Bispecific antibody-drug conjugate binding to EGFR and MUC1 and use thereof

Abstract

Provided are a bispecific antibody-drug conjugate binding to EGFR and MUC1 and use thereof. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises a bispecific antibody binding to EGFR and MUC1 or an antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug moiety. Provided are a preparation method for the antibody-drug conjugate or the pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising same. The antibody-drug conjugate has extremely high inhibitory activity against the proliferation of tumor cells both in vivo and in vitro.

Description

A bispecific antibody-drug conjugate combining EGFR and MUC1 and its applications

[0001] This application claims priority to Chinese patent application 2025101209722, filed on January 24, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of biomedicine, specifically relating to a bispecific antibody-drug conjugate that combines EGFR and MUC1 and its applications. Background Technology

[0003] EGFR is a member of the transmembrane receptor tyrosine kinase family. It readily binds to its ligands, typically leading to receptor dimerization, activating the activity of intracellular tyrosine protein kinases, phosphorylating C-terminal tyrosine residues, and triggering downstream enzymes such as Ras, Raf, and PI3K, thereby initiating a series of complex and cross-linked signal transduction pathways downstream. EGFR regulates cell growth, differentiation, and apoptosis by mediating these signaling pathways.

[0004] Currently approved anti-EGFR drugs for clinical use are mainly divided into antibody drugs and small molecule drugs. However, the complete cure rate of these drugs is insufficient, and only a small number of patients do not develop related drug resistance after longer treatment. Moreover, EGFR is also expressed at a certain level in normal tissues, leading to significant safety concerns for EGFR monoclonal antibodies in clinical use.

[0005] Therefore, there is still considerable room for optimization and improvement in the development of drugs targeting EGFR. Summary of the Invention

[0006] This disclosure provides a bispecific antibody-drug conjugate combining EGFR and MUC1, and its application. The bispecific antibody-drug conjugate exhibits excellent endocytosis effect, proliferation inhibition activity, tumor growth inhibition activity, and good in vivo safety.

[0007] One of the technical solutions provided in this disclosure is: an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the antibody-drug conjugate or a pharmaceutically acceptable salt thereof comprises a bispecific antibody that binds to EGFR and MUC1 or an antigen-binding fragment thereof, a linker unit L, and a cytotoxic drug portion;

[0008] The bispecific 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...

[0009] The amino acid sequences of H1CDR1, H1CDR2, and H1CDR3 contained in VH1 are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; the amino acid sequences of H2CDR1, H2CDR2, and H2CDR3 contained in VH2 are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 contained in VL are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.

[0010] or,

[0011] The amino acid sequences of H1CDR1, H1CDR2, and H1CDR3 contained in VH1 are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:6, respectively; the amino acid sequences of H2CDR1, H2CDR2, and H2CDR3 contained in VH2 are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 contained in VL are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.

[0012] In some embodiments, VH1 comprises the amino acid sequences of H1CDR1, H1CDR2, and H1CDR3 of the heavy chain variable region as shown in SEQ ID NO:13 or SEQ ID NO:14; VH2 comprises the amino acid sequences of H2CDR1, H2CDR2, and H2CDR3 of the heavy chain variable region as shown in SEQ ID NO:12; and VL comprises the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO:15.

[0013] In some embodiments, the VH1 comprises an amino acid sequence as shown in SEQ ID NO:13 or SEQ ID NO:14, or has 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:13 or SEQ ID NO:14; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least equivalent antigen-binding function to the original sequence.

[0014] In some preferred embodiments, the amino acid sequence of the VH1 is shown in SEQ ID NO:13 or SEQ ID NO:14.

[0015] In some embodiments, the VH2 comprises an amino acid sequence as shown in SEQ ID NO:12, or has 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:12; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least equivalent antigen-binding function to the original sequence.

[0016] In some preferred embodiments, the amino acid sequence of the VH2 is shown in SEQ ID NO:12.

[0017] In some embodiments, the VL comprises an amino acid sequence as shown in SEQ ID NO:15, or has 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:15; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same antigen-binding function as the original sequence.

[0018] In some preferred embodiments, the amino acid sequence of the VL is as shown in SEQ ID NO:15.

[0019] In some preferred embodiments disclosed herein, the amino acid sequences of VH1, VH2 and VL are as shown in SEQ ID NO:13, SEQ ID NO:12 and SEQ ID NO:15, respectively; or, as shown in SEQ ID NO:14, SEQ ID NO:12 and SEQ ID NO:15, respectively.

[0020] In some specific embodiments, the bispecific antibody or its antigen-binding fragment includes one or more of the following:

[0021] (1) Fully human antibodies, humanized antibodies, chimeric antibodies, proantibodies, bispecific antibodies, monoclonal antibodies and polyclonal antibodies;

[0022] (2) Fab, Fab', F(ab')2, Fv or scFv;

[0023] (3) The MUC1 binding domain is a human anti-MUC1 antibody or its antigen-binding fragment; and,

[0024] (4) The EGFR binding domain is a human anti-EGFR antibody or its antigen-binding fragment.

[0025] In this disclosure, the term "proantibody" refers to a "probody drug," a special antibody drug design derived from the combination of a "prodrug" and an "antibody." Probody drugs are initially inactive, only becoming active after undergoing specific chemical reactions or enzymatic transformation within the body. This design allows probody drugs to remain inactive in normal tissues, but are activated in specific microenvironments, such as the tumor microenvironment, through the action of proteases, thereby reducing toxicity to normal tissues and improving drug targeting.

[0026] 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.

[0027] In some implementations, the antibody molecule is humanized. Different methods for humanizing antibodies are known to those skilled in the art, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13:1619-1633).

[0028] In some embodiments disclosed herein, 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 HC1, and the MUC1 binding domain includes a light chain constant region CL and a heavy chain constant region HC2.

[0029] In some embodiments, the heavy chain constant region is preferably the heavy chain constant region of human antibody IgG1, and the light chain constant region is preferably the light chain constant region of human antibody κ chain.

[0030] In some implementations, the heavy chain constant region contains a Knob-into-hole mutation.

[0031] In some implementations, HC1 contains a Knob mutation and HC2 contains a corresponding Hole mutation; or, HC1 contains a Hole mutation and HC2 contains a corresponding Knob mutation; to ensure proper pairing and stable binding of the two different heavy strands.

[0032] In some implementations, HC1 contains a Knob mutation, such as T366W, and HC2 contains a Hole mutation, such as one or more selected from T366S, L368A, and Y407V; or, HC2 contains a Knob mutation, such as T366W, and HC1 contains a Hole mutation, such as one or more selected from T366S, L368A, and Y407V.

[0033] In some implementations, the heavy chain constant regions HC1 and HC2 contain L234A / L235A mutations according to the EU numbering system.

[0034] In some embodiments, the heavy chain constant region HC1 comprises the amino acid sequence shown in SEQ ID NO:16, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16; and / or, the heavy chain constant region HC2 comprises the amino acid sequence shown in SEQ ID NO:17, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:17; the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence.

[0035] In some embodiments, the heavy chain constant region HC1 comprises the amino acid sequence shown in SEQ ID NO:17, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:17; and / or, the heavy chain constant region HC2 comprises SEQ ID NO:16, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16; the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence.

[0036] In some embodiments, the heavy chain constant region HC1 comprises the amino acid sequence shown in SEQ ID NO:50, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:50; and / or, the heavy chain constant region HC2 comprises SEQ ID NO:51, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:51; the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence.

[0037] In some embodiments, the heavy chain constant region HC1 comprises the amino acid sequence shown in SEQ ID NO:51, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:51; and / or, the heavy chain constant region HC2 comprises SEQ ID NO:50, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:50; the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence.

[0038] In some embodiments, the amino acid sequence of the heavy chain constant region HC1 is shown in SEQ ID NO:16, and the amino acid sequence of the heavy chain constant region HC2 is shown in SEQ ID NO:17.

[0039] In some embodiments, the amino acid sequence of the heavy chain constant region HC1 is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain constant region HC2 is shown in SEQ ID NO:16.

[0040] In some embodiments, the amino acid sequence of the heavy chain constant region HC1 is shown in SEQ ID NO:50, and the amino acid sequence of the heavy chain constant region HC2 is shown in SEQ ID NO:51.

[0041] In some embodiments, the amino acid sequence of the heavy chain constant region HC1 is shown in SEQ ID NO:51, and the amino acid sequence of the heavy chain constant region HC2 is shown in SEQ ID NO:50.

[0042] In some embodiments, the light chain constant region CL contains the amino acid sequence shown in SEQ ID NO:18, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:18; the amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same function as the original sequence.

[0043] In some embodiments, the amino acid sequence of the light chain constant region CL is shown in SEQ ID NO:18.

[0044] In some preferred embodiments disclosed herein, the EGFR-binding domain comprises a heavy chain H1 and a light chain L, and the MUC1-binding domain comprises a heavy chain H2 and a light chain L; wherein,

[0045] The amino acid sequences of H1, H2 and L are shown in SEQ ID NO:33, SEQ ID NO:32 and SEQ ID NO:24, respectively.

[0046] In some embodiments, the amino acid sequences of H1, H2 and L are shown as SEQ ID NO:34, SEQ ID NO:32 and SEQ ID NO:24, respectively.

[0047] In some embodiments, the amino acid sequences of H1, H2 and L are shown as SEQ ID NO:48, SEQ ID NO:47 and SEQ ID NO:24, respectively.

[0048] In some embodiments, the amino acid sequences of H1, H2 and L are shown as SEQ ID NO:49, SEQ ID NO:47 and SEQ ID NO:24, respectively.

[0049] In some specific embodiments, the cytotoxic drug portion has the structure shown in formula (A-1), its stereoisomer, pharmaceutically acceptable salt, solvate, or solvate of a salt thereof.

[0050] in,

[0051] M is -L 2 -L 1 -C(O)-;

[0052] L 2 It is -O-, and L 2 Connected to the connector unit L;

[0053] L 1 -(C(R) 1a (R)1b )) m -CH2-;

[0054] m is selected from 1, 2, 3, or 4;

[0055] Each R 1a and R 1b Independently hydrogen, halogen, hydroxyl, amino, or C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted with one or more R;

[0056] Each R is independently either hydrogen or halogen.

[0057] In some specific implementation schemes, each R 1a Independently hydrogen, halogen, or C1-C6 alkyl; and / or, each R 1b It can be hydrogen, halogen, or C1-C6 alkyl independently.

[0058] In some specific implementation schemes, L 1 for

[0059] In some specific embodiments, the cytotoxic drug portion is any of the following structures:

[0060] In some specific implementations, the connector unit L is -L a -L b -L c -; and the L c It is partially connected to the cytotoxic drug.

[0061] In some specific implementations, -L a -for Preferred The a-terminus is linked to the bispecific antibody or its antigen-binding fragment, and the b-terminus is linked to L... b Connected.

[0062] In some specific implementations, -L b - For any of the following structures:

[0063] Preferred More preferably Among them, C-end and L a Connected, d end and L c Connected.

[0064] In some specific implementations, -L c -for

[0065] In some specific implementations, the connector unit L is Preferred

[0066] In some specific embodiments, the structure of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is shown in formula (A-2):

[0067] Where p is p' or p”, p' represents the number of connections, and p' is any integer from 1 to 10; for example, the number of connections p' is any integer from 3 to 9; for example, the number of connections p' is any integer from 5 to 8; the number of connections p' is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; for example, 6 or 8; and / or, p” represents the average number of connections, and p” is any integer or decimal from 1 to 10; for example, the number of connections p” is any integer or decimal from 3 to 9; for example, the number of connections p” is any integer or decimal from 5 to 8; the average number of connections p” is an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10, for example, 5.9, 6.17, 7.93, 5.82, 6.06 or 7.92.

[0068] In the preparation of ADCs, the number of drug molecules linked to each antibody molecule is specific, i.e., p' can be an integer such as 0, 1, 2, or 3. This is because the linkage reaction is a discrete event, and the number of drug molecules linked to each antibody molecule is fixed; there will be no half-molecule cases. When considering the entire ADC sample, the number of drug molecules linked to different antibody molecules may vary due to factors such as the efficiency of the linkage reaction and differences between antibody molecules. Therefore, to describe the average linkage of the entire sample, a small value is usually used to represent the average number of drug molecules linked to each antibody molecule, i.e., p'". This value is the average calculated by analyzing data from multiple antibody molecules, and p' can be a decimal such as 5.9, 6.17, 7.93, 5.82, 6.06, or 7.92. For example, if the average number of linkages p” for an ADC sample is 5.9, it means that on average, 5.9 drug molecules are linked to each antibody molecule in the sample. This value is derived by statistically analyzing the linkage of a large number of antibody molecules; in reality, the number of linkages per antibody molecule is still an integer. The drug loading of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA, and HPLC.

[0069] In some specific implementations, Ab is a bispecific antibody or its antigen-binding fragment as defined above.

[0070] In some specific implementations, M is M as defined above.

[0071] In some specific implementations, L is the connector unit L as defined above.

[0072] In some specific embodiments, the antibody-drug conjugate or a pharmaceutically acceptable salt thereof is selected from the following structural formulas:

[0073] in,

[0074] p is p as defined in any of the preceding items.

[0075] In some specific implementations, Ab is a bispecific antibody or its antigen-binding fragment as defined above.

