Anti-EGFR and MUC1 bispecific antibody and use thereof

Abstract

Provided are an anti-EGFR and MUC1 bispecific antibody and use thereof. Specifically, provided are a bispecific antibody that specifically binds to EGFR and MUC1, or a combination fragment thereof, and a composition comprising same. Also provided are a nucleic acid molecule encoding the bispecific antibody or an antigen-binding fragment thereof, a vector and a transformant that are used for expressing the bispecific antibody or the antigen-binding fragment thereof, a chimeric antigen receptor, a genetically modified cellular antibody-drug conjugate, and a pharmaceutical composition that comprise the antibody or the antigen-binding fragment thereof, and use thereof.

Description

A bispecific antibody against EGFR and MUC1 and its uses

[0001] This application claims priority to Chinese patent application 2025101111029, filed on January 23, 2025, and Chinese patent application 2026100856240, filed on January 21, 2026. The full text of the aforementioned Chinese patent applications is incorporated herein by reference. Technical Field

[0002] This invention relates to the field of monoclonal antibodies, and more specifically to a bispecific antibody against EGFR and MUC1 and its uses. Background Technology

[0003] EGFR (epidermal growth factor receptor) is a transmembrane tyrosine kinase receptor involved in cell proliferation, differentiation, and survival. Belonging to the ErbB receptor family, it is a transmembrane glycoprotein composed of extracellular, intracellular, and cytoplasmic domains. It activates downstream signaling pathways upon binding to ligands (such as epidermal growth factor EGF), thereby promoting cell proliferation, differentiation, and survival. Abnormal activation of EGFR is a key factor in tumor development in various cancers. MUC1 (mucin 1) is a highly glycosylated transmembrane protein that plays a lubricating and protective role in normal tissues. Under normal circumstances, it is mainly expressed on the surface of epithelial cells, but its expression level is significantly upregulated in various cancers and is closely related to tumor invasiveness and poor prognosis. Both EGFR and MUC1 are common drug resistance targets. EGFR mutations, activation of signaling pathways, and MUC1-mediated signal transduction may lead to drug resistance.

[0004] EGFR / MUC1 bispecific antibodies represent an emerging cancer treatment strategy. By targeting both EGFR and MUC1, they can bypass resistance mechanisms to single targets and simultaneously block two key signaling pathways, thereby enhancing therapeutic efficacy. Compared to monoclonal antibodies, EGFR / MUC1 bispecific antibodies offer greater therapeutic potential and demonstrate significant therapeutic advantages as a novel cancer treatment strategy, providing patients with more effective treatment options. Summary of the Invention

[0005] To address the above technical problems, this invention provides a bispecific antibody against EGFR and MUC1 and its uses.

[0006] Specifically, the present invention provides an antibody or antigen-binding fragment against EGFR / MUC1, wherein the antibody or antigen-binding fragment comprises an EGFR-binding domain and a MUC1-binding domain; the EGFR-binding domain comprises a heavy chain variable region VH1 and a light chain variable region VL, and the MUC1-binding domain comprises a heavy chain variable region VH2 and a light chain variable region VL; wherein...

[0007] The VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:103, SEQ ID NO:104, and SEQ ID NO:105, respectively; or, the VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively.

[0008] The VL contains the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0009] In some embodiments, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:106, SEQ ID NO:107, and SEQ ID NO:108, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively.

[0010] In some embodiments, the VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; or, the VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:11, respectively; or, the VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:29, respectively.

[0011] In some embodiments, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:21, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:25, and SEQ ID NO:26, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:27, SEQ ID NO:20, and SEQ ID NO:26, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:28, SEQ ID NO:20, and SEQ ID NO:21, respectively. H2CDR1, H2CDR2, and H2CDR3 are shown in NO:26.

[0012] This invention uses the Kabat definition method to determine the CDR sequence.

[0013] In some embodiments, the antibody or its antigen-binding fragment is selected from the following:

[0014] (1) The VL contains the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively;

[0015] (2) VH1 and VH2 are selected from any one of the following:

[0016] The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0017] The VH1 contains the amino acid sequences H1CDR1, H1CDR2 and H1CDR3 as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:11, respectively; and the VH2 contains the amino acid sequences H2CDR1, H2CDR2 and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0018] The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively.

[0019] The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:21, respectively.

[0020] The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively.

[0021] The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:21, respectively.

[0022] The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:25, and SEQ ID NO:26, respectively.

[0023] The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:27, SEQ ID NO:20, and SEQ ID NO:26, respectively.

[0024] The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:28, SEQ ID NO:20, and SEQ ID NO:26, respectively.

[0025] The VH1 contains the amino acid sequences H1CDR1, H1CDR2 and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:29, respectively; and the VH2 contains the amino acid sequences H2CDR1, H2CDR2 and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0026] The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:11, respectively, and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0027] In some embodiments, the antibody or its antigen-binding fragment is selected from the following:

[0028] (1) The VH1 comprises an amino acid sequence as shown in any one of SEQ ID NO:31-33 or 35; and / or, the VH2 comprises an amino acid sequence as shown in any one of SEQ ID NO:30, 34 or 36-40; and / or, the VL comprises an amino acid sequence as shown in SEQ ID NO:4; or,

[0029] (2) The VH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:31-33 or 35; and / or, the VH2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:30, 34, or 36-40; and / or, the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0030] In some specific implementations, the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity does not involve changes to the CDR sequence.

[0031] In some specific implementations, the antibody or its antigen-binding fragment is selected from the following:

[0032] VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:30; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0033] VH1 contains the amino acid sequence shown in SEQ ID NO:32; VH2 contains the amino acid sequence shown in SEQ ID NO:30; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0034] VH1 contains the amino acid sequence shown in SEQ ID NO:35; VH2 contains the amino acid sequence shown in SEQ ID NO:34; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0035] VH1 contains the amino acid sequence shown in SEQ ID NO:35; VH2 contains the amino acid sequence shown in SEQ ID NO:36; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0036] VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:37; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0037] VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:36; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0038] VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:38; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0039] VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:39; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0040] VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:40; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0041] VH1 contains the amino acid sequence shown in SEQ ID NO:33; VH2 contains the amino acid sequence shown in SEQ ID NO:30; VL contains the amino acid sequence shown in SEQ ID NO:4;

[0042] The VH1 contains the amino acid sequence shown in SEQ ID NO:32; the VH2 contains the amino acid sequence shown in SEQ ID NO:30; and the VL contains the amino acid sequence shown in SEQ ID NO:4.

[0043] In some embodiments, the antibody or its antigen-binding fragment comprises one or more of the following:

[0044] (1) Fully human antibodies, humanized antibodies, chimeric antibodies, proantibodies, bispecific antibodies or multispecific antibodies;

[0045] (2) Fab, Fab', F(ab')2, Fv or scFv;

[0046] (3) The MUC1 binding domain is a human anti-MUC1 antibody or its antigen-binding fragment; and,

[0047] (4) The EGFR binding domain is a human anti-EGFR antibody or its antigen-binding fragment.

[0048] Bispecific antibodies are constructed by binding different antigen-binding fragments such as Fab, Fab', F(ab')2, Fv, or ScFv. The Fab fragment contains a complete light and heavy chain antigen-binding region. Fab' is obtained by further processing Fab. F(ab')2 is composed of two Fab' fragments linked by disulfide bonds. Fv and scFv are the smallest antibody-binding fragment and a single-chain antibody fragment, respectively. They can all be designed to bind two different antigens, thereby achieving the function of bispecific antibodies.

[0049] In some embodiments, the EGFR binding domain and the MUC1 binding domain further include a light chain constant region and a heavy chain constant region, respectively. The EGFR binding domain includes a light chain constant region CL and a heavy chain constant region CH1, and the MUC1 binding domain includes a light chain constant region CL and a heavy chain constant region CH2.

[0050] In some specific implementations, the heavy chain constant region is the heavy chain constant region of human antibody IgG1.

[0051] In some specific implementations, the light chain constant region is the light chain constant region of the human antibody κ chain.

[0052] In some embodiments, the heavy chain constant regions of the EGFR binding domain and the MUC1 binding domain contain Knob-into-hole mutations; the heavy chain constant regions CH1 and CH2 contain different Knob or Hole mutations.

[0053] In some specific implementations, CH1 contains a Knob mutation, such as T366W, and CH2 contains a Hole mutation, such as one or more selected from T366S, L368A, and Y407V; or CH2 contains a Knob mutation, such as T366W, and CH1 contains a Hole mutation, such as one or more selected from T366S, L368A, and Y407V.

[0054] In some specific implementations, CH1 and CH2 contain L234A / L235A mutations.

[0055] In some specific implementations, when CH1 contains the Hole mutation, CH1 also contains the H435R / Y436F mutation; or, when CH2 contains the Hole mutation, CH2 also contains the H435R / Y436F mutation; that is, when CH1 contains the T366S, L368A, and Y407V mutations and also contains the H435R / Y436F mutation, CH2 contains the T366W mutation; or, when CH2 contains the T366S, L368A, and Y407V mutations and also contains the H435R / Y436F mutation, CH1 contains the T366W mutation.

[0056] In some specific implementations, CH1 is shown as SEQ ID NO:76, and CH2 is shown as SEQ ID NO:77.

[0057] In some specific implementations, CH1 is shown as SEQ ID NO:77, and CH2 is shown as SEQ ID NO:76.

[0058] In some specific implementations, CH1 is shown as SEQ ID NO:85, and CH2 is shown as SEQ ID NO:86.

[0059] In some specific implementations, CH1 is shown as SEQ ID NO:86, and CH2 is shown as SEQ ID NO:85.

