Antibody-drug conjugate targeting EGFR and cmet and use thereof

Abstract

Provided are an antibody-drug conjugate targeting EGFR and cMET and the use thereof. By means of optimizing the bispecific antibody targeting EGFR and cMET, and conjugating different types of linkers and cytotoxins by means of enzymatic site-specific conjugation technology, ADC drugs having both high stability and significant efficacy are obtained.

Description

Antibody-drug conjugates targeting EGFR and cMET and their uses

[0001] Cross-references

[0002] This disclosure claims priority to Chinese invention patent application No. 2025103610784, which is incorporated herein in its entirety. Technical Field

[0003] This disclosure relates to the biopharmaceutical field, specifically to an antibody-drug conjugate targeting EGFR and cMET and its uses. Background Technology

[0004] EGFR (epidermal growth factor receptor) and cMET (hepatocyte growth factor receptor) are two important transmembrane tyrosine kinase receptors that play crucial roles in the occurrence, development, and treatment of cancer. Aberrant activation of EGFR is a significant driver of various cancers, while activation of the cMET signaling pathway can promote tumor cell proliferation, survival, and migration. Furthermore, there is a complex interaction between EGFR and cMET; both can jointly activate signaling pathways through autocrine or paracrine mechanisms, promoting tumor cell proliferation, survival, and migration.

[0005] For EGFR, several tyrosine protease inhibitors (TKIs) and monoclonal antibodies have been approved, such as gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, as well as cetuximab and panitumumab (Uribe, Marrocco, and Yarden 2021). However, despite these advancements, clinical benefits have fallen short of expectations, primarily due to primary and acquired resistance, leading to disease relapse. Up to 30% of first-line patients have reported off-target resistance to osimertinib (Chmielecki et al. 2023), with cMET amplification being the most common, occurring in approximately 15% (Engelman et al. 2007; Saw et al. 2024). Therefore, utilizing a bispecific antibody-ADC strategy to simultaneously inhibit EGFR and cMET would be more beneficial for patients.

[0006] Currently, several EGFR / c-MET-targeting drugs have been reported globally, including Johnson & Johnson's Amivantamab (approved), Jiahe Biopharma's GB-263, AmoyDx Biopharma's EMB-01, and Betta / Merus' MCLA-129. However, no EGFR / c-MET-targeting ADC drugs have been approved yet, which is related to the complexity of ADC drug development. ADC drug development involves a meticulously controlled system engineering process involving four elements: antibodies, bioactive small molecules, linkers, and conjugation methods. Among these, the conjugation method significantly influences drug-related properties such as efficacy stability, metabolic consistency, and quality control (Tsuchikama K, An Z. Antibody-drug conjugates: recent advances in conjugation and linker chemistries. Protein Cell 2018, 9, 33-46).

[0007] Currently, ADC drug conjugation methods are broadly classified into two types: traditional chemical conjugation and enzyme-directed conjugation. Traditional chemical conjugation is represented by the use of cysteine ​​residues for conjugation, such as AstraZeneca's AZD9592 EGFR-cMET dual-target ADC (patent number US20230183358A1), which is currently in Phase I clinical trials. Its conjugation results are randomized, and the stability of the connection between small molecules and antibodies is not high. Common enzyme-directed conjugation methods include sortase-mediated conjugation, glutamine transferase-mediated conjugation, and conjugation based on enzymatic glycan remodeling.

[0008] Among several conjugation methods, site-specific conjugation technology based on enzymatic glycan remodeling targets the Fc segment of the antibody. It eliminates the need for antibody modification and cell engineering, significantly reducing the difficulty and workload in development and potentially becoming a universal platform technology for antibody site-specific conjugation. Currently, there are several reports on site-specific conjugation technologies based on enzymatic glycan remodeling, primarily focusing on improving the purity of single-structure products, simplifying reaction steps, reducing byproducts, and avoiding over-digestion. However, most of these studies are based on monoclonal antibodies. For bispecific antibodies, which typically have two different heavy chains, this structural asymmetry introduces significant uncertainty into the antibody-linker conjugation reaction. Furthermore, the targeting effect of non-traditional "Y"-shaped bispecific antibodies after conjugation with the linker is even more difficult to predict. In addition, bispecific antibodies targeting different targets may require significantly different spatial conformations when binding to different targets. In this case, the selected linker needs to ensure the spatial conformation requirements of the bispecific antibody when binding to different targets. Therefore, when using bispecific antibodies to prepare ADCs, the conjugation method and the choice of linker have a crucial impact on the stability, conjugation efficiency, heterogeneity, and even the preventive or therapeutic effects of the ADC.

[0009] The complexity of ADC development has led to numerous cases where the dual-target antibody itself achieves significant therapeutic effects, while the corresponding ADC shows poor clinical efficacy. For example, Regeneron's cMet bispecific antibody-based ADC, REGN5093-M114, was discontinued due to lower-than-expected clinical efficacy. Therefore, it is necessary to optimize the bispecific antibodies used to prepare ADCs targeting EGFR and cMET, as well as the connection methods between them and the linkers. Summary of the Invention

[0010] This disclosure optimizes and screens bispecific antibodies used in the preparation of ADC drugs targeting EGFR and cMET, and uses them to conjugate different types of linkers-cytotoxins, resulting in ADC drugs with both high stability and significant efficacy.

[0011] The first aspect of this disclosure provides an antibody-drug conjugate having Ab-[LP] (t) ] z The structure;

[0012] Wherein, Ab is a bispecific antibody targeting EGFR and cMET or its antigen-binding fragment, including a first domain targeting EGFR and a second domain targeting cMET:

[0013] Wherein, the first structural domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is a variant of SEQ ID NO:1 or SEQ ID NO:1 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR2 is a variant of SEQ ID NO:2 or SEQ ID NO:2 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR3 is a variant of SEQ ID NO:3 or SEQ ID NO:3 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0014] and / or

[0015] The first domain further comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is SEQ ID NO:9 or a variant of SEQ ID NO:9 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is SEQ ID NO:10 or a variant of SEQ ID NO:10 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is SEQ ID NO:11 or a variant of SEQ ID NO:11 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0016] P is a cytotoxin;

[0017] t is any integer greater than 1, and can be any integer from 1 to 10, such as 2, 4, 6 or 8.

[0018] Ab and P are connected by connector L, which includes the structure shown in equation (I);

[0019] Equation (I) is:

[0020] in,

[0021] In equation (I), the * terminal is connected to Ab; the # terminal is connected to P.

[0022] R 0 C 1-10 alkyl;

[0023] D has the following structure:

[0024] In equation (I-1), * represents the end connected to Ab;

[0025] ** represents the sum (I) The end of the part connected to the -NH-;

[0026] R 1 R 2 R 3 R 4 R 5 R 6 Each is independently selected from H and C. 1-6 alkoxy or OH; and satisfy the following condition: R 1 and R 2 Different, R 3 and R 4 Differences and R 5 and R 6 different;

[0027] R 7 It is hydrogen or α-L-fucosylate;

[0028] Q has the following structure:

[0029] In this case, terminal (I-2)# is connected to P;

[0030] m is any integer selected from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0031] n is any integer selected from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0032] q is any integer selected from 1 to 4, such as 1, 2, 3 or 4;

[0033] p is any integer selected from 2 to 20, such as 2, 3, 4, 5, 6, 7, 9, 12, 14, 16, 18, 19 or 20;

[0034] i is any integer selected from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 9, 12, 14, 16, 18, 19 or 20;

[0035] j is any integer selected from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 9, 12, 14, 16, 18, 19 or 20;

[0036] z is an integer selected from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0037] In some embodiments, the first structural domain contains HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; the first structural domain contains LCDR1 as shown in SEQ ID NO:9, LCDR2 as shown in SEQ ID NO:10, and LCDR3 as shown in SEQ ID NO:11.

[0038] In some embodiments, the second domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is SEQ ID NO:4 or a variant of SEQ ID NO:4 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR2 is SEQ ID NO:5 or a variant of SEQ ID NO:5 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR3 is SEQ ID NO:6 or a variant of SEQ ID NO:6 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0039] and / or

[0040] The second structural domain comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is a variant of SEQ ID NO:12 or SEQ ID NO:12 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is a variant of SEQ ID NO:13 or SEQ ID NO:13 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is a variant of SEQ ID NO:14 or SEQ ID NO:14 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0041] In some embodiments, the second structural domain contains HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:5, and HCDR3 as shown in SEQ ID NO:6; the second structural domain contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14.

[0042] In some implementations, the EGFR is a human EGFR, and / or the cMET is a human cMET.

[0043] In some implementations, the first structural domain includes a heavy chain variable region and a light chain variable region.

[0044] In some embodiments, the first domain contains a heavy chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% consistency with SEQ ID NO:7, and / or, the first domain further contains a light chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% consistency with SEQ ID NO:15.

[0045] In some embodiments, the first structural domain contains the heavy chain variable region shown in SEQ ID NO:7 and the light chain variable region shown in SEQ ID NO:15.

[0046] In some implementations, the second structural domain includes a heavy chain variable region and a light chain variable region.

[0047] In some embodiments, the second domain contains a heavy chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% consistency with SEQ ID NO:8, and / or, the second domain further contains a light chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% consistency with SEQ ID NO:16.

[0048] In some embodiments, the second structural domain contains the heavy chain variable region shown in SEQ ID NO:8 and the light chain variable region shown in SEQ ID NO:16.

[0049] In some embodiments, the first domain contains a first heavy chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:21; and / or, the first domain contains a first light chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:22.

[0050] In some embodiments, the first structural domain contains a first heavy chain as shown in SEQ ID NO:21, or a first light chain as shown in SEQ ID NO:22.

[0051] In some embodiments, the second domain contains a second heavy chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:23; and / or, the second domain contains a second light chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:24.

[0052] In some embodiments, the second structural domain contains a second heavy chain as shown in SEQ ID NO:23, or a second light chain as shown in SEQ ID NO:24.

[0053] A second aspect of this disclosure provides an antibody-drug conjugate having Ab-[LP] (t) ] z The structure;

[0054] Wherein, Ab is a bispecific antibody targeting EGFR and cMET or its antigen-binding fragment, including a first domain targeting EGFR and a second domain targeting cMET:

[0055] Wherein, the second structural domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is SEQ ID NO:4 or a variant of SEQ ID NO:4 with 1-3 amino acids substituted, deleted, or added and having the same or similar function; the amino acid sequence of HCDR2 is SEQ ID NO:5 or a variant of SEQ ID NO:5 with 1-3 amino acids substituted, deleted, or added and having the same or similar function; the amino acid sequence of HCDR3 is SEQ ID NO:6 or a variant of SEQ ID NO:6 with 1-3 amino acids substituted, deleted, or added and having the same or similar function.

[0056] and / or

[0057] The second structural domain comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is a variant of SEQ ID NO:12 or SEQ ID NO:12 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is a variant of SEQ ID NO:13 or SEQ ID NO:13 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is a variant of SEQ ID NO:14 or SEQ ID NO:14 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0058] P is a cytotoxin;

[0059] t is any integer greater than 1, and can be any integer from 1 to 10, such as 2, 4, 6 or 8;

[0060] Ab and P are connected by connector L, which includes the structure shown in equation (I);

[0061] Equation (I) is:

[0062] in,

[0063] In equation (I), the * terminal is connected to Ab; the # terminal is connected to P.

[0064] R 0 C 1-10 alkyl;

[0065] D has the following structure:

[0066] In equation (I-1), * represents the end connected to Ab;

[0067] ** represents the sum (I) The end of the part connected to the -NH-;

[0068] R 1 R 2 R 3 R 4 R 5 R 6 Each is independently selected from H and C. 1-6 alkoxy or OH; and satisfy the following condition: R 1 and R 2 Different, R 3 and R 4 Differences and R 5 and R 6 different;

[0069] R 7 It is hydrogen or α-L-fucosylate;

[0070] Q has the following structure:

[0071] In this case, terminal (I-2)# is connected to P;

[0072] m is any integer selected from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0073] n is any integer selected from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0074] q is any integer selected from 1 to 4, such as 1, 2, 3 or 4;

[0075] p is any integer selected from 2 to 20, such as 2, 3, 4, 5, 6, 7, 9, 12, 14, 16, 18, 19 or 20;

[0076] i is any integer selected from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 9, 12, 14, 16, 18, 19 or 20;

[0077] j is any integer selected from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 9, 12, 14, 16, 18, 19 or 20;

[0078] z is any integer selected from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0079] In some embodiments, the second structural domain contains HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:5, and HCDR3 as shown in SEQ ID NO:6; the second structural domain contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14.

