Modified antibodies, antibody fragments thereof, and antibody-drug conjugates

Abstract

Modified antibodies, antibody fragments thereof, and antibody-drug conjugates (ADCs) are provided, in which one or more of the heavy chain disulfide bonds are moved to a region that cannot access the solvent.

Description

Technical Field

[0001] The present disclosure relates to modified antibodies, antibody fragments thereof, and antibody-drug conjugates (ADCs) in which one or more of the heavy-chain to light-chain disulfide bonds are moved to a region inaccessible to the solvent.

Background Art

[0002] Antibody-drug conjugates (ADCs) have attracted attention as an important class of biopharmaceuticals for the targeted treatment of cancer. So far, 14 ADCs have been approved by the US FDA. An ADC is a complex molecule composed of an antibody linked to a biologically active cytotoxic agent. Conventional strategies for drug conjugation (i.e., lysine- and interchain cysteine-based conjugation to achieve the average drug-to-antibody ratio (DAR) at the target level) have been plagued by the heterogeneity of the conjugation sites and the conjugated drugs. The development of site-specific conjugation has enabled the preparation of homogeneous ADCs. However, most of these strategies require re-engineering of the antibody sequence, multi-step operations, and expensive substrates, thus limiting their use in the industry.

[0003] Therefore, there is a need for modified antibodies and antibody fragments thereof that facilitate the preparation of homogeneous ADCs.

Summary of the Invention

[0004] The present disclosure relates to modified antibodies, antibody fragments thereof, and ADCs in which one or more of the heavy-chain to light-chain disulfide bonds are moved to a region inaccessible to the solvent. In some embodiments, the modified antibodies and antibody fragments thereof can facilitate the preparation of homogeneous ADCs.

[0005] In some embodiments, one or more heavy-chain-light-chain disulfide bonds are moved to a region inaccessible to the solvent, and as a result, the new disulfide bonds are not cleaved by a reducing agent under typical reducing conditions (e.g., excessive TCEP). In some embodiments, unnatural disulfide bonds are formed at the positions of heavy-chain and light-chain amino acid residues. In some embodiments, the region inaccessible to the solvent is the region where these disulfide bonds are not cleaved under typical reducing conditions. In some embodiments, the relative solvent exposure area (SASA) of the side chains of candidate amino acid pairs in the structure of the Fab fragment is calculated. In some embodiments, a disulfide bond is classified as "solvent accessible" if (1) the sum of the relative SASA of the two amino acid side chains is greater than 65%. In some embodiments, a disulfide bond is classified as "solvent inaccessible" if (1) the sum of the relative SASA of the two amino acid side chains is less than 65%. In some embodiments, the sum of the relative SASA of the two amino acid side chains is less than 65%. In some embodiments, the relative SASA of the first amino acid side chain is greater than 40%, and the relative SASA of the second amino acid side chain is less than 10%. In some embodiments, the relative SASA of the first amino acid side chain is greater than 30%, and the relative SASA of the second amino acid side chain is less than 15%. In some embodiments, the relative SASA of each of the two amino acid side chains is less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, or less than 45%.

[0006] In one aspect, the present disclosure is an antibody-drug conjugate (ADC) comprising a payload and an antibody or an antibody fragment thereof (e.g., an antigen-binding fragment), or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or the antibody fragment thereof comprises non-native disulfide bonds formed at positions of heavy-chain amino acid residues and light-chain amino acid residues, wherein the sum of (1) the relative solvent accessible surface area (SASA) of the side chains of the heavy-chain amino acid residues and (2) the relative SASA of the side chains of the light-chain amino acid residues is less than 65%, less than 60%, or less than 55%, wherein the antibody or the antibody fragment thereof does not have a disulfide bond between the amino acid residue at position 220 of the heavy-chain constant region and the amino acid residue at position 214 of the light-chain constant region, and wherein the amino acid positions are based on EU numbering, relates to an ADC or a pharmaceutically acceptable salt or solvate thereof.

[0007] In some embodiments, the relative SASA of the heavy-chain amino acid side chains is greater than 40%, and the relative SASA of the light-chain amino acid side chains is less than 10%, or the relative SASA of the light-chain amino acid side chains is greater than 40%, and the relative SASA of the heavy-chain amino acid side chains is less than 10%.

[0008] In some embodiments, the relative SASA of the heavy-chain amino acid side chains is greater than 30%, and the relative SASA of the light-chain amino acid side chains is less than 15%, or the relative SASA of the light-chain amino acid side chains is greater than 30%, and the relative SASA of the heavy-chain amino acid side chains is less than 15%.

[0009] In some embodiments, the relative SASA of each of the amino acid side chains of the heavy chain and the light chain is less than 20%, less than 25%, less than 30%, less than 35%, less than 40, or less than 45%.