[0060] In some embodiments, the antibody or its antigen-binding fragment is selected from any of the following:

[0061] (I) The heavy chain of the EGFR-binding domain comprises an amino acid sequence as shown in any one of SEQ ID NO:42, 43, 45, 46, 48, 51, 56, 57, 59, 62, 67, and 68, and the light chain of the EGFR-binding domain comprises an amino acid sequence as shown in SEQ ID NO:5; and / or, the heavy chain of the MUC1-binding domain comprises an amino acid sequence as shown in any one of SEQ ID NO:41, 44, 47, 49, 50, 52, 53, 54, 55, 58, 60, 61, 63, 64, 65, and 66, and the light chain of the MUC1-binding domain comprises an amino acid sequence as shown in SEQ ID NO:5; or,

[0062] (II) The heavy chain of the EGFR-binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:42, 43, 45, 46, 48, 51, 56, 57, 59, 62, 67, and 68; and the light chain of the EGFR-binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5; and / or, the heavy chain of the MUC1-binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5; and / or, the light chain of the EGFR-binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5; The amino acid sequence shown in any one of NO:41, 44, 47, 49, 50, 52, 53, 54, 55, 58, 60, 61, 63, 64, 65, and 66 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain of the MUC1 binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5.

[0063] In some specific implementations, the amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity does not involve changes to the CDR sequence.

[0064] In some specific implementations, the antibody or its antigen-binding fragment is selected from the following:

[0065] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:42, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:41, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0066] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:43, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:41, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0067] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:48, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:47, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0068] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:48, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:49, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0069] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:50, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0070] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:49, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0071] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:52, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0072] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:53, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0073] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:54, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0074] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:55, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0075] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:56, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:55, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0076] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:57, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:55, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0077] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:45, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:44, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0078] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:46, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:44, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0079] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:59, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:58, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0080] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:59, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:60, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0081] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:61, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0082] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:60, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0083] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:63, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0084] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:64, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0085] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:65, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0086] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:66, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0087] The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:67, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:66, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or,

[0088] The amino acid sequence of the heavy chain of the EGFR binding domain is shown in SEQ ID NO:68, the amino acid sequence of the light chain of the EGFR binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1 binding domain is shown in SEQ ID NO:66, and the amino acid sequence of the light chain of the MUC1 binding domain is shown in SEQ ID NO:5.

[0089] The present invention also provides an isolated nucleic acid that encodes the antibody or an antigen-binding fragment thereof.

[0090] The present invention also provides a recombinant expression vector, characterized in that the recombinant expression vector comprises the aforementioned nucleic acid.

[0091] In some implementations, the recombinant expression vector is selected from viral vectors and non-viral vectors.

[0092] In some embodiments, the non-viral vector is selected from plasmids, linear DNA fragments, and RNA;

[0093] In some implementations, the recombinant expression vector is a plasmid.

[0094] In some specific implementations, the backbone plasmid of the recombinant expression vector is pCDNA3.1.

[0095] The present invention also provides a transformant comprising the nucleic acid or the recombinant expression vector described above, wherein the transformant is a non-animal or non-plant variety.

[0096] In some implementations, the transformant is a eukaryotic cell.

[0097] In some specific implementations, the eukaryotic cells are mammalian cells.

[0098] The present invention also provides a method for preparing an antibody or antigen-binding fragment against EGFR / MUC1, the method comprising culturing the transformant and obtaining the antibody or antigen-binding fragment from the culture.

[0099] The present invention also provides a chimeric antigen receptor comprising the antibody or its antigen-binding fragment.

[0100] In some specific implementations, the chimeric antigen receptor further includes a co-stimulatory domain and a signal transduction domain.

[0101] The present invention also provides a genetically modified cell that expresses the chimeric antigen receptor.

[0102] In some specific implementations, the genetically modified cells are T cells or NK cells.

[0103] The present invention also provides an antibody-drug conjugate comprising a cytotoxic agent or tag, and the antibody or its antigen-binding fragment thereof.

[0104] The present invention also provides a pharmaceutical composition comprising the antibody or its antigen-binding fragment, the nucleic acid, the recombinant expression vector, the transformant, the genetically modified cell or the antibody-drug conjugate, and pharmaceutically acceptable carriers and / or excipients.

[0105] The present invention also provides the use of the antibody or antigen-binding fragment thereof, the genetically modified cell, the antibody-drug conjugate or the pharmaceutical composition thereof in the preparation of a medicament for the treatment and / or prevention of EGFR and / or MUC1-mediated diseases.

[0106] In some implementations, the disease is cancer.

[0107] In some specific implementations, the cancer is selected from one or more of skin cancer, ovarian cancer, breast cancer, and non-small cell lung cancer.

[0108] The present invention also provides a method for treating and / or preventing EGFR and / or MUC1-mediated diseases or conditions, the method comprising administering to a subject in need a therapeutic or preventative amount of the antibody or its antigen-binding fragment, the genetically modified cell, the antibody-drug conjugate, or the pharmaceutical composition.

[0109] In some implementations, the disease is cancer.

[0110] In some specific implementations, the cancer is selected from one or more of skin cancer, ovarian cancer, breast cancer, and non-small cell lung cancer.

[0111] The present invention also provides the antibody or its antigen-binding fragment, the genetically modified cell, the antibody-drug conjugate, or the pharmaceutical composition thereof for the treatment and / or prevention of EGFR and / or MUC1-mediated diseases or conditions.

[0112] In some implementations, the disease is cancer.

[0113] In some specific implementations, the cancer is selected from one or more of skin cancer, ovarian cancer, breast cancer, and non-small cell lung cancer.

[0114] The present invention also provides a kit comprising the antibody or its antigen-binding fragment, the nucleic acid, the recombinant expression vector, the transformant, the genetically modified cells, the antibody-drug conjugate, or the pharmaceutical composition thereof.

[0115] Terminology Definition

[0116] Unless otherwise stated, the present invention will be implemented using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, all of which are within the scope of the art.

[0117] To facilitate a better understanding of this invention, certain technical terms are specifically defined as follows. Unless otherwise expressly defined elsewhere in this document, the technical terms or expressions used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0118] For definitions and terminology in this field, those skilled in the art may refer at least in part to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues follow the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids. Singular forms used herein (including the claims) include their corresponding plural forms unless otherwise expressly specified herein.

[0119] The term “about” when used in conjunction with a numeric value means to encompass a range of numeric values ​​having a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as these variations are suitable for carrying out the disclosed methods.

[0120] The term “and / or” should be understood to mean any one of the options or any combination of two or more of the options.

[0121] As used herein, 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, that is, including at least one in the quantity or element list, but also including more than one, and optionally, additional unlisted items. Only when explicitly indicated by the opposite terms, 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.

[0122] Unless the context clearly indicates otherwise, the words “one” and “a” should be understood as “at least one” as used in this article.

[0123] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by these cells or the liver, which results in selective damage, destruction, or clearance from the body of invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation.

[0124] The terms “activity” or “bioactivity”, or “biological property” or “biocharacteristic”, are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize EGFR activity in vivo or in vitro, IC50, etc.50 The in vivo stability and immunogenic properties of antibodies. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics are observed, measured, or evaluated using techniques known in the art, including but not limited to ELISA, FACS, or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from various sources (including human, primate, or any other source).

[0125] The term "antibody" refers to any form of antibody that has the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camel-derived single-domain antibodies.

[0126] 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.

[0127] The term "full-length antibody" refers to an immunoglobulin molecule that, in its natural state, contains at least four peptide chains: two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region (abbreviated as CH in this document). Each light chain consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region. The light chain constant region consists of a CL domain. The VH and VL regions can be further subdivided into highly variable complementarity-determining regions (CDRs) and regions separated by more conserved regions called frame regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0128] The term "antigen-binding fragment" of an antibody ("parental antibody") includes a fragment or derivative of an antibody, typically comprising at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments well known in the art; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. In some preferred embodiments of the invention, the antigen-binding fragments of the invention are selected from Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments; and multispecific antibodies. When antigen-binding activity is expressed on a molar concentration basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative maintains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or higher of the antigen-binding affinity of the parent antibody. It is also anticipated that the antigen-binding fragment of the antibody may include conserved or non-conserved amino acid substitutions that do not significantly alter its biological activity (referred to as “conserved variants” or “functionally conserved variants” of the antibody). The term “binding compound” refers to both the antibody and its binding fragment.

[0129] The term "scFv" antibody refers to an antibody fragment containing both VH and VL domains, which are present within a single polypeptide chain. Fv polypeptides typically also include a polypeptide linker between the VH and VL domains, enabling the scFv to form the desired structure for antigen binding.

[0130] The term "multispecific antibody," used in its broadest sense, encompasses antibodies that exhibit multi-epitope specificity. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit exhibits multi-epitope specificity; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target; full-length antibodies, antibody fragments, bispecific antibodies, and triabodies, antibody fragments covalently or non-covalently linked, etc.

[0131] The term "anti-MUC1 antibody" or "MUC1-binding antibody" in this invention refers to an antibody that has the affinity to bind to the MUC1 protein or a fragment thereof, such that the antibody can be used to prepare diagnostic and / or therapeutic agents for MUC1.

[0132] The term "anti-EGFR antibody" or "EGFR-binding antibody" in this invention refers to an antibody that has the affinity to bind to the EGFR protein or fragments thereof such that the antibody can be used to prepare diagnostic and / or therapeutic agents targeting EGFR.

