody ADC-2 (HER3-ADC).

[0498] Experimental conclusion:

[0499] In the EGFR-ADC-resistant model, bispecific ADC has a better tumor inhibition effect than monospecific ADC.

[0500] 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

[0501] To investigate the inhibitory effect of ADC-6 on tumor formation in human non-small cell lung cancer cell lines in vivo, the antitumor efficacy of ADC-6 was evaluated after subcutaneous inoculation of NCI-H441 xenografts in mice and compared with that of the parental monoclonal antibody ADC.

[0502] 1. Test drugs and materials

[0503] Blank control group (control group): normal saline

[0504] ADC-1 (treatment group): 0.5 mg / kg

[0505] ADC-2 (treatment group): 0.5 mg / kg

[0506] ADC-6 (treatment group): 0.5 mg / kg

[0507] 2. Preparation method: All samples were diluted with physiological saline.

[0508] 3. Experimental Animals: Male NU / NU mice aged 6-7 weeks were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.

[0509] 4. Test method:

[0510] 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.

[0511] The experimental mice were subcutaneously inoculated with 5×10 6 NCI-H441 cells were resuspended in 1:1 PBS and Matrigel (0.1 mL / cell) and tumor growth was observed regularly.

[0512] When the tumor grows to about 139.12 mm 3 Tumor-bearing mice were randomly divided into groups using StudyDirect™. The test article was administered intravenously (IV) once starting on day 0, with a dose of 0.5 mg / kg for each group. The study endpoint was day 35 after grouping. Tumor volume and body weight were measured twice weekly and recorded.

[0513] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0514] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the major and minor diameters of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated using T / C (%). The T / C (%) percentage value is an indicator of tumor growth inhibition, where T and C represent the average tumor volume of the treatment group and the control group, respectively, on a given day. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0515] 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 a significant difference. The graphics software was GraphPad Prism.

[0516] Table 10. Evaluation of the antitumor efficacy of the test substances on the NCI-H441 cell subcutaneous xenograft tumor model

[0517] (Calculated based on tumor volume 35 days after administration)

[0518] 1. Tumor volume is expressed as mean ± standard error;

[0519] 2. Tumor growth inhibition was determined by T / C (T / C (%) = T 35 / V 35 × 100) and TGI (TGI (%) = [1-(T 35 -T0) / (V 35 -V0)]×100) reflection;

[0520] 3. 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).

[0521] The experimental results are shown in FIG7 and Table 10. In the NCI-H441 model, ADC-6 has a better tumor inhibition effect than ADC-2.

[0522] Experimental conclusion:

[0523] In a model with comparable EGFR and HER3 expression, dual-antibody ADC has better tumor inhibition effect than monoclonal antibody ADC.

[0524] Example 9: In vivo efficacy evaluation of antibody drug conjugates in mice bearing the human oral squamous cell carcinoma cell line CAL-27 with high EGFR expression and low HER3 expression

[0525] To investigate the inhibitory effect of ADC-6 on tumor formation in human oral squamous cell carcinoma (OCSCC) cells in vivo, the antitumor efficacy of ADC-6 was evaluated after subcutaneous inoculation of CAL-27 cells into xenografts in mice. The antitumor efficacy was then compared with that of the parental ADC and an EGFR monoclonal antibody.

[0526] 1. Test drugs and materials

[0527] Blank control group (control group): normal saline

[0528] ADC-1 (treatment group): 3 mg / kg

[0529] ADC-2 (treatment group): 3 mg / kg

[0530] ADC-6 (treatment group): 3 mg / kg

[0531] EGFR monoclonal antibody Zalutumumab (treatment group): 3 mg / kg

[0532] 2. Preparation method: All samples were diluted with physiological saline.

[0533] 3. Experimental animals: 6-7 week old female NOD / Scid mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0534] 4. Test method:

[0535] CAL-27 (CL-00599) cells were cultured in DMEM 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.

[0536] The experimental mice were subcutaneously inoculated with 1×10 7 CAL-27 cells were resuspended in 1:1 PBS and Matrigel (0.1 mL / cell) and tumor growth was observed regularly.

[0537] When the tumor grows to about 144.75 mm 3 Tumor-bearing mice were randomly divided into groups using StudyDirect™. The test article was administered intravenously (IV) starting on day 0, once on day 0 and once on day 14, for a total of two injections. The treatment group received a dose of 3 mg / kg. The endpoint was day 27 after grouping. Tumor volume and body weight were measured twice weekly and recorded.

[0538] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0539] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the major and minor diameters of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated using T / C (%). The T / C (%) percentage value is an indicator of tumor growth inhibition, where T and C represent the average tumor volume of the treatment group and the control group, respectively, on a given day. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0540] 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 a significant difference. The graphics software was GraphPad Prism.

[0541] Table 11. Evaluation of the antitumor efficacy of the test substances on the CAL-27 cell subcutaneous xenograft tumor model

[0542] (Calculated based on tumor volume 27 days after administration)

[0543] 1. Tumor volume is expressed as mean ± standard error;

[0544] 2. Tumor growth inhibition was determined by T / C (T / C (%) = T 27 / V 27 × 100) and TGI (TGI (%) = [1-(T 27 -T0) / (V 27 -V0)]×100) reflection;

[0545] 3. 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).

[0546] The experimental results are shown in FIG8 and Table 11. In the CAL-27 model, both ADC-1 and ADC-6 have significant tumor inhibition effects, and are superior to ADC-2 and EGFR monoclonal antibodies.

[0547] Experimental conclusion:

[0548] In the EGFR-high expression and HER3-low expression model that is sensitive to targeted EGFR, the bispecific ADC has a better tumor inhibition effect than EGFR monoclonal antibody or HER3 ADC, and is not inferior to EGFR ADC.

[0549] Example 10: In vivo efficacy evaluation of antibody drug conjugates with different DAR values ​​in NCI-H1975 and OE-19 tumor-bearing mice

[0550] To investigate the inhibitory effect of different DAR values ​​of DBXT001-01 conjugate-cytotoxin X2 on tumor formation in human non-small cell lung cancer cell line NCI-H1975 and human esophageal cancer cell line OE-19, the antitumor effects of different DAR values ​​of the dual-antibody conjugate toxin were evaluated after ectopic inoculation of NCI-H1975 or OE-19 into xenograft tumors on the right back of mice.

