Bispecific antibodies, their drug conjugates, and their uses

Abstract

Disclosed in this disclosure are bispecific antibodies, drug conjugates thereof, and their uses. 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, where VH1 comprises H1CDR1, H1CDR2, and H1CDR3 having the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; VL1 comprises L1CDR1, L1CDR2, and L1CDR3 having the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; VH2 comprises H2CDR1, H2CDR2, and H2CDR3 having the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and VL2 comprises L2CDR1, L2CDR2, and L2CDR3 having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. The bispecific antibodies and drug conjugates disclosed herein possess good endocytotic activity, growth inhibitory activity, tumor growth inhibitory activity, and in vivo safety.

Description

[Technical Field]

[0001] This application claims priority to China Patent Application No. 2023108956544 filed on 19 July 2023, China Patent Application No. 2024102449540 filed on 4 March 2024, and China Patent Application No. 2024109141579 filed on 9 July 2024, which are incorporated herein by reference in their entirety.

[0002] This disclosure relates to the field of biotechnology, specifically to bispecific antibodies containing an EGFR-binding domain and a HER3-binding domain, drug conjugates thereof, and their use. [Background technology]

[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 can form dimers after binding to ligands such as EGF and TNF-α, and activates downstream signaling pathways (such as MAPK, PI3K, and Stat) through phosphorylation, thereby maintaining cell proliferation and promoting cell division and growth. Because ErbB family receptors are conservative, EGFR can also form heterodimers with other proteins in the same family (e.g., HER2, HER3, and HER4), thereby regulating cell proliferation more broadly.

[0004] HER3 is a member of the ErbB family and plays a crucial role in cell proliferation, tumor metastasis, and drug resistance. While drugs targeting EGFR and HER3 have shown significant clinical efficacy in palliative care for various cancers, previous efforts to develop anti-HER3 antibodies for cancer treatment have repeatedly failed.

[0005] An antibody-drug conjugate (ADC) consists of three parts: an antibody or its antigen-binding fragment (targeting), a linker, and a small molecule drug. The antibody or its antigen-binding fragment conjugates with a bioactive small molecule drug, such as a cytotoxic cytotoxin, via a cleavable or non-cleavable linker. This utilizes the specificity of the antibody or its antigen-binding fragment in targeting cells of interest or binding to highly expressed antigens, as well as the high efficiency of the small molecule drug, thereby reducing or avoiding toxic side effects on non-target cells. In other words, antibody-drug conjugates used in tumors can precisely target tumor cells and reduce the impact on non-tumor cells compared to conventional oncological chemotherapy drugs.

[0006] There is still a need in this field for bispecific antibody-drug conjugates with superior affinity and specificity. [Overview of the project]

[0007] The technical problem addressed in this disclosure is to overcome the drawback of the relatively limited number of bispecific antibody-drug conjugates targeting EGFR and HER3 in the prior art. Therefore, this disclosure provides bispecific antibodies, their drug conjugates, and their uses. The bispecific antibody-drug conjugates of this disclosure have good endocytotic effects, growth inhibitory activity, tumor growth inhibitory activity, and in vivo safety.

[0008] This disclosure solves the aforementioned technical problems primarily through the following technical means.

[0009] To solve the technical problems described above, a first aspect of this disclosure 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, where VH1 comprises H1CDR1, H1CDR2, and H1CDR3 having the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and VL 1 comprises L1CDR1, L1CDR2, and L1CDR3 having the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; VH2 comprises H2CDR1, H2CDR2, and H2CDR3 having the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and VL2 comprises L2CDR1, L2CDR2, and L2CDR3 having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

[0010] In some embodiments of this disclosure, the amino acid sequence of H2CDR2 is shown in SEQ ID NO: 77 or 78.

[0011] In some preferred embodiments of the present disclosure, VH1 includes framework regions, H1FR1, H1FR2, H1FR3, and H1FR4, each having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to or to the sequences shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16;

[0012] VL1 includes a framework region, L1FR1, L1FR2, L1FR3, and L1FR4, each having an amino acid sequence that is either shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to them;

[0013] VH2 includes framework region H2FR1 having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in sequence number 21, for example having the E16D mutation on sequence number 21; framework regions H2FR2 and H2FR4 having amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequences shown in sequence number 22 and sequence number 24, respectively; and framework region H2FR3 having amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in sequence number 23, for example having the S18D mutation on sequence number 23;

[0014] VL2 includes framework region L2FR1, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25, for example, having the S9D mutation and the V15L mutation, or the S7E mutation on SEQ ID NO: 25; and framework regions L2FR2, L2FR3, and L2FR4, which are shown in SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 27, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with them.

[0015] In some embodiments of this disclosure, the amino acid sequence of VH1 is shown in SEQ ID NO: 28, the amino acid sequence of VL1 is shown in SEQ ID NO: 29, the amino acid sequence of VH2 is shown in SEQ ID NO: 30, SEQ ID NO: 79, or SEQ ID NO: 80, and the amino acid sequence of VL2 is shown in SEQ ID NO: 31, SEQ ID NO: 81, or SEQ ID NO: 82.

[0016] In some preferred embodiments of the present disclosure, the amino acid sequences of the bispecific antibodies VH1, VL1, VH2, and VL2 are shown in SEQ ID NOs. 28, 29, 30, and 31, respectively, or in SEQ ID NOs. 28, 29, 79, and 81, respectively, or in SEQ ID NOs. 28, 29, 79, and 31, respectively, or in SEQ ID NOs. 28, 29, 80, and 82, respectively.

[0017] In some embodiments of the present disclosure, the EGFR-binding domain and the HER3-binding domain further comprise a light chain constant region and a heavy chain constant region, respectively, wherein 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, where the amino acid sequences of CL1 and CL2 are 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 to them, 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, where the amino acid sequences of C1H1 and C2H1 are sequences The amino acid sequences of C1H1 and C2H1 are not identical to or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in sequence number 34 or sequence number 35; the amino acid sequences of Fc1 and Fc2 are variant sequences of the amino acid sequence shown in sequence number 36, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to it, for example, having the T146W mutation, or the S134C and T146W mutation, or the T146S, L148A and Y187V mutation, or the Y349C, T366S, L368A and Y407V mutation on sequence number 36, and the amino acid sequences of Fc1 and Fc2 are not identical.

[0018] In some preferred embodiments of the present disclosure, 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 a T146W mutation, or an S134C and T146W mutation, or a T146S, L148A and Y187V mutation, or a Y349C, T366S, L368A and Y407V mutation on SEQ ID NO: 36.

[0019] In some more preferred embodiments of the present disclosure, Fc1 and Fc2 are linked by disulfide bonds in a hinge region and a knob-into-hole structure, where Fc1 is knob-Fc and Fc2 is hole-Fc, or Fc2 is knob-Fc and Fc1 is hole-Fc.

[0020] In some more preferred embodiments of this disclosure, C1H1 and Fc1, and C2H1 and Fc2 are linked by a hinge region, where the amino acid sequence of the hinge region is shown in SEQ ID NO: 89.

[0021] In some preferred embodiments of the present disclosure, 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, where the amino acid sequence of CL1 is 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 Including, the amino acid sequence of C1H1 is either the sequence shown in SEQ ID NO: 34 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and the amino acid sequences of Fc1 and Fc2 are either variant sequences of the amino acid sequence shown in SEQ ID NO: 36 or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0022] In some preferred embodiments of this disclosure, 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.

[0023] In some preferred embodiments of the present disclosure, Fc1 and Fc2 are linked by disulfide bonds in a hinge region and a knob-into-hole structure, where Fc1 is knob-Fc and Fc is hole-Fc, or Fc2 is knob-Fc and Fc1 is hole-Fc.

[0024] In some preferred embodiments of the present disclosure, C1H1 and Fc1 are linked by a hinge region having the amino acid sequence shown in SEQ ID NO: 89; VL2 and VH2 are linked by a hinge region having the amino acid sequence shown in SEQ ID NO: 95; and VH2 and Fc2 are linked by a hinge region having the amino acid sequence shown in SEQ ID NO: 96.

[0025] In some preferred embodiments of the present disclosure, 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 shown in SEQ ID NO: 37 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and / or the amino acid sequence of L1 is shown in SEQ ID NO: 38 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and / or the amino acid sequence of H2 is shown in SEQ ID NO: 90 or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0026] In some preferred embodiments of this disclosure, the bispecific antibody is DBXT005-01, comprising heavy chain H1, light chain L1, and heavy chain H2 having the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 90, respectively. In some preferred embodiments of this disclosure, heavy chain H2 has an Fc+scFv structure.

[0027] In some embodiments of this disclosure, the bispecific antibody comprises a heavy chain H1, a light chain L1, a heavy chain H2, and a light chain L2.

[0028] In some preferred embodiments of this disclosure, the bispecific antibody is DBXT001-01, comprising heavy chain H1, light chain L1, heavy chain H2, and light chain L2, respectively, having the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40;

[0029] DBXT001-02, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 38, SEQ ID NO: 42, and SEQ ID NO: 40, respectively;

[0030] DBXT001-03, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NO: 43, SEQ ID NO: 38, SEQ ID NO: 44, and SEQ ID NO: 40, respectively;

[0031] DBXT001-04, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 38, SEQ ID NO: 46, and SEQ ID NO: 40, respectively;

[0032] DBXT001-05, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NOs. 47, 48, 49, and 50, respectively;

[0033] DBXT001-06, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 51, SEQ ID NO: 48, SEQ ID NO: 52, and SEQ ID NO: 50, respectively;

[0034] DBXT001-07, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 53, SEQ ID NO: 48, SEQ ID NO: 54, and SEQ ID NO: 50, respectively;

[0035] DBXT001-08, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 48, SEQ ID NO: 56, and SEQ ID NO: 50, respectively;

[0036] DBXT002-01, comprising heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 57, and SEQ ID NO: 58, respectively;

[0037] DBXT002-02, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 38, SEQ ID NO: 59, and SEQ ID NO: 58, respectively;

[0038] DBXT002-03, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 43, SEQ ID NO: 38, SEQ ID NO: 60, and SEQ ID NO: 58, respectively;

[0039] DBXT002-04, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 38, SEQ ID NO: 61, and SEQ ID NO: 58, respectively;

[0040] DBXT002-05, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NOs. 47, 48, 62, and 63, respectively;

[0041] DBXT002-06, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 51, SEQ ID NO: 48, SEQ ID NO: 64, and SEQ ID NO: 63, respectively;

[0042] DBXT002-07, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 53, SEQ ID NO: 48, SEQ ID NO: 65, and SEQ ID NO: 63, respectively;

[0043] DBXT002-08, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 48, SEQ ID NO: 66, and SEQ ID NO: 63, respectively;

[0044] DBXT003-01, comprising heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 57, and SEQ ID NO: 40, respectively;

[0045] DBXT003-02, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 38, SEQ ID NO: 59, and SEQ ID NO: 40, respectively;

[0046] DBXT003-03, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 43, SEQ ID NO: 38, SEQ ID NO: 60, and SEQ ID NO: 40, respectively;

[0047] DBXT003-04, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 38, SEQ ID NO: 61, and SEQ ID NO: 40, respectively;

[0048] DBXT003-05, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NOs. 47, 48, 62, and 50, respectively;

[0049] DBXT003-06, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 51, SEQ ID NO: 48, SEQ ID NO: 64, and SEQ ID NO: 50, respectively;

[0050] DBXT003-07, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 53, SEQ ID NO: 48, SEQ ID NO: 65, and SEQ ID NO: 50, respectively;

[0051] DBXT003-08 contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 48, SEQ ID NO: 66, and SEQ ID NO: 50, respectively;

[0052] DBXT004-01, comprising heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 67, and SEQ ID NO: 68, respectively;

[0053] DBXT004-02, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 41, SEQ ID NO: 38, SEQ ID NO: 69, and SEQ ID NO: 68, respectively;

[0054] DBXT004-03, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 43, SEQ ID NO: 38, SEQ ID NO: 70, and SEQ ID NO: 68, respectively;

[0055] DBXT004-04, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 45, SEQ ID NO: 38, SEQ ID NO: 71, and SEQ ID NO: 68, respectively;

[0056] DBXT004-05, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NOs. 47, 48, 72, and 73, respectively;

[0057] DBXT004-06, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 51, SEQ ID NO: 48, SEQ ID NO: 74, and SEQ ID NO: 73, respectively;

[0058] DBXT004-07, which contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2 having the amino acid sequences shown in SEQ ID NO: 53, SEQ ID NO: 48, SEQ ID NO: 75, and SEQ ID NO: 73, respectively;

[0059] DBXT004-08 contains heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NO: 55, SEQ ID NO: 48, SEQ ID NO: 76, and SEQ ID NO: 73, respectively.

[0060] 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.

[0061] To address the technical challenges described above, a second aspect of the present disclosure provides an isolated nucleic acid encoding a bispecific antibody according to the first aspect of the present disclosure.

[0062] To address the technical challenges described above, a third aspect of this disclosure provides a recombinant expression vector comprising a nucleic acid according to a second aspect of this disclosure.

[0063] To address the aforementioned technical challenges, a fourth aspect of the present disclosure provides a transformant comprising a host cell containing a nucleic acid according to the second aspect of the present disclosure or a recombinant expression vector according to the third aspect of the present disclosure.

[0064] In some preferred embodiments of this disclosure, the host cell is a mammalian cell such as a eukaryotic cell, preferably a CHO cell.

[0065] To solve the technical problems described above, a fifth aspect of the present disclosure provides a method for preparing a bispecific antibody according to a first aspect of the present disclosure, the method comprising culturing a transformant according to a fourth aspect of the present disclosure to obtain a bispecific antibody.

[0066] HC1 is the heavy chain steady region of the first heavy chain, and HC2 is the heavy chain steady region of the second heavy chain. VH1, C1H1, and Fc1 are the VH, CH1, and Fc regions of the first heavy chain H1, respectively; CL1 and VL1 are the CL and VL regions of the first light chain L1, respectively; VH2, C2H1, and Fc2 are the VH, CH1, and Fc regions of the second heavy chain H2, respectively; and CL2 and VL2 are the CL and VL regions of the second light chain L2, respectively. L1FR1, L1FR2, L1FR3, and L1FR4 are framework regions of the light chain variable region of the first light chain, and H1FR1, H1FR2, H1FR3, and H1FR4 are framework regions of the heavy chain variable region of the first heavy chain. L2FR1, L2FR2, L2FR3, and L2FR4 are framework regions of the light chain variable region of the second light chain, and H2FR1, H2FR2, H2FR3, and H2FR4 are framework regions of the heavy chain variable region of the second heavy chain.

[0067] Antibody sequences are numbered using the Kabat numbering system.

[0068] The Disclosure further provides bispecific antibody-drug conjugates comprising the following fragments, or tautomers, enantiomers, diastereoisomers, or mixtures thereof, or pharmaceutically acceptable salts thereof: the fragments comprising a bispecific antibody or its antigen-binding fragment, a linker unit L, and a cytotoxic drug, wherein the bispecific antibody or its antigen-binding fragment is as described in any one embodiment of the Disclosure.

[0069] In some embodiments, the cytotoxic agent is camptothecin and its derivatives.

[0070] In some embodiments, the cytotoxic agent is of the structure of formula (A-1), or a tautomer, enantiomer, or diastereoisomer thereof: [ka] (In the formula, M is -L , -L 1 -C(O)-; L 2 is selected from -NH-, O, and S, and L 2 is linked to the linker unit L; L 1 is -(C(R 1a )(R 1b )) m -CH2-, saturated C3-C6 cycloalkylene, or saturated 3-membered - 6-membered heterocyclylene, where the saturated C3-C6 cycloalkylene and saturated 3-membered - 6-membered heterocyclylene are each independently optionally substituted with one or more R 2a ; m is selected from 1, 2, 3, and 4; each heteroatom in the saturated 3-membered - 6-membered heterocyclylene is independently N, O, or S, and the number of heteroatoms is 1, 2, or 3; R 1a and R 1b are each independently selected from hydrogen, halogen, hydroxyl, amino, and C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with one or more halogens;<OO00494>R 2a is selected from halogen, hydroxyl, amino, and C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with one or more halogens).<0OO0495><00004l96> In certain preferred embodiments of the present disclosure, in the compounds of formula (A-1), (A-2), (A-2a) or (A-2b), certain groups are defined as follows, and groups not mentioned are as described in any one of the embodiments of the present disclosure (abbreviated as "in some embodiments").

[0072] <0OO0500>In some embodiments, L 2 is preferably -O- or -S-, more preferably -O-.

[0073] In some embodiments, L 1 is -(C(R 1a)(R 1b )) m -CH2- and R 1a R is selected from hydrogen, halogens, and C1-C6 alkyl groups; 1b This is selected from hydrogen, halogens, and C1-C6 alkyl groups.

[0074] In some embodiments, L 1 is, -(C(R 1a )(R 1b )) m -CH2- and R 1a is a C1-C6 alkyl group, preferably a C1-C3 alkyl group; R 1b The elements are selected from hydrogen and C1-C6 alkyl, preferably hydrogen and C1-C3 alkyl.

[0075] In some embodiments, L 1 is, -(C(R 1a )(R 1b )) m -CH2- and R 1a is -CH3; R 1b It is selected from hydrogen and -CH3.

[0076] In some embodiments, L 1 is, -(C(R 1a )(R 1b )) m -CH2-; m is 1 or 2, preferably 1.

[0077] In some embodiments, L 1 is, [ka] Selected from.

[0078] In some embodiments, L 1 is saturated C3-C6 cycloalkylene or saturated 3-membered-6-membered heterocyclene, preferably saturated C3-C6 cycloalkylene, where saturated C3-C6 cycloalkylene and saturated 3-membered-6-membered heterocyclene each independently have one or more R2a It is arbitrarily substituted, and each R 2a This is independently selected from halogens and C1-C6 alkyl groups.

[0079] In some embodiments, L 1 is one or more R 2a Cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, optionally substituted with each R 2a These are independently selected from halogens and C1-C6 alkyl groups.

[0080] In some embodiments, L 1 teeth, [ka] Selected from.

[0081] In some embodiments, L 1 In the definition of X1, the C3-C6 cycloalkylene is cyclobutyl or cyclohexyl, preferably cyclobutyl.

[0082] In some embodiments, L 1 In the definition, each heteroatom of a 3-membered-6-membered heterocyclylene is independently N or O, and the number of heteroatoms is preferably 1 or 2.

[0083] In some embodiments, R 1a , R 1b , and R 2a In the definition, C1-C6 alkyl is C1-C3 alkyl, and preferably methyl.

[0084] In some embodiments, R, R 1a , R 1b , and R 2a In this definition, the halogen is F, Cl, Br, or I, preferably F, Cl, or Br.

[0085] In some embodiments, in the structure of formula (A-1), M is -L 2 -L 1 -C(O)-; L 2 is -O-; L 1 is, -(C(R 1a )(R 1b )) m -CH2-, or saturated C3-C6 cycloalkylene, where saturated C3-C6 cycloalkylene is one or more R 2a It is arbitrarily replaced with; m is selected from 1 or 2; R 1a and R 1b Each of these is independently selected from hydrogen, halogen, and C1-C6 alkyl, where the C1-C6 alkyl is optionally substituted with one or more halogens; R 2a The C1-C6 alkyl group is selected from halogens and C1-C6 alkyl groups, and the C1-C6 alkyl group is optionally substituted with one or more halogens.

[0086] In some embodiments, M is: [ka] That is the case.

[0087] In some embodiments, M is [ka] That is the case.

[0088] In some embodiments, the cytotoxic agent is selected from one of the following structures: [ka]

[0089] In some embodiments, the linker unit L is -L a -L b -L c -Therefore, L cIt is linked to cytotoxic drugs; -L a -teeth, [ka] or -C 1-8 Alkylene-C(O)-, preferably, [ka] or -C 1-6 It is alkylene-C(O)-, and more preferably, [ka] and;-L a In the definition of -, each base fragment preferably has L at its rightmost end. b It is connected to; -L a - is more preferably, [ka] Therefore, end a is connected to Ab, and end b is connected to L b It is connected to; -L b - is -(polypeptide of 1 to 6 natural amino acids)-NH-, preferably -(polypeptide of 2 to 4 natural amino acids)-NH-, and more preferably the following structure: [ka] Furthermore, more preferably, [ka] One of the following is selected; -L b In the definition of -, each base fragment preferably has L at its rightmost end. c It is connected to; -L b - is more, [ka] Therefore, the c-end is L a It is connected to and the d end is L c It is connected to; -L c - is C1-6 alkylene, preferably C1-3 alkylene, more preferably, [ka] That is the case.

[0090] In some embodiments, the linker unit L is [ka] Preferably, [ka] The present disclosure provides a bispecific antibody-drug conjugate.