[0133] The term "knob-into-hole structure" refers to a mutation in the hydrophobic amino acid of the CH3 domain of the antibody Fc. One CH3 side chain amino acid is mutated to form a larger hydrophobic amino acid (knob) to enhance hydrophobic interaction; the other CH3 side chain amino acid is mutated to form a smaller hydrophobic amino acid (hole) to reduce steric barriers. The mutated CH3 with knob and with hole interacts hydrophobically to form a knob-into-hole structure (KiH). This promotes the formation of heavy chain heterodimers. The KiH mutation primarily involves hydrophobic amino acids within the CH3 domain; the exposed hydrophobic amino acids remain almost unchanged, therefore not affecting the effector function of the Fc or the resulting immunogenicity. The term "heavy chain constant region-knob" refers to the inclusion of a T366W point mutation in the antibody heavy chain constant region to form a knob-like spatial structure. Accordingly, "heavy chain constant region - hole" refers to the inclusion of point mutations T366S, L368A, and Y407V in the antibody heavy chain constant region to form a hole-like spatial structure. To weaken the binding to protein A, point mutations H435R and Y436F can also be introduced into the heavy chain constant region - hole (the numbering in this section is based on the EU numbering system, also known as the EU index, such as Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0134] In this application, the term "LALA" mutations (L234A and L235A, also known as "Ala-Ala" or "LALA", where the numbering is based on the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991) are used. These mutations reduce C1q and FcγR binding, resulting in reduced effector function.

[0135] The bispecific antibody of the present invention comprises two Fc regions, each of which is part of a separate antibody heavy chain. Fc1 and Fc2 may have the same sequence, except that they are either spontaneously or in combination with purified heterodimers (i.e., bispecific molecules) in the CH3 domain.

[0136] The Fc region of the bispecific antibody of the present invention can be a human Fc region. The Fc region of the bispecific antibody of the present invention can be of any isotype, including but not limited to IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc regions are all of the IgG1 isotype. In some embodiments, one of the Fc regions of the antibody is of the IgG1 isotype and the other is of the IgG4 isotype.

[0137] In some embodiments, the antibodies provided herein may be further modified to contain other protein-loving moieties known in the art and readily available. Suitable moieties for antibody activity include, but are not limited to, non-limiting examples of polymers such as polyethers, polyethylene glycols, polyvinyl alcohol, poly-1,3-fluorinated polyols, poly-1,3-diols, poly-1,2-linolenic acid, polyvinyl alcohol, polyamino acid polymers (e.g., glycerol), polyvinyl alcohol / ethylene vaporized copolymers, polyvinyl alcohol, and mixtures thereof.

[0138] The term "chimeric antibody" refers to an antibody having a variable domain of a first antibody and a constant domain of a second antibody, wherein the first and second antibodies originate from different species. Typically, the variable domain is derived from antibodies from rodents, etc. ("parental antibodies"), while the constant domain sequence is derived from human antibodies, making the resulting chimeric antibody less likely to induce an adverse immune response in human subjects compared to parental rodent antibodies. In some embodiments of the invention, the rodent is a mouse or rat. In some preferred embodiments of the invention, the chimeric antibody has an affinity for the antigen that is not less than or nearly equal to that of the parental mouse antibody.

[0139] The term "humanized antibody" refers to an antibody form containing sequences derived from human and non-human (e.g., mouse, rat) antibodies. Generally, humanized antibodies contain at least one, and usually two, variable domains, where all or almost all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or almost all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Humanized antibodies may optionally contain at least a portion of the constant regions of human immunoglobulins.

[0140] The term "fully human antibody" refers to an antibody that contains only the sequence of human immunoglobulin proteins. If produced in mice, in mouse cells, or in hybridomas derived from mouse cells, a fully human antibody may contain mouse glycans. Similarly, a "mouse antibody" refers to an antibody that contains only the sequence of mouse immunoglobulins. Alternatively, if produced in rats, in rat cells, or in hybridomas derived from rat cells, a fully human antibody may contain rat glycans. Likewise, a "rat antibody" refers to an antibody that contains only the sequence of rat immunoglobulins.

[0141] The term "Fc region" is used in this document to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is based on the EU numbering system, also known as the EU index, e.g., Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0142] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. Epitopes are typically composed of various chemically active surface molecules such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. The difference between conformational and non-conformational epitopes lies in the fact that, in the presence of denaturing solvents, the former loses binding, rather than the latter.

[0143] The term “cross-reactivity” as described herein refers to the binding of antigen fragments to the same target molecule in human, monkey, and / or mouse (mouse or rat) populations. Therefore, “cross-reactivity” should be understood as the interspecies reaction between an antigen-binding molecule (e.g., an antibody) and a similar molecule (e.g., EGFR) expressed in different species. The cross-reactivity specificity of monoclonal antibodies recognizing human EGFR, monkey, and / or mouse EGFR (mouse or rat) can be determined by FACS analysis.

[0144] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (KD), which is the ratio of the dissociation rate constant to the binding rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. In some embodiments of the invention, affinity is measured using surface plasmon resonance (SPR) technology, such as the affinity between the antibody and antigen of the present invention. In some preferred embodiments of the invention, a specific method for measuring affinity is the BIAcore method described herein.

[0145] The term "non-binding" protein or cell refers to a protein or cell that does not bind to itself, or does not bind to it with high affinity, meaning that the KD of the bound protein or cell is 1.0 × 10⁻⁶. -6 M or higher, more preferably 1.0 × 10 -5 M or higher, more preferably 1.0 × 10 - 4 M or higher, 1.0×10 -3 M or higher, more preferably 1.0 × 10 -2 M or higher.

[0146] For IgG antibodies, the term "high affinity" refers to a KD of 1.0 × 10⁻⁶ for the antigen. -6 M or lower, preferably 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. For example, "high affinity" binding for the IgM subtype refers to a KD of 10. -6 M or lower, preferably 10 -7 M or lower, preferably 10 -8 M or lower.

[0147] The terms “nucleic acid,” “polynucleotide,” “nucleic acid molecule,” and “polynucleotide molecule” are used interchangeably herein (unless the context otherwise indicates) and refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless explicitly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to a reference nucleic acid and are metabolized in a manner similar to that of naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, complementary sequences, and explicitly stated sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced by a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0148] "Construction" refers to any recombinant polynucleotide molecule (such as plasmids, granules, viruses, autonomously replicating polynucleotide molecules, bacteriophages, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecules) derived from any source, capable of integration into the genome or autonomous replication, containing one or more polynucleotide molecules that are functionally linked (i.e., operably linked). In some preferred embodiments of the invention, the recombinant construct comprises the polynucleotides of the invention operably linked to transcription initiation regulatory sequences that drive and / or direct transcription of the polynucleotides of the invention in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to drive and / or direct the expression of the polynucleotides of the invention.

[0149] "Vector" refers to any recombinant polynucleotide construct that can be used for transformation purposes (i.e., introducing heterologous DNA into host cells). One type of vector is the "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is the viral vector, into which an additional DNA segment can be ligated into the viral genome. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free-living mammalian vectors). After introduction into the host cell, other vectors (e.g., non-free-living mammalian vectors) integrate into the host cell's genome and thus replicate along with the host genome. Furthermore, some vectors can guide the expression of operatively linked genes. These vectors are referred to herein as "expression vectors."

[0150] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a target gene upon transformation, transfection, or transduction into host cells. An expression vector contains one or more phenotypic selection markers and origins of replication to ensure vector maintenance and to provide amplification within the host when needed. In some preferred embodiments of the invention, the expression vector comprises the constructs of the invention and / or the polynucleotides of the invention.

[0151] The term "EGFR and / or MUC1-mediated disease" refers to cancers involving cancer cells expressing EGFR and / or MUC1, such as skin cancer, ovarian cancer, breast cancer, and non-small cell lung cancer.

[0152] As used herein, the term “treatment” or “cure” for any disease or condition, in one embodiment, means improving the disease or condition (i.e., slowing or halting or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, “treatment” or “cure” means alleviating or improving at least one bodily parameter, including those physical parameters that may not be identifiable by the patient. In yet another embodiment, “treatment” or “cure” means regulating the disease or condition physically (e.g., stabilization of identifiable symptoms), physiologically (e.g., stabilization of bodily parameters), or in both ways. Unless explicitly described herein, methods for assessing the treatment and / or prevention of disease are generally known in the art.

[0153] "Subjects" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, including non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cyno-eating macaque" refers to or is derived from the cyno-eating macaque.

[0154] "Combined" administration of one or more other therapeutic agents includes simultaneous (co-) administration and consecutive administration in any order.

[0155] "Therapeutic effective amount," "therapeutic effective dose," and "effective amount" refer to the amount of the EGFR antibody or its antigen-binding fragment of the present invention, when administered alone or in combination with other therapeutic agents to cells, tissues, or subjects, that effectively prevents or improves the symptoms of one or more diseases or conditions, or the development of such diseases or conditions. Therapeutic effective dose also refers to the amount of antibody or its antigen-binding fragment sufficient to cause symptom improvement, such as the amount that treats, cures, prevents, or improves the associated medical condition, or increases the rate of treatment, cure, prevention, or improvement of such conditions. When administered to an individual as a single active ingredient, the therapeutic effective dose refers only to that ingredient. When administered in combination, the therapeutic effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether administered in combination, sequentially, or simultaneously. The effective amount of the therapeutic agent will result in an increase of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in diagnostic criteria or parameters.

