Bispecific antibody targeting EGFR and her3, drug conjugate and use thereof

Abstract

Provided are a bispecific antibody targeting EGFR and HER3, a drug conjugate thereof, and a pharmaceutical composition containing the bispecific antibody and drug conjugate thereof and the use thereof.

Description

Bispecific antibodies against EGFR and HER3, drug conjugates and their uses

[0001] This application claims priority to Chinese Patent Application No. 202510384925.9, filed March 28, 2025; Chinese Patent Application No. 202510697029.8, filed May 28, 2025; and Chinese Patent Application No. 202510716826.6, filed May 30, 2025, all of which are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to the field of biomedicine. Specifically, this invention provides bispecific antibodies against EGFR and HER3, drug conjugates thereof, and pharmaceutical compositions comprising said bispecific antibodies and drug conjugates thereof, as well as their uses. Background Technology

[0003] Epidermal growth factor receptor (EGFR), encoded by the c-erbB proto-oncogene, is a transmembrane receptor with a molecular weight of approximately 170 kDa. It is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Upon binding to its specific ligands, including epidermal growth factor (EGF) and transforming growth factor α (TGFα), EGFR dimers, further stimulating intracellular protein tyrosine kinase activity and triggering downstream signaling cascades, leading to DNA synthesis and cell proliferation. EGFR also participates in phenotypic regulation such as cell migration, adhesion, and proliferation. EGFR overexpression is associated with many malignant tumors, and its overexpression is significantly associated with poor patient prognosis. HER3, a member of the erbB family, plays a crucial role in cell proliferation, tumor metastasis, and drug resistance. There is a need in this field for bispecific antibodies and drug conjugates that combine EGFR and HER3, offering precise targeting and superior affinity. Summary of the Invention

[0004] This article provides at least the following implementation schemes:

[0005] Implementation Scheme 1. A bispecific antibody comprising a first antigen-binding portion binding to EGFR and a second antigen-binding portion binding to HER3, wherein the first antigen-binding portion comprises at least one EGFR-specific VHH, and the at least one EGFR-specific VHH comprises HCDR1, HCDR2 and HCDR3 of any VHH selected from SEQ ID NO: 16, 17, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146 and 150.

[0006] Implementation Scheme 2. The bispecific antibody of Implementation Scheme 1, wherein at least one VHH specifically binding to EGFR comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 4, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, wherein HCDR2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 2, 5, 15, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, and wherein HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 1, 4, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, wherein HCDR2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 2, 5, 15, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, and wherein HCDR3 comprises an The amino acid sequence represented by any one of NO:3, 6, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149.

[0007] Implementation Scheme 3. A bispecific antibody of Implementation Scheme 1 or 2, wherein at least one VHH that specifically binds to EGFR comprises HCDR1, HCDR2, and HCDR3, wherein

[0008] (1) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; or

[0009] (2) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:15, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; or

[0010] (3) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; or

[0011] (4) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:79, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:80, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:81; or

[0012] (5) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:83, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:84, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:85; or

[0013] (6) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:87, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:88, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:89; or

[0014] (7) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:92, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:93; or

[0015] (8) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:94, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:96, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:97; or

[0016] (9) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:99, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:100, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:101; or

[0017] (10) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:103, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:104, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:105; or

[0018] (11) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:107, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:108, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:109; or

[0019] (12) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:111, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:112, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:113; or

[0020] (13) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:115, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:116, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:117; or

[0021] (14) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:119, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:120, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:121; or

[0022] (15) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:123, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:124, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:125; or

[0023] (16) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:127, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:128, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:129.

[0024] (17) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:131, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:132, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:133; or

[0025] (18) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:135, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:136, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:137; or

[0026] (19) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:139, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:140, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:141; or

[0027] (20) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:143, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:144, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:145; or

[0028] (21) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:147, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:148, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:149.

[0029] Implementation Scheme 4. A bispecific antibody of any one of Implementation Schemes 1-3, wherein the at least one VHH specifically binding to EGFR comprises an amino acid sequence of any one of SEQ ID NO: 16, 17, 51-64, 65-78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, and 150, or an amino acid sequence of any one of 16, 17, 51-64, 65-78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, and 150. The amino acid sequence has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, or consists of any one of the amino acid sequences 16, 17, 51-64, 65-78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, and 150.

[0030] Implementation Scheme 5. A bispecific antibody of any one of Implementation Schemes 1-4, wherein the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; preferably, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:16 or 18; more preferably, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:18.

[0031] Implementation Scheme 6. A bispecific antibody of any one of Implementation Schemes 1-4, wherein the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 or 15, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; preferably, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:17 or 76; more preferably, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:76.

[0032] Implementation Scheme 7. A bispecific antibody of any one of Implementation Schemes 1-6, wherein the second antigen-binding portion binding HER3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3; wherein the HCDR1 of the second antigen-binding portion binding HER3 comprises the amino acid sequence of SEQ ID NO:30, the HCDR2 comprises the amino acid sequence of SEQ ID NO:31, the HCDR3 comprises the amino acid sequence of SEQ ID NO:32, the LCDR1 comprises the amino acid sequence of SEQ ID NO:33, the LCDR2 comprises the amino acid sequence of SEQ ID NO:34, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:35.

[0033] Implementation Scheme 8. The bispecific antibody of Implementation Scheme 7, wherein the heavy chain variable region of the second antigen-binding portion binding to HER3 comprises the amino acid sequence of SEQ ID NO:26 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26; and / or, the light chain variable region comprises the amino acid sequence of SEQ ID NO:28 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:28.

[0034] Implementation Scheme 9. A bispecific antibody of any one of Implementation Schemes 1-8, comprising a first antigen-binding moiety binding to EGFR and a second antigen-binding moiety binding to HER3.

[0035] The first antigen-binding moiety that binds to EGFR comprises at least one EGFR-specific VHH, wherein the VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; and

[0036] The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. HCDR1 of the second antigen-binding region that binds to HER3 includes the amino acid sequence of SEQ ID NO:30, HCDR2 includes the amino acid sequence of SEQ ID NO:31, HCDR3 includes the amino acid sequence of SEQ ID NO:32, LCDR1 includes the amino acid sequence of SEQ ID NO:33, LCDR2 includes the amino acid sequence of SEQ ID NO:34, and LCDR3 includes the amino acid sequence of SEQ ID NO:35.

[0037] Implementation Scheme 10. A bispecific antibody of any one of Implementation Schemes 1-8, comprising a first antigen-binding moiety binding to EGFR and a second antigen-binding moiety binding to HER3.

[0038] The first antigen-binding moiety that binds to EGFR comprises at least one EGFR-specific VHH, wherein the VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown according to SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown according to SEQ ID NO:5 or 15, and HCDR3 comprises the amino acid sequence shown according to SEQ ID NO:6; and

[0039] The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. HCDR1 of the second antigen-binding region that binds to HER3 includes the amino acid sequence of SEQ ID NO:30, HCDR2 includes the amino acid sequence of SEQ ID NO:31, HCDR3 includes the amino acid sequence of SEQ ID NO:32, LCDR1 includes the amino acid sequence of SEQ ID NO:33, LCDR2 includes the amino acid sequence of SEQ ID NO:34, and LCDR3 includes the amino acid sequence of SEQ ID NO:35.

[0040] Implementation Scheme 11. A bispecific antibody according to any one of Implementation Schemes 1-8, comprising a first antigen-binding moiety binding to EGFR and a second antigen-binding moiety binding to HER3.

[0041] The first antigen-binding moiety that binds to EGFR includes at least one EGFR-specific VHH, said VHH containing the amino acid sequence shown in SEQ ID NO: 16 or 18, preferably containing the amino acid sequence shown in SEQ ID NO: 18; and

[0042] The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28.

[0043] Implementation Scheme 12. A bispecific antibody of any one of Implementation Schemes 1-8, comprising a first antigen-binding moiety binding to EGFR and a second antigen-binding moiety binding to HER3.

[0044] The first antigen-binding moiety that binds to EGFR includes at least one EGFR-specific VHH, said VHH containing the amino acid sequence shown in SEQ ID NO: 17 or 76, preferably containing the amino acid sequence shown in SEQ ID NO: 76; and

[0045] The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28.

[0046] Implementation Scheme 13. A bispecific antibody of any one of Implementation Schemes 1-12, wherein the bispecific antibody further comprises

[0047] i) Heavy chain constant region CH1 of an immunoglobulin; for example, the heavy chain constant region CH1 is the heavy chain constant region CH1 of human IgG1 or IgG4; preferably, the heavy chain constant region CH1 contains an amino acid sequence as shown in SEQ ID NO:22;

[0048] ii) Heavy chain hinge region of an immunoglobulin; for example, the heavy chain hinge region is the heavy chain hinge region of human IgG1 or IgG4; preferably, the hinge region comprises an amino acid sequence as shown in SEQ ID NO:23 or SEQ ID NO:24;

[0049] iii) An Fc fragment of an immunoglobulin; for example, the Fc fragment is an Fc fragment of the human IgG1 or IgG4 heavy chain; preferably, the immunoglobulin Fc fragment comprises an amino acid sequence as shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:25; and / or

[0050] iv) Light chain constant region (CL) of immunoglobulin; for example, the light chain constant region is the human κ light chain constant region; preferably, the light chain constant region CL contains an amino acid sequence as shown in SEQ ID NO:29;

[0051] v) A peptide linker; for example, a peptide linker comprising a peptide sequence of (GGGGS)n, (GS)n, or both, wherein n is an integer from 1 to 6; preferably, the linker comprises the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.

[0052] Implementation Scheme 14. A bispecific antibody of any one of Implementation Schemes 1-13, wherein the bispecific antibody has two heavy chain polypeptide chains, one heavy chain polypeptide chain containing an immunoglobulin Fc (Knob) fragment and the other heavy chain polypeptide chain containing an immunoglobulin Fc (Hole) fragment, wherein the two heavy chain polypeptide chains form a dimer through disulfide bonds in the hinge region and a Knob-into-Hole structure;

[0053] For example, the immunoglobulin Fc(knob) fragment contains the amino acid sequence shown in SEQ ID NO:20, and the immunoglobulin Fc(Hole) fragment contains the amino acid sequence shown in SEQ ID NO:21.

[0054] Implementation Scheme 15. A bispecific antibody of any one of Implementation Schemes 1-14, wherein the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain; wherein the first polypeptide chain comprises a VHH of the first antigen-binding moiety binding to EGFR, a heavy chain variable region of the second antigen-binding moiety binding to HER3, an immunoglobulin heavy chain constant region CH1, an immunoglobulin heavy chain hinge region, and an immunoglobulin Fc fragment, and optionally, further comprises the linker; and wherein the second polypeptide chain comprises a light chain variable region of the second antigen-binding moiety binding to HER3 and an immunoglobulin light chain constant region.

[0055] Implementation Scheme 16. The bispecific antibody of Implementation Scheme 15, wherein the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain; wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a VHH of the first antigen-binding portion that binds to EGFR, a linker, a heavy chain variable region of the second antigen-binding portion that binds to HER3, an immunoglobulin heavy chain constant region CH1, an immunoglobulin heavy chain hinge region, and an immunoglobulin Fc fragment.

[0056] Implementation Scheme 17. The bispecific antibody of Implementation Scheme 16, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus:

[0057] (1) VHH that binds to the first antigen-binding portion of EGFR, comprising the amino acid sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:76;

[0058] (2) A linker comprising an amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39;

[0059] (3) The heavy chain variable region of the second antigen-binding region of HER3, which contains the amino acid sequence of SEQ ID NO:26;

[0060] (4) Immunoglobulin heavy chain constant region CH1, which contains the amino acid sequence of SEQ ID NO:22;

[0061] (5) The immunoglobulin heavy chain hinge region, comprising the amino acid sequence of SEQ ID NO:23; and

[0062] (6) Immunoglobulin Fc fragment, which contains the amino acid sequence of SEQ ID NO:19,

[0063] Preferably, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:46 or SEQ ID NO:48, or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:46 or SEQ ID NO:48.

[0064] Implementation Scheme 18. The bispecific antibody of Implementation Scheme 15, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region of the second antigen-binding moiety binding to HER3, an immunoglobulin heavy chain constant region CH1, an immunoglobulin heavy chain hinge region, an Fc fragment of immunoglobulin, a linker, and VHH of the first antigen-binding moiety binding to EGFR.

[0065] Implementation Scheme 19. The bispecific antibody of Implementation Scheme 18, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus:

[0066] (1) The heavy chain variable region of the second antigen-binding region of HER3, which contains the amino acid sequence of SEQ ID NO:26;

[0067] (2) Heavy chain constant region CH1, which contains the amino acid sequence of SEQ ID NO:22;

[0068] (3) The heavy chain hinge region of an immunoglobulin, which contains the amino acid sequence of SEQ ID NO:23;

[0069] (4) Fc fragment, which contains the amino acid sequence of SEQ ID NO:19;

[0070] (5) A linker comprising the amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39; and

[0071] (6) VHH that binds to the first antigen-binding portion of EGFR, comprising the amino acid sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:76.

[0072] Preferably, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:47.

[0073] Implementation Scheme 20. A bispecific antibody of any one of Implementation Schemes 15-19, wherein the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain; wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region of the second antigen-binding moiety that binds to HER3 and a light chain constant region of the immunoglobulin.

[0074] Implementation Scheme 21. The bispecific antibody of Implementation Scheme 20, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus:

[0075] (1) The light chain variable region of the second antigen-binding moiety of HER3, which contains the amino acid sequence of SEQ ID NO:28; and

[0076] (2) The light chain constant region of immunoglobulin, which contains the amino acid sequence of SEQ ID NO:29.

[0077] Preferably, the second polypeptide chain comprises SEQ ID NO:41, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:41.

[0078] Implementation Scheme 22. A bispecific antibody according to any one of Implementation Schemes 1-14, wherein the bispecific antibody comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein the first polypeptide chain comprises the heavy chain variable region of the second antigen-binding moiety binding to HER3, the immunoglobulin heavy chain constant region CH1, the immunoglobulin heavy chain hinge region, and the immunoglobulin Fc fragment; the second polypeptide chain comprises the VHH of the first antigen-binding moiety binding to EGFR, the immunoglobulin heavy chain hinge region, and the immunoglobulin Fc fragment; and the third polypeptide chain comprises the light chain variable region of the second antigen-binding moiety binding to HER3 and the light chain constant region of immunoglobulin.

[0079] Implementation Scheme 23. The bispecific antibody of Implementation Scheme 22, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, the heavy chain variable region of the second antigen-binding moiety that binds to HER3, the immunoglobulin heavy chain constant region CH1, the immunoglobulin heavy chain hinge region, and the Fc fragment of immunoglobulin.

[0080] Implementation Scheme 24. The bispecific antibody of Implementation Scheme 23, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus:

[0081] (1) The heavy chain variable region of the second antigen-binding region of HER3, which contains the amino acid sequence of SEQ ID NO:26;

[0082] (2) Immunoglobulin heavy chain constant region CH1, which contains the amino acid sequence of SEQ ID NO:22;

[0083] (3) The heavy chain hinge region of an immunoglobulin, comprising the amino acid sequence of SEQ ID NO:23; and

[0084] (4) Immunoglobulin Fc fragment, which contains the amino acid sequence of SEQ ID NO:20,

[0085] Preferably, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:44, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:44.

[0086] Implementation Scheme 25. A bispecific antibody of any one of Implementation Schemes 22-24, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, the VHH of the first antigen-binding moiety that binds to EGFR, a linker, an immunoglobulin heavy chain hinge region, and an immunoglobulin Fc fragment.

[0087] Implementation Scheme 26. The bispecific antibody of Implementation Scheme 25, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus:

[0088] (1) VHH that binds to the first antigen-binding portion of EGFR, which contains the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:76;

[0089] (2) A linker comprising an amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39;

[0090] (3) The heavy chain hinge region of an immunoglobulin, comprising the amino acid sequence of SEQ ID NO:24; and

[0091] (4) Fc fragment, which contains the amino acid sequence of SEQ ID NO:21.

[0092] Preferably, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:45.

[0093] Implementation Scheme 27. A bispecific antibody of any one of Implementation Schemes 22-26, wherein the third polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region of the second antigen-binding moiety that binds to HER3 and an immunoglobulin light chain constant region.

[0094] Implementation Scheme 28. The bispecific antibody of Implementation Scheme 27, wherein the third polypeptide chain comprises, from the N-terminus to the C-terminus:

[0095] (1) The light chain variable region of the second antigen-binding moiety of HER3, which contains the amino acid sequence of SEQ ID NO:28; and

[0096] (2) The light chain constant region of immunoglobulin, which contains the amino acid sequence of SEQ ID NO:29.

[0097] Preferably, the third polypeptide chain comprises SEQ ID NO:41, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:41.

[0098] Implementation Scheme 29. A bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, the first polypeptide chains comprising the amino acid sequence shown in SEQ ID NO:40, and the second polypeptide chains comprising the amino acid sequence shown in SEQ ID NO:41.

[0099] Implementation Scheme 30. A bispecific antibody or antigen-binding fragment thereof targeting EGFR and HER3, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, and wherein...

[0100] i) The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:42, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41;

[0101] ii) The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:46, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41;

[0102] iii) The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:47, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41; or

[0103] iv) The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:48, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41.

[0104] Implementation Scheme 31. A bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof, wherein the bispecific antibody comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, the first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:44, the second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:45, and the third polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:41.

[0105] Implementation Scheme 32. A bispecific antibody-drug conjugate or an isomer thereof and a pharmaceutically acceptable salt thereof, wherein

[0106] The bispecific antibody-drug conjugate comprises: a bispecific antibody according to any one of embodiments 1-31 and a therapeutically active substance or pharmaceutically active ingredient D conjugated thereto.

[0107] Preferably, the bispecific antibody-drug conjugate comprises: a bispecific antibody, a linker L, and a therapeutically active substance or pharmaceutically active ingredient D, according to any one of embodiments 1-31.

[0108] Implementation Scheme 33. The bispecific antibody-drug conjugate or its isomer and its pharmaceutically acceptable salt of Implementation Scheme 32, wherein the therapeutically active substance or pharmaceutically active ingredient D is selected from cytotoxins, plant toxins, small molecule toxins, radioactive isotopes, and maytansine alkaloids, preferably cytotoxins.

[0109] Implementation Scheme 34. The bispecific antibody-drug conjugate or its isomer and pharmaceutically acceptable salt of Implementation Scheme 33, wherein the cytotoxicant is a monomethylaurestatin compound, a camptothecin compound or a maytansine alkaloid.

[0110] Preferably, the monomethyl auristatin compound is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF); or

[0111] The maytansine alkaloids mentioned are DM1, DM3, or DM4; or

[0112] The camptothecin-like compound is Or SN-38.

[0113] Implementation Scheme 35. A bispecific antibody-drug conjugate or an isomer thereof and a pharmaceutically acceptable salt thereof, any one of Implementation Schemes 32-34, wherein the bispecific antibody-drug conjugate comprises a plurality of Ds, the plurality of Ds being different or the same.

[0114] Implementation Scheme 36. A bispecific antibody-drug conjugate or an isomer thereof and a pharmaceutically acceptable salt thereof, any one of Implementation Schemes 32-35, wherein the bispecific antibody-drug conjugate has a drug / antibody ratio (DAR) of 1-15, for example, a DAR of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0115] Implementation Scheme 37. The bispecific antibody-drug conjugate or its isomer and its pharmaceutically acceptable salt of Implementation Scheme 36, wherein the DAR is an average DAR, for example, an average DAR of 1-15, preferably 1-10, more preferably 1-8.

[0116] Implementation Scheme 38. The bispecific antibody-drug conjugate or its isomers and pharmaceutically acceptable salts of Implementation Scheme 37, with an average DAR of 2 to 10, for example 2 to 8.

[0117] Implementation Scheme 39. A bispecific antibody-drug conjugate or an isomer thereof of any one of Implementation Schemes 32-38 and a pharmaceutically acceptable salt thereof, wherein the linker L is a combination of one or more L'; wherein L' is selected from carbonyl, amino, amide, aminoacyl, -(PEG)n-, -(CH2)n-, heteroatom-containing -(CH2)n-, -(C≡C)-, -(CH=CH)-, -O-, -S-, maleimide (mc), maleimide propionyl (MP), methylsulfonylpyrimidinyl, valine-citrulline (val-cit), valine-alanine (val-ala), N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), alanine-phenylalanine (ala-phe), phenylalanine- Lysine (phe-lys), p-aminobenzyloxycarbonyl (PAB), dimethyl ethylenediamine (DMED), N-succinimide-4-(2-pyridinylthio)valerate (SPP), N-succinimide-4-(N-maleimide-methyl)-cyclohexane-1-carboxylate (SMCC), N-succinimide-(4-iodo-acetyl)aminobenzoate (SIAB), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-3-(pyridin-2-yldithio)-propionate (SPDP), glycine-glycine-phenylalanine-glycine (GGFG), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-vc-PABC); wherein n is independently selected from an integer from 1 to 20;

[0118] Preferably, L can be J-L1-L2-X-L3.

[0119] The J mentioned is selected from

[0120] L1 is selected from single bonds and -(PEG). n -or-(CH2) n -, the -(CH2) n-Optionally contains 1 to 6 heteroatoms selected from N, O, and S; wherein n is independently selected as an integer from 1 to 20. Preferably, L1 is selected from -(PEG). n -, where n is independently 2, 4, 6 or 8;

[0121] L2 is selected from one or more combinations of single bonds, -O-, -S-, -CH2-, -NH-, and -C(O)-; wherein the -CH2- and -NH- may optionally be replaced by C 1～6 Alkyl or halogen substitution 1 to 3 times;

[0122] X is selected from a single bond or a combination of 1 to 4 X's, wherein the X's are independently selected from amino acids, such as glycine, alanine, phenylalanine, valine, lysine, and citrulline. For example, X is selected from a single bond or valine-citrulline (val-cit), valine-alanine (val-ala), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), glycine-glycine-phenylalanine-glycine (GGFG).

[0123] L3 is selected from one or more combinations of single bond, -O-, -S-, -CH2-, -NH-, -C(O)-, -phenyl-, -cyclopropyl-, -cyclobutyl-, and -cyclohexyl-; wherein the -CH2-, -NH-, -phenyl-, -cyclopropyl-, -cyclobutyl-, and -cyclohexyl- may optionally be C 1～6 Alkyl, halogen, cyano or hydroxyl groups are substituted 1 to 3 times.

[0124] Implementation Scheme 40. A bispecific antibody-drug conjugate or an isomer thereof, or a pharmaceutically acceptable salt thereof, from any of Implementation Schemes 32-39.

[0125] The structures LD of the linker L and the therapeutic active substance or pharmaceutical active ingredient D are shown in formulas Cpd3, Cpd5 and Cpd6:

[0126] Implementation Scheme 41. A bispecific antibody-drug conjugate or its isomer and a pharmaceutically acceptable salt thereof, as described in any one of Implementation Schemes 32-40, wherein the structure of the bispecific antibody-drug conjugate is shown in Formula (I-C3), Formula (I-C5) and Formula (I-C6):

[0127] Where m is the average number of connections, and m is independently selected from an integer or decimal number from 1 to 10; Ab is the bispecific antibody or its antigen-binding fragment.

[0128] Implementation Scheme 42. A polynucleotide comprising a polynucleotide sequence encoding a bispecific antibody of any one of Implementation Schemes 1-31.

[0129] Implementation Scheme 43. An expression vector comprising the polynucleotide of Implementation Scheme 42.

