Antibody-drug conjugates with high drug loading and uses thereof

Abstract

The invention relates to an antibody-drug conjugate with high drug loading and an application of the antibody-drug conjugate. The antibody-drug conjugate has a high drug-antibody ratio (DAR) comprising an antibody and a drug conjugation moiety, the constant region of the antibody comprising a reactive glutamine. The invention also provides a preparation method and application of the antibody-drug conjugate with high drug loading.

Description

Antibody-drug conjugates with high drug loading and uses thereof Technical Field

[0001] The present invention relates to an antibody-drug conjugate with high drug loading, a preparation method thereof and use thereof. Background Art

[0002] Antibody drug conjugates (ADCs) are composed of antigen-targeting monoclonal antibodies and small molecule cytotoxic drugs connected via a linker. They combine the lethality of traditional small molecule chemotherapy with the tumor-targeting properties of antibody drugs. ADCs consist of three components: an antibody that selectively recognizes antigens on the surface of cancer cells, a payload that kills cancer cells, and a linker that connects the antibody and payload. After recognizing antigens on the surface of tumor cells, the ADC enters the cell via endocytosis. The linker is hydrolyzed by proteases in lysosomes, releasing the payload in a biologically active form and causing cancer cell death. The amount of payload that enters the cell is determined by the number of surface antigens per cell, the ADC drug-to-payload ratio (DAR), and the time required for the antigen to return to the cell surface. The payload then permeates the cytoplasm and exerts an inhibitory effect on surrounding tumor cells, known as the bystander effect.

[0003] ADC R&D technology continues to evolve. ADC research dates back to the 1980s, but it wasn't until 2000 that the first-generation ADC, represented by Mylotarg, was successfully marketed. Due to poor tumor targeting, low localization rates, and the unstable linker used, the drug exhibited significant toxicity. Ultimately, due to severe liver damage and poor efficacy, Pfizer voluntarily withdrew the drug from the market in 2010, only reinstating it in 2017. A second-generation ADC, represented by Kadcyla, received FDA approval in February 2013 for the treatment of HER2-positive breast cancer patients who have previously failed trastuzumab and taxane chemotherapy. Kadcyla is a HER2-targeted antibody-drug conjugate consisting of the humanized anti-HER2 IgG1 antibody trastuzumab and the microtubule inhibitor DM1 (a maytansine derivative), covalently linked via a stable thioether linker (MCC). Improvements in antibody technology for second-generation ADCs have enhanced tumor cell targeting and reduced cross-reactivity with healthy cells. However, the conjugation sites of the payloads in ADCs remain nonspecific, leading to differences in the pharmacokinetics of ADCs with different numbers of payloads attached. Third-generation ADCs are developing toward specific conjugation sites and uniform DAR values. Although no ADC has yet been successfully marketed, many site-specific conjugated ADCs have demonstrated advantages in clinical practice, such as reduced toxicity, the absence of unbound monoclonal antibodies, significantly improved stability and pharmacokinetics, and enhanced efficacy.

[0004] Among the site-specific conjugation methods for antibodies and small molecule drugs, enzyme-catalyzed conjugation is a type of site-specific conjugation method that can achieve site-specific conjugation of ADCs. Enzyme-catalyzed conjugation involves using genetic engineering techniques to create specific amino acid sequences in antibodies that are specifically recognized by a certain enzyme. This enzyme then leverages the specific action of this amino acid sequence to modify specific amino acid residues within the antibody, creating active reaction sites that react with the linker to achieve site-specific conjugation. Key enzymes include transglutaminases, glycosyltransferases, and sortase A. Microbial transglutaminase (mTGase) can also catalyze transpeptidation reactions, primarily catalyzing the transfer of acyl groups between amides on glutamines in proteins or peptides and other primary amines, participating in the cross-linking of proteins or small molecules to form isopeptide bonds. Therefore, mTGase can covalently conjugate the linker-payload to a specific glutamine (Q295) in the antibody, forming a site-specific ADC with a drug-to-antibody ratio (DAR) of 2.0. For example, the antibody-drug conjugate DP303c prepared in Chinese patent application CN114901308A is an antibody-drug conjugate comprising a monoclonal IgG1 antibody (DP001) targeting HER2 and a cleavable LND1002 (linker-toxin) site-specifically conjugated to glutamine 295 in the constant region of each heavy chain of DP001. DP303c has a DAR (drug-to-antibody ratio) of about 1.8 to about 2.2, such as about 1.8, 1.9, 2.0, 2.1, or 2.2.

[0005] DS-8201a (trade name: Enhertu, generic name: Trastuzumab Deruxtecan), an antibody-drug conjugate jointly developed and commercialized by Daiichi Sankyo and AstraZeneca, has demonstrated breakthrough therapeutic potential in patients with resistance to various HER2-ADC drugs, making DS-8201a a blockbuster drug in recent years. The success of DS-8201a has led to the development of a growing number of ADC drugs with low toxicity and high DAR values ​​compared to small molecule toxins. Currently, ADC drugs prepared using mTGase enzymatic conjugation have a DAR of 2.0, which limits the number of small molecule toxins that can be conjugated. Currently, molecules with high DAR values ​​often utilize non-site-specific chemical conjugation methods, which also have certain drawbacks in terms of stability.

[0006] Summary of the Invention

[0007] In a first aspect, the present application relates to an antibody-drug conjugate with a high drug load, comprising an antibody or an antigen-binding fragment thereof and a drug-conjugated portion, wherein the constant region of the antibody or antigen-binding fragment thereof comprises one or more reactive glutamines, wherein the reactive glutamines are selected from the group consisting of: (i) a glutamine in a Q tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG and LLQGA located at the carboxyl terminus of a light chain, a heavy chain, or both a light chain and a heavy chain; (ii) a glutamine selected from the group consisting of 3QQSGA, LQSGA, EEQYGA, EEQYQSG and LLQGA located at the carboxyl terminus of a heavy chain; 54L-355Q-356G (Q438), heavy chain 445Q-G446, heavy chain 117L-118Q-119G-120G, heavy chain 310L-Q311-312G, heavy chain 399L-400Q-401G, heavy chain 268L-269Q-270G, heavy chain 252Q-253G, heavy chain 444Q-445G-446A, heavy chain 283L-V284-285Q-286G, heavy chain 285L-286Q, heavy chain 340L-34 1L-Q342-343G, heavy chain 384L-385Q-386G-387G, heavy chain 268L-269Q-270G-271G, heavy chain 325L-326Q, heavy chain 329Q-330G, heavy chain 360L-361Q-362G, heavy chain 384Q-G385-386G, heavy chain 134L-135Q-136G, reactive glutamine in heavy chain 282Q; (iii) light chain 126L-127Q-G (iv) endogenous glutamine at Q295 of the heavy chain; and (v) glutamine in the Q-tagged LQSG replacing G200-L201-S202 of the light chain, and glutamine in the Q-tagged LQSG inserted after T135 of the heavy chain.

[0008] In some embodiments, the antibody-drug conjugate comprises an antibody or an antigen-binding fragment thereof and a drug-conjugated portion, wherein the constant region of the antibody comprises two or more reactive glutamines, wherein the reactive glutamine is located at the carboxyl terminus of the light chain or heavy chain, based on the EU numbering convention, and is selected from the glutamine in the Q-tag GGLQSGA, LQSGA or GGGLQSGA, the glutamine in the Q-tag LQSG replacing the light chain G200-L201-S202, the glutamine in the Q-tag LQSG inserted after T135 of the heavy chain, the glutamine in the heavy chain S354L-R355Q-E356G (Q438), the heavy chain P445Q-G446G, the heavy chain H268L-E269Q-D270G, the heavy chain M252Q-I253G, the heavy chain S444Q-P446G, the heavy chain 5G-G446A, heavy chain V282Q, heavy chain E283L-V284V-H285Q-N286G, heavy chain H285-N286Q, heavy chain K340L-G341L-Q342Q-P343G, heavy chain N384L-G385Q-Q386G-P387G, heavy chain H268L-E269Q-D270G-P271G, heavy chain Q The drug conjugate moiety is conjugated to the antibody through a reactive glutamine site converted by amino acid modification of 438I, heavy chain Q295A, heavy chain T109L-V110Q-A111G-A112G, or light chain K126L-S127Q-G128G-T129G, or an endogenous glutamine in heavy chain Q295-V296-N297.

[0009] In a second aspect, the present application relates to an engineered antibody or antigen-binding fragment thereof, wherein the constant region of the antibody or antigen-binding fragment thereof comprises one or more reactive glutamines, wherein the reactive glutamines are selected from the group consisting of: (i) a glutamine in a Q tag selected from the group consisting of GGLQSGA, LQSGA, EEQYGA, EEQYQSG and LLQGA located at the carboxyl terminus of the light chain, the heavy chain, or both the light chain and the heavy chain; (ii) a glutamine selected from the group consisting of 354L-355Q-356G ( Q438), heavy chain 445Q-G446, heavy chain 117L-118Q-119G-120G, heavy chain 310L-Q311-312G, heavy chain 399L-400Q-401G, heavy chain 268L-269Q-270G, heavy chain 252Q-253G, heavy chain 444Q-445G-446A, heavy chain 283L-V284-285Q-286G, heavy chain 285L-286Q, heavy chain 340L-341L-Q342-3 43G, heavy chain 384L-385Q-386G-387G, heavy chain 268L-269Q-270G-271G, heavy chain 325L-326Q, heavy chain 329Q-330G, heavy chain 360L-361Q-362G, heavy chain 384Q-G385-386G, heavy chain 134L-135Q-136G, reactive glutamine in heavy chain 282Q; (iii) light chain 126L-127Q-G128-1 29G, light chain 205L-206Q-207G-208G, light chain 109L-110Q-111G-112G, light chain 199L-200Q-201S-202G converted to reactive glutamine by amino acid modification; (iv) endogenous glutamine at Q295 of the heavy chain; and (v) glutamine in the Q-tagged LQSG replacing G200-L201-S202 of the light chain, and glutamine in the Q-tagged LQSG inserted after T135 of the heavy chain.

[0010] In the above first and second aspects:

[0011] In some embodiments, the reactive glutamine is selected from: (i) a glutamine in a Q-containing tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA located at the carboxyl terminus of the light chain, the heavy chain, or both the light chain and the heavy chain; (ii) a glutamine selected from heavy chain S354L-R355Q-E356G (Q438), heavy chain P445Q-G446 (also expressed as P445Q-G446G), heavy chain S117L-A118Q-S119G-T120G, heavy chain H310L-Q311-D312G (also expressed as H310L-Q311Q-D312G), heavy chain D399L-S400Q-D401G, heavy chain H268L-E269Q- D270G, heavy chain M252Q-I253G, heavy chain S444Q-P445G-G446A, heavy chain E283L-V284-H285Q-N286G (also expressed as E283L-V284V-H285Q-N286G), heavy chain H285L-N286Q, heavy chain K340L-G341L-Q342-P343G (also expressed as K340L-G341L -Q342Q-P343G), heavy chain N384L-G385Q-Q386G-P387G, heavy chain H268L-E269Q-D270G-P271G, heavy chain N325L-K326Q, heavy chain P329Q-A330G, heavy chain K360L-N361Q-Q362G, heavy chain N384Q-G385-Q386G (can also be expressed as N384Q-G385G-Q 386G), heavy chain S134L-T135Q-S136G, heavy chain V282Q converted to reactive glutamine by amino acid modification; (iii) Q295 endogenous glutamine (also expressed as Q295Q); (iv) light chain K126L-S127Q-G128-T129G (also expressed as K126L-S127Q-G128G-T129G), light chain V205L-T 206Q-K207G-S208G, light chain T109L-V110Q-A111G-A112G, light chain Q199L-G200Q-L201S-S202G converted into reactive glutamine by amino acid modification; and (v) glutamine in the Q-tagged LQSG of the light chain G200-L201-S202 is substituted, and glutamine in the Q-tagged LQSG inserted after T135 of the heavy chain is inserted.

[0012] In some embodiments, the heavy chain Q295 endogenous glutamine is the endogenous glutamine in the heavy chain Q295-V296-N297.

[0013] In some embodiments, the constant region of the antibody or antigen-binding fragment thereof comprises one or more reactive glutamines, for example, two, three, four, five, six, seven, eight, nine, or ten reactive glutamines. In some embodiments, the constant region of the antibody or antigen-binding fragment thereof comprises one or more reactive glutamines, for example, two, four, six, eight, ten, twelve, fourteen, or sixteen reactive glutamines.

[0014] In some embodiments, the reactive glutamine comprises endogenous glutamine at Q295 of the heavy chain and a reactive glutamine selected from one or more of the following: (1) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA at the carboxyl terminus of the light chain; (2) a glutamine in a Q-tag LQSG replacing G200-L201-S202 of the light chain; (3) a glutamine in a Q-tag LQSG inserted after T135 of the heavy chain; and (4) a reactive glutamine selected from 354L-355Q-356G (Q438) of the heavy chain, 445Q-G446 of the heavy chain, and 268L-269Q-270G-271G of the heavy chain that is converted by amino acid modification. In a further embodiment, the endogenous glutamine at Q295 of the heavy chain is the endogenous glutamine at Q295-V296-N297 of the heavy chain. In a further embodiment, the Q-tag at the carboxyl terminus of the light chain is GGLQSGA.

[0015] In some embodiments, the reactive glutamine comprises endogenous glutamine at Q295 of the heavy chain and a reactive glutamine selected from one or more of the following: (1) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA at the carboxyl terminus of the light chain; (2) a glutamine in a Q-tag LQSG replacing G200-L201-S202 of the light chain; (3) a glutamine in a Q-tag LQSG inserted after T135 of the heavy chain; and (4) a reactive glutamine selected from S354L-R355Q-E356G (Q438) of the heavy chain, P445Q-G446G of the heavy chain, and H268L-E269Q-D270G-P271G of the heavy chain that is converted by amino acid modification. In a further embodiment, the endogenous glutamine at Q295 of the heavy chain is the endogenous glutamine at Q295-V296-N297 of the heavy chain. In a further embodiment, the Q-tag at the carboxyl terminus of the light chain is GGLQSGA.

[0016] In some embodiments, the heavy chain Q295 endogenous glutamine is the endogenous glutamine in the heavy chain Q295-V296-N297.

[0017] In some embodiments, the Q-containing tag at the carboxyl terminus of the light chain is GGLQSGA.

[0018] In some embodiments, the reactive glutamine comprises endogenous glutamine in Q295-V296-N297 of the heavy chain and a reactive glutamine selected from one or more of the following: (1) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA located at the carboxyl terminus of the light chain; (2) a glutamine in a Q-tag LQSG replacing G200-L201-S202 of the light chain; (3) a glutamine in a Q-tag LQSG inserted after T135 of the heavy chain; and (4) a reactive glutamine selected from S354L-R355Q-E356G (Q438) of the heavy chain, P445Q-G446G of the heavy chain, and H268L-E269Q-D270G-P271G of the heavy chain that is converted by amino acid modification. In a further embodiment, the Q-containing tag at the carboxyl terminus of the light chain is GGLQSGA.

[0019] In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA at the carboxyl terminus of the light chain and an endogenous glutamine in Q295 of the heavy chain. In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA at the carboxyl terminus of the light chain and an endogenous glutamine in Q295-V296-N297 of the heavy chain. In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA at the carboxyl terminus of the light chain and an endogenous glutamine in Q295-V296-N297 of the heavy chain.

[0020] In some embodiments, the reactive glutamine comprises (or is) a reactive glutamine converted from amino acid modification in 354L-355Q-356G (Q438) of the heavy chain and an endogenous glutamine in Q295 of the heavy chain. In some embodiments, the reactive glutamine comprises (or is) a reactive glutamine converted from amino acid modification in S354L-R355Q-E356G (Q438) of the heavy chain and an endogenous glutamine in Q295 of the heavy chain. In some embodiments, the reactive glutamine comprises (or is) a reactive glutamine converted from amino acid modification in S354L-R355Q-E356G (Q438) of the heavy chain and an endogenous glutamine in Q295-V296-N297 of the heavy chain.

[0021] In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA located at the carboxyl terminus of the light chain, an endogenous glutamine in Q295 of the heavy chain, and a reactive glutamine in 354L-355Q-356G (Q438) of the heavy chain that is converted by amino acid modification. In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA located at the carboxyl terminus of the light chain, an endogenous glutamine in Q295-V296-N297 of the heavy chain, and a reactive glutamine in 354L-355Q-356G (Q438) of the heavy chain that is converted by amino acid modification. In some embodiments, the reactive glutamine comprises (or is) the glutamine in the Q-tag of GGLQSGA located at the carboxyl terminus of the light chain, the endogenous glutamine in Q295-V296-N297 of the heavy chain, and the reactive glutamine converted from S354L-R355Q-E356G (Q438) of the heavy chain by amino acid modification.