[0091] In some embodiments, a bispecific antibody-drug conjugate of the present disclosure having the structure of formula (A-2) is provided: [ka] (wherein p represents the average number of connections, and p is one integer or decimal between 1 and 10, preferably one integer or decimal between 3 and 9, for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98, or 8; Ab and M are as defined in any one of the embodiments of this disclosure; L is a linker unit L as described in any one of the embodiments of this disclosure.

[0092] In some embodiments, a bispecific antibody-drug conjugate of the present disclosure having the structure of formula (A-2) is provided: [ka] (In the formula, p represents the average linking number, where p is either an integer or a decimal number from 1 to 10, preferably either an integer or a decimal number from 3 to 9, such as 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98, or 8; Ab is a bispecific antibody as described in any one of the embodiments of the present disclosure, or an antigen-binding fragment thereof; L is the linker unit L as described in any one of the embodiments of the present disclosure; M is -L 2 -L 1 -C(O)-; L 2 is -O- or -S-, and L2 is linked to L; L 1 is -(C(R 1a )(R 1b )) m -CH2-, saturated C3-C6 cycloalkylene, or saturated 3-membered-6-membered heterocyclylene, where the saturated C3-C6 cycloalkylene and saturated 3-membered-6-membered heterocyclylene are each independently optionally substituted with one or more R 2a ; m is 1, 2, 3, or 4; each heteroatom in the saturated 3-membered-6-membered heterocyclyl is independently N, O, or S, and the number of heteroatoms is 1, 2, or 3; R 1a and R 1b R 2a are each independently hydrogen, halogen, hydroxyl, amino, or C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with one or more halogens; R 2a is selected from halogen, hydroxyl, amino, and C1-C6 alkyl, and the C1-C6 alkyl is optionally substituted with one or more halogens).

[0093] In some embodiments, a bispecific antibody-drug conjugate of the present disclosure having the structure of formula (A-2a) or (A-2b) is provided:

Chemical formula

[0094] In some embodiments, provided are bispecific antibody-drug conjugates selected from the following:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0095] In some embodiments, Ab is selected from the bispecific antibodies of the DBXT001 series (DBXT001-01~08), DBXT002 series (DBXT002-01~08), DBXT003 series (DBXT003-01~08), and DBXT004 series (DBXT004-01~08), which include the EGFR-binding domain and HER3-binding domain of the Disclosure; preferably, Ab is a bispecific antibody of the DBXT004 series (DBXT004-01~08), which includes the EGFR-binding domain and HER3-binding domain of the Disclosure. Ab is selected from the bispecific antibodies of the BXT001 series, DBXT002 series, and DBXT005-01; more preferably, Ab is selected from the bispecific antibodies of the DBXT001 series and DBXT005-01, which include the EGFR-binding domain and HER3-binding domain of the present disclosure; even more preferably, Ab is selected from the bispecific antibodies DBXT001-01 and DBXT005-01, which include the EGFR-binding domain and HER3-binding domain of the present disclosure.

[0096] In some embodiments, Ab is a bispecific antibody DBXT005-01 comprising the EGFR-binding domain and the HER3-binding domain of the present disclosure.

[0097] In some embodiments, the bispecific antibody-drug conjugate is selected from one of the following structures: [ka] (In the formula, p represents the average number of connections, where p is an integer or decimal between 1 and 10, preferably an integer or decimal between 3 and 9, for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98, or 8).

[0098] In some embodiments, the bispecific antibody-drug conjugate is selected from one of the following structures: [ka] [ka] [ka] [ka] [ka]

[0099] The amino acid sequences of the two-specific antibodies DBXT001 (DBXT001-01~08), DBXT002 (DBXT002-01~08), DBXT003 (DBXT003-01~08), and DBXT004 (DBXT004-01~08), which constitute the EGFR-binding domain and HER3-binding domain of this disclosure, are listed in the sequence listing of this disclosure.

[0100] The antibody CDRs in this disclosure are numbered using the Kabat numbering system.

[0101] In some embodiments, the bispecific antibody-drug conjugate is as follows: [ka] (In the formula, p represents the average number of connections, where p is an integer or decimal between 1 and 10, preferably an integer or decimal between 3 and 9, more preferably an integer or decimal between 4 and 6, for example, 5.99; DBXT005-01 is an anti-EGFR / HER3 bispecific antibody containing a heavy chain H1 having the amino acid sequence shown in SEQ ID NO: 37, a light chain L1 having the amino acid sequence shown in SEQ ID NO: 38, and a heavy chain H2 having the amino acid sequence shown in SEQ ID NO: 90.

[0102] In some embodiments, the bispecific antibody-drug conjugate is selected from the following conjugates: [ka] (In the formula, p1 represents the number of connections, where p1 is one integer from 1 to 10, preferably one integer from 3 to 9, and more preferably one integer from 4 to 6, for example, 4, 5, or 6.) DBXT005-01 is an anti-EGFR / HER3 bispecific antibody containing a heavy chain H1 having the amino acid sequence shown in SEQ ID NO: 37, a light chain L1 having the amino acid sequence shown in SEQ ID NO: 38, and a heavy chain H2 having the amino acid sequence shown in SEQ ID NO: 90.

[0103] In some embodiments, the average connection number p of the present disclosure may be any integer or decimal number between 1 and 10. For example, the average connection number p may be any integer or decimal number between 3 and 9. For example, the average connection number p may be any integer or decimal number between 1 and 2, 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 7 and 8, 8 and 9, or 9 and 10. Preferably, the average connection number p is 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98, or 8.

[0104] In some embodiments, the concatenation number p1 of the present disclosure is one integer from 1 to 10. For example, the concatenation number p1 is one integer from 3 to 9. For example, the concatenation number p1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the concatenation number p1 is 4, 5, or 6.

[0105] In yet another embodiment, the present disclosure provides a method for preparing a bispecific antibody-drug conjugate according to any one embodiment of the present disclosure, comprising the steps of: mixing a bispecific antibody dissolved in a buffer with a linker unit L-cytotoxic drug dissolved in a solvent under the action of a reducing agent to obtain a bispecific antibody-drug conjugate.

[0106] In some embodiments, the preparation method includes reacting an anti-EGFR / HER3 bispecific antibody with a compound of formula X2, for example, reacting DBXT005-01 with a compound of formula X2: [ka]

[0107] In some embodiments, the reducing agent is a conventional reducing agent for such reactions in the art, such as tris(2-carboxyethyl)phosphine hydrochloride.

[0108] In some embodiments, the buffer is a conventional buffer for such reactions in the art, such as ethylenediaminetetraacetic acid.

[0109] In some embodiments, the solvent is a conventional solvent for such reactions in the art, such as dimethylacetamide.

[0110] In yet another embodiment, the Disclosure provides a bispecific antibody as described in any one embodiment of the Disclosure, a pharmaceutical composition comprising an isolated nucleic acid as described in any one embodiment of the Disclosure, a recombinant expression vector as described in any one embodiment of the Disclosure, a transformant as described in any one embodiment of the Disclosure, and / or a bispecific antibody-drug conjugate as described in any one embodiment of the Disclosure, as well as a pharmaceutically acceptable carrier or excipient.

[0111] In yet another aspect, the Disclosure provides a method for preparing a pharmaceutical composition of the Disclosure, comprising combining a bispecific antibody-drug conjugate described in any one embodiment of the Disclosure, or a pharmaceutically acceptable form thereof, or a mixture thereof, with one or more pharmaceutically acceptable carriers or excipients.

[0112] Preferred examples of pharmaceutically acceptable carriers used in pharmaceutical compositions in this disclosure are those described in Remington's Pharmaceutical Sciences (2005).

[0113] In this disclosure, pharmaceutical compositions may be administered in any form, insofar as they achieve the prevention, relief, inhibition, or cure of symptoms in human or animal patients. For example, they may be formulated in various appropriate dosage forms depending on the route of administration.

[0114] In other embodiments, the administration of any of the bispecific antibody-drug conjugates or pharmaceutical compositions described in any one embodiment of this disclosure may be combined with an additional treatment method. The additional treatment method may be selected from, but is not limited to, radiotherapy, chemotherapy, immunotherapy, or a combination thereof.

[0115] In yet another embodiment, the Disclosure provides a pharmaceutical formulation or a pharmaceutical composition according to any one embodiment of the Disclosure, comprising a bispecific antibody-drug conjugate, or a pharmaceutically acceptable form thereof, or a mixture thereof, as an active ingredient. In some embodiments, the formulation is in the form of a solid formulation, a semi-solid formulation, a liquid formulation, or a gaseous formulation.

[0116] In yet another embodiment, the Disclosure provides the use of a bispecific antibody as described in any one embodiment of the Disclosure, an isolated nucleic acid as described in any one embodiment of the Disclosure, a recombinant expression vector as described in any one embodiment of the Disclosure, a transformant as described in any one embodiment of the Disclosure, a bispecific antibody-drug conjugate as described in any one embodiment of the Disclosure, and / or a pharmaceutical composition as described in any one embodiment of the Disclosure in the preparation of a pharmacopoeia for treating and / or preventing cancer, wherein the cancer is preferably a cancer having positive expression of EGFR and / or HER3.

[0117] In yet another embodiment, the Disclosure provides a method for treating and / or preventing cancer, comprising administering to a subject in need a bispecific antibody as described in any one embodiment of the Disclosure, an isolated nucleic acid as described in any one embodiment of the Disclosure, a recombinant expression vector as described in any one embodiment of the Disclosure, a transformant as described in any one embodiment of the Disclosure, a bispecific antibody-drug conjugate as described in any one embodiment of the Disclosure, and / or a pharmaceutical composition as described in any one embodiment of the Disclosure, wherein the cancer is preferably a cancer having positive expression of EGFR and / or HER3.

[0118] In yet another embodiment, the Disclosure provides a bispecific antibody as described in any one embodiment of the Disclosure, an isolated nucleic acid as described in any one embodiment of the Disclosure, a recombinant expression vector as described in any one embodiment of the Disclosure, a transformant as described in any one embodiment of the Disclosure, a bispecific antibody-drug conjugate as described in any one embodiment of the Disclosure, and / or a pharmaceutical composition as described in any one embodiment of the Disclosure, for use in the treatment and / or prevention of cancer, wherein the cancer is preferably a cancer having positive expression of EGFR and / or HER3.

[0119] In some embodiments, the cancers of the present disclosure are 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, plasmacytoma, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma.

[0120] In some embodiments, the cancers of this disclosure are preferably selected from breast cancer, colorectal cancer, skin cancer, lung cancer, esophageal cancer, and oral squamous cell carcinoma.

[0121] In some embodiments, lung cancer is preferably non-small cell lung cancer, skin cancer is preferably cutaneous squamous cell carcinoma, and colorectal cancer is preferably rectal cancer.

[0122] In yet another embodiment, the Disclosure provides the use of a bispecific antibody described in any one embodiment of the Disclosure, an isolated nucleic acid described in any one embodiment of the Disclosure, a recombinant expression vector described in any one embodiment of the Disclosure, a transformant described in any one embodiment of the Disclosure, a bispecific antibody-drug conjugate described in any one embodiment of the Disclosure, and / or a pharmaceutical composition described in any one embodiment of the Disclosure in the preparation of an EGFR and / or HER3 inhibitor.

[0123] In some embodiments, the administration routes of the present disclosure include, but are not limited to, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intra-arterial administration, intra-articular administration (e.g., in arthritis joints), administration by inhalation or aerosol delivery, and intratumor administration.

[0124] In some embodiments, the disclosure provides co-administration of therapeutically effective doses of one or more treatments (e.g., a treatment and / or additional therapeutic agents) to a subject. In some embodiments, the treatments include surgical procedures and / or radiotherapy.

[0125] In some embodiments, the methods or uses provided herein further include administering one or more treatments (e.g., treatments and / or additional therapeutic agents) to an individual. The antibody-drug conjugates of the herein or their pharmaceutically acceptable salts may be used alone or in combination with additional therapeutic agents in treatment. For example, it may be co-administered with at least one additional therapeutic agent.

[0126] In yet another embodiment, the Disclosure provides a combination of drugs, as well as one or more additional therapeutic agents, comprising a bispecific antibody as described in any one embodiment of the Disclosure, an isolated nucleic acid as described in any one embodiment of the Disclosure, a recombinant expression vector as described in any one embodiment of the Disclosure, a transformant as described in any one embodiment of the Disclosure, a bispecific antibody-drug conjugate as described in any one embodiment of the Disclosure, and / or a pharmaceutical composition as described in any one embodiment of the Disclosure.

[0127] In yet another embodiment, the Disclosure provides a kit comprising a bispecific antibody as described in any one embodiment of the Disclosure, an isolated nucleic acid as described in any one embodiment of the Disclosure, a recombinant expression vector as described in any one embodiment of the Disclosure, a transformant as described in any one embodiment of the Disclosure, a bispecific antibody-drug conjugate as described in any one embodiment of the Disclosure, and / or a pharmaceutical composition as described in any one embodiment of the Disclosure.

[0128] Definition of Terms Unless otherwise specified, embodiments of this disclosure utilize prior art in molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the scope of the art of those skilled in the art.

[0129] Unless otherwise defined herein, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art to which this disclosure relates. For definitions and terms in the art, experts can refer in particular to *Current Protocols in Molecular Biology* (Ausubel). The abbreviations for amino acid residues are standard three-letter and / or one-letter codes commonly used in the art to indicate one of the 20 L-amino acids.

[0130] To facilitate understanding of this disclosure, several technical and scientific terms are defined below.

[0131] In this disclosure, the term "EGFR (epidermal growth factor receptor)" refers to the receptor for epidermal growth factor (EGF) cell proliferation and signaling. EGFR is a member of the ErbB receptor family, which includes EGFR (ErbB-1), HER2 / c-neu (ErbB-2), HER3 (ErbB-3), and HER4 (ErbB-4). EGFR is also known as HER1 or ErbB-1. Mutations or overexpression of EGFR are commonly associated with tumors. EGFR is a glycoprotein and a tyrosine kinase receptor. EGFR spans the cell membrane and has a molecular weight of 170 kDa. Located on the surface of the cell membrane, EGFR is activated by complexing with ligands such as EGF and TGFα (transforming growth factor α). Upon activation, EGFR transforms from a monomer to a dimer, although there is evidence that dimers existed before activation. EGFR can also be activated by dimerizing with other members of the ErbB receptor family, such as ErbB-2 / HER2 / neu.

[0132] In this disclosure, the term “human epidermal growth factor receptor 3 (HER3)” is also known as receptor tyrosine protein kinase ErbB-3 (ErbB3) and is a member of the EGFR / ErbB family. Unlike other ErbB family members, HER2 and EGFR, HER3 itself does not possess phosphorylation activity. Therefore, for HER3 to induce downstream activity as a heterodimer, it must bind to a phosphorylation-active member, either EGFR or HER2. When complexed with the native ligand NRG1, HER3 undergoes conformational change, heterodimerization, and phosphorylation, activating MAPK, PI3K / Akt, and PLCγ via signaling.

[0133] In this disclosure, the term "approximately," when used in conjunction with a numerical value, is intended to encompass numerical values ​​ranging from a lower limit of 5% less than the specified numerical value to an upper limit of 5% more than the specified numerical value, including, but not limited to, ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed method.

[0134] In this disclosure, the term "and / or" should be understood to mean any one of the options or any combination of two or more of the options.

[0135] In this disclosure, the term “or” should be understood to have the same meaning as “and / or” as defined above. For example, if the items in a list are separated, “or” or “and / or” is inclusive; that is, it should be interpreted to include not only at least one number or one of the elements in the list, but more than one, and optionally, any further unlisted items. Only when contradictory terms such as “only one,” “exactly one,” or “consisting of ~” are explicitly indicated in the claims does it refer to a single number or one element of a list that is listed alone.

[0136] In this disclosure, the terms "a" and "an" should be understood to mean "at least one" unless the context explicitly indicates otherwise.

[0137] In this disclosure, the term “antibody-drug conjugate” generally means an antibody linked to a biologically active cytotoxic drug by a stable linking unit. In this disclosure, “antibody-drug conjugate” may also be a bispecific antibody-drug conjugate, which may mean a bispecific antibody or its antigen-binding fragment linked to a biologically active cytotoxic drug fragment by a stable linking unit.

[0138] In this disclosure, the term “cytotoxic drug” generally refers to a toxic drug, which may have chemical molecules strong enough to disrupt the normal growth of tumor cells. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations. “Cytotoxic drugs” include low-molecular-weight toxins, or enzyme-active toxins derived from bacteria, fungi, plants, or animals, or radioisotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 This may include radioactive isotopes of Lu, toxic drugs, chemotherapeutic drugs, antibiotics, nucleoplasties, or derivatives thereof. For example, cytotoxic drugs may include, but are not limited to, camptothecin derivatives; for example, it may be the 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).

[0139] In this disclosure, the term “antibody (Ab)” generally refers to an immunoglobulin that is reactive with a particular protein or peptide, or a fragment thereof. Antibodies may be any class of antibodies, including but not limited to IgG, IgA, IgM, IgD, and IgE, and any dependent classes (e.g., IgG1, IgG2, IgG3, and IgG4). Antibodies may have a heavy chain constant region, selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies may also have a light chain, selected from, for example, kappa (κ) or lambda (λ). Antibodies in this disclosure may originate from any species. The term “antibody” may include intact polyclonal antibodies, intact monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecules, insofar as the antibody exhibits the desired biological activity.

[0140] In this disclosure, the terms “antigen-binding fragment” or “antigen-binding domain” generally refer to a portion of an antibody molecule containing amino acids responsible for the antibody’s specific binding to an antigen. The portion of the antigen that is specifically recognized and bound by the antibody is referred to herein as an “epitope.” As described herein, an antigen-binding domain may typically include, but does not necessarily include, both the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH). For example, an Fd fragment has two VH regions, which usually retain some of the antigen-binding function of the intact antigen-binding domain. Examples of antibody antigen-binding fragments include: (1) Fab fragment - a monovalent fragment having VL, VH, constant light chain (CL), and CH1 domains; (2) F(ab')2 fragment - a bivalent fragment having two Fab fragments linked by disulfide crosslinks in the hinge region; (3) Fd fragment - having two VH and CH1 domains; (4) Fv fragment - having the VL and VH domains of a single arm of the antibody; (5) 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) - having a VH domain; (6) isolated complementarity-determining region (CDR); and (7) single-chain Fv (scFv) - e.g., derived from scFv.The two domains of the Fv fragment—VL and VH—are encoded by separate genes, but they can be linked by recombination using synthetic linkers, which allows the VL and VH regions to be paired to form a single monovalent molecule (known as single-chain Fv (scFv)) (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)); and (8) “VHH”—this refers to the variable antigen-binding domain of heavy chain antibodies derived from camelids (camels, dromedaries, llamas, alpacas, etc.) (Nguyen VK et al, 2000, The EMBO Journal, 19, 921-930; Muyldermans See the review articles S., 2001, J Biotechnol., 74, 277-302; and Vanlandschoot P. et al., 2011, Antiviral Research 92, 389-407. VHHs are sometimes also called nanobodies (Nb).

[0141] In this disclosure, the terms “variable region,” “variable domain,” or “variable antigen-binding domain” generally refer to domains in the heavy or light chain of an antibody that are involved in the binding of the antibody to an antigen. In this disclosure, the term “variable” generally means that certain parts of the sequence of the variable domain of an antibody differ significantly, resulting in different binding and specificity of different particular antibodies to specific antigens. Variability is not uniformly distributed throughout the variable region of an antibody. It is concentrated in three fragments in the variable regions of the light and heavy chains, respectively, known as complementarity-determining regions (CDRs) or hypervariable regions (HVRs): LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. The more highly conserved portion of the variable region is known as the framework region (FR). The innate variable regions of the heavy and light chains each contain four FRs (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, and L-FR4), which mostly take the form of β-sheet structures. The FRs are linked by three CDR structural loop regions. The CDRs of each chain are held in close proximity by the FRs and, together with the CDRs of other chains, form the antigen-binding site of the antibody.

[0142] In this disclosure, the variable region of an antibody can be coded, or the CDR of an antibody can be classified in various ways. For example, see the Kabat numbering scheme and definition rules based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, fifth edition, National Institutes of Health, Bethesda, Md. (1991)), the Chothia numbering scheme and definition rules based on the position of structural loop regions (A1-Lazikani et al., J Mol Biol 273:927-48, 1997), the IMGT numbering scheme and definition rules by Lefranc et al. 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. (Mathieu Dondelinger et al., Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition, Front.Immunol., October) (See 16, 2018.)

[0143] In this disclosure, the terms “monoclonal antibody” or “mAb” refer to an antibody obtained from a substantially homogeneous population of antibodies; that is, the antibodies constituting that population are identical except for any naturally occurring variations that may be present in trace amounts. Monoclonal antibodies are highly specific and target a single antigen epitope. In contrast, conventional (polyclonal) antibody preparations typically contain multiple antibodies that target (or are specific to) different epitopes. The modifier “monoclonal” indicates that the antibody is obtained from a substantially homogeneous population of antibodies and does not imply that the antibody is produced in any particular manner.