[0076] In some specific embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt is the following conjugate or its pharmaceutically acceptable salt:

[0077] Where p is p as defined in any of the preceding terms;

[0078] And / or, DB1001, DB1002, DB1003, and DB1004 are bispecific antibodies binding to EGFR and MUC1, wherein the bispecific antibody comprises an EGFR-binding domain and a MUC1-binding domain, the EGFR-binding domain comprising a heavy chain H1 and a light chain L, and the MUC1-binding domain comprising a heavy chain H2 and a light chain L; wherein the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1001 are shown in SEQ ID NO:33, SEQ ID NO:32, and SEQ ID NO:24, respectively; the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1002 are shown in SEQ ID NO:34, SEQ ID NO:32, and SEQ ID NO:24, respectively; and the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1003 are shown in SEQ ID NO:48, SEQ ID NO:47, and SEQ ID NO:48, respectively. As shown in NO:24; the heavy chain H1, heavy chain H2 and light chain L amino acid sequences of the DB1004 are shown in SEQ ID NO:49, SEQ ID NO:47 and SEQ ID NO:24, respectively.

[0079] In some specific embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt is the following conjugate or its pharmaceutically acceptable salt:

[0080] in,

[0081] DB1001, DB1002, DB1003, and DB1004 are bispecific antibodies that bind to EGFR and MUC1. Each bispecific antibody contains an EGFR-binding domain and a MUC1-binding domain. The EGFR-binding domain comprises a heavy chain H1 and a light chain L, and the MUC1-binding domain comprises a heavy chain H2 and a light chain L. The amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1001 are shown in SEQ ID NO:33, SEQ ID NO:32, and SEQ ID NO:24, respectively. The amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1002 are shown in SEQ ID NO:34, SEQ ID NO:32, and SEQ ID NO:24, respectively. The amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1003 are shown in SEQ ID NO:48, SEQ ID NO:47, and SEQ ID NO:48, respectively. As shown in NO:24; the heavy chain H1, heavy chain H2 and light chain L amino acid sequences of the DB1004 are shown in SEQ ID NO:49, SEQ ID NO:47 and SEQ ID NO:24, respectively.

[0082] To address the aforementioned technical problems, the second technical solution provided in this application is: a method for preparing an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in one of the technical solutions of this application, wherein the preparation method comprises reacting a bispecific antibody or its antigen-binding fragment with a compound as shown in Formula II to obtain the antibody-drug conjugate or a pharmaceutically acceptable salt thereof.

[0083] L'-cytotoxic drugs

[0084] II;

[0085] L' is the connector unit L as defined in one of the technical solutions of this application. a The non-connected state of the a-terminus; the cytotoxic drug is the cytotoxic drug portion as defined in one of the technical solutions of this application.

[0086] The connector unit L typically consists of three parts, namely L a L b and L c L a L is a terminal linker of an antibody or its antigen-binding fragment. b It is a connecting arm, L c End-connected cytotoxic drugs.

[0087] Connector unit L of L aThe non-connected state at the a-terminus refers to the connection of the adapter unit L to a cytotoxic drug, but not to an antibody or its antigen-binding fragment. In this case, the segment where the adapter unit was originally connected to the antibody or its antigen-binding fragment is in the non-connected state. Under normal circumstances, L... a The terminal is linked to the antibody or its antigen-binding fragment. However, when referring to L... a When the connector unit L is in a non-connected state, it means that this end is not bound to an antibody or its antigen-binding fragment. In this case, the connector unit L only performs part of its function, namely, connecting to the cytotoxic drug, without binding to the antibody.

[0088] In some specific embodiments, the antibody-drug conjugate or its pharmaceutically acceptable salt satisfies one or more of the following conditions:

[0089] (1) The compound shown in Formula II is:

[0090] (2) The bispecific antibodies are DB1001, DB1002, DB1003 and DB1004;

[0091] DB1001, DB1002, DB1003, and DB1004 are the aforementioned bispecific antibodies binding to EGFR and MUC1. Each bispecific antibody comprises an EGFR-binding domain and a MUC1-binding domain. The EGFR-binding domain includes a heavy chain H1 and a light chain L, and the MUC1-binding domain includes a heavy chain H2 and a light chain L. The amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1001 are shown in SEQ ID NO:33, SEQ ID NO:32, and SEQ ID NO:24, respectively. The amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1002 are shown in SEQ ID NO:34, SEQ ID NO:32, and SEQ ID NO:24, respectively. The amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1003 are shown in SEQ ID NO:48, SEQ ID NO:47, and SEQ ID NO:48, respectively. As shown in NO:24; the heavy chain H1, heavy chain H2 and light chain L amino acid sequences of the DB1004 are shown in SEQ ID NO:49, SEQ ID NO:47 and SEQ ID NO:24, respectively.

[0092] The third technical solution provided in this application is: a pharmaceutical composition comprising an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in one of the technical solutions of this application, and a pharmaceutically acceptable carrier.

[0093] The fourth technical solution provided in this application is: the use of antibody-drug conjugates or their pharmaceutically acceptable salts as described in the first technical solution of this application, and / or the use of pharmaceutical compositions as described in the third technical solution of this application in the preparation of drugs for diagnosing, preventing and / or treating cancer.

[0094] In some specific implementations, the cancer is an EGFR and / or MUC1-related cancer, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer.

[0095] The fifth technical solution provided in this application is: a method for diagnosing, preventing and / or treating cancer, the method comprising administering to a subject in need an effective amount of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in the first technical solution of this application, and / or a pharmaceutical composition as described in the third technical solution of this application.

[0096] In some specific implementations, the cancer is an EGFR and / or MUC1-related cancer, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer.

[0097] The sixth technical solution provided in this application is: an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in the first technical solution of this application for the diagnosis, prevention and / or treatment of cancer, and / or a pharmaceutical composition as described in the third technical solution of this application.

[0098] In some specific implementations, the cancer is an EGFR and / or MUC1-related cancer, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer.

[0099] The seventh technical solution provided in this application is: a combination therapy comprising administering an effective amount of a first therapeutic agent and a second therapeutic agent to a subject in need, wherein the first therapeutic agent is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in one of the technical solutions of this application, and / or a pharmaceutical composition as described in three of the technical solutions of this application.

[0100] In some implementations, the first therapeutic agent is different from the second therapeutic agent.

[0101] In some specific implementations, the second therapeutic agent comprises a second drug for treating cancers associated with EGFR and / or MUC1 expression.

[0102] In some specific embodiments, the second therapeutic agent is an anti-EGFR and / or MUC1 antibody or its antigen-binding fragment.

[0103] In some specific embodiments, the anti-EGFR and / or MUC1 antibody or its antigen-binding fragment is different from the antibody-drug conjugate or its pharmaceutically acceptable salt as defined in one of the technical solutions of this application, which is a bispecific antibody or its antigen-binding fragment.

[0104] In some specific embodiments, the second therapeutic agent is an antibody-drug conjugate of an anti-EGFR and / or MUC1 antibody or its antigen-binding fragment.

[0105] In some specific embodiments, the antibody-drug conjugate of the anti-EGFR and / or MUC1 antibody or its antigen-binding fragment is different from the antibody-drug conjugate or its pharmaceutically acceptable salt described in one of the technical solutions of this application.

[0106] In some specific implementations, the cancer described in any of the preceding technical solutions is a cancer associated with EGFR and / or MUC1 expression, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer.

[0107] The eighth technical solution provided in this application is: a combination of a first therapeutic agent and a second therapeutic agent for the diagnosis, prevention and / or treatment of cancer; the first therapeutic agent is an antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in one of the technical solutions of this application, and / or a pharmaceutical composition as described in three of the technical solutions of this application.

[0108] In some specific embodiments, as described in any of the preceding technical solutions, the cancer is an EGFR and / or MUC1 expression-related cancer, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer. Based on common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred embodiments of this application.

[0109] All reagents and raw materials used in this application are commercially available.

[0110] The positive and progressive effects of this application are as follows: The bispecific antibody-drug conjugates that combine EGFR and MUC1 disclosed herein have excellent binding activity and tumor cell selectivity of both MUC1 and EGFR monoclonal antibodies. They also have co-localization effects of MUC1 and EGFR, blocking EGFR degradation caused by ligand activation. This not only enhances antibody internalization and tumor growth inhibition or reduces tumor growth, but also binds to cancer cells with higher specificity, thereby reducing the impact on normal cells and thus having better safety. Attached Figure Description

[0111] Figure 1 shows the antibody screening antigen and control antibody activity detection.

[0112] Figures 2A-2C show the ELISA method for detecting the binding activity of candidate EGFR antibodies against antigen proteins of different species; Figure 2A shows the binding activity of candidate EGFR antibodies against human EGFR antigen protein; Figure 2B shows the binding activity of candidate EGFR antibodies against monkey EGFR antigen protein; Figure 2C shows the binding activity of candidate EGFR antibodies against mouse EGFR antigen protein.

[0113] Figures 3A-3C show the ELISA method used to detect the binding activity of candidate MUC1 antibodies against glycosylated and non-glycosylated antigens. Figure 3A shows the binding ability of candidate MUC1 antibodies against STn-MUC1; Figure 3B shows the binding activity of candidate MUC1 antibodies against BSA-modified STn-MUC1; Figure 3C shows the binding activity of candidate MUC1 antibodies against non-glycosylated modified MUC1 peptides.

[0114] Figure 4 shows the FACS method for detecting the binding activity of candidate EGFR antibodies to tumor cells.

[0115] Figure 5 shows the FACS method for detecting the binding activity of candidate MUC1 antibody to MCF7 tumor cells.

[0116] Figures 6A-6B show the internalization ability of candidate EGFR antibodies detected by the Incucyte method; Figure 6A shows the internalization time curve of candidate EGFR antibodies in A431 cells; Figure 6B shows the calculation of the area under the curve for the internalization of candidate EGFR antibodies in A431 cells.

[0117] Figure 7 shows the internalization ability of candidate MUC1 antibody in OVCAR3 cells as detected by FACS method.

[0118] Figure 8 shows the ability of candidate EGFR antibodies to block hEGF activation of EGFR signaling by the HTRF method.

[0119] Figure 9 shows the tumor-selective binding ability of candidate EGFR antibodies.

[0120] Figure 10 shows the binding ability of candidate MUC1 antibodies to free MUC1 in the pleural and peritoneal fluid of breast cancer patients.

[0121] Figures 11A-11B show the binding activity of the anti-MUC1 affinity maturation antibody molecules against glycosylated and non-glycosylated antigens. Figure 11A shows the binding activity of the affinity maturation molecule against the tumor-specific antigen ST-MUC1; Figure 11B shows the binding activity of the affinity maturation molecule against non-glycosylated modified MUC1 peptides.

[0122] Figure 12 shows the binding activity of anti-human EGFR affinity maturation antibody molecules to tumor cells.

[0123] Figures 13A-13B show the binding activity of the anti-MUC1 affinity maturation antibody molecule on tumor cells. Figure 13A shows the binding activity of the affinity maturation molecule in NCI-H1975 cells; Figure 13B shows the binding activity of the affinity maturation molecule in OVCAR3 cells.

[0124] Figures 14A-14B show the internalization ability of anti-EGFR affinity maturation antibody molecules. Figure 14A shows the internalization ability of affinity maturation antibody in NCI-H1975 cells; Figure 14B shows the internalization ability of affinity maturation antibody in KYSE150 cells.

[0125] Figures 15A-15B show the internalization ability of the anti-MUC1 affinity maturation antibody molecule. Figure 15A shows the internalization ability of the affinity maturation molecule in OVCAR3 cells; Figure 15B shows the internalization ability of the affinity maturation molecule in NCI-H1975 cells.

[0126] Figure 16 shows the tumor-selective binding ability of the anti-human MUC1 affinity maturation antibody molecule.

[0127] Figures 17A-17C show the internalization ability of the candidate bispecific antibody. Figure 17A shows the internalization ability of the candidate bispecific antibody in MDA-MB-468 cells; Figure 17B shows the internalization ability of the candidate bispecific antibody in OVCAR3 cells; Figure 17C shows the internalization ability of the candidate bispecific antibody in NCI-H1975 cells.

[0128] Figure 18 shows the tumor selectivity detection of candidate bispecific antibodies.

[0129] Figures 19A-19C show the internalization ability of antibody-drug conjugates on tumor cells. Figure 19A shows the test results for MDA-MB-468 tumor cells; Figure 19B shows the test results for NCI-H1975 tumor cells; and Figure 19C shows the test results for OVCAR3 tumor cells.

[0130] Figure 20 shows the internalization ability of antibody-drug conjugates on tumor cells.

[0131] Figures 21A-21B show the internalization ability of antibody-drug conjugates on tumor cells. Figure 21A shows the test results for MDA-MB-468 tumor cells; Figure 21B shows the test results for NCI-H1650 tumor cells.

[0132] Figures 22A-22F show the in vitro proliferation inhibition test of antibody-drug conjugates on tumor cells. Figure 22A shows the test results for HCC1143 tumor cells; Figure 22B shows the test results for HCC1806 tumor cells; Figure 22C shows the test results for NCI-H2126 tumor cells; Figure 22D shows the test results for BT20 tumor cells; Figure 22E shows the test results for MDA-MB-468 tumor cells; and Figure 22F shows the test results for NCI-H1975 tumor cells.

[0133] Figures 23A-23C show the efficacy evaluation of the antibody-drug conjugate in the HCC1806 mouse model. Figure 23A shows the tumor growth curve; Figure 23B shows the animal body weight data; Figure 23C shows the endpoint tumor mass data.

[0134] Figures 24A-24C show the efficacy evaluation of the antibody-drug conjugate in the human esophageal cancer LD1-0015-362418PDX model. Figure 24A shows the tumor growth curve; Figure 24B shows the animal body weight data; and Figure 24C shows the endpoint tumor mass data.

[0135] Figures 25A-25B show the efficacy evaluation of the antibody-drug conjugate in the NCI-H2126 lung cancer CDX model. Figure 25A shows the tumor growth curve; Figure 25B shows the animal body weight data.