[0551] The tested drugs and their dosing schedules are shown in Tables 12 and 13.

[0552] Table 12. Administration routes, doses, and regimens in the human lung cancer NCI-H1975 subcutaneous xenograft model

[0553] Table 13. Administration routes, doses, and regimens in the subcutaneous xenograft model of human esophageal cancer OE-19 cells

[0554] 2. Preparation method: All samples were diluted with physiological saline.

[0555] 3. Experimental animals: 6-8 week old female Balb / c nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0556] 4. Test method:

[0557] NCI-H1975 (CL-00650) cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, while OE-19 (CL-00806) cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. Cells were harvested during the exponential growth phase and resuspended in PBS to an appropriate concentration for subcutaneous tumor inoculation in nude mice.

[0558] The experimental mice were subcutaneously inoculated with 5×10 6 NCI-H1975 cells were resuspended in PBS (0.1 mL / cell) and tumor growth was observed regularly.

[0559] The experimental mice were subcutaneously inoculated with 5×10 6 OE-19 cells were resuspended in 1:1 PBS and Matrigel (0.1 mL / cell), and tumor growth was observed regularly.

[0560] When the tumor grows to about 121.71 mm 3 (NCI-H1975) or 139.93mm 3 (OE-19) tumor-bearing mice were randomly divided into groups using StudyDirect™. Grouping was designated Day 0. Specific dosing schedules are shown in Tables 12 and 13. The study endpoint was Day 31 (NCI-H1975) or Day 35 (OE-19) after grouping. Tumor volume and body weight were measured twice weekly and recorded.

[0561] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0562] The formula for calculating tumor volume is: V = 0.5a × b 2, a and b represent the major and minor diameters of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated using T / C (%). The T / C (%) percentage value is an indicator of tumor growth inhibition, where T and C represent the average tumor volume of the treatment group and the control group, respectively, on a given day. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0563] 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 a significant difference. The graphics software was GraphPad Prism.

[0564] Table 14. Evaluation of the anti-tumor efficacy of the test substances on the NCI-H1975 cell subcutaneous xenograft tumor model

[0565] (Based on 18 days after administration 1 Tumor volume calculated)

[0566] Table 15. Evaluation of the antitumor efficacy of the test substances in the OE-19 cell subcutaneous xenograft tumor model

[0567] (Based on 21 days after administration 1 Tumor volume calculated)

[0568] 1. Because some mice in the NCI-H1975 blank control group reached the euthanasia criteria after 18 days, the statistical analysis data was incomplete. Therefore, 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, the statistical analysis data was incomplete. Therefore, the statistical analysis was performed on the 21st day when the number of animals was complete.

[0569] 2. Tumor volume is expressed as mean ± standard error;

[0570] 3. Tumor growth inhibition was determined by T / C (T / C (%) = T D / V D × 100) and TGI (TGI (%) = [1-(T D -T0) / (V D -V0)]×100) reflection;

[0571] 4. 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).

[0572] The experimental results are shown in Figures 9 and 10 and Tables 14 and 15. In the dosing regimen of the NCI-H1975 model, ADC-2, ADC-4, ADC-5, and ADC-6 all had tumor inhibitory effects.

[0573] Experimental conclusion:

[0574] The dual-antibody coupled coupler-cytotoxin X2 of the present invention targeting EGFR and HER3 has an anti-tumor effect, and the anti-tumor effect is enhanced as the DAR value increases.

[0575] Example 11: Comparison of the efficacy of the present antibody-drug conjugate and the control antibody conjugate in NCI-H1975 tumor-bearing mice

[0576] To compare the inhibitory effects of DBXT001-01-coupled linker-cytotoxin X2 and the control antibody SI-1X6.4-coupled linker-cytotoxin X2 on tumor formation in the human non-small cell lung cancer cell line NCI-H1975 in vivo, the antitumor effects of both were evaluated after NCI-H1975 xenografts were formed by subcutaneous inoculation of the NCI-H1975 cells on the right back of mice.

[0577] The tested drugs and their dosing schedules are shown in Table 16.

[0578] Table 16. Administration routes, doses, and regimens in the human lung cancer NCI-H1975 subcutaneous xenograft model

[0579] a. Molar conversion was performed according to molecular weight to ensure consistent molar concentrations among treatment groups.

[0580] 1. Preparation method: All samples were diluted with physiological saline.

[0581] 2. Experimental animals: 6-8 week old female Balb / c nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0582] 3. Test method:

[0583] 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.

[0584] The experimental mice were subcutaneously inoculated with 5×10 6NCI-H1975 cells were resuspended in 1:1 DPBS:Matrigel (0.2 mL / cell). Tumor growth was observed regularly.

[0585] When the tumor grows to about 164 mm 3 (NCI-H1975) tumor-bearing mice were randomly divided into groups using StudyDirector™. The day of grouping was designated Day 0. The specific dosing schedule is shown in Table 16. The study endpoint was Day 26 after grouping. Tumor volume and body weight were measured twice weekly and the data were recorded.

[0586] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0587] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the major and minor diameters of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated using T / C (%). The T / C (%) percentage value is an indicator of tumor growth inhibition, where T and C represent the average tumor volume of the treatment group and the control group, respectively, on a given day. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0588] 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 a significant difference. The graphics software was GraphPad Prism.

[0589] Table 17. Evaluation of the anti-tumor efficacy of the test substances on the NCI-H1975 cell subcutaneous xenograft tumor model

[0590] (Based on 17 days after administration 1 Tumor volume calculated)

[0591] 1. Because some mice in the NCI-H1975 blank control group reached the euthanasia criteria after 17 days, the statistical analysis data was incomplete. Therefore, the statistical analysis was performed on day 17 when the number of animals was complete.

[0592] 2. Tumor volume is expressed as mean ± standard error;

[0593] 3. Tumor growth inhibition was determined by T / C (T / C (%) = T 17 / V 17 × 100) and TGI (TGI (%) = [1-(T 17 -T0) / (V 17 -V0)]×100) reflection;

[0594] 4. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant)

[0595] The experimental results are shown in Figure 11 and Table 17. In the NCI-H1975 model, ADC-3, ADC-6, and ADC-10 all had significant tumor-suppressing effects in the dosing regimen. ADC-6 showed the strongest tumor-suppressing effect, and ADC-3 and ADC-10 had comparable efficacy.