[0080] In some embodiments, the first domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is a variant of SEQ ID NO:1 or SEQ ID NO:1 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR2 is a variant of SEQ ID NO:2 or SEQ ID NO:2 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR3 is a variant of SEQ ID NO:3 or SEQ ID NO:3 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0081] and / or

[0082] The first domain further comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is SEQ ID NO:9 or a variant of SEQ ID NO:9 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is SEQ ID NO:10 or a variant of SEQ ID NO:10 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is SEQ ID NO:11 or a variant of SEQ ID NO:11 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0083] In some embodiments, the first structural domain contains HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; the first structural domain contains LCDR1 as shown in SEQ ID NO:9, LCDR2 as shown in SEQ ID NO:10, and LCDR3 as shown in SEQ ID NO:11.

[0084] In some implementations, the EGFR is a human EGFR, and / or the cMET is a human cMET.

[0085] In some implementations, the first structural domain includes a heavy chain variable region and a light chain variable region.

[0086] In some embodiments, the first domain contains a heavy chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% consistency with SEQ ID NO:7, and / or, the first domain further contains a light chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% consistency with SEQ ID NO:15.

[0087] In some embodiments, the first structural domain contains the heavy chain variable region shown in SEQ ID NO:7 and the light chain variable region shown in SEQ ID NO:15.

[0088] In some implementations, the second structural domain includes a heavy chain variable region and a light chain variable region.

[0089] In some embodiments, the second domain contains a heavy chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% consistency with SEQ ID NO:8, and / or, the second domain further contains a light chain variable region having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% consistency with SEQ ID NO:16.

[0090] In some embodiments, the second structural domain contains the heavy chain variable region shown in SEQ ID NO:8 and the light chain variable region shown in SEQ ID NO:16.

[0091] In some embodiments, the first domain contains a first heavy chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:21; and / or, the first domain contains a first light chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:22.

[0092] In some embodiments, the first structural domain contains a first heavy chain as shown in SEQ ID NO:21, or a first light chain as shown in SEQ ID NO:22.

[0093] In some embodiments, the second domain contains a second heavy chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:23; and / or, the second domain contains a second light chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:24.

[0094] In some embodiments, the second structural domain contains a second heavy chain as shown in SEQ ID NO:23, or a second light chain as shown in SEQ ID NO:24.

[0095] A third aspect of this disclosure provides an antibody-drug conjugate having Ab-[LP] (t) ] z The structure;

[0096] Wherein, Ab is a bispecific antibody targeting EGFR and cMET or its antigen-binding fragment, including a first domain targeting EGFR and a second domain targeting cMET:

[0097] Wherein, the first structural domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is a variant of SEQ ID NO:1 or SEQ ID NO:1 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR2 is a variant of SEQ ID NO:2 or SEQ ID NO:2 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR3 is a variant of SEQ ID NO:3 or SEQ ID NO:3 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0098] The second domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is SEQ ID NO:4 or a variant of SEQ ID NO:4 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR2 is SEQ ID NO:5 or a variant of SEQ ID NO:5 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR3 is SEQ ID NO:6 or a variant of SEQ ID NO:6 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0099] P is a cytotoxin;

[0100] t is any integer greater than 1, and can be any integer from 1 to 10, such as 2, 4, 6 or 8;

[0101] Ab and P are connected by connector L, which includes the structure shown in equation (I);

[0102] Equation (I) is:

[0103] in,

[0104] In equation (I), the * terminal is connected to Ab; the # terminal is connected to P.

[0105] R 0 C 1-10 alkyl;

[0106] D has the following structure:

[0107] In equation (I-1), * represents the end connected to Ab;

[0108] ** represents the sum (I) The end of the part connected to the -NH-;

[0109] R1 R 2 R 3 R 4 R 5 R 6 Each is independently selected from H and C. 1-6 alkoxy or OH; and satisfy the following condition: R 1 and R 2 Different, R 3 and R 4 Differences and R 5 and R 6 different;

[0110] R 7 It is hydrogen or α-L-fucosylate;

[0111] Q has the following structure:

[0112] In this case, terminal (I-2)# is connected to P;

[0113] m is any integer selected from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0114] n is any integer selected from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8;

[0115] q is any integer selected from 1 to 4, such as 1, 2, 3 or 4;

[0116] p is any integer selected from 2 to 20, such as 2, 3, 4, 5, 6, 7, 9, 12, 14, 16, 18, 19 or 20;

[0117] i is any integer selected from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 9, 12, 14, 16, 18, 19 or 20;

[0118] j is any integer selected from 1 to 20, such as 1, 2, 3, 4, 5, 6, 7, 9, 12, 14, 16, 18, 19 or 20;

[0119] z is any integer selected from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7 or 8.

[0120] In some embodiments, the first structural domain contains HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; the second structural domain contains HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:5, and HCDR3 as shown in SEQ ID NO:6.

[0121] In some implementations, the EGFR is a human EGFR, and / or the cMET is a human cMET.

[0122] In some implementations, the first domain and / or the second domain includes a humanized heavy chain variable region.

[0123] In some embodiments, the first domain contains a heavy chain variable region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:7, and / or the second domain contains a heavy chain variable region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:8.

[0124] In some embodiments, the first structural domain contains the heavy chain variable region shown in SEQ ID NO:7, and / or the second structural domain contains the heavy chain variable region shown in SEQ ID NO:8.

[0125] In some embodiments, the first domain further comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is SEQ ID NO:9 or a variant of SEQ ID NO:9 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is SEQ ID NO:10 or a variant of SEQ ID NO:10 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is SEQ ID NO:11 or a variant of SEQ ID NO:11 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0126] In some embodiments, the second domain further comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is a variant of SEQ ID NO:12 or SEQ ID NO:12 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is a variant of SEQ ID NO:13 or SEQ ID NO:13 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is a variant of SEQ ID NO:14 or SEQ ID NO:14 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0127] In some embodiments, the first structural domain contains LCDR1 as shown in SEQ ID NO:9, LCDR2 as shown in SEQ ID NO:10, and LCDR3 as shown in SEQ ID NO:11; the second structural domain contains LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14.

[0128] In some embodiments, the first domain contains a light chain variable region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:15, and / or the second domain contains a light chain variable region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:16.

[0129] In some embodiments, the first structural domain contains the light chain variable region shown in SEQ ID NO:15, and / or the second structural domain contains the light chain variable region shown in SEQ ID NO:16.

[0130] In some embodiments, the first domain and / or the second domain contains a heavy chain constant region derived from a corresponding functional fragment of human immunoglobulin IgG1 or IgG4, or a variant having the same or similar function thereto; when the residues are numbered according to the EU numbering system, the heavy chain constant regions of the first domain and / or the second domain each have 1-7 substitution mutation sites, and the mutation sites of the heavy chain constant regions of the first domain and / or the second domain are different.

[0131] In some embodiments, when the residues are numbered according to the EU numbering system, the substitution mutation site is selected from amino acids at positions 350, 366, 368, 370, 399, 405, 407, or 409.

[0132] In some implementations, when the residues are numbered according to the EU numbering system, the substitution mutation includes K409R or F405L.

[0133] In some embodiments, the heavy chain constant region of the first structural domain has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% similarity to SEQ ID NO:17; and / or, the heavy chain constant region of the second structural domain has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% similarity to SEQ ID NO:19.

[0134] In some embodiments, the heavy chain constant region of the first domain contains the amino acid sequence shown in SEQ ID NO:17; and / or, the heavy chain constant region of the second domain contains the amino acid sequence shown in SEQ ID NO:19.

[0135] In some embodiments, the first domain contains a first heavy chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:21; and / or, the second domain contains a second heavy chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:23.

[0136] In some embodiments, the first structural domain contains a first heavy chain as shown in SEQ ID NO:21; and / or, the second structural domain contains a second heavy chain as shown in SEQ ID NO:23.

[0137] In some embodiments, the first domain contains a light chain constant region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:18; and / or, the second domain contains a light chain constant region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:20.

[0138] In some embodiments, the first structural domain contains the light chain constant region shown in SEQ ID NO:18; and / or, the second structural domain contains the light chain constant region shown in SEQ ID NO:20.

[0139] In some embodiments, the first domain contains a first light chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:22; and / or, the second domain contains a second light chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:24.

[0140] In some embodiments, the first structural domain contains a first light chain as shown in SEQ ID NO:22; and / or, the second structural domain contains a second light chain as shown in SEQ ID NO:24.

[0141] In some embodiments, the first structural domain contains a first heavy chain as shown in SEQ ID NO:21 and a first light chain as shown in SEQ ID NO:22; and / or the second structural domain contains a second heavy chain as shown in SEQ ID NO:23 and a second light chain as shown in SEQ ID NO:24.

[0142] In some embodiments described in the first, second, or third aspects above, the bispecific antibody comprises (a) or (b):

[0143] (a) A first heavy chain and a first light chain forming a first dimer, a second heavy chain and a second light chain forming a second dimer, the first dimer and the second dimer being connected by disulfide bonds;

[0144] (b) A first heavy chain and a second light chain forming a first dimer, a second heavy chain and a first light chain forming a second dimer, the first dimer and the second dimer being connected by disulfide bonds.

[0145] In some embodiments described in the first, second, or third aspects above, P is a topoisomerase inhibitor.

[0146] In some embodiments described in the first, second, or third aspects above, t is any integer from 1 to 10, such as 2, 4, 6, or 8.

[0147] In some of the embodiments described in the first, second, or third aspects above, R 0 C 1-3 Alkyl, for example, R 0 It is a methyl group.

[0148] In some embodiments described in the first, second, or third aspects above, the -NHC(O)CH2- at the * end of formula (I-1) is part of an amino acid in the Fc region of the antibody; preferably, the -NHC(O)CH2- at the * end is part of asparagine in the Fc region of the antibody; more preferably, the -NHC(O)CH2- at the * end is part of asparagine at position 297 of the Fc region of the antibody.

[0149] In some embodiments described in the first, second, or third aspects above, R in formula (I-1) 1 R 2 R 3 R 4 R 5 R 6 Each is independently selected from H, methoxy, or OH; preferably, R 1 R 2 R 3 R 4 R 5 R 6 Each is independently selected from H or OH.

[0150] In some embodiments described in the first, second, or third aspects above, R in formula (I-1) 7 It is H or α-L-fucosylate.

[0151] In some embodiments described in the first, second or third aspects above, m in formula (I) is any integer selected from 1 to 5, preferably 3.

[0152] In some embodiments described in the first, second, or third aspects above, n in formula (I) is any integer selected from 1 to 5, preferably 3.

[0153] In some embodiments described in the first, second or third aspects above, q in formula (I-2) is 1 or 2.

[0154] In some embodiments described in the first, second, or third aspects above, p in formula (I-2) is an integer selected from 2 to 10, preferably 2.

[0155] In some embodiments described in the first, second or third aspects above, i in formula (I) is an integer selected from 1 to 10, preferably 4.

[0156] In some embodiments described in the first, second or third aspects above, j in formula (I) is an integer selected from 2 to 16, preferably 12.

[0157] In some embodiments described in the first, second or third aspects above, n in formula (I) is 2 or 3.

[0158] In some embodiments described in the first, second, or third aspects above, z is 1, 2, 4, and preferably 2.

[0159] In some embodiments described in the first, second, or third aspects above, D has the following structure:

[0160] In some embodiments described in the first, second, or third aspects above, the structure of the connector L is as follows:

[0161] In some embodiments described in the first, second, or third aspects above, the structure of the connector is as follows:

[0162] In some embodiments described in the first, second, or third aspects above, each P is independently selected from DNA topoisomerase I inhibitors; preferably, the DNA topoisomerase I inhibitors include camptothecin inhibitors, such as irinotecan and topotecan; and non-camptothecin inhibitors, such as indoline benzozazepine compounds.

