Pharmaceutical composition comprising antibody-drug conjugate that specifically binds to EGFR and MUC1

Abstract

A pharmaceutical composition comprising an antibody-drug conjugate that specifically binds to EGFR and MUC1, and the use thereof in the preparation of a drug used for preventing or treating diseases.

Description

A pharmaceutical composition comprising an antibody-drug conjugate that specifically binds to EGFR and MUC1

[0001] This application claims priority to Chinese patent application CN202411442197.4, filed on October 16, 2024. Technical Field

[0002] This disclosure pertains to the field of pharmaceutical formulations, specifically relating to a pharmaceutical composition comprising an antibody-drug conjugate that specifically binds to EGFR and MUC1. Background Technology

[0003] The statements herein are provided only as background information in connection with this disclosure and do not necessarily constitute prior art.

[0004] MUC1 is a transmembrane glycoprotein rich in glycosylation. Its extracellular region is a dimer formed by two chains interacting through hydrogen bonds, namely MUC1-N and MUC1-C. MUC1-N has abundant O-glycosylation and a small amount of N-glycosylation, and its amino acid backbone is composed of repeated VNTRs. MUC1-C contains an extracellular domain, a transmembrane domain, and an intracellular domain.

[0005] In normal tissues, MUC1 exists in its full length at the apex of epithelial cells, while EGFR is located at the basal end. Tumor cells, having lost their apical and basal polarity, allow EGFR and MUC1 to be evenly distributed on their surface, resulting in spatial proximity. Furthermore, the O-glycosylation of MUC1-N on tumor cells becomes significantly sparser. As the tumor progresses, MUC1-N detaches under the catalysis of inflammatory factor-related enzymes in the tumor microenvironment, exposing MUC1-C.

[0006] Ecinotecan toxoid is a camptothecin derivative that inhibits topoisomerase I, selectively inhibiting DNA replication in proliferating tumor cells. Furthermore, eccinotecan toxoid exhibits excellent membrane permeability, allowing it to penetrate killed cancer cells and continue killing adjacent cancer cells, demonstrating a clear bystander effect in clinical practice.

[0007] Antibody-drug conjugates (ADCs) are obtained by linking antibodies to biologically active drugs via linkers. ADCs fully utilize the specificity of antibodies in binding to antigens on the surface of normal and tumor cells, as well as the high efficiency of drugs (such as cytotoxic agents), while avoiding the drawbacks of low efficacy of antibodies and excessive toxic side effects of drugs. Compared with traditional chemotherapy drugs, antibody-drug conjugates can more precisely kill tumor cells and reduce the impact on normal cells.

[0008] ADCs have a more complex heterogeneous structure than antibodies, which poses a greater challenge to ADC formulation. Summary of the Invention

[0009] This disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate that specifically binds to EGFR and MUC1. The pharmaceutical composition exhibits good therapeutic activity, safety, pharmacokinetic properties, and drug-likeness (e.g., stability).

[0010] In some embodiments, this disclosure provides a pharmaceutical composition comprising an antibody-drug conjugate that specifically binds to EGFR and MUC1 and a buffer, wherein:

[0011] The antibody-drug conjugate that specifically binds to EGFR and MUC1 has a structure as shown in the general formula (Pc-9-A):

[0012] in:

[0013] n is between 1 and 10;

[0014] Pc is an antibody that specifically binds to EGFR and MUC1. It comprises one antigen-binding module specifically binding to MUC1 and one antigen-binding module specifically binding to EGFR. The antigen-binding module specifically binding to MUC1 is a Fab, and the antigen-binding module specifically binding to EGFR is a replaced Fab containing a dimeric Titin chain and an Obscurin chain.

[0015] The antibody that specifically binds to EGFR and MUC1 comprises a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d), wherein:

[0016] Equation (a): [MUC1-VH]-[CH1]-[Fc1],

[0017] Equation (b): [MUC1-VL]-[CL],

[0018] Equation (c): [EGFR-VH]-[connector 1]-[Titin]-[Fc2],

[0019] Equation (d): [EGFR-VL]-[connector 2]-[Obscurin],

[0020] in:

[0021] Linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist.

[0022] The structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end;

[0023] The buffer is a histidine buffer, an acetate buffer, or a succinate buffer.

[0024] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the buffer is a histidine-histidine hydrochloride buffer, an acetate buffer, or a succinate buffer. In some embodiments, the buffer is a histidine-histidine hydrochloride buffer, an acetate-sodium acetate buffer, or a succinate-sodium succinate buffer. In some embodiments, the buffer is a histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer. In some embodiments, the buffer is a histidine-histidine hydrochloride buffer.

[0025] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the antigen-binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the antigen-binding module that specifically binds to MUC1 comprises a heavy chain variable region MUC1-VH and a light chain variable region MUC1-VL; wherein:

[0026] The EGFR-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129; the EGFR-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121; and

[0027] The HCDR1 of the MUC1-VH contains the amino acid sequence of SEQ ID NO: 12, the HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 14; the LCDR1 of the MUC1-VL contains the amino acid sequence of SEQ ID NO: 15, the LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and the LCDR3 contains the amino acid sequence of SEQ ID NO: 17.

[0028] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the antigen-binding module that specifically binds to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the antigen-binding module that specifically binds to MUC1 comprises a heavy chain variable region MUC1-VH and a light chain variable region MUC1-VL; wherein:

[0029] The amino acid sequences of HCDR1 of the EGFR-VH are shown in SEQ ID NO: 116, HCDR2 in SEQ ID NO: 117, and HCDR3 in SEQ ID NO: 129; the amino acid sequences of LCDR1 of the EGFR-VL are shown in SEQ ID NO: 119, LCDR2 in SEQ ID NO: 120, and LCDR3 in SEQ ID NO: 121; and

[0030] The amino acid sequences of HCDR1 of MUC1-VH are shown in SEQ ID NO: 12, HCDR2 in SEQ ID NO: 13, and HCDR3 in SEQ ID NO: 14; the amino acid sequences of LCDR1 of MUC1-VL are shown in SEQ ID NO: 15, LCDR2 in SEQ ID NO: 16, and LCDR3 in SEQ ID NO: 17.

[0031] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 138, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 149; and

[0032] The MUC1-VH comprises an amino acid sequence of SEQ ID NO: 36, 37 or 38 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 36, 37 or 38, and the MUC1-VL comprises an amino acid sequence of SEQ ID NO: 39, 40, 41 or 42 or an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with SEQ ID NO: 39, 40, 41 or 42.

[0033] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149; and

[0034] The MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, 37 or 38, and the MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, 40, 41 or 42.

[0035] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149; and

[0036] The MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, and the MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, 40, 41, or 42; or

[0037] The MUC1-VH contains the amino acid sequence of SEQ ID NO: 37, and the MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, 40, 41, or 42; or

[0038] The MUC1-VH contains the amino acid sequence of SEQ ID NO: 38, and the MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, 40, 41 or 42.

[0039] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149; and

[0040] The MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, and the MUC1-VL contains the amino acid sequence of SEQ ID NO: 39.

[0041] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the amino acid sequence of said EGFR-VH is as shown in SEQ ID NO: 138, and the amino acid sequence of said EGFR-VL is as shown in SEQ ID NO: 149; and

[0042] The amino acid sequence of MUC1-VH is shown in SEQ ID NO: 36, and the amino acid sequence of MUC1-VL is shown in SEQ ID NO: 39.

[0043] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 165, and the obscurin chain comprises the amino acid sequence of SEQ ID NO: 166.

[0044] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the antibody that specifically binds to EGFR and MUC1 comprises an Fc region, wherein the Fc region is an IgG1 Fc region. In some embodiments, the Fc region comprises one or more amino acid substitutions capable of reducing the binding of the Fc region to the Fcγ receptor.

[0045] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the antibody that specifically binds to EGFR and MUC1 comprises an Fc region, the Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, the Fc1 and Fc2 each independently having one or more amino acid substitutions that reduce homodimerization of the Fc region.

[0046] In some embodiments, such as the pharmaceutical composition described in any of the preceding embodiments, Fc1 has a raised structure according to the mortar and pestle technique, and Fc2 has a hole structure according to the mortar and pestle technique; or, Fc2 has a raised structure according to the mortar and pestle technique, and Fc1 has a hole structure according to the mortar and pestle technique.

[0047] In some embodiments, in the pharmaceutical composition described in any of the preceding claims, the amino acid C at position 358 of Fc1 is C, and the amino acid W at position 370; and the amino acid C at position 357 of Fc2 is C, the amino acid S at position 374, the amino acid A at position 376, and the amino acid V at position 415, numbered according to the EU index; or, the amino acid C at position 358 of Fc2 is C, and the amino acid W at position 370; and the amino acid C at position 357 of Fc1 is C, the amino acid S at position 374, the amino acid A at position 376, and the amino acid V at position 415, numbered according to the EU index.

[0048] In some embodiments, in the pharmaceutical composition of any of the preceding claims, Fc1 comprises the amino acid sequence of SEQ ID NO: 169 and Fc2 comprises the amino acid sequence of SEQ ID NO: 170; or Fc1 comprises the amino acid sequence of SEQ ID NO: 182 and Fc2 comprises the amino acid sequence of SEQ ID NO: 183; or Fc2 comprises the amino acid sequence of SEQ ID NO: 169 and Fc1 comprises the amino acid sequence of SEQ ID NO: 170; or Fc2 comprises the amino acid sequence of SEQ ID NO: 182 and Fc1 comprises the amino acid sequence of SEQ ID NO: 183.

[0049] In some embodiments, in the pharmaceutical composition described in any of the preceding embodiments, said linker 1 and linker 2 are peptide linkers known in the art, provided that the antigen-binding molecule can exhibit the desired antigen-binding activity. For example, the peptide linker may be a flexible peptide having 1-50 or 3-20 amino acid residues. In some embodiments, said peptide linkers each independently have L1-(GGGGS). t The structure is -L2, wherein L1 is a bond, A, G, GS, GGG, GGS, or GGGGS (SEQ ID NO: 168), t is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, L2 is a bond, G, GG, GGG, or GGGG (SEQ ID NO: 181), and the peptide linker is not a bond. In some embodiments, linker 1 and linker 2 are identical, and their amino acid sequences are as shown in SEQ ID NO: 168.

[0050] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the CH1 is the CH1 sequence of IgG. In some embodiments, the CH1 is the CH1 of IgG1. In some embodiments, the CH1 comprises the amino acid sequence of SEQ ID NO: 167.

[0051] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the CL is a light chain constant region of the antibody. In some embodiments, the CL is a κ or λ light chain constant region. In some embodiments, the CL comprises the amino acid sequence of SEQ ID NO: 70.

[0052] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the antibody that specifically binds to EGFR and MUC1 has a first strand comprising the amino acid sequence of SEQ ID NO: 171, a second strand comprising the amino acid sequence of SEQ ID NO: 74, a third strand comprising the amino acid sequence of SEQ ID NO: 174, and a fourth strand comprising the amino acid sequence of SEQ ID NO: 173; or

[0053] The antibody that specifically binds to EGFR and MUC1 has a first chain containing the amino acid sequence of SEQ ID NO: 178, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 179, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173.

[0054] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the antibody that specifically binds to EGFR and MUC1 has a first chain containing the amino acid sequence of SEQ ID NO: 171, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 174, and a fourth chain containing the amino acid sequence of SEQ ID NO: 173.

[0055] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the antibody that specifically binds to EGFR and MUC1 has a first chain with an amino acid sequence as shown in SEQ ID NO: 171, a second chain with an amino acid sequence as shown in SEQ ID NO: 74, a third chain with an amino acid sequence as shown in SEQ ID NO: 174, and a fourth chain with an amino acid sequence as shown in SEQ ID NO: 173.

[0056] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein n is the average number of pharmaceutical modules per antibody that specifically binds to EGFR and MUC1, and may be an integer or a decimal. In some embodiments, n is 1-10, or 2-10, or 3-10, or 4-10, or 5-10, or 6-10, or 7-10, or 8-10, or 1-9, or 2-9, or 3-9, or 4-9, or 5-9, or 6-9, or 7-9, or 1-8, or 2-8, or 3-8, or 4-8, or 5-8, or 6-8, or 1-7, or 2-7, or 3-7, or 4-7, or 5-7, or 1-6, or 2-6, or 3-6, or 4-6, or 1-5, or 2-5, or 3-5, or 1-4, or 2-4, or 1-3. In some embodiments, n is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10.

[0057] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein n is 2 to 8. In some embodiments, n is 4 to 8. In some embodiments, n is about 6. In some embodiments, n is 6.

[0058] In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is from 1 mg / mL to 50 mg / mL. In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is from 5 mg / mL to 40 mg / mL. In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is from 5 mg / mL to 30 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is from 5 mg / mL to 20 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is from 8 mg / mL to 12 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is from 9 mg / mL to 11 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is about 5 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate that specifically binds to EGFR and MUC1 is about 10 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate that specifically binds to EGFR and MUC1 is about 20 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate that specifically binds to EGFR and MUC1 is about 1 mg / mL, about 5 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, or about 50 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is 1 mg / mL, 5 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, or 50 mg / mL, or any range between these values. In some embodiments, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is 5 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is 10 mg / mL. In some embodiments, the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is 20 mg / mL.

[0059] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the buffer is from 1 mM to 50 mM. In some embodiments, the concentration of the buffer is from 5 mM to 40 mM. In some embodiments, the concentration of the buffer is from 5 mM to 30 mM. In some embodiments, the concentration of the buffer is from 5 mM to 20 mM. In some embodiments, the concentration of the buffer is from 8 mM to 12 mM. In some embodiments, the concentration of the buffer is from 9 mM to 11 mM. In some embodiments, the concentration of the buffer is about 5 mM. In some embodiments, the concentration of the buffer is about 10 mM. In some embodiments, the concentration of the buffer is about 20 mM. In some embodiments, the concentration of the buffer is about 1 mM, about 5 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of the buffer is 1 mM, 5 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM, or any range between these values. In some embodiments, the concentration of the buffer is 5 mM. In some embodiments, the concentration of the buffer is 10 mM. In some embodiments, the concentration of the buffer is 20 mM.

[0060] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the histidine-histidine hydrochloride buffer is from 1 mM to 50 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 40 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 30 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 5 mM to 20 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 8 mM to 12 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is from 9 mM to 11 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 5 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 10 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 20 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is about 1 mM, about 5 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 1 mM, 5 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM, or any range between these values. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 5 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 10 mM. In some embodiments, the concentration of the histidine-histidine hydrochloride buffer is 20 mM.

[0061] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments has a pH of 4.5 to 8.0. In some embodiments, the pharmaceutical composition has a pH of 4.5 to 6.5. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.5. In some embodiments, the pharmaceutical composition has a pH of 5.0 to 6.0. In some embodiments, the pharmaceutical composition has a pH of 4.5 to 6.0. In some embodiments, the pharmaceutical composition has a pH of 4.5 to 5.5. In some embodiments, the pharmaceutical composition has a pH of 4.7 to 5.3. In some embodiments, the pharmaceutical composition has a pH of 4.8 to 5.2. In some embodiments, the pharmaceutical composition has a pH of 4.9 to 5.1. In some embodiments, the pharmaceutical composition has a pH of about 4.5. In some embodiments, the pharmaceutical composition has a pH of about 5.0. In some embodiments, the pharmaceutical composition has a pH of about 5.5. In some embodiments, the pharmaceutical composition has a pH of about 6.0. In some embodiments, the pharmaceutical composition has a pH of about 6.5. In some embodiments, the pH of the pharmaceutical composition is about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 7.0, about 7.5, or about 8.0. In some embodiments, the pH of the pharmaceutical composition is 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 7.0, 7.5, or 8.0, or any range between these values. In some embodiments, the pH of the pharmaceutical composition is 4.5. In some embodiments, the pH of the pharmaceutical composition is 5.0. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is 6.0. When point values ​​are mentioned in this disclosure, it should be understood that the point value includes a range of error. This range of error is due to factors such as laboratory environment, human operation, instrumentation, methodology, measurement error, etc. Taking pH as an example, when the measured value is about 5.0, it should be understood that it includes a range of error. As an example, when measuring a formulation using an industrial pH meter, "about 5.0" means 5.0 ± 0.3 (i.e., pH from 4.7 to 5.3).

[0062] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises a surfactant. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from polysorbates (e.g., polysorbate 80, polysorbate 20), poloxamer (e.g., poloxamer 188), Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-sarcosine, linoleyl-sarcosine, myristyl-sarcosine, linoleyl-betaine, myristyl The surfactants include methyl betaine, cetyl betaine, lauramidopropyl betaine, cocaramide propyl betaine, linoleamide propyl betaine, myristamidopropyl betaine, palmitoamide propyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitoamide propyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl, sodium methyl oleate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc. In some embodiments, the surfactant is polysorbate or poloxamer. In some embodiments, the surfactant is polysorbate 80 or poloxamer 188. In some embodiments, the surfactant is poloxamer 188. In some embodiments, the surfactant is polysorbate 80.

[0063] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the surfactant is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of the surfactant is from 0.1 mg / mL to 1 mg / mL. In some embodiments, the concentration of the surfactant is from 0.1 mg / mL to 0.3 mg / mL. In some embodiments, the concentration of the surfactant is from 0.3 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the surfactant is from 0.1 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of the surfactant is from 0.4 mg / mL to 1 mg / mL. In some embodiments, the concentration of the surfactant is about 0.1 mg / mL. In some embodiments, the concentration of the surfactant is about 0.2 mg / mL. In some embodiments, the concentration of the surfactant is about 0.4 mg / mL. In some embodiments, the concentration of the surfactant is about 1 mg / mL. In some embodiments, the surfactant concentration is about 0.01 mg / mL, about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the surfactant concentration is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the surfactant concentration is 0.1 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL. In some embodiments, the surfactant concentration is 0.4 mg / mL. In some embodiments, the surfactant concentration is 1 mg / mL.

[0064] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of polysorbate 80 is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.1 mg / mL to 1 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.1 mg / mL to 0.3 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.3 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.1 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is from 0.4 mg / mL to 1 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.1 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.2 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 1 mg / mL. In some embodiments, the concentration of polysorbate 80 is about 0.01 mg / mL, about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of polysorbate 80 is 0.1 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.2 mg / mL. In some embodiments, the concentration of polysorbate 80 is 0.4 mg / mL. In some embodiments, the concentration of polysorbate 80 is 1 mg / mL.

[0065] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of poloxamer 188 is from 0.01 mg / mL to 2 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 0.1 mg / mL to 1 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 0.1 mg / mL to 0.3 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 0.3 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 0.1 mg / mL to 0.4 mg / mL. In some embodiments, the concentration of poloxamer 188 is from 0.4 mg / mL to 1 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 0.1 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 0.2 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 0.4 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 1 mg / mL. In some embodiments, the concentration of poloxamer 188 is about 0.01 mg / mL, about 0.02 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL, about 1.5 mg / mL, or about 2.0 mg / mL. In some embodiments, the concentration of poloxamer 188 is 0.01 mg / mL, 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, or 2.0 mg / mL, or any range between these values. In some embodiments, the concentration of poloxamer 188 is 0.1 mg / mL. In some embodiments, the concentration of poloxamer 188 is 0.2 mg / mL. In some embodiments, the concentration of poloxamer 188 is 0.4 mg / mL. In some embodiments, the concentration of poloxamer 188 is 1 mg / mL.

[0066] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises a sugar. In some embodiments, the sugar is selected from conventional compositions (CH2O)n and their derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. In some embodiments, the sugar is selected from sucrose, trehalose, mannitol, sorbitol, glucose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabinitol, sylitol, mesquite, maltotriose, mesquite, mannitol, stachyose, maltose, lactulose, maltitol, maltitol, lactitol, isomaltulose, etc. In some embodiments, the sugar is sucrose, trehalose, mannitol, or sorbitol. In some embodiments, the sugar is sucrose.

[0067] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the sugar concentration is from 10 mg / mL to 120 mg / mL. In some embodiments, the sugar concentration is from 20 mg / mL to 110 mg / mL. In some embodiments, the sugar concentration is from 20 mg / mL to 100 mg / mL. In some embodiments, the sugar concentration is from 20 mg / mL to 95 mg / mL. In some embodiments, the sugar concentration is from 20 mg / mL to 80 mg / mL. In some embodiments, the sugar concentration is from 30 mg / mL to 50 mg / mL. In some embodiments, the sugar concentration is from 32 mg / mL to 48 mg / mL. In some embodiments, the sugar concentration is from 36 mg / mL to 44 mg / mL. In some embodiments, the sugar concentration is from 60 mg / mL to 100 mg / mL. In some embodiments, the sugar concentration is from 65 mg / mL to 95 mg / mL. In some embodiments, the sugar concentration is from 64 mg / mL to 96 mg / mL. In some embodiments, the sugar concentration is from 72 mg / mL to 88 mg / mL. In some embodiments, the sugar concentration is about 20 mg / mL. In some embodiments, the sugar concentration is about 40 mg / mL. In some embodiments, the sugar concentration is about 80 mg / mL. In some embodiments, the sugar concentration is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 32 mg / mL, about 36 mg / mL, about 40 mg / mL, about 44 mg / mL, about 48 mg / mL, about 50 mg / mL, about 60 mg / mL, about 64 mg / mL, about 65 mg / mL, about 70 mg / mL, about 72 mg / mL, about 80 mg / mL, about 88 mg / mL, about 90 mg / mL, about 95 mg / mL, about 96 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL. In some embodiments, the sugar concentration is about 80 mg / mL. In some embodiments, the sugar concentration is 10 mg / mL, 20 mg / mL, 30 mg / mL, 32 mg / mL, 36 mg / mL, 40 mg / mL, 44 mg / mL, 48 mg / mL, 50 mg / mL, 60 mg / mL, 64 mg / mL, 65 mg / mL, 70 mg / mL, 72 mg / mL, 80 mg / mL, 88 mg / mL, 90 mg / mL, 95 mg / mL, 96 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the sugar concentration is 20 mg / mL. In some embodiments, the sugar concentration is 40 mg / mL.In some embodiments, the concentration of the sugar is 80 mg / mL.