[0596] Experimental conclusion:

[0597] The dual-antibody coupled linker-cytotoxin X2 (DAR8) of the present invention targeting EGFR and HER3 has a stronger tumor inhibition effect than the control antibody SI-1X6.4 coupled linker-cytotoxin X2 (DAR8).

[0598] Example 12: Comparison of the efficacy of the present antibody-drug conjugate and the control antibody conjugate in OE-19 tumor-bearing mice

[0599] To compare the inhibitory effects of DBXT001-01-conjugated linker-cytotoxin X2 or linker-cytotoxin X1 with those of the control antibody SI-1X6.4-conjugated linker-cytotoxin X2 on tumor formation in the human esophageal cancer cell line OE-19 in vivo, the antitumor effects in vivo were evaluated after subcutaneous inoculation of OE-19 into xenografts on the right back of mice.

[0600] The tested drugs and their dosing schedules are shown in Table 18.

[0601] Table 18. Administration routes, doses, and regimens in the human esophageal cancer OE-19 subcutaneous xenograft model

[0602] a. Molar conversion was performed according to molecular weight to ensure consistent molar concentrations among treatment groups.

[0603] 1. Preparation method: All samples were diluted with physiological saline.

[0604] 2. Experimental animals: 6-8 week old female Balb / c nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0605] 3. Test method:

[0606] OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2 mM L-Glutamine.

[0607] The experimental mice were subcutaneously inoculated with 5×10 6 OE-19 cells were resuspended in 1:1 PBS and Matrigel (0.1 mL / cell), and tumor growth was observed regularly.

[0608] When the tumor grows to about 138 mm 3 Tumor-bearing mice were randomly divided into groups using StudyDirect™. The day of grouping was designated Day 0. The specific dosing schedule is shown in Table 18. The experimental endpoint was Day 21 after grouping. Tumor volume and body weight were measured twice a week and the data were recorded.

[0609] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0610] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the major and minor diameters of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated using T / C (%). The T / C (%) percentage value is an indicator of tumor growth inhibition, where T and C represent the average tumor volume of the treatment group and the control group, respectively, on a given day. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0611] 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 a significant difference. The graphics software was GraphPad Prism.

[0612] Table 19. Evaluation of the anti-tumor efficacy of the test substances in the OE-19 cell subcutaneous xenograft tumor model

[0613] (Calculated based on tumor volume 21 days after administration)

[0614] 1. Tumor volume is expressed as mean ± standard error;

[0615] 2. Tumor growth inhibition was determined by T / C (T / C (%) = T 21 / V 21 × 100) and TGI (TGI (%) = [1-(T 21 -T0) / (V 21 -V0)]×100) reflects.

[0616] 3. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant)

[0617] The experimental results are shown in Figure 12 and Table 19. In the dosing regimen of the OE-19 model, ADC-3, ADC-6, ADC-9, and ADC-10 all had tumor inhibitory effects, among which ADC-6 showed the strongest tumor inhibitory effect. ADC-3, ADC-9, and ADC-10 had comparable efficacy.

[0618] Experimental conclusion:

[0619] In a model of EGFR-targeted therapy resistance, the dual-antibody conjugate linker-cytotoxin X2 (DAR8) of the present invention targeting EGFR and HER3 can overcome EGFR resistance and has a stronger tumor inhibitory effect than the control antibody SI-1X6.4 conjugate linker-cytotoxin X2 (DAR8).

[0620] Example 13: In vivo efficacy evaluation of antibody drug conjugate linker-cytotoxin X1 in NCI-H1975 tumor-bearing mice

[0621] To investigate the inhibitory effect of DBXT001-01 conjugate with cytotoxin X1 (DAR8) on tumor formation in the human non-small cell lung cancer cell line NCI-H1975, the anti-tumor efficacy of this antibody conjugate was evaluated after subcutaneous inoculation of NCI-H1975 xenografts on the right back of mice.

[0622] The tested drugs and their dosing schedules are shown in Table 20.

[0623] Table 20. Administration routes, doses, and regimens in the human lung cancer NCI-H1975 subcutaneous xenograft model

[0624] 2. Preparation method: All samples were diluted with physiological saline.

[0625] 3. Experimental animals: NCI-H1975: 6-8 week old female CB-17 SCID rats, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.;

[0626] 4. Test method:

[0627] NCI-H1975 cells (cell bank of the Chinese Academy of Sciences) were cultured in RPMI1640 medium containing 10% fetal bovine serum. Cells in the exponential growth phase were collected and resuspended in DPBS to a suitable concentration for subcutaneous tumor inoculation in nude mice.

[0628] The experimental mice were subcutaneously inoculated with 5×10 6 NCI-H1975 cells (resuspend the cells in DPBS and mix with BD Matrigel at a ratio of 1:1, Catalogue: 356234, 0.2 mL / cell), and observe the tumor growth regularly.

[0629] When the tumor grows to about 201.31 mm 3 Tumor-bearing mice were randomly divided into groups using StudyDirector™. The day of grouping was designated Day 0. The specific dosing schedule is shown in Table 20. The experimental endpoint was Day 20 after grouping. Tumor volume and body weight were measured twice a week and the data were recorded.

[0630] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0631] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the major and minor diameters of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated using T / C (%). The T / C (%) percentage value is an indicator of tumor growth inhibition, where T and C represent the average tumor volume of the treatment group and the control group, respectively, on a given day. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0632] 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 a significant difference. The graphics software was GraphPad Prism.

[0633] Table 21. Evaluation of the antitumor efficacy of the test substances on the NCI-H1975 cell subcutaneous xenograft tumor model

[0634] (Based on 18 days after administration 1 Tumor volume calculated)

[0635] 1. Because some mice in the NCI-H1975 blank control group reached the euthanasia criteria after 18 days, the statistical analysis data was incomplete. Therefore, the statistical analysis was performed on the 18th day when the number of animals was complete.

[0636] 2. Tumor volume is expressed as mean ± standard error;

[0637] 3. Tumor growth inhibition was determined by T / C (T / C (%) = T 18 / V 18 × 100) and TGI (TGI (%) = [1-(T 18 -T0) / (V 18 -V0)]×100) reflection;

[0638] 4. 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).