[0144] In the present disclosure, the term "multispecific antibody" refers to an antibody that contains two or more antigen-binding domains and can bind to two or more different epitopes (e.g., two, three, four, or more different epitopes) that may be on the same antigen or different antigens. Examples of multispecific antibodies include "bispecific antibodies" (abbreviated as BsAbs) or "bispecific molecules" that bind to two different antigens or two different epitopes. In the present specification, a bispecific antibody targeting EGFR and HER3 may refer to, for example, an "anti-EGFR / HER3" or "EGFR×HER3" bispecific molecule, or a bispecific antibody containing an EGFR-binding domain and a HER3-binding domain, or other similar terms.

[0145] In the present disclosure, the term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that constitutes at least a part of the constant region. This term includes the Fc region of the native sequence and the mutant Fc region. In some embodiments, the Fc region of the human IgG heavy chain 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 (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991)).

[0146] In the present disclosure, the term "knob-into-hole structure" refers to a mutation of hydrophobic amino acids in the CH3 of an antibody Fc. The side-chain amino acids of CH3 in one chain are mutated to form a larger hydrophobic amino acid molecule (knob), enhancing the hydrophobic force. The side-chain amino acids of CH3 in the other chain are mutated to form a smaller amino acid (hole) to reduce steric hindrance. After mutation, the CH3 with a knob and the CH3 with a hole hydrophobically form a knob-into-hole structure (KiH), promoting the formation of heavy-chain heterodimers. The KiH mutation mainly occurs in the hydrophobic amino acids inside the spatial structure of the CH3 domain, and the amino acids exposed outside change little after mutation, so it has no impact on the effector function of Fc and the induced immunogenicity. The term "knob-Fc" refers to introducing a point mutation of T366W into the Fc region of an antibody to form a spatial structure like a knob. Correspondingly, "hole-Fc" refers to introducing point mutations of T366S, L368A, and Y407V into the Fc region of an antibody to form a spatial structure like a hole. By further introducing point mutations of S354C and Y349C into knob-Fc and hole-Fc respectively, the formation of heterodimers by disulfide bonds can be further promoted. At the same time, by further introducing point mutations of H435R and Y436F into hole-Fc, the binding to protein A can be reduced.

[0147] In the present disclosure, the term "humanized antibody" refers to an antibody form that includes the sequences of both human antibodies and non-human antibodies (e.g., mouse and rat). Generally, a humanized antibody includes substantially all, typically two, variable domains, and all or substantially all of the variable domains correspond to those of non-human immunoglobulins, while all or substantially all of the framework regions (FRs) are those of human immunoglobulin sequences. A humanized antibody may optionally include at least a part of the human immunoglobulin constant region (Fc).

[0148] In this disclosure, “isotype” of an antibody refers to the antibody type (e.g., IgM, IgE, and IgG (IgG1, IgG2, or IgG4, etc.)) provided by a heavy chain constant region gene. Isotypes also include modified forms of any of these types that are modified to alter Fc function, such as by enhancing or reducing effector function or binding to the Fc receptor.

[0149] In this disclosure, the term “cross-reactivity” refers to the binding of the same target molecule to an antigenic fragment from humans, monkeys, and / or murid animals (mice or rats). Therefore, “cross-reactivity” should be understood as an interspecies reaction between an antigen-binding molecule (e.g., an antibody) and a similar molecule expressed in different species (e.g., BDCA2). The cross-reactivity specificity of monoclonal antibodies that recognize human BDCA2, as well as BDCA2 from monkeys and / or murid animals (mice or rats), can be determined by FACS analysis.

[0150] In this disclosure, "affinity" or "binding affinity" refers to an intrinsic binding affinity that reflects the interaction between the members involved in binding. The affinity of molecule X for its counterpart Y is generally expressed as the ratio of the dissociation rate constant to the association rate constant (K, respectively). off (Kd) and K on The affinity can be expressed by the equilibrium dissociation constant (KD), which is (Ka). Affinity can be measured by common methods known in the art. In some embodiments of this disclosure, affinity, for example, affinity between the antibody and antigen of this disclosure, is measured using surface plasmon resonance (SPR) techniques. In some preferred embodiments of this disclosure, one particular method for measuring affinity is the BIAcore method.

[0151] In this disclosure, the term “not binding” to a protein or cell means not binding to a protein or cell, or not binding with high affinity, i.e., 1.0 × 10⁻⁶ -6 M or more, preferably 1.0 × 10 -5 M or higher, comfort level 1.0 × 10 -4M or larger or 1.0 × 10 -3 M or more, more preferably 1.0 × 10 -2 A KD of M or higher means that the protein or cell binds to the cell.

[0152] In this disclosure, the term "high affinity" for an IgG antibody means that its KD for the antigen is 1.0 × 10⁻⁶. -6 M or less, preferably 5.0 × 10 -8 M or less, more preferably 1.0 × 10 -8 M or less or 5.0 × 10 -9 M or less, more preferably 1.0 × 10 -9 This refers to being M or less. "High affinity" binding may differ for other dependent antibodies. For example, the "high affinity" binding of dependent IgM is 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 This refers to KD ratios of M or lower.

[0153] In this disclosure, the terms “percent (%) amino acid sequence identity” or simply “identity” are defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to amino acid residues in a reference amino acid sequence after the amino acid sequence has been aligned (with gaps introduced as necessary) to achieve maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Sequence alignment for the purpose of determining percent amino acid sequence identity can be performed using various methods within the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the entire length of the aligned sequence.

[0154] In this disclosure, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine, which may be, for example, fluorine or chlorine.

[0155] In this disclosure, the term “alkyl” generally refers to a residue derived from an alkane by the removal of a hydrogen atom. The term “alkyl” generally refers to a saturated linear or branched aliphatic hydrocarbon group having a residue derived from a parent alkane by the removal of a hydrogen atom from the same carbon atom or from two different carbon atoms, which may be a linear or branched group containing 1 to 20 carbon atoms (e.g., 1 to 12 carbon atoms), or an alkyl chain containing 1 to 6 carbon atoms (e.g., an alkyl chain containing 1 to 3 carbon atoms). Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, and butyl. Alkyl may be substituted or unsubstituted, replaced or unreplaced; for example, if substituted, substitution by substituents may occur at any available bond site.

[0156] In this disclosure, the term “alkylene” generally refers to a saturated linear or branched aliphatic hydrocarbon group having two residues derived from a parent alkane by the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms, which may be a linear or branched group containing 1 to 20 carbon atoms; for example, the term “methylene” may refer to a residue derived from a single carbon atom group by the removal of two hydrogen atoms. Methylene may be substituted or unsubstituted, replaced or unreplaced. For example, alkylene is an alkylene containing 1 to 12 carbon atoms, e.g., an alkylene containing 1 to 6 carbon atoms. Non-limiting examples of alkylenes 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-).

[0157] In this disclosure, the term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, where 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 cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl. Examples of polycyclic cycloalkyls include spirocycloalkyls, condensed cycloalkyls, and crosslinked cycloalkyls.

[0158] The term "cycloalkylene" refers to a divalent group that is linked to the rest of the molecule by two single bonds, and the rest of the definition is the same as the definition of the term "cycloalkyl".

[0159] In this disclosure, the term “partially unsaturated” generally means that a cyclic structure contains at least one double or triple bond between ring molecules. The term “partially unsaturated” encompasses cyclic structures having multiple unsaturated sites, but is not intended to include aromatic or heteroaromatic rings as defined herein. The term “unsaturated” means that the site has one or more degrees of unsaturation.

[0160] In this disclosure, the term “heterocyclyl” means a saturated or partially unsaturated monocyclic or polycyclic light substituent containing 3 to 20 ring atoms, where one or more ring atoms are heteroatoms selected from nitrogen, oxygen, and sulfur, and the remaining ring atoms are carbon atoms. Preferably, the heterocyclyl contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the heterocyclyl contains 3 to 8 ring atoms, of which 1 to 3 are heteroatoms; even more preferably, the heterocyclyl contains 3 to 6 ring atoms, of which 1 to 3 are heteroatoms; and most preferably, the heterocyclyl contains 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, tetrahydropyranil, piperidinyl, morpholinil, thiomorpholinil, and homopiperazinyl. Non-exclusive examples of polycyclic heterocyclies include spiroheterocyclies, condensed heterocyclies, and cross-linked heterocyclies. The heterocyclyl ring may be condensed with an aryl, heteroaryl, or cycloalkyl ring, and the ring linked to the parent structure is a heterocyclyl.

[0161] The term "heteroalkylene" refers to a divalent group that is linked to the rest of the molecule by two single bonds, and the rest of the definition is the same as the definition of the term "cycloalkylene."

[0162] In this disclosure, the term “independently” generally means that the variables apply in all cases, whether they are present or absent, to the same compound, with the same or different definitions. For example, a variable may refer to the type or number of substituents in the compound, the type of atom in the compound, and so on. For example, if R appears twice in a compound and R is defined as “independently carbon or nitrogen,” both Rs may be carbon, both Rs may be nitrogen, or one R may be carbon and the other R may be nitrogen.

[0163] In this disclosure, the terms “optional” or “optionally” generally mean that the events or circumstances described thereafter may occur, but are not necessarily required, and that the descriptions include cases in which the events or circumstances may or may not occur. For example, “optionally alkyl-substituted heterocyclyl group” means that alkyl substitution may be present, but is not necessary, and that the description includes cases in which the heterocyclyl group is alkyl-substituted or not.

[0164] In this disclosure, the term “substituted” generally means that one or more hydrogen atoms in the group, e.g., up to five (e.g., one to three) hydrogen atoms, are each independently substituted with a corresponding number of substituents. Substituents exist only in their possible chemical positions, and those skilled in the art will be able to determine possible or impossible substituents (by experiment or theory) without strenuous effort. For example, an amino or hydroxyl group with free hydrogen may be unstable if it is bonded to a carbon atom with an unsaturated (e.g., olefinic) bond.

[0165] In this disclosure, unless otherwise specified, the “linking” between bases may generally be in any orientation. The “linking” between base X and base Y may generally be in any orientation, which generally means that when base X is used in linkers Y and base Z, two or more linking sites of base X may be arbitrarily linked to either base Y or base Z.

[0166] In this disclosure, as is known to those skilled in the art, under certain circumstances, notations indicating the number of atoms present in the group may precede terms such as "alkyl" and "cycloalkyl," and the subscript number following "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group containing three carbon atoms (e.g., n-propyl or isopropyl); C 1-10 Therefore, a member of a group may have any number of carbon atoms between 1 and 10.

[0167] In the present disclosure, the compounds or antibody-drug conjugates of the present disclosure include their tautomers, intermediates, racemates, enantiomers, and / or diastereoisomers. In the present disclosure, the term "diastereoisomer" generally refers to stereoisomers having two or more chiral centers and the molecules are not mirror images of each other. Diastereoisomers may have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. In the present disclosure, the terms "tautomer" and "tautomeric form" are used interchangeably and generally refer to structural isomers of different energies that can be converted into each other by crossing a low energy barrier. For example, proton tautomers (also known as proton transfer tautomers) include proton transfer-mediated interconversions such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions by recombination of some bonding electrons. In the present disclosure, the term "mesomer" generally means that its molecule contains chiral atoms but has zero total optical rotation due to the presence of a symmetry factor. The term "racemate" or "racemic mixture" refers to a composition of two enantiomeric substances in equimolar amounts.

[0168] In the present disclosure, the term "linker unit" or "linker structure" generally refers to a chemical structure fragment or bond that is linked to a ligand at one end and linked to a cytotoxic drug at the other end or linked to another linker before being linked to the cytotoxic drug. Direct or indirect linkage to the ligand may mean that the group is directly linked to the ligand by a covalent bond or may also be linked to the ligand by a linker structure. For example, a chemical structure fragment or bond including an acid-labile linker structure (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker structure, a light-labile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure may be used as the linker structure.

[0169] In this disclosure, the term “arbitrarily linked to other molecular parts” generally means that the structure is not linked to any other chemical structure, or that the structure is linked (e.g., by chemical bonds or linker structures) to one or more other chemical structures different from its own (e.g., ligands described herein).

[0170] In this disclosure, the term “drug load” generally refers to the average number of cytotoxic drugs loaded onto each ligand, and may also be expressed as the drug-to-antibody ratio (DAR), where the cytotoxic drug load can range from 0 to 12 (e.g., 1 to 10) cytotoxic drugs per ligand (Ab). In embodiments of this disclosure, the drug load is expressed as p or p1, and may exemplify an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The drug load per ADC molecule after the complexation reaction can be characterized by conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA, and HPLC.

[0171] In this disclosure, certain atoms of the compounds or antibody-drug conjugates of this disclosure may exist in one or more isotopic forms. For example, hydrogen may be protium ( 1 H), deuterium ( 2 H), and tritium ( 3 It may exist as H), and carbon has three different isotopes ( 12 C, 13 C, and 14 It may occur naturally as C). Examples of isotopes that can be incorporated into the compounds of this disclosure 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, 125This also includes isotopes I, or similar isotopes. Therefore, one or more such isotopes may be enriched in the compounds or antibody-drug conjugates of this disclosure compared to their natural abundance. Such isotope-enriched compounds can be used for a variety of purposes, as is known to those skilled in the art. For example, deuterium ( 2 Substitution with heavy isotopes such as H) may offer certain procedural advantages, possibly due to higher metabolic stability. For example, deuterium ( 2 The natural abundance of H) is approximately 0.015%. Therefore, of approximately 6500 hydrogen atoms, one is a deuterium atom. Accordingly, the amount of deuterium in one or more sites (as may be) in the deuterium-containing compounds or antibody-drug conjugates of this disclosure is greater than 0.015%. 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 having the same structure as those disclosed herein, except for the substitution of a hydrogen atom with deuterium or tritium, or the substitution of a carbon atom with carbon-13 or carbon-14, are included within the scope of this disclosure.

[0172] In this disclosure, the term “pharmaceutical composition” generally refers to a mixture comprising one or more of the compounds described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, as well as other chemical components such as physiologically / pharmaceutically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to a living organism, enhance the absorption of the active ingredient, and thereby exert biological activity. Conventional methods for preparing pharmaceutical compositions can be found in various national pharmacopoeias. Pharmaceutical compositions may be in the form of sterile aqueous or oily suspensions for intramuscular and subcutaneous administration. Suspensions can be formulated according to known techniques using suitable dispersants or wetting agents and suspending agents. Sterile injectable formulations may also be sterile injectable solutions or suspensions prepared in solutions prepared in parenterally acceptable non-toxic diluents or solvents, such as 1,3-butanediol. Furthermore, sterile non-volatile oils may be conveniently used as solvents or suspension media. For example, any mixed non-volatile oils, including synthetic monoglycerides or diglycerides, can be used. Furthermore, fatty acids such as oleic acid are sometimes used in the preparation of injectable drugs.

[0173] In this disclosure, the term “pharmaceutically acceptable salt” generally refers to a salt of the compounds or antibody-pharmaceutical conjugates of this disclosure that, when used in mammals, may be safe and / or effective and possess the desired biological activity. The compounds or antibody-pharmaceutical conjugates of this disclosure may form salts with acids.

[0174] In this disclosure, the term “pharmaceutically acceptable carrier” generally refers to a carrier or medium for providing therapeutic agents such as antibodies, polypeptides, and genes. The term refers to any pharmaceutical carrier that does not induce the production of antibodies harmful to the individual receiving the composition and can be administered without causing excessive toxicity. Preferred carriers may be large, slow-metabolizing polymers such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. Such 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, or pH buffering agents, may also be present in these carriers.

[0175] In this disclosure, the terms “treatment” and “to treat” generally refer to methods for achieving beneficial or desired outcomes, including but not limited to therapeutic benefits. Therapeutic benefits include, but not limited to, the elimination, inhibition, reduction, or improvement of the underlying disorder being treated. Furthermore, therapeutic benefits are achieved by eliminating, inhibiting, reducing, or improving one or more physiological symptoms associated with the underlying disorder, so that improvement is observed in the patient, but the patient may still be suffering from the underlying disorder.

[0176] In this disclosure, the terms “prevention” and “preventing” generally refer to methods for achieving beneficial or desired outcomes, including but not limited to preventive benefits. For the purpose of a preventive benefit, a pharmaceutical composition may be administered to a patient at risk of developing a particular disease, or to a patient who reports having one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

[0177] In this disclosure, the terms “Subject” or “Patient” generally include human beings (i.e., males or females of any age group, e.g., pediatric subjects (e.g., infants, children, or adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)), and / or other primates (e.g., crab-eating macaques or rhesus macaques); 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.

[0178] In this disclosure, the terms “therapeutic dose,” “therapeutic dosage,” and “effective dose” refer to the amount of the compound or antibody-drug conjugate of this disclosure that, when administered to cells, tissues, or subjects alone or in combination with other therapeutic agents, is effective in preventing or improving one or more symptoms of a disease or condition, or in preventing or improving the onset of a disease or condition. The therapeutic dose also refers to an amount sufficient to cause improvement in symptoms, for example, an amount to treat, cure, prevent or improve a condition in question, or an amount to promote the treatment, cure, prevention or improvement of such condition. When an active ingredient is administered to an individual alone, the therapeutic dose refers to the amount of that ingredient alone. In the case of combination therapy, the therapeutic dose refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether those active ingredients are administered together, sequentially, or concurrently. The therapeutic dose of a therapeutic agent results in an increase of at least 10%, generally at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in a diagnostic indicator or parameter.

[0179] In this disclosure, the term “cancer” refers to a group of cells exhibiting abnormally high levels of proliferation and growth. Cancer may be benign (also called a benign tumor), premalignant, or malignant. Cancer cells may be solid cancer cells or leukemia cells. In this disclosure, the term “tumor” refers to one or more cells that make up cancer. In this disclosure, the term “tumor proliferation” is used to refer to the proliferation or growth of one or more cells that make up cancer that result in a corresponding increase in the size or extent of the cancer. The terms “cancer with positive EGFR and / or HER3 expression” and “cancer expressing EGFR and / or HER3” have the same definition and refer to cancer in which cancer cells express EGFR and / or HER3, preferably on the surface of the cancer cells.

[0180] The amino acid numbers of the antibodies in this disclosure are based on the natural order from the N-terminus to the C-terminus, according to the antibody sequence.

[0181] Based on general knowledge in the art, the above preferred conditions may be arbitrarily combined to obtain preferred embodiments of the present disclosure.

[0182] All reagents and starting materials used in this disclosure are commercially available.

[0183] The positive and progressive effects of this disclosure are as follows:

[0184] The bispecific antibodies of this disclosure have the following advantages:

[0185] 1. It can bind to both EGFR antigen and HER3 antigen, as well as to EGFR / HER3 cell lines, and exhibits superior antitumor activity compared to monoclonal antibody administration; compared to the parental MAB and control antibody SI-1X6.4, the bispecific antibody of this disclosure shows superior endocytosis against human tumor cell lines T47D and NCI-H1975, which express both EGFR and HER3, as well as the mouse cell line CT26.

[0186] 2. Compared to combination therapy with related monoclonal antibodies, the bispecific antibodies of this disclosure have the advantages of good medication adherence and controllable quality.

[0187] 3. The stability characteristics of the bispecific antibody disclosed herein are primarily reflected in studies of monomer purity and thermal stability. After affinity purification and molecular sieving, the monomer content of the bispecific antibody can reach 95%. Structural and activity analyses of the antibody after heat treatment demonstrate that the antibody can maintain good molecular conformation and complete biological activity even under harsh conditions, which is beneficial for industrial production, packaging, and storage. Overall, the anti-EGFR / HER3 bispecific antibody disclosed herein exhibits good molecular stability and has significantly superior in vitro activity (binding at the molecular and cellular levels) compared to cetuximab and EGFR. In vivo data show that the antitumor activity of the bispecific antibody is superior to that of the combined administration group of EGFR and HER3 monoclonal antibodies in EGFR+HER3 tumor cells. Thus, the bispecific antibody disclosed herein has broad application potential due to its excellent development potential and activity.