[0068] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of sucrose is from 10 mg / mL to 120 mg / mL. In some embodiments, the concentration of sucrose is from 20 mg / mL to 110 mg / mL. In some embodiments, the concentration of sucrose is from 20 mg / mL to 100 mg / mL. In some embodiments, the concentration of sucrose is from 20 mg / mL to 95 mg / mL. In some embodiments, the concentration of sucrose is from 20 mg / mL to 80 mg / mL. In some embodiments, the concentration of sucrose is from 30 mg / mL to 50 mg / mL. In some embodiments, the concentration of sucrose is from 32 mg / mL to 48 mg / mL. In some embodiments, the concentration of sucrose is from 36 mg / mL to 44 mg / mL. In some embodiments, the concentration of sucrose is from 60 mg / mL to 100 mg / mL. In some embodiments, the concentration of sucrose is from 65 mg / mL to 95 mg / mL. In some embodiments, the concentration of sucrose is from 64 mg / mL to 96 mg / mL. In some embodiments, the concentration of sucrose is from 72 mg / mL to 88 mg / mL. In some embodiments, the concentration of sucrose is about 20 mg / mL. In some embodiments, the concentration of sucrose is about 40 mg / mL. In some embodiments, the concentration of sucrose is about 80 mg / mL. In some embodiments, the concentration of the sucrose is about 10 mg / mL, about 20 mg / mL, about 30 mg / mL, about 32 mg / mL, about 36 mg / mL, about 40 mg / mL, about 44 mg / mL, about 48 mg / mL, about 50 mg / mL, about 60 mg / mL, about 64 mg / mL, about 65 mg / mL, about 70 mg / mL, about 72 mg / mL, about 80 mg / mL, about 88 mg / mL, about 90 mg / mL, about 95 mg / mL, about 96 mg / mL, about 100 mg / mL, about 110 mg / mL, or about 120 mg / mL. In some embodiments, the concentration of sucrose is 10 mg / mL, 20 mg / mL, 30 mg / mL, 32 mg / mL, 36 mg / mL, 40 mg / mL, 44 mg / mL, 48 mg / mL, 50 mg / mL, 60 mg / mL, 64 mg / mL, 65 mg / mL, 70 mg / mL, 72 mg / mL, 80 mg / mL, 88 mg / mL, 90 mg / mL, 95 mg / mL, 96 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL, or any range between these values. In some embodiments, the concentration of sucrose is 20 mg / mL. In some embodiments, the concentration of sucrose is 40 mg / mL. In some embodiments, the concentration of sucrose is 80 mg / mL.

[0069] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments further comprises an excipient. In some embodiments, the excipient is selected from one or more of the group consisting of ethylenediaminetetraacetic acid (EDTA) or a salt thereof, EDTA hydrate or a salt thereof, glycine, DTPA (diethylenetriaminepentaacetic acid), arginine hydrochloride, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, and calcium chloride. In some embodiments, the excipient is selected from one or two of the group consisting of a sodium salt of EDTA hydrate and glycine. In some embodiments, the excipient is selected from one or two of the group consisting of disodium EDTA dihydrate and glycine. In some embodiments, the excipient is a sodium salt of EDTA hydrate. In some embodiments, (I) the excipient is disodium EDTA dihydrate; or (II) the excipient is disodium EDTA dihydrate and glycine. In some embodiments, the excipient is disodium EDTA dihydrate. In some embodiments, the excipients are disodium ethylenediaminetetraacetate dihydrate and glycine. In some embodiments, the excipient is glycine.

[0070] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of the excipient is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of the excipient is from 0.01 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the excipient is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of the excipient is from 0.01 mg / mL to 0.05 mg / mL. In some embodiments, the concentration of the excipient is from 0.01 mg / mL to 0.03 mg / mL. In some embodiments, the concentration of the excipient is about 0.01 mg / mL. In some embodiments, the concentration of the excipient is about 0.02 mg / mL. In some embodiments, the concentration of the excipient is about 0.1 mg / mL. In some embodiments, the concentration of the excipient is about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, or any range between these values. In some embodiments, the concentration of the excipient is 0.01 mg / mL. In some embodiments, the concentration of the excipient is 0.02 mg / mL. In some embodiments, the concentration of the excipient is 0.1 mg / mL.

[0071] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 1 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.1 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.05 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.03 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.01 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.02 mg / mL. In some embodiments, the concentration of the disodium ethylenediaminetetraacetate dihydrate is about 0.1 mg / mL. In some embodiments, the concentration of the disodium ethylenediaminetetraacetate dihydrate is about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, or about 1 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL, or any range between these values. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is 0.01 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is 0.02 mg / mL. In some embodiments, the concentration of disodium ethylenediaminetetraacetate dihydrate is 0.1 mg / mL.

[0072] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of the excipient is from 0.01 mg / mL to 30 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of the excipient is from 0.01 mg / mL to 20 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, wherein the concentration of the excipient is from 1 mg / mL to 20 mg / mL. In some embodiments, the concentration of the excipient is from 5 mg / mL to 20 mg / mL. In some embodiments, the concentration of the excipient is from 8 mg / mL to 12 mg / mL. In some embodiments, the concentration of the excipient is from 9 mg / mL to 11 mg / mL. In some embodiments, the concentration of the excipient is about 5 mg / mL. In some embodiments, the concentration of the excipient is about 10 mg / mL. In some embodiments, the concentration of the excipient is about 20 mg / mL. In some embodiments, the concentration of the excipient is about 0.01 mg / mL, about 1 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, or about 20 mg / mL. In some embodiments, the concentration of the excipient is 0.01 mg / mL, 1 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL, or any range between these values. In some embodiments, the concentration of the excipient is 5 mg / mL. In some embodiments, the concentration of the excipient is 10 mg / mL. In some embodiments, the concentration of the excipient is 20 mg / mL.

[0073] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of glycine is from 0.01 mg / mL to 30 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of glycine is from 0.01 mg / mL to 20 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments, wherein the concentration of glycine is from 1 mg / mL to 20 mg / mL. In some embodiments, the concentration of glycine is from 5 mg / mL to 20 mg / mL. In some embodiments, the concentration of glycine is from 8 mg / mL to 12 mg / mL. In some embodiments, the concentration of glycine is from 9 mg / mL to 11 mg / mL. In some embodiments, the concentration of glycine is about 5 mg / mL. In some embodiments, the concentration of glycine is about 10 mg / mL. In some embodiments, the concentration of glycine is about 20 mg / mL. In some embodiments, the concentration of glycine is about 0.01 mg / mL, about 1 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, or about 20 mg / mL. In some embodiments, the concentration of glycine is 0.01 mg / mL, 1 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, or 20 mg / mL, or any range between these values. In some embodiments, the concentration of glycine is 5 mg / mL. In some embodiments, the concentration of glycine is 10 mg / mL. In some embodiments, the concentration of glycine is 20 mg / mL.

[0074] In some embodiments, the pharmaceutical composition as described in any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 1 mg / mL, and the concentration of glycine is from 0.01 mg / mL to 20 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 1 mg / mL, and the concentration of glycine is from 5 mg / mL to 20 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 1 mg / mL, and the concentration of glycine is from 8 mg / mL to 12 mg / mL. In some embodiments, the pharmaceutical composition as described in any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 1 mg / mL, and the concentration of glycine is about 10 mg / mL.

[0075] In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.1 mg / mL, and the concentration of glycine is from 0.01 mg / mL to 20 mg / mL. In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.1 mg / mL, and the concentration of glycine is from 5 mg / mL to 20 mg / mL. In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.1 mg / mL, and the concentration of glycine is from 8 mg / mL to 12 mg / mL. In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.1 mg / mL, and the concentration of glycine is about 10 mg / mL.

[0076] In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.02 mg / mL, and the concentration of glycine is from 0.01 mg / mL to 20 mg / mL. In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.02 mg / mL, and the concentration of glycine is from 5 mg / mL to 20 mg / mL. In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.02 mg / mL, and the concentration of glycine is from 8 mg / mL to 12 mg / mL. In some embodiments, in the pharmaceutical composition of any of the preceding claims, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.02 mg / mL, and the concentration of glycine is about 10 mg / mL.

[0077] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0078] (a) The antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 1 mg / mL to 50 mg / mL

[0079] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0080] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0081] (d) Disodium ethylenediaminetetraacetate dihydrate, ranging from 0.01 mg / mL to 1 mg / mL.

[0082] (e) Glycine at concentrations ranging from 0.01 mg / mL to 20 mg / mL, and

[0083] (f) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0.

[0084] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0085] (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL

[0086] (b) 0.1 mg / mL to 1 mg / mL of polysorbate or poloxamer,

[0087] (c) Sucrose at concentrations ranging from 20 mg / mL to 100 mg / mL,

[0088] (d) Disodium ethylenediaminetetraacetate dihydrate, ranging from 0.01 mg / mL to 0.1 mg / mL.

[0089] (e) Glycine at concentrations of 5 mg / mL to 20 mg / mL, and

[0090] (f) a 5 mM to 20 mM histidine-histidine hydrochloride buffer, an acetate-sodium acetate buffer, or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.5.

[0091] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0092] (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL

[0093] (b) 0.1 mg / mL to 1 mg / mL of polysorbate 80 or poloxamer 188,

[0094] (c) Sucrose at concentrations ranging from 20 mg / mL to 100 mg / mL,

[0095] (d) Disodium ethylenediaminetetraacetate dihydrate, ranging from 0.01 mg / mL to 0.1 mg / mL.

[0096] (e) Glycine at concentrations of 5 mg / mL to 20 mg / mL, and

[0097] (f) A 5 mM to 20 mM histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0.

[0098] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0099] (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL

[0100] (b) 0.1 mg / mL to 1 mg / mL of polysorbate 80 or poloxamer 188,

[0101] (c) Sucrose at concentrations of 20 mg / mL to 80 mg / mL,

[0102] (d) Disodium ethylenediaminetetraacetate dihydrate, ranging from 0.01 mg / mL to 0.1 mg / mL.

[0103] (e) Glycine at concentrations of 5 mg / mL to 20 mg / mL, and

[0104] (f) A 5 mM to 20 mM histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0.

[0105] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0106] (a) The antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations of 8 mg / mL to 12 mg / mL

[0107] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.3 mg / mL,

[0108] (c) Sucrose at concentrations of 30 mg / mL to 50 mg / mL,

[0109] (d) Disodium ethylenediaminetetraacetate dihydrate, ranging from 0.01 mg / mL to 0.1 mg / mL.

[0110] (e) Glycine at concentrations of 8 mg / mL to 12 mg / mL, and

[0111] (f) an 8 mM to 12 mM histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3.

[0112] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0113] (a) Approximately 10 mg / mL of the antibody-drug conjugate specifically binding to EGFR and MUC1,

[0114] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0115] (c) Approximately 40 mg / mL of sucrose,

[0116] (d) Approximately 0.02 mg / mL of disodium ethylenediaminetetraacetate dihydrate,

[0117] (e) Approximately 10 mg / mL of glycine, and

[0118] (f) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0119] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0120] (a) 10 mg / mL of the antibody-drug conjugate that specifically binds to EGFR and MUC1,

[0121] (b) 0.2 mg / mL of polysorbate 80,

[0122] (c) 40 mg / mL sucrose,

[0123] (d) 0.02 mg / mL of disodium ethylenediaminetetraacetate dihydrate,

[0124] (e) 10 mg / mL glycine, and

[0125] (f) 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0126] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0127] (a) The antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 1 mg / mL to 50 mg / mL

[0128] (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL,

[0129] (c) Sugars ranging from 10 mg / mL to 120 mg / mL,

[0130] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 1 mg / mL, and

[0131] (e) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0.

[0132] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0133] (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL

[0134] (b) 0.1 mg / mL to 1 mg / mL of polysorbate or poloxamer,

[0135] (c) Sucrose at concentrations ranging from 20 mg / mL to 100 mg / mL,

[0136] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0137] (e) a 5 mM to 20 mM histidine-histidine hydrochloride buffer, an acetate-sodium acetate buffer, or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.5.

[0138] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0139] (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL

[0140] (b) 0.1 mg / mL to 1 mg / mL of polysorbate 80 or poloxamer 188,

[0141] (c) Sucrose at concentrations ranging from 20 mg / mL to 100 mg / mL,

[0142] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0143] (e) a 5 mM to 20 mM histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0.

[0144] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0145] (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL

[0146] (b) 0.1 mg / mL to 1 mg / mL of polysorbate 80 or poloxamer 188,

[0147] (c) Sucrose at concentrations of 20 mg / mL to 80 mg / mL,

[0148] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0149] (e) a 5 mM to 20 mM histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0.

[0150] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0151] (a) The antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations of 8 mg / mL to 12 mg / mL

[0152] (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.3 mg / mL,

[0153] (c) Sucrose at concentrations of 60 mg / mL to 100 mg / mL,

[0154] (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and

[0155] (e) 8 mM to 12 mM histidine-histidine hydrochloride buffer or acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3.

[0156] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0157] (a) Approximately 10 mg / mL of the antibody-drug conjugate specifically binding to EGFR and MUC1,

[0158] (b) Approximately 0.2 mg / mL of polysorbate 80,

[0159] (c) Approximately 80 mg / mL of sucrose,

[0160] (d) Approximately 0.02 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0161] (e) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0162] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments comprises the following components:

[0163] (a) 10 mg / mL of the antibody-drug conjugate that specifically binds to EGFR and MUC1,

[0164] (b) 0.2 mg / mL of polysorbate 80,

[0165] (c) 80 mg / mL sucrose,

[0166] (d) 0.02 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and

[0167] (e) 10 mM histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.0.

[0168] In some embodiments, the pharmaceutical composition as described in any of the preceding embodiments is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water.

[0169] This disclosure also provides a method for preparing a lyophilized formulation, including the step of lyophilizing a pharmaceutical composition as described in any of the preceding claims.

[0170] This disclosure also provides a lyophilized formulation obtained by the method described above.

[0171] This disclosure also provides a lyophilized formulation comprising the pharmaceutical composition as described in any of the preceding claims.

[0172] This disclosure also provides a lyophilized formulation, which, upon reconstitution, can form a pharmaceutical composition as described in any of the preceding claims.

[0173] This disclosure also provides a reconstituted solution, characterized in that the reconstituted solution is prepared by reconstituted a lyophilized formulation as described in any of the preceding claims.

[0174] This disclosure also provides a reconstituted solution, which is a reconstituted form of the lyophilized formulation as described in any of the preceding claims.

[0175] In some embodiments, the reconstituted solution as described in any of the preceding claims has the same components and contents as the pharmaceutical composition as described in any of the preceding claims.

[0176] This disclosure also provides an article of manufacture comprising a container containing a pharmaceutical composition as described in any of the preceding claims, or a lyophilized formulation as described in any of the preceding claims, or a reconstituted solution as described in any of the preceding claims.

[0177] In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is an intravenous injection formulation, a subcutaneous injection formulation, an intraperitoneal injection formulation, or an intramuscular injection formulation. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is an intravenous injection formulation.

[0178] In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is suitable for intravenous, subcutaneous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is suitable for intravenous injection.

[0179] In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is used to prepare a drug for intravenous, subcutaneous, intraperitoneal, or intramuscular injection. In some embodiments, the pharmaceutical composition, lyophilized formulation, or reconstituted solution described in any of the preceding embodiments is used to prepare a drug for intravenous injection.

[0180] This disclosure also provides a method for treating or preventing a disease, the method comprising administering to a subject a therapeutically effective amount of a pharmaceutical composition as described in any of the preceding claims, or a lyophilized formulation as described in any of the preceding claims, or a reconstituted solution as described in any of the preceding claims.

[0181] This disclosure also provides the use of the pharmaceutical composition as described in any of the preceding claims, or the lyophilized formulation as described in any of the preceding claims, or the reconstituted solution as described in any of the preceding claims, in the preparation of a medicament for the treatment or prevention of a disease.

[0182] This disclosure also provides pharmaceutical compositions as described in any of the preceding claims, or lyophilized formulations as described in any of the preceding claims, or reconstituted solutions as described in any of the preceding claims, for use as medicaments. In some embodiments, the medicaments are used to treat or prevent disease.

[0183] In some implementations, the disease described in any of the preceding embodiments is a tumor or cancer.

[0184] In some implementations, the disease described in any of the preceding embodiments is a solid tumor or a hematologic malignancy.

[0185] In some implementations, the diseases described in any of the preceding embodiments are selected from: astrocytoma, glioblastoma, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, fallopian tube cancer, gallbladder cancer, gastric cancer, head and neck cancer, idiopathic myelofibrosis, kidney cancer, leukemia, liver cancer, esophageal cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, neuroblastoma, oligodendroglioma, ovarian cancer, peritoneal tumor, pancreatic cancer, polycythemia vera, primary neuroectodermal tumor, prostate cancer, retinoblastoma, sarcoma, squamous cell carcinoma, thyroid cancer, endometrial cancer, vestibular schwannoma, germ cell tumor, vulvar cancer, thymoma, testicular cancer, bile duct cancer, pheochromocytoma, paraganglioma, and adenoid cystic carcinoma.

[0186] In some implementations, the diseases described in any of the preceding embodiments are selected from lung cancer, head and neck cancer, esophageal cancer, breast cancer, pancreatic cancer, prostate cancer, thyroid cancer, stomach cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, and bladder cancer. Attached Figure Description

[0187] Figures 1 and 2 show the results of the in vitro internalization experiment of chimeric antibodies on T47D cells;

[0188] Figures 3A to 3E show the experimental results of determining the affinity of chimeric and humanized antibodies for HCC827-human-MUC1-C cells based on FACS.

[0189] Figures 3F to 3H show the experimental results of determining the affinity of chimeric and humanized antibodies for CHOK1-human-MUC1-C cells based on FACS.

[0190] Figures 3I to 3P show the experimental results of determining the affinity of chimeric and humanized antibodies for T47D (MUC1 high expression, EGFR low expression) cells based on FACS.

[0191] Figures 4A to 4F show the results of the in vitro internalization experiment of T47D cells with MUC1-C humanized antibody;

[0192] Figure 5 shows a schematic diagram of the molecular form of the EGFR-MUC1 bispecific antibody;

[0193] Figures 6A and 6B show the experimental results of the killing activity of EGFR-MUC1 bispecific anti-ADC (conjugated with M toxin) with different mutations on tumor cell lines HCC827 (EGFR high expression, MUC1 low expression) and HCC70 (EGFR medium expression, MUC1 medium expression).

[0194] Figure 7A shows the tumor volume changes with the number of study days in different groups of the HCC827 CDX mouse model.

[0195] Figure 7B shows the curves of body weight change with the number of study days in different groups of the HCC827 CDX mouse model.

[0196] Figure 8A shows the tumor volume changes with the number of study days in different groups of the HPAC CDX mouse model.

[0197] Figure 8B shows the curves of body weight change with the number of study days in different groups of HPAC CDX mouse model. Detailed Implementation

[0198] the term

[0199] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0200] The singular forms “a,” “an,” and “the” used in this disclosure include plural references unless the context clearly indicates otherwise.

[0201] Unless the context clearly requires otherwise, the words “comprising,” “having,” “including,” etc., in the patent specification and claims should be understood as “including but not limited to,” rather than as exclusive or exhaustive.

[0202] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur.

[0203] Those skilled in the art will understand that when used as a reference range, cutoff value, or specific value, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range with a difference of up to 20% (i.e., ±20%). Since many of the values ​​used herein were determined experimentally, those skilled in the art will understand that such determinations can vary between different experiments and are generally true across experiments. Due to this inherent variability, the values ​​used herein should not be unduly restricted. Therefore, the term "about" is used to cover variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% or less from a specified value.

[0204] Although this disclosure provides content ranges or content values, those skilled in the art will understand that the content ranges or content values ​​cover the acceptable range of error for the specific values ​​measured.

[0205] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem., 243, p3558 (1968).

[0206] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., ortholeucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids.

[0207] The term "antibody" is used in the broadest sense and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies); full-length antibodies and antigen-binding fragments (or antigen-binding portions), as long as they exhibit the desired antigen-binding activity. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is a heterotetraglycoprotein of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bound by disulfide bonds. From the N to C terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or heavy chain variable region, followed by a heavy chain constant region. The IgG heavy chain constant region (CH) typically contains three constant domains (CH1, CH2, and CH3); similarly, from the N to C terminus, each light chain has a variable region (VL), also called a variable light domain or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

[0208] The term "variable region" or "variable domain" refers to the domain in the antibody heavy or light chain involved in antibody-antigen binding. In this paper, the antibody heavy chain variable region (VH) and light chain variable region (VL) each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. The VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; the VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus (also called the N-terminus) to the carboxyl terminus (also called the C-terminus): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen-binding specificity.

[0209] The amino acid sequence boundaries of CDRs can be determined using various well-known schemes, such as the "Kabat" numbering rule, the "Chothia" numbering rule, the "ABM" numbering rule, the "contact" numbering rule, and the ImMunoGenTics (IMGT) numbering rule. The correspondence between these numbering systems is well known to those skilled in the art and is exemplified as shown in Table 1 below.

[0210] Table 1. Relationship between CDR numbering systems

[0211] Unless otherwise stated, the variable regions and CDR sequences in this disclosure embodiment are subject to the "Kabat" numbering rule.

[0212] The term "monoclonal antibody" refers to a group of antibodies that are substantially homogeneous, meaning that the antibody molecules contained in this group have the same amino acid sequence, except for the possible small number of naturally occurring mutations. In contrast, polyclonal antibodies typically comprise a variety of different antibodies with varying amino acid sequences in their variable domains, and they generally target different epitopes specifically. "Monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the production of antibodies through any particular method.

[0213] The antibodies disclosed herein may be derived from animals (such as antibodies from mice, birds, rabbits, camels, monkeys, etc.), chimeric antibodies, or humanized antibodies.

[0214] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remaining portion of the heavy chain and / or light chain is derived from another different source or species.

[0215] The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including both native and modified Fc regions. An Fc region comprises two Fc subunits that bind together to form the Fc region. In some embodiments, the Fc region comprises two distinct Fc subunits. In some embodiments, the Fc region comprises two identical Fc subunits. In some embodiments, the Fc region of a human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. In some embodiments, the Fc region of the antibody includes the Fc regions of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. In some embodiments, the boundaries of the Fc region may also vary, for example, by omitting the C-terminal lysine (residue 447 according to the EU numbering system) or omitting both the C-terminal glycine and lysine (residues 446 and 447 according to the EU numbering system). Unless otherwise stated, the Fc region is numbered according to the EU numbering system, also known as the EU index.

[0216] The term "linker" refers to a connecting unit that links two polypeptide fragments. In this document, linkers appearing in the same structural formula may be the same or different. Linkers may be "peptide linkers," containing one or more amino acids, typically about 1-30, 2-24, or 3-15 amino acids. Linkers used in this document may be the same or different. When a "-" appears in a structural formula, it indicates that the units on either side are directly connected by a covalent bond.