[0130] Implementation Scheme 44. A host cell comprising the polynucleotide of Implementation Scheme 42 or the expression vector of Implementation Scheme 43.

[0131] Implementation Scheme 45. A method for generating bispecific antibodies, comprising the following steps:

[0132] a) Culture the host cells of implementation scheme 44 under suitable conditions to express bispecific antibodies; and

[0133] b) Isolate and / or purify bispecific antibodies from host cells or their cultures.

[0134] Implementation Scheme 46. A pharmaceutical composition comprising a bispecific antibody of any one of Implementation Schemes 1-31, a bispecific antibody-drug conjugate of any one of Implementation Schemes 32-41, a polynucleotide of Implementation Scheme 42 and / or an expression vector of Implementation Scheme 43, and a pharmaceutically acceptable vector.

[0135] Implementation Scheme 47. Use of any bispecific antibody of Implementation Scheme 1-31, any bispecific antibody-drug conjugate of Implementation Scheme 32-41, polynucleotide of Implementation Scheme 42, expression vector of Implementation Scheme 43, and / or pharmaceutical composition of Implementation Scheme 46 in the preparation of a medicament for the treatment and / or prevention of a disease in a subject in need.

[0136] Implementation Scheme 48. A method for treating and / or preventing a disease in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any one of Implementation Schemes 1-31, a bispecific antibody-drug conjugate of any one of Implementation Schemes 32-41, a polynucleotide of Implementation Scheme 42, an expression vector of Implementation Scheme 43, and / or a pharmaceutical composition of Implementation Scheme 46.

[0137] Implementation Scheme 49. The use of Implementation Scheme 47 or the method of Implementation Scheme 48, wherein the disease is cancer and / or tumor, for example, the disease is cancer and / or tumor expressing EGFR and / or HER3 (EGFR and / or HER3 positive).

[0138] Implementation Scheme 50. The use or method of Implementation Scheme 49, wherein the cancer and / or tumor is selected from: lung cancer, breast cancer, skin cancer, gastric cancer, colorectal cancer, nasopharyngeal cancer, esophageal cancer, brain cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, gastroesophageal junction cancer, digestive tract cancer, uterine cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, thyroid cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, glioblastoma, osteosarcoma, sarcoma, chordoma, squamous cell carcinoma, oral squamous cell carcinoma, lymphoma, mesothelioma, urothelial carcinoma, skin cancer, melanoma, or hematologic malignancy.

[0139] The bispecific antibody and drug conjugate targeting EGFR and HER3 described in this invention have the following advantages:

[0140] 1. It can bind to tumor cells that are positive for EGFR and / or Her3;

[0141] 2. Mediates more bispecific antibody / bispecific antibody ADC internalization;

[0142] 3. It showed good tumor-suppressing effects on tumor models with different EGFR / Here3 expression levels;

[0143] 4. The optimized drug-antibody ratio (DAR) improves tolerability and increases the therapeutic window while maintaining good antitumor activity. Attached Figure Description

[0144] Figure 1: Schematic diagram of the structure of candidate bispecific antibodies.

[0145] Figure 2: Affinity of the bispecific antibody of the present invention to the cell line; Figure 2A uses the MDA-MB-468 cell line, Figure 2B uses the HCC-827 cell line, and Figure 2C uses the HCC1569 cell line.

[0146] Figure 3: Affinity of bispecific antibody molecules to BXPC-3 cells.

[0147] Figure 4: Affinity of bispecific antibody molecules to A375 cells.

[0148] Figure 5: Bispecific antibody blocking EGF binding.

[0149] Figure 6: In vivo efficacy evaluation of antibody-drug conjugates on HCC1569 tumor-bearing mice with low EGFR expression and HER3 expression.

[0150] Figure 7: In vivo efficacy evaluation of antibody-drug conjugates on HCC-827 tumor-bearing mice, a human non-small cell lung cancer cell line expressing EGFR and HER3.

[0151] Figure 8: Binding activity of humanized bispecific antibodies to overexpressing cell lines; Figure 8A shows the EGFR binding assay, and Figure 8B shows the HER3 binding assay.

[0152] Figure 9: Binding activity of humanized bispecific antibodies and their drug conjugates to EGFR cell lines; Figures 9A-9B use the MDA-MB-468 cell line, and Figures 9C-9D use the HCC-827 cell line.

[0153] Figure 10: Binding activity of humanized bispecific antibodies and their drug conjugates with HER3 cell lines; Figures 10A-10B use A375 cell line, and Figures 10C-10D use HCC-1569 cell line.

[0154] Figure 11: Detection of endocytosis of humanized bispecific antibodies and their drug conjugates in EGFR cell lines; Figures 11A-11B use MDA-MB-468 cells, and Figures 11C-11D use HCC-827 cells.

[0155] Figure 12: Detection of endocytosis of humanized bispecific antibodies and their drug conjugates in HER3 cell line; Figures 12A-12B use HCC-1569 cells.

[0156] Figure 13: Antitumor effects of candidate bispecific antibodies and their drug conjugates on human breast cancer xenograft models.

[0157] Figure 14: Antitumor activity of candidate bispecific antibodies and their drug conjugates in a human non-small cell lung cancer xenograft model. Figure 15: Effect of candidate bispecific antibody-drug conjugates on tumor growth curves of human colorectal cancer SW620 cell subcutaneous xenografts in mice.

[0158] Figure 16: Effect of candidate bispecific antibody-drug conjugates on tumor growth curves in a human lung adenocarcinoma PDX (LD1-0025-215621) mouse model.

[0159] Figure 17: Effect of candidate bispecific antibody-drug conjugates on tumor growth curves in human breast cancer PDX (LD1-0009-410668) mouse model.

[0160] Figure 18: Effect of candidate bispecific antibody-drug conjugates on tumor growth curves in a human breast cancer PDX (LD1-2009-362721) mouse model.

[0161] Figure 19: FACS detection results of antigen binding of candidate EGFR VHH that can be used for bispecific antibodies. Detailed Implementation

[0162] definition

[0163] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0164] As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “essentially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “essentially composed of” and “composed of” are encompassed within the meaning of the expression “comprising.”

[0165] As used herein, the connecting term “and / or” between multiple referred elements should be understood to include both individual and combined options. In other words, “and / or” includes both “and” and “or”. For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. Further elements qualified by “and / or” are understood in a similar manner and include any one of them and any combination thereof.

[0166] Unless otherwise stated, any numerical value or range, such as concentration or concentration range, shall in any case be understood to be modified by the term “about”. Thus, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions within that range, unless the context clearly indicates otherwise.

[0167] The term "EGFR (Epidermal Growth Factor Receptor)" refers to the receptor for epidermal growth factor (EGF) cell proliferation and signal transduction. EGFR belongs to 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, and mutations or overexpression of EGFR generally lead to tumorigenesis. EGFR is a glycoprotein, a tyrosine kinase receptor, permeable to the cell membrane, with a molecular weight of 170 kDa. EGFR is located on the cell membrane surface and is activated by binding to ligands, including EGF and TGFα (transforming growth factor α).

[0168] The term "human epidermal growth factor receptor 3 (HER3)," also known as receptor tyrosine protein kinase ErbB-3 (ErbB3), is a member of the EGFR / ErbB family. Unlike other ErbB family members HER2 and EGFR, HER3 itself does not possess kinase activity. Therefore, HER3 must bind to its kinase-active members EGFR or HER2 as a heterodimer to trigger its downstream activities. Upon binding to its natural ligand NRG1, HER3 undergoes a conformational change, heterodimerization, and phosphorylation, subsequently activating MAPK, PI3K / Akt, and PLCγ via signal transduction.

[0169] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0170] The term “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted inclusively, i.e., including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when the opposite is explicitly indicated, such as “only one” or “exactly one” or when “consisting of” is used in the claims, will it refer to only one number or one element of the list. Unless the context clearly indicates the opposite, the words “a” and “an” should be understood as “at least one” in this invention.

[0171] As used herein, the terms "first antigen-binding moiety" and similar terms such as "second antigen-binding moiety" are used for distinction purposes only and are not intended to limit the order or connection of these antigen-binding moieties. Individual antigen-binding moieties may be the same or different from each other. If necessary, two or more first antigen-binding moieties may be included, which may be the same or different. The above terms may be used the same or different in different schemes.

[0172] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, that include both amino and acid functional groups and can be included in polymers of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and congeners; amino acid analogs with variable side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D- or L-optical isomers and peptide-like compounds.

[0173] The terms “polypeptide chain,” “polypeptide,” “peptide,” and “protein” (if single-chain), and “antigen-binding moiety,” used interchangeably herein, refer to a polymer of amino acids of any length. This polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acid components. The term also covers polymers containing modified amino acids, where the modification is, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other operation, such as conjugation with a labeled component. Polypeptides may be isolated from natural sources, produced from eukaryotic or prokaryotic hosts via recombinant techniques, or may be products of synthetic methods.

[0174] As used herein, “antibody” refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen-binding site. Antibody encompasses antibody fragments. As used herein, the term “antibody” includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, intracellular antibodies, camelid antibodies, and antibody fragments, such as, but not limited to, VHH, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b).

[0175] As used herein, the terms "VHH," "VHH chain," "nanobody," and "single-domain antibody" have the same meaning and are used interchangeably. A VHH is a small, stable, and highly efficient antigen-recognizing unit formed by a single heavy chain variable domain, consisting of only one chain from the C-terminus to the N-terminus: FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1. VHH-specific binding to epitopes does not require recognition by other antigen-binding domains (unlike conventional tetrapeptide chain antibodies, where epitopes are recognized by the VL and VH structure pair). Nanobodies possess excellent biological properties, with a molecular weight of 12-15 kDa, one-tenth the size of a complete antibody. They exhibit excellent tissue penetration, high specificity, and good water solubility. Due to their unique structural properties, they combine the advantages of traditional antibodies and small molecule drugs, almost overcoming the shortcomings of traditional antibodies such as long development cycles, low stability, and stringent storage conditions. They are gradually becoming an emerging force in next-generation antibody therapy, showing broad application prospects in immunodiagnosis and treatment. VHH includes, but is not limited to, natural antibodies produced by camelids, or antibodies produced by camelids that have been humanized, or antibodies obtained through phage display technology or yeast display technology.

[0176] As used herein, an antibody “antibody fragment” or “antigen-binding fragment” or “antigen-binding portion” refers to any portion of a full-length antibody that is less than the full length but contains at least a portion of the antibody’s variable region (e.g., one or more CDRs and / or one or more antibody-binding sites) that binds to the antigen, and thus retains binding specificity and at least a portion of the full-length antibody’s specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as the derived antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, VHH, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody frame (e.g., by replacing the corresponding region), acquires an antibody that specifically binds to the antigen.

[0177] The term "monoclonal antibody" refers to an antibody derived from a basic homogeneous group of antibodies, meaning that the individual antibodies comprising this group are identical except for the possibility of naturally occurring mutations, which may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically comprise a large number of antibodies targeting different epitopes (or specific to different epitopes). The modifier "monoclonal" indicates the characteristic of antibodies derived from a basic homogeneous group of antibodies and should not be construed as requiring the production of antibodies through any particular method.

[0178] The term "multispecific antibody" refers to an antibody containing two or more antigen-binding domains, capable of binding to two or more different epitopes (e.g., two, three, four, or more different epitopes), which may be on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" or "bispecific molecules" that bind to two different antigens or two different epitopes. Bispecific antibodies targeting EGFR and HER3 may be referred to as, for example, "anti-EGFR and HER3," "anti-EGFR / HER3," or "EGFR×HER3" bispecific molecules, or bispecific antibodies containing both an EGFR-binding domain and a HER3-binding domain, or other similar terms.

[0179] As used herein, a "bispecific antibody" refers to an antibody that specifically binds to two different antigens. The bispecific antibody of this invention comprises an antigen-binding moiety that specifically binds to two different antigens, EGFR and HER3.

[0180] "Fc fragment" generally refers to a crystallizable fragment of a conventional antibody or heavy chain antibody after papain digestion. Also known as the "Fc region," it contains at least a portion of the C-terminal region of the immunoglobulin heavy chain, representing a constant region. Generally, the Fc fragment of IgG and heavy chain antibodies may contain a partial hinge region, CH2, and CH3. In this article, the Fc fragment may contain at least a partial hinge region (e.g., all or part of the hinge region), CH2, and CH3.

[0181] The term "Knob into Hole structure" refers to the mutation of the hydrophobic amino acid in the CH3 region of the antibody Fc domain. One CH3 side chain amino acid is mutated to form a larger hydrophobic amino acid (knob) to strengthen hydrophobic interactions; the other CH3 side chain amino acid is mutated to form a smaller amino acid (hole) to reduce steric hindrance. The mutated CH3 with knob and with hole interact hydrophobically to form a Knob into Hole structure (KiH), which is beneficial for the formation of heavy chain heterodimers. The KiH mutation mainly occurs in the internal hydrophobic amino acids of the CH3 domain; the exposed amino acids remain almost unchanged, so it does not affect the effector function of the Fc or the resulting immunogenicity. The term "Fc(Knob)" refers to the inclusion of a T366W point mutation in the antibody Fc region to form a knob-like spatial structure. Correspondingly, "Fc(Hole)" refers to the inclusion of T366S, L368A, and Y407V point mutations in the antibody Fc region to form a hole-like spatial structure.

[0182] The term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat, camelid) antibodies. Generally, humanized antibodies contain at least one, and usually two, variable domains, where all or almost all of the variable domains correspond to the variable domains of non-human immunoglobulins, and all or almost all of the frame regions (FRs) are the frame regions of human immunoglobulin sequences. Humanized antibodies may optionally contain at least a portion of the constant regions (Fc) of human immunoglobulins.

[0183] The term "cross-reactivity" refers to the binding of antigen fragments to the same target molecule in humans, monkeys, and / or mice (mice or rats).

[0184] The term "isotype" antibody refers to a class of antibodies (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4) provided by heavy chain constant region genes. Isotypes also include modified forms of one of these classes, where modifications have been generated to alter Fc function, such as to enhance or weaken effector function or binding to the Fc receptor.

[0185] Light chains are classified as κ or λ (Kappa or Lambda). Each heavy chain class can bind to a κ or λ light chain. Typically, light and heavy chains are covalently linked, and when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the "tail" portions of the two heavy chains are linked together via covalent disulfide bonds or non-covalent connections. In the heavy chain, amino acids extend from the N-terminus at the bifurcation end of the Y-configuration to the C-terminus at the base of each chain. The basic structure of some antibodies (e.g., IgG antibodies) comprises two heavy chain subunits and two light chain subunits covalently linked by disulfide bonds to form a "Y" structure.

[0186] Both light and heavy chains are divided into regions with structural and functional homology. The terms "constant" and "variable" are used from a functional perspective. In this regard, it should be understood that the variable regions of the variable light (VL) chain or variable heavy (VH) chain portion determine antigen recognition and specificity. Conversely, the constant regions of the light chain (CL) and heavy chain (CH1, CH2, or CH3) endow biological properties such as secretion, transplacental movement, Fc receptor binding, complement binding, etc. By convention, the numbering of constant region domains increases with distance from the antigen-binding site or the N-terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxyl termini of the heavy and light chains, respectively.

[0187] As described above, variable regions (i.e., “binding domains”) allow antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, for example, the VL and / or VH domains of an antibody, or a subset of complementarity-determining regions (CDRs), combine to form variable regions that define three-dimensional antigen-binding sites. More specifically, the antigen-binding site of a conventional antibody is defined by three CDRs on each VH and VL chain. These “complementarity-determining regions,” or “CDRs,” are discontinuous short sequences of amino acids that are specifically localized to form binding domains as the antibody adopts its three-dimensional conformation in an aqueous environment. The remaining amino acids in the binding domain, referred to as “framework (FR)” regions, exhibit minor intermolecular differences. The binding domain formed by the localized CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its complementary epitope. The amino acids constituting the CDRs and framework regions of any given heavy or light chain variable region can be identified by conventional methods (see “Sequences of Proteins of Immunological Interest”, Kabat, E. et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their full text). In this paper, the CDRs (CDRL or LCDR) of the light chain variable region may be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region may be referred to as HCDR1, HCDR2, and HCDR3.

[0188] In this paper, the amino acid sequence of CDR can be represented according to the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)) based on the antibody's three-dimensional structure and the topology of the CDR ring; Kabat (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242) based on antibody sequence variability; and AbM (Martin, ACR and J. Allen (2007) "Bioinformatics tools for antibody engineering," in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH). Verlag, pp. 95–118), Contact (MacCallum, R.M et al., (1996) J.Mol.Biol. 262: 732-745), IMGT (Lefranc, M.-P., 2011(6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc. and Lefranc, M.-P. et al., Dev.Comp.Immunol., 27, 55-77 (2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It should be understood by those skilled in the art that, unless otherwise specified, the terms “CDR” and “complementarity-determining region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementarity-determining region defined as in any of the known schemes described herein.

[0189] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0190] As used herein, the terms “frame region” and “architecture region” are used interchangeably. As used herein, the terms “frame region,” “architecture region,” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR sequence as defined above.

[0191] An antigen-binding "Fv" fragment can be formed by non-covalent interaction between a VH and a VL. A "single-chain Fv (scFv)" can also be obtained by linking VH and VL via peptide linkers. Furthermore, introducing disulfide bonds into Fv or scFv can yield "disulfide-stabilized Fv (dsFv)" or "single-chain disulfide-stabilized Fv (scdsFv or dsscFv)" respectively.

[0192] As used herein, the Fab fragment is an antibody fragment obtained by digesting a full-length immunoglobulin with papain, or a fragment with the same structure synthesized, for example, through recombinant methods. It contains a complete antibody light chain (VL-CL), a variable region of the antibody heavy chain, and a constant region of the heavy chain (VH-CH1, also known as Fd). Linking the CL and CH1 regions of “Fab” with peptide linkers yields a single-chain “Fab(scFab)”. “F(ab')2” is an antibody fragment resulting from digestion of an immunoglobulin with pepsin at pH 4.0–4.5, or a fragment with the same structure synthesized, for example, through recombinant methods. It essentially consists of two Fab fragments linked by disulfide bonds in their hinge regions. “Fab'” is half of F(ab')2 and can be obtained by reducing the disulfide bonds in the hinge region of F(ab')2.

[0193] As used in this article, the term "hinge region" refers to the heavy chain portion of the molecule that connects the CH1 and CH2 domains. This hinge region is flexible, thus allowing the two N-terminal antigen-binding regions to move independently.

[0194] As used in this article, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge a second thiol group.

[0195] As used herein, the term "percentage (%) sequence identity" or "sequence identity" has a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a known algorithm). This can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0196] In this document, amino acid sequences “derived from” or “from” a reference amino acid sequence are partially or wholly identical or homologous to the reference amino acid sequence. For example, an amino acid sequence derived from the heavy chain constant region of human immunoglobulin may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the wild-type sequence from which the heavy chain constant region of human immunoglobulin from which it is derived.

[0197] Non-critical regions of peptides (e.g., CDR regions of antibodies, non-critical amino acids in framework regions, amino acids in constant regions) can be modified, for example by substituting, adding, and / or deleting one or more amino acids, without altering the peptide's function. Suitable conserved amino acid substitutions in peptides or proteins are known to those skilled in the art and can generally be performed without changing the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a peptide do not substantially alter its biological activity.

[0198] "Affinity" or "binding affinity" measures the strength of the binding between an antibody and an antigen through non-covalent interactions. Affinity can be determined using conventional techniques known in the art, such as biomembrane interference techniques (e.g., using the Octet Fortebio detection system), radioimmunoassay, surface plasmon resonance assay, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS).

[0199] For IgG antibodies, the term "high affinity" refers to a KD of 1.0 × 10⁻⁶ for the antigen. -6 M or lower is preferred, 5.0×10 -8 M or lower, more preferably 1.0 × 10 -8 M or lower, 5.0×10 -9 M or lower, more preferably 1.0 × 10 -9 M or lower. For other antibody subtypes, "high affinity" binding may vary.

[0200] "Specific binding" generally refers to a binding molecule, such as an antibody or its fragments, variants, or derivatives, binding to an epitope through its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope. By this definition, a binding molecule is said to "specifically bind" to an epitope when it is easier for it to bind to an epitope through its antigen-binding domain than to a random, unrelated epitope.

[0201] As used in this article, the term "EC" 50 The "half-maximal effective concentration" (MCI) refers to the concentration of a drug, antibody, or toxicant that induces a 50% response between baseline and maximum after a specific exposure time.

[0202] As used herein, the term "T cell-dependent cytotoxicity" or "TDCC" refers to a form of cytotoxicity in which T cells specifically bind to target cells carrying antigens and subsequently kill the target cells. To determine the TDCC activity of a molecule of interest, in vitro and in vivo TDCC assays can be performed using methods known in the art, such as lactate dehydrogenase (LDH) release assays.

[0203] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to polymers of deoxyribonucleotides (DNA) or polymers of ribonucleotides (RNA). The terms "polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the sequence of nucleotides in a polynucleotide. Those skilled in the art will understand that the DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, with the deoxythymidine nucleotide in the DNA coding strand sequence corresponding to the uridine nucleotide in its encoded RNA sequence.

[0204] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0205] As used herein, the term “expression” includes the transcription and / or translation of nucleotide sequences. Therefore, expression can involve the production of transcripts and / or peptides.

[0206] As used herein, a "vector" is a medium used to introduce exogenous polynucleotides into host cells, whereby the exogenous polynucleotides are amplified or expressed when the vector is transformed into a suitable host cell. Vectors typically remain free, but can be designed to integrate genes or portions thereof into the chromosome of the genome. As used herein, the definition of a vector encompasses plasmids, linearized plasmids, viral vectors, granules, phage vectors, phage particles, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, poxvirus vectors, and baculovirus vectors, etc.

[0207] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0208] The term "prevention" generally refers to a method of obtaining a beneficial or desired outcome, including but not limited to preventive benefits. For the purpose of preventing benefits, 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.

[0209] Terms such as “treatment,” “curing,” “with treatment,” “relief,” or “with relief” refer to therapeutic measures that cure, alleviate, or reduce the symptoms of an existing diagnosed pathological condition or disorder, and / or halt or slow the progression of an existing diagnosed pathological condition or disorder. Terms such as “prevention,” “defense,” “avoidance,” or “containment” refer to preventive or preventative measures that prevent the progression of an undiagnosed target pathological condition or disorder. Therefore, “subjects in need” may include subjects who already have the disease; subjects who are susceptible to the disease; and subjects who need to prevent the disease.

[0210] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0211] The term "therapeuticly effective amount" refers to the amount of an antibody, peptide, polynucleotide, small organic molecule, or other drug that is effective for the disease or condition in the "treated" subject or mammal. In the case of cancer, a therapeutically effective amount of drug may reduce the number of cancer cells; inhibit or stop cancer cell division, reduce or stop tumor size growth; inhibit, for example, suppress, block, prevent, stop, delay, or reverse cancer cell infiltration into surrounding organs, including, for example, cancer spread to soft tissues and bone; inhibit, for example, suppress, block, prevent, shrink, stop, delay, or reverse tumor metastasis; inhibit, for example, suppress, block, prevent, stop, delay, or reverse tumor growth; alleviate one or more cancer-related symptoms to some extent, reduce morbidity and mortality; improve quality of life; or a combination of these effects. In terms of the extent to which a drug prevents the growth of and / or kills existing cancer cells, it may refer to inhibition of cell growth and / or cytotoxicity.