[0188] A bispecific antibody-drug conjugate described in any one embodiment of this disclosure has an in vivo antitumor effect, which manifests specifically as follows: (1) It has a superior antitumor effect against the human esophageal cancer cell line OE-19, which has low EGFR expression and moderate HER3 expression; (2) It has a superior antitumor effect against the human non-small cell lung cancer cell line NCI-H441, which has moderate EGFR expression and moderate HER3 expression; (3) It has a superior antitumor effect against the human oral squamous cell carcinoma cell line CAL-27, which is characterized by high EGFR expression and low HER3 expression; (4) It exhibits superior antitumor efficacy in a human esophageal cancer OE-19 subcutaneous xenograft model that is resistant to EGFR ADCs; (5) In a human skin cancer A431 subcutaneous xenograft model, it exhibits superior antitumor effects accompanied by high EGFR expression and low HER3 expression; (6) In a human colon cancer SW620 subcutaneous xenograft model that does not express EGFR but expresses only HER3, it exhibits superior antitumor efficacy; (7) In a human colon cancer SW48 subcutaneous xenograft model, it exhibits a superior antitumor effect accompanied by moderate EGFR expression and low HER3 expression; and (8) It exhibits superior antitumor efficacy in NCI-H1975 subcutaneous xenograft models of non-small cell lung cancer (EGFR L858R / T790M / C797S triple mutation) that are resistant to third-generation TKIs. [Brief explanation of the drawing]

[0189] [Figure 1] This demonstrates the binding of bispecific antibodies and their corresponding parental mAbs to cell lines with different EGFR / HER3 expression levels. [Figure 2] This demonstrates the binding of bispecific antibodies and their corresponding parental mAbs to cell lines with different EGFR / HER3 expression levels. [Figure 3] This shows the endocytosis activity of the bispecific antibody and its corresponding parent mAb (pHrodo method). [Figure 4] The endocytosis activity of the bispecific antibody, its corresponding parent mAb, and the control antibody is shown (incucyte method). [Figure 5] The endocytosis activity of the bispecific antibody, its corresponding parent mAb, and the control antibody is shown (incucyte method). [Figure 6] This study evaluates the in vivo efficacy of antibody-drug conjugates in mice carrying the human esophageal cancer cell line OE-19, which exhibits low EGFR expression and moderate HER3 expression. [Figure 7] This study evaluates the in vivo efficacy of antibody-drug conjugates in mice carrying the human non-small cell lung cancer cell line NCI-H441, which exhibits moderate EGFR and HER3 expression. [Figure 8] This study evaluates the in vivo efficacy of antibody-drug conjugates in mice possessing the human oral squamous cell carcinoma cell line CAL-27, characterized by high E GFR expression and low HER3 expression. [Figure 9] This study evaluates the in vivo efficacy of bispecific antibody-drug conjugates with different DAR values ​​in mice carrying the NCI-H1975 tumor. [Figure 10] This study evaluates the in vivo efficacy of bispecific antibody-drug conjugates with different DAR values ​​in OE-19 tumor-bearing mice. [Figure 11] This document compares the in vivo efficacy of the bispecific antibody-drug conjugates and irradiant antibody-drug conjugates of this disclosure in NCI-H1975 tumor-bearing mice. [Figure 12] This document compares the in vivo efficacy of the bispecific antibody-drug conjugates and control antibody-drug conjugates described herein in OE-19 tumor-bearing mice. [Figure 13] This document demonstrates the in vivo efficacy of the bispecific antibody drug of this disclosure and the linker-cytotoxin X1 conjugate in NCI-H1975 tumor-bearing mice. [Figure 14] This disclosure demonstrates the in vivo efficacy of the bispecific antibody drug conjugate in human esophageal cancer OE-19-bearing mice. [Figure 15] This disclosure demonstrates the in vivo efficacy of the bispecific antibody-drug conjugate in mice with human skin cancer A431 xenograft tumors accompanied by high EGFR expression and low HER3 expression. [Figure 16] This disclosure demonstrates the in vivo efficacy of the bispecific antibody-drug conjugate in mice carrying xenograft tumors of the human colon cancer cell line SW620, which do not express EGFR but express only HER3. [Figure 17] This disclosure demonstrates the in vivo efficacy of the bispecific antibody-drug conjugate in mice with xenograft tumors of the human colon cancer cell line SW48, characterized by moderate EGFR expression and low HER3 expression. [Figure 18] This disclosure demonstrates the in vivo efficacy of the bispecific antibody-drug conjugate in mice with xenograft tumors of non-small cell lung cancer NCI-H1975 (EGFR L858R / T790M / C797S triple mutation). [Modes for carrying out the invention]

[0190] This disclosure is further illustrated by the following embodiments. However, these embodiments should not be construed as limiting this disclosure. Experimental procedures that do not specify conditions in the following embodiments were carried out according to conventional procedures and conditions or according to the product instructions.

[0191] Sample analysis This disclosure includes all combinations of the specific embodiments described herein. Further embodiments and the full scope of applicability of this disclosure will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples are provided for illustrative purposes only, as while they illustrate preferred embodiments of this disclosure, various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from the detailed description. All publications, patents, and patent applications cited herein, including the cited documents, are incorporated herein by reference in their entirety for all purposes. [Examples]

[0192] The following embodiments are provided to illustrate and further describe some preferred embodiments and aspects of the present disclosure and should not be construed as limiting the scope of the present disclosure.

[0193] Example 1: Preparation of bispecific antibodies In the anti-EGFR × HER3 bispecific antibodies of this disclosure, the anti-HER3 antibody or its antigen-binding fragment was prepared with reference to clone 2 of hu3F8 (PCT / CN2022 / 098929), and the anti-EGFR antibody or its antigen-binding fragment was prepared with reference to Zalutumumab (WO2002100348). Meanwhile, mutations were introduced into some amino acids in the CDRs / FRs / Fc regions of these antibodies. The CDRs of the bispecific antibodies were determined according to the Kabat numbering scheme.

[0194] PCR primers were designed to construct the VH / VK gene fragment according to the amino acid sequence of the antibody variable region, and the variable region was obtained. Next, the antibody variable region was conjugated with the constant region gene fragment to construct an intact bispecific antibody sequence. After transfection of CHO cells, anti-EGFR mAbs and anti-HER3 mAbs, as well as bispecific antibodies DBXT001-01~08, DBXT002-01~08, DBXT003-01~08, DBXT004-01~08, and DBXT005-01 were obtained according to conventional expression and purification methods.

[0195] The mAb cetuximab was prepared using conventional methods. The control antibody SI-1X6.4 was prepared according to WO2023083381A1.

[0196] The names and sequences of the antibodies are shown in Table 1. [Table 1-1] [Table 1-2]

[0197] Antibody expression and purification 1. CHO-K1 cells from ECACC were thawed, inoculated into culture medium (Shanghai OPM Biosciences Co., Ltd., Cat. No. C673017), and subcultured at 37°C and 8% CO2. During transfection, the cell density was 6.0 × 10⁶. 6 Prepared to cells / mL. 1.45 × 10 8 Individual cells were taken, centrifuged, and the supernatant was removed.

[0198] 2. Add 0.5 mL of electroporation buffer to the cell pellet and mix thoroughly.

[0199] 3. Four types of plasmids in the ratio H1:L1:H2:L2=2:3:2:3 were added, the cell-plasmid suspension was thoroughly mixed, placed in an electroporation cuvette, and then set in an electroporation apparatus for electroporation.

[0200] 4. After electroporation was complete, the cells in the electroporated cuvette were transferred to a shaking lasco containing 20 mL of culture medium and incubated at room temperature for 40 minutes without shaking.

[0201] 5. After the culture was complete, the shaking flask was placed in a shaker and cultured at 37°C, 110 rpm, and 8% CO2. After 24 hours, feed / sodium butyrate / antibiotics (penicillin + streptomycin) were added, and the culture was continued for another 4 days.

[0202] 6. Four days after transfection, the supernatant was centrifuged and collected. It was purified by elution using a protein A affinity chromatography column (Bestchrom) and sodium acetate buffer (pH 3.4), collected in separate tubes, and subjected to SEC analysis.

[0203] 7. The fractionated eluates that met the conditions were further purified by gel filtration chromatography (Bestchrom 200, specifications: 16 mm × 700 mm) and elution with 1 × PBS, collected in separate tubes, and subjected to SEC analysis. The eluates in the separate tubes that met the conditions were buffered with buffer solution (25 mM H Is-HCl, 6% sucrose, pH 6.0) and stored at -20°C.

[0204] Example 2: Antibody affinity analysis (SPR) 2.1 Objective: The affinity between anti-EGFR / HER3 BsAbs and parent mAbs was measured using Biacore T200 (Cytiva).

[0205] Experimental procedure: 1. EGFR-HER3 BsAb (1 μg / mL) was captured using a ProA tip. The subjects of analysis were as follows: human EGFR (His-Avi Tag, Kactus Biosystem, EGF-HM401) serially diluted to a total of 8 concentrations ranging from 3.125 to 400 nM; human HER3 (His-Avi Tag, Kactus Biosystem, HER-HM403) serially diluted to a total of 8 concentrations ranging from 6.25 to 200 nM; monkey EGFR (rhesus monkey EGFR, C-His Tag, Kactus Biosystem, EGF-CM101) serially diluted to a total of 8 concentrations ranging from 6.25 to 400 nM; and monkey HER3 (rhesus monkey HER3, C-His Tag, Kactus Biosystem, HER-CM403) serially diluted to a total of 8 concentrations ranging from 3.125 to 200 nM.

[0206] 2. EGFR parent mAb (1 μg / mL) was captured using a ProA tip. The analyte was human EGFR (His-Avi Tag, Kactus Biosystem, EGF-HM401) serially diluted to a total of 8 concentrations ranging from 6.25 to 400 nM. HER3 parent mAb (3 μg / mL) was captured using a ProA tip. The analyte was human HER3 (His-Avi Tag, Kactus Biosystem, HER-HM403) serially diluted to a total of 8 concentrations ranging from 3.125 to 100 nM.

[0207] 3. Affinity data was 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). The affinity results are shown in Table 2. [Table 2]

[0208] Experimental conclusions: The affinity of BsAb DBXT001-01 for human EGFR was approximately 16 times weaker than its affinity for HER3. The affinity of BsAb DBXT001-01 for human EGFR was approximately 3 times weaker than its affinity for the parental EGFR antibody, and its affinity for HER3 was comparable to that of the parental HER3 antibody. Furthermore, the affinity of BsAb DBXT001-01 for human EGFR or HER3 was equivalent to its affinity for monkey EGFR or HER3. Such affinity design of BsAb can effectively reduce the toxicity of BsAb to normal tissues that widely express EGFR.

[0209] 2.2 Antibody affinity analysis (SPR) Objective: We measured the affinity of anti-EGFR / HER3 BsAbs using Biacore T200 (Cytiva).

[0210] Experimental procedure: 1. SI-1X6.4 (7.5 μg / mL) was captured using a ProA tip. The analytes were: human EGFR (His-Avi Tag, Kactus Biosystem, EGF-HM401) serially diluted to a total of 5 concentrations in the range of 1.17 to 18.75 nM; and human HER3 (His-Avi Tag, Kactus Biosystem, HER-HM403) serially diluted to a total of 5 concentrations in the range of 4.69 to 75 nM. Similarly, DBXT005-01 (6 μg / mL) was captured using a ProA tip. The analytes were: human EGFR (His-Avi Tag, Kactus Biosystem, EGF-HM401) serially diluted to a total of 5 concentrations in the range of 9.38 to 150 nM; and human HER3 (His-Avi Tag, Kactus Biosystem, HER-HM403) serially diluted to a total of 5 concentrations in the range of 4.69 to 75 nM.

[0211] 2. Affinity data was 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). The affinity results are shown in Table 3. [Table 3]

[0212] Experimental conclusions: The affinity of the BsAb DBXT005-01 of this disclosure for human EGFR was approximately 15 times weaker than that of the control antibody SI-1X6.4, and its affinity for HER3 was 25 times stronger than that of the control antibody SI-1X6.4. Such a BsAb affinity design can reduce the appropriate toxicity of BsAbs to normal tissues that broadly express EGFR, and increase affinity to tumors with high EGFR expression and low HER3 expression.

[0213] Example 3: Binding of BsAb and its corresponding parental mAb to cell lines with different EGFR / HER3 expression levels. 3.1 Objectives of the analysis: Flow cytometry will be used to compare the affinity of DBXT001-01 and its parent mAb to cell lines with different EGFR / HER3 expression levels.

[0214] Experimental procedure: 1. We established a mouse CT26 monoclonal cell line (Cobioer Biosciences) that stably expresses human EGFR and HER3. Tumor cell lines NCI-H441, NCI-H1975, and T47D were obtained from ATCC.

[0215] 2. All cell lines were cultured in complete medium at 37°C and 5% CO2.

[0216] 3. Cells were collected during the logarithmic growth phase, and cell viability, measured by trypan blue exclusion, remained above 90%. The cells were centrifuged at 1000 r / min for 5 minutes, and the supernatant was discarded. The cells were washed once with PBS and resuspended in FACS buffer to obtain a single-cell suspension. The cell density was 5 × 10⁶. 6 The concentration was adjusted to cells / mL.

[0217] 50 μL of cell suspension was added to each well of a 4.96-well plate. The diluted standard solution of the test sample was diluted fivefold to an initial concentration of 100 nM, resulting in a total of six concentrations, which were added to the plate. The mixture was thoroughly mixed and incubated at 4°C for 40 minutes.

[0218] 5. The cells were washed three times with 400 μL of FACS buffer each time, then centrifuged at 1000 r / min for 5 minutes, and finally resuspended in 100 μL of FACS buffer.

[0219] 6.2 μL of PE-labeled secondary antibody (PE anti-human IgG Fc antibody) was added, the mixture was thoroughly mixed, and incubated at 4°C in the dark for 40 minutes.

[0220] 7. The cells were washed three times with 400 μL of FACS buffer each time, then centrifuged at 1000 r / min for 5 minutes, and finally resuspended in 250 μL of FACS buffer.

[0221] 8. Fluorescence values ​​were detected using a flow cytometer.

[0222] The experimental results are shown in Figure 1 and Table 4. [Table 4]

[0223] Conclusion of the experiment: The affinity of DBXT001-01 to cell lines overexpressing either EGFR or HER3 alone was lower than that of the bivalent parent mAb, and its affinity to EGFR-overexpressing cell lines was also lower than that of cetuximab. However, the affinity of DBXT001-01 to tumor cell lines expressing both EGFR and HER3 was not weaker than that of the bivalent parent mAb. This suggests that BsAbs may help enhance binding to tumor tissues expressing both EGFR and HER3 while reducing binding to normal tissues.

[0224] 3.2 Affinity of different BsAbs to cell lines with different EGFR / HER3 expression levels Experimental Objective: To compare the affinity of different BsAbs and parental mAbs to cell lines with different EGFR / HER3 expression levels using flow cytometry.

[0225] Experimental procedure: 1. Tumor cell lines BT-474 and MDA-MB-468 were obtained from ATCC, and mouse CT26 monoclonal cell lines stably expressing human EGFR or human HER3 were obtained from Cobioer Biosciences.

[0226] 2. All cell lines were cultured in complete medium at 37°C and 5% CO2.

[0227] 3. Cells were collected during the logarithmic growth phase, and cell viability, measured by trypan blue exclusion, remained above 90%. The cells were centrifuged at 1000 r / min for 5 minutes, and the supernatant was discarded. The cells were washed once with PBS and resuspended in FACS buffer to obtain a single-cell suspension. The cell density was 5 × 10⁶. 6 The concentration was adjusted to cells / mL.

[0228] 50 μL of cell suspension was added to each well of a 4.96-well plate. The diluted standard solution of the test sample was diluted fourfold to an initial concentration of 150 nM, resulting in a total of eight concentrations, which were added to the plate. The mixture was thoroughly mixed and incubated at 4°C for 40 minutes.

[0229] 5. The cells were washed three times with 400 μL of FACS buffer each time, then centrifuged at 1000 r / min for 5 minutes, and finally resuspended in 100 μL of FACS buffer.

[0230] 6.2 μL of PE-labeled secondary antibody (PE anti-human IgG Fc antibody) was added, the mixture was thoroughly mixed, and incubated at 4°C in the dark for 40 minutes.

[0231] 7. The cells were washed three times with 400 μL of FACS buffer each time, then centrifuged at 1000 r / min for 5 minutes, and finally resuspended in 250 μL of FACS buffer.

[0232] 8. Fluorescence values ​​were detected using a flow cytometer.

[0233] The codes for the test samples are shown in Table 5.

[0234] The experimental results are shown in Figure 2 and Table 6. [Table 5] [Table 6]

[0235] Experimental conclusions: DBXT005-01 demonstrated a strong ability to saturate target antigens in cell lines with different EGFR and HER3 expression levels compared to the control antibody SI-1X6.4. It also demonstrated a strong antigen-saturating ability compared to the corresponding parent mAb.

[0236] Example 4: Endocytosis activity of antibodies (pHrodo method) Analysis purpose The endocytotic effects of the antibody drugs of this disclosure, which target EGFR and HER3, were analyzed on T47D cells expressing both EGFR and HER3, and their endocytotic activity was compared with that of parent mAbs. Cells were co-cultured with a constant concentration of the antibody drug and the endocytosis indicator pHrodo, and the endocytotic activity of the antibody drug was evaluated by observing the intracellular fluorescence signals produced by pHrodo, which entered the cells together with the antibody drug at different time points.

[0237] Experimental Procedure 1. Cell culture: T47D cells were cultured in RPMI-1640 medium containing 0.2 units / mL of bovine insulin and 10% FBS.

[0238] 2. Cell preparation: Logarithmic T47D cells were harvested, washed once with PBS, and digested for 2-3 minutes. After the cells were completely digested, 10-15 mL of cell culture medium was added to eluate the digested cells. The eluate was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The obtained cells were resuspended in cell culture medium, and the viable cell density was set to 3 × 10⁶. 5 A single-cell suspension was obtained, adjusted to the desired cell / mL concentration.

[0239] 3. Cell Plating: The cell suspension was added to 96-well cell culture plates at a rate of 50 μL / well. The culture plates were incubated in an incubator for 48 hours (37°C, 5% CO2).

[0240] 4. Sample addition: The test antibody drug was co-cultured with Fab-pHrodo to form a complex, which was then adjusted to a concentration of 6 nM or 60 nM and added to cells at a rate of 50 μL / well.

[0241] 5. Cell culture: The culture plates were incubated in an incubator for 48 hours (37°C, 5% CO2).

[0242] 6. Plate reading: At the corresponding time point, the 96-well cell culture plate was removed, the cells were digested, and the cell count and fluorescence values ​​were read using FACS.

[0243] The experimental results are shown in Figure 3.

[0244] Conclusion of the experiment: The antibody drugs of this disclosure, which target EGFR and HER3, showed superior endocytosis efficacy against T47D cells expressing both EGFR and HER3 compared to the parental MAB.

[0245] Example 5: Endocytosis activity of antibodies (Incucyte method) 5.1 Purpose of analysis: The endocytosis efficiency of the antibody drugs of this disclosure targeting EGFR and HER3 was analyzed in NCI-H1975 cell lines expressing both EGFR and HER3, and in CT26 cell lines overexpressing both EGFR and HER3, compared with another antibody drug targeting EGFR and HER3. Cells were co-cultured with a constant concentration of the antibody drug conjugate and the endocytosis indicator reagent Incucyte® Fabfluor-pH, and the endocytic activity of the antibody drug conjugate was evaluated by continuously observing changes in the fluorescence signal of live cells for 48 hours.

[0246] Experimental procedure: 1. Cell Culture: NCI-H1975 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. The medium for CT26 cell lines stably transfected with both EGFR and HER3 was 1640 + 10% FBS + 10 μg / mL puromycin + 20 μg / mL blasticidin.

[0247] 2. Cell preparation: Cells were collected during the logarithmic growth phase, washed once with PBS, and then digested for 2-3 minutes. After the cells were completely digested, 10-15 mL of cell culture medium was added to eluate the digested cells. The eluate was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The obtained cells were resuspended in cell culture medium, and the viable cell density was set to 1 × 10⁶. 5 A single-cell suspension was obtained, adjusted to the desired cell / mL concentration.

[0248] 3. Cell Plating: The cell suspension was added to 96-well cell culture plates at a rate of 50 μL / well. The plates were incubated overnight in an incubator (37°C, 5% CO2).

[0249] 4. Labeling of test antibody-drug conjugates: For high-concentration antibody labeling, the stock solution of the test sample was diluted with a 240 nM 4x dilution standard solution (final concentration 60 nM), and the stock solution of Incucyte® Fabfluor-pH was diluted with a 720 nM 4x dilution standard solution (final concentration 180 nM). The two dilution standard solutions were then thoroughly mixed and incubated at 37°C in the dark for 15 minutes. For low-concentration antibody labeling, the stock solution of the test sample was diluted with a 40 nM 4x dilution standard solution (final concentration 10 nM), and the stock solution of Incucyte® Fabfluor-pH was diluted with a 120 nM 4x dilution standard solution (final concentration 30 nM). The two dilution standard solutions were then thoroughly mixed and incubated at 37°C in the dark for 15 minutes.