[0163] Preferably, the DNA topoisomerase I inhibitor is selected from the following structures:

[0164] In some embodiments described in the first, second, or third aspects above, the LP (t) It has any of the following structures:

[0165] In some embodiments described in the first, second, or third aspects above, the antibody conjugate has the structure shown in any one of (III-1) or (III-2) below:

[0166] Among them, R 7 It is hydrogen or α-L-fucosylate;

[0167] Ab is a bispecific antibody comprising a first domain targeting EGFR and a second domain targeting cMET; the first domain contains a first heavy chain as shown in SEQ ID NO:21 and a first light chain as shown in SEQ ID NO:22; the second domain contains a second heavy chain as shown in SEQ ID NO:23 and a second light chain as shown in SEQ ID NO:24.

[0168] In some embodiments described in the first, second, or third aspects above, the antibody conjugate has the structure shown in (III-1):

[0169] Among them, R 7 It is hydrogen or α-L-fucosylate;

[0170] Ab is a bispecific antibody comprising a first domain targeting EGFR and a second domain targeting cMET; the first domain contains a first heavy chain as shown in SEQ ID NO:21 and a first light chain as shown in SEQ ID NO:22; the second domain contains a second heavy chain as shown in SEQ ID NO:23 and a second light chain as shown in SEQ ID NO:24.

[0171] In some embodiments described in the first, second, or third aspects above, the antibody conjugate has the structure shown in (III-2):

[0172] Among them, R 7 It is hydrogen or α-L-fucosylate;

[0173] Ab is a bispecific antibody comprising a first domain targeting EGFR and a second domain targeting cMET; the first domain contains a first heavy chain as shown in SEQ ID NO:21 and a first light chain as shown in SEQ ID NO:22; the second domain contains a second heavy chain as shown in SEQ ID NO:23 and a second light chain as shown in SEQ ID NO:24.

[0174] In some embodiments described in the first, second, or third aspects above, the Ab is linked to the remainder of the antibody-drug conjugate via an enzyme or a mutant thereof; wherein the enzyme or mutant thereof is an N-acetylglucosamine endonuclease or a mutant thereof.

[0175] Preferably, the N-acetylglucosamine endohydrolase is selected from Endo H, Endo D, Endo F2, Endo M, Endo Om, Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd), Endo CC (Endoglycosidase-CC), Endo CC1, and Endo CC2, or mutants thereof.

[0176] More preferably, the N-acetylglucosamine endohydrolase is selected from Endo F3, Endo S and Endo S2, or mutants thereof.

[0177] This disclosure provides a fourth aspect of a pharmaceutical composition comprising an antibody-drug conjugate as described in any of the foregoing embodiments; preferably, it further comprises at least one pharmaceutically acceptable carrier.

[0178] This disclosure provides, in its fifth aspect, the use of an antibody-drug conjugate according to any one of the embodiments of the first, second, or third aspect, or a pharmaceutical composition according to any one of the embodiments of the fourth aspect, in the preparation of a drug for inhibiting tumor growth or producing tumor regression.

[0179] In some embodiments, the tumor contains EGFR-positive cells and / or c-MET-positive cells.

[0180] In some implementations, the tumor includes tumors from patients with non-small cell lung cancer.

[0181] The sixth aspect of this disclosure provides a method for treating or preventing cancer, comprising administering to a subject in need a therapeutically effective amount of an antibody-drug conjugate or a pharmaceutical composition as described in any one of the embodiments of the first, second, or third aspect, or any one of the embodiments of the fourth aspect.

[0182] In some implementations, the cancer is EGFR positive and / or c-MET positive.

[0183] In some implementations, the cancer includes non-small cell lung cancer. Detailed Implementation

[0184] I. General Definition

[0185] Based on the above content of this disclosure, and in accordance with common technical knowledge and practices in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical ideas of this disclosure.

[0186] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0187] In this disclosure, the term "compound" can be asymmetric, for example, having one or more stereoisomers. Unless otherwise stated, all stereoisomers are included, such as enantiomers and diastereomers. Compounds containing asymmetric carbon atoms of this disclosure can be isolated in optically active pure form or in racemic form. Optically active pure form can be resolved from racemic mixtures or synthesized using chiral starting materials or chiral reagents. Racemates, diastereomers, and enantiomers are all included within the scope of this disclosure.

[0188] In this disclosure, unless the context clearly specifies otherwise, the singular forms such as “a type” and “the kind” include the plural forms. The expressions “a type or multiple types” or “at least one type” may mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0189] In this disclosure, when a quantity, concentration, or other numerical value or parameter is described in the form of a range, preferred range, or preferred upper or lower limit, it should be understood as equivalent to specifically disclosing any range formed by combining any upper or preferred value with any lower or preferred value, whether or not the range is explicitly stated. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range as well as all integers and fractions (decimals) within the range. For example, the statement "i is an integer from 1 to 20" means that i is any integer from 1 to 20, such that i can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. Other similar expressions such as j, g, k, etc., should also be understood in a similar manner.

[0190] In this disclosure, "etc." refers to the position of the substituent bond.

[0191] In this disclosure, the term "about" means within an acceptable range of error for a particular value as determined by those skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular measurement, result, or embodiment, unless otherwise expressly stated in the embodiments or elsewhere in the specification, "about" means within a standard deviation according to convention in the art.

[0192] In this disclosure, the terms “first,” “second,” “third,” and other referential terms are used only to distinguish different elements, features, or events, and do not imply any difference in importance between the elements or features, nor do they imply a sequential order of execution of the corresponding events.

[0193] In this disclosure, the terms "EU numbering system," "EU index," and "EU numbering rule" are used interchangeably. It is a standardized residue numbering scheme for the constant region (Fc region) of the antibody heavy chain. Originally derived from the Eu sequence (European) of the IgG1 antibody determined in the 1970s, the EU number is obtained by comparing the amino acid sequences of different antibodies with the Eu sequence, primarily covering the CH1, hinge, CH2, and CH3 domains. This system is crucial in antibody engineering because many functionally critical sites (such as the N297 glycosylation site and the P329 FcγR binding site) and common mutations (such as L234A / L235A) are described using EU numbers. Complementing the Kabat or IMGT numbers for the variable region, they together constitute the standard language of antibody structure.

[0194] In this disclosure, the term "knob-into-hole mutation" refers to a heavy chain heterodimerization method designed for the production of bispecific antibodies. This method utilizes the complementary spatial effect between the two chains to preferentially form heterodimers rather than homodimers by introducing a large hydrophobic mutation (forming a "handle" knot) into the CH3 domain of one heavy chain and a smaller hydrophobic mutation (forming a "hole") at the corresponding position on the other heavy chain. This significantly improves the correct assembly rate of bispecific antibodies.

[0195] In this disclosure, the term "TM mutation (Triple Mutation)" refers to a functional elimination method designed for the Fc region of an antibody, primarily used to inhibit antibody-mediated cytotoxicity (ADCC) and complement activation (CDC). In optional embodiments, the mutation may include one or more of three key sites: L234F, L235E, and P331S, significantly reducing the affinity of the Fc segment for the Fc receptor (FcγR) and complement C1q by altering its spatial structure. This design is commonly used in the development of therapeutic antibodies that require preservation of antigen-binding activity but must avoid damaging host cells or triggering excessive immune responses.

[0196] In this disclosure, the term "Controlled Fab-arm Exchange (cFAE)" refers to a technique that utilizes the natural properties of IgG4 antibodies. IgG4 can exchange half-molecules under specific conditions, and in some embodiments of this disclosure, this exchange can be controlled by introducing specific mutations (such as K409R or F405L) into the CH3 region of the antibody. This technique can exchange the Fab arms of two monoclonal antibodies to generate bispecific or multispecific antibodies. Its advantages include high yield (>95%) using existing IgG production methods and a simple preparation process.

[0197] In this disclosure, a range of numbers refers to individual integers within a given range. For example, "C 1-6 "" means that the group can have 1, 2, 3, 4, 5, or 6 carbon atoms; "C 1-3 "" means that the group can have 1 carbon atom, 2 carbon atoms or 3 carbon atoms.

[0198] In this disclosure, the terms "substituted" or "replaced" used to describe non-protein compounds or compound structures mean that any one or more hydrogen atoms on a particular atom or group are replaced by a substituent, provided that the valence state of the particular atom or group is normal and the substituted compound is stable. For example, when the substituent is a ketone group (i.e., =O), it means that two hydrogen atoms are replaced. Unless otherwise specified, the type and number of substituents can be arbitrary on a chemically feasible basis. Unless otherwise stated, the relevant substituents are selected from alkyl, halogen, amino, monoalkylamino, dialkylamino, nitro, cyano, formyl, alkylcarbonyl, carboxyl, alkyloxycarbonyl, alkylcarbonyloxy, aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, formylamino, alkylcarbonylamino, formyl(monoalkyl)amino, or alkylcarbonyl(monoalkyl)amino. When the terms “substituted” or “replaced” are used to describe amino acid residues of a protein or polypeptide, it means that an amino acid residue at a specific site in the amino acid sequence of the original protein or polypeptide is replaced with one or more other amino acids or their derivatives or analogues.

[0199] In this disclosure, when any variable (e.g., substituent R) n When a substituent (and cytotoxin P) appears more than once in the composition or structure of a compound, its definition is independent in each case. Therefore, for example, if a group is substituted by one to three Rs, the group may optionally be substituted by up to three Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and / or their variants are only permitted if such combinations produce a stable compound.

[0200] In this disclosure, the term "alkyl" refers to a straight-chain or branched saturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, connected to the rest of the molecule by single bonds. Alkyl groups can have 1-20 carbon atoms, referring to "C1-C2". 20Alkyl, such as C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, C3 alkyl, C4 alkyl, C3-C6 alkyl. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1 -Dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl, or 1,2-dimethylbutyl, or their isomers. A divalent radical is a group obtained by removing a hydrogen atom from a carbon atom with free valence electrons of the corresponding monovalent radical. A divalent radical has two linkage sites attached to the rest of the molecule. For example, "alkylene" or "alkylene group" refers to a saturated straight-chain or branched divalent hydrocarbon group. Examples of "alkylene" include, but are not limited to, methylene (-CH2-), ethylene (-C2H4-), propylene (-C3H6-), butylene (-C4H8-), and pentylene (-C5H6-). 10 -), Hexyl (-C6H) 12 -), 1-methylethylidene (-CH(CH3)CH2-), 2-methylethylidene (-CH2CH(CH3)-), methylpropylidene or ethylpropylidene, etc.

[0201] In this disclosure, the term "cycloalkyl" refers to a cyclic saturated aliphatic group consisting of carbon and hydrogen atoms, which is connected to the rest of the molecule by a single bond. Cycloalkyl groups can have 3-10 carbon atoms, i.e., "C3-C4". 10 "Cycloalkyl" refers to compounds such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or cyclodecyl. "Cycloalkylene" refers to divalent cycloalkyl groups.

[0202] In this disclosure, the term "heterocyclic" refers to a cycloalkyl group in which one or more carbon atoms are replaced by a heteroatom selected from nitrogen, oxygen, and sulfur, such as azirmonopropyl, azirmonopropyl, or thiacyclopropyl, azirmonobutyl, pyrrolidinyl, pyrazolyl, imidazoalkyl, tetrahydrofuranyl, tetrahydrothiophenyl, piperidinyl, piperazineyl, tetrahydropyranyl, or tetrahydrothiaranyl. "Hypocyclic" refers to a divalent cycloalkyl group.

[0203] In this disclosure, when a group is combined with another group, the connection between the groups can be linear or branched, provided that a chemically stable structure is formed. The structure formed by such combination can be connected to other parts of the molecule by any suitable atoms in the structure, preferably by specified chemical bonds. For example, when two or more atoms selected from -CR1 R 2 C 1-10 Alkylene, C 4-10 Cycloalkylene, C 4-10 Heterocyclic groups and -(CO)- divalent groups combine to form a complex; two or more divalent groups can form linear linkages, such as -CR. 1 R 2 -C 1-10 Alkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkylene-(CO)-, -CR 1 R 2 -C 4-10 Cycloalkyl-C 1-10 Alkylene-(CO)-, -CR 1 R 2 -CR 1 R 2′ -(CO)-、-CR 1 R 2 -CR 1′ R 2′ -CR 1″ R 2″ -(CO)- etc. The resulting divalent structure can be further linked to other parts of the molecule. When multiple identical letters representing chemical groups appear in the same chemical structural formula, they are chosen independently and are not necessarily the same.