[0136] Figures 26A-26B show the efficacy evaluation of the antibody-drug conjugate in the SW480 colorectal cancer CDX model. Figure 26A shows the tumor growth curve; Figure 26B shows the animal body weight data.

[0137] Figures 27A-27B show the efficacy evaluation of antibody-drug conjugates with different DAR values ​​in the NCI-H1975 lung cancer CDX model. Figure 27A shows the tumor growth curve; Figure 27B shows the animal body weight data.

[0138] Figures 28A-28B show the efficacy evaluation of antibody-drug conjugates with different DAR values ​​in the NCI-H1650 lung cancer CDX model. Figure 28A shows the tumor growth curve; Figure 28B shows the animal body weight data. Detailed Implementation

[0139] In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational procedures used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all routine procedures widely used in their respective fields. To better understand this application, definitions and explanations of relevant terms are provided below:

[0140] In this application, the letters in the amino acid sequence represent single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0141] In this application, the term "and / or" should be understood to mean any one of the options or any combination of two or more of the options.

[0142] In this application, 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 the opposite term is explicitly indicated, such as "only one" or "exactly one" or when "consisting of" is used in the claims, will it refer to only one number or one element of the list.

[0143] In this application, the term "antibody-drug conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug via a stable linker unit. In this application, "antibody-drug conjugate" can also refer to an antibody or its antigen-binding fragment linked to a biologically active cytotoxic drug portion via a stable linker unit.

[0144] In this application, the term "cytotoxic drug" generally refers to a toxic drug that possesses a strong chemical molecule within tumor cells that disrupts their normal growth. Cytotoxic drugs can kill tumor cells at sufficiently high concentrations. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins derived from bacteria, fungi, plants, or animals, and radioactive isotopes (e.g., At). 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32(or radioactive isotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics or ribolysins, or their derivatives, for example, can be toxic drugs, including but not limited to camptothecin derivatives, such as camptothecin derivative essanotecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione).

[0145] In this disclosure, the term “MUC1” refers to the full-length MUC1 protein, and the amino acid sequences of MUC1 molecules from human and non-human species (e.g., mice, monkeys, rabbits, dogs, pigs, etc.) are available from public resources. MUC1 can be isolated from cells or tissues expressing them or synthesized using techniques well known in the art, see, for example (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

[0146] (2) Fumoto, M.; Hinou, H.; Ohta, T.; Ito, T.; Yamada, K.; Takimoto, A.; Kondo, H.; Shimizu, H.; Inazu, T.; Nakahara, Y.; glycopeptides.J.Am.Chem.Soc.2005,127,11804-11818

[0147] (3)Dalziel, M.;Whitehouse,C.;McFarlane,I.;Brockhausen,I.;Gschmeissner,S.;Schwientek,T.;Clausen,H.;Burchell,JM;TaylorPapadimitriou,J. MUC1.J.Biol.Chem.2001,276,11007-11015.

[0148] The term "EGFR" as used herein refers to the epidermal growth factor receptor (or ErbB-1 or HER1) and may refer to the wild-type receptor or a receptor containing one or more mutations. The terms "activity" or "biological activity," or "biological properties" or "biological characteristics," 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, and IC50. 50 The in vivo stability of antibodies and the immunogenic properties of antibodies.

[0149] In this application, the term "antibody" generally refers to an immunoglobulin that reacts to a specified protein or peptide or fragment thereof. Antibodies can be from any class, including but not limited to IgG, IgA, IgM, IgD, and IgE, and antibodies from any subclass (e.g., IgG1, IgG2, IgG3, and IgG4). Antibodies may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies may also have a light chain selected from, for example, kappa (κ) or lambda (λ). The antibodies of this application may be derived from any species. The term "antibody" may include complete polyclonal antibodies, complete monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antibodies, and any other modified immunoglobulin molecules, provided that these antibodies exhibit the desired biological activity.

[0150] In this application, the term "antigen-binding fragment" generally refers to a portion of an antibody molecule containing amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen that is specifically recognized and bound by the antibody is called an "epitope." As described above, an antigen-binding domain typically includes a variable region (VL) and a variable region (VH) of the antibody light chain; however, it is not necessary to include both. Fd fragments, for example, have two VH regions and typically retain some of the antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of antibodies include (1) Fab fragments, monovalent fragments having VL, VH, constant light chain (CL) and CH1 domains; (2) F(ab′)2 fragments, bivalent fragments having two Fab fragments connected by disulfide bridges in the hinge region; (3) Fd fragments having two VH and CH1 domains; (4) Fv fragments having VL and VH domains in an antibody single arm; (5) dAb fragments (Ward et al., “Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted From Escherichia coli”, Nature 341:544-546 (1989), which are incorporated herein by reference in their entirety), having a VH domain; and (6) single-chain Fv (scFv), for example derived from scFV-libraries. Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be joined together using a recombination method via a synthetic linker. The synthetic linker allows it to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called single-chain Fv (scFv)) (see, for example, Huston et al., “Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli,” Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)).

[0151] In this application, the term "variable region" or "variable domain" generally refers to the domain of the antibody heavy or light chain involved in antibody-antigen binding. In this application, the term "variable" generally means that certain portions of the sequence of the variable domain of an antibody vary significantly, resulting in various specific antibody binding and specificity to its specific antigen. This variability is not uniformly distributed throughout the entire variable region of the antibody. It is concentrated in three segments within the light and heavy chain variable regions, referred to as complementarity-determining regions (CDRs) or hypervariable regions (HVRs), namely LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), mostly in a β-sheet configuration, connected by three CDR loop regions. The CDRs in each chain are closely packed together through the FR region and together with the CDRs from the other chain, they form the antigen-binding site of the antibody.

[0152] In this application, the amino acid sequences of the listed CDRs are all in accordance with the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia et al. (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)), based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined by any of the known schemes described herein. While the scope of protection claimed in this application is based on the sequence shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this application. Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this application, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this application due to the application of different schemes (e.g., different assignment system rules or combinations).

[0153] The sequence identity between sequences is calculated as follows. To determine the percentage of identity between two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in the first and second amino acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being compared is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue at the corresponding position in the second sequence, the molecules are identical at that position. Sequence comparison and the calculation of the percentage of identity between two sequences can be performed using mathematical algorithms. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program, which is integrated into the GCG software package. The Blossum 62 matrix or PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6 are used to determine the percentage of identity between two amino acid sequences. A particularly preferred set of parameters (and one set of parameters that should be used unless otherwise specified) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. Alternatively, the PAM120 weighted remainder table, gap length penalty of 12, and gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17), which has been incorporated into the ALIGN program (version 2.0). Additionally or alternatively, the protein sequence described in this application can be further used as a "query sequence" to perform a search against public databases to, for example, identify other family member sequences or related sequences.

[0154] In this application, the term "full-length antibody" is used interchangeably to refer to a glycoprotein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated as VH in this application) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this application) and a light chain constant region (abbreviated as CL in this application). The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of Y consists of the second and third constant regions (and a fourth constant region for IgE and IgM) of two heavy chains linked together, with disulfide bonds (interchain) forming hinges. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains, and hinge regions for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y includes a variable region of a single heavy chain and a first constant region that binds to a variable and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0155] In this application, "Fab" consists of a light chain and a heavy chain, CH1, and a variable region. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc" region contains two heavy chain segments containing the CH2 and CH3 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain. "Fab'" contains a portion of a light chain and a heavy chain containing the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains of the two Fab's to form the F(ab')2 molecule. "F(ab')2" contains two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains, thereby allowing interchain disulfide bonds to form between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains. The term "Fv" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody, but lacking the constant region.

[0156] In this application, 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 that 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 by any particular method.

[0157] In this application, the term "multispecific antibody" refers to an antibody containing two or more antigen-binding domains and capable of binding to two or more different epitopes (e.g., two, three, four, or more different epitopes), which may be on the same or different antigens. Examples of multispecific antibodies include "bispecific antibodies" (abbreviated as biantibodies) or "bispecific molecules" that bind to two different antigens or two different epitopes. A bispecific antibody targeting EGFR and MUC1 may be referred to, for example, as "anti-EGFR and MUC1," or a bispecific antibody containing both an EGFR-binding domain and a MUC1-binding domain, or other similar terms.

[0158] In this application, the terms "knob into Hole," "knob mutation," and "hole mutation" refer to the mutation of the hydrophobic amino acid in the CH3 region of the antibody Fc. One CH3 side-chain amino acid is mutated to form a larger hydrophobic amino acid (knob), such as T366W, to strengthen the hydrophobic interaction; the other CH3 side-chain amino acid is mutated to form a smaller amino acid (hole), such as T366S, L368A, or Y407V, to reduce steric hindrance. After mutation, the CH3 with the knob and the CH3 with the hole form a knob into Hole structure (KiH) through hydrophobic interactions, which is beneficial for the formation of heavy chain heterodimers. The KiH mutation mainly occurs in the internal hydrophobic amino acids of the CH3 domain; the exposed amino acids remain almost unchanged after mutation, so it does not affect the effector function of the Fc or the resulting immunogenicity. To further promote heterodimer formation, point mutations of S354C and Y349C can be introduced into the two CH3 regions, respectively, to further promote heterodimer formation through disulfide bonds. (The numbering in this paragraph 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).

[0159] In this application, the term 'LALA' is mutated as L234A and L235A, also referred to as 'Ala-Ala', 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. These mutations reduce C1q and FcγR binding, and consequently reduce effector function.

[0160] In this application, 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 substantially all of at least one, typically two, variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially 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 region (Fc) of human immunoglobulins.

[0161] "Isotype" antibodies refer to antibody classes provided by heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where modifications have been generated to alter Fc function, such as to enhance or weaken effector function or binding to the Fc receptor.

[0162] In this application, "affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can generally be determined by the equilibrium dissociation constant (K0). D The equilibrium dissociation constant represents the dissociation rate constant and the binding rate constant (k, k, k) respectively. dis and k on The ratio of affinity to antigen. Affinity can be measured by common methods known in the art. In some embodiments of this application, surface plasmon resonance (SPR) technology is used to measure affinity, such as the affinity between the antibody and the antigen in this application. In some preferred embodiments of this application, a specific method for measuring affinity is the BIAcore method described herein.

[0163] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine, or iodine, for example, it can be fluorine or chlorine.

[0164] In this application, the term "alkyl" generally refers to a residue derived from an alkane by removing a hydrogen atom. Alkyl groups can be substituted or unsubstituted, substituted or non-substituted. The term "alkyl" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having residues derived from the removal of hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane, and can be a straight-chain or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, such as a chain alkyl containing 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, butyl, etc.

[0165] In this application, the term "independently" generally means that the variable applies to any situation, regardless of whether there are variables with the same or different definitions in the same compound. For example, the variable may refer to the type or number of substituents in the compound, or the type of atoms in the compound. For example, when R appears twice in a compound and R is defined as "independently carbon or nitrogen", both Rs can be carbon, both Rs can be nitrogen, or one R can be carbon and the other R can be nitrogen.

[0166] In this application, the terms “optional” or “optionally” generally mean that the event or environment described below may but does not have to occur, and the description includes the occasion in which the event or environment occurs or does not occur. For example, “C1-C6 alkyl group optionally substituted with one or more R” means that R may but does not have to be present, and the description can include the case where the C1-C6 alkyl group is substituted with R and the case where the C1-C6 alkyl group is not substituted with R.

[0167] In this application, the term "substituted" generally refers to one or more hydrogen atoms in a group, for example, up to five, or for example, one to three hydrogen atoms, independently substituted by the corresponding number of substituents. Substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when bonded to a carbon atom with an unsaturated bond (such as an alkene).

[0168] In this application, as those skilled in the art will know, terms such as "alkyl," "alkenyl," and "cycloalkyl" may be preceded by an identifier indicating the number of atoms present in the group under specific conditions, for example, C1-C4 alkyl, C3-C7 cycloalkoxy, C1-C4 alkylcarbonylamino, etc., and the subscript number following "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl); C 1-10 In this context, the members of the group can have any number of carbon atoms falling within the range of 1-10.

[0169] In this application, the compounds or antibody-drug conjugates of this application include their tautomers, meso compounds, racemates, enantiomers, and / or diastereomers. In this application, the term "diastereomer" generally refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. In this application, the terms "tautomer" or "tautomer form" are used interchangeably and generally refer to structural isomers with different energies that can be interconverted through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via the rearrangement of some bonding electrons. In this application, the term "meta-polymorph" generally refers to a molecule containing asymmetric atoms but possessing symmetry factors that result in zero total optical rotation within the molecule. The term "racemic mixture" or "racemic mixture" refers to a composition consisting of two enantiomers in equimolar amounts.

[0170] In this application, the terms "connector unit" or "connector structure" generally refer to a chemical structural fragment or bond that is connected to a ligand at one end and to a cytotoxic drug at the other end. It may also refer to connecting other connectors before being connected to the cytotoxic drug. The direct or indirect connection to the ligand can refer to the group directly connecting to the ligand via a covalent bond, or it can refer to the connection of the ligand via a connector structure. For example, chemical structural fragments or bonds containing acid-labile connector structures (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) connector structures, light-labile connector structures, dimethyl connector structures, or disulfide-containing connector structures can be used as connector structures.