[0250] 5. Acquisition of analytical images: Labeled diluted standard solutions of the test samples were transferred to the corresponding wells of the experimental plate. The experimental plate was then transferred to the Incucyte live cell analyzer. The scanning and photography programs were set up, and images were captured using the Incucyte live cell analyzer. Quantification was performed at 2-hour intervals over 48 hours. The analysis results were expressed as total fluorescence integrated intensity (RCU X μm). 2 (represented by / image)

[0251] The experimental results are shown in Figures 4A and 4B.

[0252] Conclusion of the experiment: The antibody drugs of this disclosure, targeting EGFR and HER3, showed superior endocytotic efficacy compared to the parent mAb in cells expressing both EGFR and HER3, at both high and low concentrations. Simultaneously, the antibody drugs of this disclosure, targeting EGFR and HER3, showed superior endocytotic efficacy compared to the control antibody SI-1X6.4, at both high and low concentrations.

[0253] 5.2 Endocytosis of different BsAbs in cell lines with different EGFR / HER3 expression levels (Incucyte method) Purpose of analysis: The endocytosis efficiency of the antibody drugs of this disclosure targeting EGFR and HER3 was analyzed in A431 and NCI-H1975 cell lines with different EGFR and HER3 expression levels, as well as in the CT26 cell line overexpressing both EGFR and HER3, compared with another antibody drug targeting EGFR and HER3. Cells were co-cultured with a constant concentration of the antibody-drug conjugate and the endocytosis indicator reagent Incucyte® Fabfluor-pH, and the endocytic activity of the antibody-drug conjugate was evaluated by continuously observing changes in the fluorescence signal of live cells for 24 hours.

[0254] Experimental procedure: 1. Cell Culture: A431 cells were cultured in DMEM medium containing 10% FBS. NCI-H1975 cells were cultured in RPMI1640 medium containing 10% FBS. The medium for the CT26 cell line, which was stably transfected with both EGFR and HER3, was 1640 + 10% FBS + 10 μg / mL puromycin + 20 μg / mL blasticidin.

[0255] 2. Cell preparation: Cells were collected during the logarithmic growth phase, washed once with PBS, and then digested for 2-3 minutes. After the cells were completely digested, 10-15 mL of cell culture medium was added to eluate the digested cells. The eluate was centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The obtained cells were resuspended in cell culture medium, and the viable cell density was set to 1 × 10⁶. 5 A single-cell suspension was obtained, adjusted to the desired cell / mL concentration.

[0256] 3. Cell Plating: The cell suspension was added to 96-well cell culture plates at a rate of 50 μL / well. The plates were incubated overnight in an incubator (37°C, 5% CO2).

[0257] 4. Labeling of antibody-drug conjugates: Diluted standard solutions of the test sample / control sample and a diluted standard solution of the labeling reagent were mixed in a molar ratio of 1:3 and a volume ratio of 1:1. The mixture was then cultured in a cell culture incubator (37°C, 5% CO2) in the dark for 15 minutes to allow sufficient binding.

[0258] 5. Acquisition of analytical images: Labeled diluted standard solutions of the test samples were transferred to the corresponding wells of the experimental plate. The experimental plate was then transferred to the Incucyte live cell analyzer. The scanning and photography programs were set up, and images were captured using the Incucyte live cell analyzer. Quantification was performed at 2-hour intervals over 24 hours. The analysis results were expressed as total fluorescence integrated intensity (RCU X μm). 2 (represented by / image)

[0259] The experimental results are shown in Figures 5A, 5B, and 5C, and in Table 7. [Table 7]

[0260] Conclusion of the experiment: The EGFR and HER3-targeting antibody DBXT005-01 of this disclosure showed superior endocytosis efficacy against tumor cells with different EGFR or HER3 expression levels compared to the control antibody.

[0261] Example 6: Preparation of bispecific antibody-drug conjugates (ADCs) 6.1 Preparation of Linker-Cytotoxins (Linker-Loaded Drugs) Linker - Preparation of onboard drug X1 [ka]

[0262] Step 1 Benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a solution of 27a (5.00 g, 43.0 mmol) and NaHCO3 (10.9 g, 129 mmol) in DMF (50 mL) under a nitrogen atmosphere, and the mixture was reacted at 25°C for 17 hours. After the reaction was complete as detected by TLC (PE / EA=2 / 1), the reaction mixture was added to water (500 mL) and extracted twice with EA (250 mL). The organic phase was separated, washed with saturated sodium chloride aqueous solution (500 mL), dried on anhydrous Na2SO4, concentrated, and the residue was subjected to column chromatography (PE:EA=3:2) to obtain a colorless liquid (5.1 g, yield: 57.1%).

[0263] Step 2 A 10 mL solution of 27b (4.50 g, 21.8 mmol) in THF was added dropwise to a 30 mL solution of KI2 (4.00 g, 10.9 mmol) and TsOH (800 mg, 4.65 mmol) in THF under a nitrogen atmosphere at 0°C, and the mixture was reacted at 25°C for 2 hours. After the reaction was complete as detected by TLC (PE / EA=1 / 2), the reaction solution was added to water (200 mL) and extracted twice with EA (200 mL). The organic phase was separated, dried on anhydrous Na2SO4, concentrated, and the residue was subjected to column chromatography (PE / EA=3 / 2) to obtain a white solid (1.56 g, yield: 26%).

[0264] Step 3 Pd / C (80 mg) was added to a mixed solution of 27c (800 mg, 1.55 mmol) in EtOH (8 mL) / EA (8 mL) under a hydrogen atmosphere at 0°C, and the mixture was stirred at 0°C for 2.5 hours. After the reaction was complete as detected by LC-MS, the reaction solution was filtered through Celite, and the filter cake was washed with EA (200 mL). The filtrate was concentrated, and the residue was dissolved in THF (20 mL). The resulting solution was concentrated in a rotary evaporator until dry to obtain a white solid (600 mg, yield: 91%).

[0265] Step 4 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) under a nitrogen atmosphere at 0°C, and the mixture was reacted at 0°C for 2 hours. After the reaction was complete as detected by LC-MS, the reaction solution was added to an aqueous citric acid solution (pH=4) (150 mL), and the mixture was filtered. The filtered cake was washed with water (175 mL), dried by filtration, and dried with an oil pump to obtain a brown solid (260 mg, yield: 66%).

[0266] Step 5 Diethylamine (8 mL) was added dropwise to a 27e (260 mg, 0.309 mmol) DCM (30 mL) solution at 0°C under a nitrogen atmosphere, and the mixture was reacted at 0°C for 3 hours. After the reaction was complete as detected by LC-MS, the reaction solution was added to a petroleum ether solution (600 mL) at 0°C to precipitate a solid. The resulting mixture was allowed to stand until the solid was adsorbed to the bottom of the flask, and then the solution was poured out. The residue was dried with an oil pump to obtain a brown solid (90 mg, yield: 47.1%).

[0267] Step 6 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) under a nitrogen atmosphere at 0°C, and the mixture was reacted at 0°C for 2 hours. After the reaction was substantially complete as detected by LC-MS, the reaction solution was added to an aqueous solution of citric acid at pH 4 (30 mL) at 0°C, and the agglomerating solid was precipitated. The resulting mixture was filtered and subjected to preparative chromatography (DCM / MecOH = 10 / 1) to obtain X1 as a pale yellow solid (9.2 mg, yield: 6%).

[0268] MS m / z(ESI):1074[M+1].

[0269] 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).

[0270] Linker - Preparation of the mounted drug X2 [ka]

[0271] Step 1 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 to the resulting solution. The mixture was reacted at 25°C for 17 hours. After the starting materials were completely consumed as detected by TLC (PE / EA=3 / 1), the reaction solution was added to water (200 mL) and extracted with EA (250 mL). The organic phase was separated, washed with saturated NaCl, dried over anhydrous Na2SO4, concentrated, and the residue was subjected to column chromatography (PE:EA=2:1) ​​to obtain 34b as a colorless liquid (8.7 g, 93% yield). MS-ESI: m / z 195.1 [M+H] + .

[0272] Step 2 34c (7.3g, 19.8 mmol) and TsOH (1.46g, 8.5 mmol) were dissolved in THF (20 mL), and the mixture was cooled to 0°C under a nitrogen atmosphere. A solution of 43b (7.7g, 39.6 mmol) in THF (10 mL) was added dropwise, and the mixture was reacted at 0°C for 2 hours. After most of the starting material had been consumed as detected by TLC (PE / EA=2 / 1), the reaction solution was poured into water (100 mL) and extracted with DCM (100 mL). The organic phase was separated, washed with saturated NaCl, dried on anhydrous Na2SO4, and subjected to column chromatography (PE / EA=1 / 1) to obtain 34d as a colorless viscous substance (3.9g, yield: 39%). MS-ESI: m / z 503.3[M+H] + .

[0273] Step 3 Pd / C (1 g, 10 wt.%) was added to a mixed solution of 34d (1.9 g, 3.78 mmol) in EtOH (100 mL) / EA (100 mL) under a hydrogen atmosphere at 0°C, and the mixture was reacted at 0°C for 3 hours. After the reaction was complete as detected by TLC (PE / EA = 2 / 1), the reaction solution was filtered through Celite, and the filter cake was washed with EA / EtOH (1:1, 100 mL x 3). The filtrate was concentrated. The residue was dissolved in THF (50 mL x 3), and the resulting solution was concentrated to dryness using a rotary evaporator. This procedure was repeated three times to obtain 34e as a gray solid (1 g, yield: 64%). MS-ESI: m / z 435.2 [M + Na] + .

[0274] Step 4 To a 20 mL 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), DIEA (303 mg, 2.35 mmol) was added dropwise under a nitrogen atmosphere at 0°C. The mixture was then reacted at 0°C for 2 hours. After the reaction was complete and detectable by LC-MS, the reaction solution was added dropwise to 300 mL of water. The mixture was stirred, allowed to stand for 5 minutes, filtered, and the filtered cake was dissolved in a 100 mL solution of DCM / MeOH (10:1). The resulting solution was dried and concentrated using a rotary evaporator until dry. The residue was mixed with silica gel and subjected to column chromatography (EA:MeOH = 30:1) to obtain 34f as a yellow solid (600 mg, yield: 77%). MS-ESI: m / z 830.3 [M+H] + .

[0275] Step 5 Diethylamine (5 mL) was added dropwise to a 34 f (150 mg, 0.18 mmol) DCM (5 mL) solution under a nitrogen atmosphere at 0°C, and the mixture was reacted at 0°C for 2 hours. After the reaction was complete, as detected by LC-MS, 6 x 100 mL solutions of petroleum ether were added to the reaction mixture. A solid precipitate formed. The resulting mixture was allowed to stand until the solid settled, and then the solution was poured out. The residue was dried using an oil pump, and 34 g was obtained as a white powder (120 mg, yield: 76%). The product content, as measured by LC-MS, was 70%. MS-ESI: m / z 608.3 [M+H] + .

[0276] Step 6 A 1 mL solution of HATU (45 mg, 0.118 mmol) in DMF was added to 34 g (60 mg, 0.099 mmol), 43 h (51 mg, 0.108 mmol), and 1 mL solution of DIEA (32 mg, 0.25 mmol) in DMF under a nitrogen atmosphere at 0°C, and the mixture was reacted at 0°C for 2 hours. After the starting materials were completely consumed as detected by LC-MS, the reaction solution was subjected to reverse-phase column chromatography (eluent: (MeCN / MeOH=1 / 1):H2O=60%:40%) to obtain X2 as a yellow solid (14.8 mg, yield 14%).

[0277] MS-ESI:m / z1062.4[M+H] + .

[0278] 1H NMR (400MHz, methanol-d4) δ7.69-7.61(m,2H),7.22-7.16(m,2H),7.16-7.09(m,3H),6.76(s,2H),5.70-5.64(m,1H),5.60( d,J=16.4Hz,1H),5.40-5.31(m,2H),5.26(d,J=19.0Hz,1H),4.65-4.50(m,7H),4.25-4.16(m,1H),3.87(d,J=16.7Hz,1H) ,3.83-3.76(m,3H),3.72(d,J=17.0Hz,2H),3.44(t,J=7.1Hz,2H),3.25-3.17(m,2H),3.10-3.02(m,1H),2.92-2.83(m,1 H),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).

[0279] Linker - Preparation of the mounted drug X3 [ka]

[0280] Step 1 Bromopropene (960 mg, 7.92 mmol) was added to a solution of 32a (2.00 g, 6.6 mmol) and K2CO3 (1.82 g, 13.2 mmol) in MeCN (20 mL), and the mixture was stirred at 20°C for 5 hours. After the reaction was complete as detected by TLC (PE / EA=1 / 2), the reaction solution was poured into water (100 mL), adjusted to pH 5, and extracted three times with EA (100 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated to dryness using a rotary evaporator, and the residue was purified by column chromatography (PE / EA=2 / 1) to obtain 32b as a white solid (1.83 g, yield: 81%).

[0281] Step 2 TFA (10 mL) was added to a solution of 32b (1.38 g, 4.02 mmol) in DCM (10 mL), and the mixture was stirred at 25°C for 17 hours. After the reaction was complete as detected by TLC (PE / EA = 1 / 3), the reaction solution was concentrated in a rotary evaporator until dry, and 32c was obtained as a yellow viscous substance (0.91 g, yield not calculated).

[0282] Step 3 41d (1.92 g, 4.87 mmol) was added to a DME / H2O (20 mL / 10 mL) solution of 32c (910 mg, 4.87 mmol) and NaHCO3 (613 mg, 7.3 mmol), and the mixture was stirred at 25°C for 3 hours. After the reaction was complete as detected by TLC (DCM / MeOH=1 / 1), the reaction solution was poured into water (100 mL), adjusted to pH 5 with aqueous HCl (1N), and extracted twice with EA (150 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness using a rotary evaporator. The residue was purified by column chromatography (DCM / MeOH=20 / 1) to obtain 32e as a white solid (1.53 g, yield: 67%). MS-ESI: m / z 467.4 [M+H] + .

[0283] Step 4 Pd / C (600 mg) was added to a 32f (3 g, 5.83 mmol) MeOH (50 mL) solution, and the mixture was stirred at 25°C for 5 hours under a hydrogen atmosphere. After the reaction was complete as detected by TLC (EA), the reaction mixture was filtered, and the filtrate was concentrated to dryness using a rotary evaporator to obtain 32 g as a white solid (1.9 g, yield: 77%).

[0284] Step 5 HATU (707 mg, 1.86 mmol) was added to a solution of 32 g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol), and triethylamine (342 mg, 3.38 mmol) in DMF (10 mL), and the mixture was stirred at 0°C for 3.5 hours. After the reaction was complete as detected by TLC (EA), the reaction solution was poured into H2O (80 mL) and extracted twice with EA (100 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness using a rotary evaporator. The residue was purified by column chromatography (EA) to obtain 32 h as a white solid (1.186 g, yield: 83%). MS-ESI: m / z 842.3 [M+H] + .

[0285] Step 6 A 32h (1.186 g, 1.41 mmol) DCM / diethylamine (20 mL, 20 / 1) solution was stirred at 25°C for 17 hours. After the reaction was complete as detected by TLC (DCM / MeOH = 10 / 1), the reaction solution was poured into petroleum ether (200 mL). The resulting mixture was filtered to obtain 32i as a white solid (768 mg, yield: 88%). MS-ESI: m / z 620.3 [M+H] + .

[0286] Step 7 HATU (414 mg, 1.09 mmol) was added to a 10 mL 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), and the mixture was stirred at 20°C for 17 hours. After the reaction was complete as detected by TLC (PE / EA = 1 / 5), the reaction solution was poured into water (30 mL). The resulting mixture was filtered, and the filtered cake was purified by column chromatography (DCM / MeOH = 50 / 1) to obtain 32j as a white solid (511 mg, yield: 44%). MS-ESI: m / z 1068.3 [M+H] + .

[0287] Step 8 A solution of 32j (482 mg, 0.451 mmol) diethylamine / DCM (10 mL, 1 / 5) was stirred at 10°C for 17 hours. After the reaction was complete as detected by TLC(EA), the reaction solution was poured into PE (300 mL). The resulting mixture was filtered to obtain 32k as a white solid (301 mg, yield not calculated).

[0288] Step 9 Morpholine (93 mg, 1.07 mmol) was added to a solution of 32k (301 mg, 0.356 mmol) and Pd(PPh3)4 (82 mg, 0.071 mmol) in THF (5 mL), and the mixture was stirred at 25°C for 5 hours. After the reaction was complete, as detected by LC-MS, the reaction solution was subjected to preparative chromatography, and 32 L was obtained as a white solid (108 mg, yield: 38%). MS-ESI: m / z 806.3 [M+H] + .

[0289] Step 10 Bromoacetyl bromide (27 mg, 0.134 mmol) was added to 32 L (108 mg, 0.134 mmol) and triethylamine (41 mg, 0.402 mmol) in a THF (2 mL) / DMF (2 mL) solution, and the mixture was stirred at 0°C for 1 hour. After the reaction was complete as detected by TLC (DCM / MeOH = 10 / 1), the reaction solution was subjected to direct preparative chromatography to obtain X3 as a white solid (15 mg, yield: 12%).

[0290] MS-ESI:m / z926.3[M+H] + .

[0291] 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).

[0292] Linker - Preparation of onboard drug X4 [ka]

[0293] Step 1 Pd / C (400 mg, 10 wt.%) was added to 33a (2.00 g, 2.58 mmol) in MeOH (20 mL), and the mixture was stirred at 20°C for 5 hours. After the reaction was complete as detected by TLC(EA), the reaction solution was filtered, and the filtrate was concentrated in a rotary evaporator until dry to obtain 33b as a white solid (1.3 g, yield: 74%).

[0294] Step 2 HATU (305 mg, 0.802 mmol) was added 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), and the mixture was stirred at 0°C for 2 hours. After the reaction was complete and detectable by TLC (DCM / MeOH = 1 / 10), the reaction solution was poured into water (40 mL). The resulting mixture was filtered to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 20 / 1) to obtain 33c as a yellow solid (360 mg, yield: 41%).

[0295] Step 3 Diethylamine (2 mL) was added to a solution of 33c (360 mg, 0.326 mmol) in DCM (10 mL), and the mixture was stirred at 25°C for 17 hours. After the reaction was complete, as detected by TLC (DCM / MeOH = 5 / 1), the reaction solution was poured into PE (100 mL). The resulting mixture was filtered to obtain 33d as a white solid (205 mg, yield: 71%). MS-ESI: m / z 881.3 [M+H] + .

[0296] Step 4 A solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL) was added to a solution of 33d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) in water (1 mL), and the mixture was stirred at 0°C for 1 hour. The reaction solution was subjected to direct preparative chromatography to obtain X4 as a white solid (15 mg, yield: 6%).

[0297] MS-ESI:m / z1001.2[M+H] + .

[0298] 1 H 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).

[0299] 6.2 Preparation of Antibody-Drug Conjugates [Table 8]

[0300] Isotype IgG1. Source: Purchased from Biointron (Taicang).

[0301] Preparation of DBXT001-01-X1 [ka]

[0302] ADC-7 (DBXT001-01-X1-DAR4) To a buffer solution for antibody DBXT001-01 (PBS, pH 7.2; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM MEDTA solution (0.875 mL) and a prepared tris(2-carboxyethyl)phosphine hydrochloride solution (3.48 mM, 0.379 mL, 1.32 μmol) were added. The mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and reacted at 22°C for 2 hours, after which the reaction was stopped.

[0303] X1 (2.61 mg, 2.43 μmol) was dissolved in 1.75 mL of DMA, and the resulting solution was added to the above solution. The resulting mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and shaken at 22°C for 2 hours, after which the reaction was stopped. After filtration with activated carbon, the reaction solution was desalted and purified on an AKTA using a G-25 gel column (desalting column: HiPrep 26 / 10 Desalting column, 53 mL; eluent: 30 mM histidine-hydrochloride pH 5.5), and ultrafiltration concentration was performed using a 30 KD ultrafiltration tube to obtain a solution of exemplary product ADC-7 (30 mM histidine-hydrochloride pH 5.5; 68 mg, 22 mg / mL, yield: 97%), which was stored at -80°C.

[0304] HIC DAR analysis was performed, and the calculated DAR values ​​were obtained from n=4.06; the purity of the SEC main peak was 98.2%.

[0305] ADC-8 (DBXT001-01-X1-DAR6) To a buffer solution of antibody DBXT001-01 (PBS pH 7.2; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM MEDTA solution (0.875 mL) and a prepared tris(2-carboxyethyl)phosphine hydrochloride solution (3.48 mM, 0.598 mL, 2.08 μmol) were added. The mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and reacted at 22°C for 2 hours, after which the reaction was stopped.