[0204] In this disclosure, the term "alkoxy" can be linear, branched, or cyclic. The number of carbon atoms in an alkoxy group is not particularly limited, but is preferably from 1 to 20. Specific examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, i-propyloxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, neopentoxy, isopentoxy, n-hexyloxy, 3,3-dimethylbutoxy, 2-ethylbutoxy, n-octoxy, n-nonoxy, n-decoxy, etc.

[0205] In this disclosure, the terms "carbonyl" or "carboxyl" include structures in compounds and segments in which carbon atoms are connected to oxygen atoms via double bonds. Examples of carbonyl moieties include aldehydes, ketones, carboxylic acids, amides, esters, acid anhydrides, etc.

[0206] In this disclosure, the term "acyl" refers to a carbonyl structure in which a carbonyl group is linked to a hydrogen atom (i.e., a formyl group), an aliphatic group (C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, such as an acetyl group), a cycloalkyl group (C3-C8 cycloalkyl), a heterocyclic group (C3-C8 heterocyclic alkyl and C5-C6 heteroaryl), or an aryl group (C6 aryl, such as a benzoyl group). The acyl group can be unsubstituted or substituted (e.g., a salicylyl group).

[0207] In this disclosure, examples of halogen groups may include fluorine, chlorine, bromine, or iodine.

[0208] In this disclosure, the term "aryl" refers to an aromatic ring group consisting of a monocyclic or fused polycyclic aromatic ring with a conjugated π-electron system, obtained by removing a hydrogen atom from a single carbon atom of the parent aromatic ring system. It includes saturated, partially unsaturated rings, or bicyclic aromatic rings with fused aromatic carbon rings. Specific examples include, but are not limited to, phenyl or naphthyl groups.

[0209] In this disclosure, the term "heteroaryl" refers to a monovalent aryl group comprising at least one heteroatom independently selected from nitrogen, oxygen, and sulfur. The heteroaryl group can be monocyclic or polycyclic, such as bicyclic, wherein two or more rings exist in the form of fused rings, bridged rings, or spirocyclic rings, and at least one ring contains one or more heteroatoms. Specific examples of heteroaryl groups include, but are not limited to, pyridyl, thiophene, imidazolyl, pyrimidinyl, furanyl, pyrazinyl, thiazolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, imidazopyridyl, benzofuranyl, pyridazinyl, isoyindolyl, and pyridinoneyl.

[0210] In this disclosure, the term "heterocyclic" refers to a 5-12 member saturated non-aromatic system having a ring carbon atom and one to two ring heteroatoms, wherein the heteroatoms are independently selected from nitrogen, sulfur, or oxygen atoms. In heterocyclic groups containing one or more nitrogen atoms, the connecting point can be a carbon or nitrogen atom, provided the valence allows. The heterocycle can be a monocyclic or polycyclic system, such as a bicyclic system, wherein two or more rings exist in the form of fused rings, bridged rings, or spirocyclic rings, wherein at least one ring contains one or more heteroatoms. In spirocyclic heterocyclic groups, two different rings share a common atom; an example of a spirocyclic heterocyclic group is an azeropeptyl group, but it is not limited thereto.

[0211] In this disclosure, the term "partially unsaturated bicyclic heterocycle" refers to a bicyclic group comprising at least one of a C atom and a heteroatom such as N, O, or S as a ring member, wherein the bicyclic group is partially unsaturated and contains at least one C-C double bond. The most unsaturated heterocycle contains the maximum number of C-C double bonds allowed by the ring size, as well as double bonds between the C atom and the heteroatom. The partially unsaturated bicyclic heterocycle contains fewer double bonds than allowed by the ring size. Specific examples of partially unsaturated bicyclic heterocycles include, but are not limited to, benzofuranyl, benzothiophenyl, quinoxalinyl, quinazolinyl, isoindolinone, pteridinyl, etc.

[0212] In this disclosure, the term "targeting molecule" refers to a molecule that has an affinity for a specific target (e.g., receptors, cell surface proteins, cytokines, tumor-specific antigens, etc.). Targeting molecules can deliver a payload to a specific site in the body via targeted delivery. Targeting molecules can recognize one or more targets. The specific target is defined by the target it recognizes. For example, a targeting molecule that targets a receptor can deliver a cytotoxin to a site containing a large number of receptors. Examples of targeting molecules include, but are not limited to, antibodies, binding proteins to a given antigen, antibody mimics, scaffold proteins with affinity for a given target, ligands, etc.

[0213] In this disclosure, the term "EGFR" belongs to the HER family and consists of four subtypes (EGFR1-4). It primarily promotes cell proliferation, survival, and migration by binding to ligands such as epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) to activate signal transduction pathways, including the PI3K-AKT and MAPK pathways. EGFR is overexpressed or mutated in various cancers, such as non-small cell lung cancer (NSCLC), and its abnormal activation is closely related to the malignancy of the tumor.

[0214] In this disclosure, the term "cMET," composed of an α-chain and a β-chain, is a transmembrane dimeric heterodimer protein that primarily promotes cell proliferation, survival, and migration by binding to hepatocyte growth factor (HGF) and activating signaling pathways such as the PI3K-AKT and MAPK pathways. cMET is overexpressed or amplified in various solid tumors, such as lung cancer, and its aberrant activation is closely associated with tumor invasiveness and drug resistance.

[0215] In this disclosure, the term "antibody" is used broadly and its definition encompasses conventional antibodies, recombinant / genetically engineered antibodies, and particularly includes complete monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they possess the desired biological activity. Antibodies can be any subtype (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass and can be derived from any suitable species. In some embodiments, the antibody is of human or murine origin. Antibodies can also be fully human antibodies, humanized antibodies, or chimeric antibodies prepared by recombinant methods. As used herein, a monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that, except for a few possible natural mutations, the individual antibodies constituting the population are identical. Monoclonal antibodies exhibit high specificity for a single antigenic site.

[0216] In this disclosure, the term "bispecific antibody" refers to an engineered antibody having two distinct antigen-binding sites. In one embodiment, this disclosure relates to multiple ADCs containing a bispecific antibody capable of binding EGFR and cMET. The bispecific antibody can be a complete antibody or an antigen-binding fragment derived from a complete antibody, such as a nanobody VHH. A complete antibody or full-length antibody essentially comprises an antigen-binding variable region and a light chain constant region (CL) and a heavy chain constant region (CH), which may include CH1, CH2, CH3, and CH4, depending on the antibody subtype. The antigen-binding variable region (also called a fragment variable region, Fv fragment) typically comprises a light chain variable region (VL) and a heavy chain variable region (VH). The constant region can be a constant region having a native sequence (e.g., a constant region having a human native sequence) or a variant of its amino acid sequence. The variable region recognizes and interacts with a target antigen. The constant region can be recognized and interacted with by the immune system. The antibody fragment may comprise a portion of a complete antibody, preferably its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab′, F(ab′)2, Fd fragments consisting of VH and CH1 domains, Fv fragments, single-domain antibody (dAb) fragments, and isolated complementarity-determining regions (CDRs). Fab fragments are antibody fragments obtained by digesting full-length immunoglobulins with papain, or fragments with the same structure generated, for example, through recombinant expression. Fab fragments comprise a light chain (containing VL and CL) and another chain, wherein the other chain contains a variable region (VH) of the heavy chain and a constant region (CH1) of the heavy chain. F(ab′)2 fragments are antibody fragments obtained by digesting immunoglobulins with pepsin at pH 4.0–4.5, or fragments with the same structure generated, for example, through recombinant expression. F(ab′)2 fragments essentially comprise two Fab fragments, each heavy chain portion containing several additional amino acids, including cysteine ​​residues that form the disulfide bonds connecting the two fragments. Fab′ fragments are fragments containing half of the F(ab′)2 fragment (one heavy chain and one light chain). The antibody fragment may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Examples of antibody fragments also include single-chain Fv (scFv), Fv, dsFv, bispecific antibodies, Fd and Fd′ fragments, and other fragments, including modified fragments. Antibody fragments typically contain at least or about 50 amino acids, and usually at least or about 200 amino acids. Antigen-binding fragments may include any antibody fragment that, when inserted into an antibody frame (e.g., by replacing the corresponding region), yields an antibody that specifically binds to the antigen.

[0217] In this disclosure, the term "antibody variable region" includes both the heavy chain variable region (VH) and the light chain variable region (VL) in a complete antibody, which can be bound together by non-covalent bonds and interchain disulfide bonds to form an antigen-binding site (Fab fragment). In nanobodies, only the heavy chain variable region is present. Typically, each heavy chain variable region (VH) consists of one variable region (VH) and three or four constant regions (CH1, CH2, CH3, CH4). The N-terminus of the heavy chain variable region (VH) and the N-terminus of the light chain variable region (VL) can together form an antigen-binding pocket.

[0218] In this disclosure, the term "CDR (Complementarity-Determining Regions)" refers to the three hypervariable regions (CDR1, CDR2, and CDR3) contained in each VH and VL. These regions have highly variable amino acid sequences and directly participate in antigen binding. Heavy chain CDRs are typically abbreviated as HCDR1, HCDR2, and HCDR3, while light chain CDRs are abbreviated as LCDR1, LCDR2, and LCDR3. CDR numbering rules involve various standardization systems, with the Kabat, Chothia, and IMGT schemes being the most widely used. This disclosure adopts the Kabat numbering rule.

[0219] In this disclosure, the term "nanobody" (Nb) refers to the smallest antigen-binding fragment or single variable domain (VHH) derived from a natural heavy chain antibody, as is known to those skilled in the art. They are derived from heavy chain-only antibodies found in camels. Immunoglobulins lacking light polypeptide chains are found in the "camelidae" family. "Camelidae" includes Old World camels (Bactrian and Dromedary camels) and New World camels. Single variable domain heavy chain antibodies are referred to herein as nanobodies or VHH antibodies.

[0220] In this disclosure, antibody-drug conjugates are obtained by conjugating a cytotoxin to the C-terminus of an antibody light chain via a linker. In other embodiments, the antibody-drug conjugates are obtained by site-directed conjugation at any naturally occurring N-glycosylation modification site containing the antibody's Fc region. Molecules containing glycans in the antibody's Fc region (including but not limited to antibodies / bispecific antibodies / Fc fusion proteins / single-chain antibodies / nanobodies, etc.) are prepared in a one-step process using the oligosaccharide-containing linker loadings of this disclosure. Therefore, the antibodies of this disclosure are not particularly limited; their Fc regions only need to contain glycans, and they can be natural antibodies. Furthermore, the antibodies of this disclosure can also be prepared using techniques well known in the art, such as the following techniques or combinations thereof: recombinant technology / genetic engineering technology, phage display technology, synthetic technology, or other techniques known in the art. For example, genetically engineered recombinant antibodies can be expressed using a suitable culture system (e.g., *E. coli* or mammalian cells). The genetic engineering can refer to, for example, the introduction of a ligase-specific recognition sequence at its end. As used herein, the term "targeting molecule-drug conjugate" is referred to as a "conjugate." Examples of conjugates include, but are not limited to, antibody-drug conjugates. Small molecule compounds are molecules whose size is comparable to that of organic molecules commonly used in pharmaceuticals. This term does not cover biological macromolecules (such as proteins and nucleic acids), but it does cover low molecular weight peptides or their derivatives, such as dipeptides, tripeptides, tetrapeptides, and pentapeptides. Typically, the molecular weight of small molecule compounds can be, for example, approximately 100–2000 Da, approximately 200–1000 Da, approximately 200–900 Da, approximately 200–800 Da, approximately 200–700 Da, approximately 200–600 Da, or approximately 200–500 Da. The terms "antibody-drug conjugate" and "antibody-drug conjugate" have the same meaning.

[0221] In this disclosure, the term "cytotoxic" refers to a substance that inhibits or prevents the expression activity, cellular function, and / or causes cell damage. Currently, cytotoxic agents commonly used in ADCs are more toxic than chemotherapeutic drugs. Examples of cytotoxic agents include, but are not limited to, drugs targeting the following targets: microtubule cytoskeleton, DNA, RNA, kinin-mediated protein transport, and regulation of apoptosis. Drugs targeting DNA may be, for example, drugs that directly disrupt DNA structure or topoisomerase inhibitors. Examples of topoisomerase inhibitors include, but are not limited to, exatecan and its derivatives (e.g., DX8951f, DXd-(1), and DXd-(2)), camptothecins, and anthracyclines.