[0171] In this application, the term "drug loading" generally refers to the average number of cytotoxic drugs loaded on each ligand (DAR), which can be called the "average number of links p" or expressed as the ratio of cytotoxic drug to antibody. The range of cytotoxic drug loading can be 0-12 links per ligand (Ab), for example, 1-10 cytotoxic drugs. The drug loading of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization. The "average number of links p" can be an integer or decimal from 1 to 10. For example, the "average number of links p" can be an integer or decimal from 2 to 8. For example, the "average number of links p" can be an integer or decimal from 3 to 9. For example, the "average number of links p" can be an integer or decimal from 5 to 8. For example, the "average number of links p" can be an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0172] In some embodiments of this application, antibody-drug conjugates refer to compounds containing the same DAR distribution. The term "drug loading" refers to the number of cytotoxic drugs loaded on each ligand, which can be called the number of links or expressed as the ratio of cytotoxic drug to antibody. The range of cytotoxic drug loading can be 0-12 links per ligand (Ab), for example, 1-10 cytotoxic drugs. The number of links p' can be any integer from 1 to 10. For example, the number of links p' can be any integer from 3 to 9. For example, the number of links p' can be any integer from 5 to 8. For example, the number of links p' can be 4, 5, 6, 7, or 8.

[0173] In this application, certain atoms of the compound or antibody-drug conjugate may appear in more than one isotopic form. For example, hydrogen may appear as protium (…). 1 H), deuterium ( 2 H) and tritium ( 3 Carbon exists in the form of H, and it may exist in three different isotopes (H). 12 C 13 C and 14 C) Naturally occurring. Examples of isotopes that may be incorporated into the compounds of this application include, but are not limited to, those that exist naturally. 15 N、 18 O、 17 O、 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125I, or similar isotopes. Therefore, the compounds or antibody-drug conjugates of this application can be enriched in one or more of these isotopes relative to their natural abundance. As those skilled in the art will know, such isotope-enriched compounds can be used for a variety of purposes. For example, with heavy isotopes such as deuterium (I, or similar isotopes). 2 H) substitution may offer certain therapeutic advantages, possibly due to greater metabolic stability. For example, deuterium (H) 2 The natural abundance of deuterium (H) is approximately 0.015%. Therefore, there is approximately one deuterium atom for every 6500 hydrogen atoms in nature. Consequently, the deuterium abundance of the deuterium-containing compounds or antibody-drug conjugates of this application is greater than 0.015% at one or more positions (as the case may be). Unless otherwise specified, the structures described in this application may also include compounds or antibody-drug conjugates that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds or antibody-drug conjugates whose structures are identical to those of this application except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon-13 or carbon-14, are within the scope of this application.

[0174] In this application, the term "pharmaceutical composition" generally refers to a mixture containing one or more of the compounds or antibody-drug conjugates described in this application, or their physiologically / pharmaceutical acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmaceutical acceptable carriers and excipients. Pharmaceutical compositions can facilitate administration to an organism, promote the absorption of the active ingredient, and thus exert its biological activity. Conventional preparation methods for pharmaceutical compositions can be found in the Chinese Pharmacopoeia. Pharmaceutical compositions can be in the form of sterile injectable aqueous or oil suspensions for intramuscular and subcutaneous administration. Such suspensions can be formulated using suitable dispersants or wetting agents and suspending agents as described above, according to known techniques. Sterile injectable formulations can also be sterile injectable solutions or suspensions prepared in non-toxic, parenteral-acceptable diluents or solvents, such as solutions prepared in 1,3-butanediol. Furthermore, sterile fixative oils can conveniently be used as solvents or suspension media. For example, any blended fixative oil, including synthetic mono- or diglycerides, can be used. Additionally, fatty acids such as oleic acid can also be used to prepare injectable formulations.

[0175] In this application, the terms "pharmaceutically acceptable salt" or "pharmaceutically usable salt" generally refer to salts of compounds or antibody-drug conjugates of this application, or salts of compounds or antibody-drug conjugates described in this application, which may be safe and / or effective when used in mammals and may have the desired biological activity. The compounds or antibody-drug conjugates of this application may form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0176] In this application, a pharmaceutically acceptable carrier is any of those conventionally used carriers, limited only by physicochemical considerations (such as solubility and lack of reactivity with anti-EGFR and MUC1 antibodies) and by route of administration. Pharmaceutically acceptable carriers described herein, such as mediators, adjuvants, excipients, and diluents, are well known to those skilled in the art and are readily available to the public. In one aspect, a pharmaceutically acceptable carrier is a carrier that is chemically inert to the active ingredient of a pharmaceutical composition and does not have adverse side effects or toxicity under the conditions of use. In some embodiments, the carrier does not produce adverse, allergic, or other inappropriate reactions when administered to animals or humans. In some aspects, the pharmaceutical composition is free of pyrogens and other impurities that would be harmful to humans or animals. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonics, and absorption delay agents, etc.; their uses are well known in the art.

[0177] The term "treatment" generally refers to a method for achieving a beneficial or desired outcome, including but not limited to therapeutic benefits. Therapeutic benefits include, but are not limited to, eradicating, suppressing, reducing, or improving the underlying disorder being treated. Furthermore, therapeutic benefits are achieved by eradicating, suppressing, reducing, or improving one or more physiological symptoms associated with the underlying disorder, thereby observing improvement in the patient, although the patient may still have the underlying disorder.

[0178] The term "prevention" generally refers to a method of obtaining a beneficial or desired outcome, including but not limited to preventive benefits. For the purpose of preventing benefits, a pharmaceutical composition may be administered to a patient at risk of developing a particular disease or to a patient who reports having one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

[0179] The terms “subject” or “patient” or “individual” generally refer to humans (i.e., males or females of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or older adults)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs; and / or birds, including commercially relevant birds such as chickens, ducks, geese, quails, and / or turkeys.

[0180] As used in this application, the term "effective amount" means the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" means the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0181] As used herein, “cancer disease” or “cancer” includes diseases characterized by abnormally regulated cell growth, proliferation, differentiation, adhesion, and / or migration. Cancer cells are abnormal cells that grow through rapid, uncontrolled cell proliferation and continue to grow even after the stimuli that initiated new growth have ceased.

[0182] As used herein, the term "EGFR and / or MUC1 expression-related cancer" refers to cancer involving cancer cells expressing EGFR and / or MUC1, preferably on the surface of said cancer cells.

[0183] The present application is further illustrated below by way of embodiments, but these embodiments are not intended to limit the scope of the present application. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected in accordance with the product instructions.

[0184] Example 1: Preparation of anti-EGFR maternal monoclonal antibody and anti-MUC1 maternal monoclonal antibody

[0185] 1.1 Preparation of recombinant proteins for screening and activity detection of anti-MUC1 antibodies

[0186] The peptides and glycosylated peptides used for MUC1 antibody screening were all synthesized internally, and the synthesis methods are referenced in the literature. [1-3] The report states that the synthetic antigen contains a polypeptide sequence derived from MUC1, HGVTSAPDTRPAPGSTAPPA (SEQ ID NO:25).

[0187] The types of antigens synthesized are shown in Table 1.

[0188] Table 1

[0189] 1.2 Preparation of recombinant protein for detecting anti-EGFR antibody activity

[0190] The EGFR antibody screening antigens GSV0->EGFR-P00533-25-645-chis and GSV0->EGFR-P00533-25-645-Fc were synthesized, prepared, and their activity was detected internally using conventional methods. The antigen activity detection control antigens Human EGFR his and Human EGFR Fc were purchased from ACRO, catalog numbers EGR-H5222 and EGR-H5252, respectively. The amino acid sequence of the human EGFR extracellular domain is amino acids 25-645 of P00533 (sequence shown in SEQ ID NO:26), and was used for the activity detection of the antibodies in this application.

[0191] The antigens Mouse-EGFR-his and Rhesus macaque EGFR-His were purchased from ACRO, with catalog numbers EGR-M5224 and EGR-C52H1, respectively.

[0192] The human-mouse chimeric antibody Cetuximab (light chain as shown in SEQ ID NO:37, heavy chain as shown in SEQ ID NO:38) and the humanized antibody hCetuximab (sequence derived from WO2021247798, light chain as shown in SEQ ID NO:40, heavy chain as shown in SEQ ID NO:39) were used as the EGFR control antibodies in this application and were obtained using conventional antibody expression and purification methods. The results of antigen and antibody activity detection are shown in Figure 1.

[0193] Gatipotuzumab (sequence from US8779102B2, heavy chain sequence SEQ ID NO:41, light chain sequence SEQ ID NO:42), 1B2 (sequence from US20120040375A1, heavy chain sequence SEQ ID NO:43, light chain sequence SEQ ID NO:44), and HT186-D11 (sequence from US20110318757A1, heavy chain sequence SEQ ID NO:45, light chain sequence SEQ ID NO:46) were used as control antibodies for MUC1 and produced using conventional recombinant antibody production processes.

[0194] SEQ ID NO:26 P00533

[0195] SEQ ID NO:38 Cetuximab heavy chain

[0196] SEQ ID NO:37 Cetuximab Light Chain

[0197] SEQ ID NO:39 hCetuximab heavy chain

[0198] SEQ ID NO:40 hCetuximab light chain

[0199] SEQ ID NO:41 Gatipotuzumab heavy chain

[0200] SEQ ID NO:42 Gatipotuzumab light chain

[0201] SEQ ID NO:43 1B2 heavy chain

[0202] SEQ ID NO:44 1B2 light chain

[0203] SEQ ID NO:45 HT186-D11 heavy chain

[0204] SEQ ID NO:46 HT186-D11 Light Chain

[0205] 1.3 Phage Display Library Screening

[0206] In this embodiment, a conventional phage display library was used, and the library was screened with antigens prepared in Examples 1.1 and 1.2 to obtain multiple antibody molecules that specifically bind to human glycosylated MUC1 and multiple antibody molecules that specifically bind to human EGFR.

[0207] (1) Screening of antibody gene phage display libraries using magnetic bead method

[0208] 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.

[0209] (2) Screening of antibody gene phage display libraries using the immunotube method

[0210] Immunotube screening involves coating antigen proteins onto the surface of highly absorbent immunotubes. A phage-displaying antibody library is added to the immunotube, and the mixture is incubated, washed, and eluted with the adsorbed antigen proteins. After 2-4 rounds of screening, specific monoclonal antibodies against the antigen are 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.

[0211] (3) Monoclonal antibody screening

[0212] 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.

[0213] Initial ELISA monoclonal 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, screening for maternal antibodies. 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.

[0214] Table 2. Amino acid sequence of the variable region of anti-EGFR antibody

[0215] ELISA monoclonal antibody screening used the antigen proteins STn-MUC1, STn-MUC1-BSA, STn-MUC1-KLH, MUC1, and MUC1-BSA to obtain antibodies capable of binding to different forms of antigens, and their sequences were determined. Based on antigen binding ability, maternal antibodies were screened. The amino acid sequences of the CDR regions of the obtained antibodies VH and VL are shown in Table 3. The CDR sequences were determined using the Kabat definition method.

[0216] Table 3. Amino acid sequence of the variable region of anti-MUC1 antibody

[0217] 1.4 Construction and expression of fully human antibodies

[0218] The coding sequences for the constant regions of the heavy and light chains (κ) 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 EGFR or MUC1 antibody 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 fully human anti-EGFR and fully human MUC1 antibodies, respectively. The heavy chain of the fully human EGFR antibody is SEQ ID NO:30, and the light chain is SEQ ID NO:24; the heavy chain of the fully human MUC1 antibody is SEQ ID NO:27, and the light chain is SEQ ID NO:24.

[0219] SEQ ID NO:20 Human clone heavy chain constant region

[0220] SEQ ID NO:19 Human clonal heavy chain constant region with LALA mutation

[0221] SEQ ID NO:18 Human clone light chain constant region κ

[0222] SEQ ID NO:24 Light Chain

[0223] SEQ ID NO:30 B307 heavy chain:

[0224] SEQ ID NO:27 B82 heavy chain:

[0225] 1.5 Detection of fully human antibody-antigen binding activity

[0226] 1.5.1 Detection of Anti-EGFR Fully Human Antibody Binding Activity with Antigen

[0227] Dilute the antigens (GSVO->EGFR-P00533-25-645-chis, Rhesus macaqueEGFR-His, Mouse-EGFR-his) with 1×PBS to a concentration of 2 μg / mL, and add 30 μL / well to a 96-well ELISA plate. Incubate 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 three times with PBST, add 30 μL / well of 1% PBSM to dilute to 1 μg / mL sample, 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 measure OD. 450 .

[0228] The results are shown in Figures 2A, 2B, 2C and Table 4. All candidate antibodies were able to bind to EGFR in humans, monkeys and mice.

[0229] Table 4. Antigen-binding activity of anti-EGFR antibodies (ECG) 50 )

[0230] 1.5.2 Detection of Anti-human MUC1 Fully Human Antibody Binding Activity to Antigen

[0231] Dilute the antigens (STn-MUC1, STn-MUC1-BSA, MUC1) to 4 μg / mL with 1×PBS, and add 30 μL / well to each well of a 96-well ELISA plate. Coat overnight at 4°C. Wash the plate three times with PBST, add blocking buffer (5% PBSM), and block for 2 h at room temperature. After washing three times with PBST, add serially diluted antibody (1% PBSM) at 30 μL / well and incubate for 60 min at room temperature. Wash the plate three times with PBST, add secondary antibody (Anti-human-IgG-Fc-HRP (abcam; ab97225)), and incubate for 60 min at room temperature. 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 measure OD. 450 .

[0232] The results are shown in Figures 3A-3C and Table 5. The candidate antibody can bind to the tumor-specific glycosylated modified antigen STn-MUC1 (Figure 3A), and its binding ability to the multivalent antigen STn-MUC1-BSA is significantly enhanced (Figure 3B). The detection results of non-glycosylated MUC1 show that the candidate antibody has a weak binding signal (Figure 3C), indicating that it has the selective binding characteristics of glycosylated antigens.