[0212] The term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker unit. In this invention, "antibody-drug conjugate" can be a bispecific antibody-drug conjugate, which refers to a bispecific antibody or its antigen-binding fragment linked to a biologically active cytotoxic drug fragment via a stable linker unit.

[0213] The compounds or antibody-drug conjugates of the present invention comprise their tautomers, mesosomes, racemates, enantiomers, and / or diastereomers. In this invention, the term "diastereomer" generally refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. In this invention, the terms "tautomer" or "tautomeric form" are used interchangeably. The term "mesosome" generally refers to a molecule containing asymmetric atoms but possessing symmetry factors that result in zero total optical rotation within the molecule. The term "racemate" or "racemic mixture" refers to a composition consisting of two enantiomers in equimolar amounts.

[0214] The term "linker" or "linker structure" generally refers to a chemical structural fragment or bond that is linked at one end to a ligand (such as an antibody) and at the other end to a cytotoxic drug. It can also be linked to other linkers before being linked to a cytotoxic drug. The direct or indirect linking to the ligand (such as an antibody) can mean that the group is directly linked to the ligand via a covalent bond, or that the ligand is linked via a linker structure.

[0215] The term "drug loading" typically refers to the average amount of cytotoxic drug (payload) loaded onto each ligand, and can also be expressed as the drug / antibody ratio (DAR). The range of cytotoxic drug loading can be 0-12 cytotoxic drugs per ligand (Ab), for example, 1-10 cytotoxic drugs. In embodiments of the invention, drug loading is expressed as p or p1, and can exemplary be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The drug loading of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0216] Certain atoms of the compounds or antibody-drug conjugates of the present invention may appear in more than one isotopic form. For example, hydrogen may appear as protium (…). 1 H), deuterium ( 2 H) and tritium ( 3 Carbon exists in the form of H, and it may exist in three different isotopes (H). 12 C 13 C and 14 C) Naturally occurring. Examples of isotopes that can be incorporated into the compounds of this invention include, but are not limited to, those found in nature. 15 N、 18 O、 17 O、 18 F, 32 P, 33 P, 12 9I、 131 I, 123 I, 124 I, 125 I, or similar isotopes. Therefore, the compounds or antibody-drug conjugates of the present invention can be enriched in one or more of these isotopes relative to their natural abundance. As those skilled in the art will know, such isotope-enriched compounds can be used for a variety of purposes. For example, substitution with a heavy isotope such as deuterium (2H) may provide certain therapeutic advantages, which could be due to greater metabolic stability. For example, the natural abundance of deuterium (2H) is about 0.015%. Therefore, there is approximately one deuterium atom for every 6500 hydrogen atoms in nature. Therefore, the deuterium-containing compounds or antibody-drug conjugates of the present invention have a deuterium abundance greater than 0.015% at one or more sites (as the case may be). Unless otherwise specified, the structures described in the present invention may also include compounds or antibody-drug conjugates that differ only in the presence or absence of one or more isotope-enriched atoms. For example, compounds or antibody-drug conjugates whose remaining parts are identical to the structure of the present invention, except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon-13 or carbon-14, are all within the scope of the present invention.

[0217] The term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds described in this invention or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to the organism, promote the absorption of the active ingredient, and thus exert its biological activity. Conventional methods for preparing pharmaceutical compositions can be found in national pharmacopoeias. Pharmaceutical compositions can be in the form of sterile injectable aqueous or oil suspensions for intramuscular and subcutaneous administration. These suspensions can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions prepared in non-toxic, parenteral-acceptable diluents or solvents. Furthermore, sterile fixative oils can be conveniently used as solvents or suspension media. For example, any blended fixative oil, including synthetic mono- or diglycerides, can be used. Additionally, fatty acids such as oleic acid can also be used to prepare injectable formulations.

[0218] The terms “pharmaceutically acceptable salt” or “medicinal salt” generally refer to salts of the compounds or antibody-drug conjugates of the present invention, which are safe and / or effective when used in mammals and have the desired biological activity. The compounds or antibody-drug conjugates of the present invention can form salts with acids.

[0219] The term "pharmaceutically acceptable carrier" generally refers to a carrier or delivery system for administering therapeutic agents, such as antibodies or peptides, genes, and other therapeutic agents. This term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without causing excessive toxicity. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polyamino acids, amino acid copolymers, lipid aggregates, and inactivated viral particles. These carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions may include liquids such as water, saline, glycerol, and ethanol. These carriers may also contain excipients such as wetting agents or emulsifiers, pH buffers, etc.

[0220] As used in this article, the term "subject" refers to a mammal, such as a human.

[0221] The term "cancer" refers to a group of cells exhibiting an abnormally high level of proliferation and growth. Cancer can be benign (also called a benign tumor), pre-malignant, or malignant. Cancer cells can be solid cancer cells or leukemia cancer cells. In this invention, the term "tumor" refers to one or more cells containing cancer. In this invention, the term "tumor growth" refers to the proliferation or growth of one or more cells containing cancer, resulting in a corresponding increase in the size or extent of the cancer. The terms "EGFR and / or HER3 positive expression cancer" and "EGFR and / or HER3 expressing cancer" have the same definition and are cancers in which cancer cells express EGFR and / or HER3, preferably on the surface of cancer cells.

[0222] The antibody designations used herein (such as 1G6, 2B13, b0bd-hu2B13, etc.) are for the purpose of distinguishing or identifying antibodies or products only, and are not intended to indicate that such designations are characteristic of the antibodies or products of this invention. Those skilled in the art will understand that, for example, other antibodies or products may also use such designations for the purpose of distinction or identification, but these do not refer to the same or equivalent antibodies or products. Similarly, similar designations used in the examples are merely for illustrative purposes, and the antibodies or products of this invention are defined by the features described in the appended claims.

[0223] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0224] All reagents and raw materials not specified in this invention are commercially available.

[0225] Bispecific antibodies against EGFR and HER3

[0226] In one aspect, the present invention provides a bispecific antibody comprising a first antigen-binding portion that binds to EGFR and a second antigen-binding portion that binds to HER3. Preferably, the binding is a specific binding.

[0227] Methods for constructing bispecific antibodies using antibodies or antigen-binding fragments of interest are well known to those skilled in the art (see, for example, WO1993008829; Suresh MR, et al. Bispecific monoclonal antibodies from hybrid hybridomas. Methods Enzymol. 1986; 121:210-28.; and Traunecker A, et al. Bispecific single chain molecules (Janusins) target cytotoxic lymphocytes on HIV infected cells. EMBO J. 1991 Dec; 10(12):3655-9). Bispecific antibodies can be generated and isolated using a variety of techniques known in the art. For example, a polynucleotide encoding a bispecific antibody can be obtained using recombinant DNA technology, optionally cloned into an expression vector, and then transformed into a host cell with the polynucleotide or expression vector. The transformed host cell can be cultured under suitable conditions to allow expression of the polynucleotide or expression vector, and finally the bispecific antibody can be isolated and purified from the host cell or culture medium. Alternatively, the individual parts of the bispecific antibody can be obtained separately, for example, the first antigen-binding part and the second antigen-binding part as described herein can be obtained separately, and then the individual parts can be optionally coupled through a linker by enzymatic or chemical conjugation techniques to obtain a bispecific antibody that specifically binds to EGFR and HER3.

[0228] As used herein, “first antigen-binding part” and “second antigen-binding part” refer to amino acid sequences containing antigen-binding sites that can bind to antigen epitopes, and their definitions fall within the scope of the meaning of antibody or antigen-binding fragment.

[0229] The first antigen-binding part can be any form of antibody or antigen-binding fragment, including but not limited to VHH, Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', and F(ab')2.

[0230] In some preferred embodiments, the first antigen-binding portion comprises at least one (e.g., one) VHH that specifically binds to EGFR.

[0231] In some embodiments, the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3 in any of the VHHs in SEQ ID NO: 16, 17, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, and 150.

[0232] In some implementations, the CDR is a CDR defined according to Kabat, AbM, Chothia, North, IMGT, or Contact, or a combination thereof.

[0233] In some embodiments, the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 4, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147; HCDR2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 2, 5, 15, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148; and HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 1, 4, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147; and HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 1, 4, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 136, 140, 144, 148; and HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 1, 4, 79, 83 The amino acid sequence represented by any one of NO:3, 6, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149.

[0234] In some embodiments, the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3, wherein

[0235] (1) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; or

[0236] (2) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; or

[0237] (3) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:15, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; or

[0238] (4) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:79, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:80, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:81; or

[0239] (5) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:83, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:84, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:85; or

[0240] (6) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:87, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:88, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:89; or

[0241] (7) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:92, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:93; or

[0242] (8) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:94, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:96, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:97; or

[0243] (9) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:99, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:100, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:101; or

[0244] (10) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:103, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:104, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:105; or

[0245] (11) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:107, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:108, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:109; or

[0246] (12) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:111, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:112, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:113; or

[0247] (13) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:115, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:116, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:117; or

[0248] (14) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:119, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:120, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:121; or

[0249] (15) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:123, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:124, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:125; or

[0250] (16) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:127, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:128, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:129.

[0251] (17) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:131, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:132, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:133; or

[0252] (18) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:135, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:136, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:137; or

[0253] (19) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:139, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:140, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:141; or

[0254] (20) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:143, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:144, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:145; or

[0255] (21) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:147, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:148, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:149.

[0256] In some embodiments, the at least one EGFR-specific VHH of the present invention is humanized, for example, partially or fully humanized. In some embodiments, the PTM (post-translational modification) site of the at least one EGFR-specific VHH is removed.

[0257] In some embodiments, the at least one VHH that specifically binds to EGFR

[0258] (1) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:16, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:16, or consisting of SEQ ID NO:16; or

[0259] (2) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:17, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:17, or consisting of SEQ ID NO:17; or

[0260] (3) An amino acid sequence comprising any one of the amino acid sequences shown in SEQ ID NO:76-78, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the amino acid sequences in SEQ ID NO:76-78, or composed of any one of the amino acid sequences in SEQ ID NO:76-78; or

[0261] (4) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:82, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:82, or composed of SEQ ID NO:82; or

[0262] (5) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:86, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:86, or composed of SEQ ID NO:86; or

[0263] (6) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:90, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:90, or consisting of SEQ ID NO:90; or

[0264] (7) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:94, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:94, or composed of SEQ ID NO:94; or

[0265] (8) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:98, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:98, or consisting of SEQ ID NO:98; or

[0266] (9) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:102, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:102, or composed of SEQ ID NO:102; or

[0267] (10) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:106, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:106, or consisting of SEQ ID NO:106; or

[0268] (11) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:110, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:110, or composed of SEQ ID NO:110; or

[0269] (12) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:114, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:114, or composed of SEQ ID NO:114; or

[0270] (13) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:118, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:118, or composed of SEQ ID NO:118; or

[0271] (14) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:122, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:122, or composed of SEQ ID NO:122; or

[0272] (15) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:126, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:126, or composed of SEQ ID NO:126; or

[0273] (16) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:130, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:130, or consisting of SEQ ID NO:130; or

[0274] (17) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:134, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:134, or composed of SEQ ID NO:134; or

[0275] (18) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:138, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:138, or composed of SEQ ID NO:138; or

[0276] (19) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:142, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:142, or composed of SEQ ID NO:142; or

[0277] (20) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:146, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:146, or consisting of SEQ ID NO:146; or

[0278] (21) An amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:150, or having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:150, or consisting of SEQ ID NO:150.

[0279] In some embodiments, the at least one EGFR-specific VHH comprises an amino acid sequence as shown in any of SEQ ID NO:51-64.

[0280] In some embodiments, the at least one EGFR-specific VHH comprises an amino acid sequence as shown in any of SEQ ID NO:65-78.

[0281] In some preferred embodiments, the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3. In some preferred embodiments, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:16 or 18. In some preferred embodiments, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:18.

[0282] In some preferred embodiments, the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 or 15, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6. In some preferred embodiments, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:17 or 76. In some preferred embodiments, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:76.

[0283] In some embodiments, the second antigen-binding moiety binding HER3 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3; wherein HCDR1 of the second antigen-binding moiety binding HER3 includes the amino acid sequence of SEQ ID NO:30, HCDR2 includes the amino acid sequence of SEQ ID NO:31, HCDR3 includes the amino acid sequence of SEQ ID NO:32, LCDR1 includes the amino acid sequence of SEQ ID NO:33, LCDR2 includes the amino acid sequence of SEQ ID NO:34, and LCDR3 includes the amino acid sequence of SEQ ID NO:35.

[0284] In some embodiments, the heavy chain variable region of the second antigen-binding portion of HER3 comprises the amino acid sequence of SEQ ID NO:26 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26; and / or, the light chain variable region comprises the amino acid sequence of SEQ ID NO:28 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:28.

[0285] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding moiety that binds to EGFR and a second antigen-binding moiety that binds to HER3.

[0286] The first antigen-binding portion comprises at least one EGFR-specific VHH, wherein the VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; and

[0287] The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. HCDR1 of the second antigen-binding region that binds to HER3 includes the amino acid sequence of SEQ ID NO:30, HCDR2 includes the amino acid sequence of SEQ ID NO:31, HCDR3 includes the amino acid sequence of SEQ ID NO:32, LCDR1 includes the amino acid sequence of SEQ ID NO:33, LCDR2 includes the amino acid sequence of SEQ ID NO:34, and LCDR3 includes the amino acid sequence of SEQ ID NO:35.

[0288] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding moiety that binds to EGFR and a second antigen-binding moiety that binds to HER3.

[0289] The first antigen-binding portion comprises at least one EGFR-specific VHH, wherein the VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 or 15, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; and

[0290] The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. HCDR1 of the second antigen-binding region that binds to HER3 includes the amino acid sequence of SEQ ID NO:30, HCDR2 includes the amino acid sequence of SEQ ID NO:31, HCDR3 includes the amino acid sequence of SEQ ID NO:32, LCDR1 includes the amino acid sequence of SEQ ID NO:33, LCDR2 includes the amino acid sequence of SEQ ID NO:34, and LCDR3 includes the amino acid sequence of SEQ ID NO:35.

[0291] In some embodiments, the bispecific antibody of the present invention comprises a first antigen-binding moiety that binds to EGFR and a second antigen-binding moiety that binds to HER3.

[0292] The first antigen-binding portion comprises at least one EGFR-specific VHH, said VHH comprising the amino acid sequence shown in SEQ ID NO:16 or 18, preferably comprising the amino acid sequence shown in SEQ ID NO:18; and

[0293] The second antigen-binding moiety that binds to HER3 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28. In some embodiments, the bispecific antibody of the present invention includes a first antigen-binding moiety that binds to EGFR and a second antigen-binding moiety that binds to HER3.

[0294] The first antigen-binding portion comprises at least one EGFR-specific VHH, said VHH comprising the amino acid sequence shown in SEQ ID NO:17 or 76, preferably comprising the amino acid sequence shown in SEQ ID NO:76; and

[0295] The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28.

[0296] In some embodiments, the bispecific antibody of the present invention comprises: (a) a first antigenic region that specifically binds to human EGFR, the first antigenic region comprising VHH containing SEQ ID NO:16; and (b) a second antigenic region that specifically binds to HER3, the second antigenic region comprising a heavy chain variable region containing SEQ ID NO:26 and a light chain variable region containing SEQ ID NO:28.

[0297] In some embodiments, the bispecific antibody of the present invention comprises: (a) a first antigenic region that specifically binds to human EGFR, the first antigenic region comprising VHH containing SEQ ID NO:17; and (b) a second antigenic region that specifically binds to HER3, the second antigenic region comprising a heavy chain variable region containing SEQ ID NO:26 and a light chain variable region containing SEQ ID NO:28.

[0298] In some embodiments, the bispecific antibody of the present invention comprises: (a) a first antigenic region that specifically binds to human EGFR, the first antigenic region comprising VHH containing SEQ ID NO:18; and (b) a second antigenic region that specifically binds to HER3, the second antigenic region comprising a heavy chain variable region containing SEQ ID NO:26 and a light chain variable region containing SEQ ID NO:28.

[0299] In some embodiments, the bispecific antibody of the present invention comprises: (a) a first antigenic region that specifically binds to human EGFR, the first antigenic region comprising VHH containing SEQ ID NO:76; and (b) a second antigenic region that specifically binds to HER3, the second antigenic region comprising a heavy chain variable region containing SEQ ID NO:26 and a light chain variable region containing SEQ ID NO:28.

[0300] In some implementations, the antibody sequence of the present invention is defined using the Kabat numbering system.

[0301] In some embodiments, the bispecific antibody of the present invention further comprises an immunoglobulin constant region. The immunoglobulin constant region can be the heavy chain constant region (CH) and light chain constant region (CL) of an immunoglobulin of any species. The heavy chain constant region can be derived from the heavy chain constant region of any subtype (e.g., IgA, IgD, IgE, IgG, and IgM), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b) of an immunoglobulin, or a combination thereof. For example, the heavy chain constant region of IgG1 may include: all or part of the hinge region -CH2-CH3 or CH1-hinge region -CH2-CH3. The light chain constant region can be derived from the λ (Lambda) light chain or the κ (Kappa) light chain. In a preferred embodiment, the heavy chain constant region is the heavy chain constant region of human IgG1.

[0302] In some embodiments, the bispecific antibody further comprises the heavy chain constant region CH1 of an immunoglobulin. In some embodiments, the heavy chain constant region CH1 is the heavy chain constant region CH1 of human IgG1 or IgG4. In some embodiments, the heavy chain constant region CH1 comprises an amino acid sequence as shown in SEQ ID NO:22.

[0303] In some embodiments, the bispecific antibody further comprises a heavy chain hinge region of an immunoglobulin. In some embodiments, the heavy chain hinge region is a heavy chain hinge region of human IgG1 or IgG4. In some embodiments, the hinge region comprises an amino acid sequence as shown in SEQ ID NO:23 or SEQ ID NO:24.

[0304] In some embodiments, the bispecific antibody further comprises an immunoglobulin Fc fragment. In some embodiments, the Fc fragment is an Fc fragment of the human IgG1 or IgG4 heavy chain. In some embodiments, the immunoglobulin Fc fragment comprises an amino acid sequence as shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:25.

[0305] In some embodiments, the immunoglobulin Fc fragment contains the T366W mutation.

[0306] In some embodiments, the immunoglobulin Fc fragment contains a T366S mutation.

[0307] In some embodiments, the immunoglobulin Fc fragment contains an L368A mutation.

[0308] In some embodiments, the immunoglobulin Fc fragment contains a Y407V mutation.

[0309] In some embodiments, the immunoglobulin Fc fragment is an immunoglobulin Fc(knob) fragment containing the T366W mutation. In some embodiments, the immunoglobulin Fc fragment is an immunoglobulin Fc(Hole) fragment containing the T366S, L368A, and Y407V mutations.

[0310] In some embodiments, the bispecific antibody further comprises a light chain constant region (CL) of an immunoglobulin. In some embodiments, the light chain constant region is a human κ light chain constant region. In some embodiments, the light chain constant region CL comprises an amino acid sequence as shown in SEQ ID NO:29.

[0311] The first antigen-binding portion and the second antigen-binding portion may optionally be connected via a linker. In some embodiments, the first antigen-binding portion and the second antigen-binding portion are not connected via a linker. In other embodiments, the first antigen-binding portion and the second antigen-binding portion are connected via a linker, such as a peptide linker or a chemical bond. Preferably, the first antigen-binding portion and the second antigen-binding portion are connected via a peptide linker. Exemplary peptide linkers may include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S), or combinations thereof, such as GGAS, GGGS, GGGSG, (GS). n Or (G4S) n , where n is an integer from 1 to 20.

[0312] In some embodiments, the bispecific antibody further includes a linker; the first antigen-binding portion and the second antigen-binding portion are connected via a peptide linker; the peptide linker is exemplary and may be selected from (GGGGS). n (GS) n The peptide sequence may be obtained by splicing either the peptide or the peptide, where n is an integer from 1 to 6. In some embodiments, the linker is as shown in the amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.

[0313] In some embodiments, the bispecific antibody of the present invention has two heavy chain polypeptide chains, one of which contains an Fc (Knob) fragment and the other contains an Fc (Hole) fragment, the two heavy chain polypeptide chains forming a dimer through disulfide bonds in the hinge region and a Knob-into-Hole structure.

[0314] In some embodiments, the Fc fragment on one heavy chain polypeptide chain is designated as Fc(Knob), and the Fc fragment on the other heavy chain polypeptide chain is designated as Fc(Hole).

[0315] In some embodiments, the immunoglobulin Fc(knob) fragment contains the T366W mutation, and in some embodiments, the immunoglobulin Fc(Hole) fragment contains the T366S, L368A, and Y407V mutations.

[0316] In some embodiments, the immunoglobulin Fc(knob) fragment comprises an amino acid sequence as shown in SEQ ID NO:20.

[0317] In some embodiments, the immunoglobulin Fc(Hole) fragment comprises an amino acid sequence as shown in SEQ ID NO:21.

[0318] In some embodiments, the bispecific antibody of the present invention comprises a first polypeptide chain and a second polypeptide chain; wherein the first polypeptide chain comprises the VHH of the first antigen-binding moiety binding to EGFR, the heavy chain variable region of the second antigen-binding moiety binding to HER3, the immunoglobulin heavy chain constant region CH1, the immunoglobulin heavy chain hinge region, and the immunoglobulin Fc fragment, and optionally, further comprises a linker; and wherein the second polypeptide chain comprises the light chain variable region of the second antigen-binding moiety binding to HER3 and the immunoglobulin light chain constant region. The heavy chain constant region CH1, the immunoglobulin heavy chain hinge region, the immunoglobulin Fc fragment, the linker, and / or the immunoglobulin light chain constant region may be as defined herein.

[0319] In some embodiments, the bispecific antibody of the present invention comprises two first polypeptide chains and two second polypeptide chains. In some embodiments, the bispecific antibody of the present invention comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0320] In some embodiments, the bispecific antibody of the present invention comprises a first polypeptide chain and a second polypeptide chain; wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, the VHH of the first antigen-binding moiety binding to EGFR, a linker, the heavy chain variable region of the second antigen-binding moiety binding to HER3, the immunoglobulin heavy chain constant region CH1, the immunoglobulin heavy chain hinge region, and the Fc fragment of immunoglobulin.

[0321] In some specific implementations, the first polypeptide chain comprises, from the N-terminus to the C-terminus:

[0322] (1) VHH that binds to the first antigen-binding portion of EGFR, comprising the amino acid sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:76;

[0323] (2) A linker comprising an amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39;

[0324] (3) The heavy chain variable region of the second antigen-binding region of HER3, which contains the amino acid sequence of SEQ ID NO:26;

[0325] (4) Immunoglobulin heavy chain constant region CH1, which contains the amino acid sequence of SEQ ID NO:22;

[0326] (5) Immunoglobulin heavy chain hinge region, which contains the amino acid sequence of SEQ ID NO:23;

[0327] (6) Immunoglobulin Fc fragment, which contains the amino acid sequence of SEQ ID NO:19.

[0328] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:46 or SEQ ID NO:48, or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:46 or SEQ ID NO:48.

[0329] In some embodiments, the bispecific antibody comprises two identical first polypeptide chains and two identical second polypeptide chains; wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region of the second antigen-binding moiety binding to HER3, an immunoglobulin heavy chain constant region CH1, an immunoglobulin heavy chain hinge region, an Fc fragment of immunoglobulin, a linker, and a VHH of the first antigen-binding moiety binding to EGFR.