[0217] The term "humanized" antibody refers to an antibody that retains the reactivity of a non-human antibody while exhibiting lower immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR region and replacing the rest of the antibody with its human counterpart (i.e., the frame region portion of the constant region and the variable region).

[0218] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). Unless otherwise specified, as used herein, binding "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can typically be represented by the dissociation constant (KD). Affinity can be measured using conventional methods known in the art.

[0219] The term "antigen" refers to a molecule or molecular moiety that can be bound by a selective binder of an antigen-binding protein (such as an antibody). An antigen may have one or more epitopes that can interact with different antigen-binding proteins (such as antibodies).

[0220] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody or its antigen-binding fragment. Epitopes can be formed from consecutive amino acids (linear epitopes) or contain non-consecutive amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., tertiary folding of the antigen as a protein), which allows non-consecutive amino acids to be spatially close. The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, Western blotting, peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen, and cross-blocking.

[0221] The terms "capable of specific binding," "specific binding," or "binding" refer to the ability of an antibody to bind to a specific antigen or epitope with a higher affinity than other antigens or epitopes. Typically, antibodies bind at an affinity of approximately 1 × 10⁻⁶. -7An equilibrium dissociation constant (KD) of M or less binds to an antigen or epitope. In some embodiments, the KD of antibody binding to an antigen is 10% or less (e.g., 1%) of the KD of the antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using known methods, such as by FACS or surface plasmon resonance assays. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, such as the cynomolgus (cyno), the chimpanzee (chimp), or the common marmoset (marmoset).

[0222] The term "sequence identity" refers to the degree (percentage) to which the amino acids / nucleic acids of two sequences are identical at equivalent positions when two sequences are optimally aligned, with gaps introduced where necessary to obtain the maximum percentage of sequence identity, without considering any conserved substitutions as part of sequence identity. To determine the percentage of sequence identity, alignment can be performed using techniques known in the art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0223] Antibody drug conjugates (ADCs) are conjugates obtained by linking an antibody (or its antigen-binding fragment) directly or through a linker to a drug.

[0224] A "drug" (abbreviated as D) is any substance that has biological or detectable activity (e.g., therapeutic agents, detectable markers, binders, etc.) and its prodrugs, which are metabolized in the body to become active agents. Examples of therapeutic agents include cytotoxic agents, chemotherapeutic agents, cell growth inhibitors, and immunomodulators. Chemotherapeutic agents are chemical compounds that can be used to treat cancer. Representative therapeutic agents include cytotoxins, cytotoxic agents, and cell growth inhibitors.

[0225] Cytotoxicity refers to the loss, elimination, and / or killing of target cells. Cytotoxic agents are drugs that have cytotoxic and / or cell growth-inhibiting effects on cells. Cell growth inhibition refers to the inhibition of cell proliferation. Cell growth inhibitors are drugs that have a cell growth-inhibiting effect on cells, thereby inhibiting the growth and / or expansion of specific subgroups of cells.

[0226] Additional representative therapeutic agents include radioisotopes, chemotherapeutic agents, immunomodulators, anti-angiogenic agents, antiproliferative agents, apoptosis-promoting agents, and cell-lysing enzymes (e.g., RNase). These drug descriptive terms are not mutually exclusive, and therefore, one or more of the aforementioned terms may be used to describe a therapeutic agent. For example, the selected radioisotope may also be a cytotoxic agent. Therapeutic agents can be prepared as pharmaceutically acceptable salts, acids, or derivatives of any of the above. Generally, conjugates containing a radioisotope as a drug are called radioimmunoconjugates, and those containing a chemotherapeutic agent as a drug are called chemoimmunoconjugates.

[0227] Examples of cytotoxic agents include, but are not limited to, anthracycline, orrisstatin, CC-1065, dolastatin, docalmicin, enediyne, geldanamycin, maytansine, puromycin, taxane, vinca alkaloids, SN-38, tubulolysin, hemiasterlin, eribulin, trabectedin, lurbinectedin, and their stereoisomers, isosteres, analogues, or derivatives. Chemotherapy agents, phytotoxicants, other bioactive proteins, enzymes (i.e., ADEPT), radioactive isotopes, and photosensitizers (i.e., for photodynamic therapy) may also be used.

[0228] The terms "connector unit" and "connector" refer to a chemical structural fragment or bond that is linked to an antibody at one end and a drug at the other. Connectors can also be attached to other connectors before being linked to an antibody or drug. Connector attachment to antibodies can be accomplished in various ways, such as via surface lysine residues, reductive coupling to oxidized carbohydrates, release of cysteine ​​residues via reducing interchain disulfide bonds, modification of reactive cysteine ​​residues at specific sites, and tags containing acyl donor glutamine, or modification of peptides to make them reactive endogenous glutamine in the presence of transglutaminase and amines. Various ADC linker systems are known in the art, including hydrazone-, disulfide-, and peptide-based links.

[0229] The connector may comprise one or more connector elements. Exemplary connector elements include 6-maleiminohexanoyl (“MC”), maleiminopropionyl (“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimino-4-(2-pyridylthio)valerate (“SPP”), N-succinimino-4-(N-maleiminomethyl)cyclohexane-1-carboxylate (“SMCC”, also referred to herein as “MCC”), and N-succinimino-(4-iodo-acetyl)aminobenzoate (“SIAB”).

[0230] The linker may be selected from the following elements or combinations thereof: extensions, spacers, and amino acid units. The linker may be synthesized by methods known in the art, such as those described in US20050238649A1. The linker may be a “cleavable linker” that facilitates drug release into cells. For example, acid-labile linkers (e.g., hydrazones), protease-sensitive linkers (e.g., peptidase-sensitive linkers), photostable linkers, dimethyl linkers, or disulfide-containing linkers may be used (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020).

[0231] “LD” is the connector-drug section formed when the drug (D) is connected to the connector (L).

[0232] "Drug loading," also known as drug-to-antibody ratio (DAR), refers to the average number of drugs conjugated to each antibody in an ADC. It can range from about 1 to about 10 drugs per antibody, and in some embodiments, from about 1 to about 8 drugs per antibody, preferably from the ranges of 2-8, 2-7, 2-6, 2-5, 2-4, 1-3, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. The general formula of the ADC disclosed herein includes a set of antibody-drug conjugates within the aforementioned range. In embodiments disclosed herein, drug loading may be expressed as n, which can be a decimal or an integer. Drug loading can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, HIC, and RP-HPLC.

[0233] "Surfactant" refers to a surface-active agent, preferably a nonionic surfactant. Surfactants can reduce protein aggregation and / or particle formation in formulations. The amount of surfactant added is such that it reduces protein aggregation and minimizes particle formation in the formulation.

[0234] "Poloxamer" is an α-hydro-ω-hydroxy poly(oxyethylene) a -Poly(oxypropylene) b - Poly(ethylene oxide) a Block copolymers. These are formed by reacting propylene oxide and propylene glycol to form polyoxypropylene glycol, followed by the addition of ethylene oxide to form a block copolymer. Here, 'a' represents the number of ethylene oxide units, and 'b' represents the number of propylene oxide units. Examples of poloxamers include poloxamer 188. Specifically, poloxamer 188 has 75–85 ethylene oxide units (a) and 25–30 propylene oxide units (b) in the copolymer, with an ethylene oxide (EO) content of 79.9%–83.7% and an average molecular weight of 7680–9510.

[0235] In this disclosure, "disodium edetate", "disodium ethylenediaminetetraacetate dihydrate" and "Na2EDTA·2H2O" can be used interchangeably, and they are all substances with CAS number: 6381-92-6.

[0236] "Viscosity modifier" is a conventional pharmaceutical excipient added to adjust the viscosity of a formulation. Viscosity modifiers can be inorganic salts and amino acid salts. Preferably, the inorganic salt is selected from sodium chloride, calcium chloride, and magnesium chloride. More preferably, the amino acid salt is selected from arginine hydrochloride, histidine hydrochloride, lysine hydrochloride, histidine acetate, etc.

[0237] "Buffer" refers to a buffering agent that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that maintain pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate (also known as tartrate), tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0238] "Histidine buffer" is a buffer containing histidine. Examples of histidine buffers include histidine-histidine acetate, histidine-histidine hydrochloride, histidine-histidine phosphate, and histidine-histidine sulfate buffers. For example, a histidine-histidine hydrochloride buffer may be prepared by histidine and hydrochloric acid, or by histidine and histidine hydrochloride.

[0239] "Citrate buffers" are buffers that contain citrate ions. Examples of citrate buffers include sodium citrate, potassium citrate, calcium citrate, magnesium citrate, etc.

[0240] "Succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include sodium succinate, potassium succinate, and calcium succinate. A preferred succinate buffer is sodium succinate. Exemplarily, the sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0241] Phosphate buffers are buffers that contain phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and disodium hydrogen phosphate-citric acid.

[0242] "Acetate buffers" are buffers that contain acetate ions. Examples of acetate buffers include sodium acetate, histidine-histidine acetate, potassium acetate, calcium acetate, magnesium acetate, etc.

[0243] "Displacement" refers to the replacement of the solvent system in which the protein is dissolved. For example, a high-salt or hypertonic solvent system containing the protein may be replaced by a physical process in which the buffer system of a stabilizing agent is used, thereby allowing the protein to remain in the stabilizing agent. The physical process includes, but is not limited to, ultrafiltration, dialysis, or centrifugation.

[0244] "Pharmaceutical composition" means a mixture containing one or more antibody-drug conjugates or their physiologically / pharmacologically acceptable salts or prodrugs described herein, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0245] The term "pharmaceutically acceptable salt" or "medicinal salt" refers to the salts of the antibody-drug conjugates disclosed herein, which are safe and effective when used in subjects and possess the intended biological activity. As an example, the antibody-drug conjugates disclosed herein contain at least one amino group and can therefore form salts with acids. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0246] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated using well-known conventional methods.

[0247] "Lyophilized formulation" refers to a pharmaceutical composition or formulation obtained by vacuum freeze-drying a liquid or solution preparation. Typically, freeze-drying includes pre-freezing, primary drying, and secondary drying. Pre-freezing aims to freeze the product to obtain a crystalline solid; in some embodiments, the pre-freezing temperature is set to -45°C, and the pre-freezing rate is set to 1°C / min. Primary drying, also known as main drying, is the main stage of sample freeze-drying and aims to remove ice from the product while maintaining its shape and minimizing damage. Improper selection of the temperature and vacuum level during primary drying can lead to product collapse; higher temperatures and vacuum levels increase freeze-drying efficiency but also increase the risk of product collapse. In some embodiments, the primary drying temperature can be a temperature conventional in the art, such as -30°C to 0°C. Secondary drying, also known as desorption drying, is the main step of removing bound water from the product by applying an ultimate vacuum (0.01 mbar) and increasing the temperature (20°C to 40°C). Because most biological products are temperature-sensitive, the secondary drying temperature is often chosen at the lower end of the temperature range, such as 25°C. The freeze-drying time depends on the freezer, the dosage of the freeze-dried formulation, and the container of the freeze-dried drug. Such adjustments are well known to those skilled in the art.

[0248] Unless otherwise specified, the solvent in the solution form of the pharmaceutical compositions described in this disclosure is water.

[0249] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0250] The pharmaceutical compositions disclosed herein achieve a stable effect: the antibody-drug conjugates therein substantially retain their physical and / or chemical stability and / or biological activity after storage; preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, various analytical techniques are available for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0251] Stable formulations include those in which no significant changes are observed when stored at refrigerated temperatures (2°C–8°C) for at least 1 month, at least 3 months, at least 6 months, preferably 1 year, and even more preferably up to 2 years. Stable liquid formulations also include those that exhibit the desired characteristics after storage at temperatures including 25°C for periods of 1 month, 3 months, or 6 months. Furthermore, stable liquid formulations also include those that exhibit the desired characteristics after storage at 40°C for periods of 4 weeks, 1 month, 3 months, or 6 months. Typical examples of stability include antibody-drug conjugates showing aggregation or degradation typically not exceeding about 10%, preferably not exceeding about 5%, as determined by SEC-HPLC. Visually, the formulation is a pale yellow, nearly colorless, clear liquid or a colorless, clear liquid, or clear to slightly milky white. The concentration, pH, weight, and molecular osmotic pressure of the formulation exhibit aggregation of not more than about 10%, preferably not more than about 5%.

[0252] If, after visual inspection of color and / or clarity, or by means of UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody-drug conjugate does not show significant increase in aggregation, precipitation, and / or denaturation, then the antibody-drug conjugate "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

[0253] If an antibody-drug conjugate does not exhibit significant chemical changes, then the antibody-drug conjugate "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying the chemically altered form of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and CE-SDS), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, and isofpartate measurement), and isomerization (evaluated by measuring isofpartate content, peptide mapping, etc.).

[0254] If the biological activity of an antibody-drug conjugate at a given time is within a predetermined range of the biological activity exhibited when the drug formulation is prepared, then the antibody-drug conjugate "retains its biological activity" in the drug formulation.

[0255] "Administration," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Administration," "giving," and "treatment" also mean, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo, such as cells. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0256] "Treatment" means administering an oral or topical therapeutic agent, such as a pharmaceutical composition comprising any of the substances disclosed herein, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves symptoms of one or more diseases, in order to induce the regression of such symptoms or inhibit their progression to any clinically measurable degree. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments disclosed herein (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0257] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate a diagnosis. The effective amount used on a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects disclosed herein may be animal or human subjects.

[0258] The pharmaceutical compositions disclosed herein may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is required, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration may be carried out via any suitable route, such as by injection, such as intravenous or subcutaneous injection. Various dosing schedules are considered herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulsatile infusion. In some embodiments, the pharmaceutical compositions disclosed herein are administered by intravenous injection.

[0259] The pharmaceutical compositions disclosed herein will be formulated, administered, and applied in accordance with good medical practice. Factors considered in this context include the specific condition being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to a medical practitioner. Optionally, the pharmaceutical composition may also be formulated with one or more other agents for the prevention or treatment of said condition. The effective amount of such other agents depends on the amount of antibody-drug conjugate present in the pharmaceutical composition, the type of condition or treatment, and other factors. It may be used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and via any route determined empirically / clinically as appropriate.

[0260] Details of one or more embodiments of this disclosure are set forth in the foregoing description. While any methods and materials similar to or the same as those described herein may be used to implement or test this disclosure, preferred methods and materials are described below. Other features, objects, and advantages of this disclosure will be apparent from the description and claims. In the description and claims, the singular form includes plural references unless the context clearly indicates otherwise. Unless otherwise defined, all technical and scientific terms used herein have their general meaning as understood by one of ordinary skill in the art to which this disclosure pertains. All patents and publications referenced in the description are incorporated herein by reference. The following embodiments are presented to illustrate preferred embodiments of this disclosure more fully. These embodiments should not be construed in any way as limiting the scope of this disclosure, which is defined by the claims.

[0261] Example

[0262] The present disclosure is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0263] Experimental methods not specifying specific conditions in the examples or test cases disclosed herein are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Reagents not specifying their source are commercially available, standard reagents.

[0264] I. Antibody Preparation

[0265] The antibodies that specifically bind to EGFR and MUC1 disclosed herein are derived from CN202310394160.8, CN202311772280.3, and PCT / CN2024 / 087447 (included herein in their entirety by reference).

[0266] Example 1: Preparation of MUC1 antigen

[0267] Using the UniProt MUC1 antigen (human MUC1 protein, Uniprot number: P15941) as a template for MUC1, the amino acid sequences of the antigen and detection protein disclosed herein were designed. Optionally, different tags, such as His tags or Fc tags, were fused to the MUC1 protein. The sequences were cloned into the pTT5 vector (Biovector, CAT#102762), transiently expressed in 293 cells, and purified to obtain the antigen and detection protein disclosed herein.

[0268] The His-tagged extracellular domain of the MUC1-C protein (abbreviated as MUC1-CL-6xHis) sequence is used as an immunogenic antigen and detection reagent.

[0269] Note: The underlined part is the 6×His tag, and the bold part is the extracellular domain of the MUC1 protein.

[0270] The extracellular domain of the MUC1-C protein and the sequence of the Human-IgG1-Fc fusion protein (abbreviated as hMUC1-CL-Fc) serve as an immunogen;

[0271] Note: The underlined part is the Human-IgG1-Fc portion, and the bolded part is the extracellular domain of the MUC1 protein.

[0272] The sequence of the extracellular domain of Cyno-MUC1-C protein fusion protein with Human-IgG1-Fc (abbreviated as Cyno MUC1-C-Fc) was also used as a detection reagent.

[0273] Note: The underlined part is the Human-IgG1-Fc portion, and the bolded part is the extracellular domain of the Cyno-MUC1 protein.

[0274] Example 2: Purification of MUC1-related recombinant proteins

[0275] 1. Purification steps for His-tagged recombinant proteins

[0276] Cell expression supernatant samples were centrifuged at high speed to remove impurities. The nickel column was equilibrated with PBS solution containing 20 mM imidazole and washed 2-5 column volumes. The replaced cell supernatant samples were loaded onto a Ni Sepharose Excel column (GE, 17-3712-02). The column was washed with PBS solution until the A280 reading dropped to baseline. The column was then washed with PBS + 20 mM imidazole to remove non-specifically bound proteins, and the eluent was collected. The target protein was then eluted with PBS solution containing 300 mM imidazole, and the elution peak was collected. The target protein was then transferred to PBS using a concentration tube and concentrated to an appropriate concentration. The obtained protein was identified by electrophoresis, peptide mapping, and LC-MS and then aliquoted for use. MUC1-CL-6xHis, tagged with His, was obtained and used as the detection reagent for the antibody disclosed herein.

[0277] 2. Purification steps of the MUC1-CL-Fc fusion protein

[0278] Cell expression supernatant samples were centrifuged at high speed to remove impurities. The supernatant was then subjected to MabSelect Sure (GE, 17-5438-01) affinity chromatography. The MabSelect Sure column was first regenerated with 0.2M NaOH, then equilibrated with PBS. After binding the supernatant, the column was washed with PBS until the A280 reading returned to baseline. The target protein was eluted with 0.1M acetate buffer at pH 3.5 and neutralized with 1M Tris-HCl. The target protein was then transferred to PBS using a concentration tube and concentrated to an appropriate concentration. The obtained protein was identified by electrophoresis and LC-MS and then aliquoted for use. This method was used to purify the MUC1-CL-Fc fusion protein, and it can also be used to purify the antibody protein disclosed herein.

[0279] 3. Purification steps for hybridoma screening antibody sample expression:

[0280] Cell expression supernatant samples were centrifuged at high speed to remove impurities. Protein A magnetic bead packing material (SM003100) was added to the supernatant and incubated at room temperature with shaking for 3 hours. The packing material was washed three times with PBS, followed by one wash with ultrapure water. The target protein was eluted with 0.1M acetate buffer at pH 3.0 and neutralized with 1M Tris-HCl. The target protein was transferred to PBS using a concentration tube and concentrated to an appropriate concentration. The obtained protein was identified by electrophoresis and LC-MS and then aliquoted for use.

[0281] Example 3: Mouse immunization regimen for mouse anti-MUC1-C antibody and acquisition of hybridoma antibody

[0282] 1. Mouse immunization

[0283] Anti-human MUC1-C antibodies were generated by immunizing mice. Experimental Balb / c and SJL white mice, female, 6 - 8 weeks old (Shanghai Slake Experimental Animal Co., Ltd., Animal Production License No.: SCXK(Shanghai)2017 - 0005). Feeding environment: SPF level. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle adjustment, temperature 20 - 25 °C; humidity 40 - 60%. The mice that had adapted to the environment were immunized according to the following protocol.

[0284] Immunization protocol 1: Immunize using the protein antigen (hMUC1 C-L-Fc). The protein antigen (hMUC1C-L-Fc) was used Gold Adjuvant (Sigma) and Thermo Alum (Thermo) adjuvants for cross-immunization. The protein antigen (hMUC1 C-L-Fc) and the adjuvant ( Gold Adjuvant) were in a ratio of 1:1, and after emulsification, inoculation was carried out. The protein antigen (hMUC1 C-L-Fc) and the adjuvant (Thermo Alum) were in a ratio of 3:1, and after shaking and mixing evenly, inoculation was carried out. 50 μg / animal / time (primary immunization), 25 μg / animal / time (routine immunization), 50 μg / animal / time (booster immunization). The immunization times were 0, 14, 34, 48, 78 days. Blood was taken on the 26th, 40th, 57th, 82nd days to measure the antibody titer in the mouse serum. After the 4th - 5th immunizations, the antibody titer in the mouse serum was determined by the ELISA method, and mice with a high antibody titer in the serum and a titer tending to plateau were selected for spleen cell fusion. Three days before spleen cell fusion, booster immunization was carried out, with an intraperitoneal (i.p.) injection of 50 μg / animal of the protein antigen (hMUC1 C-L-Fc) and an antigen solution prepared with physiological saline or an intraperitoneal (i.p.) injection of 1×10 7 cells / animal of the cell antigen solution resuspended with phosphate buffer solution.

[0285] Immunization protocol 2: Immunize using the protein antigen (hMUC1-C-L-His) and the cell antigen (CHO-K1-MUC1-C) for cross-immunization. Among them, the protein antigen (hMUC1-C-L-His) was used Gold Adjuvant (Sigma) and Thermo Alum (Thermo) adjuvants for cross-immunization. The protein antigen (hMUC1-C-L-His) and the adjuvant ( Gold Adjuvant) were in a ratio of 1:1, and after emulsification, inoculation was carried out. The antigen hMUC1-C-L-Fc and the adjuvant (Thermo The ratio of alum (to aspirin) is 3:1. After shaking to mix, inoculate. Administer 50 μg / animal / dose (primary immunization), 25 μg / animal / dose (routine immunization), and 50 μg / animal / dose (booster immunization); for cell antigen (CHO-K1-MUC1-C) immunization, use 1×103 7 Cells / mouse / inoculation. Cell antigens were resuspended in phosphate-buffered saline before inoculation. Inoculation was performed on days 0, 14, 34, 48, 69, and 86. Blood samples were collected on days 26, 40, 57, 82, and 96. After the 5th to 9th immunizations, the antibody titer in mouse serum was determined by ELISA. Mice with high antibody titers in serum that were trending towards a plateau were selected, and their spleens were harvested for spleen cell fusion. A booster immunization was administered 3 days before spleen cell fusion, with an intraperitoneal (ip) injection of 1×10⁻⁶ cells / mouse. 7 Cell antigen solution resuspended in phosphate buffer for cells / animals.