[0233] Table 5. Antigen-binding activity of MUC1 antibody (EC5) 50 )

[0234] 1.6 Affinity kinetics of anti-human MUC1 fully human monoclonal antibody

[0235] The device used was a Biacore T200 (Cytiva) with a Protein A chip to capture antibody molecules onto the chip. The mobile phase consisted of serially diluted antigens. The parameters were set according to the table below for detection.

[0236] Table 6 Biacore Detection Parameter Settings

[0237] The results are shown in Table 7. All candidate antibodies have the ability to bind to the antigen, and their binding ability is comparable to or slightly weaker than that of the control antibody 1B2.

[0238] Table 7. Affinity of MUC1 Antibody to Antigen

[0239] 1.7 Detection of the binding ability of fully human antibodies to tumor cells

[0240] The binding activity of MUC1 antibody and EGFR antibody to MCF7 and A431 cells, respectively, was detected using the FACS method, as follows:

[0241] MCF7 cells (human breast cancer cells) or A431 cells (human epidermal cancer cells) were prepared into cell suspensions and their density was adjusted 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.

[0242] The results are shown in Figures 4 and 5 and Tables 8 and 9. Both the candidate MUC1 antibody and the EGFR antibody showed cell binding ability.

[0243] Table 8 shows the binding ability of human EGFR monoclonal antibodies to A431 cells.

[0244] Table 9. Binding ability of fully human MUC1 antibody to MCF7 cells.

[0245] 1.8 Detection of the internalization capacity of fully human monoclonal antibodies

[0246] 1.8.1 Detection of the internalization ability of fully human anti-EGFR antibody on A431 cells:

[0247] Incucyte Method: Take a clean, sterile 3599 cell culture plate and add 80 μL of cell suspension. Allow the cells to adhere for 4 hours. Mix an equal volume of the test antibody with Fabfluor-pH Dyes (Sartorius, Cat. #4722) (molar ratio: 1:3 of test antibody to labeling Fab), and incubate at room temperature in the dark for 15 minutes. Slowly add 20 μL of antibody-Fabfluor mix below the surface of the liquid. Gently shake the plate horizontally back and forth and left and right to mix the antibody-Fabfluor mix with the cells. Place the plate into an IncuCyte (Sartorius, Incucyte S3) container. Set the imaging program and take pictures.

[0248] The results, as shown in Figures 6A and 6B, indicate that the anti-EGFR antibody B307 can be internalized into A431 cells, and is superior to the control antibody hCetuximab.

[0249] 1.8.2 Detection of the internalization ability of fully human anti-human MUC1 antibody on tumor cells:

[0250] Flow cytometry: Prepare a sufficient amount of antibody at a working concentration of 4× in cell culture medium, and pipette 25 μL of the 4× working concentration antibody solution into a 96-well plate. Prepare a 4× working concentration Zenon pHrodo iFL IgG labeling reagent, and pipette 25 μL of the 4× working concentration into the 96-well plate containing the antibody. Incubate at room temperature for 5 min to allow the labeling complex to form. The molar ratio of antibody to labeling reagent is 1:3. Add cells of appropriate density to the 96-well plate. After cell attachment, adjust the culture medium volume to 50 μL per well and add 50 μL of labeled antibody. Incubate under standard cell culture conditions (37°C) for 48 h, and then analyze using flow cytometry.

[0251] As shown in Figure 7, the anti-MUC1 antibody B82 exhibited good internalization ability in OVCAR3 (human ovarian cancer cells).

[0252] 1.9 Detection of the ability of fully human anti-EGFR antibodies to block the binding of EGF and EGFR on cells

[0253] NCI-H292 (human lung adenocarcinoma cells) were cultured routinely at 37°C and 5% CO2. Cells in the logarithmic growth phase were counted after digestion. The cell suspension was adjusted to a suitable density, and 90 μL of cell suspension was added at a density of 50,000 cells / well. The cells were incubated overnight. The next day, 1 μM EGF and 1 μM sample stock solution were prepared. The test antibody was added to the cell culture wells at a final concentration of 10 nM, and the cells were incubated for 30 minutes. Then, 10 nM EGF was added, and the cells were incubated for another 30 minutes. Samples were processed according to the HTRF HUMAN PAN PHOSPHO-EGFR DETECTION KITS: 500 tests (64HR1PEG) manufacturer's instructions. The signals at 665 nm and 620 nm were detected using an EnVision microplate reader, and the signal ratio at the two wavelengths was calculated.

[0254] As shown in Figure 8, the anti-EGFR antibody B307 was able to block the signal activation caused by EGF and was superior to the control antibody Cetuximab.

[0255] 1.10 Detection of tumor-selective binding ability of fully human anti-EGFR antibodies

[0256] 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 (Invitrogen, C34554) was added, and the cells were incubated at room temperature in the dark for 20 min for staining. MCF10A 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 to achieve a cell ratio of 1:30. 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 (Biolegend, 410708) 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.

[0257] As shown in Figure 9, the B307 antibody exhibited superior tumor-selective binding ability compared to the control antibody molecule Cetuximab.

[0258] 1.11 Detection of the binding ability of fully human anti-human MUC1 antibody to free MUC1 in pleural and peritoneal fluid of breast cancer patients

[0259] The breast and peritoneal fluid samples were obtained from Beijing Ketu Medical. Biotin solution was added to the MUC1 detection antibody (MUC1(VU4H5)mouse mAb, CST#4538) solution at a biotin to antibody molar ratio of 50:1. After ultrafiltration to elute free biotin, Biotin-labeled MUC1 mAb was obtained.

[0260] MUC1 capture mAb (MUC1(D9O8K)XP Rabbit mAb, catalog number CST#4161, used as a positive control) and three test antibodies (B82, Gatipotuzumab, and 1B2) were diluted with PBS at 250 ng / mL. An IgG control (Sino Biological#HG1K, used as a negative control, "no capture antibody") was diluted to the same concentration. 200 μL of MUC1 Capture mAb, the three test antibodies, and IgG were added to each well of a 96-well plate and incubated overnight at 4°C. The plate was washed four times with PBST: 280 μL of PBS-Tween 20 (0.05%) was added to each well, and the remaining buffer was gently blotted dry on absorbent paper. Subsequent washing was performed in the same manner. 200 μL of 1% BSA-PBS solution was added, and the plate was incubated at 37°C for 1 hour for blocking. Four washes with PBST were then performed. According to the experimental design, the following samples were added to the plate: four groups of 200 μL pleural effusion samples, T47D cell lysis buffer samples (30 μg / well), and negative wells containing 0.1% BSA-PBS. The plates were incubated at room temperature for 3 hours, followed by 4 washes with PBST. Biotin-labeled MUC1 mAb was diluted 1:1000 with PBS, and 100 μL of Biotin-labeled MUC1 mAb was added to each well. The plates were incubated at room temperature for 1 hour, followed by 4 washes with PBST. HRP Streptavidin (SA-HRP) was diluted 1:2000 with PBS, and 100 μL of diluted SA-HRP was added to each well. The plates were incubated at room temperature for 30 minutes, followed by 4 washes with PBST. 100 μL of TMB substrate solution was added to each well, and after incubation at room temperature for 20 minutes, 50 μL of 2M H2SO4 was added to each well to stop the color development. The OD values ​​of the 96-well plate were then read using a microplate reader. 450 value.

[0261] As shown in Figure 10, the candidate MUC1 antibody B82 showed low binding signals to all four pleural and peritoneal fluid samples, indicating that the candidate antibody was only weakly affected by peripheral free MUC1.

[0262] 1.12 Construction and screening of antibody affinity maturation libraries

[0263] Anti-MUC1 antibody B82 and anti-EGFR antibody B307 were selected and engineered using phage display technology to obtain candidate molecules with higher activity.

[0264] Mutation primers were designed according to the library construction strategy. Using a 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.

[0265] Phage display libraries are screened using corresponding antigens. The resulting phage display mutant libraries or output sets are then processed through a series of steps, including inoculation, phage assistance infection, 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 antigens are enriched.

[0266] Based on the detection results of the initial screening output set, suitable library outputs were selected for monoclonal ELISA screening. The process involved a series of steps, including target antigen coating, blocking, primary antibody incubation, secondary antibody addition, color development, termination, and OD value detection. Clones with OD values ​​higher than the parent clone were selected as preferred clones and submitted for testing. Through ELISA screening and sequencing analysis, affinity-mature candidate molecules were identified. The amino acid sequences of the CDR regions of the obtained antibodies VH and VL are shown in Table 10. The CDR sequences were determined using the Kabat definition method.

[0267] Table 10. Sequences of VH and VL, candidate molecules for affinity maturation.

[0268] 1.13 Construction and Expression of Affinity-Maturing Antibodies

[0269] The affinity maturation candidate antibodies were expressed, purified, and subjected to quality testing according to the method described in Example 1.4. Specifically, the heavy chain of the anti-EGFR affinity maturation antibody B307mut3 is SEQ ID NO:31, and the light chain is SEQ ID NO:24; the heavy chain of the anti-MUC1 affinity maturation antibody B82mut4 is SEQ ID NO:28, and the light chain is SEQ ID NO:24; and the heavy chain of the anti-MUC1 affinity maturation antibody B82mut4 (LALA) is SEQ ID NO:29, and the light chain is SEQ ID NO:24.

[0270] SEQ ID NO:31 B307mut3 heavy chain:

[0271] SEQ ID NO:28 B82mut4 heavy chain:

[0272] SEQ ID NO:29 B82mut4(LALA) heavy chain:

[0273] 1.14 Detection of Affinity Maturation Antibody-Antigen Binding Activity

[0274] The binding ability of the anti-human MUC1 affinity maturation antibody molecule to the antigen was detected according to the method in Example 1.5.2.

[0275] The experimental results are shown in Figures 11A and 11B and Table 11. The mature affinity molecule B82mut4 showed significant binding activity with ST-MUC1, and the binding ability was higher than that of the parent antibody B82. Furthermore, the binding ability with non-glycosylated MUC1 remained at a low level.

[0276] Table 11 Affinity-matured antibody-antigen binding activity

[0277] 1.15 Detection of the binding ability of maturing antibodies to tumor cells

[0278] The binding ability of the affinity maturation antibody to tumor cells was detected according to the method in Example 1.7.

[0279] The results are shown in Figure 12 and Table 12. The anti-human EGFR affinity mature antibody molecule has a significant binding ability with A431 cells, which is comparable to that of the maternal antibody B307.

[0280] Table 12. Binding activity of human EGFR affinity maturation antibodies to A431 cells.

[0281] The results are shown in Figures 13A and 13B and Table 13. The anti-human MUC1 affinity mature antibody molecule has a significant binding ability with NCI-H1975 and OVCAR3 cells, and the binding ability is higher than that of the maternal antibody B82.

[0282] Table 13. Binding ability of human MUC1 affinity maturation antibody to NCI-H1975 and OVCAR3 cells.

[0283] 1.16 Affinity Maturation Antibody Internalization Ability Detection

[0284] Refer to Example 1.8 The method was to detect the internalization ability of anti-EGFR affinity maturation antibody molecules in NCI-H1975 (human lung adenocarcinoma cells) and KYSE150 (human esophageal squamous cell carcinoma cells), and the internalization ability of anti-MUC1 affinity maturation antibody molecules in OVCAR3 and NCI-H1975, respectively.

[0285] The results are shown in Figures 14A, 14B, 15A, and 15B. All the affinity-matured antibodies were able to internalize into the detection cells. Among them, the internalization ability of B307mut3 was comparable to that of the maternal antibody B307, while the internalization ability of B82mut4 was stronger than that of the maternal antibody B82.

[0286] 1.17 Detection of tumor-selective binding ability of anti-human MUC1 affinity maturation antibody molecules

[0287] The tumor-selective binding ability of the anti-human MUC1 affinity maturation antibody molecule was detected using the method described in Example 1.10.

[0288] As shown in Figure 16, the affinity maturation-preferred antibody B82mut4 (LALA) showed superior tumor-selective binding ability compared to the control antibody molecules Gatipotuzumab and HT186-D11.

[0289] Example 2: Preparation of anti-EGFR / MUC1 bispecific antibody

[0290] M1231 (sequence from WO2021247798, heavy chain sequences are SEQ ID NO:35 and 36, light chain sequence is SEQ ID NO:40) and control molecule M4H1L1-ZalH4 (sequence from WO2024213106A1, chain 1 sequence is SEQ ID NO:52, chain 2 sequence is SEQ ID NO:53, chain 3 sequence is SEQ ID NO:54, chain 4 sequence is SEQ ID NO:55) were compared.

[0291] The control antibody, used as a bispecific antibody, was produced using conventional recombinant antibody production processes.

[0292] SEQ ID NO:35 M1231analog heavy chain 1

[0293] SEQ ID NO:36 M1231analog heavy chain 2

[0294] SEQ ID NO:40 M1231analog light chain

[0295] SEQ ID NO:52 M4H1L1-ZalH4 chain 1

[0296] SEQ ID NO:53 M4H1L1-ZalH4 chain 2

[0297] SEQ ID NO:54 M4H1L1-ZalH4 chain 3

[0298] SEQ ID NO:55 M4H1L1-ZalH4 chain 4

[0299] 2.1 Construction and expression of bispecific antibodies

[0300] The coding sequences for the human clonal heavy chain constant region (LALA-hole), heavy chain constant region (LALA-knob), and light chain constant region κ were introduced into the pCDNA3.1 plasmid. The coding sequences for the heavy chain variable regions and light chain variable regions of anti-human EGFR and MUC1 antibodies were introduced into the pCDNA3.1 plasmid containing the 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 anti-human EGFR / MUC1 bispecific antibodies.