[0639] The experimental results are shown in FIG13 and Table 21. In the NCI-H1975 model, ADC-9 had a significant tumor inhibitory effect in the dosing regimen, while the blank control group and ADC-12 had no tumor inhibitory effect.

[0640] Experimental conclusion:

[0641] The antibody-drug conjugate of the present invention comprising a bispecific antibody conjugate and a cytotoxin X1 targeting EGFR and HER3 has a significant effect of inhibiting tumor growth.

[0642] Example 14 Comparison of the efficacy of the bispecific ADC of the present invention and the control ADC in a mouse model resistant to EGFR ADC

[0643] To compare the in vivo efficacy of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) in an EGFR ADC-resistant model, the anti-tumor effects of the two ADCs were compared after subcutaneous inoculation of OE-19 xenografts in mice.

[0644] 1. The test drugs and their dosing regimens are shown in Table 22.

[0645] Table 22. Administration routes, doses, and regimens in the human esophageal cancer OE-19 subcutaneous xenograft model

[0646] 2. Preparation method: All samples were diluted with physiological saline.

[0647] 3. Experimental animals: 6-7 week old female BALB / c nude mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0648] 4. Test method:

[0649] OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2 mM 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.

[0650] The experimental mice were subcutaneously inoculated with 5×10 6 OE-19 cells were resuspended in 1:1 PBS and Matrigel (0.1 mL / cell), and tumor growth was observed regularly.

[0651] When the tumor grows to about 151 mm 3 Tumor-bearing mice were randomly divided into groups using StudyDirect™. The test article was injected 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. Tumor volume and body weight were measured twice a week and the data were recorded.

[0652] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0653] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the major and minor diameters of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated using T / C (%). The T / C (%) percentage value is an indicator of tumor growth inhibition, where T and C represent the average tumor volume of the treatment group and the control group, respectively, on a given day. The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i -T0) / (V i -V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0654] One-way ANOVA was used to compare the sample groups, and P < 0.05 was considered significant. GraphPad Prism was used for graphing and statistical analysis.

[0655] Table 23. Evaluation of the antitumor efficacy of the test substances in the OE-19 cell subcutaneous xenograft tumor model

[0656] (Calculated based on tumor volume 23 days after administration)

[0657] 1. Tumor volume is expressed as mean ± standard error;

[0658] 2. Tumor growth inhibition was determined by T / C (T / C (%) = T 23 / V 23 × 100) and TGI (TGI (%) = [1-(T 23 -T0) / (V 23 -V0)]×100) reflection;

[0659] 3. 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).

[0660] The experimental results are shown in FIG14 and Table 23. In the EGFR ADC resistance model, the antibody conjugate of the present invention has a better tumor inhibition effect than the control ADC-14.

[0661] 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

[0662] To compare the inhibitory effects of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) on tumor formation in EGFR-overexpressing and HER3-underexpressing cell lines in vivo, the anti-tumor effects of the two ADCs were compared after subcutaneous inoculation of A431 xenografts in mice.

[0663] 1. The test drugs and dosing regimens are shown in Table 244.

[0664] Table 24. Administration routes, doses, and regimens in the human skin cancer A431 subcutaneous xenograft model

[0665] 2. Preparation method: All samples were prepared by diluting with auxiliary solvents.

[0666] 3. Experimental animals: Female NCG mice aged 6-8 weeks were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0667] 4. Test method:

[0668] A431 (CRL-1555) cells were cultured in DMEM supplemented with 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.

[0669] The experimental mice were subcutaneously inoculated with 5×10 6 A431 cells, and tumor growth was observed regularly.

[0670] When the tumor grows to about 137 mm 3 The tumor-bearing mice were randomly divided into groups and the test article was injected intravenously (iv) once 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.

[0671] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0672] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated by TGI (%). The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i / T0) / (V i / V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0673] One-way ANOVA was used to compare the sample groups, and P < 0.05 was considered significant. GraphPad Prism was used for graphing and statistical analysis.

[0674] Table 25. Evaluation of the anti-tumor efficacy of the test substances on the A431 cell subcutaneous xenograft tumor model

[0675] (Calculated based on tumor volume 21 days after administration)

[0676] 1. Tumor volume is expressed as mean ± standard error;

[0677] 2. Tumor growth inhibition was determined by T / C (T / C (%) = T 21 / V 21 × 100) and TGI (TGI (%) = [1-(T 21 / T0) / (V 21 / V0)]×100) reflection;

[0678] 3. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant). Comparison of tumor volume between each experimental group and the blank control group.

[0679] 4. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant). Comparison of tumor volume between the dual-antibody ADC and control ADC experimental groups in this example.

[0680] The experimental results, as shown in Figure 15 and Table 25, show that in a tumor model with high EGFR expression and low HER3 expression, at the same dosage, the antibody conjugate of the present invention exhibited superior tumor inhibition compared to the control ADC. The control ADC failed to inhibit tumor growth at this dosage concentration. Mice in all dosing groups tolerated the treatment well, with no significant weight loss.

[0681] Example 16 Comparison of the efficacy of the dual-antibody ADC of this example and the control ADC in a mouse model of human colon cancer cell line xenograft tumors that do not express EGFR but only express HER3

[0682] To compare the inhibitory effects of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) on in vivo tumor formation in human colon cancer cell lines that do not express EGFR but only express HER3, the anti-tumor effects of the two drugs were compared after subcutaneous inoculation of SW620 cells into xenografts in mice.

[0683] 1. The test drugs and dosing regimens are shown in Table 26.

[0684] Table 26. Administration routes, doses, and regimens in the human colon cancer SW620 subcutaneous xenograft model

[0685] 2. Preparation method: All samples were prepared by diluting with auxiliary solvents.

[0686] 3. Experimental animals: Female NCG mice aged 6-8 weeks were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0687] 4. Test method:

[0688] SW620 (CCL-227) cells were cultured in DMEM supplemented with 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.

[0689] The experimental mice were subcutaneously inoculated with 5×10 6 SW620 cells, and tumor growth was observed regularly.

[0690] When the tumor grows to about 131 mm 3The tumor-bearing mice were randomly divided into groups and the test article was injected intravenously (iv) starting on the same day (day 0). The end time 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.