[0156] "Pharmaceutically acceptable carriers" refer to components in a pharmaceutical preparation or composition that are non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0157] The precise amino acid sequence boundary of the variable region CDR of the antibody of the present invention can be determined using any of many well-known schemes, including those based on the three-dimensional structure of the antibody and the topology of the CDR loop, such as Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), and those based on antibody sequence variability, such as Kabat et al. (Sequences of Proteins of Immunological Interest, 4th edition, USDapartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), and the International ImMunoGeneTics database (IMGT) (1999 Nucleic Acids). Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0158] Unless otherwise stated, the CDR of the antibodies of the present invention can be determined by those skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0159] It should be noted that the boundaries of the CDRs of the variable region of the same antibody may differ based on different assignment systems. That is, the CDR sequences of the variable region of the same antibody defined under different assignment systems may differ. Therefore, when referring to antibodies defined with the specific CDR sequence of 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 the specific CDR boundaries defined by this invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0160] Antibodies with different specificities (i.e., different binding sites against different antigens) have different CDRs. However, although CDRs differ between antibodies, only a limited number of amino acid sites within a CDR are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, a minimal overlapping region can be determined, thus providing a “minimum binding unit” for antigen binding. The minimum binding unit can be a sub-part of a CDR. As will be apparent to those skilled in the art, the residues of the remaining portion of the CDR sequence can be determined by the antibody’s structure and protein folding. Therefore, the present invention also contemplates any variants of the CDRs given herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit may remain unchanged, while the remaining CDR residues as defined by Kabat or Chothia may be substituted with conserved amino acid residues.

[0161] In this invention, the three CDR regions of the heavy chain variable region VH1 are denoted as H1CDR1, H1CDR2, and H1CDR3; the three CDR regions of the heavy chain variable region VH2 are denoted as H2CDR1, H2CDR2, and H2CDR3; and the three CDR regions of the light chain variable region VL are denoted as LCDR1, LCDR2, and LCDR3. This invention involves mixing and pairing various chimeric heavy and light chain expression plasmids for transfection into expression cells, resulting in an anti-EGFR chimeric antibody.

[0162] The term "percentage (%) amino acid sequence identity," or simply "identity," is defined as the percentage of identical amino acid residues in a candidate amino acid sequence to a reference amino acid sequence after aligning the amino acid sequences (and, where necessary, introducing gaps) to obtain the maximum percentage sequence identity, without considering any conserved substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percentage amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm required to obtain the maximum alignment of the full length of the sequences being compared.

[0163] In some implementations, it may be necessary to produce cysteine-engineered antibodies, such as "thioMAb", in which one or more residues of the antibody are replaced with cysteine ​​residues.

[0164] In some embodiments, the antibodies provided herein may be further modified to contain other non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0165] This invention includes all combinations of the specific embodiments described. Further embodiments of the invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. For all purposes, all disclosures, patents, and patent applications cited herein, including in citations, are incorporated herein by reference in their entirety. The compounds of this invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other methods, and equivalent substitutions well known to those skilled in the art, with preferred embodiments including but not limited to the embodiments of this invention.

[0166] The reagents and raw materials used in this invention are all commercially available.

[0167] The positive and progressive effects of this invention are as follows: the bispecific antibody or its antigen-binding fragment provided by this invention has binding activity to MUC1 and EGFR and exhibits significant internalization ability, which is comparable to or better than that of the control molecule. Compared with monoclonal antibodies, it also exhibits better tumor selectivity. Attached Figure Description

[0168] Figures 1-3 show the binding activity of the parent antibody and antigen in the ELISA method. Figure 1 shows the binding activity of the MUC1 candidate antibody with BSA-modified STn-MUC1; Figure 2 shows the detection of the binding activity of the MUC1 candidate antibody with the MUC1 peptide; Figure 3 shows the binding activity of the EGFR candidate antibody with human, monkey, and mouse EGFR.

[0169] Figures 4 and 5 show the binding activity of the maternal antibody to cells as detected by FACS. Figure 4 shows the binding activity of the MUC1 candidate antibody to MCF7 cells; Figure 5 shows the binding activity of the EGFR candidate antibody to A432 cells.

[0170] Figures 6-8 show the internalization ability tests of the maternal antibody and bispecific antibody. Figure 6 shows the internalization ability test of the MUC1 candidate monoclonal antibody; Figure 7 shows the internalization ability test of the EGFR candidate monoclonal antibody; Figure 8 shows the internalization ability test of the bispecific antibody.

[0171] Figures 9 and 10 show the antigen-binding activity assays of the preferred MUC1 antibody B82 mutant. Figure 9 shows the binding activity of the B82 mutant with ST-MUC1; Figure 10 shows the binding activity of the B82 mutant with the MUC1 peptide.

[0172] Figure 11 shows the ELISA method for detecting the binding activity of affinity maturation antibodies to human EGFR antigen protein.

[0173] Figures 12-15 show the internalization ability of the preferred MUC1 and EGFR antibody mutants and their bispecific antibodies. Figure 12 shows the internalization ability of the B82 mutant in OVCAR3 and NCI-H1975 cells; Figure 13 shows the internalization ability of the B307 mutant in NCI-H1975 cells; Figures 14-15 show the internalization ability of the bispecific antibodies.

[0174] Figure 16 shows the tumor selectivity detection of candidate bispecific antibodies.

[0175] Figures 17 and 18 show the detection of the binding ability of bispecific antibodies to different tumor cells. Detailed Implementation

[0176] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. The detailed description below shows and describes only exemplary embodiments of this application. As those skilled in the art will recognize, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in this application are merely exemplary and not restrictive.

[0177] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0178] The control antibody Cetuximab used in this invention is a human-mouse chimeric monoclonal antibody obtained using conventional antibody expression and purification methods according to the amino acid sequence of Cetuximab (light chain as shown in SEQ ID NO:95, heavy chain as shown in SEQ ID NO:96), which is derived from US6217866B1.

[0179] The control antibody Gatipotuzumab used in this invention has a sequence derived from US8779102B2, with the light chain sequence being SEQ ID NO:97 and the heavy chain sequence being SEQ ID NO:98, and is produced using conventional recombinant antibody production processes.

[0180] The control antibody 1B2 used in this invention has a sequence derived from US20120040375A1, with the light chain sequence being SEQ ID NO:99 and the heavy chain sequence being SEQ ID NO:100. It was produced using conventional recombinant antibody production processes.

[0181] The control antibody hCetuximab used in this invention represents humanized Cetuximab. Its sequence is derived from WO2021247798A1, with the light chain sequence being SEQ ID NO:101 and the heavy chain sequence being SEQ ID NO:102. It is produced using conventional recombinant antibody production processes.

[0182] The control antibody M1231 used in this invention has a sequence derived from WO2021247798A1, with heavy chain 1 sequence as SEQ ID NO:109, heavy chain 2 sequence as SEQ ID NO:110, and light chain sequence as SEQ ID NO:111. It was produced using conventional recombinant antibody production processes.

[0183] Example 1: Antibody Construction and Expression

[0184] 1.1 Material Preparation

[0185] 1) References on the synthesis methods of peptides and glycosylated peptides used for MUC1 antibody screening. [1-3] The report states that the synthetic antigen contains a polypeptide derived from MUC1, with the sequence HGVTSAPDTRPAPGSTAPPA (SEQ ID NO:69).

[0186] The types of antigens synthesized are shown in Table 1.

[0187] Table 1

[0188] 2) EGFR antibody screening antigens GSV0->EGFR-P00533-25-645-chis and GSV0->EGFR-P00533-25-645-Fc were prepared and their activity was detected using conventional methods. Antigen detection antigens Mouse-EGFR-his and Rhesus ma caque EGFR-His were purchased from ACRO, catalog numbers EGR-M5224 and EGR-C52H1, respectively. The amino acid sequence of the human EGFR extracellular domain is amino acids 25-645 of P00533, used for the activity detection of the antibodies in this application.

[0189] P00533 (SEQ ID NO:112)

[0190] 1.2 Phage Display Library Screening

[0191] 1) Screening of anti-MUC1 antibody molecules using phage display library: Using conventional phage display library technology, and using the above-prepared biotinylated and non-biotinylated STn-MUC1, STn-MUC1-BSA, and STn-MUC1-KLH as antigens, the library was screened. Using the following screening method, a number of antibody molecules that specifically bind to human glycosylated MUC1 were obtained.

[0192] 1.2.1 Screening of antibody gene phage display libraries using magnetic bead method

[0193] Magnetic bead screening involves biotin-labeling antigen proteins and then binding them to magnetic beads conjugated with streptavidin. The process involves incubating, washing, and eluting the antigen-bound magnetic beads and a phage display library containing antibody genes. Typically, 3-4 rounds of screening are performed, resulting in a large enrichment of specific monoclonal antibodies against the antigen. In this embodiment, biotin-labeled antigen proteins were used for phage display library screening, with three rounds of screening. Each round used a 3-fold gradient of antigen concentration decreasing to obtain antibody clones with high affinity.

[0194] 1.2.2 Screening of antibody gene phage display libraries using the immunotube method

[0195] Immunotube screening involves coating the antigen proteins GSV0->EGFR-P00533-25-645-chis and GSV0->EGFR-P00533-25-645-Fc onto the surface of an immunotube with high adsorption capacity. A phage-displaying antibody library is added to the immunotube, and the protein adsorbed on the immunotube surface is incubated, washed, and eluted. After 2-4 rounds of screening, specific monoclonal antibodies against the antigen are finally enriched. In this example, three rounds of screening are performed, with each round using a 3-fold gradient of decreasing antigen concentration to obtain antibody clones with high affinity.

[0196] 2) Screening of anti-EGFR antibody molecules using phage display library: Using conventional phage display library technology, and using the antigen protein prepared in 1.1 Material Preparation 2) as the screening antigen, the library was screened. Using the screening methods in 1.2.1 and 1.2.2 above, a number of antibody molecules that specifically bind to human EGFR were obtained.