[0301] Table 14 Heavy and Light Chains of Bispecific Antibodies

[0302] SEQ ID NO:16 Heavy chain constant region (LALA-hole)

[0303] SEQ ID NO:17 Heavy chain constant region (LALA-knob)

[0304] SEQ ID NO:50 Heavy chain constant region (hole)

[0305] SEQ ID NO:51 Heavy chain constant region (knob)

[0306] SEQ ID NO:32 B82mut4 heavy chain (LALA-knob)

[0307] SEQ ID NO:33 B307 Heavy Chain (LALA-hole)

[0308] SEQ ID NO:34 B307mut3 heavy chain (LALA-hole)

[0309] SEQ ID NO:47 B82mut4 heavy chain (knob)

[0310] SEQ ID NO:48 B307 Heavy chain (hole)

[0311] SEQ ID NO:49 B307mut3 heavy chain (hole)

[0312] 2.2 Detection of Bispecific Antibody Internalization Ability

[0313] The internalization ability of the bispecific antibody was detected using the Incucyte method described in Example 1.8.

[0314] The results are shown in Figures 17A, 17B, and 17C. The constructed bispecific antibody molecule DB1001 showed significant internalization ability in MDA-MB-468, OVCAR-3, and NCI-H1975 cells, and its internalization ability was comparable to or better than that of the control molecule M1231analog.

[0315] 2.3 Detection of tumor-selective binding ability of bispecific antibodies

[0316] The tumor-selective binding ability of the bispecific antibody was detected using the method described in Example 1.10.

[0317] As shown in Figure 18, the bispecific antibody DB1001 exhibited superior tumor-selective binding ability compared to the parental EGFR monoclonal antibody B307.

[0318] 2.4 Affinity detection of bispecific antibody with recombinant human EGFR protein

[0319] 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 target 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.

[0320] The experimental results were analyzed using Biacore Insight Evaluation Software.

[0321] 2.5 Detection of the internalization ability of bispecific antibodies on different tumor cells

[0322] The internalization ability of the antibody on MDA-MB-468 (breast cancer cells, source: ATCC), OVCAR-3 (ovarian cancer cells, source: ATCC) and NCI-H1975 (lung cancer cells, source: ATCC) was detected by flow cytometry, referring to the flow cytometry method in Example 1.8.2.

[0323] The results are shown in Figures 19A-19C. The bispecific antibody DB1001 showed better cell internalization ability compared to the control molecule.

[0324] Example 3: Preparation of bispecific antibody-drug conjugates

[0325] Connector-cytotoxin X2 is synthesized according to patent WO2022068878A1.

[0326] 3.1 Preparation method of antibody-drug conjugates

[0327] The reducing agent and the protective agent were prepared separately using ultrapure water as follows: 5 mmol / L TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo) aqueous solution and 100 mmol / L EDTA (disodium ethylenediaminetetraacetate, manufacturer: Sigma) aqueous solution.

[0328] Take 200 mg of antibody (25.00 mg / ml) and place it in a 50 mL centrifuge tube. Dilute the antibody concentration to 10 mg / ml with 20 mM His-HCl, pH 6.0 buffer. Add 100 mM EDTA aqueous solution at 5% of the total reaction volume. After vortexing and mixing, add 5 mmol / L TCEP aqueous solution for antibody reduction. The molar ratio of TCEP to antibody is 10:1. After vortexing and mixing, react in a refrigerated thermostat at 37°C for 2 h. Add the aforementioned adapter-cytotoxic X2 DMSO solution at a final drug-to-antibody molar ratio of 12:1. Add DMSO at 10% of the total reaction volume. After vortexing and mixing, react in a refrigerated thermostat at 4°C for 1 h. The sample preservation buffer was replaced using an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore). The sample was first ultrafiltered three times with 20mM His-HCl buffer containing 10% DMSO at pH 6.0, and then ultrafiltered six times with 20mM His-HCl buffer without DMSO at pH 6.0 to obtain the antibody-drug conjugate. The drug loading (DAR / p) of the antibody-drug conjugate was detected by RP-HPLC and SEC-HPLC.

[0329] 3.1.1 Preparation of ADC1

[0330] Using the preparation method described in Example 3.1 above, the molar ratio of TCEP to antibody was adjusted to 4:1, and the final molar ratio of drug to antibody concentration was 10:1. Antibody-drug conjugate ADC1 was prepared by conjugating antibody DB1001 with adapter-cytotoxin X2. The DAR / p ratio was measured to be 5.9.

[0331] 3.1.2 Preparation of ADC2

[0332] Using the preparation method described in Example 3.1 above, the molar ratio of TCEP to antibody was adjusted to 4:1, and the final molar ratio of drug to antibody concentration was 10:1. Antibody-drug conjugate ADC2 was prepared by conjugating antibody DB1002 with adapter-cytotoxin X2. The DAR / p ratio was measured to be 6.17.

[0333] 3.1.3 Preparation of ADC3

[0334] Using the preparation method described in Example 3.1 above, antibody-drug conjugate ADC3 was prepared by conjugating antibody DB1001 with linker-cytotoxin X2. The DAR / p ratio was measured to be 7.93.

[0335] 3.1.4 Preparation of ADC4

[0336] Using the preparation method described in Example 3.1 above, the molar ratio of TCEP to antibody was adjusted to 4:1, and the final molar ratio of drug to antibody concentration was 10:1. Antibody-drug conjugate ADC4 was prepared by conjugating antibody DB1003 with adapter-cytotoxin X2. The DAR / p ratio was measured to be 5.82.

[0337] 3.1.5 Preparation of ADC5

[0338] Using the preparation method described in Example 3.1 above, the molar ratio of TCEP to antibody was adjusted to 4:1, and the final molar ratio of drug to antibody concentration was 10:1. Antibody-drug conjugate ADC5 was prepared by conjugating antibody DB1004 with adapter-cytotoxin X2. The DAR / p ratio was measured to be 6.06.

[0339] 3.1.6 Preparation of ADC6

[0340] Using the preparation method described in Example 3.1 above, antibody-drug conjugate ADC6 was prepared by conjugating antibody DB1003 with linker-cytotoxin X2. The DAR / p ratio was measured to be 7.92.

[0341] The structure of the control molecule M4H1L1-ZalH4-9-A (prepared according to WO2024213106A1) is as follows, n=6:

[0342] 3.2 Detection of the internalization ability of antibody-drug conjugates on tumor cells

[0343] Refer to Example 1.8.1 The method is used to detect the internalization ability of antibody-drug conjugates.

[0344] The internalization ability of antibody-drug conjugates was detected by flow cytometry according to Example 1.8.2.

[0345] As shown in Figure 20, the antibody-drug conjugate ADC1 exhibited significant internalization ability in different cells, and its internalization ability was comparable to that of the parent antibody, indicating that the ADC conjugation process did not change the antibody's internalization ability.

[0346] The results are shown in Figures 21A and 21B. The antibody-drug conjugate ADC1 showed significant internalization ability in different cells, which was superior to that of the control molecule.

[0347] 3.3 In vitro assay of antibody-drug conjugates to inhibit tumor cell proliferation

[0348] use The chemiluminescence immunoassay (CTG method) was used to evaluate the inhibitory effect of anti-EGFR / MUC1 bispecific antibody-drug conjugates ADC1 and ADC2 on cell proliferation after incubation for 7 days in tumor cells expressing EGFR and MUC1.

[0349] Log-phase cells were collected, and tumor cells were seeded at optimized cell densities in 50 μL wells. The cells were incubated overnight at 37°C with 5% CO2. On the second day, ADC1, ADC2, and ADC1 naked anti-DB1001 were diluted 3-fold with complete culture medium to obtain eight concentration gradients (starting with the highest concentration of 1000 nM). 50 μL / well of each drug was added to the cell culture plate, with complete culture medium used as a blank control. Two replicates were set up. The cells were incubated at 37°C with 5% CO2 for 7 days. After incubation, the cell culture plates were removed and allowed to equilibrate to room temperature. 50 μL of CTG assay reagent (Promega, Cat#: G7573) was added to each well, vortexed, and incubated in the dark for 10 minutes. The signal values ​​were then read using a microplate reader. GraphPad Prism software was used to plot sigmoid dose-response curves using a nonlinear regression model, and the IC50 value was calculated. Cell viability calculation formula = (Lum test drug - Lum blank control) / (Lum solvent blank control - Lum blank control) × 100%.

[0350] The results are shown in Figures 22A, 22B, 22C, 22D, 22E, and 22F, and Table 15. The experimental results show that ADC1 and ADC2 have strong inhibitory activity against the proliferation of various tumor cells, while the unconjugated antibody did not show any inhibitory effect on tumor cell growth.

[0351] Table 15 Inhibitory activity of antibody-drug conjugates against tumor cell proliferation in vitro.

[0352] 3.4 Evaluation of the efficacy of antibody-drug conjugates in the HCC1806 mouse model

[0353] To investigate the inhibitory effect of the bispecific antibody-drug conjugate against EGFR and MUC1 on tumor growth, a homologous transplantation mouse model was constructed in CB-17SCID mice using HCC1806 breast cancer cells to evaluate the antitumor effect of the bispecific antibody-drug conjugate ADC1.

[0354] 1. Test drug and materials

[0355] G1: Blank control group (control group): DPBS

[0356] G2:ADC1: 3mg / kg

[0357] G3:ADC1: 6mg / kg

[0358] 2. Preparation method: All samples were prepared by diluting with DPBS.

[0359] 3. Experimental animals: CB-17SCID mice, female, 6-8 weeks old, weighing approximately 16-20g. Purchased from Vital Rivers.

[0360] 4. Experimental Methods: HCC1806 cell resuscitation and culture. HCC1806 cells in the logarithmic growth phase (5th generation after resuscitation) were collected, the culture medium was removed, and the cells were washed twice with DPBS before seeding (cell viability 96.34%). Seeding density: 5 × 10⁶ cells / year. 6 200 μL of cells (1:1 with added Matrigel) were subcutaneously injected into the right side of mice. The tumor volume reached 150 mm. 3 Groups were formed, with the grouping day defined as D1, and drug administration began on D1. The coefficient of variation (CV) for tumor volume should not exceed 1 / 3. The test substance ADC1 was administered intravenously (iv) once a week for a total of one injection, at doses of 3 mg / kg and 6 mg / kg. The experiment ended after 24 days of drug administration. Tumor volume and body weight were measured 2–3 times per week, and the data were recorded. The tumor inhibition rate was calculated by measuring tumor volume.

[0361] At the end of the experiment, the mice were euthanized, and the tumor inhibition rate (TGI) was calculated (TGI(%) = [1 - (Ti - T1) / (Vi - V1)] × 100). Ti: mean tumor volume on day i of treatment and positive control; T1: mean tumor volume on day 1 of treatment and positive control; Vi: mean tumor volume on day i of negative control; V1: mean tumor volume on day 1 of negative control.

[0362] The tumor growth curve, animal body weight, and terminal tumor weight data are shown in Figure 23A, Figure 23B, and Figure 23C, respectively. The results showed that the terminal TGI of ADC1 was 106.5% and 106.8% at the doses of 3 mg / kg and 6 mg / kg, respectively.

[0363] 3.5 Pharmacodynamic Evaluation of Antibody-Drug Conjugate in Human Esophageal Cancer LD1-0015-362418 PDX Model

[0364] To study the inhibitory effect of the bispecific antibody-drug conjugate against EGFR and MUC1 of this application on tumor growth, a human esophageal cancer PDX animal model LD1-0015-362418 was constructed in NU / NU mice to evaluate the anti-tumor effect of the bispecific antibody-drug conjugate ADC1.

[0365] 1. Test Drugs and Materials

[0366] G1: Blank control group (control group): Normal saline

[0367] G2: ADC1: 3 mg / kg

[0368] G3: ADC1: 6 mg / kg

[0369] 2. Preparation method: All samples were diluted and prepared with normal saline.

[0370] 3. Experimental animals: NU / NU mice, female, 6 - 8 weeks old, body weight 18 - 22 g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (SCXK(Beijing)2021 - 0006).

[0371] 4. Experimental method: The tumor mass of the successfully revived FP2+5 generation LD1-0015-362418 human esophageal cancer orthotopic transplantation tumor was cut into small pieces about 3 mm × 3 mm × 3 mm, and inoculated subcutaneously on the right back of NU / NU mice using a tumor mass inoculation needle (the pharmacodynamic experiment generation was FP1+4). Each animal was inoculated with about 60 - 75 mg of tumor tissue and 25 μL of Matrigel was added. Observe the mice after inoculation and monitor the tumor growth. On the 24th day after inoculation, when the average tumor volume of the tumor-bearing mice was 149.56 mm 3 at this time, grouping and dosing were carried out, and the day of grouping and dosing was defined as day 0. The test substance ADC1 was injected intravenously (i.v.) at the doses of 3 mg / kg and 6 mg / kg, and each was injected once on day 0 and day 10, respectively. The experiment ended after 35 days of dosing. The tumor volume and body weight were measured 2 times a week, and the data were recorded. The tumor inhibition rate was calculated by measuring the tumor volume.

[0372] At the end of the experiment, the mice were euthanized. Tumor growth inhibition rate TGI (%) = [1-(Ti-T0) / (Vi-V0)]×100, where Ti is the average tumor volume after the start of drug administration in the treatment group, T0 is the average tumor volume at the first drug administration in the treatment group, V0 is the average tumor volume at the first drug administration in the vehicle control group, and Vi is the average tumor volume after the start of drug administration in the vehicle control group.

[0373] Tumor growth curves, animal body weight, and endpoint tumor weight data are shown in Figures 24A, 24B, and 24C, respectively. The results show that ADC1 can inhibit tumor cell growth in a dose-dependent manner.