[0330] In some specific implementations, the first polypeptide chain comprises, from the N-terminus to the C-terminus:

[0331] (1) The heavy chain variable region of the second antigen-binding region of HER3, which contains the amino acid sequence of SEQ ID NO:26;

[0332] (2) Heavy chain constant region CH1, which contains the amino acid sequence of SEQ ID NO:22;

[0333] (3) The heavy chain hinge region of an immunoglobulin, which contains the amino acid sequence of SEQ ID NO:23;

[0334] (4) Fc fragment, which contains the amino acid sequence of SEQ ID NO:19;

[0335] (5) A linker comprising an amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39;

[0336] (6) VHH that binds to the first antigen-binding portion of EGFR, which contains the amino acid sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:76.

[0337] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:47.

[0338] In some embodiments, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain; wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region of the second antigen-binding moiety that binds to HER3 and a light chain constant region of the immunoglobulin.

[0339] In some specific implementations, the second polypeptide chain comprises, from the N-terminus to the C-terminus:

[0340] (1) The light chain variable region of the second antigen-binding moiety of HER3, which contains the amino acid sequence of SEQ ID NO:28;

[0341] (2) The light chain constant region of an immunoglobulin, which contains the amino acid sequence of SEQ ID NO:29.

[0342] In some embodiments, the second polypeptide chain comprises SEQ ID NO:41, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:41.

[0343] In some embodiments, the bispecific antibody comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein the first polypeptide chain comprises a heavy chain variable region of the second antigen-binding moiety binding to HER3, an immunoglobulin heavy chain constant region CH1, an immunoglobulin heavy chain hinge region, and an immunoglobulin Fc fragment; the second polypeptide chain comprises a VHH of the first antigen-binding moiety binding to EGFR, an immunoglobulin heavy chain hinge region, and an immunoglobulin Fc fragment; and the third polypeptide chain comprises a light chain variable region of the second antigen-binding moiety binding to HER3 and an immunoglobulin light chain constant region. The heavy chain constant region CH1, the immunoglobulin heavy chain hinge region, the immunoglobulin Fc fragment, and / or the immunoglobulin light chain constant region may be as defined herein.

[0344] In some embodiments, the bispecific antibody comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region of the second antigen-binding moiety that binds to HER3, an immunoglobulin heavy chain constant region CH1, an immunoglobulin heavy chain hinge region, and an immunoglobulin Fc fragment.

[0345] In some implementations, the first polypeptide chain comprises, from the N-terminus to the C-terminus:

[0346] (1) The heavy chain variable region of the second antigen-binding region of HER3, which contains the amino acid sequence of SEQ ID NO:26;

[0347] (2) Immunoglobulin heavy chain constant region CH1, which contains the amino acid sequence of SEQ ID NO:22;

[0348] (3) The heavy chain hinge region of an immunoglobulin, which contains the amino acid sequence of SEQ ID NO:23;

[0349] (4) Immunoglobulin Fc fragment, which contains the amino acid sequence of SEQ ID NO:20.

[0350] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:44, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:44.

[0351] In some embodiments, the bispecific antibody comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region of the first antigen-binding moiety that binds to EGFR, a linker, an immunoglobulin heavy chain hinge region, and an immunoglobulin Fc fragment.

[0352] In some implementations, the second polypeptide chain comprises, from the N-terminus to the C-terminus:

[0353] (1) The heavy chain variable region that binds to the first antigen-binding region of EGFR, which contains the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:76;

[0354] (2) A linker comprising an amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39;

[0355] (3) The heavy chain hinge region of an immunoglobulin, which contains the amino acid sequence of SEQ ID NO:24;

[0356] (4) Fc fragment, which contains the amino acid sequence of SEQ ID NO:21.

[0357] In some embodiments, the second polypeptide chain comprises the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:45.

[0358] In some embodiments, the bispecific antibody comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein the third polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region of the second antigen-binding moiety that binds to HER3 and an immunoglobulin light chain constant region.

[0359] In some implementations, the third polypeptide chain comprises, from the N-terminus to the C-terminus:

[0360] (1) The light chain variable region of the second antigen-binding moiety of HER3, which contains the amino acid sequence of SEQ ID NO:28;

[0361] (2) The light chain constant region of an immunoglobulin, which contains the amino acid sequence of SEQ ID NO:29.

[0362] In some embodiments, the third polypeptide chain comprises SEQ ID NO:41, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:41.

[0363] Bispecific antibody 1G6-Pach

[0364] In one aspect, the present invention provides a bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof.

[0365] The antibody contains two first polypeptide chains and two second polypeptide chains.

[0366] The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:40;

[0367] The second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41.

[0368] Bispecific antibody 2B13-Pach

[0369] In one aspect, the present invention provides a bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof.

[0370] The antibody contains two first polypeptide chains and two second polypeptide chains.

[0371] The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:42;

[0372] The second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41.

[0373] Bispecific antibody b0bd-hu2B13-1

[0374] In one aspect, the present invention provides a bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof.

[0375] An antibody contains a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain.

[0376] The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:44;

[0377] The second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:45;

[0378] The third polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41.

[0379] Bispecific antibody b0bd-hu2B13-2

[0380] In one aspect, the present invention provides a bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof.

[0381] The antibody contains two first polypeptide chains and two second polypeptide chains.

[0382] The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:46;

[0383] The second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41.

[0384] Bispecific antibody b0bd-hu2B13-3c

[0385] In one aspect, the present invention provides a bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof.

[0386] The antibody contains two first polypeptide chains and two second polypeptide chains.

[0387] The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:47;

[0388] The second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41.

[0389] Bispecific antibody b0bd-hu1G6-2

[0390] In one aspect, the present invention provides a bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof.

[0391] The antibody contains two first polypeptide chains and two second polypeptide chains.

[0392] The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:48;

[0393] The second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41.

[0394] In some embodiments, the bispecific antibody or its antigen-binding fragment of the present invention has at least one of the following characteristics:

[0395] 1) Tumor cells that bind to EGFR and / or Her3 positive expression;

[0396] 2) Mediates the internalization of bispecific antibodies / bispecific antibody-drug conjugates;

[0397] 3) It showed good tumor-suppressing effects on tumor models with different EGFR / Here3 expression levels.

[0398] In some embodiments, the bispecific antibody of the present invention is capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90% or higher.

[0399] Bispecific antibody-drug conjugates targeting EGFR and HER3 and their preparation

[0400] The present invention further provides a bispecific antibody-drug conjugate or its isomer and a pharmaceutically acceptable salt thereof, wherein the bispecific antibody-drug conjugate comprises: a first antigen-binding moiety binding to EGFR, a second antigen-binding moiety binding to HER3, a linker L and a therapeutically active substance or pharmaceutically active ingredient D; wherein the first antigen-binding moiety binding to EGFR and the second antigen-binding moiety binding to HER3 are as described above in the present invention.

[0401] The present invention further provides a bispecific antibody-drug conjugate or its isomer and a pharmaceutically acceptable salt thereof, wherein the bispecific antibody-drug conjugate comprises: the bispecific antibody of the present invention and a therapeutically active substance or pharmaceutically active ingredient D conjugated thereto.

[0402] The present invention further provides a bispecific antibody-drug conjugate or an isomer thereof and a pharmaceutically acceptable salt thereof, wherein the bispecific antibody-drug conjugate comprises: the bispecific antibody of the present invention, a linker L, and a therapeutically active substance or pharmaceutically active ingredient D.

[0403] In some embodiments, the therapeutically active substance or pharmaceutically active ingredient D is a cytotoxin, plant toxin, small molecule toxin, radioactive isotope, maytansine alkaloid, etc. Cytotoxins are preferred.

[0404] In some embodiments, the cytotoxin is a monomethylaurestatin compound, a camptothecin compound, or a maytansine alkaloid.

[0405] In some embodiments, the monomethyl auristatin compound may be monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0406] In some implementations, the maytansine alkaloids may be DM1, DM3 or DM4.

[0407] In some implementations, camptothecin compounds can be used as... Or SN-38.

[0408] In some embodiments, the bispecific antibody-drug conjugate comprises a plurality of Ds, which may be a combination of different therapeutic active substances or pharmaceutical active ingredients, or a combination of the same therapeutic active substance or pharmaceutical active ingredient.

[0409] In some embodiments, the bispecific antibody-drug conjugate has a drug-to-antibody ratio (DAR) of 1-15, for example, a DAR of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0410] In some implementations, the DAR is the average DAR.

[0411] In some implementations, the average DAR is 1-15, for example, 1-10.

[0412] In some implementations, the average DAR is preferably 1 to 8.

[0413] As a specific implementation method, the average DAR of the bispecific antibody-drug conjugate of the present invention is preferably 2 to 10, for example 2 to 8.

[0414] In some embodiments, the linker L is a combination of one or more L's; the L' is selected from carbonyl, amino, amide, aminoacyl, -(PEG)n-, -(CH2)n-, heteroatom-containing -(CH2)n-, -(C≡C)-, -(CH=CH)-, -O-, -S-, maleimide (mc), maleimide propionyl (MP), methylsulfonylpyrimidinyl, valine-citrulline (val-cit), valine-alanine (val-ala), N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), p-aminobenzyloxycarbonyl ( PAB), dimethyl ethylenediamine (DMED), N-succinimide-4-(2-pyridinylthio)valerate (SPP), N-succinimide-4-(N-maleimide-methyl)-cyclohexane-1-carboxylate (SMCC), N-succinimide-(4-iodo-acetyl)aminobenzoate (SIAB), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-3-(pyridin-2-yldithio)propionate (SPDP), glycine-glycine-phenylalanine-glycine (GGFG), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-vc-PABC); wherein n is independently selected from an integer from 1 to 20.

[0415] In some implementations, L can be J-L1-L2-X-L3; J is selected from...

[0416] In some implementations, L1 is selected from single bonds, -(PEG) n -or-(CH2)n -, the -(CH2) n -Optionally contains 1 to 6 heteroatoms selected from N, O, and S; wherein n is independently selected as an integer from 1 to 20. Preferably, L1 is selected from -(PEG). n - where n is independently 2, 4, 6 or 8.

[0417] In some embodiments, L2 is selected from one or more combinations of single bonds, -O-, -S-, -CH2-, -NH-, and -C(O)-; wherein the -CH2- and -NH- may optionally be replaced by C 1～6 Alkyl or halogen substitution 1 to 3 times.

[0418] In some embodiments, X is selected from a single bond or a combination of 1 to 4 X's, wherein each X' is independently selected from amino acids, such as glycine, alanine, phenylalanine, valine, lysine, and citrulline. In some embodiments, X is selected from a single bond or valine-citrulline (val-cit), valine-alanine (val-ala), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), glycine-glycine-phenylalanine-glycine (GGFG).

[0419] In some embodiments, L3 is selected from one or more combinations of single bond, -O-, -S-, -CH2-, -NH-, -C(O)-, -phenyl-, -cyclopropyl-, -cyclobutyl-, and -cyclohexyl-; wherein the -CH2-, -NH-, -phenyl-, -cyclopropyl-, -cyclobutyl-, and -cyclohexyl- may optionally be C 1～6 Alkyl, halogen, cyano or hydroxyl groups are substituted 1 to 3 times.

[0420] In some embodiments, the structures LD of the linker L and the therapeutically active substance or pharmaceutically active ingredient D are as shown in formulas Cpd3, Cpd5, and Cpd6:

[0421] In some embodiments, the bispecific antibody-drug conjugates of the present invention have structures as shown in formulas (I-C3), (I-C5), and (I-C6):

[0422] in,

[0423] m is the average number of connections, and m is independently selected from an integer or decimal number between 1 and 10;

[0424] Ab is the bispecific antibody or its antigen-binding fragment of the present invention.

[0425] In some implementations, the average number of connections m can be any integer or decimal from 1 to 10. For example, the average number of connections m can be any integer or decimal from 2 to 9. For example, the average number of connections m can be any decimal or integer from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10. For example, the average number of connections can be 4, 4.01, 4.10, 4.99, 6, 6.01, 6.10, 5.99, 7.99, 7.98, 8, or 8.05.

[0426] In some implementations, the average number of connections m is any integer from 1 to 10. For example, the average number of connections m is 1, 2, 3, 4, 5, 6, 7, or 8.

[0427] In some embodiments, the bispecific antibody or its antigen-binding fragment refers to b0bd-hu2B13-1, b0bd-hu2B13-2, b0bd-hu2B13-3c, and B0bd-hu1G6-2.

[0428] In some embodiments, the bispecific antibody-drug conjugate of the present invention preferably has the following structure:

[0429] Or a combination thereof.

[0430] In some embodiments, the bispecific antibody-drug conjugate of the present invention is capable of inhibiting tumor growth by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%, more preferably at least about 90% or higher.

[0431] In another aspect, the present invention provides a method for preparing the bispecific antibody-drug conjugate of the present invention, comprising the following steps: mixing the bispecific antibody of the present invention dissolved in a buffer solution with a linker-therapeutic active substance or pharmaceutical active ingredient (LD) dissolved in a solvent under the action of a reducing agent to obtain the bispecific antibody-drug conjugate.

[0432] In some embodiments, the preparation method includes reacting the bispecific antibody against EGFR and HER3 of the present invention with compounds of the aforementioned formulas Cpd3, Cpd5 and Cpd6.

[0433] In some embodiments, the reducing agent is a reducing agent conventional for this type of reaction in the art. In some embodiments, the solvent is a solvent conventional for this type of reaction in the art. In some embodiments, the buffer solution is a buffer solution conventional for this type of reaction in the art.

[0434] Polynucleotides, vectors and host cells

[0435] In another aspect, the present invention provides a polynucleotide comprising a polynucleotide sequence encoding the bispecific antibody against EGFR and HER3 of the present invention.

[0436] The polynucleotides of the present invention can be obtained using methods known in the art. For example, the polynucleotides of the present invention can be isolated from phage display libraries, yeast display libraries, immunized animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemically synthesized. The polynucleotides of the present invention can be codon-optimized for the host cell used for expression.

[0437] In another aspect, the present invention also provides expression vectors comprising the polynucleotides of the present invention. The expression vectors may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The polynucleotides of the present invention may be present in one or more expression vectors. In some embodiments, the polynucleotides of the present invention are prepared as recombinant nucleic acids. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR) techniques), etc.

[0438] The present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention. The polynucleotide or expression vector of the present invention can be introduced into a suitable host cell using various methods known in the art. These methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.

[0439] In a preferred embodiment, the host cell is used to express the bispecific antibody of the present invention that binds to EGFR and HER3. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as Escherichia coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Suitable mammalian host cells for antibody expression include, but are not limited to, exogenous human cervical cancer cells (HeLa cells), human embryonic kidney cells (HEK cells, such as HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

[0440] The present invention also provides a method for generating the bispecific antibody against EGFR and HER3 of the present invention, comprising the following steps:

[0441] a) Culturing the host cells of the present invention under suitable conditions to express the bispecific antibodies of the present invention; and

[0442] b) Isolate and / or purify the bispecific antibodies of the present invention from host cells or their cultures.

[0443] Pharmaceutical Composition

[0444] The present invention also provides a pharmaceutical composition comprising the bispecific antibody against EGFR and HER3 of the present invention and / or the bispecific antibody-drug conjugate of the present invention and / or the polynucleotide of the present invention and / or the expression vector of the present invention, as well as a pharmaceutically acceptable vector. In some specific embodiments, the pharmaceutical composition comprises the bispecific antibodies 1G6-Pach, 2B13-Pach, b0bd-hu2B13-1, b0bd-hu2B13-2, b0bd-hu2B13-3C, b0bd-hu1G6-2, or a combination thereof.

[0445] The pharmaceutical compositions described herein can be in various dosage forms, including but not limited to solid, semi-solid, liquid, powder, or lyophilized forms. For compositions containing antibodies or antigen fragments thereof, preferred dosage forms are typically, for example, injection solutions and lyophilized powders.

[0446] The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, such as systemic or local administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavitary), local (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or local). Preferably, the pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Administration methods may include, for example, injection or infusion.

[0447] Those skilled in the art will understand that the exact dosage will depend on various factors, such as the pharmacokinetic properties of the pharmaceutical composition, the duration of treatment, the excretion rate of a particular compound, the therapeutic purpose, the route of administration, and the condition of the subject, such as the patient's age, health status, weight, sex, diet, medical history, and other factors known in the medical field. As a general guideline, the dosage range for the bispecific antibodies against EGFR and HER3 of the present invention, the bispecific antibody-drug conjugates of the present invention, or the pharmaceutical compositions of the present invention is about 0.0001-1000 mg / kg. Administration may be by subcutaneous or intravenous injection.

[0448] The present invention also provides a kit comprising the bispecific antibody against EGFR and HER3 of the present invention, the bispecific antibody-drug conjugate of the present invention, the polynucleotide of the present invention, the expression vector of the present invention, and / or the pharmaceutical composition of the present invention.

[0449] treat

[0450] In another aspect, the present invention relates to the use of the bispecific antibodies against EGFR and HER3 of the present invention, the bispecific antibody-drug conjugates of the present invention, the polynucleotides of the present invention, the expression vectors of the present invention, and / or the pharmaceutical compositions of the present invention in the preparation of medicaments for treating and / or preventing diseases in subjects in need.

[0451] This invention also relates to the bispecific antibodies against EGFR and HER3 of this invention, the bispecific antibody-drug conjugates of this invention, the polynucleotides of this invention, the expression vectors of this invention, and / or the pharmaceutical compositions of this invention, for the treatment and / or prevention of diseases.

[0452] The present invention also relates to the bispecific antibodies against EGFR and HER3 of the present invention, the bispecific antibody-drug conjugates of the present invention, the polynucleotides of the present invention, the expression vectors of the present invention, and / or the pharmaceutical compositions of the present invention, which are used as inhibitors of EGFR and / or HER3.

[0453] The present invention also provides a method for treating and / or preventing disease in a subject in need, the method comprising administering to the subject a therapeutically effective amount of the present invention’s bispecific antibody against EGFR and HER3, the present invention’s bispecific antibody-drug conjugate, the present invention’s polynucleotide, the present invention’s expression vector, and / or the present invention’s pharmaceutical composition.

[0454] In some embodiments, the disease is cancer and / or tumor. In some embodiments, the disease is cancer and / or tumor expressing EGFR and / or HER3 (EGFR and / or HER3 positive).

[0455] As used herein, cancers and / or tumors include, but are not limited to: lung cancer, breast cancer, skin cancer, stomach cancer, colorectal cancer, nasopharyngeal cancer, esophageal cancer, brain cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, gastroesophageal junction cancer, digestive tract cancer, uterine cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, thyroid cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, glioblastoma, osteosarcoma, sarcoma, chordoma, squamous cell carcinoma, oral squamous cell carcinoma, lymphoma, mesothelioma, urothelial carcinoma, skin cancer, melanoma, or hematologic malignancy.

[0456] Combination therapy

[0457] For cancer treatment, the bispecific antibodies against EGFR and HER3 of the present invention, the bispecific antibody-drug conjugates of the present invention, the polynucleotides of the present invention, the expression vectors of the present invention, and / or the pharmaceutical compositions of the present invention can be used in combination with other treatment methods, including but not limited to: surgery, chemotherapy, radiotherapy, immunotherapy, hormone therapy, angiogenesis inhibition, and palliative treatment.

[0458] The bispecific antibodies against EGFR and HER3 of the present invention, the bispecific antibody-drug conjugates of the present invention, the polynucleotides of the present invention, the expression vectors of the present invention, and / or the pharmaceutical compositions of the present invention can also be administered in combination with at least one or more of the therapeutic agents described herein. There are no limitations on the manner of combined administration.

[0459] In some embodiments, the bispecific antibodies against EGFR and HER3 of the present invention, the bispecific antibody-drug conjugates of the present invention, the polynucleotides of the present invention, the expression vectors of the present invention, and / or the pharmaceutical compositions of the present invention used in combination comprise bispecific antibodies 1G6-Pach, 2B13-Pach, b0bd-hu2B13-1, b0bd-hu2B13-2, b0bd-hu2B13-3C, b0bd-hu1G6-2 or combinations thereof, or their respective antibody-drug conjugates. Beneficial effects

[0460] The bispecific antibody and its antibody-drug conjugate targeting EGFR and HER3 of this invention can simultaneously bind to EGFR and HER3 antigens and EGFR / HER3 cell lines, exhibiting better antitumor efficacy than monospecific antibodies. Specifically, in HCC827 lung cancer and H1569 breast cancer models, the bispecific antibody ADC of this invention showed better efficacy than both the monospecific antibody ADC and the control bispecific antibody ADC SI-1X6.4.

[0461] The bispecific antibody in this invention exhibits excellent stability. After affinity purification, the antibody purity can reach 95%. The structure and activity of the antibody remain well maintained after heat treatment, demonstrating that the antibody can maintain a good molecular conformation and complete biological activity even under harsh environments, which is beneficial for industrial production and packaging storage.

[0462] Example

[0463] A further understanding of the invention can be obtained by referring to the specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of the invention in any way. Obviously, many modifications and variations can be made to the invention without departing from its spirit, and therefore, these modifications and variations are also within the scope of protection claimed in this application. All proportions used herein include percentages, and unless otherwise specified, are by weight.

[0464] Example 1: Preparation and Characterization of Bispecific Antibodies Against EGFR and HER3

[0465] 1.1 Antibody Preparation

[0466] Preparation of control antibodies: This application uses anti-HER3 VHH 7D12, HER3 monospecific antibody Patritumab, EGFR-HER3 bispecific antibody SI-1X6.4 (also known as BL-B01D1) and its antibody-drug conjugate (DAR8), bispecific antibody DB-YE-X20 (also known as DB1418) and its antibody-drug conjugate (DAR6), and EGFR monospecific antibody Cetuximab as positive control antibodies.

[0467] 7D12 was prepared according to the sequence disclosed in patent application WO2007042289 (VHH being SEQ ID NO. 81 in that application), Patritumab according to the sequence disclosed in WO2007077028 (VH and VL being SEQ ID NO. 70 and SEQ ID NO. 72 in that patent application, respectively), and SI-1X6.4 according to WO2023083381 (especially SEQ ID NO. 2 and SEQ ID NO. 4 in that patent application), and was prepared according to the disclosed method. DB-YE-X20 was prepared according to WO2025016453A1 (especially SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 90 in that patent application as the target sequence, X2 being the target linker-toxin), and was prepared according to the patented method. Cetuximab monoclonal antibody (Cetuximab antibody sequence as shown in SEQ ID NO: 151 and SEQ ID NO: 152 of this application) was prepared according to conventional methods.

[0468] 1.2 Antibody Configuration and Construction

[0469] This embodiment describes the structure and expression vector construction of anti-EGFR and HER3 bispecific antibodies. Six anti-EGFR and HER3 bispecific antibodies were designed and constructed. The antibody conformations are shown in Figures 1A, 1B, and 1C, and the corresponding amino acid sequences are shown in Table 2. The specific antibody conformations are described below:

[0470] Antibodies 1G6-Pach, 2B13-Pach, b0bd-hu2B13-2, and b0bd-hu1G6-2 contain two identical first polypeptide chains and two identical second polypeptide chains, as shown in Figure 1A. The anti-EGFR VHH is linked to the N-terminus of the heavy chain of the anti-HER3 antibody. This configuration constitutes a bispecific bivalent antibody.