[0222] In this disclosure, the term "spacer" refers to a structure located between different structural modules that can spatially separate the structural modules. The definition of a spacer does not limit whether it has a specific function or whether it can be cleaved or degraded in vivo. Examples of spacers include, but are not limited to, amino acid and non-amino acid structures, wherein non-amino acid structures may be, but are not limited to, amino acid derivatives or analogs. "Spacer sequence" refers to an amino acid sequence that serves as a spacer, and examples include, but are not limited to, sequences containing a single amino acid or sequences containing multiple amino acids, such as sequences containing two amino acids, such as GA. A self-cleaving spacer (e.g., self-cleaving spacer Sp1) is a covalent component that causes the activation of a protective moiety in a precursor, resulting in the sequential cleavage of two chemical bonds: the protective moiety (e.g., the cleavable sequence) is removed upon activation, triggering a cascade of decomposition reactions that lead to the sequential release of smaller molecules. Examples of self-cleaving spacers include, but are not limited to, PABC (p-aminobenzyloxycarbonyl), acetals, heteroacetals, and combinations thereof.

[0223] In this disclosure, the term "amino acid" includes both "natural amino acids" and "non-natural amino acids." The term "natural amino acid" refers to an amino acid that is a component of proteins, including the twenty common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), and the less common selenocysteine ​​and pyrrolidone. As used herein, the term "non-natural amino acid" refers to an amino acid that is not a component of proteins. Specifically, this term refers to an amino acid that is not a natural amino acid as defined above.

[0224] In this disclosure, the term "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms that are suitable for use in human and animal tissues to the extent of reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0225] In this disclosure, the term "treatment" includes the suppression, relief, prevention, or elimination of one or more symptoms or side effects associated with the treated disease, condition, or disorder. The term "effective amount" or "therapeutic effective amount" refers to a dose sufficient to treat, suppress, or reduce one or more symptoms of the treated disease state or otherwise provide the desired pharmacological and / or physiological effect. Precise dosages will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder, and the treatment administered. The effect of an effective amount can be relative to a control. These controls are known in the art and discussed herein, and can be, for example, the condition of the subject before or without administration of the drug or combination of drugs, or, in the case of a combination of drugs, the combined effect can be compared to the effect of administration of only one drug.

[0226] In this disclosure, the term "prevention" should be understood to include complete prevention, partial prevention, and reducing an individual's risk of developing the disease. The term should also be understood to include the administration of medication through early detection of disease symptoms or potential symptoms.

[0227] In this disclosure, Fc glycosylation refers to an important biological modification in immunoglobulin G (IgG), primarily occurring at the Asn297 site of the CH2 domain of the heavy chain. This glycosylation site is conserved and exhibits diverse modification forms, including N-acetylglucosamine (GlcNAc), fucose, sialic acid (Sia), and mannose (Man). Common types of Fc glycosylation include a core heptasaccharide structure, consisting of four N-acetylglucosamine (GlcNAc) and three mannose (Man) residues, and may also include fucose (Fuc) and sialic acid (Sia). Different Fc glycosylation modifications not only affect the immunogenicity of antibodies (such as ADCC, CDC, and ADCP), but are also closely related to their half-life, stability, and immunogenicity, as follows: Sialization: Changes in sialylation levels significantly affect the binding affinity of the Fc segment to the Fcγ receptor. High sialylation usually enhances the ADCC effect, while desialylation enhances the CDC effect. Fucosylation: Fucosylation affects the binding affinity of the Fc segment to FcγRIIIa, while defucosylation can enhance the ADCC effect. Galactosylation: Terminal galactosylation plays an important role in the conformational formation of the Fc segment, but core fucosylation alone does not significantly affect the conformation. Bibranched structure: Some Fc glycosylation modifications may form bibranched structures, which can affect their function in some cases.

[0228] In this disclosure, "enzyme-mediated site-specific conjugation" refers to the use of the high selectivity of enzymes to precisely link drug molecules to specific sites on antibody molecules, thereby generating ADCs with homogeneity and a defined drug loading number (DAR). Common methods include glycosylase methods (such as... Utilizing the glycosylation sites (such as the N297 site) in the Fc region of an antibody, most of the glycan chain is cleaved by an endoglucosidase, introducing an azide group, and then the drug is linked via click chemistry. Transglutaminase (MTGase): Recognizes specific glutamine residues on the antibody heavy chain and directly covalently links amine-containing drugs to that site. Sortase A: Recognizes specific short peptide sequences (LPXTG) and links drug molecules with glycine terminals, such as...

[0229] In this disclosure, complete remission (CR) is defined as: in an animal model, the disappearance of all target lesions and a reduction in the short axis of any pathological lymph node (whether target or non-target) to <10 mm; partial remission (PR) is defined as: in an animal model, the total diameter of target lesions is reduced by at least 30% relative to the baseline total diameter; disease progression (PD) is defined as: in an animal model, the total diameter of target lesions increases by at least 20% relative to the minimum total diameter of lesions in the study (if the baseline total is the minimum in the study, then the baseline total is included); and / or in addition to a relative increase of 20%, the total diameter must also achieve an absolute increase of at least 5 mm; and / or the appearance of one or more new lesions is also considered disease progression; stable disease (SD) is defined as: in an animal model, the reduction in diameter does not meet the PR criteria and the increase in diameter does not meet the PD criteria, relative to the minimum total diameter in the study.

[0230] II. Detailed Description of the Invention

[0231] Bispecific antibodies

[0232] In some embodiments, the Ab-[LP] provided in this disclosure (t) ] z The Ab structure shown targets EGFR and cMET, including a light chain-heavy chain dimer (first domain) targeting EGFR and a light chain-heavy chain dimer (second domain) targeting cMET.

[0233] In some implementations, the EGFR is a human-derived EGFR, and / or the cMET is a human-derived cMET. Accordingly, the first domain and / or the second domain includes a humanized heavy chain variable region.

[0234] In some embodiments, the heavy chain of the first domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is a variant of SEQ ID NO:1 or SEQ ID NO:1 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR2 is a variant of SEQ ID NO:2 or SEQ ID NO:2 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR3 is a variant of SEQ ID NO:3 or SEQ ID NO:3 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0235] In some embodiments, the heavy chain of the second domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is SEQ ID NO:4 or a variant of SEQ ID NO:4 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR2 is SEQ ID NO:5 or a variant of SEQ ID NO:5 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR3 is SEQ ID NO:6 or a variant of SEQ ID NO:6 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0236] In some embodiments, the first domain contains a heavy chain variable region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:7, and / or the second domain contains a heavy chain variable region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:8.

[0237] In some embodiments, the first domain further comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is SEQ ID NO:9 or a variant of SEQ ID NO:9 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is SEQ ID NO:10 or a variant of SEQ ID NO:10 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is SEQ ID NO:11 or a variant of SEQ ID NO:11 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0238] In some embodiments, the second structural domain comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is a variant of SEQ ID NO:12 or SEQ ID NO:12 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is a variant of SEQ ID NO:13 or SEQ ID NO:13 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is a variant of SEQ ID NO:14 or SEQ ID NO:14 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

[0239] In some embodiments, the first domain contains a light chain variable region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:15, and / or the second domain contains a light chain variable region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:16.

[0240] In some embodiments, the first structural domain contains the light chain variable region shown in SEQ ID NO:15, and / or the second structural domain contains the light chain variable region shown in SEQ ID NO:16.

[0241] In some embodiments, the first domain and / or the second domain contains a heavy chain constant region derived from a corresponding functional fragment of human immunoglobulin IgG1 or IgG4, or a variant having the same or similar function thereto; when the residues are numbered according to the EU numbering system, the heavy chain constant regions of the first domain and / or the second domain each have 1-7 substitution mutation sites, and the mutation sites of the heavy chain constant regions of the first domain and / or the second domain are different.

[0242] In some embodiments, when residues are numbered according to the EU index, the substitution mutation site is selected from amino acids at positions 350, 366, 368, 370, 399, 405, 407, or 409. For example, the substitution mutation includes K409R or F405L.

[0243] In some embodiments, the heavy chain constant region of the first structural domain has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% similarity to SEQ ID NO:17; and / or, the heavy chain constant region of the second structural domain has at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% similarity to SEQ ID NO:19.

[0244] In some embodiments, the first domain contains a first heavy chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:21; and / or, the second domain contains a second heavy chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:23.

[0245] In some embodiments, the first domain contains a light chain constant region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:18; and / or, the second domain contains a light chain constant region that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:20.

[0246] In some embodiments, the first domain contains a first light chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:22; and / or, the second domain contains a second light chain that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO:24.

[0247] In some embodiments, the bispecific antibody comprises (a) or (b):

[0248] (a) A first heavy chain and a first light chain forming a first dimer, a second heavy chain and a second light chain forming a second dimer, the first dimer and the second dimer being connected by disulfide bonds;

[0249] (b) A first heavy chain and a second light chain forming a first dimer, a second heavy chain and a first light chain forming a second dimer, the first dimer and the second dimer being connected by disulfide bonds.

[0250] connector

[0251] In some embodiments, the connector L provided in this disclosure has the following structure:

[0252] In some embodiments, the connector has the following structure:

[0253] Cytotoxin P

[0254] In some embodiments of this disclosure, the cytotoxin used is selected from one or more of the following DNA topoisomerase I inhibitors:

[0255] Coupling methods

[0256] In some embodiments of this disclosure, the Ab is linked to the remainder of the antibody-drug conjugate via an enzyme or a mutant thereof; wherein the enzyme or mutant thereof is an N-acetylglucosamine endonuclease or a mutant thereof.

[0257] Preferably, the N-acetylglucosamine endohydrolase is selected from Endo H, Endo D, Endo F2, Endo M, Endo Om, Endo S (Streptococcus pyogenes endoglycosidase-S), Endo F3 (Elizabethkingia miricola endoglycosidase-F3), Endo S2 (Endoglycosidase-S2), Endo Sd (Endoglycosidase-Sd), Endo CC (Endoglycosidase-CC), Endo CC1, and Endo CC2, or mutants thereof.

[0258] More preferably, the N-acetylglucosamine endohydrolase is selected from Endo F3, Endo S and Endo S2, or mutants thereof.

[0259] Traditional conjugation methods using lysine or cysteine ​​often result in random conjugation sites, high product heterogeneity, and unstable small molecule-antibody linkage, leading to off-target toxicity. In some embodiments of this disclosure, site-specific conjugation using enzymatic antibody glycans is expected to overcome the limitations of high heterogeneity in traditional conjugation methods, without requiring additional antibody engineering.

[0260] III. Examples

[0261] The present disclosure is further illustrated below with reference to embodiments. The description of specific exemplary embodiments of the present disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit the present disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the teachings of this specification. The exemplary embodiments were chosen and described in order to explain the specific principles of the present disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the present disclosure, as well as various different choices and variations.

[0262] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The abbreviations and full names of the reagents used are listed below.

[0263] Table 1. Abbreviation-Full Name Comparison Table

[0264] (I) Preparation of Linker-Supported Material (LP)

[0265] Example 1: Preparation of LP1

[0266] The LP1 structure is as follows:

[0267] The synthesis method is the same as that of LP-6 in Example 9 of WO2023232144A1.

[0268] Example 2 LP2

[0269] LP2 was purchased from Shanghai Xiyao Pharmaceutical Technology Co., Ltd., 20mg, purity: >95%.

[0270] Example 3: Preparation of LP3

[0271] Preparation of compound 3-1

[0272] 3-1 was synthesized using a solid-phase polypeptide synthesis method, following the synthesis procedure of LP-6-2 in patent (WO2023232144A1), except that Asp added in the first and third steps was replaced with Glu. The crude product after cleavage was prepared by Prep-HPLC. After lyophilization, 3-1 was obtained. 76 H 125 N9O 31 2+ [M+2H] 2+ MS(ESI) of 2: Calculated value 829.92, actual value 830.56.

[0273] Preparation of compound 3-2

[0274] Step A: Preparation of compound 3-2b

[0275] Weigh 3-1 (21.4 g, 12.9 mmol) and 3-2a (0.1-10 equivalents of 3-1) into a round-bottom flask, dissolve in DMF (450 mL), cool to 0-5 °C, stir for 5 min, add HATU to dissolve, and finally add 2,4,6-trimethylpyridine. React at 0-5 °C for 1 h, then return to room temperature and stir for another 1 h. After confirming complete reaction of the starting material, prepare by Prep-HPLC. Lyophilize to obtain 3-2b (31.5 g, yield 81.6%, white solid). C 144 H 223 N 24 O 49 3+

[0276] [M+3NH4] 3+ MS(ESI) of / 3: Calculated value 1024.19, actual value 1024.72.