[0022] In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA located at the carboxyl terminus of the light chain, endogenous glutamine in Q295 of the heavy chain, and reactive glutamine modified from 354L-355Q-356G (Q438) of the heavy chain and 445Q-G446 of the heavy chain. In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA located at the carboxyl terminus of the light chain, endogenous glutamine in Q295-V296-N297 of the heavy chain, and reactive glutamine modified from 354L-355Q-356G (Q438) and 445Q-G446 of the heavy chain. In some embodiments, the reactive glutamine comprises (or is) the glutamine in the GGLQSGA Q-tag at the carboxyl terminus of the light chain, the endogenous glutamine in Q295-V296-N297 of the heavy chain, and the reactive glutamine in heavy chain S354L-R355Q-E356G (Q438), or heavy chain P445Q-G446G converted by amino acid modification. In some embodiments, the amino acid modification of heavy chain Q438I is further included.

[0023] In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG and LLQGA located at the carboxyl terminus of the light chain, the endogenous glutamine of Q295 of the heavy chain, the reactive glutamine in 354L-355Q-356G (Q438) of the heavy chain converted by amino acid modification, and the LQSG Q-tag glutamine inserted after T135 of the heavy chain. In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA located at the carboxyl terminus of the light chain, an endogenous glutamine in Q295-V296-N297 of the heavy chain, 354L-355Q-356G (Q438) of the heavy chain, and a glutamine in the Q-tag LQSG inserted after T135 of the heavy chain. In some embodiments, the reactive glutamine comprises (or is) a glutamine in the Q-tag of GGLQSGA located at the carboxyl terminus of the light chain, an endogenous glutamine in Q295-V296-N297 of the heavy chain, a reactive glutamine in S354L-R355Q-E356G (Q438) of the heavy chain that is converted by amino acid modification, and a glutamine in the Q-tag LQSG inserted after T135 of the heavy chain.

[0024] In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG and LLQGA located at the carboxyl terminus of the light chain, the endogenous glutamine of Q295 of the heavy chain, the reactive glutamine in 354L-355Q-356G (Q438) of the heavy chain converted by amino acid modification, and the glutamine in the Q-tag replacing LQSG of G200-L201-S202 of the light chain. In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG and LLQGA located at the carboxyl terminus of the light chain, an endogenous glutamine in Q295-V296-N297 of the heavy chain, a reactive glutamine in 354L-355Q-356G (Q438) of the heavy chain converted by amino acid modification, and a glutamine in the Q-tag LQSG replacing G200-L201-S202 of the light chain. In some embodiments, the reactive glutamine comprises (or is) the glutamine in the Q-tag of GGLQSGA located at the carboxyl terminus of the light chain, the endogenous glutamine in Q295-V296-N297 of the heavy chain, the reactive glutamine in S354L-R355Q-E356G (Q438) of the heavy chain converted by amino acid modification, and the glutamine in the Q-tag of LQSG replacing G200-L201-S202 of the light chain.

[0025] In some embodiments, the reactive glutamine comprises (or is) a reactive glutamine in heavy chain 354L-355Q-356G (Q438) and heavy chain 445Q-G446 that is converted to a reactive glutamine by amino acid modification, and the constant region of the antibody or antigen-binding fragment thereof further comprises an amino acid modification of 438I. In some embodiments, the reactive glutamine comprises (or is) a reactive glutamine in heavy chain S354L-R355Q-E356G (Q438) and heavy chain P445Q-G446G that is converted to a reactive glutamine by amino acid modification, and the constant region of the antibody or antigen-binding fragment thereof further comprises an amino acid modification of Q438I.

[0026] In some embodiments, the reactive glutamine comprises (or is) a reactive glutamine in heavy chain 354L-355Q-356G (Q438) and heavy chain 445Q-G446 that is converted to a reactive glutamine by amino acid modification, and the antibody or antigen-binding fragment thereof further comprises an amino acid modification of 295A. In some embodiments, the reactive glutamine comprises (or is) a reactive glutamine in heavy chain S354L-R355Q-E356G (Q438) and heavy chain P445Q-G446G that is converted to a reactive glutamine by amino acid modification, and the antibody or antigen-binding fragment thereof further comprises an amino acid modification of Q295A.

[0027] In some embodiments, the reactive glutamine comprises a reactive glutamine in 445Q-G446 of the heavy chain converted by amino acid modification, and K is removed from the C-terminus of the heavy chain.

[0028] In some embodiments, the reactive glutamine comprises (or is) a glutamine in a Q-containing tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA located at the carboxyl terminus of the heavy chain and / or light chain. In some embodiments, K is removed from the C-terminus of the heavy chain before the Q-containing tag is attached.

[0029] In some embodiments, the specific amino acid mutation scheme of the reactive glutamine is shown in Table 1.

[0030] In some embodiments, the specific amino acid mutation scheme of the antibody or antigen-binding fragment thereof is shown in Table 1.

[0031] In some embodiments, both heavy chains and / or both light chains of the antibody have the Q-containing tag inserted therein or contain the amino acid modification. In some embodiments, the Q-containing tag or the amino acid modification is identical in both heavy chains and / or both light chains. In some embodiments, the Q-containing tag and the amino acid modification are identical in both heavy chains. In some embodiments, the Q-containing tag and the amino acid modification are identical in both light chains. In some embodiments, the Q-containing tag and the amino acid modification are identical in both heavy chains and both light chains.

[0032] In some embodiments, the antibody moiety is linked to the drug conjugate moiety via a linker comprising an amino group that is covalently linked to a reactive glutamine in the antibody.

[0033] In some embodiments, the antibody-drug conjugate has the following structure: A-(NH-LX)d, wherein A is an antibody or antigen-binding fragment described herein, -NH-L- is a linker, X is a drug molecule, and d is 1-16, preferably 1-12, more preferably 1-8, such as an integer or decimal of 1, 2, 3, 4, 5, 6, 7, or 8.

[0034] In some embodiments, wherein -L- comprises a stretching unit, and / or a reaction unit, and / or a cleavable unit, and / or a self-cleaving unit, and / or a branching unit, and / or a hydrophilic unit.

[0035] In some embodiments, -L- has the following structure: -L1-RG-B-(L2-L3)s-, wherein L1 is an extension unit, RG is a reaction unit or bond, B is a branching unit or bond, L2 is a cleavable unit or bond, L3 is a self-cleavable unit or bond, when B is a bond, s is 1, when B is a branching unit, s is selected from 2 or 3, wherein L1, B, L2, and L3 are optionally connected to a hydrophilic unit.

[0036] In some embodiments,

[0037] Wherein, -L1- is selected from:

[0038] -(CH2CH2O) n CH2C(O)-、-(CH2CH2O) n CH2CH2C(O)-、-(CH2CH2O) n CH2CH2-, n is selected from an integer of 1-24, preferably an integer of 3-12, more preferably an integer of 3-8, most preferably an integer of 3-6,

[0039] RG is selected from a bond or:

[0040] B is selected from a bond or:

[0041] wherein each u is independently selected from an integer of 1-8, such as 1, 2, 3, 4, 5, 6, 7, or 8.

[0042] L2 is selected from a bond or:

[0043] Wherein the * end is connected to B;

[0044] L3 is selected from a bond or:

[0045] The N-terminus is connected to L2.

[0046] In some embodiments, wherein X is selected from:

[0047] In some embodiments, the antibody-drug conjugate has the following structure:

[0048] In some embodiments, in the antibody-drug conjugate, the antibody portion and the drug conjugate portion are connected via a linker, and the linker is a cleavable linker or a non-cleavable linker. In some embodiments, when the linker is a cleavable linker, the cleavable linker includes but is not limited to NH2-(CH2CH2O) n -Val-Cit-PABC-X and NH2-(CH2CH2O) n -(Val-Cit-PABC-X)2, wherein X is a drug molecule, and n is an integer of at least 1 (such as any one of 2, 3, 4, 6, 8, 10, 12, 16, 20 or 24), PABC refers to p-aminobenzyloxycarbonyl, Val refers to valine, and Cit refers to citrulline. In some embodiments, when the linker is a non-cleavable linker, the non-cleavable linker includes but is not limited to NH2-RX, NH2NH-RX and NH2-ORX, wherein R is an alkyl group or a polyethylene glycol group (PEG), X is a drug molecule, and the polyethylene glycol group may have -(CH2CH2O) n -, wherein n is an integer of at least 1 (such as any one of 2, 4, 6, 8, 10, 12, 16, 20 or 24).

[0049] In some embodiments, the linker is selected from the group consisting of acetyl-lysine-glycine, aminohexanoic acid, acetyl-lysine-β-alanine, amino-PEG2-C2, amino-PEG3-C2, amino-PEG6-C2, acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl, amino-PEG6-C2-Val-Cit-PABC, amino-PEG3-C2-Val-Cit-PABC, aminohexanoyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-di]bis-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine.

[0050] In some embodiments, the cleavable linker is a structure shown in Formula I to Formula IV:

[0051] In some embodiments, the antibody in the antibody-drug conjugate is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a minibody, or a bispecific antibody.

[0052] In some embodiments, the ratio of the drug molecule to the antibody is about 2-16:1, for example, about 2:1, 4:1, 6:1, 8:1, 10:1, 12:1, 14:1, or 16:1.

[0053] In some embodiments, the drug molecule is an anticancer drug, for example, a cytotoxic drug, an immunopotentiator, or a radioisotope.

[0054] In some embodiments, the cytotoxic drug is selected from the group consisting of a tubulin inhibitor, a DNA topoisomerase inhibitor, a DNA damaging agent, an immunopotentiator, an antimetabolite, and an antitumor antibiotic.

[0055] In some embodiments, the tubulin inhibitor is selected from auristatin derivatives (e.g., MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (e.g., DM1, DM4, Ansamitocin, Mertansine, and dolastatin and its derivatives).

[0056] In some embodiments, the DNA topoisomerase inhibitor is selected from camptothecin analogs and DNA topoisomerase I inhibitors and their derivatives, for example, DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxycamptothecin, topotecan, lertotecan, belotecan, exitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4 -dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide and their derivatives.

[0057] In some embodiments, the DNA damaging agent is selected from the group consisting of calicheamicins, duocarmycins, and anthramycin derivatives PBD (pyrrolobenzodiazepine).

[0058] In some embodiments, the immune enhancer is selected from levamisole, pidotimod, imiquimod, isoprinosine, polyinosinic-cytidylic acid, and polyinosinic-uridylic acid.

[0059] In some embodiments, the antimetabolite is selected from methotrexate, 6-mercaptopurine, and 5-fluorouracil.

[0060] In some embodiments, the anti-tumor antibiotic is selected from polypeptide antibiotics (eg, actinomycin D or bleomycin) and anthraquinone drugs (eg, doxorubicin or mitoxantrone hydrochloride).

[0061] In some embodiments, the radioisotope is selected from 211 At 131 I. 125 I. 90 Y. 186 Re、 188 Re、 153 Sm, 212 Bi, 32 P. 60 Co and 177 Lu.

[0062] In some embodiments, the antibody is an anti-HER2, anti-Nectin 4, or anti-ROR1 antibody.

[0063] In some embodiments, the antibodies encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, humanized antibodies, human antibodies (e.g., fully human antibodies), single chain (ScFv), bispecific antibodies, multispecific antibodies, mutants thereof, fusion proteins comprising antibody portions, and any other modified configuration of an immunoglobulin molecule that comprises an antigen recognition site of the desired specificity.

[0064] In some embodiments, the anti-HER2 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO: 1, a heavy chain CDR2 as shown in SEQ ID NO: 2, and a heavy chain CDR3 as shown in SEQ ID NO: 3, and the light chain variable region comprises a light chain CDR1 as shown in SEQ ID NO: 4, a light chain CDR2 as shown in SEQ ID NO: 5, and a light chain CDR3 sequence as shown in SEQ ID NO: 6.

[0065] In some embodiments, the anti-HER2 antibody comprises the heavy chain variable region shown in SEQ ID NO: 7, and / or comprises the light chain variable region sequence shown in SEQ ID NO: 8.

[0066] In some embodiments, the anti-HER2 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is subjected to a reactive glutamine mutation based on the heavy chain constant region sequence shown in SEQ ID NO: 9, and / or the light chain constant region is subjected to a reactive glutamine mutation based on the light chain constant region sequence shown in SEQ ID NO: 10.

[0067] In some embodiments, the anti-HER2 antibody is based on the light and heavy chain amino acid sequences of DP001 or trastuzumab and undergoes reactive glutamine mutation.

[0068] In some embodiments, the heavy chain constant region of the anti-HER2 antibody comprises one or more reactive glutamines selected from the following: (i) a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA at the carboxyl terminus of the light chain, the heavy chain, or both; (ii) a glutamine selected from heavy chain S354L-R355Q-E356G (Q438), heavy chain P445Q-G446, heavy chain S117L-A118Q-S119G-T120G, heavy chain H310L-Q311-D312G, heavy chain D399L-S400Q-D401G, heavy chain H268L-E269Q-D270G, heavy chain M252Q-I253G, Heavy chain S444Q-P445G-G446A, heavy chain E283L-V284-H285Q-N286G, heavy chain H285L-N286Q, heavy chain K340L-G341L-Q342-P343G, heavy chain N384L-G385Q-Q386G-P387G, heavy chain H268L-E269Q-D270G-P271G, heavy chain N325L-K326Q, heavy chain P329Q-A330G, heavy chain K360L-N361Q-Q362G, heavy chain N384Q-G385-Q386G, heavy chain S134L-T135Q-S136G, heavy chain V28 2Q converted to a reactive glutamine by amino acid modification; (iii) endogenous glutamine in Q295; (iv) reactive glutamine by amino acid modification in light chain K126L-S127Q-G128-T129G, light chain V205L-T206Q-K207G-S208G, light chain T109L-V110Q-A111G-A112G, light chain Q199L-G200Q-L201S-S202G; and (v) glutamine in the Q-tagged LQSG replacing G200-L201-S202 of the light chain, and glutamine in the Q-tagged LQSG inserted after T135 of the heavy chain.

[0069] In some embodiments, the anti-nectin 4 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO:11, a heavy chain CDR2 as shown in SEQ ID NO:12, and a heavy chain CDR3 as shown in SEQ ID NO:13, and the light chain variable region comprises a light chain CDR1 as shown in SEQ ID NO:14, a light chain CDR2 as shown in SEQ ID NO:15, and a light chain CDR3 sequence as shown in SEQ ID NO:16.

[0070] In some embodiments, the anti-nectin 4 antibody comprises the heavy chain variable region shown in SEQ ID NO: 17, and / or comprises the light chain variable region sequence shown in SEQ ID NO: 18.

[0071] In some embodiments, the anti-nectin 4 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is subjected to a reactive glutamine mutation based on the heavy chain constant region sequence shown in SEQ ID NO: 19, and / or the light chain constant region is subjected to a reactive glutamine mutation based on the light chain constant region sequence shown in SEQ ID NO: 20.

[0072] In some embodiments, the anti-nectin 4 antibody is based on the light and heavy chain amino acid sequences of Enfortumab and undergoes reactive glutamine mutations.

[0073] In some embodiments, the heavy chain constant region of the anti-nectin 4 antibody comprises one or more reactive glutamines selected from the group consisting of: (i) a glutamine in a Q-tag selected from the group consisting of GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA at the carboxyl terminus of the light chain, the heavy chain, or both; (ii) a glutamine in a Q-tag selected from the group consisting of S354L-R355Q-E356G (Q438) of the heavy chain, P445Q-G446 of the heavy chain, S11 7L-A118Q-S119G-T120G, heavy chain H310L-Q311-D312G, heavy chain D399L-S400Q-D401G, heavy chain H268L-E269Q-D270G, heavy chain M252Q-I253G, heavy chain S444Q-P445G-G446A, heavy chain E283L-V284-H285Q-N286G, heavy chain H285L-N28 6Q, heavy chain K340L-G341L-Q342-P343G, heavy chain N384L-G385Q-Q386G-P387G, heavy chain H268L-E269Q-D270G-P271G, heavy chain N325L-K326Q, heavy chain P329Q-A330G, heavy chain K360L-N361Q-Q362G, heavy chain N384Q-G385-Q386G, heavy chain S (iii) endogenous glutamine in Q295; (iv) reactive glutamine in the light chain K126L-S127Q-G128-T129G, light chain V205L-T206Q-K207G-S208G, light chain T109L-V110Q-A111G-A112G, light chain Q199L-G200Q-L201S-S202G; and (v) glutamine in the Q-tag LQSG replacing G200-L201-S202 of the light chain, and glutamine in the Q-tag LQSG inserted after T135 of the heavy chain.

[0074] In some embodiments, the anti-ROR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO:21, a heavy chain CDR2 as shown in SEQ ID NO:22, and a heavy chain CDR3 as shown in SEQ ID NO:23, and the light chain variable region comprises a light chain CDR1 as shown in SEQ ID NO:24, a light chain CDR2 as shown in SEQ ID NO:25, and a light chain CDR3 sequence as shown in SEQ ID NO:26.

[0075] In some embodiments, the anti-ROR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO:21, a heavy chain CDR2 as shown in SEQ ID NO:29, and a heavy chain CDR3 as shown in SEQ ID NO:23, and the light chain variable region comprises a light chain CDR1 as shown in SEQ ID NO:24, a light chain CDR2 as shown in SEQ ID NO:25, and a light chain CDR3 sequence as shown in SEQ ID NO:26.