[0691] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0692] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated by TGI (%). The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i / T0) / (V i / V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0693] One-way ANOVA was used to compare the sample groups, and P < 0.05 was considered significant. GraphPad Prism was used for graphing and statistical analysis.

[0694] Table 27. Evaluation of the antitumor efficacy of the test substances on the SW620 cell subcutaneous xenograft tumor model

[0695] 1. Tumor volume is expressed as mean ± standard error;

[0696] 2. Tumor growth inhibition was determined by TGI (TGI (%) = [1-(T 31 / T0) / (V 31 / V0)]×100) reflection;

[0697] 3. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant). Comparison of tumor volume between each experimental group and the blank control group.

[0698] 4. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant). Comparison of tumor volume between the dual-antibody ADC and control ADC experimental groups in this example.

[0699] 5.N / A indicates that mice in the control group were euthanized on day 31 in accordance with animal welfare principles.

[0700] The experimental results, as shown in Figure 16 and Table 27, show that in a tumor model in which EGFR is absent but HER3 is expressed, all experimental groups demonstrated significant tumor growth inhibition compared to the blank control group. At the same dose, the antibody conjugates of the present invention demonstrated superior tumor inhibition compared to the control ADC, achieving complete tumor regression that persisted up to day 59. The control ADC failed to achieve complete tumor regression and failed to inhibit tumor regrowth. Mice in all treatment groups tolerated the drug well, with no significant weight loss.

[0701] Example 17 Comparison of the efficacy of the bispecific ADC of the present invention and the control ADC in a mouse model of human colon cancer cell line xenograft tumors expressing EGFR and low expression of HER3

[0702] To compare the inhibitory effects of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) on in vivo tumor formation in human colon cancer cell lines expressing EGFR and low HER3 expression, the anti-tumor effects of the two were compared after subcutaneous inoculation of SW48 cells into xenografts in mice.

[0703] 1. The test drugs and dosing regimens are shown in Table 28.

[0704] Table 28. Administration routes, doses, and regimens in the human colon cancer SW48 subcutaneous xenograft model

[0705] 2. Preparation method: All samples were prepared by diluting with auxiliary solvents.

[0706] 3. Experimental animals: Female NCG mice aged 6-8 weeks were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0707] 4. Test method:

[0708] SW48 (CCL-231) cells were cultured in DMEM supplemented with 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.

[0709] The experimental mice were subcutaneously inoculated with 1×10 7 SW48 cells, and tumor growth was observed regularly.

[0710] When the tumor grows to about 131 mm 3 The tumor-bearing mice were randomly divided into groups and the test article was injected 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.

[0711] There were 5 mice in each control group or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0712] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated by TGI (%). The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i / T0) / (V i / V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0713] One-way ANOVA was used to compare the sample groups, and P < 0.05 was considered significant. GraphPad Prism was used for graphing and statistical analysis.

[0714] Table 29. Evaluation of the antitumor efficacy of the test substances on the SW48 cell subcutaneous xenograft tumor model

[0715] 1. Tumor volume is expressed as mean ± standard error;

[0716] 2. Tumor growth inhibition was determined by TGI (TGI (%) = [1-(T 21 -T0) / (V 21 -V0)]×100) reflection;

[0717] 3. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant). Comparison of tumor volume between each experimental group and the blank control group.

[0718] 4. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant). Comparison of tumor volume between the dual-antibody ADC and control ADC experimental groups in this example.

[0719] 5.N / A indicates that mice in the control group were euthanized on day 21 in accordance with animal welfare principles.

[0720] The experimental results, as shown in Figure 17 and Table 29, show that in a tumor model with EGFR expression and low HER3 expression, all experimental groups demonstrated significant tumor growth inhibition compared to the blank control group. At the same dose, the antibody conjugates of the present invention demonstrated superior tumor inhibition compared to the control ADC, achieving complete tumor regression in some mice and sustained tumor growth inhibition up to day 45. The control ADC failed to achieve complete tumor regression and failed to inhibit tumor recurrence. Mice in all treatment groups tolerated the treatment well, with no significant weight loss.

[0721] Example 18: Comparison of the efficacy of the bispecific ADC of the present invention and the control ADC in an osimertinib-resistant human non-small cell lung cancer cell line xenograft mouse model

[0722] To compare the inhibitory effects of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) on tumor formation in osimertinib-resistant non-small cell lung cancer cell lines in vivo, the anti-tumor effects of the two drugs were compared after subcutaneous inoculation of NCI-H1975 (EGFR triple mutation L858R / T790M / C797S) xenografts in mice.

[0723] 1. The test drugs and dosing regimens are shown in Table 30.

[0724] Table 30. Administration route, dose and regimen in the subcutaneous xenograft model of human non-small cell lung cancer NCI-H1975 (EGFR triple mutation L858R / T790M / C797S)

[0725] 2. Preparation method: All samples were prepared by diluting with auxiliary solvents.

[0726] 3. Experimental animals: 6-7 week old female NOD / SCID mice were purchased from Jicui Yaokang Biotechnology Co., Ltd.

[0727] 4. Test method:

[0728] NCI-H1975 EGFR L858R / T790M / C797S (CL-01195) cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum and 100 μg / mL hygromycin. Exponentially growing NCI-H1975 cells were harvested and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in NOD / SCID mice. Mice were inoculated subcutaneously with 1×10 7 NCI-H1975 EGFR L858R / T790M / C797S cells were resuspended in a 1:1 ratio of PBS to Matrigel (0.2 mL / cell) and tumor growth was observed regularly.

[0729] When the tumor grows to about 169.46 mm 3 The tumor-bearing mice were randomly divided into groups and the test article was injected 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.

[0730] There were 6 mice in each control or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume.

[0731] The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The anti-tumor efficacy of the compound was evaluated by TGI (%). The tumor growth inhibition rate was calculated using the following formula: TGI (%) = [1-(T i / T0) / (V i / V0)]×100, where T i V is the average tumor volume of a certain medication group on a certain day, and T0 is the average tumor volume of this medication group at the beginning of medication; i For one day (with T i V0 is the average tumor volume of the vehicle control group at the beginning of drug administration.

[0732] One-way ANOVA was used to compare the sample groups, and P < 0.05 was considered significant. GraphPad Prism was used for graphing and statistical analysis.