[0197] 1.3 Monoclonal Antibody Screening

[0198] The enrichment effect was evaluated by ELISA detection of the phage pools eluted in each round. Clones were randomly selected from the phage pools selected in each round for sequence analysis. The enrichment effect and the reproducibility ratio of the measured sequences were combined to select an appropriate round for single clone selection.

[0199] 1) MUC1 Monoclonal Antibody Screening: For initial ELISA monoclonal screening, the antigen proteins STn-MUC1, STn-MUC1-BSA, STn-MUC1-KLH, MUC1, and MUC1-BSA were used to obtain antibodies capable of binding to different forms of the antigen, and their sequences were determined. Based on antigen binding ability, MUC1-specific antibodies were screened. The amino acid sequences of the CDR regions of the obtained antibodies VH and VL are shown in Table 2. The CDR sequences were determined using the Kabat definition method.

[0200] The coding sequences for the Fc region of the heavy chain and the κ region of the light chain of the human clone were introduced into the pCDNA3.1 plasmid. The coding sequences for the variable regions of the heavy and light chains of the anti-human MUC1 antibody were then introduced into the pCDNA3.1 plasmid containing the already incorporated constant region coding sequences, and the correct clones were confirmed by sequencing. Various chimeric heavy and light chain expression plasmids were mixed and paired for transfection into Expi CHO expression cells to obtain the fully human anti-human MUC1 antibodies A11, B82, and B70.

[0201] The full-length light chain sequences of the fully human anti-human MUC1 antibodies A11, B82, and B70 are shown in SEQ ID NO:5, and the full-length heavy chain sequences are shown in Table 2.

[0202] 2) EGFR monoclonal antibody screening: Initial ELISA screening used the antigen protein GSV0 -> EGFR-P00533-25-645-chis. The obtained clones were further verified using ELISA to confirm binding to Mouse-EGFR-his and Rhesus macaque EGFR-His, and EGFR-specific antibodies were screened. The amino acid sequences of the CDR regions of the obtained antibodies VH and VL are shown in Table 2. The CDR sequence was determined using the Kabat definition method.

[0203] The coding sequences for the Fc region of the heavy chain and the κ region of the light chain of the human clone were introduced into the pCDNA3.1 plasmid. The coding sequences for the variable regions of the heavy and light chains of the aforementioned anti-human EGFR antibodies were synthesized. The coding sequences for the variable regions of the heavy and light chains of various anti-human EGFR antibodies were introduced into the pCDNA3.1 plasmid containing the already introduced constant region coding sequences, and the correct clones were confirmed by sequencing. Various chimeric heavy and light chain expression plasmids were mixed and paired for transfection into Expi CHO expression cells to obtain the fully human anti-human EGFR antibodies B307 and A73. The constant region of the B307 antibody was mutated using L234A and L235A mutations (according to the Kabat definition) to obtain the B307-FC mutant antibody.

[0204] The full-length light chain sequences of the B307, A73, and B307-FC mutant anti-human EGFR fully human antibody are shown in SEQ ID NO:5, and the full-length heavy chain sequences are shown in Table 2.

[0205] Table 2: CDR and variable region amino acid sequences of anti-MUC1 antibodies (KABAT protocol)

[0206] Example 2: Detection of binding activity between maternal monoclonal antibody and antigen

[0207] The binding activity of MUC1 antibody and EGFR antibody to the antigen was detected by ELISA, as follows.

[0208] To detect the binding activity of MUC1 antibody to antigen: Dilute the antigen (STn-MUC1, STn-MUC1-BSA, MUC1) with 1×PBS to a concentration of 4 μg / mL, and add 30 μL / well to a 96-well ELISA plate. Coat overnight at 4°C. Wash the plate three times with PBST, add blocking buffer (5% PBSM), and block at room temperature for 2 h. After washing the plate three times with PBST, add 30 μL / well of sample diluted with 1% PBSM and incubate at room temperature for 60 min. Wash the plate three times with PBST, add secondary antibody (Anti-human-IgG-Fc-HRP (purchased from: abcam; catalog number: ab97225)), and incubate at room temperature for 60 min. Wash the plate three times with PBST, and add 30 μL TMB to each well. Stop the reaction by adding 2M stop solution and simultaneously detect OD450.

[0209] To detect the binding activity of EGFR antibodies to antigens: Antigens (GSV0->EGFR-P00533-25-645-chis, Rhesus macaque EGFR-His, Mouse-EGFR-his) were diluted with 1×PBS, serially diluted from 1 μg / mL, and added to 96-well ELISA plates at 30 μL / well. The plates were incubated overnight at 4°C. After washing three times with PBST, blocking buffer (5% PBSM) was added and the plates were blocked at room temperature for 2 h. After washing three times with PBST, 1% PBSM was added to dilute the sample to 1 μg / mL at 30 μL / well, and the plates were incubated at room temperature for 60 min. After washing three times with PBST, secondary antibody (Anti-human-IgG-Fc-HRP (purchased from abcam; catalog number: ab97225)) was added and the plates were incubated at room temperature for 60 min. After washing three times with PBST, 30 μL of TMB was added to each well. The reaction was stopped by adding 2M stop solution, and OD450 was measured simultaneously.

[0210] The results are shown in Figures 1-3 and Tables 3-4. The MUC1 antibody used in this invention can bind to the tumor-specific glycosylated modified antigen STn-MUC1-BSA (Figure 1), but has a weaker binding ability to non-glycosylated MUC1 (Figure 2). The EGFR antibody used in this invention has cross-binding ability with human, monkey and mouse EGFR (Figure 3).

[0211] Table 3: ELISA binding activity of the parental monoclonal antibody to MUC1

[0212] Table 4: ELISA binding activity of parental monoclonal antibody to EGFR

[0213] Example 3: Detection of the binding ability of the maternal monoclonal antibody to tumor cells

[0214] The binding activity of MUC1 antibody and EGFR antibody to MCF7 and A431 cells was detected by FACS method, as follows.

[0215] Prepare a cell suspension of MCF7 or A431 cells and adjust the density to 1×10⁻⁶. 6 Cells / mL. Take a 96-well round-bottom plate and add 100 μL of cell suspension to each well using a 100 μL pipette. Centrifuge at 300 g / min for 5 min. Discard the supernatant. Dilute the antibody with FACS Buffer to prepare eight concentration gradients: 20.00000, 5.00000, 1.25000, 0.31250, 0.07813, 0.01953, 0.00488, and 0.00122 μg / mL. Add 100 μL of antibody dilution to each well using a 100 μL pipette and incubate at 4°C for 60 min. Wash the plate twice with FACS Buffer. Secondary antibody PE labelled anti-human IgG Fc (purchased from Abcam; catalog number: 98596) was diluted 1:300 with FACS Buffer and added to each well at 100 μL. The plates were incubated at 4°C for 30 min. The plates were washed twice with FACS buffer. Flow cytometry was used for analysis.

[0216] The results are shown in Figures 4-5 and Tables 5-6. The MUC1 antibody and EGFR antibody used in this invention both have cell binding ability.

[0217] Table 5: Binding of MUC1 antibody to MCF7 cells

[0218] Table 6: Binding of EGFR antibody to A431 cells

[0219] Example 4: Construction of a bispecific molecule

[0220] The bispecific antibody prepared in this embodiment contains two heavy chains and two light chains. The first heavy chain and the first light chain form an EGFR binding domain, and the second heavy chain and the second light chain form a MUC1 binding domain. The variable region sequences of the first heavy chain and the first light chain are derived from antibody A73 and B307, and the variable region sequences of the second heavy chain and the second light chain are derived from antibody A11, B82, and B70. Based on the amino acid sequence of the antibody variable region, primers were designed and PCR was constructed to obtain the VH / VK gene fragment, and the variable region was obtained. The constant regions of the heavy chain and the light chain were selected from the human clone heavy chain constant region and the light chain constant region κ, respectively. Furthermore, a T366S / L368A / Y407V mutation (hole) and an H435R / Y436F mutation are introduced into the heavy chain constant region of the antibody EGFR binding domain, and a T366W mutation (knob) is introduced into the heavy chain constant region of the antibody MUC1 binding domain. Alternatively, a T366S / L368A / Y407V mutation (hole) and an H435R / Y436F mutation are introduced into the heavy chain constant region of the antibody MUC1 binding domain, and a T366W mutation (knob) is introduced into the heavy chain constant region of the antibody EGFR binding domain. For example, in this embodiment, the heavy chain constant region of the antibody EGFR binding domain is selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO:76, the heavy chain constant region of the antibody MUC1 binding domain is selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO:77, and the light chain constant region is selected from the human light chain constant region as shown in SEQ ID NO:78. Gene fragments from the variable and constant regions were combined to construct complete bispecific antibody sequences. After transfection into CHO cells, the bispecific antibodies A11A73, B82A73, B70B307, and B82B307 were obtained using standard expression and purification methods. The sequences are shown below:

[0221] Heavy chain constant region - hole (with H435R / Y436F mutation, SEQ ID NO:76)

[0222] Heavy chain constant region - knob (SEQ ID NO:77)

[0223] CL (SEQ ID NO:78)

[0224] The first and second light chain sequences of A11A73, B82A73, B70B307 and B82B307 are as follows:

[0225] Light chain variable region-1, light chain variable region-2: (SEQ ID NO:4)

[0226] Light chain-1, light chain-2: (SEQ ID NO:5)

[0227] The first and second heavy chain sequences of A11A73, B82A73, B70B307 and B82B307 are as follows:

[0228] A11A73 Heavy Chain Variable Region-1: (SEQ ID NO:35)

[0229] A11A73 Heavy Chain Variable Region-2:(SEQ ID NO:34)

[0230] B82A73 Heavy Chain Variable Region-1 (SEQ ID NO:35)

[0231] B82A73 Heavy Chain Variable Region-2 (SEQ ID NO:36)

[0232] B70B307 Heavy Chain Variable Region-1 (SEQ ID NO:31)

[0233] B70B307 Heavy Chain Variable Region-2 (SEQ ID NO:37)

[0234] B82B307 Heavy Chain Variable Region-1 (SEQ ID NO:31)

[0235] B82B307 Heavy Chain Variable Region-2 (SEQ ID NO:36)

[0236] A11A73 Heavy Chain-1 (SEQ ID NO:48)

[0237] A11A73 Heavy Chain-2 (SEQ ID NO:47)

[0238] B82A73 Heavy Chain-1 (SEQ ID NO:48)

[0239] B82A73 Heavy Chain-2 (SEQ ID NO:49)

[0240]

[0241] B70B307 Heavy Chain-1 (SEQ ID NO:51)

[0242] B70B307 Heavy Chain-2 (SEQ ID NO:50)

[0243] B82B307 Heavy Chain-1 (SEQ ID NO:51)

[0244] B82B307 Heavy Chain-2 (SEQ ID NO:49) Note: The bold underlined portion in the above sequence represents the CDR region (KABAT scheme).

[0245] Example 5: Detection of the internalization ability of maternal monoclonal antibody and bispecific antibody

[0246] The internalization ability of MUC1 monoclonal antibody in NCI-H1975 cells was detected by flow cytometry as follows: Sufficient 4X working concentration of MUC1 antibody was prepared in cell culture medium, and 25 μL of the 4X working concentration antibody solution was added to a 96-well plate. 25 μL of 4X working concentration Zenon pHrodo iFL IgG labeling reagent was added to the 96-well plate containing the antibody, and incubated at room temperature for 5 min to allow the labeling complex to form. The molar ratio of antibody to labeling reagent was 1:3. Cells of appropriate density were added to the 96-well plate, and after attachment, the culture medium volume per well was adjusted to 50 μL, and 50 μL of labeled antibody was added. The cells were incubated under standard cell culture conditions (37℃) for 48 h, and then detected by flow cytometry.

[0247] The internalization capacity of EGFR monoclonal and bispecific antibodies was detected using the Incucyte method, as follows: Sufficient 4X working concentration antibody was prepared in cell culture medium, and 25 μL of the 4X working concentration antibody solution was added to a 96-well plate. 25 μL of 4X working concentration Zenon pHrodo iFL IgG labeling reagent was added to the 96-well plate containing the antibody, and incubated at room temperature for 5 min to allow the labeling complex to form. The molar ratio of antibody to labeling reagent was 1:3. Cells of appropriate density were added to the 96-well plate, and after attachment, the culture medium volume per well was adjusted to 50 μL, and 50 μL of labeled antibody was added. The plate was incubated under standard cell culture conditions (37℃) for 48 h, and the results were analyzed using flow cytometry.

[0248] The results are shown in Figures 6-8. MUC1 antibodies A11, B82, and B70, along with EGFR antibodies A73 and B307, were all internalized into tumor cells (Figures 6 and 7). The bispecific antibody molecules A11A73, B82A73, B70B307, and B82B307 constructed using the shown monoclonal antibodies all exhibited significant internalization ability in MDA-MB-468, OVCAR-3, and NCI-H1975 cells, and their internalization ability was comparable to or better than that of the control molecules (Figure 8).

[0249] Example 6: Modification of MUC monoclonal antibodies and EGFR monoclonal antibodies

[0250] MUC1 monoclonal antibody B82 and EGFR antibody B307 were selected as preferred clones, and antibody engineering was performed using phage display technology to obtain candidate molecules with higher activity.

[0251] Mutation primers were designed according to the library construction strategy. Using the correctly sequenced maternal antibody plasmid as a template, single- or double-point mutations were introduced into the target gene using molecular cloning technology, and the target fragment was amplified. The antibody gene was then recombined using in vitro ligation. After obtaining the antibody gene, the vector and gene sequence were digested with enzymes, and the antibody nucleotide sequence was constructed into a phage display vector. The constructed vector was then electroporated into *E. coli* to obtain a phage display library. The phage display library was screened using the corresponding antigen. The phage display mutant library or the obtained output set was processed through a series of steps, including inoculation, phage infection assistance, phage amplification, phage precipitation and resuspension, to prepare enriched phages for the next round of screening. After 3-4 rounds of screening, specific monoclonal antibodies against the antigen were enriched. ELISA screening and sequencing analysis were used to screen for the following specific antibodies. The amino acid sequences of the CDR regions of the obtained antibodies VH and VL are shown in Table 7. The CDR sequence was determined using the Kabat definition method.

[0252] Table 7: CDR and variable region amino acid sequences of affinity maturation candidate molecules (KABAT scheme)

[0253] The affinity maturation candidate antibody was expressed, purified, and its quality was tested in accordance with the method described in Example 1.

[0254] The full-length light chain sequence of the fully human anti-EGFR antibody or the fully human anti-MUC1 antibody is shown in SEQ ID NO:5, and the full-length heavy chain sequence is shown in Table 8.

[0255] Table 8: Full-length amino acid sequence of the heavy chain of anti-EGFR antibody

[0256] Example 7: Detection of Antigen Binding Activity of Monoclonal Antibody Mutant

[0257] The binding activity of the anti-human MUC1 monoclonal antibody mutant to the antigen was detected by referring to the method in Example 2. The experimental results are shown in Figures 9-10 and Table 9. The mature molecule with high affinity showed significant binding activity to ST-MUC1, and the binding ability was higher than that of the parent antibody B82. Moreover, the binding ability with non-glycosylated MUC1 remained at a low level.

[0258] Table 9: Antigen-binding activity of the preferred MUC1 antibody B82 mutant

[0259] The binding activity of anti-human EGFR monoclonal antibody mutants to antigens was detected by referring to the method in Example 2. The experimental results are shown in Figure 11 and Table 10. All the mature molecules with affinity showed significant binding activity to the antigen. Among them, B307mut3, B307mut1 and B307mut2 showed better binding activity to the antigen than B307.

[0260] Table 10: Binding activity of affinity-matured antibodies to human EGFR antigen protein

[0261] Example 8: Construction of bispecific molecules

[0262] The bispecific antibody prepared in this embodiment contains two heavy chains and two light chains. The first heavy chain and the first light chain form an EGFR binding domain, and the second heavy chain and the second light chain form a MUC1 binding domain. The variable region sequences of the first heavy chain and the first light chain are derived from antibody B307, B307mut2, or B307mut3, and the variable region sequences of the second heavy chain and the second light chain are derived from antibody B82mut1, B82mut2, B82mut3, or B82mut4. Based on the amino acid sequence of the antibody variable region, primers were designed and PCR was constructed to obtain the VH / VK gene fragment and obtain the variable region. The constant regions of the heavy chain and the light chain were selected from the human clone heavy chain constant region and the light chain constant region κ, respectively. Furthermore, T366S / L368A / Y407V (hole) and L234A / L235A mutations are introduced into the heavy chain constant region of the antibody EGFR binding domain, and T366W (knob) and L234A / L235A mutations are introduced into the heavy chain constant region of the antibody MUC1 binding domain; alternatively, T366S / L368A / Y407V (hole) and L234A / L235A mutations are introduced into the heavy chain constant region of the antibody MUC1 binding domain. Mutations were introduced into the heavy chain constant region of the antibody EGFR binding domain, specifically the T366W (knob) and L234A / L235A mutations. For example, in this embodiment, the variable region sequence of the antibody EGFR binding domain (first heavy chain and first light chain) is derived from antibody B307 or B307mut3, the variable region sequence of the antibody MUC1 binding domain (second heavy chain and second light chain) is derived from antibody B82mut4, the heavy chain constant region of the antibody EGFR binding domain is selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO:85, the heavy chain constant region of the antibody MUC1 binding domain is selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO:86, and the light chain constant region is selected from the human light chain constant region as shown in SEQ ID NO:78. The variable and constant region gene fragments were combined to construct a complete bispecific antibody sequence. After transfection into CHO cells, the bispecific antibodies DB-142120 and DB-142123 were obtained using conventional expression and purification methods. The amino acid sequences of the first and second light chain variable regions of DB-142120 and DB-142123 are both shown in SEQ ID NO:4, and the amino acid sequences of the first and second light chains of DB-142120 and DB-142123 are both shown in SEQ ID NO:5. The heavy chain amino acid sequences of DB-142120 and DB-142123 are as follows:

[0263] DB-142120 Heavy Chain Variable Region-1 (SEQ ID NO:31)

[0264] DB-142120 Heavy Chain Variable Region-2 (SEQ ID NO:30)

[0265] DB-142123 Heavy Chain Variable Region-1 (SEQ ID NO:32)

[0266] DB-142123 Heavy Chain Variable Region-2 (SEQ ID NO:30)

[0267] DB-142120 Heavy Chain-1 (SEQ ID NO:42)

[0268] DB-142120 Heavy Chain-2 (SEQ ID NO:41)

[0269] DB-142123 Heavy Chain-1 (SEQ ID NO:43)

[0270] DB-142123 Heavy Chain-2 (SEQ ID NO:41)

[0271] Example 9: Antibody mutants and their bispecific antibody internalization ability detection

[0272] The internalization capacity of bispecific antibodies composed of MUC1, EGFR antibody mutants and MUC mutants in cells was detected using the method described in Example 5.