[0010] In some embodiments, the amino acid residue at position 220 of the heavy-chain constant region is a non-cysteine residue, or the amino acid residue at position 214 of the light-chain constant region is a non-cysteine residue, and wherein the amino acid positions are based on EU numbering.

[0011] In some embodiments, the non-natural disulfide bond is resistant to reduction by reducing agents such as tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and / or dithiothreitol (DTT).

[0012] In some embodiments, when an antibody or antibody fragment is reduced with 10 to 30 equivalents of TCEP per single antibody molecule, more than 90% or more than 95% of the non-natural disulfide bonds remain intact.

[0013] In some embodiments, the concentration of the antibody or antibody fragment is 0.1 to 50 mg / ml during the reduction reaction.

[0014] In some embodiments, non-natural disulfide bonds remain stable under reducing conditions that can reduce more than 90% of the hinge region disulfide bonds of an antibody or antibody fragment.

[0015] In some embodiments, at least one non-cysteine ​​amino acid residue in the heavy chain is mutated to a cysteine ​​residue.

[0016] In some embodiments, the non-cysteine ​​amino acid residue is located at a position selected from the group consisting of 126, 168, 170, 173, 134, 133, 130, and 141, where the amino acid position is based on EU numbering.

[0017] In some embodiments, the antibody or antibody fragment contains one of the following mutations in the heavy chain constant region: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C.

[0018] In some embodiments, the antibody or antibody fragment comprises two heavy chains, each containing one or more of the following mutations in the heavy chain constant region: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C.

[0019] In some embodiments, the antibody or antibody fragment comprises two heavy chains, with only one heavy chain containing one of the following mutations in the heavy chain constant region: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C.

[0020] In some embodiments, at least one non-cysteine ​​amino acid in the constant region of the light chain of the antibody or an antibody fragment is mutated to a cysteine ​​residue.

[0021] In some embodiments, at least one non-cysteine ​​amino acid is located at a position selected from the group consisting of 124, 164, 162, 116, 209, 117, and 118.

[0022] In some embodiments, the antibody or antibody fragment contains one or more of the following mutations in the light chain constant region: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C.

[0023] In some embodiments, the antibody or antibody fragment comprises two light chains, each containing one or more of the following mutations in the light chain constant region: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C.

[0024] In some embodiments, the antibody or antibody fragment comprises two light chains, and only one of the light chains contains one or more of the following mutations in its constant region: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C.

[0025] In some embodiments, the antibody or antibody fragment comprises one or more of the following sets of mutations: In the heavy chain, F126C, and in the light chain, Q124C, In the heavy chain, H168C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, S162C, In the heavy chain, V173C, and in the light chain, S162C, In the heavy chain, S134C, and in the light chain, F116C, In the heavy chain, K133C, and in the light chain, F209C, In the heavy chain, K133C, and in the light chain, I117C, In the heavy chain, P130C, and in the light chain, F118C, In the heavy chain, A141C is used, and in the light chain, F116C is used.

[0026] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, and only one pair of these heavy and light chains contains one of the following sets of mutations: In the heavy chain, F126C, and in the light chain, Q124C, In the heavy chain, H168C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, S162C, In the heavy chain, V173C, and in the light chain, S162C, In the heavy chain, S134C, and in the light chain, F116C, In the heavy chain, K133C, and in the light chain, F209C, In the heavy chain, K133C, and in the light chain, I117C, In the heavy chain, P130C, and in the light chain, F118C, In the heavy chain, A141C, and in the light chain, F116C.

[0027] In some embodiments, the antibody or antibody fragment comprises two pairs, a heavy chain and a light chain, each pair of which contains one or more of the following sets of mutations: In the heavy chain, F126C, and in the light chain, Q124C, In the heavy chain, H168C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, S162C, In the heavy chain, V173C, and in the light chain, S162C, In the heavy chain, S134C, and in the light chain, F116C, In the heavy chain, K133C, and in the light chain, F209C, In the heavy chain, K133C, and in the light chain, I117C, In the heavy chain, P130C, and in the light chain, F118C, In the heavy chain, A141C, and in the light chain, F116C.

[0028] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain comprising F126C and each light chain comprising Q124C.

[0029] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain containing H168C and each light chain containing T164C.

[0030] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain containing F170C and each light chain containing T164C.

[0031] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain comprising F170C and each light chain comprising S162C.

[0032] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain comprising V173C and each light chain comprising S162C.

[0033] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain containing S134C and each light chain containing F116C.

[0034] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain containing K133C and each light chain containing F209C.

[0035] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain containing K133C and each light chain containing I117C.

[0036] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain containing P130C and each light chain containing F118C.

[0037] In some embodiments, the antibody or antibody fragment comprises two pairs of heavy and light chains, each heavy chain containing A141C and each light chain containing F116C.