[0076] In some embodiments, the anti-ROR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO:21, a heavy chain CDR2 as shown in SEQ ID NO:31, and a heavy chain CDR3 as shown in SEQ ID NO:23, and the light chain variable region comprises a light chain CDR1 as shown in SEQ ID NO:24, a light chain CDR2 as shown in SEQ ID NO:25, and a light chain CDR3 sequence as shown in SEQ ID NO:26.

[0077] In some embodiments, the anti-ROR1 antibody comprises the heavy chain variable region shown in SEQ ID NO: 27, and / or comprises the light chain variable region sequence shown in SEQ ID NO: 28.

[0078] In some embodiments, the anti-ROR1 antibody comprises the heavy chain variable region shown in SEQ ID NO: 30, and / or comprises the light chain variable region sequence shown in SEQ ID NO: 28.

[0079] In some embodiments, the anti-ROR1 antibody heavy chain comprises the heavy chain variable region shown in SEQ ID NO: 32, and / or comprises the light chain variable region sequence shown in SEQ ID NO: 28.

[0080] In some embodiments, the anti-ROR1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region sequence as shown in SEQ ID NO: 33, and the light chain comprises a light chain constant region sequence as shown in SEQ ID NO: 34.

[0081] In some embodiments, the heavy chain constant region of the anti-ROR1 antibody comprises one or more reactive glutamines selected from the group consisting of: (i) a glutamine in a Q-tag selected from the group consisting of GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA at the carboxyl terminus of the light chain, the heavy chain, or both; (ii) a glutamine selected from the group consisting of heavy chain S354L-R355Q-E356G (Q438), heavy chain P445Q-G446, heavy chain S117L-A118Q-S119G-T120G, heavy chain H310L-Q311-D312G, heavy chain D399L-S400Q-D40 1G, heavy chain H268L-E269Q-D270G, heavy chain M252Q-I253G, heavy chain S444Q-P445G-G446A, heavy chain E283L-V284-H285Q-N286G, heavy chain H285L-N286Q, heavy chain K340L-G341L-Q342-P343G, heavy chain N384L-G385Q-Q386G-P387G, heavy chain H268L-E269Q-D270G-P271G, heavy chain N325L-K326Q, heavy chain P329Q-A330G, heavy chain K360L-N361Q-Q362G, Reactive glutamines converted by amino acid modification in heavy chain N384Q-G385-Q386G, heavy chain S134L-T135Q-S136G, and heavy chain V282Q; (iii) endogenous glutamine in Q295; (iv) reactive glutamines converted by amino acid modification in light chain K126L-S127Q-G128-T129G, light chain V205L-T206Q-K207G-S208G, light chain T109L-V110Q-A111G-A112G, and light chain Q199L-G200Q-L201S-S202G; and (v) glutamine in the Q-tag LQSG replacing G200-L201-S202 of the light chain, and glutamine in the Q-tag LQSG inserted after T135 of the heavy chain. The variable region and CDR numbering in this application adopts the Kabat numbering convention.

[0082] The constant regions in this application use the EU numbering convention.

[0083] In some embodiments, the drug molecule is DXD or a derivative thereof, MMAE or a derivative thereof.

[0084] In some embodiments, wherein the linker is NH2-(CH2CH2O) n -Val-Cit-PABC-X, X is a drug molecule; preferably, n is 3.

[0085] In some embodiments, wherein the linker is NH2-(CH2CH2O) n -Val-Cit-PABC-X, X is DXD or a derivative thereof, MMAE or a derivative thereof; preferably, n is 3.

[0086] In some embodiments, the linker can be directly coupled to the antibody in a one-step process using mTGase enzymatic method, and the hydrophilicity can be adjusted using PEG.

[0087] In some embodiments, the drug conjugated moiety is LND1002, whose structure is shown in Formula V:

[0088] In some embodiments, the drug conjugated moiety is JSSW038, whose structure is shown in Formula VI:

[0089] In some embodiments, the drug conjugation moiety structure is shown in Formula VII:

[0090] In some embodiments, the antibody-drug conjugate is AB5-ADC, MFC8-ADC, MFC10-ADC, DP28-ADC, DP35-ADC, DP36-ADC, DP35-JSSW038, DP36-JSSW038, MFC8-JSSW038, DP28-JSSW038, Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, or Hu17-H4L1-4LND1002.

[0091] Exemplary drug conjugation moiety structures can also be found in Figures 2A-2B.

[0092] In a third aspect, the present application relates to a method for preparing the above-mentioned antibody-drug conjugate, comprising the step of performing one or more insertion or substitution mutations on the constant region of the antibody to obtain reactive glutamine.

[0093] In some embodiments, the reactive glutamine obtained by insertion or substitution mutation is as described in the first aspect above.

[0094] In some embodiments, the above-mentioned method of preparing the target antibody-drug conjugate further comprises the steps of preparing the antibody and enzymatically coupling the drug conjugate moiety by mTGase to obtain ADC.

[0095] The present application also relates to a method for preparing an antibody-drug conjugate with a high drug load, which comprises the steps of preparing the antibody-drug conjugate by the aforementioned method, and then further coupling the drug conjugation moiety by a chemical method to obtain an ADC with a higher DAR value.

[0096] In some embodiments, the chemical coupling comprises one or more of click chemistry, amide condensation, and Michael addition. In one embodiment, the chemical method is click chemistry achieved by azide-cycloalkyne addition reaction.

[0097] In some embodiments, the ADC may be prepared by the aforementioned chemical method first, and then the drug conjugate moiety may be coupled by the aforementioned mTGase enzymatic method to obtain an ADC with a higher DAR value.

[0098] In some embodiments, the drug conjugation moiety is JSSW-SY.

[0099] In some embodiments, the step of preparing a monoclonal antibody comprises constructing the mutated sequence into a eukaryotic cell protein expression plasmid vector, extracting the plasmid and transiently transfecting CHO cells to express the monoclonal antibody, and enzymatically coupling the obtained antibody to obtain ADC by mTGase.

[0100] In a fourth aspect, the present application relates to the use of the above-mentioned antibody-drug conjugate or antibody in the preparation of a drug for treating cancer.

[0101] In a fifth aspect, the present application relates to a method for using the above-mentioned antibody-drug conjugate or antibody to treat cancer.

[0102] In a sixth aspect, the present application relates to a method for treating cancer, comprising administering a therapeutically effective amount of the aforementioned antibody-drug conjugate or antibody.

[0103] In a seventh aspect, the present application relates to an acyl-donor substrate containing a Q tag for transglutaminase, wherein the Q tag is selected from: (i) GGLQSGA, LQSGA, EEQYGA and EEQYQSG located at the carboxyl terminus of the light chain, the heavy chain or the light chain and the heavy chain; (ii) heavy chain 354L-355Q-356G (Q438), heavy chain 445Q-G446, heavy chain 117L-1 18Q-119G-120G, heavy chain 310L-Q311-312G, heavy chain 399L-400Q-401G, heavy chain 268L-269Q-270G, heavy chain 252Q-253G, heavy chain 444Q-445G-446A, heavy chain 283L-V284-285Q-286G, heavy chain 285L-286Q, heavy chain 340L-341L-Q 342-343G, heavy chain 384L-385Q-386G-387G, heavy chain 268L-269Q-270G-271G, heavy chain 325L-326Q, heavy chain 329Q-330G, heavy chain 360L-361Q-362G, heavy chain 384Q-G385-386G, heavy chain 134L-135Q-136G, heavy chain 282Q; (iii) light light chain 126L-127Q-G128-129G, light chain 205L-206Q-207G-208G, light chain 109L-110Q-111G-112G, and light chain 199L-200Q-201S-202G; and (iv) a Q-tagged LQSG replacing G200-L201-S202 of the light chain, and a Q-tagged LQSG inserted after T135 of the heavy chain.

[0104] In some embodiments, the Q-containing tag is selected from the group consisting of: (i) GGLQSGA, LQSGA, EEQYGA, EEQYQSG, and LLQGA located at the carboxyl terminus of the light chain, the heavy chain, or both; (ii) heavy chain S354L-R355Q-E356G (Q438), heavy chain P445Q-G446, heavy chain S117L-A118Q-S119G-T120G, heavy chain H310L-Q3 11-D312G, heavy chain D399L-S400Q-D401G, heavy chain H268L-E269Q-D270G, heavy chain M252Q-I253G, heavy chain S444Q-P445G-G446A, heavy chain E283L-V284-H285Q-N286G, heavy chain H285L-N286Q, heavy chain K340L-G341L-Q342-P343G, heavy chain N384L-G3 85Q-Q386G-P387G, heavy chain H268L-E269Q-D270G-P271G, heavy chain N325L-K326Q, heavy chain P329Q-A330G, heavy chain K360L-N361Q-Q362G, heavy chain N384Q-G385-Q386G, heavy chain S134L-T135Q-S136G, heavy chain V282Q; (iii) Q295 endogenous glutamine; (i v) light chain K126L-S127Q-G128-T129G, light chain V205L-T206Q-K207G-S208G, light chain T109L-V110Q-A111G-A112G, light chain Q199L-G200Q-L201S-S202G; and (v) Q-tagged LQSG replacing light chain G200-L201-S202, Q-tagged LQSG inserted after T135 of the heavy chain.

[0105] In the fourth to seventh aspects:

[0106] In some embodiments, the cancer is a solid tumor or a hematological tumor.

[0107] In some embodiments, the solid tumor is a HER-2-positive cancer, such as breast cancer, colorectal cancer (also known as colon and rectal cancer, and including colon cancer and rectal cancer), ovarian cancer, gastric cancer (also known as gastric adenocarcinoma), urinary tract cancer, or lung cancer (including small cell lung cancer and non-small cell lung cancer). Preferably, it is breast cancer.

[0108] In some embodiments, the solid tumor is a Nectin 4-positive cancer, such as breast cancer, pancreatic cancer, lung cancer, ovarian cancer, or gastric cancer.

[0109] In some embodiments, the solid tumor or hematological tumor is a ROR1-positive cancer, such as hematological cancers such as chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and solid tumors (ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma and colorectal cancer, etc.).

[0110] In some embodiments, the antibody-drug conjugates or antibodies of the present application can be used alone.

[0111] In some embodiments, the antibody-drug conjugates or antibodies of the present application can be used in combination with other therapies, and can be combined with surgery, radiation therapy, hormone therapy, and the like.

[0112] In some embodiments of various aspects of the present invention, the antibodies or functional fragments thereof of the present application have ADCC activity.

[0113] In some embodiments of various aspects of the present invention, the antibodies or functional fragments thereof of the present application have CDC activity.

[0114] In some embodiments of various aspects of the present invention, the antibody or antigen-binding fragment thereof of the present application is an IgG1 antibody.

[0115] The present application uses the amino acid sequence of the antibody as the skeleton. For example, by mutating the amino acids in the constant region of IgG1, the glutamine at position 295 (EU numbering) in the Fc region of the antibody is used as the coupling site for mTGase (DAR is 2), and an mTGase enzyme-catalyzed coupling site is added to obtain an antibody-drug conjugate with a high drug load.

[0116] The constant region of the antibody is modeled by computer-aided drug design, glutamine mutation is performed on the point where solvent accessibility is high and spatial structure is exposed, or the amino acid around the original glutamine of the antibody is changed into the amino acid with smaller spatial structure, thereby increasing the exposure of glutamine, so that glutamine can become the coupling site catalyzed by mTGase enzyme, thereby improving the DAR value of ADC drugs. The sequence after mutation is constructed into a eukaryotic protein expression plasmid vector, and the plasmid is transiently transfected and expressed in cells (for example, CHO cells) to express monoclonal antibodies, and the obtained antibody is coupled to obtain ADC by mTGase enzymatic method. The antibody-drug conjugate with high drug load obtained in the present application has good homogeneity, enhanced tumor killing function, and good safety simultaneously. The exemplary reaction schematic diagram of ADC coupling carried out by mTGase enzymatic method is shown in Figure 1. Description of the drawings:

[0117] Figure 1: Schematic diagram of mTGase enzymatic ADC conjugation reaction.

[0118] 2A-2B: Exemplary drug conjugation moiety structures.

[0119] Figure 3A-3E: In vitro plasma stability results of enzymatically coupled samples.

[0120] Figure 4: ADC endocytosis assay results.

[0121] Figure 5: In vivo efficacy of ADC on H1975 human lung adenocarcinoma xenografts.

[0122] Figure 6: In vivo efficacy of ADC on HCC1187 human breast cancer xenografts.

[0123] Figure 7: ADC concentration-time curve after three doses in cynomolgus monkeys.

[0124] Figure 8: Dose-time curve of free small molecules after three doses in cynomolgus monkeys.

[0125] Figure 9: Total drug resistance curve.

[0126] Figure 10: Drug-time curve of MMAE. Example:

[0127] It will be understood that the embodiments and implementations described herein are for illustrative purposes only and that various modifications or changes thereto will be apparent to those skilled in the art and are intended to be included within the spirit and scope of this application.

[0128] This application uses only a limited number of antibodies and a limited number of drug-conjugated moieties as examples to validate experimental data for ADC molecules designed in this application by coupling reactive glutamine-bearing antibodies to drug-conjugated moieties. This application aims to modify antibody constant regions to increase reactive glutamines, thereby increasing the number of drug-conjugated moieties that can be conjugated to the antibodies. The scheme designed in this application to increase reactive glutamines in antibody constant regions is applicable to any antibody, any target, and any drug-conjugated moiety.

[0129] definition

[0130] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0131] The antibody-drug conjugate (ADC) described herein refers to a cytotoxic drug conjugated to an antibody, wherein the antibody's antigen is expressed on the surface of cancer cells and the antibody also binds to a cell surface antigen that can be internalized by the cell, thereby selectively delivering the drug to the cancer cell, thereby causing the drug to accumulate in the cancer cell and killing the cancer cell. Targeting ligands such as antibodies (e.g., monoclonal antibodies) and antigen-binding fragments are linked to biologically active molecules through chemical linker compounds.

[0132] The antibody-drug conjugate (ADC) with a high drug load described in this application refers to: modeling the constant region of the antibody through computer-aided drug design, glutamine mutation at points of solvent accessibility and spatial structural exposure, or changing the amino acids surrounding the original glutamine of the antibody to amino acids with a smaller spatial structure, thereby increasing the exposure of glutamine, making glutamine available as a coupling site for mTGase enzyme catalysis, thereby obtaining an ADC drug with an improved DAR value; the antibody and drug conjugate moiety can also be further coupled through chemical methods, such as click chemistry achieved by an azide-cycloalkyne addition reaction, to further increase the DAR value of the ADC drug; the DAR value can be increased to 2 or more, for example, 4, 6, 8, 10, 12, 14 or 16.

[0133] The term "linker-drug compound" or "linker-drug compound" refers to the partial structure of an antibody-drug conjugate consisting of a linker compound and a biologically active compound.

[0134] As used herein, the terms "comprise," "include," and "contain" are used interchangeably to include not only closed definitions but also semi-closed and open definitions. In other words, the terms include "consisting of," "consisting essentially of."

[0135] As used herein, "HER2" refers to human epidermal growth factor receptor 2. "HER2-positive cancer" refers to a cancer that overexpresses HER2 on cancer cells compared to non-cancerous normal cells. HER2 status can be determined using known HER2 tests (including immunohistochemistry (IHC) testing, fluorescence in situ hybridization (FISH) testing, subtraction probe technique chromogenic in situ hybridization (SPoT-LightHER2 CISH) testing, and Inform double in situ hybridization (Inform HER2 Dual ISH) testing). HER2-positive cancers include cancers that test as 2+ (critical) or 3+ (positive) in the IHC test. HER2-positive cancers also include cancers that test as positive in HER2FISH testing, SPoT-Light HER2 CISH testing, and / or Inform HER2 Dual ISH testing. "HER22+ cancer" refers to a cancer that tests as 2+ in the IHC test. "HER23+ cancer" refers to a cancer that tests as 3+ in the IHC test.

[0136] As used herein, nectin is a novel cell adhesion molecule belonging to the immunoglobulin superfamily that can be expressed independently of Ca 2+ Nectin proteins participate in intercellular interactions and form intercellular adhesion junctions. The nectin family of proteins consists of four members: nectin-1, nectin-2, nectin-3, and nectin-4. Nectins 1 to 3 are widely expressed in normal human tissues. Nectins 1 to 2 are commonly found in immune organs (bone marrow, thymus, spleen, and lymph nodes), while nectin-3 is primarily expressed in the testis and placenta. Unlike other nectin family members, which are widely expressed in normal human tissues, nectin-4 is highly expressed in normal embryonic and fetal tissues and very low in healthy adult tissues. However, abnormally high expression of nectin-4 has been detected in various cancer samples. Nectin-4 participates in the formation of intercellular adhesion junctions, mediates intercellular connections, and participates in regulating physiological processes such as cell proliferation, differentiation, and migration. Although the precise molecular mechanisms underlying tumorigenesis and progression remain unclear, multiple studies have shown that nectin-4 can induce angiogenesis, promote cancer cell proliferation and migration, and induce epithelial-mesenchymal transition, implicating it in the progression of various cancers. Considering the expression pattern and level of Nectin-4, Nectin-4 becomes an attractive target for the treatment of various tumor diseases.