[0286] Immunization regimen 3: Immunization uses cross-immunization with protein antigen (hMUC1-CL-Fc) and cell antigen (CHO-K1-MUC1-C). The protein antigen (hMUC1-CL-Fc) is used... Gold Adjuvant (Sigma) and Thermo Alum (Thermo) adjuvant cross-immunization. Protein antigen (hMUC1-CL-Fc) and adjuvant ( The ratio of Gold Adjuvant (hMUC1-CL-Fc) was 1:1, and the mixture was emulsified before inoculation. The ratio of alum (to aspirin) is 3:1. After shaking to mix, inoculate. Administer 50 μg / animal / dose (primary immunization), 25 μg / animal / dose (routine immunization), and 50 μg / animal / dose (booster immunization); for cell antigen (CHO-K1-MUC1-C) immunization, use 1×103 7 Cells / mouse / inoculation. Cell antigen was resuspended in phosphate buffer before inoculation. Inoculation was performed on days 0, 12, 25, 40, 55, 112, 127, and 156. Blood samples were collected on days 21, 35, 49, 63, 108, and 154. After the 5th to 8th immunizations, the antibody titer in mouse serum was determined by ELISA. Mice with high antibody titers in serum that were approaching a plateau were selected, and their spleens were harvested for spleen cell fusion. Three days before spleen cell fusion, a booster immunization was performed by intraperitoneal (ip) injection of 50 μg / mouse of the protein antigen (hMUC1-CL-Fc) and an antigen solution prepared with physiological saline.

[0287] Immunization Protocol 4: Immunization uses cross-immunization with protein antigen (hMUC1-CL-Fc) and cell antigen (HEK293-MUC1-C). The protein antigen (hMUC1-CL-Fc) is used... Gold Adjuvant (Sigma) and Thermo Alum (Thermo) adjuvant cross-immunization. Protein antigen (hMUC1-CL-Fc) and adjuvant ( The ratio of Gold Adjuvant (hMUC1-CL-Fc) was 1:1, and the mixture was emulsified before inoculation. The ratio of alum (e.g., 3:1) is used, and the mixture is shaken to mix thoroughly before inoculation. 50 μg / animal / dose (primary immunization), 25 μg / animal / dose (routine immunization), 50 μg / animal / dose (boost immunization). Cellular antigen (HEK293-MUC1-C) immunization uses 1×103 7 Cells / mouse / inoculation; cell antigen was resuspended in phosphate buffer before inoculation. Inoculation was performed on days 0, 14, 28, and 42. Blood samples were collected on days 10 and 38. After the 3rd and 4th immunizations, the antibody titer in mouse serum was determined using ELISA. Mice with high antibody titers that tended to plateau were selected, and their spleens were harvested for spleen cell fusion. A booster immunization was performed 3 days prior to spleen cell fusion, with an intraperitoneal (ip) injection of 50 μg / mouse of the protein antigen (hMUC1-CL-Fc) and an antigen solution prepared with physiological saline.

[0288] 2. Spleen cell fusion

[0289] An optimized electrofusion method was used to fuse splenic lymphocytes with myeloma Sp2 / 0 cells (…). CRL-8287 TM Hybridoma cells are obtained by fusing them together.

[0290] Based on the spleen cell count, the fused hybridoma cells were resuspended at a density of 3-4 × 10^5 / mL in complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI), and seeded at 150 μL / well in 96-well plates. After incubation at 37°C and 5% CO2 for 4-5 days, the supernatant was removed, and 200 μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI) was added. After incubation at 37°C and 5% CO2 for 2 days, ELISA detection was performed.

[0291] Example 4: Screening of mouse anti-MUC1-C hybridoma antibodies

[0292] 1. Hybridoma supernatant antibody-hMUC1-CL-his protein ELISA assay

[0293] Human hMUC1-CL-his protein was diluted to a concentration of 1 μg / mL with PBS buffer (pH 7.4). 100 μL of this solution was added to each well of a 96-well microplate (Corning, catalog number CLS3590-100EA) and incubated at 4°C for 16-18 hours. After discarding the solution, 200 μL of blocking buffer (Sangon Biotech, catalog number A600669-0250) diluted in PBS was added to each well, and the plate was incubated at 37°C for 1.5 hours. After blocking, the blocking buffer was discarded, and the plate was washed three times with PBST buffer (pH 7.4 PBS containing 0.1% Tween-20). 100 μL of hybridoma supernatant was added to each well, and the plate was incubated at 37°C for 1 hour. After incubation, wash the plate 5 times with PBST, add 50 μL / well of secondary antibody diluted with 2% MPBS (Jackson ImmunoResearch, catalog number 1115-035-003), and incubate at 37°C for 1 hour. After washing 5 times with PBST, add 50 μL / well of TMB chromogenic substrate (KPL, Cat No. 52-00-03), incubate at room temperature for 5-10 min, and stop the reaction by adding 50 μL / well of 1M H2SO4. Read the absorbance at 450 nm using a VERSAmax microplate reader (Molecular Devices). The results are shown in the table below.

[0294] Table 2. Results of ELISA assay for binding hybridoma supernatant to human MUC1-CL-his antigen

[0295] 2. Hybridoma supernatant antibody binding to human MUC1-C cell line mirrorball experiment

[0296] HCC827-human-MUC1-C (internally constructed overexpression stable transgenic cell line), CHOK1-cyno-MUC1-C (internally constructed overexpression stable transgenic cell line), or CHOK1-WT cells were digested and washed once with PBS buffer. Afterwards, they were centrifuged at 1000 rpm for 5 min and then rinsed with CellTracker solution diluted in PBS buffer to a final concentration of 50 nM. TMCells were resuspended in Green CMFDA dye (Thermo Fisher Scientific (China) Co., Ltd., catalog number C7025) at a density of 1E6 / mL and incubated for 30 minutes. After incubation, the cells were centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS containing 1% FBS (Gibco, catalog number 10100147). After centrifugation, the cells were resuspended in PBS buffer (final density 1-2E5 / mL), and APC-labeled fluorescent secondary antibody (BD Biosciences, catalog number 550826) was added at a dilution of 1:200. The cell mixture was added to 384-well plates (Corning, catalog number 3764), 20 μL of cells per well, i.e., 2000 to 4000 cells per well. Except for the positive and negative control wells, 20 μL of hybridoma supernatant was added to each well of the 384-well plate. The plates were incubated at room temperature in the dark for 2 hours, and the readings were taken using a Mirrorball instrument (Sptlabtech).

[0297] Table 3. Results of mirrorball experiment using hybridoma supernatant Note: "+" indicates that there is a combination, and "-" indicates that there is no combination.

[0298] 3. Screening of hybridoma cells for antibodies against MUC1-C

[0299] Four hybridomas obtained after qualitative detection by ELISA and mirrorball were sequenced, and the corresponding antibodies were named after sequencing. The antibody from hybridoma 17F3 was named M4, the antibody from hybridoma 28E4 was named M6, the antibody from hybridoma 3H6 was named F4-1, and the antibody from hybridoma 36G9 was named F4-18.

[0300] The four obtained antibodies were sequenced, and the sequences were classified according to their CDRs. The murine variable region sequences were then ligated with the human antibody constant region sequences, and chimeric antibodies were expressed. The amino acid sequences of the heavy and light chain variable regions of the screened antibodies are as follows:

[0301] >M4 heavy chain variable region sequence (M4 mVH):

[0302] >M4 light chain variable region sequence (M4 mVL):

[0303] >M6 heavy chain variable region sequence (M6 mVH):

[0304] >M6 light chain variable region sequence (M6 mVL):

[0305] >F4-1 heavy chain variable region sequence (F4-1 mVH):

[0306] >F4-1 light chain variable region sequence (F4-1 mVL):

[0307] >F4-18 heavy chain variable region sequence (F4-18 mVH):

[0308] >F4-18 light chain variable region sequence (F4-18 mVL):

[0309] Note: In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The underlined part is the CDR sequence determined according to the Kabat numbering system, and the ununderlined part is the FR sequence.

[0310] The heavy and light chain CDR region sequences of murine antibodies M4, M6, F4-1, and F4-18 are shown in the table below:

[0311] Table 4. Antibody heavy chain and light chain CDR sequences Note: The CDRs in the table are CDRs determined according to the Kabat numbering system.

[0312] Example 5: Humanization of anti-MUC1-C murine antibody

[0313] The MOE software was used to compare the genetic sequences of the heavy and light chain variable regions with the IMGT human antibody heavy and light chain variable regions germline genes that showed high homology with M4, M6, F4-1, and F4-18, respectively, as templates. The CDRs of these four murine antibodies were then transplanted into their corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Exemplarily, in the following specific embodiments, the CDR amino acid residues were determined and annotated using the Kabat numbering system.

[0314] 1. Humanization of M4 mouse antibodies

[0315] The humanized light chain templates for murine antibody M4 are IGKV1-39*01, IGKV6-21*02, IGKV3-11*01, and IGKJ4*01, while the humanized heavy chain templates are IGHV1-46*01 and IGHJ6*01. The CDRs of murine antibody M4 were transplanted into their respective humanized templates. Then, the amino acid FR portion of the humanized antibody underwent reverse mutation modification, considering the removal of potential isomerization and other chemical modification sites, the formation of N-terminal pyroglutamic acid, and the reduction of potential immunogenicity. The light chain FR portion includes one or more mutations of 3, 43, 47, 49, or 60 (the positions of these mutation sites are determined according to Kabat numbering rules), and the heavy chain FR portion includes one or more mutations of 1, 28, 38, 40, 48, 71, 73, 76, and 82a (the positions of these mutation sites are determined according to Kabat numbering rules). The amino acid substitutions in the variable region of the humanized antibody M4 are shown in the table below.

[0316] Table 5. Amino acid substitutions in the variable region of M4 humanized antibody Note: Graft represents the insertion of murine antibody CDR into the human FR region; the location of the mutation site is determined according to the Kabat numbering rules, such as "S82aR" which means that the S at position 82a (also known as 82A) is mutated to R according to the Kabat numbering system.

[0317] The heavy chain variable region / light chain variable region sequences of the M4 humanized antibody are as follows:

[0318] >huM4VH1

[0319] >huM4VH2

[0320] >huM4VH3

[0321] >huM4VL1

[0322] >huM4VL2

[0323] >huM4VL3

[0324] >huM4VL4

[0325] In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The underlined part in the sequence is the CDR sequence determined according to the Kabat numbering system, and the non-underlined part is the FR sequence.

[0326] 2. Humanization of M6 mouse antibodies

[0327] The humanized light chain templates for murine antibody M6 are IGKV4-1*01 / IGKV3-11*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-46*01 and IGHJ6*01. The CDRs of murine antibody M6 were transplanted into their respective humanized templates. Then, the amino acid FR portion of the humanized antibody underwent reverse mutation modification, considering the removal of potential isomerization and other chemical modification sites, the formation of N-terminal pyroglutamic acid, and the reduction of potential immunogenicity. The light chain FR portion includes one or more mutations of 1, 4, 45, 68, or 83 (the positions of the mutation sites are determined according to Kabat numbering rules), and the heavy chain includes one or more mutations of 1, 28, 30, 39, 40, 43, 69, 71, 76, 82b, 83, 84, and 97 (the positions of the mutation sites are determined according to Kabat numbering rules). The amino acid substitutions in the variable region of the humanized antibody M6 are shown in the table below.

[0328] Table 6. Amino acid substitutions in the variable region of the M6 ​​humanized antibody Note: Graft represents the insertion of murine antibody CDR into the human FR region; the location of the mutation site is determined according to the Kabat numbering rules, such as "G68R" which means that the 68th G is mutated to R according to the Kabat numbering system.

[0329] The heavy chain variable region / light chain variable region sequences of the M6 ​​humanized antibody are as follows:

[0330] >huM6VH1

[0331] >huM6VH2

[0332] >huM6VH3

[0333] >huM6VH4

[0334] >huM6VH5

[0335] >huM6VH6

[0336] >huM6VH7

[0337] >huM6VL1

[0338] >huM6VL2

[0339] >huM6VL3

[0340] The CDRs of the M6 ​​humanized antibody are as follows:

[0341] Table 7. CDRs of M6 humanized antibodies

[0342] In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the underlined part is the CDR sequence determined according to the Kabat numbering system, and the non-underlined part is the FR sequence.

[0343] 3. Humanization of F4-1 mouse antibody

[0344] The humanized light chain templates for murine antibody F4-1 are IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ6*01. The CDRs of murine antibody F4-1 were transplanted into their respective humanized templates. Then, the amino acid FR portion of the humanized antibody underwent reverse mutation modification, considering the removal of potential isomerization and other chemical modification sites, the formation of N-terminal pyroglutamic acid, and the reduction of potential immunogenicity. The light chain FR portion includes mutations at positions 4, 36, 42, 43, 47, 60, 70, and 75 (the positions of these mutation sites are determined according to Kabat numbering rules), and the heavy chain FR portion includes one or more mutations at positions 1, 2, 12, 40, 44, 47, 48, 69, 71, and 76 (the positions of these mutation sites are determined according to Kabat numbering rules). The amino acid substitutions in the variable region of the humanized antibody F4-1 are shown in the table below.

[0345] Table 8. Amino acid substitutions in the variable region of F4-1 humanized antibody Note: Graft represents the insertion of a murine antibody CDR into the human FR region; the location of the mutation site is determined according to the Kabat numbering rules. For example, "Y36F" means that the 36th Y is mutated to F according to the Kabat numbering system.

[0346] The heavy chain variable region / light chain variable region sequences of the F4-1 humanized antibody are as follows:

[0347] >huF4-1VH1

[0348] >huF4-1VH2

[0349] >huF4-1VH3

[0350] >huF4-1VL1

[0351] >huF4-1VL2

[0352] >huF4-1VL3

[0353] >huF4-1VL4

[0354] In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the underlined part is the CDR sequence determined according to the Kabat numbering system, and the non-underlined part is the FR sequence.

[0355] 4. Humanization of F4-18 mouse antibody

[0356] The humanized light chain templates for murine antibody F4-18 are IGKV1-39*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-3*01 and IGHJ1*01. The CDRs of murine antibody F4-18 were transplanted into their respective humanized templates. Then, the amino acid reversion mutations of the FR portion of the humanized antibody were performed, considering the removal of potential isomerization and other chemical modification sites, the formation of N-terminal pyroglutamic acid, and the reduction of potential immunogenicity. The FR portion of the light chain includes mutations at positions 4, 36, 39, 42, 44, 46, 60, 66, 69, and 71 (the positions of these mutation sites are determined according to Kabat numbering rules). The heavy chain includes one or more mutations at positions 12, 20, 24, 40, 44, 48, 69, 71, 96, and 101 (the positions of these mutation sites are determined according to Kabat numbering rules). The amino acid substitutions in the variable region of the humanized antibody F4-18 are shown in the table below.

[0357] Table 9-1. Amino acid substitutions in the variable region of F4-18 humanized antibody Note: Graft represents the insertion of a murine antibody CDR into the human FR region; the location of the mutation site is determined according to the Kabat numbering rules. For example, "M4L" indicates that the 4th M is mutated to L according to the Kabat numbering system.

[0358] The heavy chain variable region / light chain variable region sequences of the F4-18 humanized antibody are as follows:

[0359] >huF4-18VH1

[0360] >huF4-18VH2

[0361] >huF4-18VH3

[0362] >huF4-18VH4

[0363] >huF4-18VH5

[0364] >huF4-18VL1

[0365] >huF4-18VL2

[0366] >huF4-18VL3

[0367] >huF4-18VL4

[0368] The CDRs for the F4-18 humanized antibody are as follows:

[0369] Table 9-2. CDRs of F4-18 humanized antibody

[0370] In the above sequence, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the underlined part is the CDR sequence determined according to the Kabat numbering system, and the non-underlined part is the FR sequence.

[0371] 5. Construction and expression of anti-MUC1-C humanized antibody IgG1 form

[0372] Primers were designed for PCR to construct the VH / VK gene fragments of each humanized antibody. These fragments were then homologously recombinated with the expression vector pTT5 (containing a signal peptide and a constant region gene (CH1-FC / CL) fragment, constructed in the laboratory) to construct the full-length antibody expression vector VH-CH1-FC-pTT5 / VK-CL-pTT5. The heavy chain constant region of the antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or their variants, and the light chain constant region can be selected from the light chain constant region of human κ, λ chains, or their variants. For example, in the following embodiments, the antibody heavy chain constant region is selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO: 69 or 186, and the light chain constant region is selected from the human light chain constant region as shown in SEQ ID NO: 70.

[0373] Human IgG1 heavy chain constant region sequence:

[0374] Human IgG1 heavy chain constant region sequence (with LALA mutation):

[0375] Human light chain constant region sequence:

[0376] By linking the carboxyl terminus of the heavy chain variable region of the previously screened murine antibodies M4, M6, F4-1, and F4-18 to the amino terminus of the human heavy chain constant region as shown in SEQ ID NO: 69, and simultaneously linking the carboxyl terminus of the light chain variable region of the murine antibodies to the amino terminus of the human light chain constant region as shown in SEQ ID NO: 70, the corresponding chimeric antibodies can be obtained. Specifically, the chimeric antibodies of M4, M6, F4-1, and F4-18 are represented as ChiM4, ChiM6, ChiF4-1, and ChiF4-18, respectively.

[0377] The carboxyl terminus of the variable region of the humanized antibody heavy chain of M4, M6, F4-1, and F4-18 constructed above is linked to the amino terminus of the constant region of the human heavy chain as shown in SEQ ID NO: 69 to form the full-length antibody heavy chain. The carboxyl terminus of the variable region of the humanized antibody light chain of M4, M6, F4-1, and F4-18 is linked to the amino terminus of the constant region of the human light chain as shown in SEQ ID NO: 70 to form the full-length antibody light chain. The humanized antibodies shown in Tables 10-13 below can be obtained.

[0378] Table 10. Humanized Antibodies for M4 Note: In the table, “M4-H1L1” indicates that the heavy chain variable region is huM4VH1 (SEQ ID NO: 36), the light chain variable region is huM4VL1 (SEQ ID NO: 39), and the heavy chain constant region is as shown in SEQ ID NO: 69, and the light chain constant region is as shown in SEQ ID NO: 70, and so on.

[0379] Table 11. Humanized Antibodies for M6 Note: In the table, “M6-H1L1” indicates that the heavy chain variable region is huM6VH1 (SEQ ID NO: 43), the light chain variable region is huM6VL1 (SEQ ID NO: 50), and the heavy chain constant region is as shown in SEQ ID NO: 69, and the light chain constant region is as shown in SEQ ID NO: 70, and so on.

[0380] Table 12. Humanized Antibodies for F4-1 Note: In the table, "F4-1-H1L1" indicates that the heavy chain variable region is huF4-1VH1 (SEQ ID NO: 53), the light chain variable region is huF4-1VL1 (SEQ ID NO: 56), and the heavy chain constant region is as shown in SEQ ID NO: 69, and the light chain constant region is as shown in SEQ ID NO: 70, and so on.

[0381] Table 13. Humanized Antibodies for F4-18 Note: In the table, "F4-18-H1L1" indicates that the heavy chain variable region is huF4-18VH1 (SEQ ID NO: 60), the light chain variable region is huF4-18VL1 (SEQ ID NO: 65), and the heavy chain constant region is as shown in SEQ ID NO: 69, and the light chain constant region is as shown in SEQ ID NO: 70, and so on.

[0382] An example of a humanized antibody heavy / light chain full-length sequence is shown below:

[0383] M4-H1L1 heavy chain sequence:

[0384] M4-H1L1 light chain sequence:

[0385] M4-H2L1 heavy chain sequence:

[0386] M4-H2L1 light chain sequence:

[0387] M4-H1L2 heavy chain sequence:

[0388] M4-H1L2 light chain sequence:

[0389] M4-H2L2 heavy chain sequence:

[0390] M4-H2L2 light chain sequence:

[0391] M6-H1L1 heavy chain sequence:

[0392] M6-H1L1 light chain sequence:

[0393] M6-H3L1 heavy chain sequence:

[0394] M6-H3L1 light chain sequence:

[0395] M6-H2L2 heavy chain sequence:

[0396] M6-H2L2 light chain sequence:

[0397] M6-H3L2 heavy chain sequence:

[0398] M6-H3L2 light chain sequence:

[0399] M6-H4L2 heavy chain sequence:

[0400] M6-H4L2 light chain sequence:

[0401] >F4-1-H1L1 heavy chain sequence:

[0402] >F4-1-H1L1 light chain sequence:

[0403] >F4-1-H1L2 heavy chain sequence:

[0404] >F4-1-H1L2 light chain sequence:

[0405] >F4-1-H2L2 heavy chain sequence:

[0406] >F4-1-H2L2 light chain sequence:

[0407] >F4-1-H1L4 heavy chain sequence:

[0408] >F4-1-H1L4 light chain sequence:

[0409] >F4-18-H1L1 heavy chain sequence:

[0410] >F4-18-H1L1 light chain sequence:

[0411] >F4-18-H2L1 heavy chain sequence:

[0412] >F4-18-H2L1 light chain sequence:

[0413] >F4-18-H2L2 heavy chain sequence:

[0414] >F4-18-H2L2 light chain sequence:

[0415] >F4-18-H2L3 heavy chain sequence:

[0416] >F4-18-H2L3 light chain sequence:

[0417] >F4-18-H4L3 heavy chain sequence:

[0418] >F4-18-H4L3 light chain sequence:

[0419] >F4-18-H1L4 heavy chain sequence:

[0420] >F4-18-H1L4 light chain sequence:

[0421] >F4-18-H2L4 heavy chain sequence:

[0422] >F4-18-H2L4 light chain sequence:

[0423] >F4-18-H4L4 heavy chain sequence:

[0424] >F4-18-H4L4 light chain sequence:

[0425] Note: In the full-length antibody sequence above, the underlined portion is the variable region sequence, and the ununderlined portion is the constant region sequence.