[0306] X1 (4.30 mg, 4.00 μmol) was dissolved in 1.75 mL of DMA, and the resulting solution was added to the above solution. The resulting mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and reacted by shaking at 22°C for 2 hours, after which the reaction was stopped. After filtration with activated carbon, the reaction solution was desalted and purified on an AKTA using a G-25 gel column (desalting column: HiPrep 26 / 10 Desalting column, 53 mL; eluent: 30 mM histidine-hydrochloride pH 5.5), and ultrafiltration concentration was performed using a 30 KD ultrafiltration tube to obtain a solution of exemplary product ADC-8 (30 mM histidine-hydrochloride pH 5.5; 69 mg, 23 mg / mL, yield: 99%), which was stored at -80°C.

[0307] HIC DAR analysis was performed, and the calculated DAR values ​​were obtained from n=6.11; the purity of the SEC main peak was 99.2%.

[0308] ADC-9 (DBXT001-01-X1-DAR8) To a buffer solution of antibody DBXT001-01 (PBS pH 7.2; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM MEDTA solution (0.875 mL) and a prepared tris(2-carboxyethyl)phosphine hydrochloride solution (3.48 mM, 1.12 mL, 3.89 μmol) were added. The mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and reacted at 22°C for 2 hours, after which the reaction was stopped.

[0309] X1 (5.05 mg, 4.70 μmol) was dissolved in 1.75 mL of DMA, and the resulting solution was added to the above solution. The resulting mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and shaken at 22°C for 2 hours, after which the reaction was stopped. After filtration with activated carbon, the reaction solution was desalted and purified on an AKTA using a G-25 gel column (desalting column: HiPrep 26 / 10 Desalting column, 53 mL; eluent: 30 mM histidine-hydrochloride pH 5.5), and ultrafiltration concentration was performed using a 30 KD ultrafiltration tube to obtain a solution of exemplary product ADC-9 (30 mM histidine-hydrochloride pH 5.5; 68 mg, 22.4 mg / mL, yield: 97%), which was stored at -80°C.

[0310] HIC DAR analysis was performed, and the calculated DAR values ​​were obtained for n=7.99; the SEC main peak purity was 99.0%.

[0311] Preparation of DBXT001-01-X2 [ka]

[0312] ADC-4 (DBXT001-01-X2-DAR4) To a buffer solution of antibody DBXT001-01 (PBS pH 7.4; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM MEDTA solution (0.875 mL) and a prepared tris(2-carboxyethyl)phosphine hydrochloride solution (3.48 mM, 0.379 mL, 1.32 μmol) were added. The mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and reacted at 37°C for 2 hours, after which the reaction was stopped.

[0313] X2 (2.75 mg, 2.59 μmol) was dissolved in 1.75 mL of DMA, and the resulting solution was added to the above solution. The resulting mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and shaken at 22°C for 2 hours, after which the reaction was stopped. After filtration with activated carbon, the reaction solution was desalted and purified on an AKTA using a G-25 gel column (desalting column: HiPrep 26 / 10 Desalting column, 53 mL; eluent: 30 mM histidine-hydrochloride pH 5.5), and ultrafiltration concentration was performed using a 30 KD ultrafiltration tube to obtain a solution of exemplary product ADC-4 (30 mM histidine-hydrochloride pH 5.5; 67.2 mg, 22 mg / mL, yield: 96%), which was stored at -80°C.

[0314] HIC DAR analysis was performed, and the calculated DAR values ​​were obtained from n=4.10; the purity of the SEC main peak was 98.4%.

[0315] ADC-5 (DBXT001-01-X2-DAR6) To a buffer solution of antibody DBXT001-01 (PBS pH 7.4; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM MEDTA solution (0.875 mL) and a prepared tris(2-carboxyethyl)phosphine hydrochloride solution (3.48 mM, 0.598 mL, 2.08 μmol) were added. The mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and reacted at 37°C for 2 hours, after which the reaction was stopped.

[0316] X2 (4.58 mg, 4.31 μmol) was dissolved in 1.75 mL of DMA, and the resulting solution was added to the above solution. The resulting mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and shaken at 22°C for 2 hours, after which the reaction was stopped. After filtration with activated carbon, the reaction solution was desalted and purified on an AKTA using a G-25 gel column (desalting column: HiPrep 26 / 10 Desalting column, 53 mL; eluent: 30 mM histidine-hydrochloric acid pH 5.5), and ultrafiltration concentration was performed using a 30 KD ultrafiltration tube to obtain a solution of exemplary product ADC-5 (30 mM histidine-hydrochloric acid pH 5.5; 66.5 mg, 25 mg / mL, yield: 95%), which was stored at -80°C.

[0317] HIC DAR analysis was performed, and the calculated DAR values ​​were obtained from n=6.05; the SEC main peak purity was 98.9%.

[0318] ADC-6 (DBXT001-01-X2-DAR8) To a buffer solution of antibody DBXT001-01 (PBS pH 7.2; 70 mg, 8.52 mg / mL, 0.47 μmol), 10 mM MEDTA solution (0.875 mL) and a prepared tris(2-carboxyethyl)phosphine hydrochloride solution (3.48 mM, 1.12 mL, 3.89 μmol) were added. The mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and reacted at 22°C for 2 hours, after which the reaction was stopped.

[0319] X2 (5.49 mg, 5.17 μmol) was dissolved in 1.75 mL of DMA, and the resulting solution was added to the above solution. The resulting mixture was placed in a constant temperature stirrer at a stirring speed of 60 rpm and shaken at 22°C for 2 hours, after which the reaction was stopped. After filtration with activated carbon, the reaction solution was desalted and purified on an AKTA using a G-25 gel column (desalting column: HiPrep 26 / 10 Desalting column, 53 mL; eluent: 30 mM histidine-hydrochloride pH 5.5), and ultrafiltration concentration was performed using a 30 KD ultrafiltration tube to obtain a solution of exemplary product ADC-6 (30 mM histidine-hydrochloride pH 5.5; 67.9 mg, 29.2 mg / mL, yield: 97%), which was stored at -80°C.

[0320] HIC DAR analysis was performed, and the calculated DAR values ​​were n=7.98; the SEC main peak purity was 99.0%.

[0321] ADC-13 (DBXT005-01-X2, DAR6) The antibody DBXT005-01 was reduced with 20 equivalents of tris(2-carboxyethyl)phosphine (TCEP) in PBS7.4 buffer (PBS7.4, 2mMEDTA, pH7.4). The reaction was carried out at 22°C for 3 hours with shaking (shaking speed 60 rpm).

[0322] After intermediate monitoring, 5 equivalents of tris(2-carboxyethyl)phosphine (TCEP) were added, and the mixture was reacted at 22°C for 1 hour while shaking in a shaker. The reaction solution was used directly in the next complexation reaction without removing any excess TCEP.

[0323] To the reductive antibody, a 200 mM histidine hydrochloride solution (pH 6.0) in dimethyl sulfoxide containing 0.1% v / v acetate (so that the final concentration of the complex reaction solution was 20 mM histidine hydrochloride), and a linker-cytotoxin (linker-carrying drug) solution (10 equivalents, 10 mM x 2 solution (in dimethyl sulfoxide containing 0.1% v / v acetate)) were slowly added at room temperature. The reaction system contained 3% v / v dimethyl sulfoxide (DMSO) containing 0.1% v / v acetate. After thorough mixing, the complex reaction solution was reacted at 22°C for 1.5 hours with shaking (shaking speed 60 rpm).

[0324] After intermediate monitoring, linker-cytotoxin (linker-loaded drug) (2 equivalents, 10 mM x 2 solution (in 0.1% v / v dimethyl sulfoxide containing acetate)) was added. After thorough mixing, the complex reaction mixture was further reacted at 22°C for 2 hours with shaking in a shaker (shaking speed 60 rpm).

[0325] After the reaction was complete, the ADC solution was buffer-replaced using a desalting column (40K) with a storage buffer (20 mM histidine hydrochloric acid, pH 6.0). A sample was taken and sent for detection, yielding the ADC product.

[0326] The detection results are as follows: [Table 9]

[0327] ADC-14 (BL-B01D1, DAR8) was prepared based on patent WO2023083381A1, and the details are as follows:

[0328] The antibody SI-1X6.4 was reduced with 15 equivalents of tris(2-carboxyethyl)phosphine (TCEP) in PBS7.4 buffer (PBS7.4, 2mMEDTA, pH7.4). The reaction was carried out at 22°C for 17.5 hours with shaking (shaking speed 60 rpm). The reaction solution was used directly in the subsequent complexation reaction without removing excess TCEP.

[0329] Dimethyl sulfoxide (DMSO) and linker-cytotoxin (linker-loaded drug) (10 equivalents, 10 mM Ed-04 DMSO solution) were slowly added to the reductive antibody at room temperature. The reaction system contained 10% v / v dimethyl sulfoxide (DMSO). After thorough mixing, the complex reaction mixture was reacted at 22°C for 2 hours while shaking in a shaker (shaking speed 60 rpm).

[0330] After intermediate monitoring, the linker-cytotoxin (linker-loaded drug) (2 equivalents, 10 mM Ed-04 DMSO solution) was added. After thorough mixing, the complex reaction mixture was further reacted at 22°C for 1.5 hours with shaking in a shaker (shaking speed 60 rpm).

[0331] After the reaction was complete, 200 mM histidine hydrochloride pH 5.5 was added to the reaction mixture to achieve a final concentration of 20 mM histidine hydrochloride. Next, the reaction mixture was filtered using a 0.22 μm PES syringe filter. Subsequently, the ADC solution was buffer-exchanged with a storage buffer (20 mM histidine hydrochloride, pH 5.5) using an AKTA equipped with a desalting column (HiPrep® 26 / 10 Desalting). The reaction mixture was concentrated using Amicon (30K MWCO) and finally filtered using a 0.22 μm PES syringe filter to obtain the ADC product. Samples were collected and sent for detection.

[0332] The detection results are as follows: [Table 10]

[0333] ADC-1 to 3, ADC-10, and ADC-11 were prepared following the preparation steps of DBXT001-01-X2, except that the corresponding antibody clones in Table 8 were replaced.

[0334] By replacing antibody clone DBXT001-01 in Table 8 with DBXT001-02~08, DBXT003-01~08, and DBXT004-01~08, the corresponding bispecific antibody-drug conjugates can be prepared by referring to the experimental procedure described above.

[0335] Example 7: Evaluation of the in vivo efficacy of an antibody-drug conjugate in mice with the human esophageal cancer cell line OE-19, characterized by low EGFR expression and moderate HER3 expression. To investigate the inhibitory effect of ADC-6 on in vivo tumor formation induced by human esophageal cancer cell lines, mice were subcutaneously inoculated with OE-19 to induce xenograft tumors. The antitumor effect of ADC-6 was then evaluated and compared with that of parental MAb ADC.

[0336] 1. Test reagents and materials Blank control group (control group): Physiological saline ADC-1 (treatment group): Day 0: 1 mg / kg, Day 12: 3 mg / kg ADC-2 (treatment group): Day 0: 1 mg / kg, Day 12: 3 mg / kg ADC-6 (treatment group): Day 0: 1 mg / kg, Day 12: 3 mg / kg

[0337] 2. Preparation method: All samples were prepared by diluting them with physiological saline.

[0338] 3. Experimental animals: 6-7 week old female BALB / c nude mice, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0339] 4. Experimental procedure: OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine. Exponentially growing OE-19 cells were harvested and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into mice.

[0340] In the experimental mice, 5 × 10 6 Each mouse was subcutaneously inoculated with 0.1 mL of OE-19 cells (resuspended in a 1:1 mixture of PBS and Matrigel) into the right side of its back. Tumor growth was monitored periodically.

[0341] The tumor was approximately 140.15 mm in size. 3 Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector™. Intravenous (IV) injection of the test sample was started on the day of group assignment (day 0), with a total of two injections given on day 0 and day 12. The treatment group received 1 mg / kg on day 0 and 3 mg / kg on day 12. The experimental endpoint was 25 days after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0342] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0343] The tumor volume is calculated using the following formula: V = 0.5a × b 2 The formula was used to calculate the tumor's longest and shortest diameters (where a and b represent the tumor's longest and shortest diameters, respectively). The antitumor effect of the compound was evaluated as T / C (%). The percentage value of T / C (%) is an index that reflects the tumor growth inhibition rate, where T and C represent the average tumor volume on a specific day in the treatment group and the control group, respectively. The tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T iThe calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0344] Comparisons between the two groups of samples were performed using independent sample t-tests, and the data were analyzed using SPSS. A p-value of <0.05 indicates a statistically significant difference. Plots were created using GraphPad Prism software. [Table 11]

[0345] The experimental results are shown in Figure 6 and Table 9. The results showed that in the OE-19 model, mAb ADC-1 (EGFR-ADC) could not inhibit tumor growth, while BsAb ADC-6 (EGFR-HER3 ADC) had a superior antitumor effect compared to mAb ADC-2 (HER3-ADC).

[0346] Conclusion of the experiment: In an EGFR ADC resistance model, BsAb ADCs showed superior antitumor efficacy compared to mAb ADCs.

[0347] Example 8: Evaluation of the in vivo efficacy of an antibody-drug conjugate in mice possessing the human non-small cell lung cancer cell line NCI-H441 with moderate EGFR and HER3 expression. To investigate the inhibitory effect of ADC-6 on in vivo tumor formation induced by human non-small cell lung cancer cell lines, mice were subcutaneously inoculated with NCI-H441 to induce xenograft tumors. The antitumor effect of ADC-6 was then evaluated and compared with that of parental MAb ADC.

[0348] 1. Test reagents and materials Blank control group (control group): Physiological saline ADC-1 (treatment group): 0.5 mg / kg ADC-2 (treatment group): 0.0.5 mg / kg ADC-6 (treatment group): 0.0.5 mg / kg

[0349] 2. Preparation method: All samples were prepared by diluting them with physiological saline.

[0350] 3. Experimental animals: 6-7 week old male NU / NU mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0351] 4. Experimental procedure: NCI-H441 (CL-00759) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. NCI-H441 cells in the exponential growth phase were harvested and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into mice.

[0352] In the experimental mice, 5 × 10 6 Each mouse was subcutaneously inoculated with 0.1 mL of NCI-H441 cells (resuspended in a 1:1 mixture of PBS and Matrigel) into the right side of its back. Tumor growth was monitored periodically.

[0353] The tumor was approximately 139.12 mm in size. 3 Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector®. Intravenous (IV) injection of the test sample was started on the day of group assignment (day 0), with a total of one injection. The treatment group received a dose of 0.5 mg / kg on day 0. The experimental endpoint was 35 days after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0354] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0355] The tumor volume is calculated using the following formula: V = 0.5a × b 2The formula was used to calculate the tumor's longest and shortest diameters (where a and b represent the tumor's longest and shortest diameters, respectively). The antitumor effect of the compound was evaluated as T / C (%). The percentage value of T / C (%) is an index that reflects the tumor growth inhibition rate, where T and C represent the average tumor volume on a specific day in the treatment group and the control group, respectively. The tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0356] Comparisons between the two groups of samples were performed using independent sample t-tests, and the data were analyzed using SPSS. A p-value of <0.05 indicates a statistically significant difference. Plots were created using GraphPad Prism software. [Table 12]

[0357] The experimental results are shown in Figure 7 and Table 10. The results showed that in the NCI-H441 model, ADC-6 had a superior antitumor effect compared to ADC-2.

[0358] Conclusion of the experiment: In models with similar EGFR and HER3 expression levels, BsAb ADCs showed superior antitumor efficacy compared to mAb ADCs.

[0359] Example 9: Evaluation of in vivo efficacy of antibody-drug conjugate in mice possessing the human oral squamous cell carcinoma cell line CAL-27, characterized by high EGFR expression and low HER3 expression. To investigate the inhibitory effect of ADC-6 on in vivo tumor formation induced by human oral squamous cell carcinoma cell lines, mice were subcutaneously inoculated with CAL-27 to induce xenograft tumors. The antitumor effect of ADC-6 was then evaluated and compared with that of parental MAb ADC and anti-EGFR mAbs.

[0360] 1. Test reagents and materials Blank control group (control group): Physiological saline ADC-1 (treatment group): 3 mg / kg ADC-2 (treatment group): 3 mg / kg ADC-6 (treatment group): 3 mg / kg 3 mg / kg Anti-EGFR mAb zalutumumab (treatment group): 3 mg / kg

[0361] 2. Preparation method: All samples were prepared by diluting them with physiological saline.

[0362] 3. Experimental animals: Female NOD / Scid mice, 6-7 weeks old, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0363] 4. Experimental procedure: CAL-27 (CL-00599) cells were cultured in DMEM medium containing 10% fetal bovine serum. CAL-27 cells in the exponential growth phase were harvested and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into mice.

[0364] In the experimental mice, 1 × 10 7 Each mouse was subcutaneously inoculated with 0.1 mL of CAL-27 cells (resuspended in a 1:1 mixture of PBS and Matrigel) into the right side of its back. Tumor growth was monitored periodically.

[0365] The tumor was approximately 144.75 mm in size. 3Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector™. Intravenous (IV) injection of the test sample was started on the day of group assignment (day 0), with a total of two injections given on day 0 and day 14. The treatment group was administered 3 mg / kg. The experimental endpoint was 27 days after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0366] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0367] The tumor volume is calculated using the following formula: V = 0.5a × b 2 The formula was used to calculate the tumor's longest and shortest diameters (where a and b represent the tumor's longest and shortest diameters, respectively). The antitumor effect of the compound was evaluated as T / C (%). The percentage value of T / C (%) is an index that reflects the tumor growth inhibition rate, where T and C represent the average tumor volume on a specific day in the treatment group and the control group, respectively. The tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0368] Comparisons between the two groups of samples were performed using independent sample t-tests, and the data were analyzed using SPSS. A p-value of <0.05 indicates a statistically significant difference. Plots were created using GraphPad Prism software. [Table 13]

[0369] The experimental results are shown in Figure 8 and Table 11. The results showed that in the CAL-27 model, both ADC-1 and ADC-6 had significant antitumor effects and were superior to ADC-2 and anti-EGFR mAbs.

[0370] Conclusion of the experiment: In a high-EGFR expression + low-HER3 expression model sensitive to EGFR targeting, BsAb ADCs demonstrated superior antitumor efficacy compared to anti-EGFR mAbs or HER3 ADCs, and were not inferior to EGFR ADCs.

[0371] Example 10: Evaluation of in vivo efficacy of antibody-drug conjugates with different DAR values ​​in NCI-H1975 and OE-19 tumor-bearing mice. To investigate the inhibitory effects of DBXT001-01 and linker-cytotoxin X2 complexes with different DAR values ​​on in vivo tumor formation induced by human non-small cell lung cancer cell line NCI-H1975 and human esophageal cancer cell line OE-19, NCI-H1975 or OE-19 were subcutaneously ectopically inoculated into the right side of the back of mice to induce xenograft tumors, and then the antitumor effects of BsAb-toxin complexes with different DAR values ​​were evaluated.

[0372] The test drugs and administration regimens are shown in Tables 12 and 13. [Table 14] [Table 15]

[0373] 2. Preparation method: All samples were prepared by diluting them with physiological saline.

[0374] 3. Experimental animals: Balb / c nude mice aged 6-8 weeks, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0375] 4. Experimental procedure: NCI-H1975 (CL-00650) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum, and OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2 mML-glutamine. Cells were harvested during the exponential growth phase and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into nude mice.

[0376] In the experimental mice, 5 × 10 6 Each mouse was subcutaneously inoculated with 0.1 mL of NCI-H1975 cells (resuspended in PBS) into the right side of its back. Tumor growth was monitored periodically.

[0377] In the experimental mice, 5 × 10 6 Each mouse was subcutaneously inoculated with 0.1 mL of OE-19 cells (resuspended in a 1:1 mixture of PBS and Matrigel) into the right side of its back. Tumor growth was monitored periodically.

[0378] The tumor was approximately 121.71 mm in size. 3 (NCI-H1975) or 139.93mm 3 At the stage of growth (OE-19), tumor-bearing mice were randomly assigned to groups using StudyDirector™. The day of group assignment was designated as day 0. Specific administration regimens are shown in Tables 12 and 13. The experimental endpoint was defined as day 31 after group assignment (NCI-H1975) or day 35 after group assignment (OE-19). Tumor volume and body weight were measured twice a week, and the data were recorded.

[0379] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0380] The tumor volume is calculated using the following formula: V = 0.5a × b 2The formula was used to calculate the tumor's longest and shortest diameters (where a and b represent the tumor's longest and shortest diameters, respectively). The antitumor effect of the compound was evaluated as T / C (%). The percentage value of T / C (%) is an index that reflects the tumor growth inhibition rate, where T and C represent the average tumor volume on a specific day in the treatment group and the control group, respectively. The tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0381] Comparisons between the two groups of samples were performed using independent sample t-tests, and the data were analyzed using SPSS. A p-value of <0.05 indicates a statistically significant difference. Plots were created using GraphPad Prism software. [Table 16] [Table 17]

[0382] The experimental results are shown in Figures 9 and 10, and in Tables 14 and 10. The results showed that ADC-2, ADC-4, ADC-5, and ADC-6 all had antitumor effects in the NCI-H1975 model administration regimen.