[0137] ROR1, short for receptor-tyrosine-kinase-like orphan receptor, is a type I single-pass transmembrane protein and a member of the receptor tyrosine kinase (RTK) family. It is lowly expressed during embryonic development but highly expressed in a variety of malignant tumors and tissues, such as chronic lymphocytic leukemia (CLL), allergic lymphocytic leukemia (ALL), breast cancer, ovarian cancer, melanoma, and lung adenocarcinoma. Numerous data indicate that ROR1 plays a significant role in promoting tumor growth and metastasis, inducing tumor cell drug resistance, and inhibiting apoptosis. The human ROR1 molecule consists of an extracellular domain, including an immunoglobulin-like domain (Ig-like), a cysteine-rich domain or frizzled (CRD or FZD), and a Kring (KNG) domain (KRD). The intracellular domain contains a tyrosine kinase domain (TKD), two serine / threonine-rich domains (Ser / ThrD), and a proline-rich domain (PRD). Overexpression of ROR1 during cancer progression can promote tumor growth. ROR1 can bind to Wnt5a, participate in Wnt signaling, and interact with signaling pathways such as EGFR and Met, promoting tumor cell growth, proliferation, and migration. ROR1 and CK1ε (casein kinase 1) activate AKT / PI3K, which in turn activates the pCREB pathway, leading to cell proliferation; ROR1 enhances EGFR signaling and induces epithelial-mesenchymal transition (EMT); ROR1 is highly expressed in most hematological tumors and solid tumors. For example, the proportion of ROR1+ in CLL patients exceeds 90%; in some solid tumors, such as lung cancer, pancreatic cancer, and colorectal cancer cells, ROR1 expression is also significantly increased and is closely related to disease progression and treatment efficacy. Therefore, ROR1 can be used as a specific targeted marker for the development of tumor treatment drugs.

[0138] The term "antibody" is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity or function. As used herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0139] As used herein, "full-length antibody" refers to the molecules that constitute the natural biological form of an antibody, including variable and constant regions. For example, in most mammals (including humans and mice), the full-length antibody of the IgG isotype is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having a light chain (L or LC) and a heavy chain (H or HC), each light chain comprising an immunoglobulin domain light chain variable region (VL) and a light chain constant region (CL), and each heavy chain comprising an immunoglobulin domain heavy chain variable region (VH), a heavy chain constant region CH1, CH2, and CH3. In some mammals, such as camels and llamas, IgG antibodies can consist of only two heavy chains, each of which comprises a variable domain attached to the Fc region. Unless otherwise indicated, in the present application's Examples, LC refers to a light chain and HC refers to a heavy chain.

[0140] As used herein, "Fc region" refers to a polypeptide comprising the constant region of an antibody heavy chain and excluding the first constant region immunoglobulin domain. For IgG, the Fc region may comprise immunoglobulin domains CH2 and CH3 and a hinge between CH1 and CH2.

[0141] Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). It will be understood by those skilled in the art that, unless otherwise specified, the numbering of amino acid residues in the Fc region or constant region of a given antibody according to the EU numbering system is to be understood as encompassing the numbering of amino acid residues in the corresponding Fc region or constant region defined by any other numbering system. Although the scope of protection claimed in the claims of the present application is shown based on the definition rules of the EU numbering system, the amino acid numbers corresponding to the definition rules of other numbering systems should also fall within the scope of protection of the present application.

[0142] The term "constant region" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence relative to the rest of the immunoglobulin (the variable domain), which contains the antigen-binding site. The constant domain contains the CH1, CH2, and CH3 domains (collectively referred to as CH) of the heavy chain and the CHL (or CL) domain of the light chain.

[0143] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain may be referred to as "VH." The variable domain of the light chain may be referred to as "VL." These domains are generally the most variable part of an antibody and contain the antigen binding site.

[0144] As used herein, the term "CDR" or "complementarity determining region" is intended to refer to the non-contiguous antigen combining sites found within the variable regions of both heavy and light chain polypeptides. These specific regions have been described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., U. S. Pat. of Health and Human Services, “Sequences of proteins of immunological interest” (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273:927-948 (1997); MacCallum et al., J. Mol. Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Lefranc et al., J. Mol. MP et al., Dev. Comp. Immunol., 27: 55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309: 657-670 (2001), wherein the definitions include overlapping or subsets of amino acid residues when compared to each other. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated by reference in their entireties for use in this application and for possible inclusion in one or more claims herein.

[0145] As used herein, the term "antigen-binding fragment" of an antibody refers to an antibody fragment that retains the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragment, consisting of VH and CH1 domains; Fv fragment, consisting of the VL and VH domains of a single arm of an antibody; dAb fragment (Ward et al., 1989 Nature 341: 544-546), consisting of a VH domain; and isolated CDRs. Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be recombinantly linked via a synthetic linker, allowing them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv); see, for example, Bird et al., 1988 Science 242:423-426; Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). In this application, single-chain antibodies are also encompassed within the scope of "antigen-binding fragments." "Diabodies" refer to small molecule antibody fragments with two antigen-binding sites, which contain a heavy chain variable region (VH) and a light chain variable region (VL) linked on a single polypeptide chain (VH-VL). A very short linker is used, which prevents the two domains on the same chain from pairing and forces them to pair with complementary domains on another chain, thereby forming two antigen-binding sites. Diabodies are described in more detail in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993). Antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art.

[0146] The term "solvent accessibility" is described as the accessible surface area (ASA), or solvent accessibility surface area (SASA), which is the surface area of ​​a biomolecule accessible to solvent. ASA is typically measured in square angstroms (a standard unit of measurement in molecular biology) and is often calculated using the "rolling ball method."

[0147] "Kabat numbering" and similar terms are generally recognized in the art and refer to a system for numbering the amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding fragment. In some aspects, CDR can be determined according to the Kabat numbering system (see, for example, Kabat EA and Wu TT (1971) Ann NY Acad Sci 190: 382-391; and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH publication number 91-3242). Using the Kabat numbering system, the CDR in the antibody heavy chain molecule is typically present at amino acid positions 31 to 35, and the CDR optionally can include one or two additional amino acids (referred to as 35A and 35B in the Kabat numbering scheme) (CDR1) after position 35; amino acid positions 50 to 65 (CDR2); and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, the CDRs in the antibody light chain molecule are typically present at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein are determined according to the Kabat numbering scheme.

[0148] "IMGT numbering": The CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7: 132-136 and Lefranc MP et al. (1999) Nucleic Acids Res 27: 209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 97.

[0149] "Chothia numbering": the Chothia CDR-H1 loop occurs at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop occurs at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop occurs at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop occurs at light chain amino acids 24 to 34, the Chothia CDR-L2 loop occurs at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop occurs at light chain amino acids 89 to 97.

[0150] " Transglutaminase " is used interchangeably with " TGase " in this article, refers to the enzyme that can implement transglutamination reaction. As used herein, the term " transglutaminase " refers to such reaction, wherein the γ-glutaminyl group of the acceptor glutamine residue from protein / peptide is transferred to an amine group, such as the ε-amino group of primary amine or lysine. Prior art has disclosed several types of transglutaminases from various living organisms (including microorganisms), such as TGase from guinea pig liver (GTGase), fish liver (FTGase) and microorganism (mTGase) and the TGase (rTGase) of any restructuring. Suitable TGase includes, but is not limited to bacterial transglutaminase (BTG), such as the enzyme (protein-glutamine-γ-glutamyl transferase) with EC index number EC 2.3.2.13. In some embodiments, TGase is from Streptomyces radakaensis (TG_SL, SEQ ID NO:35). In some embodiments, TGase is from Streptomyces mobaraensis (TG_SM, SEQ ID NO:36). In some embodiments, the TGase is a recombinant TGase from Streptomyces ladakai (TG_SL, SEQ ID NO: 37).

[0151] In some embodiments, the transglutaminase used in the methods described herein is a recombinant protein produced using recombinant technology.

[0152] In some embodiments, the transglutaminase is a wild type, for example, a TGase having a sequence of SEQ ID NO: 35. In some embodiments, the transglutaminase is a recombinant TGase comprising a sequence of SEQ ID NO: 36, wherein the recombinant TGase further comprises an additional proline at the N-terminus and an optional purification tag (such as a polyhistidine tag). In some embodiments, the transglutaminase is a recombinant TGase having a sequence of SEQ ID NO: 37.

[0153] In some embodiments, the transglutaminase is engineered. In some embodiments, the transglutaminase is an engineered transglutaminase specifically designed to catalyze the transglutamination reaction at an acceptor glutamine near an N-glycosylation site.

[0154] The term "acceptor glutamine residue", when referring to an amino acid residue of a polypeptide or protein, refers to a glutamine residue that, under appropriate conditions, is recognized by TGase and can be cross-linked to a conjugated portion comprising a donor amine group by TGase via a reaction between glutamine and a donor amine group (such as lysine or a structurally related primary amine such as aminopentyl).

[0155] "Endogenous glutamine" refers to naturally occurring glutamine that has not been engineered, such as glutamine in the variable domain, CDR, or constant region.

[0156] "Becoming reactive glutamine" refers to glutamine that has become susceptible, exposed or reactive to amine-donating reagents through antibody engineering (e.g., amino acid modification, etc.), for example, in the presence of transglutaminase, the amide group on the reactive glutamine can undergo acyl transfer reaction with other primary amines.

[0157] "Reactive glutamine" refers to a glutamine that is susceptible, exposed or reactive to an amine-donating reagent, for example, the amide group on the reactive glutamine is capable of undergoing an acyl transfer reaction with other primary amines in the presence of a transglutaminase.

[0158] "Amino acid modification" refers to an amino acid sequence change in a predetermined amino acid sequence. Exemplary modifications include amino acid substitutions, insertions and / or deletions. Preferred amino acid modifications herein are insertions or substitutions. "Amino acid substitution" refers to replacing at least one amino acid residue present in a predetermined amino acid sequence with another different "substitution" amino acid residue. "Amino acid insertion" refers to the incorporation of at least one amino acid into a predetermined amino acid sequence, and "insertion" in this application includes larger "peptide insertions", such as insertions of about three to about five or even up to about ten amino acid residues. "Amino acid deletion" refers to the removal of at least one amino acid from the original amino acid sequence.

[0159] Herein, "capital letter 1 + number + capital letter 2" indicates that the amino acid residue at the position of the number is substituted from a certain amino acid residue 1 to another amino acid residue 2, for example, "R355Q" indicates that the arginine residue at the amino acid position numbered 355 is substituted with a glutamine residue. In any of the above embodiments, "number + capital letter 3" indicates that the amino acid residue at the position of the number is substituted from a certain amino acid residue to another amino acid residue 3, for example, "355Q" indicates that the amino acid at the amino acid position numbered 355 is substituted with a glutamine residue.

[0160] As used herein, "capital letter + number" indicates the amino acid residue at that position. For example, "G200" indicates that the amino acid residue at position 200 (EU numbering) is glycine and has not been substituted by any other amino acid residue. "Capital letter 1 + number + capital letter 1," such as "G200G," indicates that the amino acid residue at position 200 (EU numbering) is glycine and has not been substituted by any other amino acid residue.

[0161] In any of the above embodiments, "capital letter 1 + number 1 + capital letter 2 - capital letter 3 + number 2 + capital letter 4" means that the amino acids numbered 1 and 2 are mutated simultaneously, wherein the amino acid residue numbered 1 is mutated from the amino acid residue represented by capital letter 1 to the amino acid residue represented by capital letter 2, and the amino acid residue numbered 2 is mutated from the amino acid residue represented by capital letter 3 to the amino acid residue represented by capital letter 4. For example, "S354L-R355Q-E356G" means that the serine residue at amino acid number 354 of the antibody is mutated to a leucine residue, the arginine residue at amino acid number 355 is mutated to a glutamine residue, and the glutamic acid residue at amino acid number 356 is mutated to a glycine residue.

[0162] As used herein, the term "354L-355Q-356G (Q438)" or "S354L-R355Q-E356G (Q438)" refers to an antibody constant region comprising the heavy chain mutation 354L-355Q-356G (or S354L-R355Q-E356G). This mutation exposes the glutamine at position Q438, converting the glutamine at position Q438 to a reactive glutamine. Reactive glutamine 354L-355Q-356G is synonymous with 354L-355Q-356G (Q438); reactive glutamine S354L-R355Q-E356G is synonymous with S354L-R355Q-E356G (Q438).

[0163] The term "containing Q tag", "containing Q tag", "with Q tag" or "with Q tag short peptide" refers to an amino acid sequence comprising a reactive glutamine residue, when the glutamine residue is incorporated into (e.g., attached to) a polypeptide sequence under suitable conditions, it is recognized by transglutaminase ("TGase") and cross-linked by TGase via the reaction between the amino acid side chain and the reactive group in the amino acid sequence. The recognition tag can be a peptide sequence that does not naturally occur in the polypeptide. In certain embodiments, the TGase recognition tag comprises at least one glutamine. In certain embodiments, the TGase recognition tag comprises the amino acid sequence XXQX, wherein X is any amino acid or does not exist (e.g., conventional amino acids Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys or Trp or unconventional amino acids).

[0164] In the present invention, the 20 conventional amino acids refer to alanine (Ala or A), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W) and tyrosine (Tyr or Y).

[0165] "UC961-vc-MMAE," also known as VLS-101, is an ADC derived from the conjugation of the tubulin polymerization inhibitor MMAE to the cysteine ​​residue of the antibody Cirmtuzumab (UC961) using the cleavable linker MC-Val-Cit-PAB. The average DAR is 4. The antibody sequence can be found in sequences 3-6 of CN111587124A.

[0166] As used herein, the term "NH2-PEG3-Val-Cit-pABC" refers to the following structure:

[0167] Among them, the * position is connected to the bioactive molecule X such as MMAE. When it is connected to the antibody, it has the following structure:

[0168] The amino group (-NH-) is used to connect the antibody.

[0169] The term "MMAE" refers to monomethyl auristatin E, which has the following structure:

[0170] When connected to the connector, it has the following structure:

[0171] Exemplary routes of administration of ADCs include, but are not limited to, oral, intravenous, intracavitary, intratumoral, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, ocular, topical, intraperitoneal, intracranial, intrapleural, and epidermal routes, or delivered to a lymph gland, body space, organ, or tissue known to contain cancer cells. In some embodiments, the ADC is administered intravenously. In some embodiments, the ADC is administered by infusion. In some embodiments, the ADC is administered by injection.

[0172] The conjugate portion of the ADC described herein comprises a toxin, such as a cytotoxic agent that can be used for cancer therapy. In some embodiments, the toxin is a chemotherapeutic agent. In some embodiments, the toxin is a small molecule drug. Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatins, dolastatin, CC-1065, duocarmycins, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, SN-38, tubulysin, hemiasterlin and its stereoisomers, isosteres, analogs or derivatives. In some embodiments, the conjugate portion comprises monodansylcadaverine (MDC). In some embodiments, the conjugate portion comprises TAM1. In some embodiments, the conjugate portion comprises monomethyl auristatin E (MMAE) or a derivative thereof.

[0173] Anthracyclines are derived from the bacterium Streptomyces and have been used to treat a wide range of cancers, such as leukemias, lymphomas, breast cancer, uterine cancer, ovarian cancer, and lung cancer. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

[0174] Dolastatins and their peptide analogs and derivatives, auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, for example, U.S. Patent No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42: 2961-2965 (1998). Exemplary dolastatins and auristatins include, but are not limited to, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD, MMAF, MMAE, and 5-benzoylvaleric acid-AE ester (AEVB).

[0175] Duocarmycin and CC-1065 are DNA alkylating agents with cytotoxic potency. See Boger and Johnson, PNAS 92:3642-3649 (1995). Exemplary dolastatins and auristatins include, but are not limited to (+)-duocarmycin A and (+)-duocarmycin SA and (+)-CC-1065.

[0176] Enediynes are a class of antitumor bacterial products characterized by nine- and ten-membered rings or ring systems with conjugated triple-double-triple bonds. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, and dynemicin.

[0177] The geldanamycins are benzoquinone ansamycin antibiotics that bind to Hsp90 (heat shock protein 90) and have been used as antitumor drugs. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0178] Maytansine or its derivatives, maytansinoids, inhibit cell proliferation by inhibiting microtubule formation by inhibiting tubulin polymerization during mitosis. See Remillard et al., Science 189: 1002-1005 (1975). Exemplary maytansines and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives and ansamitocin.

[0179] Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g., ) and docetaxel

[0180] Vinca alkaloids are also anti-tubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0181] Example 1: Point mutation design

[0182] The constant region of a monoclonal IgG1 antibody targeting HER2 (DP001) was modeled by computer-aided drug design. Glutamine mutations were performed at points with high solvent accessibility and exposed spatial structure. Alternatively, the amino acids surrounding the antibody glutamine were changed to amino acids with a smaller spatial structure, or short peptides with Q amino acid residues were inserted to increase glutamine exposure and the number of reactive glutamines. These reactive glutamines became coupling sites catalyzed by the mTGase enzyme (SEQ ID NO: 35, which was used in all examples), thereby increasing the DAR value of the ADC drug. The point mutations designed in this application are shown in Table 1 below.