[0426] Example 6: Modification of anti-EGFR antibodies

[0427] Molecules that specifically bind to EGFR can be derived from any suitable antibody, such as zalutumumab or its variants, wherein:

[0428] Table 14. CDR sequences of Zalutumumab

[0429] >Zalutumumab heavy chain variable region sequence (abbreviated as "ZalVH"):

[0430] >Zalutumumab light chain variable region sequence (abbreviated as "ZalVL"):

[0431] >Zalutumumab heavy chain sequence:

[0432] >Zalutumumab light chain sequence:

[0433] By mutating amino acids at positions 31, 33, 52A, 56, 60, 97, and / or 99 of the heavy chain variable region of Zalutumumab, and / or by mutating amino acid at position 1 of the light chain variable region, a total of 15 anti-EGFR antibodies were obtained, namely: ZalH', ZalH1, ZalH2, ZalH3, ZalH4, ZalH5, ZalH6, ZalH7, ZalH8, ZalH9, ZalH10, ZalH11, ZalH12, ZalH13, and ZalH14, with the specific sequences as follows:

[0434] Table 15. Amino acid sequences of the replaced CDRs Note: Antibody ZalH1 indicates an amino acid mutation at position 31 (HCDR1) of the heavy chain variable region and position 1 of the light chain variable region of zalutumumab; antibody ZalH7 indicates a mutation at positions 31 and 52A (HCDR1 and HCDR2) of the heavy chain variable region and position 1 of the light chain variable region of zalutumumab; ZalH' indicates a mutation only at position 1 of the light chain variable region of zalutumumab; the antibody heavy chain constant region is selected from the human IgG1 heavy chain constant region as shown in SEQ ID NO: 69, and the light chain constant region is selected from the human light chain constant region as shown in SEQ ID NO: 70. Other antibodies follow the same pattern.

[0435] The heavy chain variable region sequence of ZalH1 (abbreviated as "ZalVH1"):

[0436] The heavy chain variable region sequence of ZalH2 (abbreviated as "ZalVH2"):

[0437] The heavy chain variable region sequence of ZalH3 (abbreviated as "ZalVH3"):

[0438] The heavy chain variable region sequence of ZalH4 (abbreviated as "ZalVH4"):

[0439] The heavy chain variable region sequence of ZalH5 (abbreviated as "ZalVH5"):

[0440] The heavy chain variable region sequence of ZalH6 (abbreviated as "ZalVH6"):

[0441] The heavy chain variable region sequence of ZalH7 (abbreviated as "ZalVH7"):

[0442] The heavy chain variable region sequence of ZalH8 (abbreviated as "ZalVH8"):

[0443] The heavy chain variable region sequence of ZalH9 (abbreviated as "ZalVH9"):

[0444] The heavy chain variable region sequence of ZalH10 (abbreviated as "ZalVH10"):

[0445] The heavy chain variable region sequence of ZalH11 (abbreviated as "ZalVH11"):

[0446] The heavy chain variable region sequence of ZalH12 (abbreviated as "ZalVH12"):

[0447] The heavy chain variable region sequence of ZalH13 (abbreviated as "ZalVH13"):

[0448] The heavy chain variable region sequence of ZalH14 (abbreviated as "ZalVH14"):

[0449] >Light chain variable region sequences of ZalH' and ZalH1 to ZalH14 (hereinafter referred to as "ZalVL1"):

[0450] Heavy chain sequence of ZalH': SEQ ID NO: 124

[0451] Heavy chain sequence of ZalH1:

[0452] Heavy chain sequence of ZalH2:

[0453] Heavy chain sequence of ZalH3:

[0454] The heavy chain sequence of ZalH4:

[0455] Heavy chain sequence of ZalH5:

[0456] The heavy chain sequence of ZalH6:

[0457] Heavy chain sequence of ZalH7:

[0458] Heavy chain sequence of ZalH8:

[0459] The heavy chain sequence of ZalH9:

[0460] Heavy chain sequence of ZalH10:

[0461] Heavy chain sequence of ZalH11:

[0462] Heavy chain sequence of ZalH12:

[0463] Heavy chain sequence of ZalH13:

[0464] Heavy chain sequence of ZalH14:

[0465] >Light chain sequences of ZalH' and ZalH1 to ZalH14:

[0466] Note: In the above antibody sequences, the underlined part is the antibody variable region sequence, the double underlined part is the antibody CDR sequence, the ununderlined part is the antibody constant region sequence, and the bolded letters are mutant amino acids.

[0467] Example 7: Construction of anti-EGFR-MUC1 bispecific antibody

[0468] The EGFR-MUC1 bispecific antibody is produced in a 1:1 molecular form. The MUC1 arm is selected as M4-H1L1 (hereinafter referred to as "M4H1L1"), which is assembled with EGFR antibodies ZalH', ZalH4, ZalH8, ZalH10, and ZalH13 to form the bispecific antibody. The VH of the EGFR antibody is combined with titin, and the VL is combined with obscurin. Furthermore, S358C and T370W mutations (knobs) are introduced into the MUC1 antibody heavy chain, and Y357C, T374S, L376A, and Y415V mutations (holes) are introduced into the EGFR antibody heavy chain. The format is an asymmetric molecule containing four chains.

[0469] Chain 1: [VH(anti-MUC1)]-[IgG1(CH1)]-[Fc(Knob)];

[0470] Chain 2: [VL(anti-MUC1)]-[CL];

[0471] Chain 3: [VH(anti-EGFR)]-[connector 1]-[Titin]-[Fc(Hole)];

[0472] Chain 4: [VL(anti-EGFR)]-[connector 2]-[Obscurin], the schematic diagram of which is shown in Figure 5 (where T represents Titin and O represents Obscurin).

[0473] Table 16. Bispecific antibodies disclosed herein Note: For example, M4H1L1-ZalH' indicates that the molecule uses the variable region of M4-H1L1 as the MUC1 binding domain and the variable region of ZalH' as the EGFR binding domain. The Format shown in Figure 5 is the molecular structure, and so on.

[0474] Titin Chain:

[0475] Obscurin chain:

[0476] CH1:

[0477] >CL:SEQ ID NO:70

[0478] Connector 1 and connector 2: GGGGS (SEQ ID NO: 168)

[0479] >Fc(knob):

[0480] >Fc(hole):

[0481] The full-length sequence is as follows:

[0482] M4H1L1-ZalH' sequence:

[0483] Chain 1 (huM4VH1-CH1-Fc(Knob)):

[0484] Chain 2 (huM4VL1-CL):

[0485] Chain 3:

[0486] Chain 4:

[0487] M4H1L1-ZalH4 sequence:

[0488] Chain 1: SEQ ID NO: 171

[0489] Chain 2: SEQ ID NO: 74

[0490] Chain 3:

[0491] Chain 4: SEQ ID NO: 173

[0492] M4H1L1-ZalH8 sequence:

[0493] Chain 1: SEQ ID NO: 171

[0494] Chain 2: SEQ ID NO: 74

[0495] Chain 3:

[0496] Chain 4: SEQ ID NO: 173

[0497] M4H1L1-ZalH10 sequence:

[0498] Chain 1: SEQ ID NO: 171

[0499] Chain 2: SEQ ID NO: 74

[0500] Chain 3:

[0501] Chain 4: SEQ ID NO: 173

[0502] M4H1L1-ZalH13 sequence:

[0503] Chain 1: SEQ ID NO: 171

[0504] Chain 2: SEQ ID NO: 74

[0505] Chain 3:

[0506] Chain 4: SEQ ID NO: 173

[0507] Note: In the antibody sequences above, the underlined part is the antibody variable region sequence, the ununderlined part is the antibody constant region sequence, and the wavy underline is the linker sequence.

[0508] The VH / VL sequence of the negative control antibody IgG1 used in this disclosure is derived from patent US6114143A, with the heavy chain constant region and light chain constant region sequences being SEQ ID NO: 69 and SEQ ID NO: 70, respectively. Their full-length sequences are as follows:

[0509] IgG1 heavy chain:

[0510] IgG1 light chain:

[0511] Note: The underlined parts in the sequence are variable regions, and the italicized parts are constant regions.

[0512] Example 8: M4H1L1-ZalH4-LALA bispecific antibody

[0513] The M4H1L1-ZalH4-LALA bispecific antibody is developed based on the M4H1L1-ZalH4 bispecific antibody by introducing L238A and L239A mutations into strand 1 and L242A and L243A mutations into strand 3, while leaving strands 2 and 4 unchanged. This results in M4H1L1-ZalH4-LALA, which consists of four strands, with the specific sequences as follows:

[0514] M4H1L1-ZalH4-LALA sequence:

[0515] Chain 1:

[0516] Chain 2: SEQ ID NO: 74

[0517] Chain 3:

[0518] Chain 4: SEQ ID NO: 173

[0519] >Fc(knob)':

[0520] >Fc(hole)':

[0521] Note: In the antibody sequences above, the underlined part is the antibody variable region sequence, the ununderlined part is the antibody constant region sequence, the wavy underline is the linker sequence, and the bold letters are mutant amino acids.

[0522] II. Preparation of ADC

[0523] The ADC disclosed herein is derived from CN202311772280.3 and PCT / CN2024 / 087447 (included in this document by reference in their entirety).

[0524] Example 9: EGFR-MUC1 dual antibody conjugated with M toxin

[0525] M4H1L1-ZalH'-M

[0526] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 6.3 μL, 63 nmol) was added to the PBS buffered aqueous solution of antibody M4H1L1-ZalH' (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 0.38 mL, 25.7 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL. Compound M (0.245 mg, 257 nmol) was dissolved in 24 μL of acetonitrile and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain histidine-acetic acid buffer (0.3 mg / mL, 10.2 mL) of M4H1L1-ZalH'-M, which was stored at 4 °C. The average DAR was calculated by MS: n = 3.87.

[0527] M4H1L1-ZalH4-M

[0528] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 8.4 μL, 84 nmol) was added to the PBS buffered aqueous solution of antibody M4H1L1-ZalH4 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 0.5 mL, 33.8 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0529] Compound M (0.32 mg, 338 nmol) was dissolved in 32 μL of acetonitrile and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain histidine-acetic acid buffer (0.42 mg / mL, 10.7 mL) of M4H1L1-ZalH4-M, which was stored at 4 °C. The average DAR was calculated by MS: n = 4.8.

[0530] M4H1L1-ZalH8-M

[0531] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 8.25 μL, 82.5 nmol) was added to the PBS buffered aqueous solution of antibody M4H1L1-ZalH8 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 0.5 mL, 33.8 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0532] Compound M (0.32 mg, 338 nmol) was dissolved in 32 μL of acetonitrile and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain histidine-acetic acid buffer (0.39 mg / mL, 10.7 mL) of M4H1L1-ZalH8-M, which was stored at 4 °C. The average DAR was calculated by MS: n = 4.13.

[0533] M4H1L1-ZalH10-M

[0534] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 4.57 μL, 45.7 nmol) was added to the PBS buffered aqueous solution of antibody M4H1L1-ZalH10 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 0.276 mL, 18.6 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0535] Compound M (0.18 mg, 186 nmol) was dissolved in 18 μL of acetonitrile and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain the title product M4H1L1-ZalH10-M in histidine-acetic acid buffer (0.22 mg / mL, 10.2 mL), which was stored at 4 °C. The average DAR was calculated by MS: n = 4.33.

[0536] M4H1L1-ZalH13-M

[0537] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 7.85 μL, 78.5 nmol) was added to the PBS buffered aqueous solution of antibody M4H1L1-ZalH13 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 0.47 mL, 32 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0538] Compound M (0.306 mg, 320 nmol) was dissolved in 31 μL of acetonitrile and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain histidine-acetic acid buffer (0.42 mg / mL, 10.2 mL) of M4H1L1-ZalH13-M, which was stored at 4 °C. The average DAR was calculated by MS: n = 4.3.

[0539] IgG1-M

[0540] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 8.11 μL, 81.1 nmol) was added to a PBS buffered aqueous solution of antibody IgG1 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 0.5 mL, 33.8 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0541] Compound M (0.32 mg, 338 nmol) was dissolved in 32 μL of acetonitrile and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was then purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain histidine-acetic acid buffer of IgG1-M (0.48 mg / mL, 10.2 mL), which was stored at 4 °C. The average DAR was calculated by MS: n = 4.37.

[0542] Example 10: Antibody-conjugated 9-A toxin

[0543] 1. EGFR-MUC1 dual antibody conjugated with 9-A toxin

[0544] ADC-1

[0545] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 851 μL, 8.51 μmol) was added to a PBS buffered aqueous solution of antibody M4H1L1-ZalH4 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 30 mL, 2.027 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to a pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0546] Compound 9-A (26.1 mg, 24.32 μmol) was dissolved in 1.5 mL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain histidine-acetic acid buffer of ADC-1 (7.1 mg / mL, 39 mL), which was stored at 4 °C. The average DAR calculated by HIC was n = 5.78.

[0547] ADC-2

[0548] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 509.5 μL, 5.095 μmol) was added to the PBS buffered aqueous solution of antibody M4H1L1-ZalH4 (pH = 6.3, 0.05 M PBS buffer; 10.0 mg / mL, 30.8 mL, 2.074 μmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0549] Compound 9-A (22.3 mg, 20.74 μmol) was dissolved in 1.5 mL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain histidine-acetic acid buffer of ADC-2 (5.8 mg / mL, 47.6 mL), which was stored at 4 °C. The average DAR was calculated by HIC: n = 4.5.

[0550] ADC-3

[0551] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 29.3 μL, 293 nmol) was added to the PBS buffered aqueous solution of antibody M4H1L1-ZalH4-LALA (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.11 mL, 75 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0552] Compound 9-A (0.967 mg, 900 nmol) was dissolved in 55 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain histidine-acetic acid buffer of ADC-3 (0.96 mg / mL, 10.5 mL), which was stored at 4 °C. The average DAR was calculated by MS: n = 5.51.

[0553] 2. EGFR antibody conjugated with 9-A toxin

[0554] ZalH4-9-A

[0555] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 35 μL, 350 nmol) was added to the PBS buffered aqueous solution of antibody ZalH4 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.57 mL, 106 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0556] Compound 9-A (1.37 mg, 1.272 μmol) was dissolved in 80 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain ZalH4-9-A in histidine-acetic acid buffer (1.1 mg / mL, 12.8 mL), which was stored at 4 °C. The average DAR calculated by RP-HPLC was n = 5.94.

[0557] 3. MUC1 antibody conjugated to 9-A toxin

[0558] M4H1L1-9-A

[0559] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 31.2 μL, 312 nmol) was added to the PBS buffered aqueous solution of antibody M4H1L1 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 1.4 mL, 94.6 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath, and then the solution was transferred to pH 5.0 30 mM histidine-acetic acid buffer using a Sephadex G25 gel column and concentrated to 10 mg / mL.

[0560] Compound 9-A (1.22 mg, 1.135 μmol) was dissolved in 70 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: pH 5.0 30 mM histidine-acetic acid buffer) to obtain histidine-acetic acid buffer (0.94 mg / mL, 12.5 mL) of M4H1L1-9-A, which was stored at 4 °C. RP-HPLC calculated average: n = 6.6.

[0561] 4. Negative control antibody conjugated with 9-A toxin

[0562] IgG1-9-A

[0563] At 37°C, a prepared aqueous solution of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP.HCl) (10 mM, 81.6 μL, 816 nmol) was added to a PBS buffered aqueous solution of antibody IgG1 (pH = 6.3, 0.05 M PBS buffered aqueous solution; 10.0 mg / mL, 3.9 mL, 263 nmol). The solution was placed in a water bath and shaken at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was then cooled to 25°C in a water bath.

[0564] Compound 9-A (3.4 mg, 3.162 μmol) was dissolved in 200 μL of dimethyl sulfoxide and added dropwise to the above reaction solution. The mixture was placed in a water bath and shaken at 25 °C for 3 hours, after which the reaction was stopped. The reaction solution was purified by desalting using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous solution at pH 6.3 containing 0.001 M EDTA) to obtain PBS buffered solution of the title product IgG-9-A (2.42 mg / mL, 14.7 mL), which was stored at 4 °C. RP-HPLC calculated average: n = 6.15.

[0565] Test Example 1: Determination of chimeric antibody affinity based on FACS

[0566] HCC827-human-MUC1-C (internal overexpression stable transgenic cell line), CHOK1-cyno-MUC1-C (internal overexpression stable transgenic cell line), and T47D cells (human breast duct carcinoma cells) were digested separately, and 1E5 cells were added to each well of a 96-well plate. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS buffer containing 2% FBS. Antibody was started at a concentration of 20 μg / mL and serially diluted 5-fold in PBS buffer containing 2% FBS, resulting in 8 concentration gradients. 100 μL of antibody sample was added to each well, the cells were resuspended, and incubated at 4°C for 1 hour. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS buffer containing 2% FBS. Secondary antibody (Alexa) was added. 488g anti-human IgG (H+L) 1:1000 (Invitrogen, A11013) was incubated at 4°C for 40 minutes. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS buffer containing 2% FBS. The cells were resuspended in 100 μL of PBS buffer containing 2% FBS and then analyzed. The FACS results are as follows.

[0567] Table 17. Results of FACS-based detection of chimeric antibody-cell affinity.

[0568] The results showed that the chimeric antibody disclosed herein has a strong affinity for stable cell lines or tumor cell lines.

[0569] Test Example 2: Internalization test of tumor cells against MUC1-C chimeric antibody

[0570] I. Purpose of the Test

[0571] The purpose of this experiment was to detect the endocytic activity of MUC1 chimeric monoclonal antibody in tumor cells. Cells were treated with different concentrations of MUC1 chimeric monoclonal antibody in vitro, and after 3 days of culture, CTG (Cellular Transplantation) was used. The Luminescent Cell Viability Assay (Promega, catalog number: G7573) reagent is used to detect the proliferation of tumor cells, based on IC50. 50 The endocytic activity of the antibody was evaluated.

[0572] II. Experimental Methods

[0573] The following example, using the in vitro proliferation inhibition assay of T47D cells, illustrates the method for testing the endocytic activity of the disclosed MUC1 chimeric monoclonal antibody drug on tumor cells. This method is also applicable to, but not limited to, testing the in vitro proliferation inhibition activity of other tumor cells.

[0574] 1. Cell culture: T47D cells were cultured in 1640 medium (GE, catalog number SH30024.01) containing 10% FBS and 2 μg / mL human Insulin (Yisheng, catalog number 40112ES60).

[0575] 2. Cell preparation: Take T47D cells in logarithmic growth phase, wash them once with PBS (phosphate-buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3 mL of trypsin (0.25% Trypsin-EDTA (1x), Gibco, Life Technologies) to digest for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to wash off the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 mL of cell culture medium containing 20% ​​ultra-low IgG fetal bovine serum (Bosheng Biotechnology, catalog number BS-0007-500) to resuspend the cells and prepare a single-cell suspension.

[0576] 3. Cell plating: Mix the T47D single-cell suspension thoroughly, and adjust the viable cell density to 4 × 10⁶ cells / cells using cell culture medium. 4 Cells / mL. Mix the density-adjusted cell suspension thoroughly and add 50 μL / well to a 96-well cell culture plate. Add only 100 μL of culture medium to the outer wells of the 96-well plate. Incubate the plate in an incubator for 24 hours (37°C, 5% CO2).

[0577] 4. Preparation of MUC1 chimeric monoclonal antibody: Prepare a × concentration of DT3C (a fusion of diphtheria toxin fragment A and group G streptococcal 3C fragment, with a molar concentration 6 times that of the antibody) using serum-free RPMI 1640 medium. Prepare a 4× concentration of antibody using the same medium. Mix DT3C and antibody at a 1:1 volume ratio and incubate at room temperature for 30 min. Then perform serial dilutions.

[0578] 5. Sample addition procedure: Add the diluted antibody to the cells at a 1:1 ratio, 50 μL / well. Perform two replicates per sample. Incubate the culture plate in an incubator for 3 days (37℃, 5% CO2).

[0579] 6. Color development procedure: Take out the 96-well cell culture plate, add 50 μL of CTG solution to each well, and incubate at room temperature for 10 minutes.

[0580] 7. Plate reading procedure: Take out the 96-well cell culture plate and place it in an ELISA reader (PE, Envision) to measure the chemiluminescence.

[0581] III. Data Analysis

[0582] The data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. The results of the example are shown in the table below and Figures 1 and 2.

[0583] Table 18-1. In vitro internalization experiment of MUC1-C chimeric antibody on T47D cells

[0584] Table 18-2. In vitro internalization experiment of MUC1-C chimeric antibody on T47D cells.

[0585] The results showed that the chimeric antibody disclosed herein has strong endocytic activity against the T47D tumor cell line.

[0586] Test Example 3: FACS Evaluation of Chimeric and Humanized Antibodies

[0587] The experimental procedure was the same as in Test Example 1, "Assessment of Chimeric Antibody Affinity Based on FACS". Additionally, CHOK1-human-MUC1-C cells (internal constructed overexpression stable transgenic cell line) were used for evaluation. The experimental results are shown in the table below and Figures 3A to 3P.

[0588] Table 19-1. FACS results of chimeric and humanized antibodies Note: "+" indicates a combination.

[0589] Table 19-2. FACS results of chimeric and humanized antibodies Note: "+" indicates a combination.

[0590] Table 19-3. FACS results of chimeric and humanized antibodies Note: "+" indicates a combination.

[0591] Table 19-4. FACS results of chimeric and humanized antibodies Note: "+" indicates a combination.

[0592] The results showed that the chimeric and humanized antibodies disclosed herein have different strengths of affinity for stable cell lines or tumor cell lines, and all of them have human-monkey cross-binding activity.

[0593] Test Example 4: Internalization test of tumor cells against humanized MUC1-C antibody

[0594] I. Purpose of the Test

[0595] The purpose of this experiment was to detect the endocytic activity of the humanized MUC1 monoclonal antibody in tumor cells. Cells were treated with different concentrations of the humanized MUC1 monoclonal antibody in vitro, and after 3 days of culture, CTG (Cellular Transplantation) was used to detect the endocytic activity of the cells. The Luminescent Cell Viability Assay (Promega, catalog number: G7573) reagent is used to detect the proliferation of tumor cells, based on IC50. 50 The endocytic activity of the antibody was evaluated.

[0596] II. Experimental Methods

[0597] The following example, using the in vitro proliferation inhibition assay of T47D cells, illustrates the method for testing the endocytic activity of the disclosed MUC1 humanized monoclonal antibody drug against tumor cells. This method is also applicable to, but not limited to, testing the in vitro proliferation inhibition activity of other tumor cells.

[0598] 1. Cell culture: T47D cells were cultured in 1640 medium (GE, catalog number SH30024.01) with 10% FBS and 2ug / mL human Insulin (Yisheng, catalog number 40112ES60).

[0599] 2. Cell preparation: Take T47D cells in logarithmic growth phase, wash them once with PBS (phosphate-buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3 mL of trypsin (0.25% Trypsin-EDTA (1x), Gibco, Life Technologies) to digest for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to wash off the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 mL of cell culture medium containing 20% ​​ultra-low IgG fetal bovine serum (Bosheng Biotechnology, catalog number BS-0007-500) to resuspend the cells and prepare a single-cell suspension.