[0733] Table 31. Evaluation of the anti-tumor efficacy of the test substances in the NCI-H1975 EGFR L858R / T790M / C797S cell subcutaneous xenograft tumor model

[0734] (Calculated based on tumor volume 35 days after administration)

[0735] 1. Tumor volume is expressed as mean ± standard error;

[0736] 2. Tumor growth inhibition was determined by TGI (TGI (%) = [1-(T 35 / T0) / (V 35 / V0)]×100) reflection;

[0737] 3. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant). Comparison of tumor volume between each experimental group and the blank control group.

[0738] 4. P values ​​were calculated based on tumor volume (* represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, ns represents not significant). Comparison of tumor volume between the dual-antibody ADC and control ADC experimental groups in this example.

[0739] The experimental results are shown in Figure 18 and Table 31. In a non-small cell lung cancer tumor model resistant to the third-generation TKI osimertinib, all experimental groups showed significant tumor growth inhibition compared with the blank control group. At the same dosage, the antibody conjugate of the present invention had a better tumor inhibition effect than the control ADC. The mice in each drug-treated group tolerated the drug well and did not experience significant weight loss.

[0740] sequence

[0741] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A bispecific antibody comprising an EGFR binding domain and a HER3 binding domain, characterized in that: 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 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, respectively; the amino acid sequences of L2CDR1, L2CDR2 and L2CDR3 contained in the VL2 are shown as SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively.

2. The bispecific antibody according to claim 1, characterized in that The amino acid sequence of the H2CDR2 is shown in SEQ ID NO: 77 or 78; Preferably, the amino acid sequences of the framework regions H1FR1, H1FR2, H1FR3 and H1FR4 comprised by the VH1 are as 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; The amino acid sequences of the framework regions L1FR1, L1FR2, L1FR3 and H1FR4 comprised in the VL1 are shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, or are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; The VH2 comprises a framework region H2FR1 having an amino acid sequence as shown in SEQ ID NO:21 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, for example, having an E16D mutation on SEQ ID NO:21; the framework regions H2FR2 and H2FR4 having amino acid sequences as shown in SEQ ID NO:22 and SEQ ID NO:24, respectively, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the framework region H2FR3 having an amino acid sequence as shown in SEQ ID NO:23 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, for example, having an S18D mutation on SEQ ID NO:23; The amino acid sequence of the framework region L2FR1 comprised by 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, having S9D and V15L mutations, or S7E mutations 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.

3. The bispecific antibody according to claim 1 or 2, characterized in that: The amino acid sequence of the VH1 is shown in SEQ ID NO:28, the amino acid sequence of the VL1 is shown in SEQ ID NO:29, the amino acid sequence of the VH2 is shown in SEQ ID NO:30, SEQ ID NO:79 or SEQ ID NO:80, and the amino acid sequence of the VL2 is shown in SEQ ID NO:31, SEQ ID NO:81 or SEQ ID NO:82; Preferably, the amino acid sequences of VH1, VL1, VH2 and VL2 of the bispecific antibody are as shown in SEQ ID NO: 28, As shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31; 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.

4. The bispecific antibody according to any one of claims 1 to 3, characterized in that 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 includes a light chain constant region CL1 and a heavy chain constant region HC1, and the HER3 binding domain includes 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 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the amino acid sequences of CL1 and CL2 are not the same sequence; and / or, HC1 includes C1H1 and Fc1, and HC2 includes 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 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and the amino acid sequences of C1H1 and C2H1 are not identical sequences; the amino acid sequences of Fc1 and Fc2 are variant sequences of the amino acid sequence as shown in SEQ ID NO:36 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, for example, having T146W, or S134C and T146W, or T146S, L148A and Y187V, or Y349C, T366S, L368A and Y407V mutations in SEQ ID NO:36, and the amino acid sequences of Fc1 and Fc2 are not identical sequences; Preferably, the amino acid sequences of CL1 and CL2 are shown in SEQ ID NO:32 or SEQ ID NO:33, respectively, the amino acid sequences of C1H1 and C2H1 are shown in SEQ ID NO:34 or SEQ ID NO:35, respectively, 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 in SEQ ID NO:36; More preferably, 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; Further more preferably, 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.

5. The bispecific antibody according to any one of claims 1 to 3, characterized in that 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 heavy chain constant region HC2; wherein 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; and / or, HC1 comprises C1H1 and Fc1, and HC2 comprises Fc2; wherein the amino acid sequence of C1H1 is as shown in SEQ ID NO:34 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; the amino acid sequences of Fc1 and Fc2 are as shown in SEQ ID NO: A variant sequence of the amino acid sequence shown in NO:36 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; Preferably, the amino acid sequence of CL1 is as shown in SEQ ID NO:32, the amino acid sequence of C1H1 is as shown in SEQ ID NO:34, and the amino acid sequences of Fc1 and Fc2 are as shown in SEQ ID NO:93 and 94, respectively; More preferably, Fc1 and Fc2 are connected via a disulfide bond and a Knob into Hole structure in the hinge region, and the Fc1 is a knob- Fc, wherein Fc2 is hole-Fc, or Fc2 is knob-Fc, and Fc1 is hole-Fc; Further more preferably, C1H1 and Fc1 are connected by a hinge region with an amino acid sequence as shown in SEQ ID NO:89; VL2 and VH2 are connected by a hinge region with an amino acid sequence as shown in SEQ ID NO:95; and VH2 and Fc2 are connected by a hinge region with an amino acid sequence as shown in SEQ ID NO:

96.

6. The bispecific antibody according to any one of claims 1 to 3 or 5, characterized in that The bispecific antibody comprises a heavy chain H1, a light chain L1 and a heavy chain H2, wherein the amino acid sequence of H1 is as shown in SEQ ID NO: 37, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or the amino acid sequence of L1 is as shown in SEQ ID NO: 38, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or the 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; Preferably, the amino acid sequences of the heavy chain H1, light chain L1 and heavy chain H2 are shown as SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 90, respectively.