[0183] Table 1 Sequences of point mutations containing glutamine

[0184] Example 2: Design and detection of Q-tagged short peptides

[0185] Q-tagged peptides were added to the C-terminus of the heavy chain (HC) of the anti-nectin 4 antibody Enfortumab (amino acid sequence shown in Table 24-1). The modification rates of the Q on each Q-tagged peptide were compared: EvHcQ1 (K was removed from the C-terminus of the HC and LQSGA was added), EvHcQ2 (K was removed from the C-terminus of the HC and EEQYGA was added), EvHcQ3 (K was removed from the C-terminus of the HC and EEQYQSG was added), and EvHcQ04 (K was removed from the C-terminus of the HC and LLQGA was added). After plasmid construction, antibody expression, and purification (using the same methods as in Examples 3-5 below), the peptides were conjugated with LND1002 to prepare ADCs. After 12 hours of conjugation, the conjugation solution was collected, reduced, and then the amount of unconjugated antibody was determined by RP-HPLC to reflect the modification rate of each Q-tagged peptide.

[0186] RP-HPLC detection method: Dithiothreitol was added to the modification solution at a final concentration of 20 mM. The sample was incubated at 30°C in a water bath for 30 minutes to cleave the interchain disulfide bonds of the ADC. The sample was then analyzed by HPLC. The HPLC system was e2695 (Waters), the column was HiChrome, the column temperature was 60°C, and the mobile phase A was 0.1% trifluoroacetic acid (TFA) in water. The mobile phase B was 0.1% trifluoroacetic acid (TFA) in acetonitrile. The sample volume was 50 μL, and the mobile phase A gradient program was: 70%-32% (0-6 minutes), 68%-65% (6-17 minutes), 65%-63% (17-20 minutes), 63%-61% (20-21 minutes), 61%-60% (21-25 minutes), 60%-58% (25-28 minutes), 58%-50% (28-31 minutes), 50%-10% (31-37 minutes), 10%-70% (37-38 minutes), and 70% (38-41 minutes). The drug-linked light chain (L1) and heavy chain (H1) eluted in the order of L0, L1, H0, and H1 relative to the unlinked light chain (L0) and heavy chain (H0). The modification rate was calculated based on the peak area at 280 nm.

[0187] Table 2 ADC modification rate of Q-tagged short peptides

[0188] The results showed that after 12 hours of conjugation, the proportion of unconjugated H0 and L0 molecules in the EvHcQ1 antibody was the lowest at 1.09%. After 12 hours of conjugation with LND1002, the proportion of unconjugated H0 and L0 molecules in the EvHcQ2 antibody was 2.27%. After 12 hours of conjugation with the EvHcQ3 antibody, the proportion of unconjugated H0 and L0 molecules was 4.55%. After 12 hours of conjugation with the EvHcQ04 antibody, the proportion of unconjugated H0 and L0 molecules was 2.86%. The EvHcQ1 antibody conjugated with LND1002 had the highest modification rate, indicating that it had the highest DAR and more uniform ADC.

[0189] Example 3: Expression vector construction and plasmid preparation

[0190] 1) PCR clone the target sequence and gel electrophoresis and gel recovery to obtain the target fragment.

[0191] 2) The recovered target fragment was ligated with the plasmid vector PCDNA3.4 (containing the ampicillin resistance gene) to generate a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli (E. coli) and plated onto an ampicillin-resistant plate to screen for positive colonies.

[0192] 3) Randomly select 4 positive colonies for sequence detection, select the positive bacterial liquid of the correct clone for sequencing, expand the culture, and extract the plasmid to prepare for monoclonal antibody expression.

[0193] Example 4: Monoclonal Antibody Expression

[0194] 1) Take 145×10 CHO cells 6 Centrifuge and remove the supernatant.

[0195] 2) Add 0.5 ml of electroporation solution to the cells, mix well, and then add an appropriate amount of plasmid (concentration 500 ng / ul).

[0196] 3) After the above cell plasmid suspension is thoroughly mixed, 1 ml is added to the electroporation tube, and the electroporation tube is placed in the electroporator for electroporation.

[0197] 4) After the electroporation is completed, the cells in the electroporated tube are divided into a shake flask containing 20 ml of culture medium prepared in advance and incubated statically for 40 minutes.

[0198] 5) After incubation, place the shake flask at 37°C, 270 rpm, and 8% CO2. After 24 hours, add feed / sodium butyrate / antibody and continue incubation for 3-7 days. On the 5th day, collect samples for ELISA testing.

[0199] Example 5: Monoclonal Antibody Purification

[0200] 1) Using an AKTA purifier, equilibrate the column with 1x PBS pH 7.0 at a flow rate of 1 ml / min for 20 ml.

[0201] 2) Sample loading: flow rate 1 ml / min;

[0202] 3) Washing: 1× PBS pH 7.0, flow rate 1 ml / min, 20 ml;

[0203] 4) Elution: Sodium acetate buffer (pH 3.4), 1 ml / min, collect approximately 500 μl per tube. Collect 10 tubes in total and read the absorbance at 280 nm using a NanoDrop instrument.

[0204] 5) Dialysis: Pipette the high-concentration protein into a dialysis bag and place it into a beaker of 1×PBS pH 7.0 for dialysis.

[0205] Example 6: ADC conjugation and purification

[0206] Add 20 times the molar equivalent of the antibody to a linker or linker-drug (e.g., LND1002, JSSW038, or linker 1), an antibody (e.g., DP001, Hu17-H2L1, Hu17-H3L1, or Hu17-H4L1, whose amino acid sequences are shown in Tables 24-1 and 24-2), mTGase, and HO to an EP tube. Seal and mix thoroughly, then place at 30°C for no more than 4 days. mTGase catalyzes the reaction between the antibody and the linker-toxin to form an antibody-drug conjugate. When the heavy chain coupling rate exceeds 95%, the conjugation reaction is considered complete and purified immediately. The ADC purification method is the same as that in Example 5.

[0207] Among them, mTGase is wild-type mTGase, and its amino acid sequence is shown in SEQ ID NO:35.

[0208] Example 7: Analysis and identification of ADC physical and chemical properties

[0209] 1. Enzymatic coupling LND1002 DAR value determination

[0210] The antibodies for the ADCs in Table 3-1 were modified using the Her2-targeting monoclonal antibody (DP001) as a template according to the corresponding numbers in Example 1. The drug conjugate portion was LND1002. These antibodies were obtained by coupling and purification according to the methods of Example 6. All antibodies with the same name in the following examples were prepared using this method. The antibodies for the ADCs in Table 3-2 were modified using the ROR1-targeting monoclonal antibody (sequence shown in Table 24-2) as a template according to the corresponding numbers in Example 1. The drug conjugate portion was LND1002. The ADCs were obtained by coupling and purification according to the methods of Example 6.

[0211] A chromatographic column (Agilent PLRP-S, 5μm, 2.1mm*50mm) was selected. Mobile phase A consisted of 0.1% TFA in water and mobile phase B consisted of 0.1% acetonitrile. Gradient elution was performed with a constant ratio of A to B at a flow rate of 0.25mL / min, a column temperature of 80°C, and a detection wavelength of 280nm. The DAR was calculated using the area normalization method, or the intact molecular weight of the ADC was determined by MS. The results are shown in Tables 3-1 and 3-2 below.

[0212] Table 3-1 Summary of DAR values ​​for enzyme coupling

[0213] Table 3-2 Summary of DAR values ​​for enzymatic coupling

[0214] 2. Enzymatic coupling JSSW038 DAR value determination

[0215] The ADC antibodies in Table 4 were modified using the Her2-targeting monoclonal antibody (DP001) as a template according to the corresponding numbers in Example 1. The drug conjugate portion was JSSW038. The ADCs were obtained by coupling and purification according to the method of Example 6. The DAR values ​​were determined according to the method in Example 7.

[0216] Table 4 Summary of DAR values ​​of JSSW038 enzymatically coupled

[0217] The results are shown in Tables 3-1, 3-2, and 4, indicating that the ADCs obtained by enzymatic conjugation of some monoclonal antibodies designed with glutamine point mutations can meet the DAR value requirements. In particular, AB5-ADC, MFC2-ADC, MFC6-ADC, MFC8-ADC, MFC8-JSSW038, MFC9-ADC, MFC10-ADC, MFC12-ADC, MFC13-ADC, MFC14-ADC, MFC15-ADC, DP5-ADC, DP9-ADC, DP12-ADC, DP14-ADC, DP22-ADC, DP24-ADC, DP25-ADC, DP26-ADC, DP28-ADC, DP28-JSSW038, DP35-ADC, DP36-ADC, DP35-JSSW038, DP36-JSSW038, Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002 are close to the theoretical DAR values.

[0218] 3. Coupling site detection

[0219] The ADC sample was enzymatically hydrolyzed using protease (the antibody was modified using the Her2-targeting monoclonal antibody (DP001) as a template according to the corresponding number in Example 1, and the drug conjugated portion was LND1002, prepared according to Example 6). The enzymatically hydrolyzed peptide samples were detected using an ultrahigh performance liquid chromatography (ULC) and a high-resolution mass spectrometer. The original liquid-mass spectrometry (LC-MS / MS) files were analyzed using UNIFI software in UPLC-MS / MS to clarify the modification sites and related modification intensities of the test samples.

[0220] Take 100 μg of this product, add guanidine hydrochloride solution to a final concentration of 6 M, add 1 M dithiothreitol (DTT) to a final concentration of 20 mM, mix well, and react at 37°C for 90 minutes. After cooling to room temperature, add 1 M iodoacetamide (final concentration 50 mM), mix well, and react at room temperature in the dark for 45 minutes. Add 2 M urea (Tris-HCl, pH 7.5) to the upper layer of a 10 kDa ultrafiltration membrane, centrifuge at 13,000 rpm for 10 minutes, discard the waste solution, and repeat the replacement twice. Add trypsin at a ratio of enzyme:protein = 1:25 (w:w), mix well, and react at 37°C for 4 hours. After the reaction is complete, add 0.5 μL of FA to the enzymatic hydrolysis solution to terminate the reaction. Centrifuge and collect the supernatant for analysis.

[0221] Ultra-high performance liquid chromatography (ULC) was performed using an ACQUITY UPLC H-Class PLUS (Waters). Parameters were: column temperature 50°C, flow rate 0.3 ml / min, detection wavelength 214 nm, mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile. A high-resolution mass spectrometer was used, using a Vion Q-TOF (Waters), in MSE acquisition mode, capillary voltage (kV) 3 V, MSE collision energy 20-45 eV, and ion source temperature 120°C. LC-MS / MS raw data were analyzed using UNIFI software.

[0222] The percentage of modified sites is calculated as follows: Percent Q(X)=Area Q(X) / (Area(X)+Area Q(X))×100%. Note: Percent Q(X) is the percentage of amino acid sites X where modifications occur.

[0223] Targeting the HER2 target, the inventors determined the modification rates of each site of DP303c, AB5-ADC, MFC8-ADC, DP21-ADC, MFC14-ADC, DP22-ADC, DP14-ADC, DP23-ADC, DP28-ADC, DP24-ADC, DP25-ADC, DP26-ADC, and DP27-ADC.

[0224] Targeting the Trop2 target, the inventors determined the modification rates of each site of AB5-sacituzumab-ADC, MFC8-sacituzumab-ADC, MFC14-sacituzumab-ADC, DP28-sacituzumab-ADC, DP35-sacituzumab-ADC, and DP36-sacituzumab-ADC.

[0225] Targeting the EGFR target, the inventors determined the modification rates of each site of AB5-cexitumab-ADC, MFC8-cexitumab-ADC, MFC14-cexitumab-ADC, DP28-cexitumab-ADC, DP35-cexitumab-ADC, and DP36-cexitumab-ADC.

[0226] Targeting the TF (Tissue factor) target, the inventors determined the modification rates of each site of AB5-tisotumab-ADC, MFC8-tisotumab-ADC, MFC14-tisotumab-ADC, DP28-tisotumab-ADC, DP35-tisotumab-ADC, and DP36-tisotumab-ADC.

[0227] Targeting the ROR1 target, the inventors determined the modification rates of each site of AB5-cirmtuzumab-ADC, MFC8-cirmtuzumab-ADC, MFC14-cirmtuzumab-ADC, DP28-cirmtuzumab-ADC, DP35-cirmtuzumab-ADC, and DP36-cirmtuzumab-ADC.

[0228] AB5-antibody-ADC, indicating that a GGLQSGA peptide is inserted into the carboxyl terminus of the LC of the antibody constant region; MFC8-antibody-ADC, indicating that the following amino acid modifications are present in the HC of the constant region of the antibody: S354L-R355Q-E356G; MFC14-antibody-ADC, indicating that a GGLQSGA peptide is inserted into the carboxyl terminus of the LC of the antibody constant region and the following amino acid modifications are present in the HC: S354L-R355Q-E356G; DP28-antibody-ADC, indicating that a GGLQSGA peptide is inserted into the carboxyl terminus of the LC of the antibody constant region and the following amino acid modifications are present in the HC: S354L -R355Q-E356G, Q438I and P445Q-G446; DP35-antibody-ADC, indicating that a GGLQSGA peptide is inserted into the carboxyl terminus of the LC of the constant region of the antibody and the following amino acid modifications are present in the HC: S354L-R355Q-E356G and LQSG is inserted after T135; DP36-antibody-ADC, indicating that a GGLQSGA peptide is inserted into the carboxyl terminus of the LC of the constant region of the antibody and the following amino acid modifications are present in the HC: S354L-R355Q-E356G and the following amino acid modifications are present in the LC: G200-L201-S202 are replaced by the LQSG peptide.

[0229] After being coupled to ADC, the modification rate of each site was detected, which showed that the modification rate of each coupling site was stable and uniform.

[0230] Table 5 Modification rate of each coupling site Note: “-” indicates no mutation to glutamine

[0231] In order to identify the coupling modification rate of each mutation position of ADC after coupling purification, peptide mapping analysis was performed on the enzymatically hydrolyzed peptide samples. The results are shown in Table 5, indicating that each mutation site can achieve a high coupling modification rate.

[0232] For DAR4 ADCs, which incorporate a reactive glutamine moiety, such as AB5-ADC (with GGLQSGA inserted at the carboxyl terminus of the LC), the modification rate of the Q-tagged glutamine in the LC (hereinafter referred to as the terminal Qtag) was 100%, and the modification rate of the endogenous glutamine (hereinafter referred to as Q295) in the HC Q295-V296-N297 was 99%. The modification rate at Q295 of MFC8-ADC (HC S354L-R355Q-E356G) was 98%. The S354L-R355Q-E356G mutation exposed the glutamine at Q438, resulting in a modification rate of 89%. The modification rate at Q438 of DP21-ADC (HC P445Q-G446) was 99% at Q295 and 81% at 445Q.

[0233] For DAR6ADC, that is, ADC molecules with two reactive glutamines added, such as MFC14-ADC (HC S354L-R355Q-E356G and LC carboxyl-terminal insertion of GGLQSGA), the modification rate of the terminal Qtag was 100%, the modification rate of Q295 was 93%, and the modification rate of Q438 was 89%; DP22-ADC (HC P445Q-G446 and LC carboxyl-terminal insertion of GGLQSGA) had a terminal Qtag modification rate of 100%, a modification rate of Q295 of 92%, and a modification rate of 445Q of 86%; DP14-ADC (HC S354L-R355Q-E356G and HC P445Q-G446) had a modification rate of Q295 of 99%, but the modification rate of Q438 dropped to 61%, and the modification of 445Q was 100%; DP23-ADC (HC: Q295A and HC For DP28-ADC (HC: Q295, S354L-R355Q-E356G, Q438I, P445Q-G446, and LC with GGLQSGA inserted at the carboxyl terminus), the terminal Qtag modification rate was 100%, the Q438 modification rate was 56%, and the 445Q modification rate was 99.9%. For DP28-ADC (HC: Q295, S354L-R355Q-E356G, Q438I, P445Q-G446, and LC with GGLQSGA inserted at the carboxyl terminus), the terminal Qtag modification rate was 100%, the Q295 modification rate was 97%, and the 445Q modification rate was 100%.