[0600] 3. Cell plating: Mix the T47D single-cell suspension thoroughly, and adjust the viable cell density to 4 × 10⁶ cells / cells using cell culture medium. 4 Cells / mL. Mix the density-adjusted cell suspension thoroughly and add 50 μL / well to a 96-well cell culture plate. Add only 100 μL of culture medium to the outer wells of the 96-well plate. Incubate the plate in an incubator for 24 hours (37°C, 5% CO2).

[0601] 4. Preparation of MUC1 chimeric monoclonal antibody: Prepare 4X concentration DT3C (a fusion of diphtheria toxin fragment A and group G streptococcal 3C fragment, with a molar concentration 6 times that of the antibody) using serum-free RPMI 1640 medium. Prepare 4X concentration antibody using the same medium, mixing DT3C and antibody at a 1:1 volume ratio and incubating at room temperature for 30 min. Then perform serial dilutions.

[0602] 5. Sample addition procedure: Add the diluted antibody to the cells at a 1:1 ratio, 50 μL / well. Perform two replicates per sample. Incubate the culture plate in an incubator for 3 days (37℃, 5% CO2).

[0603] 6. Color development procedure: Take out the 96-well cell culture plate, add 50 μL of CTG solution to each well, and incubate at room temperature for 10 minutes.

[0604] 7. Plate reading procedure: Take out the 96-well cell culture plate and place it in an ELISA reader (PE, Envision) to measure the chemiluminescence.

[0605] III. Data Analysis

[0606] The data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. The results of the example are shown in Table 20 and Figures 4A to 4F.

[0607] Table 20. In vitro internalization experiment of MUC1-C humanized antibody on T47D cells.

[0608] The results showed that the humanized antibody disclosed herein has strong endocytic activity against the T47D tumor cell line.

[0609] Test Example 5: Biacore Affinity Test for Anti-MUC1-C Antibody

[0610] 1. Experimental instruments: Biacore 8K, Cytiva

[0611] 2. Experimental materials: Protein A biosensor chip (Cat.#29127556, Cytiva)

[0612] 3. Test reagents:

[0613] 1) 10*HBS-EP+ buffer solution (pH 7.4) (Cat.#BR-1006-69, Cytiva)

[0614] 2)10mM Glycine-HCl (pH 1.5)(Cat.#BR-1003-54,Cytiva)

[0615] 3) MUC1-CL-His (Homemade)

[0616] 4) Antibody sample to be tested (self-made)

[0617] The affinity of the disclosed antibody for the human MUC1-CL-His antigen was tested using a Biacore 8K instrument.

[0618] The method is as follows: Antibody molecules were affinity-captured using a Protein A biosensor chip, and then a certain concentration of antigen molecules was flowed through the chip surface. The injection was continuous for 180 seconds, followed by natural dissociation for 600 seconds. The reaction signal was monitored in real-time using a Biacore 8K instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl (pH 1.5). The Kinetics 1:1 binding model was used for data fitting. The experimental results are shown in Table 21.

[0619] Table 21. Affinity test results of anti-MUC1 antibodies to human MUC1 antigen

[0620] The results showed that the disclosed anti-MUC1-C antibody has a high affinity for the human MUC1-C antigen.

[0621] Test Example 6: Biacore Affinity Assay for Anti-EGFR Antibody

[0622] 1. Experimental instruments: Biacore 8K, Cytiva

[0623] 2. Experimental materials: Protein A biosensor chip (Cat.#29127556, Cytiva)

[0624] 3. Test reagents:

[0625] 1) 10*HBS-EP+ buffer solution (pH 7.4) (Cat.#BR-1006-69, Cytiva)

[0626] 2)10mM Glycine-HCl (pH 1.5)(Cat.#BR-1003-54,Cytiva)

[0627] 3) EGFR-His (homemade)

[0628] 4) Antibody sample to be tested (prepared in-house)

[0629] The affinity of the disclosed antibody for the human EGFR-His antigen was tested using a Biacore 8K instrument.

[0630] The method is as follows: Antibody molecules were affinity-captured using a Protein A biosensor chip, and then a certain concentration of antigen molecules was flowed through the chip surface. The injection was continuous for 180 seconds, followed by natural dissociation for 600 seconds. The reaction signal was monitored in real-time using a Biacore 8K instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl (pH 1.5). The Kinetics 1:1 binding model was used for data fitting. The experimental results are shown in Table 22.

[0631] Table 22. Affinity test results of anti-EGFR antibodies to human EGFR antigen

[0632] The results showed that the mutant of the anti-EGFR antibody disclosed herein has different strengths of affinity for human EGFR antigen.

[0633] Test Example 7: Biacore assay for affinity of anti-EGFR-MUC1 bispecific antibodies

[0634] 1. Experimental instruments: Biacore 8K, Cytiva

[0635] 2. Experimental materials: Protein A biosensor chip (Cat.#29127556, Cytiva)

[0636] 3. Test reagents:

[0637] 1) 10*HBS-EP+ buffer solution (pH 7.4) (Cat.#BR-1006-69, Cytiva)

[0638] 2)10mM Glycine-HCl (pH 1.5)(Cat.#BR-1003-54,Cytiva)

[0639] 3) EGFR-His (homemade)

[0640] 4) Antibody sample to be tested (prepared in-house)

[0641] The affinity of the disclosed antibody for the human EGFR-His antigen was tested using a Biacore 8K instrument.

[0642] The method is as follows: Antibody molecules were affinity-captured using a Protein A biosensor chip, and then a certain concentration of antigen molecules was flowed through the chip surface. The injection was continuous for 180 seconds, followed by natural dissociation for 600 seconds. The reaction signal was monitored in real time using a Biacore 8K instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl (pH 1.5). The Kinetics 1:1 binding model was used for data fitting. The results are shown in Table 23.

[0643] Table 23. Affinity test results of anti-EGFR-MUC1 bispecific antibodies to human EGFR antigen

[0644] The results showed that the disclosed anti-EGFR-MUC1 bispecific antibody has different strengths of affinity for human EGFR antigen.

[0645] Test Example 8: Determination of Affinity for Anti-EGFR-MUC1 Bispecific Antibody Based on FACS

[0646] HCC827 cells were digested, and 1E5 cells were added to each well of a 96-well plate. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS buffer containing 2% FBS. Antibody was started at a concentration of 20 μg / mL and serially diluted 5-fold in PBS buffer containing 2% FBS, resulting in 8 concentration gradients. 100 μL of antibody sample was added to each well, the cells were resuspended, and incubated at 4°C for 1 hour. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS buffer containing 2% FBS. Secondary antibody (Alexa) was added. 488g anti-human IgG (H+L) 1:1000 (Invitrogen, A11013) was incubated at 4°C for 40 minutes. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS buffer containing 2% FBS. The cells were resuspended in 100 μL of PBS buffer containing 2% FBS and the data were read. FACS results are shown in Table 24.

[0647] Table 24. Results of FACS-based detection of cell affinity between anti-EGFR-MUC1 bispecific antibodies and cells. Note: / indicates that it cannot be detected.

[0648] The results showed that the disclosed anti-EGFR-MUC1 bispecific antibody had different strengths of affinity for HCC827 tumor cells.

[0649] Test Example 9: In vitro killing activity assay of anti-EGFR-MUC1 bispecific antibody ADC

[0650] 1. Experimental Objective: To detect the inhibitory effect of EGFR-MUC1 bispecific antibody ADC on the proliferation of HCC827 / HCC70 cells.

[0651] 2. Test materials:

[0652] HCC827 cells: purchased from the Chinese Academy of Sciences, catalog number TCHU153

[0653] HCC70 cells: purchased from ATCC, catalog number CRL-2315

[0654] 3. Test methods:

[0655] 1) Cell culture: HCC827 and HCC70 cells were cultured in 1640 medium (Meilun Biotechnology, catalog number PWL015-L) containing 10% FBS.

[0656] 2) Cell preparation: Take HCC827 and HCC70 cells in the logarithmic growth phase, wash them once with PBS (phosphate-buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3 mL of trypsin (0.25% Trypsion-EDTA (100 mL), Meilun Biotechnology, catalog number PWL060) to digest for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to wash off the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 mL of cell culture medium to resuspend the cells to prepare a single-cell suspension.

[0657] 3) Cell plating: Mix HCC827 and HCC70 single-cell suspensions thoroughly, and adjust the viable cell density to 3.7 × 10⁻⁶ cells / mL using cell culture medium. 3 Cells / mL. Mix the density-adjusted cell suspension thoroughly and add 135 μL / well to a 96-well cell culture plate. Add only 150 μL of culture medium to the outer wells of the 96-well plate. Incubate the plate in an incubator for 24 hours (37°C, 5% CO2).

[0658] 4) ADC drug preparation. The ADC drug was diluted with PBS (phosphate-buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.). The ADC to be tested was diluted to 10 μM, and then diluted 5-fold with PBS, resulting in 9 wells. The final 10th well was a drug-free well. Different concentration gradients of the drug were prepared.

[0659] 5) Sample addition procedure. Add 15 μL of the prepared test samples at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37℃, 5% CO2). The final ADC drug concentration gradient in the cell plate is 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, 0 nM.

[0660] 6) Color development procedure: Take out the 96-well cell culture plate, add 75 μL of CTG solution to each well, and incubate at room temperature for 10 minutes.

[0661] 7) Plate reading procedure: Take out the 96-well cell culture plate and place it in a microplate reader (PE, Envision) to measure chemiluminescence.

[0662] The results are shown in Table 25 and Figures 6A and 6B.

[0663] Table 25. Results of in vitro cytotoxic activity assay of EGFR-MUC1 bispecific antibody ADC

[0664] The results showed that the disclosed anti-EGFR-MUC1 bispecific antibody exhibited different levels of killing activity against HCC827 and HCC70 tumor cell lines.

[0665] Test Example 10: Biacore detection of human-monkey cross-activity of M4H1L1-ZalH4

[0666] 1. Test instruments: Biacore T200, Cytiva

[0667] 2. Experimental materials: Protein A biosensor chip (Cat.#29127556, Cytiva)

[0668] 3. Test reagents:

[0669] 1) 10*HBS-EP+ buffer solution (pH 7.4) (Cat.#BR-1006-69, Cytiva)

[0670] 2)10mM Glycine-HCl (pH 1.5)(Cat.#BR-1003-54,Cytiva)

[0671] 3) hEGFR-His, hMUC1-C-His (homemade)

[0672] 4) Cynomolgus MUC1 his (Cat. #MU1-C52H5, Acro), Cynomolgus EGFR-his (Cat. #90285-C08H, Sino Biological)

[0673] 5) Antibody sample to be tested (prepared in-house)

[0674] The affinity of the disclosed antibody for human, monkey EGFR, and MUC1-C antigens was tested using a Biacore T200 instrument. The method is as follows:

[0675] Antibody molecules were affinity-captured using a Protein A biosensor chip, followed by the flow of a specific concentration of antigen molecules onto the chip surface. The injection was continuous for 180 seconds, followed by natural dissociation for 600 seconds. The Biacore T200 instrument monitored the reaction signal in real time to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl (pH 1.5). The Kinetics 1:1 binding model was used for data fitting. The results are shown in Table 26.

[0676] Table 26. Results of antibody affinity tests for human and monkey EGFR and MUC1-C antigens

[0677] The results showed that the disclosed anti-EGFR-MUC1 bispecific antibody M4H1L1-ZalH4 exhibited good cross-binding activity with human and monkey EGFR and MUC1-C antigens.

[0678] Test Example 11: Biacore detects that M4H1L1-ZalH4 has the ability to bind to two targets simultaneously.

[0679] 1. Experimental instruments: Biacore 8K, Cytiva

[0680] 2. Experimental materials: Protein A biosensor chip (Cat.#29127556, Cytiva)

[0681] 3. Test reagents:

[0682] 1) 10*HBS-EP+ buffer solution (pH 7.4) (Cat.#BR-1006-69, Cytiva)

[0683] 2)10mM Glycine-HCl (pH 1.5)(Cat.#BR-1003-54,Cytiva)

[0684] 3) hEGFR-His, hMUC1-C-His (homemade)

[0685] 4) Antibody sample to be tested (prepared in-house)

[0686] The affinity of the disclosed antibody for simultaneously binding to human EGFR and MUC1-C antigens was tested using a Biacore 8K instrument.

[0687] The method is as follows: Antibody molecules were affinity-captured using a Protein A biosensor chip. A high concentration of the first antigen molecule (Ag1, blank indicates no antigen, only solvent) was then passed through the chip surface for 180 seconds. Following this, the second antigen (dissolved in the first antigen or blank solvent) was introduced, and then dissociated in the high concentration of the first antigen (or blank solvent) for 360 seconds. The reaction signal was monitored in real time using a Biacore 8K instrument to obtain binding and dissociation curves. After dissociation in each experimental cycle, the biosensor chip was washed and regenerated with 10 mM Glycine-HCl (pH 1.5). The Kinetics 1:1 binding model was used for data fitting. The results are shown in Table 27.

[0688] Table 27. Affinity test results of antibodies simultaneously binding to human EGFR and MUC1-C antigens Note: Ag1 and Ag2 represent the primary antigen and the secondary antigen, respectively.

[0689] The results showed that the affinity of the disclosed anti-EGFR-MUC1 bispecific antibody M4H1L1-ZalH4 for binding hMUC1-C-His after binding to hEGFR-His (1.96E-08M) was basically consistent with the affinity for binding only to hEGFR-His (1.80E-08M); similarly, the affinity of M4H1L1-ZalH4 for binding hEGFR-His after binding to hMUC1-C-His (3.52E-09M) was basically consistent with the affinity for binding only to hMUC1-C-His (2.56E-09M), indicating that the EGFR-MUC1 bispecific antibody M4H1L1-ZalH4 has the ability to bind to both human EGFR and MUC1-C antigens simultaneously, and that binding to Ag1 does not affect the binding to Ag2.

[0690] Test Example 12: Determining the affinity of monoclonal antibodies, bispecific antibodies, and ADCs for tumor cells based on FACS

[0691] Discard the culture medium from the cell culture flask, wash once with PBS, and then add an appropriate amount of trypsin (Invitrogen, catalog number 25200072) to digest the cells. Add HCC827, T47D, and HPAC cells at a rate of 100,000 cells / well to a 96-well plate. Centrifuge at 300g for 5 minutes, discard the supernatant, and wash the cells once with PBS buffer containing 2% FBS. The antibody was initially diluted 5-fold in PBS buffer containing 2% FBS at a starting concentration of 20 μg / mL, resulting in 8 concentration gradients. Add 100 μL of antibody sample to each well, resuspend the cells, and incubate at 4°C for 1 hour. Centrifuge at 300g for 5 minutes, discard the supernatant, and wash the cells twice with PBS buffer containing 2% FBS. Add secondary antibody (Alexa). 488g anti-human IgG (H+L) 1:1000 (Invitrogen, A11013) was incubated at 4°C for 40 minutes. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS buffer containing 2% FBS. The cells were resuspended in 100 μL of PBS buffer containing 2% FBS and then analyzed. FACS results are shown in Table 28.

[0692] Table 28. Results of FACS-based assays for the affinity of monoclonal antibodies, bispecific antibodies, and ADCs with tumor cells. Note: / indicates that it cannot be detected.

[0693] The results showed that the disclosed ADC and the corresponding naked antibody had considerable affinity for tumor cell lines, and toxin conjugation did not affect the antibody's affinity for tumor cells.

[0694] Test Example 13: Assay of M4H1L1-ZalH4 Tumor Cell Endocytosis Activity

[0695] 1. Experimental Objective: The purpose of this experiment is to indirectly reflect the endocytosis of EGFR-MUC1 bispecific antibodies by observing the cytotoxic effects of activated DT3C protein after it enters the cell. This is based on the IC50 assay. 50 Emax was used to evaluate the endocytic activity of the antibody.

[0696] 2. Experimental Principle: DT3C is a recombinant fusion protein composed of fragment A (toxin only) of diphtheria toxin and fragment 3C (IgG binding part) of group G streptococcus. This protein has a high affinity for the Fc region of the antibody and enters the cell along with the antibody during endocytosis. Under the action of intracellular furin protease, it releases the toxic DT3C, which inhibits EF2-ADP ribosylation activity, blocking protein translation and ultimately leading to cell death. DT3C that does not enter the cell does not have cell-killing activity. The endocytic activity of the antibody is evaluated based on the cell-killing effect.

[0697] 3. Test materials:

[0698] HCC827 cells: purchased from the Chinese Academy of Sciences, catalog number TCHU153

[0699] HPAC cells: purchased from Nanjing Kebai, catalog number CBP60539

[0700] T47D cells: purchased from ATCC, catalog number HTB-133

[0701] 4. Test methods:

[0702] 1) Prepare a cell suspension using fresh cell culture medium containing 20% ​​FBS (low IgG), add 2000 cells / well / 50μL to a 96-well cell culture plate, and incubate at 37°C with 5% carbon dioxide for 16 hours.

[0703] 2) Prepare DT3C at a concentration of 4× using serum-free medium (its molar concentration is 6 times that of the antibody). Prepare antibody at a concentration of 4× using serum-free medium. Mix 80 μL of DT3C and 80 μL of antibody at a volume ratio of 1:1, incubate at room temperature for 30 min.

[0704] 3) Dilute the mixture 4-fold with serum-free medium, for a total of 9 dilutions, with the 10th point being pure medium. The final concentrations are 200, 50, 12.5, 3.125, 0.7812, 0.1953, 0.0488, 0.0122, 0.00305, and 0 nM.

[0705] 4) Add 50 μL of diluted antibody to 50 μL of cells and incubate in a 5% carbon dioxide incubator at 37°C for 72 h.

[0706] 5) Take the plate, add 50 μL of CTG to each well, incubate at room temperature in the dark for 10 min, and read the chemiluminescence on a Pherastar.

[0707] 6) Data processing.

[0708] The results are shown in Table 29.

[0709] Table 29. Results of the assay for detecting antibody-mediated tumor cell endocytosis activity

[0710] The experimental results showed that the disclosed anti-EGFR-MUC1 bispecific antibody M4H1L1-ZalH4 exhibited good endocytic activity against HCC827, T47D, and HPAC tumor cell lines. In the EGFR-dominant tumor cell line HCC827, the endocytic activity was comparable to that of the EGFR monoclonal antibody ZalH4. In the MUC1-dominant tumor cell line T47D, the endocytic activity was comparable to that of the MUC1 monoclonal antibody M4H1L1. In the HPAC tumor cell line with high expression of both EGFR and MUC1, the endocytic activity was superior to that of the EGFR monoclonal antibody ZalH4 and the MUC1 monoclonal antibody M4H1L1.

[0711] Test Example 14: Assay of EGFR-MUC1 Bispecific Antibody ADC Tumor Cell Killing Activity

[0712] 1. Experimental Objective: To detect the inhibitory effect of EGFR-MUC1 bispecific antibody ADC on the proliferation of HCC827 / T47D / HPAC cells.

[0713] 2. Test materials:

[0714] HCC827 cells: purchased from the Chinese Academy of Sciences, catalog number TCHU153

[0715] HPAC cells: purchased from Nanjing Kebai, catalog number CBP60539

[0716] T47D cells: purchased from ATCC, catalog number HTB-133

[0717] 3. Test methods:

[0718] 1) Cell culture: HCC827, HPAC, and T47D cells were cultured in 1640 medium (Meilun Biotechnology, catalog number PWL015-L) containing 10% FBS, DMEM / F12 (1:1) medium (Meilun Biotechnology, catalog number PWL005) containing 10% FBS and 0.002 mg / mL insulin and 0.005 mg / mL transferrin and 40 ng / mL hydrocortisone, respectively, and 1640 medium (Meilun Biotechnology, catalog number PWL015-L) containing 10% FBS and 10 ug / mL human insulin.

[0719] 2) Cell preparation: Take HCC827, HPAC and T47D cells in logarithmic growth phase, wash them once with PBS (phosphate buffer, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3 mL of trypsin (0.25% trypsin-EDTA disodium (100 mL), Meilun Biotechnology) to digest for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to wash off the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 mL of cell culture medium to resuspend the cells to make a single-cell suspension.

[0720] 3) Cell plating: Mix HCC827, HPAC, and T47D single-cell suspensions thoroughly, and adjust the viable cell density to 3.7 × 10⁻⁶ cells / cells using cell culture medium. 3 Cells / mL, 3.7 × 10 3 Cells / mL, 7.4 × 10 3 Cells / mL. Mix the density-adjusted cell suspension thoroughly and add 135 μL / well to a 96-well cell culture plate. Add only 150 μL of culture medium to the outer wells of the 96-well plate. Incubate the plate in an incubator for 24 hours (37°C, 5% CO2).

[0721] 4) ADC drug preparation. The ADC drug was diluted with PBS (phosphate-buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.). The ADC to be tested was diluted to 10 μM, and then diluted 5-fold with PBS, resulting in 9 wells. The final 10th well was a drug-free well. Different concentration gradients of the drug were prepared.

[0722] 5) Sample addition procedure. Add 15 μL of the prepared test samples at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37℃, 5% CO2). The final ADC drug concentration gradient in the cell plate is 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, 0 nM.

[0723] 6) Color development procedure: Take out the 96-well cell culture plate, add 75 μL of CTG solution to each well, and incubate at room temperature for 10 minutes.

[0724] 7) Plate reading procedure: Take out the 96-well cell culture plate and place it in a microplate reader (PE, Envision) to measure chemiluminescence.

[0725] The results are shown in Table 30.

[0726] Table 30. Results of in vitro killing activity assay of EGFR-MUC1 bispecific antibody ADC

[0727] The results showed that the disclosed anti-EGFR-MUC1 bispecific antibody ADC-1 exhibited good killing activity against HCC827, T47D, and HPAC tumor cell lines. In the HCC827 tumor cell line, where EGFR expression was dominant, the killing activity of the anti-EGFR-MUC1 bispecific antibody ADC-1 was comparable to that of the EGFR monoclonal antibody ADC ZalH4-9-A. In the T47D tumor cell line, where MUC1 expression was dominant, the killing activity of the anti-EGFR-MUC1 bispecific antibody ADC-1 was comparable to that of the MUC1 monoclonal antibody ADC M4H1L1-9-A. In the HPAC tumor cell line, where both EGFR and MUC1 expression were high, the killing activity of the anti-EGFR-MUC1 bispecific antibody ADC-1 was comparable to that of the MUC1 monoclonal antibody ADC M4H1L1-9-A.