[0383] Conclusion of the experiment: The BsAb complex of the present disclosure, which targets EGFR and HER3, and linker-cytotoxin X2, exhibits an antitumor effect, which was enhanced with increasing DAR levels.

[0384] Example 11: Comparison of in vivo effects of the antibody-drug conjugates and control antibody conjugates of this disclosure in NCI-H1975 tumor-bearing mice. To compare the inhibitory effects of the DBXT001-01-linker-cytotoxin X2 complex and the control antibody SI-1X6.4-linker-cytotoxin X2 complex on in vivo tumor formation by the human non-small cell lung cancer cell line NCI-H1975, NCI-H1975 was subcutaneously ectopically inoculated into the right side of the back of mice to form xenograft tumors, and the antitumor effects of the two complexes were then evaluated.

[0385] The test drugs and administration regimens are shown in Table 16. [Table 18]

[0386] 1. Preparation method: All samples were prepared by diluting them with physiological saline.

[0387] 2. Experimental animals: Balb / c nude mice aged 6-8 weeks, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0388] 3. Experimental Procedure NCI-H1975 (CL-00650) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Cells were harvested during the exponential growth phase and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into nude mice.

[0389] In the experimental mice, 5 × 10 6 Each mouse was subcutaneously inoculated with 0.2 mL of NCI-H1975 cells (resuspended in a 1:1 mixture of DPBS and Matrigel) into the right side of its back. Tumor growth was monitored periodically.

[0390] The tumor was approximately 164 mm in size. 3 Once the tumors had grown to the stage of (NCI-H1975), StudyDirector® was used to randomly assign the tumor-bearing mice to groups. The day of group assignment was designated as day 0. Specific administration regimens are shown in Table 16. The experimental endpoint was 26 days after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0391] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0392] The tumor volume is calculated using the following formula: V = 0.5a × b 2 The formula was used to calculate the tumor's longest and shortest diameters (where a and b represent the tumor's longest and shortest diameters, respectively). The antitumor effect of the compound was evaluated as T / C (%). The percentage value of T / C (%) is an index that reflects the tumor growth inhibition rate, where T and C represent the average tumor volume on a specific day in the treatment group and the control group, respectively. The tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0393] Comparisons between the two groups of samples were performed using independent sample t-tests, and the data were analyzed using SPSS. A p-value of <0.05 indicates a statistically significant difference. Plots were created using GraphPad Prism software. [Table 19]

[0394] The experimental results are shown in Figure 11 and Table 17. The results showed that in the NCI-H1975 model administration regimen, ADC-3, ADC-6, and ADC-10 all had significant antitumor effects, with ADC-6 showing the strongest antitumor effect, while ADC-3 and ADC-10 had comparable efficacy.

[0395] Conclusion of the experiment: The BsAb conjugate of the present disclosure, which targets EGFR and HER3, with linker-cytotoxin X2 (DAR8) exhibited a stronger antitumor effect compared to the conjugate of the control antibody SI-1X6.4 with linker-cytotoxin X2 (DAR8).

[0396] Example 12: Comparison of in vivo effects of the antibody-drug conjugates and control antibody conjugates of this disclosure in OE-19 tumor-bearing mice. The antitumor effects of conjugates of DBXT001-01 with linker-cytotoxin X2 or linker-cytotoxin X1, and conjugates of the control antibody SI-1X6.4 with linker-cytotoxin X2 against in vivo tumor formation induced by the human esophageal cancer cell line OE-19 were evaluated after inducing xenograft tumors by subcutaneous ectopic inoculation of OE-19 into the right side of the back of mice.

[0397] Table 18 shows the test drugs and administration regimens. [Table 20]

[0398] 1. Preparation method: All samples were prepared by diluting them with physiological saline.

[0399] 2. Experimental animals: Balb / c nude mice aged 6-8 weeks, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0400] 3. Experimental Procedure OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine.

[0401] In the experimental mice, 5 × 10 6 Each mouse was subcutaneously inoculated with 0.1 mL of OE-19 cells (resuspended in a 1:1 mixture of PBS and Matrigel) into the right side of its back. Tumor growth was monitored periodically.

[0402] The tumor was approximately 138 mm in size.3 Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector®. The day of group assignment was designated as day 0. The specific administration regimens are shown in Table 18. The experimental endpoint was 21 days after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0403] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0404] The tumor volume is calculated using the following formula: V = 0.5a × b 2 The formula was used to calculate the tumor's longest and shortest diameters (where a and b represent the tumor's longest and shortest diameters, respectively). The antitumor effect of the compound was evaluated as T / C (%). The percentage value of T / C (%) is an index that reflects the tumor growth inhibition rate, where T and C represent the average tumor volume on a specific day in the treatment group and the control group, respectively. The tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0405] Comparisons between the two groups of samples were performed using independent sample t-tests, and the data were analyzed using SPSS. A p-value of <0.05 indicates a statistically significant difference. Plots were created using GraphPad Prism software. [Table 21]

[0406] The experimental results are shown in Figure 12 and Table 19. The results showed that in the OE-19 model administration regimen, ADC-3, ADC-6, ADC-9, and ADC-10 all showed antitumor effects, with ADC-6 showing the strongest antitumor effect, and ADC-3, ADC-9, and ADC-10 having comparable efficacy.

[0407] Conclusion of the experiment: In a model showing resistance to EGFR-targeted therapy, the BsAb conjugate of the present disclosure, which targets EGFR and HER3, with linker-cytotoxin X2 (DAR8) was able to overcome EGFR resistance and exhibit a stronger antitumor effect compared to the conjugate of the control antibody SI-1X6.4 with linker-cytotoxin X2 (DAR8).

[0408] Example 13: Evaluation of the in vivo efficacy of an antibody drug and a linker-cytotoxin X1 conjugate in NCI-H1975 tumor-bearing mice. To investigate the inhibitory effect of the DBXT001-01 and linker-cytotoxin X1 (DAR8) complex on in vivo tumor formation induced by the human non-small cell lung cancer cell line NCI-H1975, NCI-H1975 was subcutaneously ectopically inoculated into the right side of the back of mice to induce xenograft tumors, and the antitumor effect of the antibody complex was then evaluated.

[0409] The test drugs and administration regimens are shown in Table 20. [Table 22]

[0410] 2. Preparation method: All samples were prepared by diluting them with physiological saline.

[0411] 3. Laboratory animals: NCI-H1975: Female CB-17 SCID cubs aged 6-8 weeks, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0412] 4. Experimental procedure: NCI-H1975 (Cell Bank of Chinese Academy of Sciences) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum. Cells in the exponential growth phase were harvested and resuspended in DPBS to a concentration suitable for subcutaneous tumor inoculation into nude mice.

[0413] In the experimental mice, 5 × 10 6 Each mouse was subcutaneously inoculated with 0.2 mL of NCI-H1975 cells (resuspended in a 1:1 mixture of DPBS and BD Matrigel (Cat. No. 356234)) into the right side of its back. Tumor growth was observed periodically.

[0414] The tumor was approximately 201.31 mm in size. 3 Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector®. The day of group assignment was designated as day 0. Specific administration regimens are shown in Table 20. The experimental endpoint was 20 days after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0415] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0416] The tumor volume is calculated using the following formula: V = 0.5a × b 2 The formula was used to calculate the tumor's longest and shortest diameters (where a and b represent the tumor's longest and shortest diameters, respectively). The antitumor effect of the compound was evaluated as T / C (%). The percentage value of T / C (%) is an index that reflects the tumor growth inhibition rate, where T and C represent the average tumor volume on a specific day in the treatment group and the control group, respectively. The tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0417] Comparisons between the two groups of samples were performed using independent sample t-tests, and the data were analyzed using SPSS. A p-value of <0.05 indicates a statistically significant difference. Plots were created using GraphPad Prism software. [Table 23]

[0418] The experimental results are shown in Figure 13 and Table 21. The results showed that in the NCI-H1975 model administration regimen, ADC-9 showed a significant antitumor effect, while the blank control group and ADC-12 did not show any antitumor effect.

[0419] Conclusion of the experiment: The antibody-pharmaceutical conjugate of the BsAb and linker-cytotoxin X1 described herein, targeting EGFR and HER3, showed a significant inhibitory effect on tumor growth.

[0420] Example 14: Comparison of in vivo efficacy of the BsAb ADC and control ADC of this disclosure in an EGFR ADC-resistant mouse model. To compare the in vivo efficacy of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) in an EGFR ADC resistance model, mice were subcutaneously inoculated with OE-19 to induce xenograft tumors, and then the antitumor effects of the two ADCs were compared.

[0421] 1. The test drugs and administration regimens are shown in Table 22. [Table 24]

[0422] 2. Preparation method: All samples were prepared by diluting them with physiological saline.

[0423] 3. Experimental animals: 6-7 week old female BALB / c nude mice, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0424] 4. Experimental procedure: OE-19 (CL-00806) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 2 mM L-glutamine. Exponentially growing OE-19 cells were harvested and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into mice.

[0425] In the experimental mice, 5 × 10 6 Each mouse was subcutaneously inoculated with 0.1 mL of OE-19 cells (resuspended in a 1:1 mixture of PBS and Matrigel) into the right side of its back. Tumor growth was monitored periodically.

[0426] The tumor was approximately 151 mm in size. 3 Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector™. Intravenous (IV) injection of the test sample was started on the day of group assignment (day 1), with a total of one injection administered. The experiment ended on day 23 after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0427] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0428] The tumor volume is calculated using the following formula: V = 0.5a × b 2 The formula was used to calculate the tumor's longest and shortest diameters (where a and b represent the tumor's longest and shortest diameters, respectively). The antitumor effect of the compound was evaluated as T / C (%). The percentage value of T / C (%) is an index that reflects the tumor growth inhibition rate, where T and C represent the average tumor volume on a specific day in the treatment group and the control group, respectively. The tumor growth inhibition rate is calculated using the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T iThe calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0429] Comparisons between sample groups were performed using one-way ANOVA. A P<0.05 value indicates a statistically significant difference. Plotting and statistical analysis were performed using GraphPad Prism software. [Table 25]

[0430] The experimental results are shown in Figure 14 and Table 23. The results showed that in an EGFR ADC resistance model, the antibody conjugate of this disclosure exhibited superior antitumor efficacy compared to the control ADC-14.

[0431] Example 15: Comparison of in vivo efficacy of the BsAb ADC and control ADC of this disclosure in a mouse model with high EGFR expression and low HER3 expression. To compare the inhibitory effects of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) on in vivo tumor formation induced by cell lines with high EGFR expression and low HER3 expression, mice were subcutaneously inoculated with A431 to induce xenograft tumors, and then the antitumor effects of the two ADCs were compared.

[0432] 1. The test drugs and administration regimens are shown in Table 24. [Table 26]

[0433] 2. Preparation method: All samples were prepared by diluting them with an adjuvant solvent.

[0434] 3. Experimental animals: Female NCG mice aged 6-8 weeks, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0435] 4. Experimental procedure: A431 (CRL-1555) cells were cultured in DMEM medium containing 10% fetal bovine serum and 2 mM glutamine. A431 cells in the exponential growth phase were harvested and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into mice.

[0436] In the experimental mice, 5 × 10 6 Individual A431 cells were subcutaneously inoculated into the right side of the back. Tumor growth was monitored regularly.

[0437] The tumor was approximately 137 mm in size. 3 Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector®. Intravenous (IV) injection of the test sample was started on the day of group assignment (day 0), with a total of one injection administered. The experimental endpoint was 21 days after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0438] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0439] The tumor volume is calculated using the following formula: V = 0.5a × b 2 (In the formula, a and b represent the long and short diameters of the tumor, respectively), was used to calculate the tumor growth inhibition rate. The antitumor effect of the compound was evaluated as TGI (%). The tumor growth inhibition rate is given by the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0440] Comparisons between sample groups were performed using one-way ANOVA. A P<0.05 value indicates a statistically significant difference. Plotting and statistical analysis were performed using GraphPad Prism software. [Table 27]

[0441] The experimental results are shown in Figure 15 and Table 25. The results demonstrated that, in a tumor model with high EGFR expression and low HER3 expression, the antibody conjugate of this disclosure exhibited superior antitumor efficacy compared to the control ADC at the same dose. The control ADC failed to inhibit tumor growth at this dose concentration. Mice in each treatment group showed good tolerance, and no significant weight loss was observed.

[0442] Example 16: Comparison of the efficacy of the BsAb ADC and the control ADC in this example in a mouse model carrying a human colon cancer cell line xenograft tumor that expresses only HER3 and not EGFR. To compare the inhibitory effects of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) on in vivo tumor formation induced by human colon cancer cell lines that express only HER3 and not EGFR, mice were subcutaneously inoculated with SW620 to induce xenograft tumors, and then the antitumor effects of the two ADCs were compared.

[0443] 1. The test drugs and administration regimens are shown in Table 26. [Table 28]

[0444] 2. Preparation method: All samples were prepared by diluting them with an adjuvant solvent.

[0445] 3. Experimental animals: Female NCG mice aged 6-8 weeks, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0446] 4. Experimental procedure: SW620 (CCL-227) cells were cultured in DMEM medium containing 10% fetal bovine serum and 2 mM of glutamine. SW620 cells were harvested during the exponential growth phase and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into mice.

[0447] In the experimental mice, 5 × 10 6 Individual SW620 cells were subcutaneously inoculated into the right side of the back. Tumor growth was monitored regularly.

[0448] The tumor was approximately 131 mm in size. 3 Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector®. Intravenous (IV) injection of the test sample was started on the day of group assignment (day 0). The experiment ended on day 59 after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0449] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0450] The tumor volume is calculated using the following formula: V = 0.5a × b 2 (In the formula, a and b represent the long and short diameters of the tumor, respectively), was used to calculate the tumor growth inhibition rate. The antitumor effect of the compound was evaluated as TGI (%). The tumor growth inhibition rate is given by the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0451] Comparisons between sample groups were performed using one-way ANOVA. A P<0.05 value indicates a statistically significant difference. Plotting and statistical analysis were performed using GraphPad Prism software. [Table 29]

[0452] The experimental results are shown in Figure 16 and Table 27. The results indicate that in tumor models with no EGFR expression and moderate HER3 expression, all experimental groups showed a significant inhibitory effect on tumor growth compared to the blank control group; at the same dose, the antibody conjugates of this disclosure had a superior antitumor effect compared to the control ADC, achieving complete tumor regression and sustaining tumor growth inhibition up to day 59; the control ADC failed to achieve complete tumor regression and failed to inhibit tumor regrowth. Mice in each treatment group showed good tolerance, and no significant weight loss was observed.

[0453] Example 17: Comparison of the efficacy of the BsAb ADC and control ADC in a mouse model of tumor-bearing human colon cancer cell line xenograft with moderate EGFR expression and low HER3 expression. To compare the inhibitory effects of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) on in vivo tumor formation induced by human colon cancer cell lines with moderate EGFR expression and low HER3 expression, mice were subcutaneously inoculated with SW48 to induce xenograft tumors, and then the antitumor effects of the two ADCs were compared.

[0454] 1. The test drugs and administration regimens are shown in Table 28. [Table 30]

[0455] 2. Preparation method: All samples were prepared by diluting them with an adjuvant solvent.

[0456] 3. Experimental animals: Female NCG mice aged 6-8 weeks, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0457] 4. Experimental procedure: SW48 (CCL-231) cells were cultured in DMEM medium containing 10% fetal bovine serum and 2 mM of glutamine. SW48 cells in the exponential growth phase were harvested and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into mice.

[0458] In the experimental mice, 1 × 10 7 Individual SW48 cells were subcutaneously inoculated into the right side of the back. Tumor growth was monitored regularly.

[0459] The tumor was approximately 131 mm in size. 3 Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector®. Intravenous (IV) injection of the test sample was started on the day of group assignment (day 0). The experiment ended on day 45 after group assignment. Tumor volume and body weight were measured twice a week and the data were recorded.

[0460] Five mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0461] The tumor volume is calculated using the following formula: V = 0.5a × b 2 (In the formula, a and b represent the long and short diameters of the tumor, respectively), was used to calculate the tumor growth inhibition rate. The antitumor effect of the compound was evaluated as TGI (%). The tumor growth inhibition rate is given by the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0462] Comparisons between sample groups were performed using one-way ANOVA. A P<0.05 value indicates a statistically significant difference. Plotting and statistical analysis were performed using GraphPad Prism software. [Table 31]

[0463] The experimental results are shown in Figure 17 and Table 29. The results showed that in tumor models with moderate EGFR expression and low HER3 expression, all experimental groups had a significant inhibitory effect on tumor growth compared to the blank control group; at the same dose, the antibody conjugate of this disclosure had a superior antitumor effect compared to the control ADC, achieving complete tumor regression in some mice and sustaining tumor growth inhibition up to day 45; the control ADC failed to achieve complete tumor regression and did not inhibit tumor recurrence. Mice in each treatment group showed good tolerance and no significant weight loss was observed.

[0464] Example 18: Comparison of the efficacy of the BsAb ADC and control ADC in a xenograft tumor-bearing mouse model of osimertinib-resistant human non-small cell lung cancer cell lines. To compare the inhibitory effects of DBXT005-01 ADC (ADC-13) and control ADCs (BL-B01D1, ADC-14) on in vivo tumor formation induced by osimertinib-resistant non-small cell lung cancer cell lines, mice were subcutaneously inoculated with NCI-H1975 (EGFR L858R / T790M / C797S triple mutation) to induce xenograft tumors, and the antitumor effects of the two ADCs were then compared.

[0465] 1. The test drug and administration regimen are shown in Table 30. [Table 32]

[0466] 2. Preparation method: All samples were prepared by diluting them with an adjuvant solvent.

[0467] 3. Experimental animals: Female NOD / SCID mice aged 6-7 weeks, purchased from Jiangsu GemPharmatech Co.,Ltd.

[0468] 4. Experimental procedure: NCI-H1975 EGFR L858R / T790M / C797S (CL-01195) cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 100 μg / mL hydroxymycin. NCI-H1975 cells were harvested during the exponential growth phase and resuspended in PBS to a concentration suitable for subcutaneous tumor inoculation into NOD / SCID mice. 1 × 10⁶ cells were then administered to experimental mice. 7 Each mouse was subcutaneously inoculated with 0.2 mL of NCI-H1975 EGFR L858R / T790M / C797S cells (resuspended in a 1:1 mixture of PBS and Matrigel) into the right side of its back. Tumor growth was observed periodically.

[0469] The tumor was approximately 169.46 mm in size. 3 Once the tumors had grown to a certain stage, the tumor-bearing mice were randomly assigned to groups using StudyDirector®. Intravenous (IV) injection of the test sample was started on the day of group assignment (day 0). The endpoint of the experiment was 35 days after group assignment. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0470] Six mice were used in both the control and treatment groups. Tumor inhibition rates were calculated by measuring tumor volume.

[0471] The tumor volume is calculated using the following formula: V = 0.5a × b 2 (In the formula, a and b represent the long and short diameters of the tumor, respectively), was used to calculate the tumor growth inhibition rate. The antitumor effect of the compound was evaluated as TGI (%). The tumor growth inhibition rate is given by the following formula: TGI (%) = [1 - (T i -T0) / (V i -V0)]×100(in the formula, T i V is the average tumor volume for a specific treatment group on a specific day, T0 is the average tumor volume for the treatment group at the start of administration, V i is a specific day (T i The calculation was performed using the mean tumor volume of the media control group on the same day, and V0 being the mean tumor volume of the media control group at the start of administration.

[0472] Comparisons between sample groups were performed using one-way ANOVA. A P<0.05 value indicates a statistically significant difference. Plotting and statistical analysis were performed using GraphPad Prism software. [Table 33]

[0473] The experimental results are shown in Figure 18 and Table 31. The results indicate that in a non-small cell lung cancer tumor model resistant to the third-generation TKI osimertinib, all experimental groups showed a significant inhibitory effect on tumor growth compared to the blank control group; the antibody conjugate of this disclosure had a superior antitumor effect compared to the control ADC at the same dose; and mice in each treatment group showed good tolerance and no significant weight loss was observed.

[0474] [Table 34-1] [Table 34-2] [Table 34-3] [Table 34-4] [Table 34-5] [Table 34-6] [Table 34-7] [Table 34-8] [Table 34-9] [Table 34-10] [Table 34-11] [Table 34-12] [Table 34-13] [Table 34-14] [Table 34-15]

[0475] While specific embodiments of the present disclosure have been described above, those skilled in the art will understand that these embodiments are merely illustrative, and many changes or modifications can be made to these embodiments without departing from the principles and spirit of the present disclosure. Accordingly, the scope of protection of the present disclosure is defined by the appended claims.