[0234] For DAR 8ADC, that is, ADC molecules with three reactive glutamines added, such as DP24-ADC (HC P445Q-G446 and HC S354L-R355Q-E356G and LC carboxyl terminal inserted with GGLQSGA), the modification rate of the terminal Qtag was 100%, the modification rate of Q295 was 97%, the modification rate of Q438 was 40%, and the modification rate of 445Q was 99%; DP25-ADC (HC H285L-N286Q and HC P445Q-G446 and LC carboxyl terminal inserted with GGLQSGA), the modification rate of the terminal Qtag was 100%; the modification rate of Q295 was 99%, the modification rate of 445Q was 87%, and the modification rate of 286Q was 72%; DP26-ADC (HC H268L-E269Q-D270G-P271G and HC P445Q-G446 and LC carboxyl terminus inserted GGLQSGA), the terminal Qtag modification rate was 100%; the modification rate of Q295 was 99%, the modification rate of 445Q was 89%, and the modification rate of 269Q was 95%;

[0235] For DAR 10ADC, an ADC molecule with four reactive glutamines added, such as DP27-ADC (HC H268L-E269Q-D270G-P271G and HC S354L-R355Q-E356G and HC P445Q-G446 and GGLQSGA inserted at the carboxyl terminus of LC), the modification rate of the terminal Qtag was 100%; the modification rate of Q295 was 98%, the modification rate of Q438 was 39%, the modification rate of 445Q was 99%, and the modification rate of 269Q was 88%.

[0236] In summary, it can be found that the Q438 at the exposed sites of HC P445Q-G446 and HC S354L-R355Q-E356G mutations has a mutual influence on the modification rate of the coupled small molecules due to their close spatial distance. For example, MFC8-ADC and MFC14-ADC both contain HC S354L-R355Q-E356G mutations, but do not contain HC P445Q-G446 mutations, and the modification rate of Q438 is 89%; when HC P445Q-G446 mutation is added to the HC S354L-R355Q-E356G mutation, such as DP14-ADC, DP24-ADC and DP27-ADC, due to HC The presence of P445Q-G446 caused the modification rates of Q438 to decrease to 61%, 40% and 39%, respectively. In addition, in the absence of the HC S354L-R355Q-E356G mutation, such as DP21-ADC, DP22-ADC, DP25-ADC and DP26-ADC, the modification rates of 445Q were 81%, 86%, 87% and 89%, respectively. When the HC S354L-R355Q-E356G mutation was added, such as DP14-ADC, DP24-ADC and DP27-ADC, the modification rates of 445Q increased to 100%, 99% and 99%, respectively. It can be seen that adding the S354L-R355Q-E356G mutation can increase the exposure of the Q438 and 445Q positions. However, since Q438 and 445Q are close in space, their simultaneous presence leads to a decrease in the modification rate of Q438. Therefore, DP28 can increase the modification rate of the 445Q position by converting Q438I.

[0237] It can be seen that the DAR 4 ADC molecules designed in this application, such as AB5-ADC, MFC8-ADC and DP21-ADC, DAR6 ADC molecules, such as MFC14-ADC, DP14-ADC, DP22-ADC molecules, DP28-ADC and DP23-ADC, DAR 8 ADC molecules, such as DP24-ADC, DP25-ADC and DP26-ADC, and DAR 10 ADC molecules, such as DP27-ADC, all achieve multi-site site-specific conjugation, and the modification rate of glutamine at each mutation site is high, providing a new method for the development of high-DAR value site-specific conjugation ADC drugs.

[0238] 4. ADC thermal stability test (DSC)

[0239] The thermal stability of ADC samples can be tested using differential scanning calorimetry (DSC). Before testing, the ADC sample concentration was determined using a NanoDrop ELISA. The ADC sample was diluted to approximately 1 mg / mL with 1× PBS pH 7.0 buffer, filtered through a 0.22 μm filter, and 350 μL of sample was added to each well. Testing was performed using a Malven MicroCal PEAQ-DSC differential calorimeter with the following parameters: scan start temperature: 20°C, scan end temperature: 100°C, and scan rate: 90°C / h. The results are shown in Table 6.

[0240] Table 6 Microcalorimetric differential scanning calorimetry (DSC) test results of enzyme-coupled samples

[0241] The results are shown in Table 6. The Tonset (melting point temperature) of each ADC was approximately 50-60°C, the Tm1 (melting temperature 1) was approximately 55-65°C, and the Tm2 (melting temperature 2) was 80-85°C. Glutamine mutation of the antibody followed by mTGase-catalyzed conjugation to obtain a high DAR ADC had no significant effect on the denaturation temperature of the ADC sample, indicating that the ADC samples obtained by this method have good drugability.

[0242] 5. ADC thermal stability test (DLS)

[0243] The particle size distribution of the ADC samples was determined using a WYATT Dyna Pro Plate Reader III high-throughput automated dynamic and static laser scattering analyzer. Before testing, the samples were slowly mixed and evenly distributed. 80 μL of the sample was pipetted into a 384-well plate, which was then placed in the instrument. The following parameters were used: Experiment Type: Isothermal, DLS Acquisitions per measurement: 10; DLS Aquisitions Time: 5 s; Start Temperature: 25°C. The results are shown in Table 7.

[0244] Table 7 Dynamic Light Scattering (DLS) detection results of enzyme-coupled samples

[0245] The results are shown in Table 7. After the antibody was glutamine mutated, ADCs with different DARs were obtained by mTGase coupling and subjected to DLS detection. The particle size of the ADCs increased slightly, but the increase was not large, and no large particle aggregates were produced, indicating that there was no significant effect on the thermal stability of the sample, suggesting that the ADC samples obtained by this method have good drugability.

[0246] 6. Comparison of thermal aggregation temperature between antibodies and ADCs

[0247] The ADC Hu17-H2L1-4LND1002 in this experiment was prepared by modifying the monoclonal IgG1 antibody targeting ROR1 (Hu17-H2L1) according to the corresponding numbering in Example 1. The drug conjugated portion was LND1002, which was prepared according to Example 6.

[0248] The drug conjugate portion (including the linker) of the positive control UC961-vc MMAE is shown in Formula VIII:

[0249] The preparation process of UC961-vc MMAE is as follows:

[0250] The UC961 antibody was replaced with a 7.0% PBS / 5mM EDTA buffer to a concentration of 10 mg / mL. 3 molar equivalents of TCEP reducing agent were added according to the antibody's molar concentration, and the mixture was heated in a 37°C water bath for 1 hour. 2.5 molar equivalents of vc-MMAE (Formula VIII, CAS No. 646502-53-6) were then added and allowed to react at room temperature for 30 minutes. 2.5 molar equivalents of N-acetyl-L-cysteine ​​were added, and the reaction was stopped after 30 minutes. After stopping the reaction, free small molecules and other impurities were removed by ultrafiltration and concentration, yielding UC961-vc MMAE.

[0251] The drug-antibody coupling ratio (DAR) distribution was determined by hydrophobic chromatography (HIC-HPLC), and the DAR value of UC961-vc MMAE was calculated to be 4.0. The UC961-vc MMAE used in the following examples was prepared using the method of this example.

[0252] The particle size distribution of the samples was determined using a WYATT Dyna Pro Plate Reader III high-throughput automated dynamic and static laser scattering analyzer. The effects of enzymatic and chemical coupling on thermal stability were assessed by changes in Tagg (thermal aggregation temperature). Before testing, the samples were gently mixed and uniformly distributed. 30 μL of sample was pipetted into a 384-well plate, avoiding the introduction of other particles and ensuring that no bubbles were present. The plate was then placed in the instrument for testing. The following setup steps were used: ① Create an experiment; ② Maintain a constant temperature of 25°C; ③ DLS acquisition time of 5 seconds; ④ DLS data acquisition number of 10. The results are shown in Table 8.

[0253] Table 8 Comparison of thermal aggregation temperature between antibodies and ADCs

[0254] The results are shown in Table 8. After the anti-ROR1 antibody Hu17-H2L1 was obtained by enzymatic method and coupled with four LND1002 ADCs, the Tagg (thermal aggregation temperature) decreased from 78.21°C to 76.84°C, a decrease of 1.37°C; after the anti-ROR1 antibody UC961 was obtained by chemical coupling method and coupled with four vc MMAE ADCs, the thermal aggregation temperature decreased from 74.59°C to 54.2°C, a decrease of 20.39°C; the ADC obtained by enzymatic coupling did not affect the interchain disulfide bonds of the antibody, while the ADC obtained by chemical coupling destroyed the interchain disulfide bonds of the antibody, which had a greater impact on the thermal stability of the antibody. The ADC sample obtained by enzymatic coupling had good drugability.

[0255] Example 8: Fab binding activity analysis (Fortebio)

[0256] The affinity of ADC Fab was detected by bio-layer interferometry (BLI).

[0257] Using an Octet RED96e instrument, AHC capture sensors were immobilized with 5 μg / ml of various ADC samples. HER2 antigen was serially diluted, starting at 100 nM and proceeding in a two-fold gradient, for a total of seven steps. The buffer system was PBS pH 7.4 with 0.02% Tween 20. The following steps were used: ① Baseline 60 s, ② Loading 800 s (antibody immobilization, threshold set to 0.50 nm), ③ Baseline 2120 s, ④ Association 120 s, ⑤ Dissociation 300 s, and ⑥ Regeneration 300 s. Blank buffer was used for blank analysis, and the baseline was aligned to the y-axis. Savitzky-Golay fitting was performed in Octet data analysis software. Results are shown in Table 9.

[0258] Table 9 Affinity test results of enzyme-coupled samples using bio-layer interferometry (BLI)

[0259] The results are shown in Table 9. After the antibody was mutated with glutamine to increase the conjugable sites in the constant region, an ADC sample with a high DAR value was obtained by mTGase catalysis. The ADC sample was then subjected to affinity testing with the HER2 antigen. Compared with the ADC sample DP303c without glutamine mutation, the affinity of the ADC sample with a high DAR value for the HER2 antigen was not significantly different. This indicates that glutamine mutation in the constant region and enzymatic conjugation to produce a high DAR value ADC does not affect the affinity of the antibody for the antigen.

[0260] Example 9: In vitro killing activity analysis and identification

[0261] 1) Purpose of the test

[0262] The purpose of this study was to examine the in vitro proliferation inhibitory activity of the antibody-drug conjugate of the present application against SK-BR-3 tumor cells. Cells were treated in vitro with varying concentrations of ADC samples (the ADCs in Table 11 were modified using the Her2-targeting monoclonal IgG1 antibody (DP001) as a template according to the corresponding numbering in Example 1, and the drug conjugate portion was JSSW038, prepared according to Example 6). After incubation, resazurin was added. Fluorescence values ​​at ex550nm / ex610nm were read using a microplate reader. Four-parameter fitting was used to calculate the IC50 value, from which the biological activity of the compound was calculated.

[0263] 1) Test materials and equipment

[0264] Material

[0265] equipment

[0266] 2) Test operation method

[0267] 3.1 Culture medium: DMEM, 10% FBS, 1× double antibody

[0268] 3.2 Cell culture: Take out a frozen SK-BR-3 cell from liquid nitrogen and resuscitate to 75cm 2 Cultured in culture flasks.

[0269] 3.3 Cell Harvest: Harvest SK-BR-3 cells when the culture flask is nearly confluent. Discard the culture medium and wash with PBS to remove dead cells and residual culture medium. Add 2-3 mL of 0.25% Trypsin-EDTA, gently shake the culture flask, and incubate at 37°C for 2-3 minutes to digest the cells.

[0270] 3.4 Cell density determination: Count the cells using a cell counting plate under a microscope.

[0271] 3.5 Inoculation of assay plates

[0272] Dilute the cells to 1 × 10 5 Cells were plated at 100 μL / well in the assay plate (except rows A and H). 120 μL of culture medium was added to each well of rows A and H as a blank control. The cells were incubated at 37°C, 5% CO2 for 4-6 hours to allow the cells to adhere.

[0273] 3.6 Dilution of ADC Sample

[0274] The starting concentration of the ADC sample was set to 18 μg / ml, and then diluted 3-fold to obtain a total of 11 gradient solutions, with the 12th column being the blank control.

[0275] 3.7 Dosing treatment

[0276] Add 20 μL of each ADC sample dilution from columns 1-11 of the dilution plate to columns 1-11 of the assay plate. Add 20 μL of fresh culture medium to column 12 and rows A and H. Gently shake on a plate shaker for 10-15 seconds. Incubate the plates at 37°C for 3 days for ADCs conjugated with LND1002 and 7 days for ADCs conjugated with JSSW038.

[0277] 3.8 Analysis

[0278] After incubation, add 20 μL of 0.03% resazurin (1× PBS dilution) to each well and shake gently for 10-15 seconds. After incubation at 37°C for 3-4 hours, read the plate reader and use EXCEL data to plot the IC values ​​of the reference standard and sample. 50 The unit is ng / mL, and the results are the killing percentage relative to DP303c. The results are shown in Tables 10 and 11.

[0279] Table 10 ADC in vitro killing results

[0280] Table 11 ADC in vitro killing results

[0281] The results are shown in Table 10. After the antibody was glutamine mutated, the mTGase-catalyzed conjugation with LND1002 yielded an ADC sample with a high DAR value. As the DAR value increased, the in vitro cytotoxicity of the ADC increased relative to DP303c, indicating that this method can yield ADC samples with higher cytotoxicity. The results are shown in Table 11. After the antibody was glutamine mutated, the mTGase-catalyzed conjugation with JSSW038 yielded an ADC sample with a high DAR value. As the DAR value increased, the in vitro cytotoxicity of the ADC increased relative to the unmutated DP001-JSSW038, indicating that this method can yield ADC samples with higher cytotoxicity.

[0282] Example 10: Fc affinity evaluation (Fortebio)

[0283] The affinity of ADC Fc and FcγR was detected by bio-layer interferometry (BLI).

[0284] HISIK capture sensors were immobilized with 5 μg / ml of FcRn, RcγRIa (CD64), RcγRIIa (CD32A), RcγRIIb (CD32B), RcγRIIIa (F176), or RcγRIIIa (V176). The test samples were serially diluted, starting at 100 nM and followed by a two-fold dilution series for a total of seven steps. The buffer system was PBS pH 7.4 + 0.02% Tween 20. The setup procedures were the same as in Example 8.

[0285] C1q antibody affinity assay: Coat the sample and perform a three-fold serial dilution, starting at 66.7 μg / ml, for a total of nine dilutions. The buffer system is PBS pH 7.4 + 0.1% Tween 20 + 1% BSA. Procedure: ① Coat overnight at 4°C; ② Block with 100 μl of 3% skim milk powder per well and shake at room temperature for 2 hours; ③ Wash three times with PBST, add 100 μl of 5 μg / ml C1q per well and shake at room temperature for 2 hours; ④ Wash four times with PBST, add 100 μl of 5 μg / ml Shp pAb to C1q (HRP) per well and shake at room temperature for 1 hour; ⑤ Wash four times with PBST, develop with 100 μl of TMB per well for 10 minutes; ⑥ Add 100 μl of 2 M H2SO4 stop solution to each well; ⑦ Read on a microplate reader at OD 450 / 650 nm.

[0286] Table 12 Affinity results of enzyme-coupled samples for FcγRs

[0287] The results are shown in Table 12. After the antibody was glutamine mutated and catalyzed by mTGase, ADC samples with higher DAR values ​​were obtained. There was no significant difference in the affinity of the ADC's Fc to FcγR receptors (FcRn, CD64, CD32A, CD32B, CD16PHE, CD16VAL, C1q), indicating that the conjugation of the antibody to the small molecule will not affect the function of the antibody Fc region.

[0288] Example 11: In vitro plasma stability

[0289] Rat plasma, human plasma, and PBST (negative control) were used as experimental systems. ADC samples were added to these experimental systems and incubated in vitro at 37°C. The stability of the ADC samples was evaluated by measuring the shedding rate of small molecules. Prepared plasma samples and negative control samples were incubated at 37°C. The reaction was terminated by adding 300 μL of ice-cold precipitant to the incubation system at 0 h, 24 h, 72 h, 96 h, 120 h, 144 h, and 168 h. The concentration of small molecules in plasma was determined by LC-MS / MS. The detection method was as follows: 50 μL of the sample to be processed was added to 200 μL of the internal standard working solution under light-shielding conditions. The mixture was vortexed at 2500 rpm for 3 minutes at room temperature, centrifuged at 12000 rpm for 5 minutes at 2-8°C, and 200 μL of the supernatant was sampled and analyzed at room temperature. The results are shown in Table 13.

[0290] Liquid phase conditions: Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile; Chromatographic column: Waters HSS T3 2.5μm, 2.1*50mm Column, flow rate: 0.5mL / min; injection volume: 5μL; autosampler temperature: 4±5℃; column temperature: 40±5℃; elution gradient setting: 0-0.5min: A 85%; 0.5-0.7min: A 85%~45%; 0.7-1.5min: A 45%~5%; 1.5-1.51min: A 5%~85%; 1.51-2min: A 85%;

[0291] Mass spectrometry conditions: Ion source: Electrospray Ionization (ESI); Ionization mode (Ion Mode): positive ion mode; Detection mode (Detection Mode): multiple reaction monitoring (MRM); Spray voltage (Ion Spray Voltage): 5500 V; Turbo Ion Spray Temp (Turbo Ion Spray Temp): 550°C; Curtain gas (Curtain Gas): 35 psi; Collision gas (Collision Gas): 9 psi; Nebulizer gas (Gas1): 60 psi; Auxiliary gas (Gas2): 60 psi; Data acquisition time: 2.0 min.