[0277] Steps B and C: Preparation of Compound 3-2

[0278] In a reaction flask, 3-2b (38.5 g, 12.7 mmol) was dissolved in DMF (400 mL), followed by the addition of a certain amount of Pd / C. The gas was purged with hydrogen three times, and the reaction was carried out at room temperature for 2 h. HPLC analysis was performed on the sample. After the starting material was completely reacted, Pd / C was filtered off with diatomaceous earth, and the mixture was cooled to 0-5 °C in an ice bath. 3-2d (0.1-10 equivalents of 3-2b) and PyBop were added, and after stirring until dissolved, DIEA was added dropwise. The reaction was carried out at room temperature for 2 h, and HPLC analysis was performed on the sample. After the starting material was completely reacted, DEA was added, and the reaction was carried out at room temperature for 30 min. After the reaction was completely reacted, the pH was adjusted to 5-6, and the mixture was prepared by Prep-HPLC. After lyophilization, 3-2 (27.4 g, yield 61.6%, pink solid) was obtained. 161 H 232 Cl2F2N 29 O 53 3+

[0279] [M+H+2NH4] 3+ MS(ESI) of / 3: Calculated value 1175.86, actual value 1176.45.

[0280] For the synthesis of compound 3-2a, please refer to patent WO2023232144A1.

[0281] For the synthesis of compounds 3-2d, please refer to patent CN202211428194A.

[0282] Preparation of compound 3-3

[0283] At room temperature, compound 3-2 (200 mg, 0.057 mmol) and DMF (2 mL) were dissolved in a 4 mL centrifuge tube. Then, α (0.1-10 equivalents relative to 3-2) and certain amounts of HATU and N,N-isopropylethylamine were added. The resulting reaction solution was stirred at room temperature until the reaction was detected as complete by HPLC. The reaction solution was purified by semi-preparative HPLC to give compound 3-3 (185 mg, yield 83.9%, white solid). 175 H 248 Cl2F2N 28 O 64 4+ [M+4H] 4+ MS(ESI) of 4: Calculated value 968.41, actual value 969.02.

[0284] Preparation of compound LP3

[0285] At 0°C, compound 3-3 (150 mg, 0.0388 mmol) and Tris buffer (pH = 9.0, 2 mL) were added to a 15 mL centrifuge tube. After dissolution, a certain amount of triethylamine and 2-chloro-1,3-dimethylimidazoline chloride were added. The resulting reaction solution was stirred at 0°C until the reaction was detected as complete by HPLC. The reaction solution was purified by semi-preparative HPLC to give compound LP3 (96.3 mg, yield 64.5%, white solid). 175 H 251 Cl2F2N 30 O 63 3+ [M+H+2NH4] 3+ MS(ESI) of / 3: Calculated value 1296.22, actual value 1296.56.

[0286] Example 4LP4

[0287] The connector-load 4 (LP4) structure is as follows:

[0288] LP4 was purchased from MedChemExpress 20mg, purity >95%.

[0289] (II) Synthesis and Characterization of ADCs

[0290] This disclosure synthesizes an ADC, as shown in the table below:

[0291] Table 2. ADC Composition Comparison Table

[0292] All the antibodies mentioned above were produced by Shanghai Baiying Biotechnology Co., Ltd. The production, purification and identification methods of antibodies Ab001, Abaz and AbNC2 are based on US20230183358A1, and the production, purification and identification methods of antibody AbNC1 are based on the preparation method of Example 1 of patent CN 106856656 B.

[0293] General methods for ADC fabrication

[0294] ADCs are prepared based on the coupling reaction of antibodies and linkers-loaded materials catalyzed by immobilized glycosidases. Specifically, antibodies and linker-loaded materials are thoroughly mixed in 1× restriction enzyme buffer at an appropriate molar ratio (1:1 to 1:100), and then immobilized enzyme medium is added and mixed thoroughly. The immobilized restriction enzyme on the matrix of the immobilized enzyme medium catalyzes the coupling reaction between the antibody and the linker-loaded material. The coupling reaction in the mixed state is carried out at 4-40°C for 0.5-20 hours. After the reaction is completed, the mixture is centrifuged, and the immobilized coupling reaction mixture is purified by ultrafiltration or dialysis to remove unreacted drug intermediates, yielding the ADC.

[0295] General methods for characterizing ADCs

[0296] General Method 1 (HIC) for Dar detection:

[0297] General methods for HIC detection: using HIC Butyl-NP5 4.6×100mm 5μm Non-Porous column; 1.5M ammonium sulfate + 20mM phosphate buffer, pH 7.0 as mobile phase A; 20mM phosphate buffer (pH 7.0): isopropanol = (3-10):1 (v / v) as mobile phase B; flow rate 0.5-1.0mL / min; gradient method: phase B increases from 35% to 100% within 10min; 280nm is selected as the detection wavelength to detect the DAR distribution of ADC.

[0298] General Method 2 (MS) for Dar detection:

[0299] Using ACQUITY TM A Premier Protein SEC column was used; ammonium acetate and formic acid were added to a 30% acetonitrile aqueous solution as the mobile phase for isocratic analysis, and detection was performed using high-resolution mass spectrometry; the sample was reduced and then injected for analysis. The molecules before and after coupling were confirmed by comparing the deconvolutioned molecular weight with the theoretical molecular weight, and the DAR value of the ADC drug and the distribution of the drug on both chains were analyzed.

[0300] General method (SEC) for the detection of polymer aggregates

[0301] use G3000SWXL 7.8mm ID×30cm, 5μm column or BioCore TM SEC-300 5μm, 7.8×300mm column; 2×PBS+10%ACN as mobile phase; room temperature; isocratic; flow rate 1.0mL / min; run time 15min; 280nm as detection wavelength was selected to analyze and detect the high molecular weight aggregation degree of ADC drugs.

[0302] Example 5: Preparation and characterization of Ab001-LP1 and Ab001-LP3

[0303] Ab001-LP1 and Ab001-LP3 were prepared using the general method of ADC.

[0304] SEC testing results for ADC1 (Ab001-LP1) showed that the high molecular weight polymer in the ADC drug was <5%, and the ADC sample was mainly in monomer form at 7.89 min.

[0305] The SEC test results of ADC2 (Ab001-LP3) showed that the high molecular weight polymer in the ADC drug was <3%, and the ADC sample was mainly in monomer form at 8.91 min.

[0306] Example 6: Preparation and Characterization of ADC5 (Abaz-LP2)

[0307] ADC5 (Abaz-LP2) is chemically coupled to LP2 via Abaz. The reaction buffer for Abaz is not limited to phosphate buffer, acetate buffer, Tris buffer, pH 4–10.

[0308] A typical one-pot coupling experiment is as follows: First, antibody Abaz and TCEP are added at a ratio of 1:1 to 1:20, using a phosphate buffer system as an example, to obtain a reaction mixture. The reaction mixture is placed at 37°C for 0.5–20 h. LP2 is dissolved in an organic solvent (DMA, DMSO, DMF), and LP2 is introduced at a molar ratio of antibody Abaz to LP2 of 1:1 to 1:100. The reaction is continued at 4–40°C for 0.5–20 h. Then, cysteine ​​(molar ratio of antibody Abaz to cysteine ​​of 1:1 to 1:20) is added to consume excess LP2. Subsequently, DHAA (L-Dehydroascorbic acid) is added to make the molar ratio of antibody Abaz to DHAA 1:1 to 1:20 to oxidize excess thiol groups. ADC5 (Abaz-LP2) is purified, ultrafiltered, or dialyzed to remove unreacted drug intermediates.

[0309] Dar distribution was tested using the general method 2 (MS) for Dar detection. The results showed that the DAR value for light chains was 0.97, for heavy chains 4.96, and for the total DAR value 5.93. The degree of high molecular weight aggregation of the ADC drug was analyzed using the general method for detecting polymer aggregates (SEC). The results are shown in the figure. High molecular weight aggregates in the ADC drug were <2%, fragments <4%, and the ADC sample at 8.38 min was mainly in monomer form.

[0310] Example 7: Preparation and Characterization of ADC6 (AbSa-LP4)

[0311] The typical one-pot conjugation procedure for ADC6 (AbSa-LP4) is as follows: First, antibody AbSa and TCEP are added at a molar ratio of 1:1 to 1:20, using a phosphate buffer system as an example, to obtain the reaction mixture. The reaction mixture is reacted at 2–40°C for 0.5–20 h. The small molecule LP4 is dissolved in an organic solvent (DMA, DMSO, or DMF), and LP4 is introduced at a molar ratio of antibody AbSa to small molecule LP4 of 1:1 to 1:100. The reaction is continued at 4–40°C for 0.5–20 h. Then, cysteine ​​is added (antibody AbSa:cysteine ​​molar ratio of 1:1 to 1:20) to consume the excess small molecule. The ADC is purified, ultrafiltered, or dialyzed to remove unreacted drug intermediates.

[0312] use Protein BEH C4 Column The DAR distribution of the ADC drug ADC6 (AbSa-LP4) was detected using a 3.5 μm, 4.6 mm × 150 mm column (manufacturer: Waters, PN: 186004504). The results showed that the unconjugated cytotoxic antibody AbSa was <4%; the overall DAR value of the ADC drug was approximately 7.7.

[0313] The degree of high molecular weight aggregation of the ADC drug was analyzed using the general method for detecting high molecular weight aggregates (SEC). The results showed that the high molecular weight aggregates in the ADC drug were <3%, and the ADC sample existed primarily in monomer form after 8.50 min.

[0314] (III) Affinity of ADC, in vivo and in vitro efficacy tests

[0315] Example 8: ADC binding experiment at the cellular level

[0316] NCI-H1975 cells and HCC827 cells (human lung adenocarcinoma cell line) expressing EGFR and c-MET were prepared into 1×10⁻⁶ cells using FACS buffer (1% BSA + pH 7.4 PBS). 6 / ml of cell suspension, 100μL / well added to a 96-well V-type plate ( In 3894), centrifuge at 2000 rpm for 3 min, discard the supernatant, add different concentrations of the ADC and antibody drug to be tested, 100 μL / well, and incubate at 4℃ in the dark for 30 min. After centrifugation at 2000 rpm for 3 min and washing 3 times, add 1:500 diluted Anti-Human-IgG-Fc-AF647 (IR, 169-605-098) to each well of a 96-well plate, 100 μL / well, and incubate at 4℃ in the dark for 30 min. After centrifugation at 2000 rpm for 3 min and washing 3 times, resuspend in 100 μL of PBS in each well. Geometric mean fluorescence intensity was detected using a flow cytometer to calculate the antibody binding to ECGs expressed by EGFR and c-MET. 50 The values ​​are shown in Table 3.

[0317] Table 3. Binding efficiency and maximum binding amount of the tested drugs in NC-H1975 and HCC827 cells.

[0318] The results showed that all the detected ADC drugs had good cell binding activity in HCC827 and NCI-H1975 cell lines, and there was no significant difference between the ADC and its antibody, proving that the linker selected for the antibody in this disclosure did not affect the immune response activity of the antibody.