7. The bispecific antibody according to any one of claims 1 to 4, characterized in that The 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; or, As shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:42 and SEQ ID NO:40, respectively; or, As shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:44 and SEQ ID NO:40, respectively; or, As shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:46 and SEQ ID NO:40, respectively; or, As shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49 and SEQ ID NO:50, respectively; or, As shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:50, respectively; or, As shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:54 and SEQ ID NO:50, respectively; or, As shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:56 and SEQ ID NO:50, respectively; or, As shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:57 and SEQ ID NO:58, respectively; or, As shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:59 and SEQ ID NO:58, respectively; or, As shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:60 and SEQ ID NO:58, respectively; or, As shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:61 and SEQ ID NO:58, respectively; or, As shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:62 and SEQ ID NO:63, respectively; or, As shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:64 and SEQ ID NO:63, respectively; or, As shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:65 and SEQ ID NO:63, respectively; or, As shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:66 and SEQ ID NO:63, respectively; or, As shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:57 and SEQ ID NO:40, respectively; or, As shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:59 and SEQ ID NO:40, respectively; or, As shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:60 and SEQ ID NO:40, respectively; or, As shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:61 and SEQ ID NO:40, respectively; or, As shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:62 and SEQ ID NO:50, respectively; or, As shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:64 and SEQ ID NO:50, respectively; or, As shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:65 and SEQ ID NO:50, respectively; or, As shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:66 and SEQ ID NO:50, respectively; or, As shown in SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:67 and SEQ ID NO:68, respectively; or, As shown in SEQ ID NO:41, SEQ ID NO:38, SEQ ID NO:69 and SEQ ID NO:68, respectively; or, As shown in SEQ ID NO:43, SEQ ID NO:38, SEQ ID NO:70 and SEQ ID NO:68, respectively; or, As shown in SEQ ID NO:45, SEQ ID NO:38, SEQ ID NO:71 and SEQ ID NO:68, respectively; or, As shown in SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:72 and SEQ ID NO:73, respectively; or, As shown in SEQ ID NO:51, SEQ ID NO:48, SEQ ID NO:74 and SEQ ID NO:73, respectively; or, As shown in SEQ ID NO:53, SEQ ID NO:48, SEQ ID NO:75 and SEQ ID NO:73, respectively; or, As shown in SEQ ID NO:55, SEQ ID NO:48, SEQ ID NO:76 and SEQ ID NO:73 respectively.

8. An isolated nucleic acid, characterized in that The nucleic acid encodes the bispecific antibody according to any one of claims 1 to 7.

9. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 8.

10. A transformant, characterized in that: The transformant comprises the nucleic acid of claim 8 or the recombinant expression vector of claim 9 in a host cell; Preferably, the host cell is a eukaryotic cell, preferably a mammalian cell, such as a CHO cell.

11. A method for preparing the bispecific antibody according to any one of claims 1 to 7, the method comprising culturing the transformant according to claim 10 to obtain the bispecific antibody.

12. A bispecific antibody drug conjugate, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, characterized in that: The bispecific antibody-drug conjugate comprises the following fragments: the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, a linker unit L, and a cytotoxic drug.

13. The bispecific antibody drug conjugate of claim 12, its tautomer, enantiomer, diastereomer, or isomer mixture, or a pharmaceutically acceptable salt thereof, wherein: The cytotoxic drug is camptothecin and its derivatives; Preferably, the cytotoxic drug is a structure as shown in formula (A-1), or a tautomer, enantiomer, or diastereomer thereof, in, M is -L 2 -L 1 -C(O)-; L 2 is -NH-, O or S, preferably -O- or -S-, more preferably -O-, and L 2 Connected to the connector unit L; L 1 -(C(R 1a )(R 1b )) m -CH2-, C3-C6 saturated cycloalkylene or 3-6 membered saturated heterocyclylene, wherein the C3-C6 saturated cycloalkylene and 3-6 membered saturated heterocyclylene are each independently optionally substituted by one or more R 2a replace; m 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; Each R 1a and R 1b are independently hydrogen, halogen, hydroxy, amino or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens; R 2a is selected from halogen, hydroxy, amino and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens.

14. The bispecific antibody drug conjugate of claim 13, its tautomer, enantiomer, diastereomer, or isomer mixture, or a pharmaceutically acceptable salt thereof, wherein: L 1 -(C(R 1a )(R 1b )) m -CH2-; each R 1a Selected from: hydrogen, halogen and C1-C6 alkyl; each R 1b Selected from: hydrogen, halogen and C1-C6 alkyl; or, L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a is a C1-C6 alkyl group, preferably a C1-C3 alkyl group; R 1b is selected from: hydrogen and C1-C6 alkyl, preferably selected from: hydrogen and C1-C3 alkyl; or, L 1 -(C(R 1a )(R 1b )) m -CH2-; R 1a is -CH3; R 1b Selected from: hydrogen and -CH3; or, L 1 is a C3-C6 saturated cycloalkylene or a 3-6-membered saturated heterocyclylene, preferably a C3-C6 saturated cycloalkylene, wherein the C3-C6 saturated cycloalkylene and the 3-6-membered saturated heterocyclylene are each independently optionally substituted by one or more R 2a Replace, each R 2a are independently halogen or C1-C6 alkyl; or, L 1 is optionally replaced by one or more R 2a Substituted: cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl; R 2a Each independently selected from: halogen and C1-C6 alkyl; or, L 1 for or, L 1 for 15. The bispecific antibody drug conjugate of claim 14, its tautomer, enantiomer, diastereomer, or isomer mixture, or a pharmaceutically acceptable salt thereof, wherein: In the structure shown in formula (A-1), M is -L 2 -L 1 -C(O)-; L 2 for -O-; L 1 -(C(R 1a )(R 1b )) m -CH2- or C3-C6 saturated cycloalkylene, the C3-C6 saturated cycloalkylene is optionally substituted by one or more R 2a replace; m is selected from 1 or 2; R 1a and R 1b Each is independently selected from hydrogen, halogen and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens; R 2a is selected from halogen and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens.