[0471] Antibody b0bd-hu2B13-3c: Contains two identical first polypeptide chains and two identical second polypeptide chains, with the conformation shown in Figure 1B. The anti-EGFR VHH is linked to the C-terminus of the heavy chain of the anti-HER3 antibody. This conformation constitutes a bispecific bivalent antibody.

[0472] Antibody b0bd-hu2B13-1: It contains an asymmetric first, second, and third polypeptide chain, as shown in Figure 1C. The anti-EGFR VHH is linked to the Fc region of the heavy chain of the anti-HER3 antibody, effectively replacing one of the anti-HER3 antibody Fab molecules. Using the knock-in-hole technique, a heterodimer is formed. This configuration results in a bispecific monovalent antibody.

[0473] The antibody names and sequences are shown in Table 2.

[0474] Table 2 Antibody Sequences (SEQ ID NO:)

[0475] 1.3 Antibody expression and purification

[0476] Transfection and Expression: Collect cells, centrifuge, and discard the supernatant. Add cells to the electroporation buffer, mix well, and then add an appropriate amount of plasmid. After thoroughly mixing the cell-plasmid suspension, add it to a 10ml electroporation tube and place the tube in the electroporator for electroporation. After electroporation, aliquot the cells from the electroporation tube into pre-prepared culture medium shake flasks and incubate statically for 40 minutes. After incubation, place the shake flasks in a 37°C, 270rpm, 8% CO2 incubator. After 24 hours, add feed / sodium butyrate / bispecific antibody and continue culturing for 3-7 days. Samples are taken for testing on day 5.

[0477] Sample purification: Protein A affinity chromatography column purification:

[0478] 1) Equilibration column: 1xPBS, flow rate 1ml / min, 20CV

[0479] 2) Sample loading: Retention time 4 min

[0480] 3) Washing: 1xPBS, flow rate 1ml / min, 10CV

[0481] 4) Elution: Sodium acetate buffer (pH 3.4), retention time 5 min, collect in separate tubes, and read the absorbance value at 280 nm using a NanoDrop instrument.

[0482] 5) Dialysis: Aspirate high-concentration protein into a dialysis bag and place it in a beaker with PBS and pH 7.2-7.4 for dialysis.

[0483] The bispecific antibody production and purification process of this invention is excellent.

[0484] 1.4 Purity Identification

[0485] SEC-HPLC experiments were performed using an LC-20AT high-performance liquid chromatograph and a gel chromatography column. The experimental conditions were as follows:

[0486] Replace the water with the mobile phase, and slowly increase the flow rate to 1,000 ml / min until the baseline stabilizes. Transfer 25 μL of antibody to the corresponding numbered vial, place the vial in the appropriate position on the instrument, and inject the sample for 15 min. Analyze, process, and save the data. Replace the mobile phase with deionized water and rinse for 1.5 h.

[0487] The SEC-HPLC results of the candidate bispecific antibodies are as follows: The percentages of high molecular weight polymers, antibody monomers, and low molecular weight substances in the sample were calculated using the area normalization method. The results show that the purity of the candidate bispecific antibody monomers is greater than 95%. Example 2: Antibody Affinity Detection (SPR)

[0488] In this embodiment, the affinity of the anti-EGFR and HER3 bispecific antibody of the present invention was detected using a Biacore X100 (Cytiva).

[0489] Experimental methods:

[0490] 1. Protein A chip captured b0bd-hu2B13-1 (0.5 μg / mL). The analytes were human EGFR (Human EGFR Protein, His Tag (MALS verified), ACRO, EGR-H5222), serially diluted in the range of 3.125-200 nM, for a total of 7 concentrations; human HER3 (Human HER3 / ERBB3 Protein (ECD, His Tag), HPLC-verified, Sinobiological, 10201-H08H), serially diluted in the range of 3.125-200 nM, for a total of 7 concentrations; monkey EGFR (Rhesus EGFR Protein (ECD, His Tag), Sinobiological, 90317-K08H), serially diluted in the range of 3.125-200 nM, for a total of 7 concentrations; and monkey HER3 (Rhesus HER3 / ERBB3 Protein (His Tag)). Tag), HPLC-verified, Sinobiological, 90043-K08H), with concentrations serially diluted in the range of 3.125-200 nM, for a total of 7 concentrations;

[0491] Similarly, Protein A chip captured b0bd-hu2B13-2 (0.5 μg / mL). The analytes were human EGFR (Human EGFR Protein, His Tag (MALS verified), ACRO, EGR-H5222), serially diluted in the range of 3.125-200 nM, for a total of 7 concentrations; human HER3 (Human HER3 / ERBB3 Protein (ECD, His Tag), HPLC-verified, Sinobiological, 10201-H08H), serially diluted in the range of 3.125-200 nM, for a total of 7 concentrations; monkey EGFR (Rhesus EGFR Protein (ECD, His Tag), Sinobiological, 90317-K08H), serially diluted in the range of 3.125-200 nM, for a total of 7 concentrations; and monkey HER3 (Rhesus HER3 / ERBB3 Protein (His Tag)). Tag), HPLC-verified, Sinobiological, 90043-K08H), with concentrations serially diluted in the range of 3.125-200 nM, for a total of 7 concentrations;

[0492] 2. Affinity data were analyzed and fitted using a 1:1 Langmuir binding model. Affinity (KD) is the ratio of the dissociation constant (Kd) to the binding constant (Ka). The affinity results are shown in Table 3.

[0493] Table 3 Affinity of bispecific antibodies and human antigens

[0494] Experimental conclusion: The bispecific antibody b0bd-hu2B13-1 of this invention exhibits approximately 9 times weaker affinity for human EGFR than the control antibody SI-1X6.4, while its affinity for HER3 is 29 times stronger. This affinity design of the bispecific antibody can reduce on-target toxicity to widely distributed EGFR-expressing normal tissues and enhance affinity for tumors with low HER3 expression.

[0495] Example 3: Bispecific antibody binding to EGFR / HER3 cell lines expressing different levels

[0496] This embodiment compares the affinity of b0bd-hu2B13-1 and its parent monospecific antibody for EGFR / HER3 cell lines with different expression levels using flow cytometry.

[0497] Experimental methods:

[0498] 1. The tumor cell lines MDA-MB-468 were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences, while HCC-827 and HCC1569 were obtained from Nanjing Kebai Biotechnology.

[0499] 2. All cell lines were cultured in complete medium at 37°C and 5% CO2.

[0500] 3. Harvest cells in the logarithmic growth phase and test cell viability using the trypan blue rejection method, ensuring cell viability is above 90%. Centrifuge at 200g for 5 min and discard the supernatant. Wash cells once with PBS, resuspend in FACS Buffer to prepare a single-cell suspension, and adjust the cell density to 1 x 10⁻⁶ cells / mL. 6 cells / mL;

[0501] 4. Add 100 μL of cell suspension to each well of a 96-well plate to make the initial concentration of the test sample working solution 1000 nM. Dilute 5 times to obtain a total of 8 concentrations. After mixing, incubate at 4°C for 60 min.

[0502] 5. Wash cells twice with FACS Buffer, 200 μL each time, centrifuge at 200g for 5 min, and finally resuspend cells with APC-labeled secondary antibody (Allophycocyanin (APC) Affini Pure F(ab')2 Fragment Goat Anti-Human IgG, Fcγfragment specific; Jackson, 109-136-098) diluted in 100 μL FACS Buffer and incubate at 4℃ in the dark for 30 min.

[0503] 6. Wash the cells twice with FACS Buffer, 200 μL each time, centrifuge at 200g for 5 min, and finally resuspend the cells with 150 μL of FACS Buffer.

[0504] 7. Fluorescence values ​​were detected by flow cytometry.

[0505] The test sample codes are shown in Table 4, and the test sample sources are as described in Example 1.

[0506] Table 4 Composition of bispecific or monospecific antibodies

[0507] The experimental results are shown in Figure 2 and Table 5.

[0508] Table 5. Affinity of bispecific antibodies and maternal monospecific antibodies to cell lines.

[0509] Experimental conclusions: For cell lines with different EGFR and HER3 expression levels, the saturation antigen activity of b0bd-hu2B13-1 was stronger than that of the control antibody SI-1X 6.4. The lower affinity of b0bd-hu2B13-1 helps reduce the binding of the bispecific antibody to normal tissues that widely express EGFR, thereby reducing toxic side effects. The saturation antigen activity of b0bd-hu2B13-1 was also stronger than that of the corresponding control bivalent monospecific antibody.

[0510] Example 4: Antibody endocytosis activity (temperature difference method)

[0511] This embodiment examines the endocytic effect of the bispecific antibody drug targeting EGFR and HER3 of this invention on MDA-MB-468, HCC-827, and HCC1569 cells expressing EGFR and / or HER3, and compares its endocytic activity with that of the parent monospecific antibody. Cells were co-incubated with an excess of a fixed concentration of the antibody drug, and the endocytic capacity of the antibody drug was evaluated by detecting the amount of residual antigen on the cell surface using FACS.

[0512] Experimental methods

[0513] 1. Cell culture: MDA-MB-468 cells were cultured in Leibovitz's L-15 (Gibco) medium with 10% FBS, while HCC-827 and HCC1569 cells were cultured in RPMI-1640 medium (Gibco) with 10% FBS.

[0514] 2. Cell preparation: Take MDA-MB-468, HCC-827, and HCC1569 cells in logarithmic growth phase, wash once with PBS, and digest for 2-3 minutes. After complete digestion, add 10-15 mL of cell culture medium to elute the digested cells. Centrifuge at 200g for 5 minutes, discard the supernatant, add cell culture medium to resuspend the cells into a single-cell suspension, and adjust the viable cell density to 1×10⁶ cells / mL. 6 cells / mL.

[0515] 3. Cell seeding: Add 100 μL / well to a 96-well cell culture plate. Centrifuge at 200g for 5 min and discard the supernatant.

[0516] 4. Sample addition procedure: Adjust the concentration of the antibody drug to be tested to 100 nM, add 100 μL / well to a 96-well cell culture plate and resuspend the cells.

[0517] 5. Antibody incubation: After incubating the culture plate at 4°C for 1 hour, centrifuge at 200g for 5 minutes and discard the supernatant.

[0518] 6. Washing cells: Add 200 μL of PBS to a 96-well cell culture plate to resuspend the cells, centrifuge at 200g for 5 min, and discard the supernatant. Repeat the washing process once more.

[0519] 7. Antibody internalization: Add 200 μL of cell culture medium to each well of a 96-well cell culture plate and resuspend the cells. Incubate the plate at 37°C for 2 hours, then centrifuge at 200g for 5 minutes and discard the supernatant. (Note: Samples from the 0h group should skip this step and proceed directly to step 9.)

[0520] 8. Cell washing: Add 200 μL of PBS to a 96-well cell culture plate to resuspend the cells, centrifuge at 200g for 5 min, and discard the supernatant. Repeat the washing once more.

[0521] 9. Secondary antibody incubation: Add 100 μL / well of the secondary antibody working solution (Allophycocyanin (APC), Affini Pure F(ab')2 Fragment Goat Anti-Human IgG, Fcγfragment specific; Jackson 109-136-098) to a 96-well cell culture plate and resuspend the cells. Incubate the culture plate at 4°C for 30 min, then centrifuge at 200g for 5 min and discard the supernatant.

[0522] 10. Washing cells: Add 200 μL of PBS to a 96-well cell culture plate to resuspend the cells, centrifuge at 200g for 5 min, and discard the supernatant. Repeat the washing process once more.

[0523] 11. Flow cytometry: Resuspend cells in 100 μL of PBS in a 96-well cell culture plate and detect them using a flow cytometer.

[0524] 12. Data Analysis: The MFI value obtained by flow cytometry was used to calculate the endocytosis rate of the antibody drug. The calculation formula is as follows:

[0525] Internalization rate = (1 - (MFI of the sample to be tested)) t2 -Background MFI t2 ) / (MFI of the sample to be tested) t0 -Background MFI t0 ))*100, where t0 represents antibody internalization time of 0h, t2 represents antibody internalization time of 2h, and background MFI represents MFI when only secondary antibody is added.

[0526] The experimental results are shown in Table 6.

[0527] Table 6. Endocytotic activity of monospecific and bispecific antibodies in cell lines.

[0528] Experimental conclusions: The antibody drug targeting EGFR and HER3 of this invention exhibits better endocytosis efficacy in MDA-MB-468, HCC-827, and HCC1569 cells that simultaneously express EGFR and HER3 compared to the parent monospecific antibody. Furthermore, the antibody drug targeting EGFR and HER3 of this invention demonstrates better endocytosis efficacy than the control antibody SI-1X6.4 and the bivalent monospecific antibody.

[0529] Example 5: Binding activity of bispecific antibodies to overexpressing cell lines

[0530] 5.1 Binding assay with BXPC-3 cells

[0531] BXPC-3 cell line (from the Chinese Academy of Sciences Cell Bank) was cultured in complete medium at 37℃ and 5% CO2. Cells in the logarithmic growth phase were harvested, and cell viability was assessed using the trypan blue exclusion method to ensure a viability of over 90%. After centrifugation at 200g for 5 min, the supernatant was discarded. Cells were washed once with PBS, resuspended in FACS Buffer to prepare a single-cell suspension, and the cell density was adjusted to 1 x 10⁻⁶ cells / mL. 6cells / mL; Add 100 μL of cell suspension to each well of a 96-well plate to make the initial concentration of the working solution 1000 nM, dilute 5 times, for a total of 8 concentrations; after mixing, incubate at 4℃ for 60 min; wash the cells twice with FACS Buffer, 200 μL each time, centrifuge at 200g for 5 min, and finally resuspend the cells with 100 μL of APC-labeled secondary antibody (Allophycocyanin (APC) Affini Pure F(ab')2 Fragment Goat Anti-Human IgG, Fcγfragment specific; Jackson, 109-136-098) diluted with FACS Buffer, and incubate at 4℃ in the dark for 30 min;

[0532] Cells were washed twice with 200 μL of FACS Buffer each time, centrifuged at 200g for 5 min, and finally resuspended in 150 μL of FACS Buffer. Fluorescence values ​​were detected by flow cytometry. Figure 3 shows the affinity of the bispecific antibody molecule for BXPC-3 cells. The results show that the bispecific antibody of the present invention has good affinity.

[0533] 5.2 Binding assay with A375 cells

[0534] Using the A375 cell line (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), the affinity of the bispecific antibody molecules for A375 cells was determined using FACS, a method similar to that in Example 5.1. Figure 4 records the MFI (Mean Factor Index) of the affinity of each group of bispecific antibody molecules. The results show that the bispecific antibody of the present invention has excellent affinity.

[0535] Example 6: Blocking experiment of bispecific antibody in EGFR and EGF binding

[0536] Flow cytometry (FACS) buffer was prepared by adding 2% fetal bovine serum (FBS) to phosphate-buffered saline (PBS); cells were collected by digestion with TrypLE (Gibco), washed with FACS buffer, and then resuspended in FACS buffer to a density of 2 × 10⁶ cells / mL. 6 / ml. Cells were then seeded into 96-well plates (100 μL / well), centrifuged at 300g for 5 minutes, and the supernatant was discarded. A mixture of 50 μL diluted antibody (final concentration 100 nM, diluted 5-fold with FACS buffer) and 50 μL of epidermal growth factor (EGF, final concentration 50 ng / ml) prepared with FACS buffer was added to each well, and the cells were incubated at 4°C in the dark for 1 hour. Cells were washed twice with flow cytometry buffer, centrifuged at 300g for 5 minutes, and the supernatant was discarded. Cells were stained with 100 μL of fluorescently conjugated secondary antibody (APC-conjugated goat anti-human IgG Fc, prepared 1:500 with FACS buffer) diluted in FACS buffer, and incubated at 4°C in the dark for 0.5 hours. Cells were washed twice with flow cytometry buffer, centrifuged at 300g for 5 minutes, and the supernatant was discarded. Cells were resuspended in 100 μL of FACS buffer, and the cell samples were analyzed by flow cytometry.

[0537] The results of EGF blocking with purified antibody are shown in Figure 5. The results show that the bispecific antibody of the present invention has a significant blocking effect.

[0538] Example 7: Preparation of Bispecific Antibody-Drug Conjugates (ADCs)

[0539] The linkers and toxins used in this embodiment are commercially available. The preparation of deruxtecan, GGFG-Dxd, Dxd, GGFG-Exd, and Val-Ala-PAB can also be carried out by referring to WO2014057687, WO1997046260, and CN101795711, the full text of which is incorporated herein by reference. Linker-ED04 is Gly-Mal-GGFG-Deruxtecan 2-hydroxypropanamide (commercially available, available from MCE), with CAS number 2750623-07-3; ED04 has CAS number 2577204-16-9 (available from MCE); the linker in Linker-ED04 is Gly-Mal-GGFG.

[0540] 7.1 Preparation of Linker-Therapeutic Active Substances or Pharmaceutical Active Ingredients (LDs)

[0541] The LD Cpd3 used in this invention and its preparation method are as follows:

[0542] Step 1: Take (9H-fluorene-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-(((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropyl-2-yl)amino)-1-oxobut-2-yl)carbamate (3-1, 2.5 g, 3.6 mmol) and (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl -1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-10,13-dione (3-2, 1.6 g, 3.6 mmol) and N,N-diisopropylethylamine (965 mg, 7.5 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL), and the reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by LCMS until completion, yielding 4-((S)-2-(S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutyrylamino)propionylamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (3-3). LCMS (ESI) m / z: 977.3 [M+H] + .

[0543] Step 2: Add hexahydropyridine (464 mg, 5.4 mmol) directly to the reaction solution from Step 1. Stir the reaction solution at room temperature for 2 hours. Monitor the reaction progress using LCMS. The reaction solution was then purified by C18 column chromatography to obtain 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (3-4) (1.8 g). LCMS (ESI) m / z: 755.2 [M+H] + .

[0544] Step 3: 1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonaoxa-4-aza-tetrate-31-acid (3-5g, 1.8g, 2.38mmol), 4-((S)-2-((S)-2-amino-3-methylbutyrylamino)propionylamino)benzyl((1S,9S)-9-ethyl 5-Fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (3-4, 1.8 g, 2.38 mmol), N,N-diisopropylethylamine (614 mg, 4.7 mmol) 6 mmol) was dissolved in anhydrous N,N-dimethylformamide (10 mL), and then stirred at room temperature for 3 h. The reaction was monitored by LCMS to obtain 4-((33S,36S)-1-(9H-fluorene-9-yl)-33-isopropyl-36-methyl-3,31,34-trioxo-2,7,10,13,16,19,25,25,28-nonazo-4,32 ,35-triaza-hepta-37-acylamino)-benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (3-6). LCMS(ESI) m / z: 1400.5 [M+H] + .

[0545] Step 4: Add hexahydropyridine (409 mg, 4.76 mmol) directly to the reaction solution from Step 3, and stir the reaction solution at room temperature for 2 hours. The reaction mixture was monitored by LCMS until completion. The reaction solution was directly purified by C18 column chromatography to obtain 4-((29S,32S)-1-amino-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diaza-tetrazine-33-acylamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (3-7) (1.2 g). LCMS (ESI) m / z: 1178.4 [M+H] + .

[0546] Step 5: 4-((29S,32S)-1-amino-29-isopropyl-32-methyl-27,30-dioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diaza-tetrazo-33-acylamino)benzyl((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4] [6,7]Indolezido[1,2-b]quinoline-1-yl)carbamate (3-7, 20 mg, 0.017 mmol), 2-(methanesulfonyl)pyrimidin-5-carboxylic acid (3-8, 13.7 mg, 0.068 mmol), and 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine tetrafluoroborate (22.3 mg, 0.068 mmol) were dissolved in anhydrous N,N-dimethylacetamide (3 mL), and the reaction mixture was stirred at room temperature for 16 hours. After purification by C18 column chromatography, 4-((31S,34S)-31-isopropyl-34-methyl-1-(2-(methanesulfonyl)pyrimidin-5-yl)-1,29,32-trioxo-5,8,11,14,17,20,23,26-octaoxa-2,30,33-triaza-tetrazo-35-acylamino-benzyl(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)carbamate (2.3 mg, yield: 10%), namely Cpd3, was obtained.

[0547] 1H NMR (400MHz, DMSO-d6) δ9.95(s,1H),9.37(s,1H),9.14(s,1H),8.46(s,1H),8.21(d,J=6.8Hz,1H),8.06(d,J=7.8H z,1H),7.89(d,J=8.3Hz,1H),7.78(d,J=10.9Hz,1H),7.60(d,J=8.4Hz,2H),7.36(d,J=8.1Hz,2H),7.31(s,1H),6. 53(s,1H),5.45(s,2H),5.28(s,3H),5.08(s,2H),4.42–4.34(m,1H),4.22–4.17(m,1H),3.62–3.53(m,10H),3.52– 3.46(m,28H),2.38(s,3H),2.33(s,1H),2.20(s,3H),2.01–1.81(m,4H),1.30(d,J=7.0Hz,3H),0.90–0.80(m,9H).

[0548] The LD Cpd5 used in this invention and its preparation method are as follows:

[0549] Step 1: Add 1-(9H-fluorene-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-acid (5-1,440 mg, 1.15 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazino[1,2] [-b] Quinoline-10,13-dione (500 mg, 1.15 mmol) was dissolved in anhydrous N,N-dimethylformamide (20 mL). N,N'-dicyclohexylcarbodiimide (308 mg, 1.5 mmol), N-hydroxysuccinimide (132 mg, 1.15 mmol) and N,N-diisopropylethylamine (148 mg, 1.15 mmol) were added under ice bath conditions. The reaction mixture was then stirred at room temperature for 16 hours. The reaction was monitored by LCMS until completion. The organic phase was concentrated by extraction with ethyl acetate and water, and purified by silica gel column chromatography using a methanol / dichloromethane system (1:20) to obtain (9H-fluorene-9-yl)methyl(2-(((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (5-3,650 mg). LCMS (ESI) m / z: 802.3 [M+H] + .

[0550] Step 2: Dissolve (9H-fluorene-9-yl)methyl(2-(((2-((((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate (5-3,650 mg, 0.81 mmol) in N,N-dimethylformamide (10 mL), then add hexahydropyridine (69 mg, 5.4 mmol), and stir the reaction mixture at room temperature for 2 hours. The reaction mixture was monitored by LCMS until completion. The reaction solution was directly purified by C18 column chromatography to obtain 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)acetamide (5-4,420 mg). LCMS (ESI) m / z: 580.2 [M+H] + .

[0551] Step 3: Add 2-amino-N-((2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-2-oxoethoxy)methyl)acetamide (5-4,420 mg, 0.73 mmol), (((9H-fluorene-9 370 mg (0.73 mmol) of glycyl-L-phenylalanine (5-5) was dissolved in anhydrous N,N-dimethylformamide (10 mL). N,N'-dicyclohexylcarbodiimide (195 mg, 0.95 mmol), N-hydroxysuccinimide (84 mg, 0.73 mmol), and N,N-diisopropylethylamine (94 mg, 0.73 mmol) were added under ice bath conditions. The reaction mixture was then stirred at room temperature for 16 hours. The reaction was monitored by LCMS until completion. The organic phase was concentrated by extraction with ethyl acetate and water, and purified by silica gel column chromatography using a methanol / dichloromethane system (1:15) to obtain (9H-fluorene-9-yl)methyl((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)carbamate (5-6,550 mg). LCMS (ESI) m / z: 1063.5 [M+H] + .