Claims

1. 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 VH1 comprises H1CDR1, H1CDR2, and H1CDR3 having the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; VL1 comprises L1CDR1, L1CDR2, and L1CDR3 having the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; VH2 comprises H2CDR1, H2CDR2, and H2CDR3 having the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; and VL2 comprises L2CDR1, L2CDR2, and L2CDR3 having the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.

2. The amino acid sequence of H2CDR2 is shown in SEQ ID NO: 77 or 78; Preferably, VH1 includes framework regions H1FR1, H1FR2, H1FR3, and H1FR4 having amino acid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to those shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively; The VL1 comprises framework regions L1FR1, L1FR2, L1FR3, and H1FR4, each having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to those shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or 99% identical to them; The VH2 has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 21, for example, a framework region H2FR1 having the E16D mutation on SEQ ID NO: 22 and SEQ ID NO: 24, respectively, or at least 90%, 91%, 92%, 93%, 94% identical to them. Framework regions H2FR2 and H2FR4 having amino acid sequences with 95%, 96%, 97%, 98%, or 99% identity; and framework region H2FR3 having an amino acid sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to or from SEQ ID NO: 23, for example, having the S18D mutation on SEQ ID NO: 23; The VL2 includes framework region L2FR1, which is 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 the S9D mutation and the V15L mutation or the S7E mutation on SEQ ID NO: 25; and framework regions L2FR2, L2FR3, and L2FR4, which are shown in SEQ ID NO: 18, SEQ ID NO: 26, and SEQ ID NO: 27, respectively, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. The bispecific antibody according to claim 1.

3. The amino acid sequence of VH1 is shown in SEQ ID NO: 28, the amino acid sequence of VL1 is shown in SEQ ID NO: 29, the amino acid sequence of VH2 is shown in SEQ ID NO: 30, SEQ ID NO: 79, or SEQ ID NO: 80, and the amino acid sequence of 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 shown in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively, or shown in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 79, and SEQ ID NO: 81, respectively, or shown in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 79, and SEQ ID NO: 31, respectively, or shown in SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 80, and SEQ ID NO: 82, respectively. A bispecific antibody according to claim 1 or 2.

4. The EGFR-binding domain and the HER3-binding domain each further comprise a light chain constant region and a heavy chain constant region, the EGFR-binding domain comprising a light chain constant region CL1 and a heavy chain constant region HC1, and the HER3-binding domain comprising a light chain constant region CL2 and a heavy chain constant region HC2, wherein the amino acid sequences of CL1 and CL2 are 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 to them, and the amino acid sequences of CL1 and CL2 are not identical; and / or, HC1 comprises C1H1 and Fc1, and HC2 comprises C2H1 and Fc2, wherein the amino acid sequences of C1H1 and C2H1 are shown in SEQ ID NO: The amino acid sequences of C1H1 and C2H1 are not identical sequences to those shown in SEQ ID NO: 34 or SEQ ID NO: 35, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to them; 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 shown in SEQ ID NO: 36, or to SEQ ID NO: 36 and having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity, for example having the T146W mutation, or the S134C and T146W mutation, or the T146S, L148A and Y187V mutation, or the Y349C, T366S, L368A and Y407V mutation on Sequence ID No. 36, and the amino acid sequences of Fc1 and Fc2 are not identical; Preferably, the amino acid sequences of CL1 and CL2 are those shown in SEQ ID NO: 32 or 33, respectively; the amino acid sequences of C1H1 and C2H1 are those shown in SEQ ID NO: 34 or 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 a T146W mutation, or an S134C and T146W mutation, or a T146S, L148A and Y187V mutation, or a Y349C, T366S, L368A and Y407V mutation on SEQ ID NO: 36; More preferably, Fc1 and Fc2 are connected by disulfide bonds in a hinge region, forming a knob-into-hole structure where Fc1 is knob-Fc and Fc2 is hole-Fc, or Fc2 is knob-Fc and Fc1 is hole-Fc; More preferably, C1H1 and Fc1, and C2H1 and Fc2 are connected by a hinge region, where the amino acid sequence of the hinge region is shown in Sequence ID No.

89. A bispecific antibody according to any one of claims 1 to 3.

5. 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 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 C1 The amino acid sequence of H1 is 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 variant sequences of the amino acid sequence shown in SEQ ID NO: 36, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 36; Preferably, 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; More preferably, Fc1 and Fc2 are connected by disulfide bonds in a hinge region, forming a knob-into-hole structure where Fc1 is knob-Fc and Fc2 is hole-Fc, or Fc2 is knob-Fc and Fc1 is hole-Fc; More preferably, C1H1 and Fc1 are connected by a hinge region having the amino acid sequence shown in SEQ ID NO: 89; VL2 and VH2 are connected by a hinge region having the amino acid sequence shown in SEQ ID NO: 95; and VH2 and Fc2 are connected by a hinge region having the amino acid sequence shown in SEQ ID NO:

96. A bispecific antibody according to any one of claims 1 to 3.

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

7. These are shown in sequence numbers 37, 38, 39, and 40, respectively; or These are shown in sequence numbers 41, 38, 42, and 40, respectively; or These are shown in sequence numbers 43, 38, 44, and 40, respectively; or These are shown in sequence numbers 45, 38, 46, and 40, respectively; or These are shown in sequence numbers 47, 48, 49, and 50, respectively; or These are shown in sequence numbers 51, 48, 52, and 50, respectively; or These are shown in sequence numbers 53, 48, 54, and 50, respectively; or These are shown in Sequence ID 55, Sequence ID 48, Sequence ID 56, and Sequence ID 50, respectively; or These are shown in Sequence ID 37, Sequence ID 38, Sequence ID 57, and Sequence ID 58, respectively; or These are shown in Sequence ID No. 41, Sequence ID No. 38, Sequence ID No. 59, and Sequence ID No. 58, respectively; or These are shown in SEQ ID NOs. 43, 38, 60, and 58, respectively; or These are shown in SEQ ID NOs. 45, 38, 61, and 58, respectively; or These are shown in sequence numbers 47, 48, 62, and 63, respectively; or These are shown in sequence numbers 51, 48, 64, and 63, respectively; or These are shown in SEQ ID NO: 53, SEQ ID NO: 48, SEQ ID NO: 65, and SEQ ID NO: 63, respectively; or These are shown in sequence numbers 55, 48, 66, and 63, respectively; or These are shown in sequence numbers 37, 38, 57, and 40, respectively; or These are shown in sequence numbers 41, 38, 59, and 40, respectively; or These are shown in sequence numbers 43, 38, 60, and 40, respectively; or These are shown in sequence numbers 45, 38, 61, and 40, respectively; or These are shown in sequence numbers 47, 48, 62, and 50, respectively; or These are shown in sequence numbers 51, 48, 64, and 50, respectively; or These are shown in Sequence ID 53, Sequence ID 48, Sequence ID 65, and Sequence ID 50, respectively; or These are shown in sequence numbers 55, 48, 66, and 50, respectively; or These are shown in SEQ ID NOs: 37, 38, 67, and 68, respectively; or These are shown in SEQ ID NOs: 41, 38, 69, and 68, respectively; or These are shown in SEQ ID NOs. 43, 38, 70, and 68, respectively; or These are shown in SEQ ID NOs. 45, 38, 71, and 68, respectively; or These are shown in sequence numbers 47, 48, 72, and 73, respectively; or These are shown in sequence numbers 51, 48, 74, and 73, respectively; or These are shown in Sequence ID 53, Sequence ID 48, Sequence ID 75, and Sequence ID 73, respectively; or As shown in Sequence ID 55, Sequence ID 48, Sequence ID 76, and Sequence ID 73, respectively, A bispecific antibody according to any one of claims 1 to 4, comprising a heavy chain H1, a light chain L1, a heavy chain H2, and a light chain L2 having an amino acid sequence.

8. An isolated nucleic acid encoding a bispecific antibody according to any one of claims 1 to 7.

9. A recombinant expression vector comprising the nucleic acid described in claim 8.

10. A transformant in a host cell comprising the nucleic acid according to claim 8, or the recombinant expression vector according to claim 9, wherein Preferably, the host cell is a eukaryotic cell, preferably a mammalian cell such as a CHO cell. The aforementioned transformed body.

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

12. The following fragments: a bispecific antibody according to any one of claims 1 to 7; or its antigen-binding fragment; a linker unit L; and a cytotoxic drug. A bispecific antibody-drug conjugate containing, or its tautomers, enantiomers, diastereoisomers, or mixtures thereof, or a pharmaceutically acceptable salt thereof.

13. The cytotoxic drug is camptothecin and its derivatives; Preferably, the cytotoxic agent has the structure of formula (A-1), or a tautomer, enantiomer, or diastereoisomer thereof: 【Chemistry 1】 (In the formula, M is -L ２ -L １ -C(O)-; L ２ is -NH-, O, or S, preferably -O- or -S-, more preferably -O-, L ２ It is connected to the linker unit L; L １ is -(C(R １ａ )(R １ｂ )) ｍ -CH ２ -, saturated C ３ -C ６ -cycloalkylene, or saturated 3- to 6-membered heterocyclylene, where the saturated C ３ -C ６ -cycloalkylene, and the saturated 3- to 6-membered heterocyclylene are each independently optionally substituted with one or more R ２ａ ; m is selected from 1, 2, 3, and 4; each heteroatom in the saturated 3-membered to 6-membered heterocyclylene is independently N, O, or S, and the number of heteroatoms is 1, 2, or 3; Each R １ａ and each R １ｂ These are independently hydrogen, halogen, hydroxyl, amino, and C １ -C ６ Selected from alkyl, the C １ -C ６ Alkyl is optionally substituted with one or more halogens; R ２ａ These are halogen, hydroxyl, amino, and C １ -C ６ Selected from alkyl, the C １ -C ６ (Alkyl is optionally substituted with one or more halogens.) The bispecific antibody-drug conjugate according to claim 12, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

14. L １ ga- (C(R １ａ ) (Caution １ｂ )) ｍ -CH ２ - and; each R １ａ However, hydrogen, halogens, and C １ -C ６ Selected from alkyl groups; each R １ｂ However, hydrogen, halogens, and C １ -C ６ Selected from alkyl groups; or L １ However, -(C(R １ａ ) (Caution １ｂ )) ｍ -CH ２ - and therefore, R １ａ However, C １ -C ６ Alkyl, preferably C １ -C ３ It is alkyl, R １ｂ However, hydrogen and C １ -C ６ Alkyl, preferably hydrogen and C １ -C ３ Selected from alkyl groups; or L １ ga- (C(R １ａ ) (Caution １ｂ )) ｍ -CH ２ - and therefore, R １ａ ga-CH ３ And R １ｂ However, hydrogen and -CH ３ Selected from; or L １ However, saturated C ３ -C ６ Cycloalkylene, or saturated 3-membered to 6-membered heterocyclene, preferably saturated C ３ -C ６ It is a cycloalkylene, and therein, the saturated C ３ -C ６ Cycloalkylene and the saturated 3-membered to 6-membered heterocyclene each independently have one or more R ２ａ It is arbitrarily replaced, and each R ２ａ However, independently, halogen or C １ -C ６ It is alkyl; or L １ However, one or more R ２ａ Cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, optionally substituted with each R ２ａ However, halogen and C １ -C ６ Selected independently of alkyl; or L １ but, 【Chemistry 2】 is, or L １ but, 【Transformation 3】 That is, The bispecific antibody-drug conjugate according to claim 13, or its tautomer, enantiomer, diastereoisomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.

15. In the structure of formula (A-1) above, M is -L ２ -L １ -C(O)-; L ２ is -O-; L １ is -(C(R １ａ )(R １ｂ )) ｍ -CH ２ -, or saturated C ３ -C ６ cycloalkylene, where the saturated C ３ -C ６ cycloalkylene is optionally substituted with one or more R ２ａ ; m is selected from 1 or 2; R １ａ and R １ｂ each independently is hydrogen, halogen, and C １ -C ６ alkyl selected from, said C １ -C ６ alkyl is optionally substituted with one or more halogens; R ２ａ However, halogen and C １ -C ６ Selected from alkyl, the C １ -C ６ Alkyl is optionally substituted with one or more halogens. The bispecific antibody-drug conjugate according to claim 14, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

16. M: 【Chemistry 4】 Preferably, 【Transformation 5】 That is, A bispecific antibody-drug conjugate according to any one of claims 13 to 15, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

17. The cytotoxic drug is selected from any one of the following structures, comprising a bispecific antibody-drug conjugate according to any one of claims 12 to 16, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof: 【Transformation 6】

18. The linker unit L is -L ａ -L ｂ -L ｃ -Therefore, L ｃ It is linked to the aforementioned cytotoxic drug; -L ａ -teeth, 【Transformation 7】 or -C １－８ Alkylene-C(O)-, preferably, 【Transformation 8】 or -C １－６ Alkylene-C(O)-, more preferably, 【Chemistry 9】 Furthermore, 【Chemistry 10】 (In the formula, the a-terminus is connected to Ab, and the b-terminus is connected to L) ｂ (It is linked to); -L ｂ - is -(polypeptide of 1 to 6 natural amino acids)-NH-, preferably -(polypeptide of 2 to 4 natural amino acids)-NH-, more preferably the following structure: 【Chemistry 11】 More preferably, 【Chemistry 12】 more, 【Chemistry 13】 (In the formula, the c-terminus is L) ａ It is connected, and the d-terminus is L ｃ It is selected from one of the following (connected to); -L ｃ - is C １－６ Alkylene, preferably C １－３ Alkylene, more preferably, 【Chemistry 14】 That is, A bispecific antibody-drug conjugate according to any one of claims 12 to 17, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

19. The linker unit L is 【Chemistry 15】 Preferably, 【Chemistry 16】 The bispecific antibody-drug conjugate according to claim 18, or a tautomer, enantiomer, diastereoisomer, or mixture of isomers thereof, or a pharmaceutically acceptable salt thereof.

20. The aforementioned bispecific antibody-drug conjugate is of formula (A-2): 【Chemistry 17】 (In the formula, p represents the average number of connections, where p is an integer or decimal number between 1 and 10, preferably an integer or decimal number between 3 and 9, for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98, or 8; Ab is a bispecific antibody according to any one of claims 1 to 7, or an antigen-binding fragment thereof; M is as defined in the antibody-drug conjugate according to any one of claims 13 to 17; (L is the linker unit L according to claim 18 or 19) A bispecific antibody-drug conjugate having the structure of any one of claims 12 to 19, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

21. The bispecific antibody-drug conjugate is of formula (A-2a) or (A-2b): [Chemistry 18] (In the formula, p represents the average number of connections, where p is an integer or decimal number from 1 to 10, preferably an integer or decimal number from 3 to 9, for example, selected from 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98, or 8; Ab is a bispecific antibody according to any one of claims 1 to 7, or an antigen-binding fragment thereof; L ２ is -NH-, O, or S, preferably -O- or -S-, more preferably -O-; X １ This is one, two, or three R ２ａ Saturated C, which is arbitrarily substituted with ３ -C ６ Selected from cycloalkylenes; X ２ is -(C(R １ａ ) (Caution １ｂ )) ｍ -CH ２ - Selected from; m is selected from 1 or 2; R １ａ and R １ｂ Each of these is independently a C atom substituted with hydrogen, a halogen, or one, two, or three halogens. １ -C ６ It is alkyl; R ２ａ These are halogen, hydroxyl, amino, and C １ -C ６ Selected from alkyl, the C １ -C ６ (Alkyl is optionally substituted with one or more halogens.) Having a structure, Preferably, the structure of the bispecific antibody-drug conjugate is as follows: 【Chemistry 19-1】 【Chemistry 19-2】 【Chemistry 19-3】 【Chemistry 19-4】 (In the formula, p represents the average number of connections, where p is an integer or decimal number between 1 and 10, preferably an integer or decimal number between 3 and 9, for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98, or 8; Ab is a bispecific antibody according to any one of claims 1 to 7, or an antigen-binding fragment thereof; Preferably, Ab is selected from the bispecific antibodies of the DBXT001, DBXT002, DBXT003, and DBXT004 series; more preferably, it is selected from the bispecific antibodies of the DBXT001, DBXT002, and DBXT005-01 series; more preferably, it is selected from the bispecific antibodies of the DBXT001 and DBXT005-01 series; and even more preferably, it is selected from the bispecific antibodies of DBXT001-01 and DBXT005-01. A bispecific antibody-drug conjugate according to any one of claims 12 to 20, selected from, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

22. The aforementioned bispecific antibody-drug conjugate is as follows: 【Chemistry 20】 (In the formula, p represents the average number of connections, where p is an integer or decimal number between 1 and 10, preferably an integer or decimal number between 3 and 9, for example, 4, 4.06, 4.10, 6, 6.11, 6.05, 7.99, 7.98, or 8. Choose one of the following: Preferably, the bispecific antibody-drug conjugate is as follows: 【Chemistry 21-1】 【Chemistry 21-2】 【Chemistry 21-3】 【Chemistry 21-4】 (In the formula, DBXT001-01 is an anti-EGFR / HER3 bispecific antibody containing heavy chain H1, light chain L1, heavy chain H2, and light chain L2, each having the amino acid sequences shown in SEQ ID NOs. 37, 38, 39, and 40, respectively. A bispecific antibody-drug conjugate according to claim 21, selected from any one of the following, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

23. The aforementioned bispecific antibody-drug conjugate is: 【Chemistry 22】 (In the formula, p represents the average number of connections, where p is an integer or decimal from 1 to 10, preferably an integer or decimal from 3 to 9, more preferably an integer or decimal from 4 to 6, for example, 5.99; DBXT005-01 is an anti-EGFR / HER3 bispecific antibody comprising a heavy chain H1 having the amino acid sequence shown in SEQ ID NO: 37, a light chain L1 having the amino acid sequence shown in SEQ ID NO: 38, and a heavy chain H2 having the amino acid sequence shown in SEQ ID NO:

90. A bispecific antibody-drug conjugate according to any one of claims 12 to 21, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

24. The aforementioned bispecific antibody-drug conjugate is: 【Chemistry 23】 (In the formula, p1 represents the number of connections, where p1 is one integer from 1 to 10, preferably one integer from 3 to 9, more preferably one integer from 4 to 6, for example 4, 5, or 6; DBXT005-01 is an anti-EGFR / HER3 bispecific antibody comprising a heavy chain H1 having the amino acid sequence shown in SEQ ID NO: 37, a light chain L1 having the amino acid sequence shown in SEQ ID NO: 38, and a heavy chain H2 having the amino acid sequence shown in SEQ ID NO:

90. A bispecific antibody-drug conjugate according to any one of claims 12 to 19, or a tautomer, enantiomer, diastereoisomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

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

26. The method according to claim 25, comprising reacting the anti-EGFR / HER3 bispecific antibody with a compound of formula X2, for example, reacting DBXT005-01 with a compound of formula X2. 【Chemistry 24】

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

28. Use of a bispecific antibody according to any one of claims 1 to 7, an isolated nucleic acid according to claim 8, a recombinant expression vector according to claim 9, a transformant according to claim 10, a bispecific antibody-drug conjugate according to any one of claims 12 to 24, and / or a pharmaceutical composition according to claim 27, in the preparation of a pharmacopoeia for treating and / or preventing cancer, wherein the cancer is preferably a cancer having positive expression of EGFR and / or HER3, for example, the cancer is breast cancer, skin cancer, gastric cancer, colorectal cancer, lung cancer, esophageal cancer, biliary tract cancer The method is characterized by selecting from 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, plasmacytoma, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma, and more preferably selecting from breast cancer, colorectal cancer, skin cancer, lung cancer, esophageal cancer, and oral squamous cell carcinoma, wherein 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.

29. A method for treating and / or preventing cancer, comprising administering to a subject in need 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, wherein the cancer is preferably a cancer having positive expression of EGFR and / or HER3, for example, the cancer being 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 The method is characterized by selecting from visceral 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, plasmacytoma, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma, and more preferably selecting from breast cancer, colorectal cancer, skin cancer, lung cancer, esophageal cancer, and oral squamous cell carcinoma, wherein 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.

30. The cancer is preferably a cancer having positive expression of 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, plasmacytoma, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma, preferably 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 use in the prevention and / or treatment of cancer, wherein 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.

31. A combination therapy comprising administering separately to a subject in need 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, and a second therapeutic agent, wherein Preferably, the cancer is a cancer having positive expression of 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, plasmacytoma, myeloma, glioma, osteosarcoma, sarcoma, oral squamous cell carcinoma, and melanoma, and preferably, the cancer is selected from breast cancer, colorectal cancer, skin cancer, lung cancer, esophageal cancer, and oral squamous cell carcinoma, where 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. The aforementioned combination therapy.

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

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