16. The bispecific antibody drug conjugate according to any one of claims 13 to 15, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, characterized in that: M is: Preferably 17. The bispecific antibody drug conjugate of any one of claims 12 to 16, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, wherein the cytotoxic drug is selected from any of the following structures:

18. The bispecific antibody drug conjugate according to any one of claims 12 to 17, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, characterized in that: The linker unit L is -L a -L b -L c -; where L c connected to the cytotoxic drug; -L a -for or -C 1-8 Alkylene-C(O)-, preferably or -C 1-6 Alkylene-C(O)-, more preferably Further optimization The a-end is connected to Ab, and the b-end is connected to L b connected; -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 More preferably The c-terminal and L a Connected, d end and L c connected; -L c - for C 1-6 Alkylene, preferably C 1-3 Alkylene, more preferably 19. The bispecific antibody drug conjugate of claim 18, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, wherein the linker unit L is Preferably 20. The bispecific antibody drug conjugate according to any one of claims 12 to 19, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the bispecific antibody drug conjugate is shown in formula (A-2): Wherein, 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 one of claims 1 to 7; M is defined as the antibody drug conjugate according to any one of claims 13 to 17; L is the connector unit L according to claim 18 or 19.

21. The bispecific antibody drug conjugate according to any one of claims 12 to 20, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the bispecific antibody drug conjugate is shown in formula (A-2a) or (A-2b): in, 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 one of claims 1 to 7; L2 is -NH-, O or S, preferably -O- or -S-; more preferably -O-; X1 is selected from optionally 1, 2 or 3 R 2a Substituted C3-C6 cycloalkylene; X2 is selected from -(C(R 1a )(R 1b )) m -CH2-; m is selected from 1 or 2; R 1a and R 1b Each is independently hydrogen, halogen or C1-C6 alkyl optionally substituted by 1, 2 or 3 halogens; R 2a is selected from halogen, hydroxy, amino and C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more halogens; Preferably, the structure of the bispecific antibody drug conjugate is selected from the following: in, 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 one of claims 1 to 7; Preferably, Ab is selected from the bispecific antibody DBXT001 series, DBXT002 series, DBXT003 series and DBXT004 series; preferably selected from the bispecific antibody DBXT001 series, DBXT002 series and DBXT005-01; more preferably selected from the bispecific antibody DBXT001 series and DBXT005-01; further preferably selected from the bispecific antibody DBXT001-01 and DBXT005-01.

22. The bispecific antibody drug conjugate of claim 21, its tautomer, enantiomer, diastereomer, or isomer mixture, or a pharmaceutically acceptable salt thereof, wherein: The bispecific antibody drug conjugate is selected from any one of the following: Wherein, 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; Preferably, the bispecific antibody drug conjugate is selected from any one of the following: Among them, DBXT001-01 is a bispecific antibody against EGFR and HER3, and the amino acid sequences of its heavy chain H1, light chain L1, heavy chain H2 and light chain L2 are shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

23. The bispecific antibody drug conjugate according to any one of claims 12 to 21, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, characterized in that: The bispecific antibody drug conjugate is: Wherein, 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.

24. The bispecific antibody drug conjugate according to any one of claims 12 to 19, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, characterized in that: The bispecific antibody drug conjugate is: Wherein, 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.

25. A method for preparing the bispecific antibody drug conjugate according to any one of claims 12 to 24, its tautomer, enantiomer, diastereomer, or mixture of isomers, or a pharmaceutically acceptable salt thereof, comprising the following steps: Under the action of a reducing agent, the bispecific antibody dissolved in a buffer solution is mixed with a linker unit L-cytotoxic drug dissolved in a solvent to obtain the bispecific antibody-drug conjugate; wherein the reducing agent is preferably tris(2-carbonylethyl)phosphine hydrochloride, the buffer solution is preferably ethylenediaminetetraacetic acid, and the solvent is preferably dimethylacetamide.

26. The method of claim 25, comprising reacting a bispecific antibody against EGFR and HER3 with a compound of formula X2. For example, DBXT005-01 is reacted with a compound of formula X2.

27. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 7, the isolated nucleic acid according to claim 8, the recombinant expression vector according to claim 9, the transformant according to claim 10 and / or the bispecific antibody-drug conjugate according to any one of claims 12 to 22, and a pharmaceutically acceptable carrier or excipient.

28. Use of the bispecific antibody according to any one of claims 1 to 7, the isolated nucleic acid according to claim 8, the recombinant expression vector according to claim 9, the transformant according to claim 10, the bispecific antibody-drug conjugate according to any one of claims 12 to 24, and / or the pharmaceutical composition according to claim 27 in the preparation of a medicament for treating and / or preventing cancer, preferably, the cancer is a cancer positively expressing EGFR and / or Her3, for example, the cancer is selected from breast cancer, skin cancer, gastric cancer, colorectal cancer, Lung cancer, esophageal cancer, biliary tract 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; 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 skin squamous cell carcinoma, and the colorectal cancer is preferably rectal cancer.

29. A method for treating and / or preventing cancer, comprising administering to a subject in need thereof the bispecific antibody of any one of claims 1 to 7, the bispecific antibody-drug conjugate of any one of claims 12 to 24, and / or the pharmaceutical composition of claim 27; preferably, the cancer is a cancer that positively expresses EGFR and / or Her3, for example, the cancer is selected from breast cancer, skin cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract 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; 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 skin squamous cell carcinoma, and the colorectal cancer is preferably rectal cancer.

30. A bispecific antibody according to any one of claims 1 to 7, a bispecific antibody-drug conjugate according to any one of claims 12 to 24, and / or a pharmaceutical composition according to claim 27 for preventing and / or treating cancer; preferably, the cancer is a cancer that positively expresses EGFR and / or Her3, for example, the cancer is selected from breast cancer, skin cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract 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; 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 skin squamous cell carcinoma, and the colorectal cancer is preferably rectal cancer.

31. A combination therapy, characterized in that The method comprises administering the bispecific antibody according to any one of claims 1 to 7, the bispecific antibody-drug conjugate according to any one of claims 12 to 24, and / or the pharmaceutical composition according to claim 27, and a second therapeutic agent, respectively, to a subject in need thereof; Preferably, the cancer is a cancer that positively expresses EGFR and / or Her3, for example, the cancer 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, 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; 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 skin squamous cell carcinoma, and the colorectal cancer is preferably rectal cancer.

32. Use of the bispecific antibody according to any one of claims 1 to 7, the isolated nucleic acid according to claim 8, the recombinant expression vector according to claim 9, the transformant according to claim 10, the bispecific antibody-drug conjugate according to any one of claims 12 to 24, and / or the pharmaceutical composition according to claim 27 in the preparation of an EGFR and / or HER3 inhibitor.

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