[0552] Step 4: Dissolve (9H-fluorene-9-yl)methyl((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)carbamate (5-6,550 mg, 0.52 mmol) in N,N-dimethylformamide (5 mL), then add hexahydropyridine (44 mg, 0.52 mmol), and stir the reaction mixture at room temperature for 2 hours. The reaction mixture was monitored by LCMS until completion. The reaction solution was directly purified by C18 column chromatography to obtain ((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)amine (5-7,400 mg). LCMS (ESI) m / z: 841.4 [M+H] + .

[0553] Step 5: Dissolve 1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nona-oxa-4-aza-tetrate-31-acid (347 mg, 0.52 mmol), HATU (274 mg, 0.72 mmol), and N,N-diisopropylethylamine (124 mg, 0.96 mmol) in anhydrous N,N-dimethylformamide (5 mL). Then, stir at room temperature for 1 hour, and then add ((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa -5,8,11,14-tetraazahexadecane-16-yl)amine (5-7, 400 mg, 0.48 mmol) was then stirred at room temperature for 16 h. The reaction was monitored by LCMS to indicate completion. The reaction solution was directly purified by C18 column chromatography to obtain (9H-fluorene-9-yl)methyl((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,1) 0,13,15-Hexahydro-1H,12H-Benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxaza-5,8,11,14,17-pentazatetratetradecane-44-yl)carbamate (5-9,350 mg). LCMS(ESI) m / z: 1486.6 [M+H] + .

[0554] Step 6: Add (9H-fluorene-9-yl)methyl((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,1 5,18-hexaoxo-3,21,24,27,30,33,36,39,42-nonaoxa-5,8,11,14,17-pentazatetratetradecane-44-yl)carbamate (5-9, 350 mg, 0.24 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then hexahydropyridine (20 mg, 0.24 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The reaction of the raw materials was monitored by LCMS until completion. The reaction solution was directly purified by C18 column chromatography to obtain 1-amino-N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide (5-10, 200 mg). LCMS(ESI) m / z: 1264.6 [M+H] + .

[0555] Step 7: Add 1-amino-N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-3, 6,9,12,15,18,21,24-octaoxaheptadecane-27-amide (5-10, 100 mg, 0.079 mmol), 2-(methanesulfonyl)pyrimidine-5-carboxylic acid (64 mg, 0.3 mmol), 2-chloro-4,6-dimethoxy-1,3,5-triazine (53 mg, 0.3 mmol), and N-methylmorpholine (182 mg, 1.8 mmol) were dissolved in anhydrous N,N-dimethylacetamide (5 mL), and the reaction mixture was stirred at room temperature for 16 hours. N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15,18-hexaoxo-3,21,24,27,30,33,36,39,42-nonoxa-5,8,11,14,17-pentazatetratetradecane-44-yl)-2-(methanesulfonyl)pyrimidine-5-carboxamide (5 mg), i.e., Cpd5, was obtained by C18 column chromatography separation and purification. LCMS(ESI) m / z: 1448.6 [M+H] + .

[0556] 1H NMR (400MHz, DMSO-d6) δ9.37(d,J=2.1Hz,2H),9.12(s,1H),8.63(s,1H),8.51(d,J=8.6Hz,1H),8.29(s,1H),8.21–8.07(m,2H ),7.99(s,1H),7.78(d,J=10.9Hz,1H),7.31(s,1H),7.21(t,J=15.2Hz,5H),6.52(s,1H),5.60(s,1H),5.42(s,2H),5.20(s,2H ),4.64(d,J=6.3Hz,2H),4.47(s,1H),4.02(s,2H),3.76–3.66(m,4H),3.59–3.53(m,8H),3.51–3.45(m,29H),2.39(s,5H),2.1 8(s,2H),2.00(d,J=7.9Hz,1H),1.84(dd,J=15.4,7.8Hz,2H),1.33–1.20(m,5H),1.12(d,J=6.6Hz,1H),0.87(t,J=6.3Hz,3H).

[0557] The LD Cpd6 used in this invention and its preparation method are as follows:

[0558] Step 1: (((9H-fluorene-9-yl)methoxy)carbonyl)-L-valine-L-alanine (6-1,943 mg, 2.3 mmol), (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolazido[1,2-b] Quinoline-10,13-dione (1 g, 2.3 mmol) was dissolved in anhydrous N,N-dimethylformamide (20 mL). N,N'-dicyclohexylcarbodiimide (616 mg, 3 mmol), N-hydroxysuccinimide (264 mg, 2.3 mmol) and N,N-diisopropylethylamine (297 mg, 2.3 mmol) were added under ice bath conditions. The reaction mixture was then stirred at room temperature for 16 hours. The reaction was monitored by LCMS until completion. The organic phase was concentrated by extraction with ethyl acetate and water, and purified by silica gel column chromatography using a methanol / dichloromethane system (1:20) to obtain (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (6-3,730 mg). LCMS (ESI) m / z: 828.3 [M+H] + .

[0559] Step 2: Dissolve (9H-fluorene-9-yl)methyl((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)carbamate (6-3,730 mg, 0.88 mmol) in N,N-dimethylformamide (10 mL), then add hexahydropyridine (75 mg, 0.88 mmol), and stir the reaction mixture at room temperature for 2 hours. The reaction mixture was monitored by LCMS until completion. The reaction solution was directly purified by C18 column chromatography to obtain (S)-2-amino-N-((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1-oxopropane-2-yl)-3-methylbutyramide (6-4,450 mg). LCMS (ESI) m / z: 606.2 [M+H] + .

[0560] Step 3: Dissolve 1-(9H-fluorene-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28-nonoxa-4-azatriocathane-31-acid (592 mg, 0.89 mmol), HATU (422 mg, 1.11 mmol), and N,N-diisopropylethylamine (143 mg, 1.11 mmol) in anhydrous N,N-dimethylformamide. (5 mL) was added, and then stirred at room temperature for 1 h. Then (S)-2-amino-N-((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)- 1-Oxopropane-2-yl)-3-methylbutyramide (450 mg, 0.74 mmol) was then stirred at room temperature for 4 h. The reaction was monitored by LCMS until completion. The reaction solution was directly purified by C18 column chromatography to obtain (9H-fluorene-9-yl)methyl((29S,32S)-33-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,1) 0,13,15-Hexahydro-1H,12H-Benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-29-isopropyl-32-methyl-27,30,33-trioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diaza-triatridecyl)carbamate (6-5,400 mg). LCMS(ESI) m / z: 1251.6 [M+H] + .

[0561] Step 4: Add (9H-fluorene-9-yl)methyl((29S,32S)-33-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-29-isopropyl 32-methyl-27,30,33-trioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diaza-triatrityl)carbamate (6-5, 400 mg, 0.32 mmol) was dissolved in N,N-dimethylformamide (5 mL), and then hexahydropyridine (27 mg, 0.32 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was monitored by LCMS until completion. The reaction solution was directly purified by C18 column chromatography to obtain 1-amino-N-((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-3,6,9,12,15,18,21,24-octaoxa-heptacosan-27-amide (6-6,210 mg). LCMS (ESI) m / z: 1029.6 [M+H] + .

[0562] Step 5: Add 1-amino-N-((S)-1-(((S)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-1-oxopropane-2-yl)amino)-3-methyl-1-oxobutane-2-yl)-3,6,9,12 15,18,21,24-octaoxa-heptadecane-27-amide (6-6, 100 mg, 0.097 mmol), 2-(methanesulfonyl)pyrimidine-5-carboxylic acid (78 mg, 0.39 mmol), 2-chloro-4,6-dimethoxy-1,3,5-triazine (68 mg, 0.39 mmol) and N-methylmorpholine (59 mg, 0.582 mmol) were dissolved in anhydrous N,N-dimethylacetamide (5 mL), and the reaction mixture was stirred at room temperature for 16 hours. N-((29S,32S)-33-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolazino[1,2-b]quinoline-1-yl)amino)-29-isopropyl-32-methyl-27,30,33-trioxo-3,6,9,12,15,18,21,24-octaoxa-28,31-diaza-hexatridecyl)-2-(methanesulfonyl)pyrimidine-5-carboxamide (11.5 mg), i.e., Cpd6, was purified and separated by C18 column chromatography. LCMS (ESI) m / z: 1213.6 [M+H] +

[0563] 1H NMR (400MHz, DMSO-d6) δ9.36(s,2H),9.13(s,1H),8.41(d,J=8.9Hz,1H),8.10(d,J=6.9Hz,1H),7.86(d,J=8.4Hz,1H),7. 79(d,J=11.0Hz,1H),7.30(s,1H),6.53(s,1H),5.54(s,1H),5.42(s,2H),5.24(d,J=18.9Hz,1H),5.11(d,J=19.0Hz,1H) ,4.32–4.21(m,1H),4.17–4.06(m,1H),3.58–3.52(m,8H),3.48(s,25H),3.44(s,6H),2.40(s,3H),2.29(dd,J=13.7,7.1 Hz,1H),2.13(s,2H),1.91(ddd,J=23.2,22.6,7.6Hz,4H),1.27(d,J=7.0Hz,3H),0.87(t,J=7.4Hz,3H),0.83–0.72(m,6H)

[0564] 7.2 Preparation of antibody-drug conjugates

[0565] The conventional method used in this embodiment is as follows: The antibody stock solution was removed from the refrigerator (-80℃), thawed, and added to a centrifuge tube. Then, PBS buffer, DTPA, and TCEP solution were added, and the reaction was carried out at room temperature. Then, Linker-Payload was added, and the reaction was continued at room temperature for 1-2 hours. After the reaction was complete, the solution was added to an ultrafiltration centrifuge tube and dialyzed to obtain the ADC stock solution (DAR: 4-8, SEC purity: 95%).

[0566] For example, the antibody stock solution (Patritumab, 45 mg) was removed from the refrigerator (-80°C), thawed, and added to a centrifuge tube. Then, PBS (20 mM, 2253 μL) buffer, DTPA (10 mM, 1125 μL), and TCEP (10 mM, 94 μL) solution were added, and the reaction was carried out at room temperature for 1-2 hours. Then, GGFG-DXD (15 mM, 816 μL) was added, and the reaction was continued at room temperature for another 1-2 hours. After the reaction was complete, the solution was transferred to an ultrafiltration centrifuge tube and replaced by dialysis to obtain the ADC stock solution (33 mg, DAR: 8, SEC purity: 99.2%, free drug: not detected).

[0567] Using other antibodies, such as b0bd-hu2B13-2, and other linker-payloads, such as cpd3, the ADC stock solution was prepared using the above method. The detection results are shown in Table 7 below:

[0568] Table 7 ADC Detection Results

[0569] Example 8: In vivo efficacy evaluation of antibody-drug conjugate in HCC1569 tumor-bearing mice with low EGFR expression and HER3 expression.

[0570] This study investigated the inhibitory effects of ADCs with different antibody structures on tumor formation in human breast cancer cell lines. After subcutaneous ectopic inoculation of HCC1569 in mice to form xenografts, the antitumor effects of each ADC were evaluated and compared with those of SI-1X6.4-Linker-ED04 and Patritumab-Deruxtecan.

[0571] To investigate the inhibitory effect of different DAR values ​​of b0bd-hu2B13-1 conjugate-cytotoxin Cpd3 on tumor formation in human breast cancer cell line HCC1596, HCC1596 cells were ectopically inoculated subcutaneously on the right back of mice to form xenografts, and the antitumor effect of different DAR values ​​of the bispecific antibody-conjugate toxin was evaluated.

[0572] 1. Test drug and materials

[0573] Blank control group (control group): normal saline

[0574] b0bd-hu2B13-1-CPD3-DAR4 (treatment group): Day 0 and Day 7: 1 mg / kg

[0575] b0bd-hu2B13-1-CPD3-DAR4 (treatment group): Day 0 and Day 7: 3 mg / kg

[0576] b0bd-hu2B13-1-CPD3-DAR4 (treatment group): Day 0 and Day 7: 10 mg / kg

[0577] b0bd-hu2B13-1-CPD3-DAR6 (treatment group): Day 0 and Day 7: 3 mg / kg

[0578] b0bd-hu2B13-1-CPD3-DAR6 (treatment group): Day 0 and Day 7: 10 mg / kg

[0579] b0bd-hu2B13-2-CPD3-DAR8 (treatment group): Day 0 and Day 7: 3 mg / kg

[0580] b0bd-hu2B13-2-CPD3-DAR8 (treatment group): Day 0 and Day 7: 10 mg / kg

[0581] Patritumab-Deruxtecan (treatment group): Day 0 and Day 7: 10 mg / kg

[0582] SI-1X6.4-Linker-ED04(BL-B01D1) (treatment group): Day 0 and Day 7: 10 mg / kg

[0583] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0584] 3. Experimental animals: 6-7 week old female NOD SCID mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0585] 4. Test methods:

[0586] HCC1569 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% Antibiotic-Antimycotic.

[0587] HCC1569 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0588] The experimental mice were subcutaneously inoculated with 3x10 [units of something] on their right back. 6 HCC1569 cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.1 ml / cell), and tumor growth was observed periodically.

[0589] When the tumor grew to approximately 159 mm 3 Tumor-bearing mice were randomly assigned to groups and administered the test substance intravenously (iv) starting on day 0, twice daily, once on day 0 and once on day 7, for a total of two injections. The endpoint of the experiment was day 74 after grouping. Body volume and weight were measured twice weekly, and the data were recorded.

[0590] Six mice were used in each of the control and treatment groups. The inhibition rate was calculated by measuring tumor volume. The experimental results are shown in Figure 6.

[0591] The results showed that the asymmetric configuration b0bd-hu2B13-1-CPD3 (DAR4, DAR6) had superior antitumor activity compared to the equal-dose symmetric bispecific antibodies b0bd-hu2B13-2-CPD3 (DAR8), Patritumab Deruxtecan, and SI-1X6.4-Linker-ED04 (DAR8). Specifically, at D46 & D70, at equal doses, b0bd-hu2B13-1-CPD3 (DAR4 & DAR6) showed superior tumor-suppressive activity compared to the symmetric bispecific antibodies b0bd-hu2B13-2-CPD3 (DAR8), Patritumab Deruxtecan, and SI-1X6.4-Linker-ED04 (DAR8); and the dosage was safe and tolerable.

[0592] Example 9: In vivo efficacy evaluation of antibody-drug conjugates in HCC-827 tumor-bearing mice, a human non-small cell lung cancer cell line with high EGFR expression and HER3 expression.

[0593] This study investigated the inhibitory effects of ADCs with different antibody structures and DAR values ​​on tumor formation in human non-small cell lung cancer cell lines. After subcutaneous ectopic inoculation of HCC827 cells in mice to form xenografts, the antitumor effects of each ADC were evaluated and compared with those of SI-1X6.4-Linker-ED04 and Patritumab-Deruxtecan.

[0594] 1. Test drug and materials

[0595] Blank control group (control group): normal saline

[0596] b0bd-hu2B13-1-Cpd3-DAR4 (treatment group): Day 0 and Day 7: 1 mg / kg

[0597] b0bd-hu2B13-1-Cpd3-DAR4 (treatment group): Day 0 and Day 7: 3 mg / kg

[0598] b0bd-hu2B13-1-Cpd3-DAR4 (treatment group): Day 0 and Day 7: 10 mg / kg

[0599] b0bd-hu2B13-1-Cpd3-DAR6 (treatment group): Day 0 and Day 7: 3 mg / kg

[0600] b0bd-hu2B13-1-Cpd3-DAR6 (treatment group): Day 0 and Day 7: 10 mg / kg

[0601] b0bd-hu2B13-2-CPD3-DAR8 (treatment group): Day 0 and Day 7: 3 mg / kg

[0602] b0bd-hu2B13-2-CPD3-DAR8 (treatment group): Day 0 and Day 7: 10 mg / kg

[0603] Patritumab-Deruxtecan (treatment group): Day 0 and Day 7: 10 mg / kg

[0604] SI-1X6.4-Linker-ED04(BL-B01D1) (treatment group): Day 0 and Day 7: 10 mg / kg

[0605] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0606] 3. Experimental animals: 7-8 week old female Balb / c nude mice, purchased from Shanghai Slack Laboratory Animal Co., Ltd.

[0607] 4. Test methods:

[0608] HCC827 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% Antibiotic-Antimycotic.

[0609] HCC827 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.

[0610] The experimental mice were subcutaneously inoculated with 1x10 spores on their right back. 7 HCC827 cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.2 ml / cell), and tumor growth was observed regularly.

[0611] When the tumor grew to approximately 284 mm 3 Tumor-bearing mice were randomly assigned to groups and administered the test substance intravenously (iv) starting on day 0, for a total of one injection. The treatment group received a dose of 10 mg / kg on day 0. The endpoint of the experiment was day 59 after grouping. Body volume and weight were measured twice a week, and the data were recorded.

[0612] Six mice were used in each of the control and treatment groups. The inhibition rate was calculated by measuring tumor volume. The experimental results are shown in Figure 7.

[0613] The results showed that the asymmetric configuration b0bd-hu2B13-1-CPD3 (DAR4 DAR6) inhibited the growth of human non-small cell lung cancer HCC827 xenografts in a single dose-dependent manner. At the same dose, the tumor-suppressive effect of b0bd-hu2B13-2-CPD3 (DAR8) was superior to that of b0bd-hu2B13-2-CPD3 (DAR8) and Patritumab Deruxtecan, and comparable to that of SI-1X6.4-Linker-ED04 (DAR8). The administered dose was safe and tolerable.

[0614] Example 10: Binding activity of humanized bispecific antibody to target protein

[0615] This embodiment evaluates the binding activity of humanized bispecific antibodies (ELISA).

[0616] 96-well ELISA plates were coated with 0.5 μg / mL human EGFR / HER3 antigen protein (100 μL / well) and incubated overnight at 4°C. The next day, the plates were washed three times with PBST, blocked with 2% BSA for 2 h, washed three times with PBST, and then serially diluted (100 nM, 10-fold serial dilution, 8 concentration spots) of each antibody and positive control antibody were added and incubated at 37°C for 1 h. After washing three times with PBST, secondary antibody Goat-anti-human Fc-HRP (Jackson 109-035-097) was added and incubated at 37°C for 1 h. After incubation, the plates were washed three times with PBST and TMB was added for color development. The reaction was stopped by adding 1M HCl at 37°C for 10 min, and the absorbance was read at OD450 / OD570 using a microplate reader.

[0617] The results are shown in Figures 8A and 8B. The humanized bispecific antibody molecule binds weakly to EGFR compared to the control SI-1X6.4; however, it binds better to Her3 than the control SI-1X6.4.

[0618] Example 11 Binding activity of humanized bispecific antibodies and their drug conjugates

[0619] This embodiment evaluates the affinity of humanized bispecific antibodies and their drug conjugates. EGFR-high expression cell lines MDA-MB-468 and HCC-827, as well as HER3-high expression cell lines A375 and HCC-1569, were selected. Candidate bispecific antibody molecules were b0bd-hu2B13-1, b0bd-hu2B13-2, and b0bd-hu2B13-3c. The corresponding antibody-drug conjugates were prepared using the conjugation method described in Example 6.

[0620] 11.1 Evaluation of binding activity with MDA-MB-468 and HCC-827 cells

[0621] The FACS method used in this embodiment is as follows: MDA-MB-468 cells and HCC-827 cells were taken and cultured in complete medium at 37°C and 5% CO2.

[0622] Cells in the logarithmic growth phase were harvested and cell viability was assessed using the trypan blue rejection assay to ensure a viability of over 90%. After centrifugation at 200g for 5 min, the supernatant was discarded. Cells were washed once with PBS, resuspended in FACS Buffer to prepare a single-cell suspension, and the cell density was adjusted to 1 x 10⁻⁶ cells / mL. 6 cells / mL; Add 100 μL of cell suspension to each well of a 96-well plate to make the initial concentration of the working solution 1000 nM, dilute 5-fold for a total of 8 concentrations; after mixing, incubate at 4℃ for 60 min; wash cells twice with FACS Buffer, 200 μL each time, centrifuge at 200g for 5 min; finally, resuspend cells with 100 μL of FACS Buffer diluted with APC-labeled secondary antibody (Allophycocyanin (APC) Affini Pure F(ab')2 Fragment Goat Anti-Human IgG, Fcγfragment specific; Jackson, 109-136-098), incubate at 4℃ in the dark for 30 min; wash cells twice with FACS Buffer, 200 μL each time, centrifuge at 200g for 5 min; finally, resuspend cells with 150 μL of FACS Buffer; detect fluorescence values ​​by flow cytometry.

[0623] The binding activities of candidate bispecific antibody molecules and their drug conjugates are shown in Figures 9A-9D. The results show that the EC50 of the bispecific antibody of this invention binding to EGFR or HER3 cell lines is weaker than that of the bivalent bispecific antibody SI-1X6.4; however, the binding plateau of the bispecific antibody of this invention to tumor cell lines simultaneously expressing EGFR and HER3 is higher than that of the bivalent bispecific antibody SI-1X6.4. This may be beneficial for the bispecific antibody to reduce binding to normal tissues and enhance binding to tumor cells simultaneously expressing EGFR and HER3.

[0624] 11.2 Evaluation of binding activity with A375 and HCC-1569 cells

[0625] The binding activity was evaluated on HCC-1569 cells in A375 cells using the method described in Example 11.1. The experimental results are shown in Figures 10A to 10D. The data in Figures 10A and 10B are shown in Table 8.1 below, and the data in Figures 10C and 10D are shown in Table 8.2 below.

[0626] Table 8.1 A375 cell binding FACS detection data

[0627] Table 8.2 HCC1569 cell binding FACS detection data

[0628] The results showed that the candidate bispecific antibodies and their drug conjugates had better binding than the positive control antibody.

[0629] Example 12 Detection of endocytosis of humanized bispecific antibodies and their drug conjugates

[0630] This embodiment evaluates the infiltration activity of humanized bispecific antibodies and their drug conjugates. The candidate bispecific antibody molecules are b0bd-hu2B13-1, b0bd-hu2B13-2, and b0bd-hu2B13-3c, and the corresponding antibody-drug conjugates are prepared using the conjugation method described in Example 5.

[0631] 12.1 Detection of endocytosis in MDA-MB-468 and HCC-827 cells

[0632] Internalization detection was performed using the FACS method as described in Example 4.

[0633] The experimental results are shown in Figures 11A to 11C. Data from Figure 11A is shown in Table 9.1, data from Figure 11B is shown in Table 9.2, and data from Figure 11C is shown in Table 9.3.

[0634] Table 9.1 MFI on FACS internalization detection MDA-MB-468 (100nM)

[0635] Table 9.2 MFI internalization percentage on FACS internalization detection MDA-MB-468 (100nM)

[0636] Table 9.3 MFI on FACS internalization detection HCC-827 (100nM)

[0637] The results showed that the candidate bispecific antibodies and their drug conjugates had superior endocytic activity.

[0638] 12.2 Detection of endocytosis in A375 and HCC-1569 cells

[0639] Similar to Example 12.1, endocytic activity was evaluated in HCC-1569 cells using the method described in Example 4, and the experimental results are shown in Figures 12A and 12B. The data in Figure 12A are shown in Table 10.1 below, and the data in Figure 12B are shown in Table 10.2 below.

[0640] Table 10.1 MFI on FACS internalization detection HCC-1569 (100 nM)

[0641] Table 10.2 Internalization degree (%) of HCC-1569 (100 nM) detected by FACS endocytosis detection

[0642] The results showed that the candidate bispecific antibodies and their drug conjugates had superior endocytic activity.

[0643] Example 13: Antitumor effects of candidate bispecific antibodies and their drug conjugates in a human breast cancer xenograft model.