[0292] Table 13 In vitro plasma stability results of enzyme-coupled samples

[0293] The results are shown in Table 13 and AE in Figure 3. The x-axis represents the incubation time (h) and the y-axis represents the toxin molecule shedding rate. The ADC sample was incubated at 37°C for up to 168 h, and the shedding rate in human plasma and rat plasma was less than 0.5%, indicating that the ADC sample is stable in plasma. The ADC has better safety and can avoid systemic toxicity and adverse reactions caused by premature release of toxin molecules.

[0294] Example 12: Endocytosis of ADC

[0295] MDA-MB-231 cells stably expressing ROR1 were collected and resuspended in DMEM medium. The target cells were gently pipetted several times to resuspend into a single cell suspension. Cell viability and cell count were determined using trypan blue staining. The cell density was adjusted to 1×10 5 cells / mL; 100 μL per well was inoculated in a 96-well cell culture dish, and the number of cells inoculated per well was 1×10 4 ADCs (Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002) were added to 96-well plates at a final concentration of 2 μg / mL and incubated in a 37°C, 5% CO2 incubator for 27 hours. Cells were removed at 3, 6, 9, and 27 hours, suspended by flow cytometry, and fluorescently labeled goat anti-human Alexa Fluro 488 (Thermo) was added. ADCs remaining on the cell surface were detected by flow cytometry. UC961-vc MMAE was used as a positive control.

[0296] The experimental results are shown in Figure 4. All ADCs (Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, and Hu17-H4L1-4LND1002) were internalized in MDA-MB-231-ROR1 cells, and the endocytosis rate of Hu17-H2L1-4LND1002 was better than that of UC961-vc MMAE.

[0297] Example 13: In vivo efficacy of ADC

[0298] 1. In vivo efficacy of H1975 human lung adenocarcinoma transplants

[0299] In this study, female NUNU mice of appropriate age were inoculated with H1975 cells to establish a human lung adenocarcinoma H1975 nude mouse xenograft tumor model. 3At the time of the study, 28 animals with well-growing tumors were selected and divided into four groups (D0) based on tumor volume. A: 7 animals in the solvent control group were given 0.9% sodium chloride injection (0.9% INJNS, solvent control group); B: 7 animals in the UC961-vc MMAE group; C: 7 animals in the Hu17-H2L1-4LND1002 experimental group; and D: 7 animals in the Hu17-H3L1-4LND1002 experimental group. All groups were dosed once a week, on D0 and D7, for a total of two doses, the first dose being 3 mg / kg and the second being 4 mg / kg. After dosing, the mice were weighed and the data were recorded. Tumor growth was dynamically observed by measuring tumor diameter at different times after dosing. The experiment ended on D20 (day 20). After carbon dioxide asphyxiation, the mice were removed and weighed. The experimental results are shown in Table 14 and Figure 5.

[0300] Tumor inhibition rate TWI = (tumor weight of control group - tumor weight of experimental group) / tumor weight of control group * 100%

[0301] Table 14 In vivo efficacy of ADC on H1975 human lung adenocarcinoma xenografts

[0302] ***: P<0.001; **: P<0.01; *: P<0.05

[0303] Under these experimental conditions, the tumor inhibition rates of UC961-vc MMAE, Hu17-H2L1-4LND1002, and Hu17-H3L1-4LND1002 were 51.0%, 65.8%, and 53.7%, respectively. All groups significantly inhibited tumor growth compared to the solvent control group. The Hu17-H2L1-4LND1002 group exhibited a more pronounced inhibitory effect than the positive control UC961-vc MMAE group, while the inhibitory effects of Hu17-H3L1-4LND1002 and the positive control UC961-vc MMAE group were comparable.

[0304] 2. In vivo efficacy in HCC1187 human breast cancer xenografts

[0305] In this experiment, female NUNU mice of appropriate age were inoculated with HCC1187 cells to establish a human breast cancer HCC1187 nude mouse xenograft tumor model. 3At 14:00, 28 animals with well-growing tumors were selected and divided into four groups (D0) based on tumor volume. Seven animals were in the solvent control group and seven animals were in the remaining experimental groups. Each group was intravenously administered with 0.9% sodium chloride injection (0.9% INJ NS, solvent control group), and the positive controls UC961-vc MMAE, Hu17-H2L1-4LND1002, and Hu17-H3L1-4LND1002 (administered on D0, D8, and D12, for a total of three doses). After administration, the mice were weighed and the data were recorded. Tumor growth was dynamically observed by measuring the tumor diameter at different times after administration. At the end of the experiment on D14, the mice were asphyxiated with carbon dioxide, and the tumors were removed and weighed. The results are shown in Table 15 and Figure 6.

[0306] Table 15 In vivo efficacy of ADC against HCC1187 human breast cancer xenografts

[0307] ***: P<0.001; **: P<0.01; *: P<0.05

[0308] Under these experimental conditions, UC961-vc MMAE, Hu17-H2L1-4LND1002, and Hu17-H3L1-4LND1002 achieved tumor inhibition rates of 38.1%, 55.7%, and 47.7%, respectively. All inhibited tumor growth compared to the solvent control group. Compared to the positive control UC961-vc MMAE group, the inhibitory effects of Hu17-H2L1-4LND1002 and Hu17-H3L1-4LND1002 in the experimental groups were more significant.

[0309] Example 14: Rat PK Study

[0310] Male SD rats were divided into 5 groups, with 3 rats in each group. They were given 10 mg / kg ADC drug. The amount of total antibody and MMAE was detected at 0, 0.5, 8, 24, 48, 96 (4d), 168 (7d), and 336 (14d) hours after administration. The concentrations of total antibody and small molecules in plasma were determined by LC-MS / MS method.

[0311] The method for detecting the concentration of small molecules is as in Example 11, and the method for detecting the total antibody is as follows:

[0312] Coating: Add 100 μL / well of coating solution to a 96-well plate, seal the plate with a film sealer, and incubate at 2-8°C for 16-18 hours. Washing: Add 300 μL / well of washing solution to a 96-well plate, repeat 5 times, and pat dry on absorbent paper.

[0313] Blocking: Add 200 μL of blocking solution to each reaction well of a 96-well plate and incubate at 37±2°C for 2 h±10 min.

[0314] Wash plate: Add 300 μL / well of washing solution to a 96-well plate, repeat 4 times, and pat dry on absorbent paper.

[0315] Sample incubation: Add the treated blank, standard curve and other samples into a 96-well plate, 100 μL / well, shake and incubate at 450 rpm and 37 ± 2 °C for 2 h ± 10 min.

[0316] Wash plate: Add 300 μL / well of washing solution to a 96-well plate, repeat 4 times, and pat dry on absorbent paper.

[0317] Detection antibody incubation: Add 100 μL / well of the detection antibody working solution to a 96-well plate and incubate at 450 rpm and 37 ± 2°C for 1 h ± 5 min.

[0318] Wash plate: Add 300 μL / well of washing solution to a 96-well plate, repeat 4 times, and pat dry on absorbent paper.

[0319] Streptavidin-HRP incubation: Add 100 μL of Streptavidin-HRP working solution to a 96-well plate and incubate at 37±2°C with shaking at 450 rpm for 1 h±5 min.

[0320] Wash plate: Add 300 μL / well of washing solution to a 96-well plate, repeat 4 times, and pat dry on absorbent paper.

[0321] Color development and termination: After TMB has stabilized at room temperature, add 100 μL / well to a 96-well plate and incubate at 25 ± 2°C in the dark for 5–25 min. After color development is complete, add 50 μL / well of ELISA stop buffer to terminate color development.

[0322] Read the plate: Use a microplate reader, set the detection wavelength to 450 nm, the reference wavelength to 650 nm, and complete the reading within 10 minutes.

[0323] Table 16 Total anti-pharmacokinetic parameters

[0324] Table 17 MMAE pharmacokinetic parameters

[0325] Table 18 Percentage of free small molecules in total small molecules in ADC

[0326] The results are shown in Tables 16-18 and Figures 9-10. Although the risk of MMAE release increases with the number of coupled small molecules, the rat PK results show that even when the DAR is increased to 6, the Cmax of free MMAE is significantly lower than 1 ng / mL, and the AUC of MMAE in ADCs with different DAR values ​​is (MMAE) AUC (总抗)Below 0.025, after mutation or addition of Q-tag, ADC molecules with different DARs are produced by enzymatic coupling. The free amount of small molecules is very low, and the risk of non-target toxicity is low, indicating that this type of molecule has good safety.

[0327] In the rat PK test, the pharmacokinetic parameters of total antibody showed that ADCs with different DAR values ​​exhibited different pharmacokinetic behaviors, and ADCs with the same DAR value but different conjugation sites also had different pharmacokinetic behaviors. Therefore, the appropriate conjugation site and DAR value can be selected according to the target characteristics and small molecules to achieve optimal efficacy and safety.

[0328] Example 15: Cynomolgus Monkey TK Study

[0329] Cynomolgus monkeys were given a single dose of 6 mg / kg each of the test article Hu17-H2L1-4LND1002 (represented by H2L1-LND in the figure) and the control article UC961-vc MMAE (represented by VLS in the figure). Blood was collected immediately (within 1 minute), 1 hour, 4 hours, 8 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, 168 hours, 336 hours, and 504 hours after dosing to monitor ADC and free MMAE levels. The results are shown in Tables 19 and 20 and Figures 7 and 8.

[0330] The concentration of small molecules in plasma was determined using LC-MS / MS. The assay method was as follows: 50 μL of the sample to be processed was added to 200 μL of the internal standard working solution under light-shielding conditions. The sample was vortexed at 2500 rpm for 3 minutes at room temperature. Centrifuged at 12000 rpm for 5 minutes at 2-8°C. 200 μL of the supernatant was injected and analyzed at room temperature. Liquid phase conditions: Mobile phase A: 0.1% formic acid in water; Mobile phase B: 0.1% formic acid in acetonitrile. Column: Waters HSS T3 2.5μm, 2.1*50mm Column, flow rate: 0.5mL / min; injection volume: 5μL; autosampler temperature: 4±5℃; column temperature: 40±5℃; elution gradient setting: 0-0.5min: A 85%; 0.5-0.7min: A 85%~45%; 0.7-1.5min: A 45%~5%; 1.5-1.51min: A 5%~85%; 1.51-2min: A 85%; mass spectrometry conditions: ion source: Electrospray Ionization (ESI); ionization mode (Ion Mode): positive ion mode; detection mode (Detection Mode): multiple reaction monitoring (MRM); spray voltage (Ion Spray Voltage): 5500V; vortex ion spray temperature (Turbo Ion Spray Temp): 550℃; curtain gas (Curtain Gas): 0. Gas): 35 psi; Collision Gas: 9 psi; Nebulizer Gas (Gas1): 60 psi; Auxiliary Gas (Gas2): 60 psi; Data acquisition time: 2.0 min.

[0331] Table 19 ADC pharmacokinetic parameters Note: Cmax: peak concentration; AUC 0-t , AUC 0-∞ : area under the plasma concentration-time curve; CL: clearance rate; MRT: 0- t : mean residence time; Vss: volume of distribution; Tmax: time to peak; t 1 / 2 :half life

[0332] Table 20 MMAE pharmacokinetic parameters Note: Cmax: peak concentration; AUC 0-t , AUC 0-∞ : area under the plasma concentration-time curve; CL: clearance rate; MRT: 0- t : mean residence time; Vss: volume of distribution; Tmax: time to peak; t 1 / 2 :half life

[0333] Tables 19 and 20, as well as Figures 7 and 8, show that at equivalent doses, H2L1-4LND1002 and UC961-vc MMAE ADC exposure levels were comparable, with an exposure ratio of approximately 1.0. At equivalent doses, H2L1-4LND1002 free small molecule exposure was lower than that of UC961-vc MMAE, with an exposure ratio of only 0.7. This indicates that H2L1-4LND1002 ADC exposure is comparable to that of UC961-vc MMAE ADC, with free small molecule exposure lower than that of UC961-vc MMAE, suggesting a better safety profile for H2L1-4LND1002 and no significant accumulation of free small molecules following administration.

[0334] Example 16: Safety Evaluation Study in Cynomolgus Monkeys

[0335] This study enrolled four age-appropriate cynomolgus macaques (Cynomolgus monkeys) and observed toxicity via intravenous administration of the original control substance, UC961-vc MMAE, and the test substance, Hu17-H2L1-4LND1002. The DAR for both the test and control substances was approximately 4. The dose design is shown in the table below: Three intravenous injections were administered every three weeks. The control substance, UC961-vc MMAE, was administered at doses of 6 mg / kg, 6 mg / kg, and 6 mg / kg, respectively. The experimental group, Hu17-H2L1-4LND1002, was administered at doses of 6 mg / kg, 9 mg / kg, and 9 mg / kg, respectively. Continuous observation was performed post-dose, with general observations twice daily and detailed observations daily. Body weight was observed one day before dosing and weekly thereafter, and hematological and biochemical parameters were assessed. The detailed pre-toxicology testing schedule and results are shown in Table 21 below.

[0336] Example 17: Conjugation and purification of multivalent high-drug-loaded ADCs

[0337] Linker 1, antibody (DP001, AB5, MFC8, MFC14, DP28, DP35 or DP36), mTGase, and H2O equivalent to 20 times the molar equivalent of the antibody were added to the EP tube, and mTGase catalyzed the conjugation reaction between linker 1 and the glutamine of the antibody. After sealing and mixing, place it at room temperature. The reaction time should not exceed 4 days. When the heavy chain coupling rate exceeds 95%, the conjugation reaction is considered complete and the reaction is stopped. The free small molecules and other impurities are then removed by ultrafiltration and concentration. The ADC intermediate after ultrafiltration and concentration is replaced in a buffer solution of PBS7.0 / 5mM EDTA to a concentration of 10 mg / mL. 20 molar equivalents of JSSW-SY (see the structural formula below) are added according to the molar concentration of the antibody. After sealing and mixing, place it at room temperature. After shaking for 24 hours, stop the reaction and purify immediately. The ADC purification method is the same as in Example 6.

[0338] Example 18: Determination of DAR Values ​​for Multivalent High Drug Load ADCs

[0339] The antibodies in the ADC samples in Table 22 were modified using the Her2-targeting monoclonal IgG1 antibody (DP001) as a template according to the corresponding numbers in Example 1. The drug conjugate moiety was JSSW-SY, prepared according to Example 17. The DAR values ​​were determined according to the method in Example 7.

[0340] Table 22 Summary of DAR values ​​of multivalent high drug load ADCs

[0341] Compared with Example 6, after enzymatically coupling a fragment with one or more active linkers such as azide, the active molecule with one or more conjugates is then coupled to the aforementioned fragment through a chemical reaction, which can further improve the DAR value of the target conjugate in the ADC molecule.

[0342] Example 19: Analysis and identification of the killing activity of high drug loading ADC

[0343] The inhibitory activity of the high drug-loaded antibody-drug conjugate SK-BR-3 tumor cell proliferation in vitro in Example 18 was tested using the same experimental method as in Example 9.

[0344] Table 23 In vitro killing results of high drug loading ADC

[0345] The results are shown in Table 23. After the antibody was glutamine mutated, an ADC with a higher drug load was obtained through mTGase enzyme catalysis reaction and chemical reaction. Its killing efficiency was improved compared with the unmutated DP001-JSSW-SY ADC in vitro.

[0346] Table 24-1 Antibody-related sequences involved in this application

[0347] Table 24-2 Antibody-related sequences involved in this application (anti-ROR1 antibodies) Note: Tables 21-1 and 21-2 use the Kabat numbering convention.

[0348] Table 24-3 Antibody-related sequences involved in this application

[0349] Table 24-4 Antibody-related sequences involved in this application

[0350] Table 24-5 mTGase-related sequences involved in this application

[0351] Table 24-6 Sequences of Q-tagged short peptides

Claims

1. A high drug loading antibody-drug conjugate comprising an antibody or an antigen binding fragment thereof and a drug conjugated portion, characterized in that The constant region of the antibody or antigen-binding fragment thereof comprises one or more reactive glutamines, wherein the reactive glutamines are selected from the group consisting of: (i) glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA and EEQYQSG located at the carboxyl terminus of the light chain, the heavy chain, or both the light chain and the heavy chain; (ii) selected from heavy chain 354L-355Q-356G (Q438), heavy chain 445Q-G446, heavy chain 117L-118Q-119G-120G, heavy chain 310L-Q311-312G, heavy chain 399L-400Q-401G, heavy chain 268L-269Q-270G, heavy chain 252Q-253G, heavy chain 444Q-445G-446A, heavy chain 283L-V284-285Q-286G, heavy chain 285L- 286Q, heavy chain 340L-341L-Q342-343G, heavy chain 384L-385Q-386G-387G, heavy chain 268L-269Q-270G-271G, heavy chain 325L-326Q, heavy chain 329Q-330G, heavy chain 360L-361Q-362G, heavy chain 384Q-G385-386G, heavy chain 134L-135Q-136G and heavy chain 282Q converted to reactive glutamine by amino acid modification; (iii) a reactive glutamine selected from the group consisting of light chain 126L-127Q-G128-129G, light chain 205L-206Q-207G-208G, light chain 109L-110Q-111G-112G, and light chain 199L-200Q-201S-202G; (iv) endogenous glutamine at Q295 of the heavy chain; and (v) Substitution of glutamine in the Q-tag LQSG of the light chain G200-L201-S202 and insertion of glutamine in the Q-tag LQSG after T135 of the heavy chain.