[0273] The results are shown in Figures 12-15. Candidate antibodies obtained through screening for MUC1 antibody B82 mutations all showed significant internalization signals in OVCAR-3 and NCI-H1975 cells, and compared to the wild-type B82 molecule, the mutants showed varying degrees of enhanced internalization ability (Figure 12). The EGFR antibody B307 mutant could be internalized into NCI-H1975 cells, and its internalization activity was superior to the control antibody Cetuximab (Figure 13). The bispecific antibody molecule DB-142120 showed significant internalization ability in MDA-MB-468, OVCAR-3, and NCI-H1975 cells, and its internalization ability was superior to the control molecule (Figure 14). The bispecific antibody molecule DB-142123 showed significant internalization ability in both OVCAR-3 and NCI-H1975 cells, and its internalization ability was superior to the control molecule (Figure 15).

[0274] Example 10: Detection of the tumor-selective binding ability of bispecific antibodies

[0275] HCC1143 cells (human breast cancer cells, triple negative) were digested, centrifuged, and resuspended in PBS. The cell density was adjusted to 1E6 cells / mL. 0.5 μM CFSE was added, and the cells were incubated at room temperature in the dark for 20 min for staining. Alternatively, HCC1143 cells were digested, centrifuged, and resuspended in PBS. The cell density was adjusted to 15E6 cells / mL. 40 μL of MCF10A was added to the CFSE-stained HCC1143 cells, achieving a cell ratio of 1:30 for both HCC1143 and MCF10A. A 100 nM antibody solution was prepared using FACS buffer (PBS + 2% FBS). 10 μL of the prepared solution was added to each well to a final concentration of 10 nM, and the cells were incubated at 4°C for 40 min. After antibody incubation, wash once with 100 μL PBS; dilute anti-human Fc PE with FACS buffer (PBS + 2% FBS) according to the fluorescent secondary antibody instructions, add 100 μL to each well of a U-plate, and incubate at 4°C in the dark for 30 minutes. Wash once with 100 μL of PBS solution containing 2% FBS, resuspend in 120 μL of PBS solution containing 2% FBS, and then detect the mean fluorescence intensity on HCC1143 and MCF10A cells by flow cytometry. The ratio of the mean fluorescence intensity on HCC1143 and MCF10A cells is used as the ordinate to compare the selective binding ability of different samples on tumor cells and normal cells.

[0276] As shown in Figure 16, the bispecific antibody DB-142120 exhibited superior tumor-selective binding ability compared to the parental EGFR monoclonal antibody B307.

[0277] Example 11: Affinity detection of bispecific antibody with recombinant human EGFR protein

[0278] Measure 100 mL of 10×HBS-EP+ buffer (purchased from Cytiva, catalog number: BR100826) and 900 mL of deionized water, and mix them into a 1 L buffer bottle. Click the chip switch button on the main interface of the Biacore 8K (purchased from Cytiva, catalog number: 2760952)+ control software to replace the maintenance chip with the Biacore Sensor Chip Protein A (purchased from Cytiva, catalog number: 29127556) chip. Insert the inlet tube of buffer A into the bottom of the buffer bottle, and insert the inlet tube labeled with water and reagents into the bottom of the pure water bottle. Click the solvent conversion button on the main interface of the control software to change the running buffer. Dilute the test antibody to the appropriate concentration using the running buffer. The running buffer is 1×HBS-EP+ buffer, pH 7.4, concentration 1 μg / mL, and injection time 80 s (TBD). Inject the test antibody (Fc2) at a flow rate of 10 μL / min until the capture level reaches the predetermined level. The test antibody was conjugated to the chip under the following conditions: running buffer (1×HBS-EP+buffer, pH 7.4), concentration (1 μg / mL), and injection time (80 s TBD). Human EGFR recombinant protein was diluted to nine concentrations (300, 150, 75, 37.5, 18.75, 9.38, 4.69, 2.34, 1.17, and 0 nM; or adjusted according to optimization conditions) using running buffer. Affinity was measured by injecting the analyte human EGFR recombinant protein at a flow rate of 30 μL / min under the following conditions: running buffer (1×HBS-EP+buffer, pH 7.4), binding time (180 s), dissociation time (300 s), and temperature (25 °C)). Regeneration buffer (10 mM Glycine-HCl (Titan, catalog number: 013536570), pH 1.5) was injected at a flow rate of 30 μL / min for 30 seconds to remove the ligand-analyte complex from the chip surface.

[0279] The experimental results were analyzed using Biacore Insight Evaluation Software. The results are shown in Table 11, indicating that the bispecific antibody DB-142123 exhibits significant binding activity to recombinant human EGFR protein.

[0280] Table 11: Binding activity of bispecific antibodies to recombinant human EGFR protein

[0281] Example 12: Detection of the binding ability of bispecific antibodies to different tumor cells

[0282] The binding affinity of antibodies to MDA-MB-468 (breast cancer cells, source: ATCC), OVCAR-3 (ovarian cancer cells, source: ATCC), and NCI-H1975 (lung cancer cells, source: ATCC) cells was detected by flow cytometry. Cell culture methods were as follows: MDA-MB-468 cells were cultured with L-15 (Gibco, catalog number: 11415-064) / RPMI 1640 (Gibco, catalog number: 22400-089) + 10% FBS (DL Biotech, catalog number: FBSV500); OVCAR-3 cells were cultured with RPMI 1640 + 0.01 mg / ml insulin (YEASEN, catalog number: 40112ES60) + 20% FBS; and NCI-H1975 cells were cultured with RPMI 1640 + 10% FBS. Cells were treated with trypsin and then counted, ensuring cell viability was above 90.0%. Press 2×10 into the 96-hole V-plate (purchased from Axygen, part number: WIPP02280). 5 Add 3 cell lines to each well. Wash cells twice with FACS staining buffer, centrifuge at 450g for 5 minutes, and discard the supernatant. Then add 100 μL of the test substance or Isotype (starting at 300 nM, 3-fold dilution, 8 concentration gradients) to the corresponding well and incubate at 4°C for 60 minutes. Wash cells twice with FACS staining buffer, centrifuge at 450g for 5 minutes, and discard the supernatant. Add PE anti-human IgG Fc Antibody (1:200 dilution) to each well and incubate at 4°C for 40 minutes in the dark. After incubation, wash cells twice with FACS staining buffer, centrifuge at 450g for 5 minutes, and discard the supernatant. Resuspend cells in 200 μL of FACS staining buffer before FACS detection.

[0283] FlowJo was used to analyze the data, and GraphPad was used for statistical analysis.

[0284] The results are shown in Figures 17-18 and Table 12. The bispecific antibody DB-142123 showed significant binding activity to both NCI-H1975 and MDA-MB-468 cells.

[0285] Table 12: Binding activity of bispecific antibodies to different tumor cells

[0286] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents.

[0287] References:

[0288] (1) Ohyabu, N.; Hinou, H.; Matsushita, T.; Izumi, R.; Shimizu, H.; Kawamoto, K.; Numata, Y.; Togame, H.; Takemoto, H.; Kondo, H.; Nishimura, S.-I. An essential epitope of anti-MUC1 monoclonal antibody KL-6 revealed by focused glycopeptide library. J. Am. Chem. Soc. 2009, 131, 17102-17109.

[0289] (2) Fumoto, M.; Hinou, H.; Ohta, T.; Ito, T.; Yamada, K.; Takimoto, A.; Kondo, H.; Shimizu, H.; Inazu, T.; Nakahara, Y.; Nishimura, S.-I. Combinatorial synthesis of MUC1 glycopeptides: polymer blotting facilitates chemical and enzymatic synthesis of highly complicated mucin glycopeptides. J. Am. Chem. Soc. 2005, 127, 11804-11818.

[0290] (3) Dalziel, M.; Whitehouse, C.; McFarlane, I.; Brockhausen, I.; Gschmeissner, S.; Schwientek, T.; Clausen, H.; Burchell, J.M.; TaylorPapadimitriou, J. The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1. J. Biol. Chem. 2001, 276, 11007-11015.

[0291] Sequence Listing

[0292] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An antibody against EGFR / MUC1 or its antigen-binding fragment, characterized in that, The antibody or its antigen-binding fragment comprises an EGFR-binding domain and a MUC1-binding domain; the EGFR-binding domain includes a heavy chain variable region VH1 and a light chain variable region VL, and the MUC1-binding domain includes a heavy chain variable region VH2 and a light chain variable region VL; wherein... The VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:103, SEQ ID NO:104, and SEQ ID NO:105, respectively; or, the VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively. The VL contains the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:106, SEQ ID NO:107, and SEQ ID NO:108, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively. Preferably, the VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; or, the VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:11, respectively; or, the VH1 comprises the amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:29, respectively. And / or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:21, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:25, and SEQ ID NO:26, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:27, SEQ ID NO:20, and SEQ ID NO:26, respectively; or, the VH2 comprises the amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:28, SEQ ID NO:20, and SEQ ID NO:21, respectively. H2CDR1, H2CDR2, and H2CDR3 are shown in NO:

26.