[0728] Test Example 15: Determining the Affinity of EGFR-MUC1 Bispecific Antibody to Tumor Cells Based on FACS

[0729] Discard the culture medium from the cell culture flask, wash once with PBS, then add an appropriate amount of trypsin (Invitrogen, catalog number 25200072) to digest the cells. Add HCC827, T47D, and HPAC cells to 96-well plates at a rate of 100,000 cells / well. Centrifuge at 300g for 5 minutes, discard the supernatant, and wash the cells once with PBS buffer containing 2% FBS. Use antibody at a starting concentration of 20 μg / mL, serially dilute 5-fold in PBS buffer containing 2% FBS, for a total of 8 concentration gradients. Add 100 μL of antibody sample to each well, resuspend the cells, and incubate at 4°C for 1 hour. Centrifuge at 300g for 5 minutes, discard the supernatant, and wash the cells twice with PBS buffer containing 2% FBS. Add secondary antibody (Alexa). 488g anti-human IgG (H+L) 1:1000 (Invitrogen, A11013) was incubated at 4°C for 40 minutes. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS buffer containing 2% FBS. The cells were resuspended in 100 μL of PBS buffer containing 2% FBS and then analyzed. The FACS results are shown in Table 31.

[0730] Table 31. Results of FACS-based assay for the affinity between anti-EGFR-MUC1 bispecific antibodies and tumor cells.

[0731] The results showed that the disclosed anti-EGFR-MUC1 bispecific antibody M4H1L1-ZalH4-LALA had good affinity for HCC827, T47D and HPAC tumor cell lines, and its affinity was comparable to that of M4H1L1-ZalH4.

[0732] Test Example 16: Assay of EGFR-MUC1 Bispecific Antitumor Cell Endocytosis Activity

[0733] 1. Experimental Objective: The purpose of this experiment is to indirectly reflect the endocytosis of EGFR-MUC1 bispecific antibodies by observing the cytotoxic effects of activated DT3C protein after it enters the cell. This is based on the IC50 assay. 50 Emax was used to evaluate the endocytic activity of the antibody.

[0734] 2. Experimental Principle: DT3C is a recombinant fusion protein composed of fragment A (toxin only) of diphtheria toxin and fragment 3C (IgG binding part) of group G streptococcus. This protein has a high affinity for the Fc region of the antibody and enters the cell along with the antibody during endocytosis. Under the action of intracellular furin protease, it releases the toxic DT3C, which inhibits EF2-ADP ribosylation activity, blocking protein translation and ultimately leading to cell death. DT3C that does not enter the cell does not have cell-killing activity. The endocytic activity of the antibody is evaluated based on the cell-killing effect.

[0735] 3. Test materials:

[0736] HCC827 cells: purchased from the Chinese Academy of Sciences, catalog number TCHU153

[0737] HPAC cells: purchased from Nanjing Kebai, catalog number CBP60539

[0738] T47D cells: purchased from ATCC, catalog number HTB-133

[0739] 4. Test methods:

[0740] 1) Prepare a cell suspension using fresh cell culture medium containing 20% ​​FBS (low IgG), add 2000 cells / well / 50μL to a 96-well cell culture plate, and incubate at 37°C with 5% carbon dioxide for 16 hours.

[0741] 2) Prepare DT3C at a concentration of 4× using serum-free medium (its molar concentration is 6 times that of the antibody). Prepare antibody at a concentration of 4× using serum-free medium. Mix 80 μL of DT3C and 80 μL of antibody at a volume ratio of 1:1, incubate at room temperature for 30 min.

[0742] 3) Dilute the mixture 4-fold with serum-free medium, for a total of 9 dilutions, with the 10th point being pure medium. The final concentrations are 200, 50, 12.5, 3.125, 0.7812, 0.1953, 0.0488, 0.0122, 0.00305, and 0 nM.

[0743] 4) Add 50 μL of diluted antibody to 50 μL of cells and incubate in a 5% carbon dioxide incubator at 37°C for 72 h.

[0744] 5) Take the plate, add 50 μL of CTG to each well, incubate at room temperature in the dark for 10 min, and read the chemiluminescence on a Pherastar.

[0745] 6) Data processing.

[0746] The results are shown in Table 32.

[0747] Table 32. Results of the assay for detecting antibody-mediated tumor cell endocytosis activity

[0748] The experimental results showed that the disclosed anti-EGFR-MUC1 bispecific antibody M4H1L1-ZalH4-LALA had good endocytic activity against HCC827, T47D and HPAC tumor cell lines, and its endocytic activity was comparable to that of M4H1L1-ZalH4.

[0749] Test Example 17: Assay of EGFR-MUC1 Bispecific Antibody ADC Tumor Cell Killing Activity

[0750] 1. Experimental objective: To detect the inhibitory effect of EGFR-MUC1 bispecific antibody ADC on the proliferation of HCC827 / T47D / HPAC cells.

[0751] 2. Test materials:

[0752] HCC827 cells: purchased from the Chinese Academy of Sciences, catalog number TCHU153

[0753] HPAC cells: purchased from Nanjing Kebai, catalog number CBP60539

[0754] T47D cells: purchased from ATCC, catalog number HTB-133

[0755] 3. Test methods:

[0756] 1) Cell culture: HCC827, HPAC, and T47D cells were cultured in 1640 medium (Meilun Biotechnology, catalog number PWL015-L) containing 10% FBS, DMEM / F12 (1:1) medium (Meilun Biotechnology, catalog number PWL005) containing 10% FBS and 0.002 mg / mL insulin and 0.005 mg / mL transferrin and 40 ng / mL hydrocortisone, respectively, and 1640 medium (Meilun Biotechnology, catalog number PWL015-L) containing 10% FBS and 10 ug / mL human insulin.

[0757] 2) Cell preparation: Take HCC827, HPAC and T47D cells in logarithmic growth phase, wash them once with PBS (phosphate buffer, Shanghai Yuanpei Biotechnology Co., Ltd.), add 2-3 mL of trypsin (0.25% trypsin-EDTA disodium (100 mL), Meilun Biotechnology) to digest for 2-3 minutes. After the cells are completely digested, add 10-15 mL of cell culture medium to wash off the digested cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and then add 10-20 mL of cell culture medium to resuspend the cells to make a single-cell suspension.

[0758] 3) Cell plating: Mix HCC827, HPAC, and T47D single-cell suspensions thoroughly, and adjust the viable cell density to 3.7 × 10⁻⁶ cells / cells using cell culture medium. 3 Cells / mL, 3.7 × 10 3 Cells / mL, 7.4 × 10 3 Cells / mL. Mix the density-adjusted cell suspension thoroughly and add 135 μL / well to a 96-well cell culture plate. Add only 150 μL of culture medium to the outer wells of the 96-well plate. Incubate the plate in an incubator for 24 hours (37°C, 5% CO2).

[0759] 4) ADC drug preparation. The ADC drug was diluted with PBS (phosphate-buffered saline, Shanghai Yuanpei Biotechnology Co., Ltd.). The ADC to be tested was diluted to 10 μM, and then diluted 5-fold with PBS, resulting in 9 wells. The final 10th well was a drug-free well. Different concentration gradients of the drug were prepared.

[0760] 5) Sample addition procedure. Add 15 μL of the prepared test samples at different concentrations to the culture plate, with two replicates for each sample. Incubate the culture plate in an incubator for 6 days (37℃, 5% CO2). The final ADC drug concentration gradient in the cell plate is 1000, 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, 0 nM.

[0761] 6) Color development procedure: Take out the 96-well cell culture plate, add 75 μL of CTG solution to each well, and incubate at room temperature for 10 minutes.

[0762] 7) Plate reading procedure: Take out the 96-well cell culture plate and place it in a microplate reader (PE, Envision) to measure chemiluminescence.

[0763] The results are shown in Table 33.

[0764] Table 33. Results of in vitro cytotoxic activity assay of EGFR-MUC1 bispecific antibody ADC

[0765] The results showed that the disclosed anti-EGFR-MUC1 bispecific anti-ADC ADC-3 had good killing activity against HCC827, T47D and HPAC tumor cell lines, and its killing activity was comparable to that of ADC-1.

[0766] Test Example 18: Efficacy of ADC in the in vivo HCC827 CDX model

[0767] 1. Experimental Objective: This experiment aims to evaluate the in vivo efficacy and toxic side effects of different doses of bispecific antibody ADCs and corresponding monoclonal antibody ADCs administered intraperitoneally to human non-small cell lung cancer cells HCC827 tumor-bearing mice.

[0768] 2. Experimental principle: This experiment constructs an HCC827 tumor-bearing mouse model on Balb / c nude mice, administers the test molecules intraperitoneally, evaluates the pharmacodynamics of different molecules, and compares the efficacy of bispecific antibody ADCs and monoclonal antibody ADCs.

[0769] 3. Experimental animals: Balb / c nude rats, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0770] 4. Test instruments:

[0771] CO2 Incubator: HERACELL 150i GP INCUBATOR, Thermo Fisher Scientific, USA

[0772] Biosafety cabinet: HFsafe-1800LC, Likang Company

[0773] Inverted microscope: DMi1, LEICA

[0774] Digital Vernier Caliper: 1195-200C, INSIZE

[0775] Electronic balance: MP6001, Shanghai Sunny Hengping Scientific Instruments Co., Ltd.

[0776] Centrifuge: L500-A, Hunan Xiangyi Instrument Equipment Co., Ltd.

[0777] Electric Thermostatic Water Bath: DK-S22, Shanghai Jinghong

[0778] Electronic balance: ML204T-02, Mettler

[0779] Microplate reader: Spark, TECAN

[0780] Biochemical Incubator: BSP-150, Shanghai Boxun Medical Bio-Instrument Co., Ltd.

[0781] 5. Experimental Procedure: HCC827 human non-small cell lung cancer cells (5×10⁻⁶) 6 100 μL of ADC was injected subcutaneously into the right rib area of ​​120 female nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 25 days, when the tumor grew to approximately 150.48 ± 21.32 mm3, mice were removed based on weight, tumor size (too large or too small), and tumor volume. Mice were then randomly divided into 9 groups (D0): Vehicle (PBS) control group, negative control group (IgG1-9-A), MUC1 monoclonal antibody ADC group (M4H1L1-9-A), EGFR monoclonal antibody ADC group (ZalH4-9-A), EGFR-MUC1 bispecific antibody ADC-2 group, and ADC-1 group. Each group consisted of 10 mice. ADC was administered intraperitoneally on D1 as a single dose. Tumor volume and animal weight were measured twice weekly, and data were recorded.

[0782] 6. Data processing: The tumor volume of each group of animals was expressed as mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism 10 software. Two / one way ANOVA was used for statistical analysis to calculate the tumor inhibition rate. The formula is: Tumor proliferation rate (T / C%) = (T - T0 / C - C0) × 100%.

[0783] 7. Experimental Results: After 30 days of administration, the tumor inhibition rate of the negative control group (6 mpk) was -13.71% (ns vs. blank control (Vehicle)); the tumor inhibition rate of the MUC1 monoclonal antibody ADC (3 mpk) was 100.08% (P<0.0001 vs. blank control); the tumor inhibition rate of the EGFR monoclonal antibody ADC (3 mpk) was 122.10% (P<0.0001 vs. blank control); the tumor inhibition rates of the EGFR-MUC1 bispecific antibody ADC-2 (3 mpk / 4.5 mpk) were 108.35% (P<0.0001 vs. blank control) and 123.58% (P<0.0001 vs. blank control), respectively; and the tumor inhibition rates of the EGFR-MUC1 bispecific antibody ADC-1 were... The tumor inhibition rates of 1.5mpk / 3mpk / 6mpk were 82.71% (P<0.0001 vs. blank control), 120.82% (P<0.0001 vs. blank control), and 121.83% (P<0.0001 vs. blank control), respectively. The results are shown in Table 34 and Figures 7A to 7B.

[0784] Table 34. Efficacy of ADC in the HCC827 CDX model in vivo (analysis using data from day 30) Note: D0 represents the grouping day, D1 represents the first day, and D9 represents the ninth day; / represents that it cannot be calculated; ns represents that it is not statistically significant, and the same applies below.

[0785] The results showed that the disclosed anti-EGFR-MUC1 bispecific antibody ADC-1 had a good tumor inhibition rate in the HCC827 CDX model, which was comparable to that of the same dose of EGFR monoclonal antibody ADC and superior to that of the same dose of MUC1 monoclonal antibody ADC.

[0786] Test Example 19: Efficacy of ADC in an in vivo HPAC CDX model

[0787] 1. Experimental Objective: This experiment aims to evaluate the in vivo efficacy and toxic side effects of different doses of bispecific antibody ADCs and corresponding monoclonal antibody ADCs administered intraperitoneally to human pancreatic acinar epithelial carcinoma cells (HPAC)-bearing mice.

[0788] 2. Experimental principle: This experiment constructs an HPAC tumor-bearing mouse model on Balb / c nude mice, administers the test molecules intraperitoneally, evaluates the pharmacodynamics of different molecules, and compares the pharmacodynamics of bispecific antibody ADCs and monoclonal antibody ADCs.

[0789] 3. Experimental animals: Balb / c nude mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0790] 4. Test instruments:

[0791] Benchtop Centrifuge: BECKMAN COULTER

[0792] Inverted microscope: NIKON TS100

[0793] CO2 incubator: Thermo

[0794] Refrigerator / Freezer: BCD-610WKM, Hefei Midea Refrigerator Co., Ltd.

[0795] Electronic balance: PL2001-L Mettler Toledo Instruments (Shanghai) Co., Ltd.

[0796] Biosafety cabinet: ESCO AC2-4S1

[0797] 5. Experimental Procedure: Human pancreatic acinar epithelial carcinoma cells HPAC (1×10⁻⁶) 6 200 μL of 50% Matrigel per mouse was injected subcutaneously into the right rib area of ​​100 female nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). After 7 days, the tumor grew to approximately 175.48 ± 17.18 mm. 3 Afterwards, mice were removed based on body weight, tumor size (too large or too small), and tumor volume. Mice were then randomly divided into nine groups (9 groups in total, 8 mice per group, D0): a blank control (PBS) group, a negative control group (IgG1-9-A), a MUC1 monoclonal antibody ADC group (M4H1L1-9-A), an EGFR monoclonal antibody ADC group (ZalH4-9-A), an EGFR-MUC1 bispecific antibody ADC-1 group, and an ADC-2 group. ADC was administered intraperitoneally twice, on D1 and D9. Tumor volume and animal body weight were measured twice weekly, and data were recorded.

[0798] 6. Data processing: The tumor volume of each group of animals was expressed as mean ± standard deviation (Mean ± SEM) and plotted using Graphpad Prism 10 software. Two / one way ANOVA was used for statistical analysis to calculate the tumor inhibition rate. The formula is: Tumor proliferation rate (T / C%) = (T - T0 / C - C0) × 100%.

[0799] 7. Experimental Results: After 30 days of administration, the tumor inhibition rate of the negative control group (6 mpk) was 7.22% (ns vs. blank control); the tumor inhibition rate of the MUC1 monoclonal antibody ADC (3 mpk) was 88.17% (P<0.0001 vs. blank control); the tumor inhibition rate of the EGFR monoclonal antibody ADC (3 mpk) was 50.90% (P<0.05 vs. blank control); the tumor inhibition rates of the EGFR-MUC1 bispecific antibody ADC-2 (3 mpk / 4.5 mpk) were 59.23% (P<0.001 vs. blank control) and 89.18% (P<0.0001 vs. blank control), respectively; and the tumor inhibition rates of the EGFR-MUC1 bispecific antibody ADC-1 (1.5 mpk / 3 mpk / 6 mpk) were 48.44% (P<0.05 vs. blank control). The percentages of positive rates were 80.57% (P<0.0001 vs. blank control) and 98.52% (P<0.0001 vs. blank control). The results are shown in Table 35 and Figures 8A to 8B.

[0800] Table 35. Efficacy of ADCs in the in vivo HPAC CDX model (analysis using data from day 30)

[0801] Note: D0 represents the grouping day, D1 represents the first day, and D9 represents the ninth day; / represents that it cannot be calculated.

[0802] The results showed that the disclosed anti-EGFR-MUC1 bispecific antibody ADC-1 had a good tumor inhibition rate in the HPAC CDX model, which was dose-dependent. Its tumor inhibition rate was comparable to that of the same dose of MUC1 monoclonal antibody ADC and superior to that of the same dose of EGFR monoclonal antibody ADC.

[0803] III. Preparation Examples - Antibody-Drug Conjugate Formulations Specifically Binding to EGFR and MUC1

[0804] 1) SEC size exclusion chromatography:

[0805] An analytical method for separating solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0806] SEC% polymer (SEC polymer content percentage) = A polymer / A total * 100% (A polymer is the peak area of ​​the polymer peak in the sample, and A total is the sum of the peak areas of all peaks.)

[0807] △SEC% represents the difference between the value of this test item after placement under each condition and the value at the beginning of placement.

[0808] Instrument used for SEC determination: Agilent HPLC 1260;

[0809] Pillar: Waters, BioResolve™ SEC mAb 2.5μm 7.8×300mm Column

[0810] 2) R-CE capillary gel electrophoresis:

[0811] An electrophoresis method in which a gel is transferred into a capillary as a supporting medium and then separated according to the molecular weight of the sample under a certain voltage.

[0812] R-CE% fragments (R-CE fragment content percentage) = A fragments / A total * 100% (A fragments is the peak area of ​​fragments in the sample, and A total is the sum of the peak areas of all peaks).

[0813] Instrument used for R-CE determination: Beckman capillary electrophoresis apparatus, model PA 800plus

[0814] △R-CE% represents the difference between the test item after placement under each condition and the initial placement value.

[0815] 3) Free toxins:

[0816] Based on the principle of reversed-phase separation, protein precipitation was followed by UPLC-UV analysis to detect the free toxin content in the supernatant. The concentration of the corresponding free toxin was calculated by substituting the sample peak area into the linear regression equation of the standard.

[0817] Instrument used for free toxin determination: Waters Acquity H Class.

[0818] 4) Osmotic pressure measurement:

[0819] The freezing point method for determining osmotic pressure is based on the principle that the freezing point depression is directly proportional to the molar concentration of the solution. It uses a highly sensitive temperature sensing element to measure the freezing point of the solution and converts the electrical charge into osmotic pressure.

[0820] Instrument used for osmotic pressure measurement: Loser, model OM815.

[0821] 5) Protein concentration determination:

[0822] The concentrations of the antibody-drug conjugates that specifically bind to EGFR and MUC1 in this disclosure (prepared with reference to the method described in ADC-1 above, DAR: approximately 6, hereinafter referred to as "protein") are calculated based on the concentration of the antibody portion in the antibody-drug conjugates that specifically bind to EGFR and MUC1.

[0823] Because the toxin in the antibody-drug conjugate that specifically binds to EGFR and MUC1 absorbs at the characteristic absorption wavelength of the protein, 280 nm, and also absorbs at 370 nm, the protein concentration is calculated using the following formula: A 280nm=(C drug ×E drug-280 +C mAb ×E mAb-280 )×l A 370nm =C drug ×E drug-370 ×l

[0824] Pick

[0825] Right now:

[0826] In the formula, A 280nm The average absorbance of a single sample of the test solution at a wavelength of 280 nm when the optical path length is 1 cm;

[0827] A 370nm The average absorbance of a single sample of the test solution at a wavelength of 370 nm when the optical path length is 1 cm;

[0828] E mAb-280 The mass extinction coefficient of the protein at a wavelength of 280 nm is 1.469 g. -1 cm -1 L;

[0829] E drug-280 The mass extinction coefficient of the toxin at a wavelength of 280 nm is 5.17 g. -1 cm -1 L;

[0830] E drug-370 The mass extinction coefficient of the toxin at a wavelength of 370 nm is 17.89 g. -1 cm -1 L;

[0831] R: The ratio of the toxin extinction coefficient at 370nm to 280nm is 3.46;

[0832] C mAb Protein concentration, mg / mL;

[0833] l: Optical path length, cm (the optical path length here is 1 cm).

[0834] If the test solution is diluted, the protein concentration is: C (mg / mL) = C mAb ×N, where N is the dilution factor.

[0835] Protein concentration measurement instrument: UV-Vis spectrophotometer, model: Nano Drop 2000.

[0836] Formulation Example 1, pH and Buffer System Screening

[0837] Formulations containing 20 mg / mL protein, 0.2 mg / mL polysorbate 80 (PS80), 0.02 mg / mL disodium edetate (Na2EDTA·2H2O, CAS: 6381-92-6, hereinafter the same) and 80 mg / mL sucrose were prepared using the buffer systems shown in Table 36. Forced degradation studies (incubation at 25°C for one month) were conducted on the samples, and the effects of different pH values ​​and buffer systems on protein stability were investigated, using appearance and SEC as evaluation indicators.

[0838] The results are shown in Table 36. After being placed at 25℃ for one month, the preferred buffer system is 10mM AA at pH 5.5 or 10mM His-HCl at pH 5.0 to 6.0.

[0839] Table 36. Screening Results of pH and Buffer Systems Note: AA represents acetic acid-sodium acetate; His-HCl represents histidine-histidine hydrochloride; SA represents succinic acid-sodium succinate.

[0840] Formulation Example 2, pH Range Confirmation

[0841] Formulations containing 10 mg / mL protein, 0.2 mg / mL PS80, 0.02 mg / mL Na2EDTA·2H2O, 40 mg / mL sucrose, and 10 mg / mL glycine were prepared using different pH buffer systems shown in Table 37. Forced degradation studies (incubation at 25°C for one month) were conducted on the samples, and the effects of different pH values ​​on protein stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0842] The results are shown in Table 37. After one month of storage at 25℃, the differences between groups were small. Therefore, the 10mM His-HCl formulations at pH 4.5–5.5 all exhibited good stability.

[0843] Table 37. pH Range Confirmation Results

[0844] Formulation Example 3: Screening of Buffer System Ion Concentration

[0845] A formulation containing 10 mg / mL protein, 0.2 mg / mL PS80, 0.02 mg / mL Na2EDTA·2H2O, 40 mg / mL sucrose, and 10 mg / mL glycine was prepared using a 5 mM–20 mM pH 5.0 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples. The effects of different ion concentrations on the stability of the buffer system were investigated, using appearance, SEC, and R-CE as evaluation indicators.

[0846] The results are shown in Table 38. After being placed at 40℃ for one month, the formulations prepared with 5-20 mM His-HCl pH5.0 buffer showed little difference between groups and good stability.