[0319] Example 9: ADC Internalization Experiment

[0320] NCI-H1975 and HCC827 cells expressing EGFR and c-MET were prepared into 4×10⁻⁶ cells using FACS buffer (2% FBS + pH 7.4 PBS) pre-cooled to 4°C. 6 Cell suspension at 1.1 mL / tube was added to a 15 mL centrifuge tube. The ADC to be tested was diluted to a final concentration of 400 nM using FACS buffer pre-chilled to 4°C. 1.1 mL of ADC solution was added to the centrifuge tube containing the cell suspension, mixed well, and incubated on moist ice for 1 h. After incubation, the tube was centrifuged at 2000 rpm for 3 min. The cells were washed twice with pre-chilled FACS buffer. The secondary antibody Anti-Human-IgG-Fc-AF647 (IR, 169-605-098) was diluted 1:500 using FACS buffer pre-chilled to 4°C. 2.2 mL of the secondary antibody dilution was added to the washed cell pellet, mixed well, resuspended, and incubated on moist ice for 30 min. Two separate 1.5 mL Eppendorf tubes were prepared, and 500 μL of 4 × 10⁴ ADC solution was added to each. 6Cell suspensions of cells / mL were centrifuged at 2000 rpm for 3 min at 4°C, and the supernatant was discarded. 1 mL of secondary antibody dilution buffer (NC group) and FACS buffer (BG group) were added to each, respectively, and the suspensions were resuspended and mixed well. The cells were then incubated on moist ice for 30 min. After incubation, the cells were centrifuged at 2000 rpm for 3 min. The supernatant was discarded, and the cells were washed twice with pre-chilled FACS buffer. Simultaneously, the NC and BG groups were washed. After washing, 2.2 mL of pre-chilled 4°C FACS buffer was added to a 15 mL centrifuge tube, and 1 mL of pre-chilled 4°C FACS buffer was added to a 1.5 mL Eppendorf tube. Cell suspensions from each sample were seeded at 100 μL / well in two wells into a 96-well U-plate. 3799), transfer the 96-well U-plate to a 37°C, 5% CO2 incubator and incubate for the corresponding durations (2h, 1.5h, 1.0h, 30min, 10min, 0min). After incubation, centrifuge the entire plate at 4°C and 2000rpm for 3min to pellet the cells. Discard the supernatant, add 100μL / well of pre-chilled 4°C, pH 2.5 acid wash buffer (0.15M NaCl + 0.1M Glycine) to each well, resuspend and mix, incubate on moist ice for 3min, centrifuge at 4°C and 2000rpm for 3min, and repeat the acid wash once. After acid washing, add 200μL / well of pre-chilled 4°C FACS buffer for washing, and repeat the washing twice. Additionally, take 100μL / well of cell suspension from each sample and transfer it to a 96-well U-plate, designated as the MAX group. The geometric mean fluorescence intensity was detected using a flow cytometer, and the endocytosis efficiency of the tested cells to the tested ADC was calculated. The endocytosis amount and internalization rate were calculated using the following formulas, and the results are shown in Table 4.

[0321] Internalization (I) (t) ):I (t) =MFI (Sample) -MFI (0min) ;

[0322] Internalization rate (Y) (t) ):Y (t) =[MFI (Sample) -MFI (0min) ] / [MFI (MAX) -MFI (NC) ×100%.

[0323] Table 4. Drug endocytosis rates in NCI-H1975 and HCC827 cells.

[0324] The results showed that all the tested ADC drugs had good endocytic activity in cell lines HCC827 and NCI-H1975, and there was no significant difference between the ADC and its antibody, proving that the provided ADC has excellent cytotoxic delivery effect.

[0325] Example 10: Cytotoxicity assay of ADC against different EGFR / cMET expression levels

[0326] The cell killing assay method is as follows:

[0327] a. Cell culture

[0328] (1) HCC827 (human lung adenocarcinoma cell line), NCI-H1993 (human lung adenocarcinoma cell line) and NCI-H661 (human lung large cell carcinoma cell line)

[0329] Cell suspensions were prepared using fresh cell culture medium containing 10% FBS, and 100 μL was added to each well of a 96-well black-walled transparent-bottom cell culture plate. Add 100 μL of PBS to 3603 and incubate at 37°C for 24 hours with 5% carbon dioxide.

[0330] (2) NCI-H1975 (human non-small cell lung cancer cell line) and NCI-H1650 (human non-small cell lung cancer cell line)

[0331] Cell suspensions were prepared using fresh cell culture medium containing 10% FBS, and 100 μL was added to each well of a 96-well low-adsorption U-plate. Add 100 μL of PBS to 7007, centrifuge at 200g for 5 min, and then incubate at 37°C for 24 hours with 5% carbon dioxide.

[0332] b. Preparation of different ADC samples

[0333] The ADC samples provided in Table 2 were serially diluted using cell culture medium to obtain ADC samples with concentrations of 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM, 0.00256 nM, 0.000512 nM, and 0.0001024 nM. Then, 100 μL of ADC solution was added to each well of the cell culture plate from step a, and the plates were incubated at 37°C with 5% CO2 for 6 days.

[0334] Table 5 Cell plating density and working drug concentration

[0335] The cells used in the experiment are as follows:

[0336] HCC827 (EGFR+++ / cMET++) was purchased from the Chinese Academy of Sciences Cell Bank;

[0337] NCI-H1993 (EGFR++ / cMET++) was purchased from ATCC;

[0338] NCI-H1650 (EGFR++ / cMET+) was purchased from the Chinese Academy of Sciences Cell Bank;

[0339] NCI-H1975 (EGFR++ / cMET++) was purchased from Nanjing Kebai;

[0340] NCI-H661 (EGFR weakly expressed / cMET-) was purchased from the Cell Bank of the Chinese Academy of Sciences.

[0341] The "+" sign indicates the expression levels of EGFR and cMET.

[0342] c. Cell treatment

[0343] (1) HCC827, NCI-H1993 and NCI-H661 cell lines

[0344] Remove the cells to be tested from the incubator, collect the cells, add 100 μL / well DMEM and 50 μL / well CTG, and mix well.

[0345] (2) NCI-H1975 and NCI-H1650 cell lines

[0346] Remove the cells from the incubator, collect the cells, add 100 μL / well of CellCounting-Lite 3D L Inferescent Cell Viability Assay, mix well, and then transfer 100 μL / well to a 96-well black-walled transparent cell culture plate. 3603)

[0347] d. Set the microplate reader program to a gain of 135, remove the cover from the black-walled transparent plate, place it according to the instrument's specified method, and read the data. The results are shown in Tables 6 and 7.

[0348] Table 6. Killing activity of ADCs against cells with different EGFR and cMET expression levels.

[0349] Table 7. Killing activity of ADCs against cells with different EGFR and cMET expression levels

[0350] The results showed that all the tested ADC drugs had strong target cell killing activity, and could kill HCC827, NCI-H1993, NCI-H1650 and NCI-H1975 cells expressing EGFR and cMET at low concentrations.

[0351] Example 11 NCI-H1975 CDX Experiment

[0352] Human lung adenocarcinoma cell line NCI-H1975 (10×10⁻⁶) 6 100 μL of PBS (with PBS as the buffer) was injected subcutaneously into the right scapular region of BALB / c Nude mice.

[0353] Nine days after inoculation, when the tumor volume is ~160mm 3 Mice were randomly divided into four groups: a solvent control group and an ADC group, an ADC1 (Ab001-LP1) 4 mg / kg group, an ADC3 (Abaz-LP1) 4 mg / kg group, and an ADC5 (Abaz-LP2) 4 mg / kg group, with five mice in each group. Mice were administered the drugs via tail vein injection on the day of grouping, while the control group received an equal volume of solvent. The drugs were administered once daily. Tumor growth was continuously monitored for 35 days after administration, with tumor volume and body weight measured twice weekly. Data were recorded using Excel statistical software: average values ​​were calculated as averages (avg); SEM values ​​were calculated as STDEV / SQRT (number of animals per group); and one-way ANOVA was used for statistical analysis. T / C and TGI values ​​were calculated using tumor volume. The calculation formulas are as follows:

[0354] T / C% = T RTV / C RTV ×100% (T) RTV Treatment group RTV; C RTV The relative tumor volume (RTV) was calculated based on the results of tumor measurements using the solvent control group. The formula is: RTV = V t / V0, where V0 is the average tumor volume measured at the time of grouping (i.e., D0), V t T represents the average tumor volume at a given measurement. RTV With C RTV Take data from the same day.

[0355] TGI (%): TGI (%) = [1 – mean tumor volume in the treatment group / mean tumor volume in the control group] × 100%.

[0356] The tumor volume on day 23 after drug administration was analyzed, and the results are shown in Table 8.

[0357] Table 8. Efficacy of ADC on NCI-H1975 CDX xenografts in tumor-bearing mice.

[0358] The results showed that ADC1 (Ab001-LP1) exhibited excellent tumor-suppressing effects.

[0359] Example 11: PDX trial efficacy study of osimertinib-resistant NSCLC lung cancer

[0360] The 12 non-small cell lung cancer PDX models used in this example were all derived from body fluid samples or tumor tissues from clinical treatment or surgery, and had been passaged in animals a limited number of times to maintain tumor heterogeneity. Approximately 30 mm³ of PDX was subcutaneously injected into the right scapula of each NU / NU, NOD / SCID, or NCG mouse. 3 Tumor tissue was used to create a model, and when the average tumor volume reached a suitable range (140-200 mm), the model was established. 3 Animals were randomly divided into 3-5 groups. PDX-1 and PDX-6 models had 3 animals per group, while other models had 2 animals per group. The drug was administered via tail vein on the same day, while the control group received the same volume of solvent. All animals in all groups received the drug at body weight (10 μL / g). The number of groups, dosage, and frequency of administration varied depending on the actual situation of the model; detailed protocols are shown in Table 9. After the first administration, tumor growth was continuously observed, with tumor volume and body weight measured twice weekly, and data recorded. Each animal reached the tumor volume endpoint (TV > 2000 mm²). 3 Animals were sacrificed at the designated time. For some PDX models with slow-growing tumors, the observation period was extended. Data were recorded using Excel statistical software: the mean was calculated as average; SEM values ​​were calculated as STDEV / SQRT (number of animals per group); graphs were created using GraphPad Prism software; TGI and tumor regression percentage were calculated using tumor volume. The calculation formulas are as follows:

[0361] TGI(%) = [1 – mean tumor volume in the treatment group / mean tumor volume in the control group] × 100%. The tumor data used in the calculation will be determined based on the specific circumstances of the model.

[0362] Tumor regression percentage: % regression = (T) 最佳 -T 初始 ) / T 初始 ×100%, where T 最佳 T is the smallest mean tumor volume after the last dose in the treatment group. 初始 This represents the average tumor volume at the time of grouping into different treatment groups. The results of the 12 PDX models after drug administration are shown in Table 0.

[0363] Table 9. Experimental Scheme for the PDX Trial Model

[0364] Table 10. Efficacy of ADC in 12 PDX xenografts

[0365] The results showed that in 12 non-small cell lung cancer PDX models, this embodiment introduced clinical evaluation methods into the PDX model, and used the "tumor regression ratio" parameter to more comprehensively simulate and reflect potential clinical outcomes (such as complete response rate CR, partial response rate PR, stable disease SD, and disease progression PD). Ab001-LP1 and Ab001-LP3 showed significant anti-tumor activity in this embodiment (PR+CR was better), which was superior to the other two types of ADCs.

[0366] In summary, the present disclosure achieves the following beneficial effects:

[0367] (1) This disclosure uses enzymatic site-directed coupling technology to prepare a novel EGFR-cMET dual-target ADC drug containing a DNA topoisomerase I inhibitor, overcoming the limitation of high heterogeneity in traditional coupling and requiring no additional antibody engineering modification.

[0368] (2) The ADC prepared by this disclosure using the bispecific antibody Ab001 is stable, homogeneous, and simple to prepare, and can be linearly scaled up at low cost.

[0369] (3) By combining different linkers-loaders and different antibodies, this disclosure designs a series of different ADCs, among which the combined efficacy of ADC1 (Ab001-LP1) and ADC2 (Ab001-LP3) is significantly better than that of the positive control.

[0370] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this disclosure.

[0371] sequence list

[0372] References

[0373] Chmielecki,JuLiann,Tony Mok,Yi-Long Wu,Ji-Youn Han,Myung-Ju Ahn,Suresh S.Ramalingam,Thomas John,et al.2023.“Analysis of Acquired Resistance Mechanisms to Osimertinib in Patients with EGFR-Mutated Advanced Non-Small Cell Lung Cancer from the AURA3 Trial.”Nature Communications 14(1):1071.https: / / doi.org / 10.1038 / s41467-023-35962-x.

[0374] Engelman,Jeffrey A.,Kreshnik ZejnuLlahu,Tetsuya Mitsudomi,YoungchuL Song,Courtney Hyland,Joon Oh Park,Neal Lindeman,et al.2007.“MET Amplification Leads to Gefitinib Resistance in Lung Cancer by Activating ERBB3 Signaling.”Science(New York,N.Y.)316(5827):1039–43.https: / / doi.org / 10.1126 / science.1141478.

[0375] Flynn,Patrick,Smruthi Suryaprakash,Dan Grossman,Val Panier,and John Wu.2024.“The Antibody-Drug Conjugate Landscape.”Nature Reviews.Drug Discovery 23(8):577–78.https: / / doi.org / 10.1038 / d41573-024-00064-w.