3. The antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The antibody or its antigen-binding fragment is selected from the following: (1) The VL contains the amino acid sequences LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively; (2) VH1 and VH2 are selected from any one of the following: The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively. The VH1 contains the amino acid sequences H1CDR1, H1CDR2 and H1CDR3 as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:11, respectively; and the VH2 contains the amino acid sequences H2CDR1, H2CDR2 and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively. The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, respectively. The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:21, respectively. The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively. The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:20, and SEQ ID NO:21, respectively. The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:25, and SEQ ID NO:26, respectively. The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:27, SEQ ID NO:20, and SEQ ID NO:26, respectively. The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:28, SEQ ID NO:20, and SEQ ID NO:26, respectively. The VH1 contains the amino acid sequences H1CDR1, H1CDR2 and H1CDR3 as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:29, respectively; and the VH2 contains the amino acid sequences H2CDR1, H2CDR2 and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively. The VH1 contains amino acid sequences H1CDR1, H1CDR2, and H1CDR3 as shown in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:11, respectively, and the VH2 contains amino acid sequences H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

4. The antibody or antigen-binding fragment thereof as described in any one of claims 1-3, characterized in that, The antibody or its antigen-binding fragment is selected from the following: (1) The VH1 comprises an amino acid sequence as shown in any one of SEQ ID NO:31-33 or 35; and / or, the VH2 comprises an amino acid sequence as shown in any one of SEQ ID NO:30, 34 or 36-40; and / or, the VL comprises an amino acid sequence as shown in SEQ ID NO:4; or, (2) The VH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:31-33 or 35; and / or, the VH2 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:30, 34, or 36-40; and / or, the VL comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:31-33 or 35 .... The amino acid sequence shown in NO:4 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity; preferably, the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity does not involve any alteration to the CDR sequence.

5. The antibody or antigen-binding fragment thereof as described in any one of claims 1-4, characterized in that, The antibody or its antigen-binding fragment is selected from the following: VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:30; VL contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:32; VH2 contains the amino acid sequence shown in SEQ ID NO:30; VL contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:35; VH2 contains the amino acid sequence shown in SEQ ID NO:34; VL contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:35; VH2 contains the amino acid sequence shown in SEQ ID NO:36; VL contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:37; VL contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:36; VL contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:38; VL contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:39; VL contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:31; VH2 contains the amino acid sequence shown in SEQ ID NO:40; VL contains the amino acid sequence shown in SEQ ID NO:4; VH1 contains the amino acid sequence shown in SEQ ID NO:33; VH2 contains the amino acid sequence shown in SEQ ID NO:30; VL contains the amino acid sequence shown in SEQ ID NO:4; The VH1 contains the amino acid sequence shown in SEQ ID NO:32; the VH2 contains the amino acid sequence shown in SEQ ID NO:30; and the VL contains the amino acid sequence shown in SEQ ID NO:

4.

6. The antibody or antigen-binding fragment thereof as described in any one of claims 1-5, characterized in that, The antibody or its antigen-binding fragment includes one or more of the following: (1) Fully human antibodies, humanized antibodies, chimeric antibodies, proantibodies, bispecific antibodies or multispecific antibodies; (2) Fab, Fab', F(ab')2, Fv or scFv; (3) The MUC1 binding domain is a human anti-MUC1 antibody or its antigen-binding fragment; and, (4) The EGFR binding domain is a human anti-EGFR antibody or its antigen-binding fragment.

7. The antibody or antigen-binding fragment thereof as described in any one of claims 1-6, characterized in that, The EGFR binding domain and the MUC1 binding domain further include a light chain constant region and a heavy chain constant region, respectively. The EGFR binding domain includes a light chain constant region CL and a heavy chain constant region CH1, and the MUC1 binding domain includes a light chain constant region CL and a heavy chain constant region CH2. Preferably, the heavy chain constant region is the heavy chain constant region of human antibody IgG1; and / or, the light chain constant region is the light chain constant region of human antibody κ chain.

8. The antibody or antigen-binding fragment thereof as described in any one of claims 1-7, characterized in that, The heavy chain constant regions of the EGFR binding domain and the MUC1 binding domain contain Knob-into-hole mutations; the heavy chain constant regions CH1 and CH2 contain different Knob or Hole mutations. Preferably, CH1 contains a Knob mutation, such as T366W, and CH2 contains a Hole mutation, such as one or more selected from T366S, L368A, and Y407V; or, CH2 contains a Knob mutation, such as T366W, and CH1 contains a Hole mutation, such as one or more selected from T366S, L368A, and Y407V. More preferably, CH1 and CH2 contain the L234A / L235A mutation; And / or, when CH1 contains the Hole mutation, CH1 also contains the H435R / Y436F mutation, or, when CH2 contains the Hole mutation, CH2 also contains the H435R / Y436F mutation.

9. The antibody or antigen-binding fragment thereof as described in any one of claims 1-8, characterized in that, The antibody or its antigen-binding fragment is selected from any of the following: (I) The heavy chain of the EGFR-binding domain comprises an amino acid sequence as shown in any one of SEQ ID NO:42, 43, 45, 46, 48, 51, 56, 57, 59, 62, 67, and 68, and the light chain of the EGFR-binding domain comprises an amino acid sequence as shown in SEQ ID NO:5; and / or, the heavy chain of the MUC1-binding domain comprises an amino acid sequence as shown in any one of SEQ ID NO:41, 44, 47, 49, 50, 52, 53, 54, 55, 58, 60, 61, 63, 64, 65, and 66, and the light chain of the MUC1-binding domain comprises an amino acid sequence as shown in SEQ ID NO:5; or, (II) The heavy chain of the EGFR-binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:42, 43, 45, 46, 48, 51, 56, 57, 59, 62, 67, and 68; and the light chain of the EGFR-binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5; and / or, the heavy chain of the MUC1-binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5; and / or, the light chain of the EGFR-binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5; The amino acid sequences shown in any one of NO:41, 44, 47, 49, 50, 52, 53, 54, 55, 58, 60, 61, 63, 64, 65, and 66 have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, and the light chain of the MUC1 binding domain comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5; preferably, the amino acid sequences having at least 95%, 96%, 97%, 98%, or 99% sequence identity do not involve changes to the CDR sequence; Preferably, the antibody or its antigen-binding fragment is selected from the following: The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:42, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:41, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:43, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:41, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:48, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:47, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:48, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:49, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:50, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:49, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:52, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:53, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:54, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:51, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:55, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:56, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:55, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:57, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:55, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:45, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:44, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:46, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:44, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:59, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:58, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:59, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:60, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:61, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:60, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:63, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:64, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:65, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:62, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:66, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR-binding domain is shown in SEQ ID NO:67, the amino acid sequence of the light chain of the EGFR-binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1-binding domain is shown in SEQ ID NO:66, and the amino acid sequence of the light chain of the MUC1-binding domain is shown in SEQ ID NO:5; or, The amino acid sequence of the heavy chain of the EGFR binding domain is shown in SEQ ID NO:68, the amino acid sequence of the light chain of the EGFR binding domain is shown in SEQ ID NO:5, the amino acid sequence of the heavy chain of the MUC1 binding domain is shown in SEQ ID NO:66, and the amino acid sequence of the light chain of the MUC1 binding domain is shown in SEQ ID NO:

5.

10. An isolated nucleic acid, characterized in that, The nucleic acid encodes an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9.

11. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 10; Preferably, the recombinant expression vector is selected from: viral vectors and non-viral vectors; More preferably, the non-viral vector is selected from: plasmids, linear DNA fragments, and RNA; More preferably, the recombinant expression vector is a plasmid, and the backbone plasmid of the recombinant expression vector is preferably pCDNA3.

1.

12. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 10 or the recombinant expression vector as described in claim 11, wherein the transformant is a non-animal or non-plant variety; preferably, the transformant is a eukaryotic cell; more preferably, the eukaryotic cell is a mammalian cell.

13. A method for preparing an antibody against EGFR / MUC1 or an antigen-binding fragment thereof, the method comprising culturing a transformant as described in claim 12 and obtaining the antibody or an antigen-binding fragment thereof from the culture.

14. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1-9; Preferably, the chimeric antigen receptor further includes a co-stimulatory domain and a signal transduction domain.

15. A genetically modified cell, characterized in that, The cell expresses the chimeric antigen receptor as described in claim 14; preferably, the gene-modified cell is a T cell or an NK cell.

16. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate comprises a cytotoxic agent or tag, and an antibody or antigen-binding fragment thereof as described in any one of claims 1-9.

17. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1-9, a nucleic acid as described in claim 10, a recombinant expression vector as described in claim 11, a transformant as described in claim 12, a genetically modified cell as described in claim 15, or an antibody-drug conjugate as described in claim 16, and a pharmaceutically acceptable carrier and / or excipients.

18. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-9, the genetically modified cell as described in claim 15, the antibody-drug conjugate as described in claim 16, or the pharmaceutical composition as described in claim 17 in the preparation of a medicament for the treatment and / or prevention of EGFR and / or MUC1-mediated diseases; Preferably, the disease is cancer; More preferably, the cancer is selected from one or more of skin cancer, ovarian cancer, breast cancer, and non-small cell lung cancer.

19. A method for treating and / or preventing EGFR and / or MUC1-mediated diseases or conditions, the method comprising administering to a subject in need a therapeutic or preventative effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 1-9, a genetically modified cell as described in claim 15, an antibody-drug conjugate as described in claim 16, or a pharmaceutical composition as described in claim 17. Preferably, the disease is cancer; More preferably, the cancer is selected from one or more of skin cancer, ovarian cancer, breast cancer, and non-small cell lung cancer.

20. A reagent kit, characterized in that, The kit comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1-9, a nucleic acid as described in claim 10, a recombinant expression vector as described in claim 11, a transformant as described in claim 12, a gene-modified cell as described in claim 15, an antibody-drug conjugate as described in claim 16, or a pharmaceutical composition as described in claim 17.

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