[0847] Table 38. Screening Results of Ion Concentration in Buffer Systems

[0848] Formulation Example 4, Sucrose Concentration Screening 1

[0849] Formulations containing 20–80 mg / mL sucrose, 10 mg / mL protein, 0.2 mg / mL PS80, 0.02 mg / mL Na2EDTA·2H2O, and 10 mg / mL glycine were prepared using 10 mM pH 5.0 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different sucrose concentrations on stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0850] The results are shown in Table 39. After one month of storage at 40℃, the differences between groups were small. Therefore, sucrose preparations ranging from 20 to 80 mg / mL all exhibited good stability.

[0851] Table 39. Results of Sucrose Concentration Screening 1

[0852] Formulation Example 5, Sucrose Concentration Screening 2

[0853] Formulations containing 20–80 mg / mL sucrose, 10 mg / mL protein, 0.2 mg / mL PS80, and 0.02 mg / mL Na2EDTA·2H2O were prepared using 10 mM pH 5.0 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different sucrose concentrations on stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0854] The results are shown in Table 40. After one month of storage at 40℃, the differences between groups were small. Therefore, sucrose preparations ranging from 20 to 80 mg / mL all exhibited good stability.

[0855] Table 40. Results of sucrose concentration screening 2

[0856] Formulation Example 6: Screening of Glycine Concentration

[0857] Formulations containing 0–20 mg / mL glycine, 10 mg / mL protein, 0.2 mg / mL PS80, 0.02 mg / mL Na2EDTA·2H2O, and 40 mg / mL sucrose were prepared using 10 mM pH 5.0 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples. The effects of different glycine concentrations on stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0858] The results are shown in Table 41. After one month of storage at 40℃, the differences between groups were small. Therefore, the 0–20 mg / mL glycine preparations all exhibited good stability.

[0859] Table 41. Screening Results of Glycine Concentration

[0860] Formulation Example 7: Effect of Na2EDTA·2H2O on Formulation Stability

[0861] Formulations with 20 mg / mL protein, 0.2 mg / mL PS80, 80 mg / mL sucrose, and those shown in Table 42 (without Na2EDTA·2H2O and with 0.02 mg / mL Na2EDTA·2H2O) were prepared using 10 mM His-HCl pH 5.5 buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effect of Na2EDTA·2H2O on the stability of the formulations was investigated using appearance and SEC as evaluation indicators.

[0862] The results are shown in Table 42. After being placed at 40℃ for one month, there was no significant difference between the preparation groups without Na2EDTA·2H2O and those with 0.02 mg / mL Na2EDTA·2H2O.

[0863] Table 42. Experimental results on the effect of Na2EDTA·2H2O on formulation stability Note: N / A indicates no addition

[0864] Formulation Example 8: Screening of Na2EDTA·2H2O Concentration

[0865] Formulations containing 0.01–0.1 mg / mL Na₂EDTA·2H₂O, 10 mg / mL protein, 0.2 mg / mL PS80, 40 mg / mL sucrose, and 10 mg / mL glycine were prepared using 10 mM pH 5.0 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples. The effects of different Na₂EDTA·2H₂O concentrations on stability were investigated, using appearance, SEC, and R-CE as evaluation indicators.

[0866] The results are shown in Table 43. After one month of storage at 40℃, the differences between groups were small. Therefore, the 0.01–0.1 mg / mL Na2EDTA·2H2O formulations all exhibited good stability.

[0867] Table 43. Screening Results of Na2EDTA·2H2O Concentration

[0868] Formulation Example 9: Screening of Surfactant Types

[0869] Formulations containing 20 mg / mL protein, 0.02 mg / mL Na₂EDTA·2H₂O, 80 mg / mL sucrose, and different surfactants as shown in Table 44 were prepared using 10 mM pH 5.5 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different surfactants on protein stability were investigated using appearance and SEC as evaluation indicators.

[0870] The results are shown in Table 44. After being stored at 40°C for one month, there were little difference between the formulations containing PS80 and poloxamer 188 (P188), and all showed good stability.

[0871] Table 44. Results of Surfactant Selection

[0872] Formulation Example 10, PS80 Concentration Screening

[0873] Formulations containing 0.1–1.0 mg / mL PS80, 10 mg / mL protein, 0.02 mg / mL Na₂EDTA·2H₂O, 40 mg / mL sucrose, and 10 mg / mL glycine were prepared using 10 mM pH 5.0 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different PS80 concentrations on stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0874] The results are shown in Table 45. After one month of storage at 40℃, the differences between groups were small. Therefore, the 0.1–1.0 mg / mL PS80 formulations all exhibited good stability.

[0875] Table 45. PS80 Concentration Screening Results

[0876] Formulation Example 11, Protein Concentration Screening

[0877] Formulations containing 5–20 mg / mL protein, 0.2 mg / mL PS80, 0.02 mg / mL Na2EDTA·2H2O, and 80 mg / mL sucrose were prepared using 10 mM pH 5.0 His-HCl buffer. Forced degradation studies (incubation at 40°C for one month) were conducted on the samples, and the effects of different protein concentrations on stability were investigated using appearance, SEC, and R-CE as evaluation indicators.

[0878] The results are shown in Table 46. After one month of storage at 40℃, the differences between groups were small. Therefore, the protein concentration formulations of 5–20 mg / mL all exhibited good accelerated stability.

[0879] Table 46. Protein Concentration Screening Table

[0880] Formulation Example 12: Investigation of Lyophilized Formulation

[0881] A formulation containing 20 mg / mL protein, 0.2 mg / mL PS80, 0.02 mg / mL Na2EDTA·2H2O, 40 mg / mL sucrose, and 10 mg / mL glycine was prepared using 10 mM pH 5.0 His-HCl buffer and then lyophilized. Forced degradation studies (incubation at 40°C for one month) were conducted on the lyophilized product. The stability of the formulation was assessed using appearance, SEC (securities exchange rate), and R-CE (reduced oxidative stress emission) as evaluation indicators.

[0882] The freeze-drying process is shown in Table 47 below:

[0883] Table 47. Freeze-drying process

[0884] The results are shown in Table 48. After freeze-drying, the powder compact appeared uniform and full, without any collapse.

[0885] Table 48. Freeze-drying results

[0886] The results are shown in Table 49. After being placed at 40℃ for one month, compared with D0, the reconstituted sample of this prescription was clear and transparent in appearance, and there was no significant change in purity.

[0887] Table 49. Stability data of lyophilized formulations at 40℃ (M1)

[0888] 40℃ M1: Store at 40℃ for one month.

[0889] Formulation Example 13, Prescription Confirmation

[0890] A formulation containing 10 mg / mL protein, 0.2 mg / mL PS80, 0.02 mg / mL Na2EDTA·2H2O, 40 mg / mL sucrose, and 10 mg / mL glycine was prepared using 10 mM pH 5.0 His-HCl buffer. Forced degradation studies were conducted on the samples (incubated at 40°C for one month), with appearance, SEC, and R-CE as evaluation indicators.

[0891] The results are shown in Table 50. After being placed at 40℃ for one month, the protein showed good stability.

[0892] Table 50. Stability results of the final prescription

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate that specifically binds to EGFR and MUC1 and a buffer, wherein: The antibody-drug conjugate that specifically binds to EGFR and MUC1 has a structure as shown in the general formula (Pc-9-A): in: n is between 1 and 10; Pc is an antibody that specifically binds to EGFR and MUC1. It comprises one antigen-binding module specifically binding to MUC1 and one antigen-binding module specifically binding to EGFR. The antigen-binding module specifically binding to MUC1 is a Fab, and the antigen-binding module specifically binding to EGFR is a replaced Fab containing a dimeric Titin chain and an Obscurin chain. The antibody that specifically binds to EGFR and MUC1 comprises a first chain having the structure shown in formula (a), a second chain having the structure shown in formula (b), a third chain having the structure shown in formula (c), and a fourth chain having the structure shown in formula (d), wherein: Equation (a): [MUC1-VH]-[CH1]-[Fc1], Equation (b): [MUC1-VL]-[CL], Equation (c): [EGFR-VH]-[connector 1]-[Titin]-[Fc2], Equation (d): [EGFR-VL]-[connector 2]-[Obscurin], in: Linker 1 and linker 2 may be the same or different, and are peptide linkers; or linker 1 or linker 2 may not exist. The structures shown in equations (a), (b), (c), and (d) are arranged from the N end to the C end; The buffer is a histidine-histidine hydrochloride buffer, an acetate buffer, or a succinate buffer. Preferably, the buffer is a histidine-histidine hydrochloride buffer, an acetate-sodium acetate buffer, or a succinic acid-sodium succinate buffer. More preferably, the buffer is a histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer; Most preferably, the buffer is a histidine-histidine hydrochloride buffer.

2. The pharmaceutical composition according to claim 1, wherein the antigen-binding module specifically binding to EGFR comprises a heavy chain variable region EGFR-VH and a light chain variable region EGFR-VL, and the antigen-binding module specifically binding to MUC1 comprises a heavy chain variable region MUC1-VH and a light chain variable region MUC1-VL; wherein: The EGFR-VH has HCDR1 containing the amino acid sequence of SEQ ID NO: 116, HCDR2 containing the amino acid sequence of SEQ ID NO: 117, and HCDR3 containing the amino acid sequence of SEQ ID NO: 129; the EGFR-VL has LCDR1 containing the amino acid sequence of SEQ ID NO: 119, LCDR2 containing the amino acid sequence of SEQ ID NO: 120, and LCDR3 containing the amino acid sequence of SEQ ID NO: 121; and The HCDR1 of the MUC1-VH contains the amino acid sequence of SEQ ID NO: 12, the HCDR2 contains the amino acid sequence of SEQ ID NO: 13, and the HCDR3 contains the amino acid sequence of SEQ ID NO: 14; the LCDR1 of the MUC1-VL contains the amino acid sequence of SEQ ID NO: 15, the LCDR2 contains the amino acid sequence of SEQ ID NO: 16, and the LCDR3 contains the amino acid sequence of SEQ ID NO:

17. Preferably, the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 138, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 149; and The MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, 37 or 38 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 36, 37 or 38, and the MUC1-VL contains the amino acid sequence of SEQ ID NO: 39, 40, 41 or 42 or an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 39, 40, 41 or 42; More preferably, the EGFR-VH comprises the amino acid sequence of SEQ ID NO: 138, and the EGFR-VL comprises the amino acid sequence of SEQ ID NO: 149; and The MUC1-VH contains the amino acid sequence of SEQ ID NO: 36, and the MUC1-VL contains the amino acid sequence of SEQ ID NO:

39.

3. The pharmaceutical composition according to claim 1 or 2, wherein the titin chain comprises the amino acid sequence of SEQ ID NO: 165, and the obscurin chain comprises the amino acid sequence of SEQ ID NO:

166.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the antibody that specifically binds to EGFR and MUC1 comprises an Fc region, wherein the Fc region is an IgG1 Fc region; Preferably, the Fc region comprises one or more amino acid substitutions that reduce the binding of the Fc region to the Fcγ receptor; and The Fc region includes a first subunit Fc1 and a second subunit Fc2 that can associate with each other, wherein Fc1 and Fc2 each independently have one or more amino acid substitutions that reduce homodimerization of the Fc region; More preferably, Fc1 contains the amino acid sequence of SEQ ID NO: 169, and Fc2 contains the amino acid sequence of SEQ ID NO: 170; or Fc1 contains the amino acid sequence of SEQ ID NO: 182, and Fc2 contains the amino acid sequence of SEQ ID NO: 183; or, Fc2 contains the amino acid sequence of SEQ ID NO: 169, and Fc1 contains the amino acid sequence of SEQ ID NO: 170; or Fc2 contains the amino acid sequence of SEQ ID NO: 182, and Fc1 contains the amino acid sequence of SEQ ID NO:

183.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the antibody specifically binding to EGFR and MUC1 has a first strand comprising the amino acid sequence of SEQ ID NO: 171, a second strand comprising the amino acid sequence of SEQ ID NO: 74, a third strand comprising the amino acid sequence of SEQ ID NO: 174, and a fourth strand comprising the amino acid sequence of SEQ ID NO: 173; or The antibody that specifically binds to EGFR and MUC1 has a first chain containing the amino acid sequence of SEQ ID NO: 178, a second chain containing the amino acid sequence of SEQ ID NO: 74, a third chain containing the amino acid sequence of SEQ ID NO: 179, and a fourth chain containing the amino acid sequence of SEQ ID NO:

173.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein n is 2 to 8; preferably, n is 4 to 8; more preferably, n is about 6.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the concentration of the antibody-drug conjugate specifically binding to EGFR and MUC1 is from 1 mg / mL to 50 mg / mL; Preferably, the concentration of the antibody-drug conjugate that specifically binds to EGFR and MUC1 is from 5 mg / mL to 20 mg / mL; More preferably, the concentration of the antibody-drug conjugate that specifically binds to EGFR and MUC1 is from 8 mg / mL to 12 mg / mL; Most preferably, the concentration of the antibody-drug conjugate that specifically binds to EGFR and MUC1 is about 10 mg / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the concentration of the buffer is from 1 mM to 50 mM; Preferably, the concentration of the buffer is from 5 mM to 20 mM; More preferably, the concentration of the buffer is 8 mM to 12 mM; Most preferably, the concentration of the buffer is about 10 mM.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pH of the pharmaceutical composition is 4.5 to 8.0; Preferably, the pH of the pharmaceutical composition is 4.5 to 6.5; More preferably, the pH of the pharmaceutical composition is 4.5 to 6.0; More preferably, the pH of the pharmaceutical composition is 4.7 to 5.3; Most preferably, the pH of the pharmaceutical composition is about 5.

0.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical composition further comprises a surfactant; Preferably, the surfactant is polysorbate or poloxamer; More preferably, the surfactant is polysorbate 80 or poloxamer 188; Most preferably, the surfactant is polysorbate 80.

11. The pharmaceutical composition according to claim 10, wherein the concentration of the surfactant is from 0.01 mg / mL to 2 mg / mL; Preferably, the concentration of the surfactant is from 0.1 mg / mL to 1 mg / mL; More preferably, the concentration of the surfactant is from 0.1 mg / mL to 0.3 mg / mL; Most preferably, the concentration of the surfactant is about 0.2 mg / mL.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutical composition further comprises sugar; Preferably, the sugar is sucrose, trehalose, mannitol, or sorbitol; More preferably, the sugar is sucrose.

13. The pharmaceutical composition according to claim 12, wherein the concentration of the sugar is from 10 mg / mL to 120 mg / mL; Preferably, the concentration of the sugar is from 20 mg / mL to 100 mg / mL; More preferably, the concentration of the sugar is 30 mg / mL to 50 mg / mL or 60 mg / mL to 100 mg / mL; Most preferably, the concentration of the sugar is about 40 mg / mL or about 80 mg / mL.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the pharmaceutical composition further comprises excipients; Preferably, the excipient is selected from one or more of the group consisting of ethylenediaminetetraacetic acid or its salt, ethylenediaminetetraacetic acid hydrate or its salt, glycine, DTPA, arginine hydrochloride, methionine, proline, histidine, phenylalanine, glutamic acid, aspartic acid, sodium chloride, and calcium chloride. More preferably, the excipient is selected from one or two of the group consisting of sodium salt of ethylenediaminetetraacetic acid hydrate and glycine; More preferably, the excipient is selected from one or two of the group consisting of disodium ethylenediaminetetraacetate dihydrate and glycine; Most preferably, (I) the excipient is disodium ethylenediaminetetraacetate dihydrate; or (II) The excipients are disodium ethylenediaminetetraacetate dihydrate and glycine.

15. The pharmaceutical composition according to claim 14, wherein: (I) The excipient is disodium ethylenediaminetetraacetate dihydrate, with a concentration of 0.01 mg / mL to 1 mg / mL; preferably, the concentration of disodium ethylenediaminetetraacetate dihydrate is 0.01 mg / mL to 0.1 mg / mL; more preferably, the concentration of disodium ethylenediaminetetraacetate dihydrate is about 0.02 mg / mL; or (II) The excipients are disodium ethylenediaminetetraacetate dihydrate and glycine, wherein: The concentration of the disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 1 mg / mL; preferably, the concentration of the disodium ethylenediaminetetraacetate dihydrate is from 0.01 mg / mL to 0.1 mg / mL; more preferably, the concentration of the disodium ethylenediaminetetraacetate dihydrate is about 0.02 mg / mL; and The concentration of glycine is from 0.01 mg / mL to 20 mg / mL; preferably, the concentration of glycine is from 5 mg / mL to 20 mg / mL; more preferably, the concentration of glycine is from 8 mg / mL to 12 mg / mL; most preferably, the concentration of glycine is about 10 mg / mL.

16. The pharmaceutical composition according to any one of claims 1 to 15, comprising the following components: (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 1 mg / mL to 50 mg / mL (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate, ranging from 0.01 mg / mL to 1 mg / mL. (e) Glycine at concentrations ranging from 0.01 mg / mL to 20 mg / mL, and (f) A buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0; Preferably, the pharmaceutical composition comprises the following components: (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL (b) 0.1 mg / mL to 1 mg / mL of polysorbate or poloxamer, (c) Sucrose at concentrations ranging from 20 mg / mL to 100 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate, ranging from 0.01 mg / mL to 0.1 mg / mL. (e) Glycine at concentrations of 5 mg / mL to 20 mg / mL, and (f) a 5 mM to 20 mM histidine-histidine hydrochloride buffer, an acetate-sodium acetate buffer, or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.5; More preferably, the pharmaceutical composition comprises the following components: (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL (b) 0.1 mg / mL to 1 mg / mL of polysorbate 80 or poloxamer 188, (c) Sucrose at concentrations ranging from 20 mg / mL to 100 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate, ranging from 0.01 mg / mL to 0.1 mg / mL. (e) Glycine at concentrations of 5 mg / mL to 20 mg / mL, and (f) a 5 mM to 20 mM histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0; More preferably, the pharmaceutical composition comprises the following components: (a) The antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations of 8 mg / mL to 12 mg / mL (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.3 mg / mL, (c) Sucrose at concentrations of 30 mg / mL to 50 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate, ranging from 0.01 mg / mL to 0.1 mg / mL. (e) Glycine at concentrations of 8 mg / mL to 12 mg / mL, and (f) an 8 mM to 12 mM histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3; Most preferably, the pharmaceutical composition comprises the following components: (a) Approximately 10 mg / mL of the antibody-drug conjugate specifically binding to EGFR and MUC1, (b) Approximately 0.2 mg / mL of polysorbate 80, (c) Approximately 40 mg / mL of sucrose, (d) Approximately 0.02 mg / mL of disodium ethylenediaminetetraacetate dihydrate, (e) Approximately 10 mg / mL of glycine, and (f) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.

0.

17. The pharmaceutical composition according to any one of claims 1 to 15, comprising the following components: (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 1 mg / mL to 50 mg / mL (b) Surfactants ranging from 0.01 mg / mL to 2 mg / mL, (c) Sugars ranging from 10 mg / mL to 120 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 1 mg / mL, and (e) a buffer of 1 mM to 50 mM, wherein the pH of the pharmaceutical composition is 4.5 to 8.0; Preferably, the pharmaceutical composition comprises the following components: (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL (b) 0.1 mg / mL to 1 mg / mL of polysorbate or poloxamer, (c) Sucrose at concentrations ranging from 20 mg / mL to 100 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and (e) a 5 mM to 20 mM histidine-histidine hydrochloride buffer, an acetate-sodium acetate buffer, or a succinate-sodium succinate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.

5. More preferably, the pharmaceutical composition comprises the following components: (a) Antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations ranging from 5 mg / mL to 20 mg / mL (b) 0.1 mg / mL to 1 mg / mL of polysorbate 80 or poloxamer 188, (c) Sucrose at concentrations ranging from 20 mg / mL to 100 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and (e) a 5 mM to 20 mM histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.5 to 6.0; More preferably, the pharmaceutical composition comprises the following components: (a) The antibody-drug conjugates that specifically bind to EGFR and MUC1 at concentrations of 8 mg / mL to 12 mg / mL (b) Polysorbate 80 at concentrations ranging from 0.1 mg / mL to 0.3 mg / mL, (c) Sucrose at concentrations of 60 mg / mL to 100 mg / mL, (d) Disodium ethylenediaminetetraacetate dihydrate at concentrations ranging from 0.01 mg / mL to 0.1 mg / mL, and (e) an 8 mM to 12 mM histidine-histidine hydrochloride buffer or an acetate-sodium acetate buffer, wherein the pH of the pharmaceutical composition is 4.7 to 5.3; Most preferably, the pharmaceutical composition comprises the following components: (a) Approximately 10 mg / mL of the antibody-drug conjugate specifically binding to EGFR and MUC1, (b) Approximately 0.2 mg / mL of polysorbate 80, (c) Approximately 80 mg / mL of sucrose, (d) Approximately 0.02 mg / mL of disodium ethylenediaminetetraacetate dihydrate, and (e) about 10 mM of histidine-histidine hydrochloride buffer, wherein the pH of the pharmaceutical composition is about 5.

0.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein it is an intravenous injection preparation, a subcutaneous injection preparation, an intraperitoneal injection preparation, or an intramuscular injection preparation; preferably an intravenous injection preparation.

19. A method for preparing a lyophilized formulation, comprising the step of lyophilizing the pharmaceutical composition according to any one of claims 1 to 18.

20. A method for preventing or treating a disease, the method comprising administering to a subject a preventive or therapeutically effective amount of the pharmaceutical composition according to any one of claims 1 to 18; Preferably, the disease is a tumor or cancer; More preferably, the diseases are selected from: astrocytoma, glioblastoma, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, fallopian tube cancer, gallbladder cancer, gastric cancer, head and neck cancer, idiopathic myelofibrosis, kidney cancer, leukemia, liver cancer, esophageal cancer, lung cancer, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, multiple myeloma, neuroblastoma, oligodendroglioma, ovarian cancer, peritoneal tumor, pancreatic cancer, polycythemia vera, primary neuroectodermal tumor, prostate cancer, retinoblastoma, sarcoma, squamous cell carcinoma, thyroid cancer, endometrial cancer, vestibular schwannoma, germ cell tumor, vulvar cancer, thymoma, testicular cancer, bile duct cancer, pheochromocytoma, paraganglioma, and adenoid cystic carcinoma; Most preferably, the disease is selected from lung cancer, head and neck cancer, esophageal cancer, breast cancer, pancreatic cancer, prostate cancer, thyroid cancer, stomach cancer, ovarian cancer, colorectal cancer, liver cancer, gallbladder cancer, kidney cancer, cervical cancer, and bladder cancer.

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