[0374] Table 16 Evaluation of the antitumor efficacy of the test drug in a human subcutaneous xenograft model of esophageal cancer (LD1-0015-362418). Notes: a. Mean ± SEM; b. Comparisons among the 5 groups in this experiment were performed using one-way ANOVA followed by Dunnett's multiple comparison analysis. The p-values ​​for Day 35 are shown in the table (ns: not significant, **: P < 0.01, ****: P < 0.0001). c. T-test analysis was used between two groups. The p-values ​​for Day 35 are shown in the table (**: P < 0.01).

[0375] Table 17. Endpoint tumor weight analysis after treatment with the test drug in a human esophageal cancer subcutaneous xenograft tumor model with an LD1 of 0015-362418. Notes: a. Mean ± SEM; b. Comparisons among the 5 groups in this experiment were performed using one-way ANOVA followed by Dunnett's multiple comparison analysis. The p-values ​​for Day 35 are shown in the table (ns: not significant, **: P < 0.01, ****: P < 0.0001). c. T-test analysis was used between two groups. The p-values ​​for Day 35 are shown in the table (**: P < 0.01).

[0376] 3.6 Efficacy evaluation of antibody-drug conjugates in the NCI-H2126 lung cancer CDX model

[0377] To investigate the inhibitory effect of the bispecific antibody-drug conjugates against EGFR and MUC1 on tumor growth, a homologous transplantation mouse model was constructed in CB-17SCID mice using NCI-H2126 non-small cell lung cancer cells to evaluate the antitumor effects of the bispecific antibody-drug conjugates ADC1 and ADC5.

[0378] 1. Test drug and materials

[0379] G1: Blank control group (control group): DPBS

[0380] G2:ADC5: 3mg / kg

[0381] 2. Preparation method: All samples were prepared by diluting with DPBS.

[0382] 3. Experimental animals: CB-17SCID mice, female, 6-8 weeks old, weighing approximately 17-20g. Purchased from Vital Rivers.

[0383] 4. Experimental Methods: NCI-H2126 cells were revived and cultured. NCI-H2126 cells in logarithmic growth phase (8th generation after revival) were collected, the culture medium was removed, and the cells were washed twice with DPBS before seeding (cell viability 98.06%). Seeding volume: 1×10⁷ cells / 200 μL / mouse (1:1 with added Matrigel) was subcutaneously seeded on the right side of mice. The tumor volume reached 158 mm². 3 Groups were formed, with the grouping day defined as D1, and drug administration began on D1. The coefficient of variation (CV) for tumor volume did not exceed 1 / 3. Test substances ADC1 and ADC5 were administered intravenously (iv) on days 1 and 14, respectively, at a dose of 3 mg / kg. Tumor volume and body weight were measured 2–3 times weekly, and data were recorded. The tumor inhibition rate was calculated based on tumor volume measurements.

[0384] The tumor inhibition rate (TGI) was calculated as follows: TGI(%) = [1 - (Ti - T1) / (Vi - V1)] × 100. Ti: mean tumor volume on day i of treatment and positive control; T1: mean tumor volume on day 1 of treatment and positive control; Vi: mean tumor volume on day i of negative control; V1: mean tumor volume on day 1 of negative control.

[0385] Tumor growth curves and animal body weight data are shown in Figures 25A and 25B, respectively. The results show that both ADC5 and ADC1 have good tumor growth inhibitory effects, and at the same dose, ADC5 has a more significant tumor growth inhibitory effect than ADC1.

[0386] Table 18 Antitumor efficacy of antibody-drug conjugates in the NCI-H2126 lung cancer CDX model

[0387] 3.7 Efficacy evaluation of antibody-drug conjugates in the SW480 colorectal cancer CDX model

[0388] To investigate the inhibitory effect of the bispecific antibody-drug conjugate against EGFR and MUC1 on tumor growth, a homologous transplantation mouse model was constructed in Balb / cNude mice using SW480 intestinal cancer cells to evaluate the antitumor effect of the bispecific antibody-drug conjugate ADC2.

[0389] 1. Test drug and materials

[0390] G1: Blank control group (control group): physiological saline

[0391] G2:ADC2, 6 mg / kg

[0392] G3:ADC2, 10 mg / kg

[0393] 2. Preparation method: All samples were prepared by diluting with physiological saline.

[0394] 3. Experimental animals: Balb / c Nude mice, female, 6-8 weeks old, weighing approximately 17-20g. Purchased from Vital Rivers.

[0395] 4. Experimental Methods: SW480 cells were revived and cultured. Cells in the logarithmic growth phase were collected, counted, and then used for tumor inoculation. Inoculation volume: 5 × 10⁻⁶ 6 200 μL / mouse (1:1 with added matrix gel) of cells were subcutaneously injected into mice. Mice were grouped when the tumor volume reached 166 mm³, with the day of grouping defined as D0, and drug administration began on D0. The test substance ADC2 was administered intravenously (iv) once, at doses of 6 mg / kg and 10 mg / kg. Tumor volume and body weight were measured twice weekly, and data were recorded. The tumor inhibition rate was calculated based on tumor volume measurements.

[0396] The tumor inhibition rate (TGI) was calculated as follows: TGI(%) = [1 - (Ti - T0) / (Vi - V0)] × 100. Ti: mean tumor volume on day i of treatment and positive control; T0: mean tumor volume on day 0 of treatment and positive control; Vi: mean tumor volume on day i of negative control; V0: mean tumor volume on day 0 of negative control.

[0397] Tumor growth curves and animal body weight data are shown in Figures 26A and 26B, respectively. The results showed that ADC2 had good tumor growth inhibitory effects at doses of 6 mg / kg and 10 mg / kg.

[0398] Table 19 Antitumor efficacy of antibody-drug conjugates in SW480 colorectal cancer CDX model

[0399] 3.8 Efficacy evaluation of antibody-drug conjugates with different DAR values ​​in the NCI-H1975 lung cancer CDX model

[0400] To investigate the inhibitory effect of different DAR values ​​of the bispecific antibody-drug conjugates against EGFR and MUC1 on tumor growth, a homologous transplantation mouse model was constructed in Balb / c Nude mice using NCI-H1975 non-small cell lung cancer cells to evaluate the antitumor effects of the bispecific antibody-drug conjugates ADC4 and ADC6.

[0401] 1. Test drug and materials

[0402] G1: Blank control group (control group): DPBS

[0403] G2:ADC4, 5mg / kg

[0404] G3:ADC6, 5mg / kg

[0405] 2. Preparation method: All samples were prepared by diluting with DPBS.

[0406] 3. Experimental animals: Balb / c Nude mice, female, 6-8 weeks old, weighing approximately 16-19g. Purchased from Vital Rivers.

[0407] 4. Experimental Methods: NCI-H1975 cells were revived and cultured. NCI-H1975 cells in logarithmic growth phase (3rd generation after revival) were collected, the culture medium was removed, and the cells were washed twice with DPBS before seeding (cell viability 96.08%). Seeding density: 2 × 10⁶ cells / year. 6 200 μL of cells (1:1 with added Matrigel) were subcutaneously injected into the right side of mice. The tumor volume reached 104 mm. 3 Groups were formed, with the grouping day defined as D1, and drug administration began on D1. The coefficient of variation (CV) for tumor volume was not to exceed 1 / 3. Test substances ADC4 and ADC6 were administered intravenously (iv) once, at a dose of 5 mg / kg each. The experiment ended after 22 days of administration. Tumor volume and body weight were measured 2-3 times weekly, and data were recorded. The tumor inhibition rate was calculated based on tumor volume measurements.

[0408] The tumor inhibition rate (TGI) was calculated as follows: TGI(%) = [1 - (Ti - T1) / (Vi - V1)] × 100. Ti: mean tumor volume on day i of treatment and positive control; T1: mean tumor volume on day 1 of treatment and positive control; Vi: mean tumor volume on day i of negative control; V1: mean tumor volume on day 1 of negative control.

[0409] Tumor growth curves and animal weight data are shown in Figures 27A and 27B, respectively. The results show that both ADC6 and ADC4 have good tumor growth inhibitory effects, and at the same dose, ADC6 has a stronger tumor growth inhibitory effect than ADC4.

[0410] Table 20 Antitumor effects of antibody-drug conjugates with different DAR values ​​in the NCI-H1975 lung cancer CDX model.

[0411] 3.9 Efficacy evaluation of antibody-drug conjugates with different DAR values ​​in the NCI-H1650 lung cancer CDX model

[0412] To investigate the inhibitory effect of different DAR values ​​of the bispecific antibody-drug conjugates against EGFR and MUC1 on tumor growth, a homologous transplantation mouse model was constructed in NOD SCID mice using NCI-H1650 lung cancer cells to evaluate the antitumor effects of the bispecific antibody-drug conjugates ADC1 and ADC3.

[0413] 1. Test drug and materials

[0414] G1: Blank control group (control group): DPBS

[0415] G2:ADC1, 10 mg / kg

[0416] G3:ADC3, 10 mg / kg

[0417] 2. Preparation method: All samples were prepared by diluting with DPBS.

[0418] 3. Experimental animals: NOD SCID mice, female, 6-8 weeks old, weighing approximately 16-19g. Purchased from Vital Rivers.

[0419] 4. Experimental Methods: NCI-H1650 cells were revived and cultured. NCI-H1650 cells in the logarithmic growth phase (6th generation after revival) were collected, the culture medium was removed, and the cells were washed twice with DPBS before seeding (cell viability 94.83%). Seeding density: 7 × 10⁶ cells / year. 6 200 μL of cells (1:1 with added Matrigel) were subcutaneously injected into the right side of mice. The tumor volume reached 150 mm. 3 Grouping was performed on a daily basis, with the day of grouping defined as D1, and drug administration began on D1. The coefficient of variation (CV) of tumor volume should not exceed 1 / 3. Test substances ADC1 and ADC3 were administered intravenously (iv) once, at a dose of 10 mg / kg each. The experiment ended after 26 days of administration. Tumor volume and body weight were measured 2-3 times weekly, and the data were recorded. The tumor inhibition rate was calculated based on the tumor volume measurement.

[0420] The tumor inhibition rate (TGI) was calculated as follows: TGI(%) = [1 - (Ti - T1) / (Vi - V1)] × 100. Ti: mean tumor volume on day i of treatment and positive control; T1: mean tumor volume on day 1 of treatment and positive control; Vi: mean tumor volume on day i of negative control; V1: mean tumor volume on day 1 of negative control.

[0421] Tumor growth curves and animal weight data are shown in Figures 28A and 28B, respectively. The results show that both ADC3 and ADC1 have good tumor growth inhibitory effects, and at the same dose, ADC3 has a stronger tumor growth inhibitory effect than ADC1.

[0422] Table 21 Antitumor effects of antibody-drug conjugates with different DAR values ​​in the NCI-H1650 lung cancer CDX model

[0423] 3.10 Toxicokinetic and pharmacokinetic studies of antibody-drug conjugates in rats and monkeys

[0424] 1. In vivo studies in monkeys

[0425] The antibody-drug conjugate was administered intravenously to monkeys, once every 3 weeks for a total of 2 doses. One monkey per sex per group was used to observe the nature, extent, dose-response relationship, and time-response relationship of potential toxic reactions caused by the antibody-drug conjugate, and to determine the target organs or tissues for toxicity, providing a reference for future research.

[0426] During the experiment, animals were clinically observed, and their weight, food intake, body temperature, electrocardiogram, and clinical pathology (blood cell count, coagulation function, and blood biochemistry) were monitored. Simultaneously, the concentrations of the antibody-drug conjugate (ADC) in serum and the concentrations of small molecules in plasma were measured for toxicokinetic analysis. Furthermore, a single dose at a concentration of 10 mpk was administered to test the pharmacokinetic (PK) of the ADC and small molecules in monkeys.

[0427] The results showed that no animals in any of the dosage groups died or were near death. No abnormalities related to the test product were observed in body weight, food intake, body temperature, electrocardiogram parameters and waveforms, clinical pathology (blood cell count, coagulation function, and blood biochemistry), gross anatomical examination, or histopathological examination (bone and bone marrow). This indicates that the antibody-drug conjugate provided in this application has good safety.

[0428] 2. In vivo studies in rats

[0429] SD rats were intravenously injected with 10 mg / kg of ADC1 and ADC3. Four rats were in each group (two males and two females). Serum samples were collected from rats at different time points after administration for analysis.

[0430] The results show that ADC1 and ADC3 have good Cmax and AUC as well as long half-life.

[0431] References:

[0432] (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

[0433] (2) Fumoto, M.; Hinou, H.; Ohta, T.; Ito, T.; Yamada, K.; Takimoto, A.; Kondo, H.; Shimizu, H.; Inazu, T.; Nakahara, Y.; glycopeptides.J.Am.Chem.Soc.2005,127,11804-11818

[0434] (3)Dalziel, M.;Whitehouse,C.;McFarlane,I.;Brockhausen,I.;Gschmeissner,S.;Schwientek,T.;Clausen,H.;Burchell,JM;TaylorPapadimitriou,J. MUC1.J.Biol.Chem.2001,276,11007-11015.

[0435] While specific embodiments of this disclosure 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 this disclosure. Therefore, the scope of protection of this disclosure is defined by the appended claims.