2. An engineered antibody, characterized in that The constant region of the antibody comprises one or more reactive glutamines, and the reactive glutamines are selected from the group consisting of: (i) glutamine in a Q-tag selected from the group consisting of GGLQSGA, LQSGA, EEQYGA and EEQYQSG located at the carboxyl terminus of the light chain, the heavy chain, or both the light chain and the heavy chain; (ii) selected from heavy chain 354L-355Q-356G (Q438), heavy chain 445Q-G446, heavy chain 117L-118Q-119G-120G, heavy chain 310L-Q311-312G, heavy chain 399L-400Q-401G, heavy chain 268L-269Q-270G, heavy chain 252Q-253G, heavy chain 444Q-445G-446A, heavy chain 283L-V284-285Q-286G, heavy chain 285L- 286Q, heavy chain 340L-341L-Q342-343G, heavy chain 384L-385Q-386G-387G, heavy chain 268L-269Q-270G-271G, heavy chain 325L-326Q, heavy chain 329Q-330G, heavy chain 360L-361Q-362G, heavy chain 384Q-G385-386G, heavy chain 134L-135Q-136G, and heavy chain 282Q converted to reactive glutamine by amino acid modification; (iii) a reactive glutamine selected from the group consisting of light chain 126L-127Q-G128-129G, light chain 205L-206Q-207G-208G, light chain 109L-110Q-111G-112G, and light chain 199L-200Q-201S-202G; (iv) endogenous glutamine at Q295 of the heavy chain; and (v) Substitution of glutamine in the Q-tag LQSG of the light chain G200-L201-S202 and insertion of glutamine in the Q-tag LQSG after T135 of the heavy chain.

3. The antibody-drug conjugate of claim 1 or the antibody of claim 2, wherein the reactive glutamine comprises endogenous glutamine at heavy chain Q295 and a reactive glutamine selected from one or more of the following: (1) a glutamine in a Q-tag selected from the group consisting of GGLQSGA, LQSGA, EEQYGA, and EEQYQSG located at the carboxyl terminus of the light chain; (2) replacing glutamine in the Q-tagged LQSG of the light chain G200-L201-S202; (3) glutamine in the Q-tagged LQSG inserted after T135 of the heavy chain; and (4) a reactive glutamine selected from heavy chain 354L-355Q-356G (Q438), heavy chain 445Q-G446, and heavy chain 268L-269Q-270G-271G converted by amino acid modification; preferably, the endogenous glutamine in heavy chain Q295 is the endogenous glutamine in heavy chain Q295-V296-N297.

4. The antibody-drug conjugate or antibody of any one of claims 1 to 3, wherein the reactive glutamine comprises a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA and EEQYQSG at the carboxyl terminus of the light chain and an endogenous glutamine in Q295-V296-N297 of the heavy chain.

5. The antibody-drug conjugate or antibody of any one of claims 1 to 3, wherein the reactive glutamine comprises reactive glutamine in heavy chain 354L-355Q-356G (Q438) converted by amino acid modification and endogenous glutamine in heavy chain Q295-V296-N297.

6. The antibody-drug conjugate or antibody of any one of claims 1 to 3, wherein the reactive glutamine comprises a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA and EEQYQSG located at the carboxyl terminus of the light chain, an endogenous glutamine in Q295-V296-N297 of the heavy chain, and a reactive glutamine in 354L-355Q-356G (Q438) of the heavy chain that is converted by amino acid modification.

7. The antibody-drug conjugate or antibody of any one of claims 1 to 3, wherein the reactive glutamine comprises a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA and EEQYQSG located at the carboxyl terminus of the light chain, endogenous glutamine in Q295-V296-N297 of the heavy chain, and reactive glutamine converted by amino acid modification in 354L-355Q-356G (Q438) and 445Q-G446 of the heavy chain; preferably, further comprising amino acid modification of Q438I of the heavy chain.

8. The antibody-drug conjugate or antibody of any one of claims 1 to 3, wherein the reactive glutamine comprises a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA and EEQYQSG located at the carboxyl terminus of the light chain, an endogenous glutamine in Q295-V296-N297 of the heavy chain, a reactive glutamine in 354L-355Q-356G (Q438) of the heavy chain converted by amino acid modification, and a glutamine in the LQSG Q-tag inserted after T135 of the heavy chain.

9. The antibody-drug conjugate or antibody of any one of claims 1 to 3, wherein the reactive glutamine comprises a glutamine in a Q-tag selected from GGLQSGA, LQSGA, EEQYGA and EEQYQSG located at the carboxyl terminus of the light chain, an endogenous glutamine in Q295-V296-N297 of the heavy chain, a reactive glutamine in 354L-355Q-356G (Q438) of the heavy chain converted by amino acid modification, and a glutamine in a Q-tag replacing LQSG of G200-L201-S202 of the light chain.

10. The antibody-drug conjugate or antibody according to any one of claims 1 to 9, characterized in that The reactive glutamine comprises reactive glutamine in heavy chain 354L-355Q-356G (Q438) and heavy chain 445Q-G446 converted by amino acid modification; preferably, the constant region of the antibody or antigen-binding fragment thereof further comprises 438I amino acid modification.

11. The antibody-drug conjugate or antibody according to any one of claims 1 to 10, characterized in that The reactive glutamine comprises reactive glutamine in 445Q-G446 of the heavy chain converted by amino acid modification, and K is removed from the C-terminus of the heavy chain.

12. The antibody-drug conjugate or antibody according to any one of claims 1 to 3, characterized in that The reactive glutamine comprises a glutamine in a Q-containing tag selected from GGLQSGA, LQSGA, EEQYGA and EEQYQSG located at the carboxyl terminus of the heavy chain and / or the light chain; preferably, the heavy chain is firstly removed of K and then connected to the Q-containing tag.

13. The antibody-drug conjugate or antibody according to any one of claims 1 to 12, wherein the antibody is an IgG1 type antibody.

14. The antibody-drug conjugate of any one of claims 1 and 3-13, wherein the antibody portion and the drug conjugate portion are connected via a linker, wherein the linker contains an amino group and is covalently linked to a reactive glutamine in the antibody via the amino group.

15. The antibody-drug conjugate of any one of claims 1 and 3-14, which has the following structure: A-(NH-LX)d, wherein A is the antibody or antigen-binding fragment thereof as described in any one of claims 1-14, -NH-L- is a linker, X is a drug molecule, and d is 1-16, preferably 1-12, more preferably 1-8, such as an integer or decimal of 1, 2, 3, 4, 5, 6, 7, or 8.

16. The antibody-drug conjugate of any one of claims 1 and 3-15, wherein the antibody portion is connected to the drug conjugate portion via a linker, and the linker is a cleavable linker or a non-cleavable linker; when the linker is a cleavable linker, the cleavable linker includes but is not limited to NH2-(CH2CH2O) n -Val-Cit-PABC-X and NH2-(CH2CH2O) n -(Val-Cit-PABC-X)2, wherein X is a drug molecule, and n is an integer of at least 1 (such as any one of 2, 3, 4, 6, 8, 10, 12, 16, 20 or 24), PABC refers to p-aminobenzyloxycarbonyl, Val refers to valine, and Cit refers to citrulline; when the linker is a non-cleavable linker, the non-cleavable linker includes but is not limited to NH2-RX, NH2NH-RX and NH2-ORX, wherein R is an alkyl group or a polyethylene glycol group (PEG), X is a drug molecule, and the polyethylene glycol group may have -(CH2CH2O) n -, wherein n is an integer of at least 1 (such as any one of 2, 4, 6, 8, 10, 12, 16, 20 or 24).

17. The antibody-drug conjugate or antibody of any one of claims 1 to 16, wherein the antibody is a monoclonal antibody, a polyclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a minibody, or a bispecific antibody or an antigen-binding fragment thereof.

18. The antibody-drug conjugate or antibody according to any one of claims 1 to 17, characterized in that The two heavy chains and / or the two light chains of the antibody are both inserted with the Q-tag or both contain the amino acid modification; preferably, the Q-tag or the amino acid modification is the same in the two heavy chains and / or the two light chains.

19. The antibody-drug conjugate of any one of claims 1 and 3-18, wherein the drug molecule of the drug conjugated portion is an anticancer drug, including but not limited to a cytotoxic drug, an immunopotentiator or a radioisotope; Preferably, the cytotoxic drug is selected from the group consisting of tubulin inhibitors, DNA topoisomerase inhibitors, DNA damaging agents, immunopotentiators, antimetabolites and antitumor antibiotics; Further preferably, the tubulin inhibitor is selected from auristatin derivatives (such as MMAE (Monomethyl auristatin E), MMAF (Monomethyl auristatin F)) or maytansine alkaloid derivatives (such as DM1, DM4, Ansamitocin, Mertansine and dolastatin peptide (dolastatin) and its derivatives); The DNA topoisomerase inhibitor is selected from camptothecin analogs and DNA topoisomerase I inhibitors and derivatives thereof, including but not limited to DXD, SN38, irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, 22-hydroxycamptothecin, topotecan, lortotecan, belotecan, exitecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxy)-1,2-diol 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide and their derivatives; The DNA damaging agent is selected from calicheamicin, duocarmycin and anthramycin derivative PBD (pyrrolobenzodiazepine); The immunopotentiator is selected from levamisole, pidotimod, imiquimod, isoprenoid, polyinosinic acid, and polyinosinic acid; The antimetabolite is selected from methotrexate, 6-mercaptopurine and 5-fluorouracil; The radioisotope is selected from 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P. 60 Co and 177 Lu; The anti-tumor antibiotic is selected from polypeptide antibiotics (such as actinomycin D or bleomycin) and anthraquinone drugs (such as doxorubicin or mitoxantrone hydrochloride).

20. The antibody-drug conjugate according to any one of claims 1 and 3-19, wherein the antibody-drug conjugate has the following structure:

21. The antibody-drug conjugate or antibody of any one of claims 1-20, wherein the antibody is an anti-HER2 antibody, an anti-Nectin 4 antibody, or an anti-ROR1 antibody.

22. The antibody-drug conjugate or antibody of claim 21, wherein the anti-HER2 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO: 1, a heavy chain CDR2 as shown in SEQ ID NO: 2, and a heavy chain CDR3 as shown in SEQ ID NO: 3, and the light chain variable region comprises a light chain CDR1 as shown in SEQ ID NO: 4, a light chain CDR2 as shown in SEQ ID NO: 5, and a light chain CDR3 sequence as shown in SEQ ID NO: 6; Preferably, the anti-HER2 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 7, and / or comprises a light chain variable region sequence as shown in SEQ ID NO: 8; Further preferably, the anti-HER2 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is subjected to reactive glutamine mutation based on the heavy chain constant region sequence shown in SEQ ID NO:9, and / or the light chain constant region is subjected to reactive glutamine mutation based on the light chain constant region sequence shown in SEQ ID NO:10; More preferably, the anti-HER2 antibody is based on the light and heavy chain amino acids of DP001 or trastuzumab and undergoes reactive glutamine mutation.

23. The antibody-drug conjugate or antibody of claim 21, wherein the anti-nectin 4 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO: 11, a heavy chain CDR2 as shown in SEQ ID NO: 12, and a heavy chain CDR3 as shown in SEQ ID NO: 13, and the light chain variable region comprises a light chain CDR1 as shown in SEQ ID NO: 14, a light chain CDR2 as shown in SEQ ID NO: 15, and a light chain CDR3 sequence as shown in SEQ ID NO: 16; Preferably, the anti-nectin 4 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 17, and / or comprises a light chain variable region sequence as shown in SEQ ID NO: 18; Further preferably, the anti-nectin 4 antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is subjected to reactive glutamine mutation based on the heavy chain constant region sequence shown in SEQ ID NO: 19, and / or the light chain constant region is subjected to reactive glutamine mutation based on the light chain constant region sequence shown in SEQ ID NO: 20; More preferably, the anti-Nectin4 antibody is based on the light and heavy chain amino acid sequences of Enfortumab and undergoes reactive glutamine mutation.

24. The antibody-drug conjugate or antibody of claim 21, wherein the anti-ROR1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 as shown in SEQ ID NO: 21, a heavy chain CDR2 as shown in SEQ ID NO: 22, SEQ ID NO: 29 or SEQ ID NO: 31, and a heavy chain CDR3 as shown in SEQ ID NO: 23, and the light chain variable region comprises a light chain CDR1 as shown in SEQ ID NO: 24, a light chain CDR2 as shown in SEQ ID NO: 25, and a light chain CDR3 sequence as shown in SEQ ID NO: 26; Preferably, the anti-ROR1 antibody comprises a heavy chain variable region as shown in SEQ ID NO: 27, SEQ ID NO: 30 or SEQ ID NO: 32, and / or comprises a light chain variable region sequence as shown in SEQ ID NO: 28; Further preferably, the anti-ROR1 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain constant region sequence as shown in SEQ ID NO:33, and the light chain comprises a light chain constant region sequence as shown in SEQ ID NO:

34.

25. The antibody-drug conjugate of any one of claims 1 and 3-24, wherein the drug molecule is DXD or a derivative thereof, MMAE or a derivative thereof.

26. The antibody-drug conjugate of claim 25, wherein the linker is NH2-(CH2CH2O) n -Val-Cit-PABC-X, X is a drug molecule; preferably, n is 3.

27. The antibody-drug conjugate of claim 25 or 26, wherein the drug conjugating moiety is JSSW-038 or LND1002.

28. The antibody-drug conjugate of claim 27, wherein the antibody-drug conjugate is AB5-ADC, MFC8-ADC, MFC10-ADC, DP28-ADC, DP35-ADC, DP36-ADC, DP35-JSSW038, DP36-JSSW038, MFC8-JSSW038, DP28-JSSW038, Hu17-H2L1-4LND1002, Hu17-H3L1-4LND1002, or Hu17-H4L1-4LND1002.

29. A method of preparing an antibody-drug conjugate or an antibody as claimed in any one of claims 1 to 28, comprising the step of making one or more insertion or substitution mutations to the constant region of the antibody to obtain reactive glutamine.

30. A method for preparing an antibody-drug conjugate as claimed in any one of claims 1 and 3-29, further comprising the steps of preparing an antibody and enzymatically coupling the drug conjugate moiety by mTGase to obtain an ADC.

31. A method for preparing an antibody-drug conjugate with a high drug load, the method comprising the step of preparing the antibody-drug conjugate by the method of claim 29 or 30, and further coupling the drug conjugation part by a chemical method to obtain an ADC with a higher DAR value; preferably, the chemical method is selected from one or more of click chemistry, amide condensation and Michael addition, and more preferably, the chemical method is click chemistry achieved by azide-cycloalkyne addition reaction.

32. The method of claim 31, wherein the drug conjugating moiety is JSSW-SY.

33. Use of the antibody-drug conjugate or antibody according to any one of claims 1 to 32 in the preparation of a medicament for treating cancer.

34. The use of claim 33, wherein the cancer is a solid tumor or a hematological tumor.

35. The use of claim 33 or 34, wherein the cancer is selected from: HER2-positive cancers, such as breast cancer, colorectal cancer, ovarian cancer, gastric cancer, urethral cancer and lung cancer (including small cell lung cancer and non-small cell lung cancer); Nectin 4-positive cancers, such as breast cancer, pancreatic cancer, lung cancer, ovarian cancer, gastric cancer; or ROR1-positive cancers, such as chronic lymphocytic leukemia, mantle cell lymphoma, ovarian cancer, breast cancer, prostate cancer, lung cancer, melanoma and colorectal cancer, etc.

36. An acyl-donor substrate for transglutaminase containing a Q tag, wherein the Q tag is selected from: (i) GGLQSGA, LQSGA, EEQYGA and EEQYQSG at the carboxyl terminus of the light chain, the heavy chain or both; (ii) Heavy chain 354L-355Q-356G (Q438), heavy chain 445Q-G446, heavy chain 117L-118Q-119G-120G, heavy chain 310L-Q311-312G, heavy chain 399L-400Q-401G, heavy chain 268L-269Q-270G, heavy chain 252Q-253G, heavy chain 444Q-445G-446A, heavy chain 283L-V284-285Q-286 G, heavy chain 285L-286Q, heavy chain 340L-341L-Q342-343G, heavy chain 384L-385Q-386G-387G, heavy chain 268L-269Q-270G-271G, heavy chain 325L-326Q, heavy chain 329Q-330G, heavy chain 360L-361Q-362G, heavy chain 384Q-G385-386G, heavy chain 134L-135Q-136G, heavy chain 282Q; (iii) light chain 126L-127Q-G128-129G, light chain 205L-206Q-207G-208G, light chain 109L-110Q-111G-112G, and light chain 199L-200Q-201S-202G; and (iv) LQSG containing a Q tag replacing G200-L201-S202 of the light chain, and LQSG containing a Q tag inserted after T135 of the heavy chain.

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