[0644] This study investigated the inhibitory effects of antibody-drug conjugates (ADCs) with different antibody structures on tumor formation in human breast cancer cell lines. After subcutaneous ectopic inoculation of HCC1569 into mice to form xenografts, the antitumor effects of each ADC were evaluated and compared with those of the positive references SI-1X6.4-Linker-ED04 and Patritumab-Deruxtecan.

[0645] HCC1569 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic solution. HCC1569 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice. 3 x 102 cells were subcutaneously inoculated into the right back of experimental mice. 6 HCC1569 cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.1 ml / cell), and tumor growth was observed regularly. When the tumor grew to approximately 157 mm... 3 Tumor-bearing mice were randomly assigned to groups and administered the test drug intravenously (iv) twice daily, on day 0 and day 7, for a total of two injections. The treatment group received a dose of 5 mg / kg on day 0 and day 7. The endpoint of the experiment was day 38 after grouping. Tumor volume and body weight were measured twice weekly and recorded. Each control or treatment group consisted of 5 mice. The inhibition rate was calculated by measuring tumor volume. The efficacy results are shown in Figures 13A-13B.

[0646] Example 14: Antitumor effects of candidate bispecific antibodies and their drug conjugates in a human non-small cell lung cancer xenograft model

[0647] This study investigated the inhibitory effects of antibody-drug conjugates (ADCs) with different antibody structures and different DAR values ​​on tumor formation in human non-small cell lung cancer cell lines.

[0648] After subcutaneous ectopic inoculation of HCC827 cells to form xenografts in mice, the antitumor effects of each ADC were evaluated and compared with those of SI-1X6.4-Linker-ED04 and Patritumab-Deruxtecan. HCC1569 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% Antibiotic-Antimycotic. HCC827 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice. 1 x 102 cells were subcutaneously inoculated into the right back of the experimental mice. 7 HCC827 cells were resuspended in a 1:1 mixture of PBS and matrix gel (0.2 ml / cell), and tumor growth was monitored regularly. When the tumor grew to approximately 232 mm... 3 Tumor-bearing mice were randomly assigned to groups and administered the test drug intravenously (iv) starting on day 0, for a total of one injection. The treatment group received a dose of 10 mg / kg on the day of administration. The endpoint of the experiment was day 52 after grouping. Tumor volume and body weight were measured twice a week, and the data were recorded. Each control or treatment group consisted of 6 mice. The inhibition rate was calculated by measuring tumor volume. The efficacy results are shown in Figures 14A-14B.

[0649] Experimental results show that the candidate bispecific antibody and its drug conjugate exhibit superior antitumor activity at DAR4, DAR6 and DAR8.

[0650] Example 15: Antitumor effects of candidate bispecific antibodies and their drug conjugates in a mouse xenograft model of human colorectal cancer SW620 cells.

[0651] This study investigated the antitumor effect of the candidate bispecific antibody molecule b0bd-hu2B13-1-CPD3 at different doses on a mouse xenograft model of human colorectal cancer SW620 cells. A tumor-bearing mouse model was established by subcutaneously transplanting human colorectal cancer SW620 cells into female Balb / c nude mice. The tumor volume reached 155 mm². 3 Animals were randomly assigned to groups to receive the drug, and the in vivo antitumor effect of b0bd-hu2B13-1-CPD3 on human breast cancer was evaluated. The efficacy was compared with that of the positive control drugs SI-1X6.4-Linker-ED04 and DB-YE-X20 (DB1418, see WO2025016453A1). Table 11 below records the grouping method and experimental results.

[0652] Table 11 Antitumor effects of different drug treatment groups on mouse subcutaneous xenografts of human colorectal cancer SW620 cells (D19). **** p<0.0001 vs Vehicle, *** p<0.001 vs Vehicle, #### p<0.0001vs 1mg / kg b0bd-hu2B13-1-CPD3, && p<0.001 vs 3mg / kg b0bd-hu2B13-1-CPD3, $$$ p<0.0001vs 10mg / kg SI-1X6.4-Linker-ED04, ^^^^ p<0.0001 vs 10 mg / kg DB-YE-X20, One-way ANOVA; TV: tumor volume; TW: tumor weight; TGI (%): tumor growth inhibition rate; PR: tumor volume reduction of more than 30% compared to the group; PR max : Maximum PR during the entire experimental period; IR (%): Tumor weight inhibition rate; TGI max The maximum TGI value during the entire experimental period.

[0653] The results showed that, compared with the Vehicle group, single administration of 1 mg / kg and 3 mg / kg b0bd-hu2B13-1-CPD3 significantly inhibited tumor growth rate, with TGIs of 27.81% and 70.00%, respectively. max The efficacy rates were 29.58% (D7) and 78.70% (D14), respectively, with IR rates of 30.36% and 69.40%, respectively. No animals showed partial response (PR) during the experimental period. A single dose of 10 mg / kg b0bd-hu2B13-1-CPD3 completely inhibited tumor growth and promoted tumor regression, with a total genicity index (TGI) of 93.45%. max The percentage was 94.15% (D14), PR max The success rate was 6 / 6, and at the experimental endpoint, 2 / 6 of the animals maintained PR.

[0654] The results above (see Figure 15) indicate that a single dose of 1-10 mg / kg b0bd-hu2B13-1-CPD3 can dose-dependently inhibit tumor growth and promote tumor regression. During the observation period after a single dose of 10 mg / kg SI-1X6.4-Linker-ED04 and DB-YE-X20, tumors continued to proliferate, with TGIs of 59.89% and 26.58%, respectively. maxThe effective rates were 68.40% (D14) and 26.58% (D19), respectively, and no animals showed PR during the experimental period. At the same dosage, b0bd-hu2B13-1-CPD3 had a significantly better antitumor effect than SI-1X6.4-Linker-ED04 and DB-YE-X20 (p<0.001).

[0655] Example 16: Antitumor effects of candidate bispecific antibodies and their drug conjugates in a human lung adenocarcinoma PDX mouse model

[0656] A PDX model was constructed by subcutaneously transplanting human lung adenocarcinoma tumor sample LD1-0025-215621 (source: Shanghai Lidi Biotechnology Co., Ltd.) with high EGFR expression (H-Score: 230) and moderate HER3 expression (H-Score: 120) into female C-NKG mice. When the tumor volume reached 184.88 mmHg, a PDX model was established. 3 Animals were randomly assigned to groups to receive the drug to evaluate the in vivo antitumor effect of b0bd-hu2B13-1-CPD3 in human lung adenocarcinoma, and its efficacy was compared with that of the positive control drugs SI-1X6.4-Linker-ED04 and DB-YE-X20. Table 12 below records the grouping method and experimental results.

[0657] Table 12 Antitumor effects of PDX (LD1-0025-215621) mouse model of human lung adenocarcinoma on day 21. * p<0.05, ** p<0.01 and *** p<0.001and **** p<0.0001 vs Vehicle, # p<0.05 vs 3mg / kg b0bd-hu2B13-1-CPD3, ^ p<0.05 vs 10mg / kg DB-YE-X20, One-way ANOVA; TV: tumor volume; TW: tumor weight; TGI (%): tumor growth inhibition rate; IR (%): tumor weight inhibition rate; PR: tumor volume reduction of more than 30% compared to the initial group; PR max The maximum PR value throughout the entire experimental period.

[0658] The experimental results (see Figure 16) showed that single doses of 3 mg / kg and 10 mg / kg of b0bd-hu2B13-1-CPD3 inhibited tumor growth and promoted tumor regression in a dose-dependent manner, with TGIs of 50.27% and 88.96%, respectively. maxThe efficacy rates were 51.70% (D18) and 88.96% (D21), respectively; the IR rates were 41.96% and 88.13%, respectively; and the PR rates were 0 / 4 and 4 / 5, respectively. With a single-dose regimen, 10 mg / kg b0bd-hu2B13-1-CPD3 significantly reduced terminal tumor weight and significantly improved the PR rate (4 / 5 vs 0 / 4), indicating that 10 mg / kg b0bd-hu2B13-1-CPD3 had a superior tumor-suppressive effect compared to 3 mg / kg. At the same dose, the TGI of the 10 mg / kg b0bd-hu2B13-1-CPD3 group was superior to that of the positive control drug DB-YE-X20 (p<0.01) and comparable to that of SI-1X6.4-Linker-ED04 (D28 TGI: 88.96% vs 43.47% vs 84.33%). However, the PR rate of the b0bd-hu2B13-1-CPD3 group was significantly improved (PR: 4 / 5 vs 0 / 4 vs 1 / 4), suggesting that b0bd-hu2B13-1-CPD3 has a more efficient tumor regression effect.

[0659] Example 17: Antitumor effects of candidate bispecific antibodies and their drug conjugates in a human breast cancer PDX mouse model

[0660] A PDX model was constructed by subcutaneously transplanting human breast cancer tumor samples LD1-0009-410668 expressing EGFR (H-Score: 120) and HER3 (H-Score: 116) into female Nu / Nu mice (source: Shanghai Lidi Biotechnology Co., Ltd.). When the tumor volume reached 124.83 mm3, the animals were randomly assigned to groups to receive the drug to evaluate the in vivo antitumor effect of b0bd-hu2B13-1-CPD3 in human breast cancer. The efficacy was compared with that of the positive control drugs SI-1X6.4-Linker-ED04 and DB-YE-X20, providing a reference for the selection of clinical indications, patient stratification, and clinical trial design of the test substance b0bd-hu2B13-1-CPD3. Table 13 below records the grouping method and experimental results.

[0661] Table 13 Antitumor effects of human breast cancer PDX (LD1-0009-410668) mouse model at D25 ** p<0.01 vs Vehicle, ^ p<0.05 vs 10mg / kg DB-YE-X20, One-way ANOVA; TV: tumor volume; TW: tumor weight; TGI (%): tumor growth inhibition rate; IR (%): tumor weight inhibition rate; PR: tumor volume reduction of more than 30% compared to the initial group; PR max The maximum value of PR throughout the entire experimental period.

[0662] The experimental results (see Figure 17) show that 10 mg / kg b0bd-hu2B13-1-CPD3 QW*2 completely inhibited tumor growth, potently induced tumor regression, and no significant rebound effect was observed 25 days after administration. The TGI was 95.59%. max The efficacy was 95.94% (D21), and the IR was 94.53%. The anti-EGFR and anti-HER3 expression of b0bd-hu2B13-1-CPD3 in human breast cancer showed a significant advantage over competing antibody-drug conjugates like DB-YE-X20.

[0663] Example 18: Antitumor effects of candidate bispecific antibodies and their drug conjugates in a human breast cancer PDX mouse model.

[0664] A PDX model was constructed by subcutaneously transplanting human breast cancer tumor samples LD1-2009-362721 (source: Shanghai Lidi Biotechnology Co., Ltd.) with low EGFR expression (H-Score: 30) and high HER3 expression (H-Score: 295) into female Nu / Nu mice. When the tumor volume reached 134.26 mmHg, a PDX model was established. 3 Animals were randomly assigned to groups to receive the drug to evaluate its in vivo antitumor effect on human breast cancer, and its efficacy was compared with that of the positive control drugs Patritumab-Deruxtecan (an analog of the HER3 ADC drug U3-1402), SI-1X6.4-Linker-ED04, and DB-YE-X20. Table 14 below records the grouping method and experimental results.

[0665] Table 14 Antitumor Effects of PDX (LD1-2009-362721) Mouse Model of Human Breast Cancer at D35 ** p<0.01 and **** p<0.0001 vs Vehicle, ^ p<0.05 vs 10mg / kg DB-YE-X20, One-way ANOVA; TGI (%): Antitumor efficacy of the drug; TGI max : Maximum TGI during the entire experimental period; IR (%): Tumor weight inhibition rate; CR: Tumor volume of 0 mm 3 PR: Tumor volume decreased by more than 30% compared to the initial grouping.

[0666] The experimental results (see Figure 18) show that a single dose of 10 mg / kg b0bd-hu2B13-1-CPD3 strongly induced tumor regression, completely inhibited tumor growth, and showed no significant recurrence trend until the experimental endpoint (D35), with a TGI of 99.88%.max The efficacy rate was 99.89% (D32), the IR was 99.88%, the PR rate was 4 / 4, and the CR rate was 3 / 4. At the same dose, b0bd-hu2B13-1-CPD3 showed a significant advantage over competing antibody-drug conjugates DB-YE-X20 in the anti-EGFR low-expression and HER3 high-expression human breast cancer; it was comparable to Patritumab-Deruxtecan and SI-1X6.4-Linker-ED04.

[0667] In summary, the embodiments of this application employ a monovalent VHH hu2B13 targeting EGFR combined with a monovalent Fab targeting Her3 to form a bispecific antibody. Combined with the optimized drug-antibody ratio DAR4, the tumor-suppressive effect is superior to competing products targeting the same target, SI-1X6.4-Linker-ED04 (bivalent EGFR bivalent Her3, DAR8) and DB-YE-X20 (bivalent EGFR bivalent Her3, DAR6), in different in vivo pharmacodynamic models. This demonstrates that the design of the monovalent EGFR monovalent Her3 binding arm and DAR4 in this application maintains superior pharmacodynamic activity while reducing the potential in vivo tolerance risk associated with high DAR values.

[0668] Example 19: Production and Identification of Anti-EGFR Single-Domain Antibodies

[0669] This embodiment describes the generation, screening, and identification of anti-EGFR single-domain antibodies (VHHs) that can be used in the bispecific antibodies of this invention. A portion of the obtained VHHs was used for the construction of the bispecific antibodies described in the preceding embodiments.

[0670] 19.1. Generation and Screening of Anti-EGFR Single-Domain Antibodies (VHH)

[0671] Based on the amino acid sequence information of the EGFR protein (P00533-1), a codon-optimized coding nucleic acid sequence was synthesized, cloned into the pCDNA3.4 vector (Invitrogen, A14697), and a His tag was added to the C-terminus. The resulting EGFR-His recombinant protein was then transfected into 293F cells (Gibco, A14527) for expression and purification.

[0672] Alpaca were routinely immunized with the obtained EGFR-His recombinant protein. Peripheral blood of the immunized alpaca was collected, PBMCs were isolated, and a single-domain antibody yeast display library was constructed and screened.

[0673] Positive yeast clones were subjected to PCR to obtain antibody sequences, which were then digested with SfiI and ligated into the eukaryotic expression vector pcDNA3.4-human IgG1Fc to construct an antibody expression vector. The eukaryotic expression vector was transiently transfected into 293F cells, and the antibody expression supernatant was collected. The binding of candidate antibodies and antigen proteins was detected by FACS. A total of 20 different sequences were obtained through three sorting processes. Eukaryotic expression vectors (with an Fc tag added to the C-terminus) were constructed and transfected into 293F cells to express antibodies for validation.

[0674] Candidate single-domain antibodies were named using their clone numbers: 2-2-B1-3 (2B13), 3-1-C7-2, 3-1-C7-4, 1-G6, 2-2-B1-2, 2-4-E7-3, 2-4-E7-4, 3-1-A11, 3-1-C4, 3-3-E10-2, 3-4-H11, 3-H3, 4-F5, 5-H9-3, 2-C4, 2-E4, 1-E12, 2-1-H6, 2-1-C7, and 1-C8. The CDRs of VHH were determined using the Kabat method for defining CDRs. The obtained CDRs and VHH sequences of the anti-EGFR single-domain antibodies are shown in Tables 24 and 25.

[0675] 19.2 Detection of the binding of recombinant antibody to target protein

[0676] For ELISA detection, the recombinant protein was diluted to a final concentration of 1 μg / mL using sterile CBS. 100 μL was added to each well of a 96-well microplate and incubated overnight at 4°C. The antigen coating solution was removed, and the plate was washed five times with PBST (containing 0.05% Tween 20). 200 μL / well of 3% MPBS was added, and the plate was blocked at 37°C for 2 hours. After removing the blocking buffer, the plate was washed five times with PBST. The expressed recombinant antibody was added, with 100 μL / well of purified antibody (starting at 10 μg / mL, serially diluted 3-fold for 3 spots, 100 μL / well), and incubated at room temperature for 1 hour (control wells were treated with PBS). The liquid in the wells was removed, and the plate was washed five times with PBST. Add 100 μL / well of HRP-Protein A (Boster, BA1080) antibody (1:50000 dilution) and incubate at room temperature for 1 hour; 8) After removing the liquid from the wells, wash the plate 5 times with PBST; add 100 μL / well of TMB chromogenic solution; incubate at room temperature in the dark for 10-15 minutes; add 50 μL / well of stop solution; read the OD450 value of the wells using a microplate reader. The ELISA results are shown in Table 15 below.

[0677] Table 15

[0678] 19.3 Detection of the binding of candidate antibodies to overexpressing cell lines

[0679] CHO-S and CHO-S-EGFR cell lines were resuscitated in liquid nitrogen and adjusted to the logarithmic growth phase. The cells were then divided into several fractions, each containing 3 × 10⁶ cells. 5 5) Add PE anti-human IgG (eBioscience, Cat#:12-4998-82) (1:5000 dilution), mix thoroughly, and incubate at room temperature for 30 minutes in the dark. Centrifuge at 800xg for 3 minutes, remove the supernatant containing the antibody, and wash the cells three times with PBS. 6) Add PE anti-human IgG (eBioscience, Cat#:12-4998-82) (1:5000 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes. Centrifuge at 800xg for 3 minutes, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS. Resuspend the cells in 200μL PBS and perform flow cytometry analysis.

[0680] The FACS test results are shown in Figure 19.

[0681] 19.4. ELISA assay for blocking EGFR and EGF binding

[0682] Dilute the EGF-Fc recombinant protein to a final concentration of 0.5 μg / mL using sterile CBS. Add 100 μL to each well of a 96-well microplate and incubate overnight at 4°C. Remove the antigen coating solution and wash five times with PBST (containing 0.05% Tween 20). Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours. After removing the blocking buffer, wash the plate five times with PBST. Prepare 4 μg / mL Biotin-EGFR solution, 50 μL / well. All test antibodies, starting at 60 μg / mL, are serially diluted 5-fold to 7 spots, 50 μL / well. After co-incubation at 37℃ for 0.5 h, transfer all solutions to an EGF-Fc-coated microplate and incubate at 37℃ for 1 h. Remove the liquid from the wells and wash 5 times with PBST. Add 100 μL / well of HRP-Streptavidin antibody (1:50000 dilution) and incubate at room temperature for 1 h. Remove the liquid from the wells and wash the plate 5 times with PBST. Add 100 μL / well of TMB chromogenic buffer and incubate at room temperature in the dark for 10-15 minutes. Add 50 μL / well of stop solution and read the OD450 values ​​of the wells using a microplate reader. In this experiment, Anmai Bio's EMB-01 was selected as the positive label and prepared according to WO2017136820A2. The EGF blocking results of the purified antibody are shown in Table 16 below. The results showed that the single-domain antibody of the present invention has a significant blocking effect.

[0683] Table 16

[0684] 19.5. Single-domain antibody affinity detection

[0685] Candidate antibodies were immobilized using an AHC2 sensor at a concentration of 5 μg / ml for 30 s. The buffer was PBST (PBS + 0.02% Tween 20), diluted with EGFR(25-645)-6His to 50, 25, 12.5, 6.25, 3.13, and 0 nM. Affinity was measured: equilibration for 60 s, binding for 180 s, dissociation for 180 s, and the detection temperature was 25 °C. Kinetic characterization was performed using a ForteBio OCTET R2 system. The results show the affinity of the single-domain antibody described in this invention, and Table 17 below records the affinity detection results.

[0686] Table 17

[0687] 19.6. Tumor cell binding assay

[0688] BXPC-3 cell line (from the Chinese Academy of Sciences Cell Bank) was cultured in complete medium at 37℃ and 5% CO2. Cells in the logarithmic growth phase were harvested, and cell viability was assessed using the trypan blue exclusion method to ensure a viability of over 90%. After centrifugation at 200g for 5 min, the supernatant was discarded. Cells were washed once with PBS, resuspended in FACS Buffer to prepare a single-cell suspension, and the cell density was adjusted to 1 x 10⁻⁶ cells / mL. 6 cells / mL; Add 100 μL of cell suspension to each well of a 96-well plate to make the initial concentration of the working solution 1000 nM, dilute 5 times, for a total of 8 concentrations; after mixing, incubate at 4℃ for 60 min; wash the cells twice with FACS Buffer, 200 μL each time, centrifuge at 200g for 5 min, and finally resuspend the cells with 100 μL of APC-labeled secondary antibody (Allophycocyanin (APC) Affini Pure F(ab')2 Fragment Goat Anti-Human IgG, Fcγfragment specific; Jackson, 109-136-098) diluted with FACS Buffer, and incubate at 4℃ in the dark for 30 min;

[0689] Wash cells twice with 200 μL of FACS Buffer each time, centrifuge at 200g for 5 min, and finally resuspend cells with 150 μL of FACS Buffer; detect fluorescence values ​​by flow cytometry.

[0690] Table 18 Affinity of EGFR-VHH molecules to BXPC-3 cells

[0691] 19.7 Humanization and Validation of Single-Domain Antibodies

[0692] CDR grafting

[0693] The humanization design employed the CDR grafting method. The basic approach involves replacing the camel-derived frame region (FR) with the human germline frame region (FR) selected in the preceding steps, retaining only the camel-derived CDR. To mitigate the adverse effects of humanization on the antibody's spatial conformation, activity, and function, reversion mutations were performed on some critical amino acids in the human frame region, reverting them to the corresponding amino acids in the camel-derived FR region.

[0694] The single-domain antibody 2-2-B1-3 obtained through screening was humanized to obtain 14 versions of humanized antibodies (Table 19); 1-G6 was humanized to obtain 11 versions of humanized antibodies (Table 20).

[0695] Humanized heavy chain antibody genes were cloned and expressed in prokaryotes. The supernatant was collected and purified by Protein A (Cytiva, 29127556) affinity chromatography.

[0696] Table 19

[0697] Table 20

[0698] PTM site removal

[0699] Analysis of high-risk PTM sites in heavy chain CDRs revealed a high-risk PTM site (DG) in the heavy chain variable region CDR2. After antibody humanization, the PTM site was removed from 1G6-huVHH1, 1G6-huVHH6, and 1G6-huVHH10 sequences. The PTM-removed antibodies were expressed, and their affinity was tested, yielding PTM-removed humanized antibodies with affinity comparable to the humanized antibodies. The resulting 1G6-huHCDR2 sequence is shown in SEQ ID NO:15; the VHH sequences of 1G6huVHH1-DA, 1G6-huVHH6-DA, and 1G6-huVHH10-DA are shown in SEQ ID NO:76, SEQ ID NO:77, and SEQ ID NO:78, respectively.

[0700] 19.8. Affinity Detection of Humanized Antibodies

[0701] The kinetics and affinity properties of the antibodies were tested using the Biacore 8K (Cytiva, 8K SPR system) platform. Single-concentration assays were performed to determine the affinity between the humanized antibody and the camel-derived maternal antibody.

[0702] The dissociation equilibrium constant KD reflects the affinity of an antibody for its target; a smaller value indicates stronger affinity. KD is determined by the ratio of the dissociation rate constant Kd to the association rate constant Ka, i.e., KD = Kd / Ka. The ratio of the dissociation equilibrium constant KD between humanized antibodies and camel-derived antibodies is a change factor (Fold↓), which reflects the change in antibody affinity after humanization. A change factor of 1 indicates that the affinity of the humanized antibody is equal to that of the camel-derived antibody; a change factor greater than 1 indicates that the affinity of the humanized antibody is lower than that of the camel-derived antibody; and a change factor less than 1 indicates that the affinity of the humanized antibody is higher than that of the camel-derived antibody. The affinity test results for humanized antibodies are shown in Tables 21-23.