[0376] Saw,Stephanie P.L.,Xiuning Le,Lizza E.L.Hendriks,and Jordi Remon.2024.“New Treatment Options for Patients With Oncogene-Addicted Non-Small Cell Lung Cancer Focusing on EGFR-Mutant Tumors.”American Society of Clinical Oncology Educational Book.American Society of Clinical Oncology.Annual Meeting 44(3):e432516.https: / / doi.org / 10.1200 / EDBK_432516.

[0377] Sierra,J.Rafael,and Ming-Sound Tsao.2011.“C-MET as a Potential Therapeutic Target and Biomarker in Cancer.”Therapeutic Advances in Medical Oncology 3(1Suppl):

[0378] S21-35.https: / / doi.org / 10.1177 / 1758834011422557.

[0379] Uribe,Mary Luz,Ilaria Marrocco,and Yosef Yarden.2021.“EGFR in Cancer:Signaling Mechanisms,Drugs,and Acquired Resistance.”Cancers 13(11):2748.https: / / doi.org / 10.3390 / cancers13112748.

Claims

1. An antibody-drug conjugate having Ab-[LP] (t) ] z The structure; in, Ab is a bispecific antibody targeting EGFR and cMET or its antigen-binding fragment, including a first domain targeting EGFR and a second domain targeting cMET: Wherein, the first structural domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is a variant of SEQ ID NO:1 or SEQ ID NO:1 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR2 is a variant of SEQ ID NO:2 or SEQ ID NO:2 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR3 is a variant of SEQ ID NO:3 or SEQ ID NO:3 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; and / or, The second domain comprises HCDR1, HCDR2, and HCDR3; the amino acid sequence of HCDR1 is SEQ ID NO:4 or a variant of SEQ ID NO:4 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR2 is SEQ ID NO:5 or a variant of SEQ ID NO:5 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of HCDR3 is SEQ ID NO:6 or a variant of SEQ ID NO:6 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function. P is a cytotoxin; t is any integer greater than 1; Ab and P are connected by connector L, which includes the structure shown in equation (I); Equation (I) is: in, In equation (I), the * terminal is connected to Ab; the # terminal is connected to P. R 0 C 1-10 alkyl; D has the following structure: In equation (I-1), * represents the end connected to Ab; ** represents the sum (I) The end of the part connected to the -NH-; R 1 R 2 R 3 R 4 R 5 R 6 Each is independently selected from H and C. 1-6 alkoxy or OH; and satisfy the following condition: R 1 and R 2 Different, R 3 and R 4 Differences and R 5 and R 6 different; R 7 It is hydrogen or α-L-fucosylate; Q has the following structure: In this case, terminal (I-2)# is connected to P; m is any integer selected from 1 to 8; n is any integer selected from 1 to 8; q is any integer selected from 1 to 4; p is any integer selected from 2 to 20; i is any integer selected from 1 to 20; j is any integer selected from 1 to 20; z is any integer selected from 1 to 8.

2. The antibody-drug conjugate as described in claim 1, wherein, The EGFR is a human-derived EGFR, and / or the cMET is a human-derived cMET.

3. The antibody-drug conjugate as described in claim 1 or 2, wherein, The first domain and / or the second domain contain humanized heavy chain variable regions.

4. The antibody-drug conjugate according to any one of claims 1-3, wherein, The first domain contains a heavy chain variable region that is at least about 85% or 100% identical to SEQ ID NO:7, and / or the second domain contains a heavy chain variable region that is at least about 85% or 100% identical to SEQ ID NO:

8.

5. The antibody-drug conjugate according to any one of claims 1-4, wherein, The first structural domain contains the heavy chain variable region shown in SEQ ID NO:7, and / or the second structural domain contains the heavy chain variable region shown in SEQ ID NO:

8.

6. The conjugate according to any one of claims 1-5, wherein, The first domain further comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is a variant of SEQ ID NO:9 or SEQ ID NO:9 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is a variant of SEQ ID NO:10 or SEQ ID NO:10 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is a variant of SEQ ID NO:11 or SEQ ID NO:11 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; and / or, The second structural domain comprises LCDR1, LCDR2, and LCDR3; the amino acid sequence of LCDR1 is a variant of SEQ ID NO:12 or SEQ ID NO:12 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR2 is a variant of SEQ ID NO:13 or SEQ ID NO:13 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function; the amino acid sequence of LCDR3 is a variant of SEQ ID NO:14 or SEQ ID NO:14 with 1-3 amino acids substituted, deleted, or added, and having the same or similar function.

7. The conjugate according to any one of claims 1-6, wherein, The first domain contains a light chain variable region having at least about 85% or 100% similarity to SEQ ID NO:15, and / or the second domain contains a light chain variable region having at least about 85% or 100% similarity to SEQ ID NO:

16.

8. The antibody-drug conjugate according to any one of claims 1-7, wherein, The first structural domain contains the light chain variable region shown in SEQ ID NO:15, and / or the second structural domain contains the light chain variable region shown in SEQ ID NO:

16.

9. The antibody-drug conjugate according to any one of claims 1-8, wherein, The first domain and / or the second domain contain heavy chain constant regions of corresponding functional fragments derived from human immunoglobulin IgG1 or IgG4 or variants having the same or similar functions. The heavy chain constant regions of the first domain and / or the second domain each contain 1-7 substitution mutation sites, and the mutation sites in the heavy chain constant regions of the first domain and / or the second domain are different.

10. The antibody-drug conjugate as described in claim 9, wherein, When residues are numbered according to EU numbering rules, the substitution mutation site is selected from amino acids at positions 350, 366, 368, 370, 399, 405, 407, or 409. Optionally, the substitution mutation includes K409R or F405L.

11. The antibody-drug conjugate according to any one of claims 1-10, wherein, The heavy chain constant region of the first structural domain has at least about 85% or 100% similarity to SEQ ID NO:17; and / or the heavy chain constant region of the second structural domain has at least about 85% or 100% similarity to SEQ ID NO:19; or, The first domain contains a light chain constant region that is at least about 85% or 100% identical to SEQ ID NO:18; and / or the second domain contains a light chain constant region that is at least about 85% or 100% identical to SEQ ID NO:

20.

12. The antibody-drug conjugate according to any one of claims 1-11, wherein, The heavy chain constant region of the first structural domain contains the amino acid sequence shown in SEQ ID NO:17, and / or the heavy chain constant region of the second structural domain contains the amino acid sequence shown in SEQ ID NO:19; The first structural domain contains the light chain constant region shown in SEQ ID NO:18, and / or the second structural domain contains the light chain constant region shown in SEQ ID NO:

20.

13. The antibody-drug conjugate according to any one of claims 1-12, wherein, The first domain contains a first heavy chain that is at least about 85% or 100% identical to SEQ ID NO:21; and / or the second domain contains a second heavy chain that is at least about 85% or 100% identical to SEQ ID NO:23; and / or, The first domain contains a first light chain that is at least about 85% or 100% identical to SEQ ID NO:22; and / or the second domain contains a second light chain that is at least about 85% or 100% identical to SEQ ID NO:

24.

14. The antibody-drug conjugate according to any one of claims 1-13, wherein, The first structural domain contains a first heavy chain as shown in SEQ ID NO:21, and / or the second structural domain contains a second heavy chain as shown in SEQ ID NO:23; The first structural domain contains a first light chain as shown in SEQ ID NO:22; and / or the second structural domain contains a second light chain as shown in SEQ ID NO:

24.

15. The antibody-drug conjugate according to any one of claims 1-14, wherein, The antibody conjugate includes any one or more of the following features: P is a topoisomerase inhibitor; t is 2; R 0 C 1-3 alkyl.

16. The antibody-drug conjugate according to any one of claims 1-15, wherein, In the formula (I-1), the -NHC(O)CH2- at the * end is part of the amino acid in the Fc region of the antibody; Preferably, the -NHC(O)CH2- at the * end is part of the asparagine in the Fc region of the antibody.

17. The antibody-drug conjugate according to any one of claims 1-16, wherein, In the formula (I-1) R 1 R 2 R 3 R 4 R 5 and R 6 Each is independently selected from H, methoxy, or OH; and / or R 7 It is α-L-fucosylate; Preferably, R 1 R 2 R 3 R 4 R 5 and R 6 Each is independently selected from H or OH.

18. The antibody-drug conjugate according to any one of claims 1-17, wherein, Equation (I) contains any one or more of the following features: m is any integer selected from 1 to 5, preferably 3; n is any integer selected from 1 to 5, preferably 3; i is any integer selected from 1 to 10, preferably 4; j is any integer selected from 2 to 16, preferably 12; and / or Equation (I-2) contains any one or a combination of two of the following features: q is 1 or 2; p is any integer selected from 2 to 10, preferably 2; Optionally, n is 2 or 3; z is 1, 2 or 4, preferably 2.

19. The antibody-drug conjugate according to any one of claims 1-18, wherein, D has the following structure:

20. The antibody-drug conjugate according to any one of claims 1-19, wherein, The structure of the connector L is shown below:

21. The antibody-drug conjugate according to any one of claims 1-20, wherein, The structure of the connector is shown below:

22. The antibody-drug conjugate according to claims 1-21, wherein, Each P is independently selected from DNA topoisomerase I inhibitors; Preferably, the DNA topoisomerase I inhibitor includes camptothecin inhibitors and non-camptothecin inhibitors; Preferably, the DNA topoisomerase I inhibitor is selected from the following structures:

23. The antibody-drug conjugate according to any one of claims 1-22, wherein, The LP (t) It has any of the following structures:

24. The antibody-drug conjugate according to any one of claims 1-23, wherein, The antibody-drug conjugate has the structure shown in either (III-1) or (III-2) below: Among them, R 7 It is hydrogen or α-L-fucosylate; Ab is a bispecific antibody comprising a first domain targeting EGFR and a second domain targeting cMET; the first domain contains a first heavy chain as shown in SEQ ID NO:21; the second domain contains a second heavy chain as shown in SEQ ID NO:23; the first domain contains a first light chain as shown in SEQ ID NO:22; and the second domain contains a second light chain as shown in SEQ ID NO:

24.

25. The antibody-drug conjugate according to any one of claims 1-24, wherein, The antibody-drug conjugate has the structure shown in (III-1): Among them, R 7 It is hydrogen or α-L-fucosylate; Ab is a bispecific antibody comprising a first domain targeting EGFR and a second domain targeting cMET; the first domain contains a first heavy chain as shown in SEQ ID NO:21 and a first light chain as shown in SEQ ID NO:22; the second domain contains a second heavy chain as shown in SEQ ID NO:23 and a second light chain as shown in SEQ ID NO:

24.

26. The antibody-drug conjugate according to any one of claims 1-24, wherein, The antibody conjugate has the structure shown in (III-2): Among them, R 7 It is hydrogen or α-L-fucosylate; Ab is a bispecific antibody comprising a first domain targeting EGFR and a second domain targeting cMET. The first domain contains a first heavy chain as shown in SEQ ID NO:21 and a first light chain as shown in SEQ ID NO:22, and the second domain contains a second heavy chain as shown in SEQ ID NO:23 and a second light chain as shown in SEQ ID NO:

24.

27. The antibody-drug conjugate according to any one of claims 1-26, wherein, The Ab is linked to the remainder of the antibody-drug conjugate via an enzyme or a mutant thereof; The enzyme or its mutant is N-acetylglucosamine endonuclease or its mutant; Preferably, the N-acetylglucosamine endohydrolase is selected from Endo H, Endo D, Endo F2, Endo M, Endo Om, Endo S, Endo F3, Endo S2, Endo Sd, Endo CC, Endo CC1 and Endo CC2, or mutants thereof.

28. A pharmaceutical composition comprising the antibody-drug conjugate according to any one of claims 1-27; Preferably, it also includes at least one pharmaceutically acceptable carrier.

29. Use of the antibody-drug conjugate of any one of claims 1-27, or the pharmaceutical composition of claim 28, in the preparation of a drug for inhibiting tumor growth or producing tumor regression; Preferably, the tumor contains one or both of EGFR-positive cells and c-MET-positive cells; and / or The tumor originated from a patient with non-small cell lung cancer.

30. A method of treating or preventing cancer, comprising administering to a subject in need a therapeutically effective amount of the antibody-drug conjugate of any one of claims 1-27 or the pharmaceutical composition of claim 28; Preferably, the cancer is characterized by one or both of EGFR positivity and c-MET positivity; and / or the cancer includes non-small cell lung cancer.

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