Claims

An antibody drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that, The antibody-drug conjugate or its pharmaceutically acceptable salt comprises a bispecific antibody or antigen-binding fragment that binds to EGFR and MUC1, a linker unit L, and a cytotoxic drug portion; The bispecific 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 amino acid sequences of H1CDR1, H1CDR2, and H1CDR3 contained in VH1 are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; the amino acid sequences of H2CDR1, H2CDR2, and H2CDR3 contained in VH2 are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 contained in VL are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. or, The amino acid sequences of H1CDR1, H1CDR2, and H1CDR3 contained in VH1 are shown in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:6, respectively; the amino acid sequences of H2CDR1, H2CDR2, and H2CDR3 contained in VH2 are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the amino acid sequences of LCDR1, LCDR2, and LCDR3 contained in VL are shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. The antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in claim 1 is characterized in that, The bispecific antibody or its antigen-binding fragment satisfies one or more of the following: 1) The VH1 comprises the amino acid sequence shown in SEQ ID NO:13 or SEQ ID NO:14, or has 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:13 or SEQ ID NO:14; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same antigen-binding function as the original sequence; the amino acid sequence of the VH1 is preferably as shown in SEQ ID NO:13 or SEQ ID NO:14; 2) The VH2 comprises the amino acid sequence shown in SEQ ID NO:12, or has 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:12; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same antigen-binding function as the original sequence; the amino acid sequence of the VH2 is preferably as shown in SEQ ID NO:12; and, 3) The VL comprises the amino acid sequence shown in SEQ ID NO:15, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:15; the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same antigen-binding function as the original sequence; the amino acid sequence of the VL is preferably as shown in SEQ ID NO:15; Preferably, the amino acid sequences of VH1, VH2 and VL are as shown in SEQ ID NO:13, SEQ ID NO:12 and SEQ ID NO:15, respectively; or, the amino acid sequences of VH1, VH2 and VL are as shown in SEQ ID NO:14, SEQ ID NO:12 and SEQ ID NO:15, respectively. The antibody-drug conjugate or its pharmaceutically acceptable salt as described in claim 1 or 2 is characterized in that, The bispecific antibody or its antigen-binding fragment includes one or more of the following: (1) Fully human antibodies, humanized antibodies, chimeric antibodies, proantibodies, bispecific antibodies, monoclonal antibodies or polyclonal 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. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-3, wherein 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 HC1, and the MUC1 binding domain includes a light chain constant region CL and a heavy chain constant region HC2. Preferably, the heavy chain constant region is the heavy chain constant region of human antibody IgG1, and the light chain constant region is the light chain constant region of human antibody κ chain. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to claim 4, characterized in that, The heavy chain constant region contains the Knob-into-hole mutation; Preferably, HC1 contains a Knob mutation, such as T366W, and HC2 contains a Hole mutation, such as one or more selected from T366S, L368A, and Y407V; or, HC2 contains a Knob mutation, such as T366W, and HC1 contains a Hole mutation, such as one or more selected from T366S, L368A, and Y407V. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to claim 4 or 5, characterized in that, The heavy chain constant regions HC1 and HC2 contain L234A / L235A mutations according to the EU numbering system. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 4-6, characterized in that, The bispecific antibody or its antigen-binding fragment satisfies any one of the following: 1) The heavy chain constant region HC1 contains the amino acid sequence shown in SEQ ID NO:16, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16; and / or, the heavy chain constant region HC2 contains the amino acid sequence shown in SEQ ID NO:17, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:17; the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence; 2) The heavy chain constant region HC1 contains the amino acid sequence shown in SEQ ID NO:17, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:17; and / or, the heavy chain constant region HC2 contains SEQ ID NO:16, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:16; the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence; 3) The heavy chain constant region HC1 contains the amino acid sequence shown in SEQ ID NO:50, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:50; and / or, the heavy chain constant region HC2 contains SEQ ID NO:51, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:51; the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence; and, 4) The heavy chain constant region HC1 contains the amino acid sequence shown in SEQ ID NO:51, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:51; and / or, the heavy chain constant region HC2 contains SEQ ID NO:50, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:50; the amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity maintains at least the same function as the original sequence; Preferably, the amino acid sequence of the heavy chain constant region HC1 is shown in SEQ ID NO:16, and the amino acid sequence of the heavy chain constant region HC2 is shown in SEQ ID NO:

17. or, The amino acid sequence of the heavy chain constant region HC1 is shown in SEQ ID NO:17, and the amino acid sequence of the heavy chain constant region HC2 is shown in SEQ ID NO:

16. or, The amino acid sequence of the heavy chain constant region HC1 is shown in SEQ ID NO:50, and the amino acid sequence of the heavy chain constant region HC2 is shown in SEQ ID NO:

51. or, The amino acid sequence of the heavy chain constant region HC1 is shown in SEQ ID NO:51, and the amino acid sequence of the heavy chain constant region HC2 is shown in SEQ ID NO:

50. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 4-7, characterized in that, The light chain constant region CL contains the amino acid sequence shown in SEQ ID NO:18, or has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:18; the amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity maintains at least the same function as the original sequence; Preferably, the amino acid sequence of the light chain constant region CL is shown in SEQ ID NO:

18. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-8, wherein The EGFR binding domain comprises a heavy chain H1 and a light chain L, and the MUC1 binding domain comprises a heavy chain H2 and a light chain L; wherein the bispecific antibody or its antigen-binding fragment satisfies any one of the following: 1) The amino acid sequences of H1, H2 and L are shown in SEQ ID NO:33, SEQ ID NO:32 and SEQ ID NO:24, respectively; 2) The amino acid sequences of H1, H2 and L are shown in SEQ ID NO:34, SEQ ID NO:32 and SEQ ID NO:24, respectively; 3) The amino acid sequences of H1, H2, and L are shown in SEQ ID NO:48, SEQ ID NO:47, and SEQ ID NO:24, respectively; and, 4) The amino acid sequences of H1, H2 and L are shown in SEQ ID NO:49, SEQ ID NO:47 and SEQ ID NO:24, respectively. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-9, wherein the cytotoxic drug moiety is of the structure shown in formula (A-1), a stereoisomer, a pharmaceutically acceptable salt, a solvate, or a solvate of a salt thereof, in, M is -L 2 -L 1 -C(O)-; L 2 is -O-, and L 2 is connected to the linker unit L; L 1 -(C(R) 1a (R) 1b )) m -CH2-; m is selected from 1, 2, 3, or 4; each R 1a and R 1b is independently hydrogen, halogen, hydroxyl, amino, or C1-C6alkyl optionally substituted with one or more R; Each R is independently either hydrogen or halogen. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to claim 10, characterized in that, each R 1a independently hydrogen, halogen, or Ci-C6alkyl; and / or, each R 1b independently hydrogen, halogen, or Ci-C6alkyl; and / or, L 1 To The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein wherein the cytotoxic drug moiety is any one of the following structures: The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-12, wherein wherein said linker unit L is -L a -L b -L c -; and said L c is attached to said cytotoxic drug moiety; Preferably, the connector unit L satisfies one or more of the following: 1) -L a - is Preferably wherein a is attached to the bispecific antibody or antigen binding fragment thereof and b is attached to L b ; 2) -L b - is any of the following structures: Preferably More preferably wherein the c-terminus and L a are connected, the d-terminus and L c are connected; and, 3) -L c - is The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-13, wherein wherein the linker unit L is Preferably The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-14, wherein in, The structure of the antibody drug conjugate or the pharmaceutically acceptable salt thereof is shown as formula (A-2): Where p is p' or p”, p' represents the number of connections, and p' is any integer from 1 to 10; for example, the number of connections p' is any integer from 3 to 9; for example, the number of connections p' is any integer from 5 to 8; the number of connections p' is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; for example, 6 or 8; and / or, p” represents the average number of connections, and p” is any integer or decimal from 1 to 10; for example, the number of connections p” is any integer or decimal from 3 to 9; for example, the number of connections p” is any integer or decimal from 5 to 8; the average number of connections p” is an integer or decimal from 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or, 9 to 10, for example, 5.9, 6.17, 7.93, 5.82, 6.06 or 7.92; Preferably, the antibody-drug conjugate or its pharmaceutically acceptable salt satisfies one or more of the following conditions: 1) Ab is a bispecific antibody or its antigen-binding fragment as defined in any one of claims 1-9; 2) M is M as defined in any one of claims 10-12; and, 3) L is the connector unit L as defined in claim 13 or 14. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-15, wherein The antibody-drug conjugate or its pharmaceutically acceptable salt thereof is selected from the following structural formulas: in, p is the p as defined in claim 15; And / or, Ab is the bispecific antibody or its antigen-binding fragment as defined in any one of claims 1-9. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-16, wherein The antibody-drug conjugate or its pharmaceutically acceptable salt is as follows: Wherein, p is the p as defined in claim 15; And / or, DB1001, DB1002, DB1003, and DB1004 are bispecific antibodies binding to EGFR and MUC1, wherein the bispecific antibody comprises an EGFR-binding domain and a MUC1-binding domain, the EGFR-binding domain comprising a heavy chain H1 and a light chain L, and the MUC1-binding domain comprising a heavy chain H2 and a light chain L; wherein the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1001 are shown in SEQ ID NO:33, SEQ ID NO:32, and SEQ ID NO:24, respectively; the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1002 are shown in SEQ ID NO:34, SEQ ID NO:32, and SEQ ID NO:24, respectively; and the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1003 are shown in SEQ ID NO:48, SEQ ID NO:47, and SEQ ID NO:48, respectively. As shown in NO:24; the heavy chain H1, heavy chain H2 and light chain L amino acid sequences of the DB1004 are shown in SEQ ID NO:49, SEQ ID NO:47 and SEQ ID NO:24, respectively. The antibody drug conjugate or pharmaceutically acceptable salt thereof according to any one of claims 1-17, wherein The antibody drug conjugate or pharmaceutically acceptable salt thereof is a conjugate as follows or a pharmaceutically acceptable salt thereof: in, DB1001, DB1002, DB1003, and DB1004 are bispecific antibodies binding to EGFR and MUC1. Each bispecific antibody comprises an EGFR-binding domain and a MUC1-binding domain. The EGFR-binding domain includes a heavy chain H1 and a light chain L, and the MUC1-binding domain includes a heavy chain H2 and a light chain L. The amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1001 are shown in SEQ ID NO:33, SEQ ID NO:32, and SEQ ID NO:24, respectively; the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1002 are shown in SEQ ID NO:34, SEQ ID NO:32, and SEQ ID NO:24, respectively; and the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1003 are shown in SEQ ID NO:48, SEQ ID NO:47, and SEQ ID NO:24, respectively. As shown; the heavy chain H1, heavy chain H2 and light chain L amino acid sequences of the DB1004 are shown in SEQ ID NO:49, SEQ ID NO:47 and SEQ ID NO:24, respectively. A method of preparing an antibody drug conjugate or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1-18, characterized in that, The preparation method includes reacting a bispecific antibody or its antigen-binding fragment with a compound as shown in Formula II to obtain the antibody-drug conjugate or a pharmaceutically acceptable salt thereof. L'-Cytotoxic Drug II; L' is L of the linker unit L as defined in claim 13 or 14 a the non-ligated state of the a end; the cytotoxic drug is a cytotoxic drug moiety as defined in any one of claims 10 to 12; Preferably, the antibody-drug conjugate or its pharmaceutically acceptable salt satisfies one or more of the following conditions: (1) a compound represented by Formula II: (2) The bispecific antibodies are DB1001, DB1002, DB1003 and DB1004; DB1001, DB1002, DB1003, and DB1004 are bispecific antibodies that bind to EGFR and MUC1. The bispecific antibody or its antigen-binding fragment contains an EGFR-binding domain and a MUC1-binding domain. The EGFR-binding domain includes a heavy chain H1 and a light chain L, and the MUC1-binding domain includes a heavy chain H2 and a light chain L. The amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1001 are shown in SEQ ID NO:33, SEQ ID NO:32, and SEQ ID NO:24, respectively; the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1002 are shown in SEQ ID NO:34, SEQ ID NO:32, and SEQ ID NO:24, respectively; and the amino acid sequences of the heavy chain H1, heavy chain H2, and light chain L of DB1003 are shown in SEQ ID NO:48, SEQ ID NO:47, and SEQ ID NO:48, respectively. As shown in NO:24; the heavy chain H1, heavy chain H2 and light chain L amino acid sequences of the DB1004 are shown in SEQ ID NO:49, SEQ ID NO:47 and SEQ ID NO:24, respectively. A pharmaceutical composition, characterized in that, It comprises the antibody-drug conjugate as described in any one of claims 1-18 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The use of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-18, and / or the use of the pharmaceutical composition as described in claim 20 in the preparation of medicaments for the diagnosis, prevention and / or treatment of cancer; Preferably, the cancer is an EGFR and / or MUC1 expression-related cancer, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer. A method of diagnosing, preventing and / or treating cancer, characterized in that, The method includes administering to a subject in need an effective amount of the antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-18, and / or, the pharmaceutical composition as described in claim 20; Preferably, the cancer is an EGFR and / or MUC1 expression-related cancer, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 1-18 for the diagnosis, prevention and / or treatment of cancer, and / or a pharmaceutical composition as described in claim 20; Preferably, the cancer is an EGFR and / or MUC1 expression-related cancer, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer. A combination therapy characterized in that, It includes administering an effective amount of a first therapeutic agent to a subject in need, and a second therapeutic agent; the first therapeutic agent is an antibody-drug conjugate as described in any one of claims 1-18 or a pharmaceutically acceptable salt thereof, and / or, a pharmaceutical composition as described in claim 20; Preferably, the cancer is an EGFR and / or MUC1 expression-related cancer, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer. A combination of a first therapeutic agent and a second therapeutic agent is used for the diagnosis, prevention, and / or treatment of cancer; the first therapeutic agent is an antibody-drug conjugate as described in any one of claims 1-18 or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition as described in claim 20; Preferably, the cancer is an EGFR and / or MUC1 expression-related cancer, such as one or more of lung cancer, colorectal cancer, breast cancer, and esophageal cancer.

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