[0703] Table 21. Affinity Detection of Humanized 2-2-B1-3 Antibody

[0704] Table 22. Affinity detection of humanized 1G6 antibodies

[0705] Table 23. Affinity assay of humanized 1G6 antibodies with PTM site removal.

[0706] The results showed that the affinity of multiple humanized antibodies remained essentially consistent with that of the maternal antibody. These humanized antibodies can be used to construct the bispecific antibodies in this invention.

[0707] Those skilled in the art will recognize that many modifications and variations can be made to the invention without departing from its spirit and scope. The specific embodiments described herein are provided by way of example only and are not intended to limit the scope in any way. The true scope and spirit of the invention are shown in the appended claims, and the description and embodiments are merely exemplary.

[0708] Sequence information

[0709] Table 24

[0710] Table 25

Claims

1. A bispecific antibody comprising a first antigen-binding portion binding to EGFR and a second antigen-binding portion binding to HER3, wherein the first antigen-binding portion comprises at least one EGFR-specific VHH, and the at least one EGFR-specific VHH comprises HCDR1, HCDR2 and HCDR3 of any VHH selected from SEQ ID NO: 16, 17, 76, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146 and 150.

2. The bispecific antibody of claim 1, wherein the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises an amino acid sequence as shown in any one of SEQ ID NO: 1, 4, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, wherein HCDR2 comprises an amino acid sequence as shown in any one of SEQ ID NO: 2, 5, 15, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, and wherein HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 1, 4, 79, 83, 87, 91, 95, 99, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, 147, and wherein HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 1, 5, 15, 80, 84, 88, 92, 96, 100, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, and wherein HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: The amino acid sequence represented by any one of NO:3, 6, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, 149.

3. The bispecific antibody of claim 1 or 2, wherein the at least one VHH that specifically binds to EGFR comprises HCDR1, HCDR2, and HCDR3, wherein (1) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; or (2) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:15, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; or (3) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; or (4) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:79, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:80, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:81; or (5) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:83, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:84, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:85; or (6) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:87, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:88, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:89; or (7) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:91, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:92, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:93; or (8) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:94, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:96, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:97; or (9) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:99, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:100, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:101; or (10) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:103, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:104, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:105; or (11) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:107, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:108, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:109; or (12) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:111, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:112, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:113; or (13) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:115, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:116, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:117; or (14) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:119, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:120, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:121; or (15) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:123, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:124, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:125; or (16) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:127, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:128, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

129. (17) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:131, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:132, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:133; or (18) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:135, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:136, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:137; or (19) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:139, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:140, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:141; or (20) HCDR1 comprises the amino acid sequence shown in SEQ ID NO:143, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:144, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:145; or (21) The HCDR1 contains the amino acid sequence shown in SEQ ID NO:147, the HCDR2 contains the amino acid sequence shown in SEQ ID NO:148, and the HCDR3 contains the amino acid sequence shown in SEQ ID NO:

149.

4. The bispecific antibody of any one of claims 1-3, wherein the at least one VHH specifically binding to EGFR comprises an amino acid sequence of any one of SEQ ID NO: 16, 17, 51-64, 65-78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, and 150, or an amino acid sequence of any one of 16, 17, 51-64, 65-78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, and 150. The amino acid sequence has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, or consists of any one of the amino acid sequences 16, 17, 51-64, 65-78, 82, 86, 90, 94, 98, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, 146, and 150.

5. The bispecific antibody of any one of claims 1-4, wherein the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; preferably, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:16 or 18; more preferably, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:

18.

6. The bispecific antibody of any one of claims 1-4, wherein the at least one EGFR-specific VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:5 or 15, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:6; preferably, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:17 or 76; more preferably, the at least one EGFR-specific VHH comprises the amino acid sequence shown in SEQ ID NO:

76.

7. The bispecific antibody of any one of claims 1-6, wherein the second antigen-binding moiety binding HER3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3; wherein the HCDR1 of the second antigen-binding moiety binding HER3 comprises the amino acid sequence of SEQ ID NO:30, the HCDR2 comprises the amino acid sequence of SEQ ID NO:31, the HCDR3 comprises the amino acid sequence of SEQ ID NO:32, the LCDR1 comprises the amino acid sequence of SEQ ID NO:33, the LCDR2 comprises the amino acid sequence of SEQ ID NO:34, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

35.

8. The bispecific antibody of claim 7, wherein the heavy chain variable region of the second antigen-binding portion binding to HER3 comprises the amino acid sequence of SEQ ID NO:26 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:26; and / or, the light chain variable region comprises the amino acid sequence of SEQ ID NO:28 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

28.

9. The bispecific antibody of any one of claims 1-8, comprising a first antigen-binding moiety binding to EGFR and a second antigen-binding moiety binding to HER3. The first antigen-binding moiety that binds to EGFR comprises at least one EGFR-specific VHH, wherein the VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3; and The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. HCDR1 of the second antigen-binding region that binds to HER3 includes the amino acid sequence of SEQ ID NO:30, HCDR2 includes the amino acid sequence of SEQ ID NO:31, HCDR3 includes the amino acid sequence of SEQ ID NO:32, LCDR1 includes the amino acid sequence of SEQ ID NO:33, LCDR2 includes the amino acid sequence of SEQ ID NO:34, and LCDR3 includes the amino acid sequence of SEQ ID NO:

35.

10. The bispecific antibody of any one of claims 1-8, comprising a first antigen-binding moiety binding to EGFR and a second antigen-binding moiety binding to HER3. The first antigen-binding moiety that binds to EGFR comprises at least one EGFR-specific VHH, wherein the VHH comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown according to SEQ ID NO:4, HCDR2 comprises the amino acid sequence shown according to SEQ ID NO:5 or 15, and HCDR3 comprises the amino acid sequence shown according to SEQ ID NO:6; and The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and the light chain variable region includes LCDR1, LCDR2, and LCDR3. HCDR1 of the second antigen-binding region that binds to HER3 includes the amino acid sequence of SEQ ID NO:30, HCDR2 includes the amino acid sequence of SEQ ID NO:31, HCDR3 includes the amino acid sequence of SEQ ID NO:32, LCDR1 includes the amino acid sequence of SEQ ID NO:33, LCDR2 includes the amino acid sequence of SEQ ID NO:34, and LCDR3 includes the amino acid sequence of SEQ ID NO:

35.

11. The bispecific antibody of any one of claims 1-8, comprising a first antigen-binding moiety binding to EGFR and a second antigen-binding moiety binding to HER3. The first antigen-binding moiety that binds to EGFR includes at least one EGFR-specific VHH, said VHH containing the amino acid sequence shown in SEQ ID NO: 16 or 18, preferably containing the amino acid sequence shown in SEQ ID NO: 18; and The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

28.

12. The bispecific antibody of any one of claims 1-8, comprising a first antigen-binding moiety binding to EGFR and a second antigen-binding moiety binding to HER3. The first antigen-binding moiety that binds to EGFR includes at least one EGFR-specific VHH, said VHH containing the amino acid sequence shown in SEQ ID NO: 17 or 76, preferably containing the amino acid sequence shown in SEQ ID NO: 76; and The second antigen-binding region that binds to HER3 includes a heavy chain variable region and a light chain variable region. The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

28.

13. The bispecific antibody of any one of claims 1-12, wherein the bispecific antibody further comprises i) Heavy chain constant region CH1 of an immunoglobulin; for example, the heavy chain constant region CH1 is the heavy chain constant region CH1 of human IgG1 or IgG4; preferably, the heavy chain constant region CH1 contains an amino acid sequence as shown in SEQ ID NO:22; ii) Heavy chain hinge region of an immunoglobulin; for example, the heavy chain hinge region is the heavy chain hinge region of human IgG1 or IgG4; preferably, the hinge region comprises an amino acid sequence as shown in SEQ ID NO:23 or SEQ ID NO:24; iii) An Fc fragment of an immunoglobulin; for example, the Fc fragment is an Fc fragment of the human IgG1 or IgG4 heavy chain; preferably, the immunoglobulin Fc fragment comprises an amino acid sequence as shown in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:25; and / or iv) Light chain constant region (CL) of immunoglobulin; for example, the light chain constant region is the human κ light chain constant region; preferably, the light chain constant region CL contains an amino acid sequence as shown in SEQ ID NO:29; v) A peptide linker; for example, a peptide linker comprising a peptide sequence of (GGGGS)n, (GS)n, or both, wherein n is an integer from 1 to 6; preferably, the linker comprises the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:

39.

14. The bispecific antibody of any one of claims 1-13, wherein the bispecific antibody has two heavy chain polypeptide chains, one heavy chain polypeptide chain containing an immunoglobulin Fc (Knob) fragment and the other heavy chain polypeptide chain containing an immunoglobulin Fc (Hole) fragment, the two heavy chain polypeptide chains forming a dimer through disulfide bonds in the hinge region and a Knob-into-Hole structure; For example, the immunoglobulin Fc(knob) fragment contains the amino acid sequence shown in SEQ ID NO:20, and the immunoglobulin Fc(Hole) fragment contains the amino acid sequence shown in SEQ ID NO:

21.

15. The bispecific antibody of any one of claims 1-14, wherein the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain; wherein, The first polypeptide chain comprises the VHH of the first antigen-binding moiety that binds to EGFR, the heavy chain variable region of the second antigen-binding moiety that binds to HER3, the immunoglobulin heavy chain constant region CH1, the immunoglobulin heavy chain hinge region, and the immunoglobulin Fc fragment, and optionally, also comprises the linker; and wherein the second polypeptide chain comprises the light chain variable region of the second antigen-binding moiety that binds to HER3 and the immunoglobulin light chain constant region.

16. The bispecific antibody of claim 15, wherein the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain; wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a VHH of the first antigen-binding moiety binding to EGFR, a linker, a heavy chain variable region of the second antigen-binding moiety binding to HER3, an immunoglobulin heavy chain constant region CH1, an immunoglobulin heavy chain hinge region, and an immunoglobulin Fc fragment.

17. The bispecific antibody of claim 16, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus: (1) VHH that binds to the first antigen-binding portion of EGFR, comprising the amino acid sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:76; (2) A linker comprising an amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39; (3) The heavy chain variable region of the second antigen-binding region of HER3, which contains the amino acid sequence of SEQ ID NO:26; (4) Immunoglobulin heavy chain constant region CH1, which contains the amino acid sequence of SEQ ID NO:22; (5) The immunoglobulin heavy chain hinge region, comprising the amino acid sequence of SEQ ID NO:23; and (6) Immunoglobulin Fc fragment, which contains the amino acid sequence of SEQ ID NO:19, Preferably, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:46 or SEQ ID NO:48, or an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:46 or SEQ ID NO:

48.

18. The bispecific antibody of claim 15, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region of the second antigen-binding moiety binding the HER3-binding region, an immunoglobulin heavy chain constant region CH1, an immunoglobulin heavy chain hinge region, an Fc fragment of immunoglobulin, a linker, and a VHH of the first antigen-binding moiety binding the EGFR-binding region.

19. The bispecific antibody of claim 18, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus: (1) The heavy chain variable region of the second antigen-binding region of HER3, which contains the amino acid sequence of SEQ ID NO:26; (2) Heavy chain constant region CH1, which contains the amino acid sequence of SEQ ID NO:22; (3) The heavy chain hinge region of an immunoglobulin, which contains the amino acid sequence of SEQ ID NO:23; (4) Fc fragment containing the amino acid sequence of SEQ ID NO:19; (5) A linker comprising the amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39; and (6) VHH that binds to the first antigen-binding portion of EGFR, comprising the amino acid sequence of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:

76. Preferably, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:47, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:

47.

20. A bispecific antibody according to any one of claims 15-19, wherein the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain; wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region of the second antigen-binding moiety of the HER3-binding protein and a light chain constant region of the immunoglobulin.

21. The bispecific antibody of claim 20, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus: (1) The light chain variable region of the second antigen-binding moiety of HER3, which contains the amino acid sequence of SEQ ID NO:28; and (2) The light chain constant region of immunoglobulin, which contains the amino acid sequence of SEQ ID NO:

29. Preferably, the second polypeptide chain comprises SEQ ID NO:41, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:

41.

22. The bispecific antibody of any one of claims 1-14, wherein the bispecific antibody comprises a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain; wherein, The first polypeptide chain includes the heavy chain variable region of the second antigen-binding moiety binding HER3, the immunoglobulin heavy chain constant region CH1, the immunoglobulin heavy chain hinge region, and the immunoglobulin Fc fragment; the second polypeptide chain includes the VHH of the first antigen-binding moiety binding EGFR, the immunoglobulin heavy chain hinge region, and the immunoglobulin Fc fragment; the third polypeptide chain includes the light chain variable region of the second antigen-binding moiety binding HER3 and the light chain constant region of immunoglobulin.

23. The bispecific antibody of claim 22, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region of the second antigen-binding moiety binding to HER3, an immunoglobulin heavy chain constant region CH1, an immunoglobulin heavy chain hinge region, and an Fc fragment of immunoglobulin.

24. The bispecific antibody of claim 23, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus: (1) The heavy chain variable region of the second antigen-binding region of HER3, which contains the amino acid sequence of SEQ ID NO:26; (2) Immunoglobulin heavy chain constant region CH1, which contains the amino acid sequence of SEQ ID NO:22; (3) The heavy chain hinge region of an immunoglobulin, comprising the amino acid sequence of SEQ ID NO:23; and (4) Immunoglobulin Fc fragment, which contains the amino acid sequence of SEQ ID NO:20, Preferably, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO:44, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:

44.

25. The bispecific antibody of any one of claims 22-24, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, the VHH of the first antigen-binding moiety that binds to EGFR, a linker, an immunoglobulin heavy chain hinge region, and an immunoglobulin Fc fragment.

26. The bispecific antibody of claim 25, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus: (1) VHH that binds to the first antigen-binding portion of EGFR, which contains the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:76; (2) A linker comprising an amino acid sequence of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 or SEQ ID NO:39; (3) The heavy chain hinge region of an immunoglobulin, comprising the amino acid sequence of SEQ ID NO:24; and (4) Fc fragment, which contains the amino acid sequence of SEQ ID NO:

21. Preferably, the second polypeptide chain contains the amino acid sequence of SEQ ID NO:45, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:

45.

27. The bispecific antibody of any one of claims 22-26, wherein the third polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region of the second antigen-binding moiety that binds to HER3 and an immunoglobulin light chain constant region.

28. The bispecific antibody of claim 27, wherein the third polypeptide chain comprises, from the N-terminus to the C-terminus: (1) The light chain variable region of the second antigen-binding moiety of HER3, which contains the amino acid sequence of SEQ ID NO:28; and (2) The light chain constant region of immunoglobulin, which contains the amino acid sequence of SEQ ID NO:

29. Preferably, the third polypeptide chain comprises SEQ ID NO:41, or an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:

41.

29. A bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, the first polypeptide chains comprising the amino acid sequence shown in SEQ ID NO:40, and the second polypeptide chains comprising the amino acid sequence shown in SEQ ID NO:

41.

30. A bispecific antibody against EGFR and HER3, or an antigen-binding fragment thereof, wherein the bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, and wherein... i) The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:42, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41; ii) The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:46, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41; iii) The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:47, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:41; or iv) The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO:48, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO:

41.

31. A bispecific antibody against EGFR and HER3 or an antigen-binding fragment thereof, wherein the bispecific antibody comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, the first polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:44, the second polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:45, and the third polypeptide chain comprising the amino acid sequence shown in SEQ ID NO:

41.

32. A bispecific antibody-drug conjugate or an isomer thereof and a pharmaceutically acceptable salt thereof, wherein... The bispecific antibody-drug conjugate comprises: a bispecific antibody according to any one of claims 1-31 and a conjugated therapeutically active substance or pharmaceutically active ingredient D. Preferably, the bispecific antibody-drug conjugate comprises: a bispecific antibody according to any one of claims 1-31, a linker L, and a therapeutically active substance or pharmaceutically active ingredient D.

33. The bispecific antibody-drug conjugate of claim 32 or its isomers and pharmaceutically acceptable salts thereof, wherein the therapeutically active substance or pharmaceutically active ingredient D is selected from cytotoxins, plant toxins, small molecule toxins, radioactive isotopes, and maytansine alkaloids, preferably cytotoxins.

34. The bispecific antibody-drug conjugate of claim 33 or an isomer thereof and a pharmaceutically acceptable salt thereof, wherein the cytotoxicant is a monomethylaurestatin compound, a camptothecin compound, or a maytansine alkaloid. Preferably, the monomethyl auristatin compound is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF); or The maytansine alkaloids mentioned are DM1, DM3, or DM4; or The camptothecin-like compound is Or SN-38.

35. A bispecific antibody-drug conjugate or an isomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of claims 32-34, wherein the bispecific antibody-drug conjugate comprises a plurality of Ds, the plurality of Ds being different or the same.

36. A bispecific antibody-drug conjugate or an isomer thereof, or a pharmaceutically acceptable salt thereof, according to any one of claims 32-35, wherein the bispecific antibody-drug conjugate has a drug-to-antibody ratio (DAR) of 1-15, for example, a DAR of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

37. The bispecific antibody-drug conjugate of claim 36 or an isomer thereof and a pharmaceutically acceptable salt thereof, wherein the DAR is an average DAR, for example, an average DAR of 1-15, preferably 1-10, more preferably 1-8.

38. The bispecific antibody-drug conjugate of claim 37 or an isomer thereof and a pharmaceutically acceptable salt thereof, having an average DAR of 2 to 10, for example 2 to 8.

39. The bispecific antibody-drug conjugate or its isomers and pharmaceutically acceptable salts of any one of claims 32-38, wherein the linker L is a combination of one or more L'; wherein L' is selected from carbonyl, amino, amide, aminoacyl, -(PEG)n-, -(CH2)n-, heteroatom-containing -(CH2)n-, -(C≡C)-, -(CH=CH)-, -O-, -S-, maleimide (mc), maleimide propionyl (MP), methylsulfonylpyrimidinyl, valine-citrulline (val-cit), valine-alanine (val-ala), N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC), alanine-phenylalanine (ala-phe), phenylalanine-lysine The following acids are used: p-aminobenzyloxycarbonyl (PAB), dimethylethylenediamine (DMED), N-succinimide-4-(2-pyridinylthio)valerate (SPP), N-succinimide-4-(N-maleimide-methyl)-cyclohexane-1-carboxylate (SMCC), N-succinimide-(4-iodo-acetyl)aminobenzoate (SIAB), N-succinimide-4-(2-pyridyldithio)butyrate (SPDB), N-succinimide-3-(pyridin-2-yldithio)propionate (SPDP), glycine-glycine-phenylalanine-glycine (GGFG), acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl (AcLys-vc-PABC); wherein n is independently selected from an integer from 1 to 20. Preferably, L can be J-L1-L2-X-L3. Said J is selected from L1 is selected from single bonds and -(PEG). n -or-(CH2) n -, the -(CH2) n -Optionally contains 1 to 6 heteroatoms selected from N, O, and S; wherein n is independently selected as an integer from 1 to 20. Preferably, L1 is selected from -(PEG). n -, where n is independently 2, 4, 6 or 8; L2 is selected from one or more combinations of single bonds, -O-, -S-, -CH2-, -NH-, and -C(O)-; wherein the -CH2- and -NH- may optionally be replaced by C 1～6 Alkyl or halogen substitution 1 to 3 times; X is selected from a single bond or a combination of 1 to 4 X's, wherein the X's are independently selected from amino acids, such as glycine, alanine, phenylalanine, valine, lysine, and citrulline. For example, X is selected from a single bond or valine-citrulline (val-cit), valine-alanine (val-ala), alanine-phenylalanine (ala-phe), phenylalanine-lysine (phe-lys), glycine-glycine-phenylalanine-glycine (GGFG). L3 is selected from one or more combinations of single bond, -O-, -S-, -CH2-, -NH-, -C(O)-, -phenyl-, -cyclopropyl-, -cyclobutyl-, and -cyclohexyl-; wherein the -CH2-, -NH-, -phenyl-, -cyclopropyl-, -cyclobutyl-, and -cyclohexyl- may optionally be C 1～6 Alkyl, halogen, cyano or hydroxyl groups are substituted 1 to 3 times.

40. The bispecific antibody-drug conjugate or its isomer, or a pharmaceutically acceptable salt thereof, according to any one of claims 32-39. The structures LD of the linker L and the therapeutic active substance or pharmaceutical active ingredient D are shown in formulas Cpd3, Cpd5 and Cpd6:

41. The bispecific antibody-drug conjugate or its isomer and a pharmaceutically acceptable salt thereof according to any one of claims 32-40, wherein the structure of the bispecific antibody-drug conjugate is shown in formula (I-C3), formula (I-C5) and formula (I-C6): in, m is the average number of connections, and m is independently selected from an integer or decimal number from 1 to 10; Ab is the bispecific antibody or its antigen-binding fragment.

42. A polynucleotide comprising a polynucleotide sequence encoding a bispecific antibody of any one of claims 1-31.

43. An expression vector comprising the polynucleotide of claim 42.

44. A host cell comprising the polynucleotide of claim 42 or the expression vector of claim 43.

45. A method for generating bispecific antibodies, comprising the following steps: a) Culturing the host cells of claim 44 under suitable conditions to express bispecific antibodies; and b) Isolate and / or purify bispecific antibodies from host cells or their cultures.

46. ​​A pharmaceutical composition comprising a bispecific antibody of any one of claims 1-31, a bispecific antibody-drug conjugate of any one of claims 32-41, a polynucleotide of claim 42 and / or an expression vector of claim 43, and a pharmaceutically acceptable vector.

47. Use of the bispecific antibody of any one of claims 1-31, the bispecific antibody-drug conjugate of any one of claims 32-41, the polynucleotide of claim 42, the expression vector of claim 43, and / or the pharmaceutical composition of claim 46 in the preparation of a medicament for treating and / or preventing a disease in a subject in need.

48. A method for treating and / or preventing a disease in a subject in need, the method comprising administering to the subject a therapeutically effective amount of a bispecific antibody of any one of claims 1-31, a bispecific antibody-drug conjugate of any one of claims 32-41, a polynucleotide of claim 42, an expression vector of claim 43, and / or a pharmaceutical composition of claim 46.

49. The use of claim 47 or the method of claim 48, wherein the disease is cancer and / or tumor, for example, the disease is cancer and / or tumor expressing EGFR and / or HER3 (EGFR and / or HER3 positive).

50. The use or method of claim 49, wherein the cancer and / or tumor is selected from: lung cancer, breast cancer, skin cancer, gastric cancer, colorectal cancer, nasopharyngeal cancer, esophageal cancer, brain cancer, biliary tract cancer, head and neck cancer, thyroid cancer, ovarian cancer, endometrial cancer, pancreatic cancer, prostate cancer, bladder cancer, gastrointestinal cancer, gastroesophageal junction cancer, digestive tract cancer, uterine cancer, cervical cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, kidney cancer, thyroid cancer, leukemia, malignant lymphoma, plasma tumor, myeloma, glioma, glioblastoma, osteosarcoma, sarcoma, chordoma, squamous cell carcinoma, oral squamous cell carcinoma, lymphoma, mesothelioma, urothelial carcinoma, skin cancer, melanoma, or hematologic malignancy.

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