[0038] In one embodiment, the disclosure relates to an ADC comprising a non-natural disulfide bond formed at the positions of a heavy chain amino acid residue and a light chain amino acid residue, wherein the antibody or antibody fragment has no disulfide bond between the amino acid residue at position 220 of the heavy chain constant region and the amino acid residue at position 214 of the light chain constant region, wherein when the antibody or antibody fragment is exposed to reducing conditions that reduce more than 90% or more of the hinged disulfide bond, more than 90% or more of the non-natural disulfide bond remains intact.

[0039] In some embodiments, the antibody or antibody fragment comprises one or more of the following sets of heavy chain mutations: F126C and C220S, H168C and C220S, F170C and C220S, V173C and C220S, S134C and C220S, K133C and C220S, P130C and C220S, and A141C and C220S.

[0040] In some embodiments, the antibody or antibody fragment comprises one or more of the following sets of light chain mutations: Q124C and C214S; T164C and C214S, S162C and C214S, F116C and C214S, F209C and C214S, I117C and C214S, and F118C and C214S.

[0041] In some embodiments, the antibody or antibody fragment comprises one or more of the following sets of mutations: In the heavy chain, F126C and C220S, and in the light chain, Q124C and C214S, In the heavy chain, H168C and C220S, and in the light chain, T164C and C214S, In the heavy chain, F170C and C220S, and in the light chain, T164C and C214S, In the heavy chain, F170C and C220S, and in the light chain, S162C and C214S, In the heavy chain, V173C and C220S, and in the light chain, S162C and C214S, In the heavy chain, S134C and C220S, and in the light chain, F116C and C214S, In the heavy chain, K133C and C220S, and in the light chain, F209C and C214S, In the heavy chain, K133C and C220S, and in the light chain, I117C and C214S, In the heavy chain, P130C and C220S, and in the light chain, F118C and C214S, The heavy chain contains A141C and C220S, and the light chain contains F116C and C214S.

[0042] In some embodiments, the antibody or antibody fragments include Fab fragments, (Fab)2 fragments, one-armed antibodies, and / or multispecific antibodies (e.g., bispecific antibodies).

[0043] In some embodiments, the antibody or antibody fragment is a human or humanized antibody or antibody fragment, a Fab fragment, a (Fab)2 fragment, a one-armed antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

[0044] In some embodiments, the antibody or antibody fragment is a human IgG1, IgG2, or IgG4 antibody or antibody fragment.

[0045] In some embodiments, the antibody or antibody fragment includes a fragment crystallizable region (Fc region).

[0046] In some embodiments, the heavy chain constant region is the constant region of (human) IgG1, IgG2, IgG3, or IgG4, for example, the constant region of IgG1. In some embodiments, the IgG1 heavy chain constant region suitable for the antibody or ADC of the present invention is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in Sequence ID No. 93, (ii) The amino acid sequence of SEQ ID NO: 93, or (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, and 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of Sequence ID No. 93, or comprising:

[0047] In some embodiments, the light chain steady region is the (human) kappa or lambda steady region. In some embodiments, the human kappa steady region is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of Sequence ID No. 88, (ii) The amino acid sequence of SEQ ID NO: 88, or (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, and 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of Sequence ID No. 88, or comprising:

[0048] In some embodiments, the human lambda steady-state region is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 89 (ii) The amino acid sequence of SEQ ID NO: 89, or (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, and 1 or fewer) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of Sequence ID No. 89, or comprising:

[0049] In some embodiments, the payload may be a cytotoxic or cell proliferation inhibitor, a biologically active protein, a synthetic polymer, an enzyme, a nucleic acid (e.g., DNA or RNA) or a fragment thereof.

[0050] In some embodiments, the drug-to-antibody ratio (DAR) is 3.74–4.

[0051] In some embodiments, the drug-to-antibody ratio (DAR) is between 5.70 and 6.

[0052] In some embodiments, the payload is covalently bound to an antibody or an antibody fragment via a thiol-based conjugation.

[0053] In one embodiment, the present disclosure relates to an antibody or antibody fragment comprising a non-natural disulfide bond formed at the positions of a heavy chain amino acid residue and a light chain amino acid residue, wherein (1) the sum of the relative solvent exposure area (SASA) of the heavy chain amino acid residue side chain and (2) the relative SASA of the light chain amino acid residue side chain is less than 65%, less than 60%, or less than 55%, wherein the antibody or antibody fragment does not have a disulfide bond between the amino acid residue at position 220 of the heavy chain constant region and the amino acid residue at position 214 of the light chain constant region, wherein the amino acid positions are based on EU numbering.

[0054] In some embodiments, the relative SASA of the heavy amino acid side chain is greater than 40% and the relative SASA of the light amino acid side chain is less than 10%, or the relative SASA of the light amino acid side chain is greater than 40% and the relative SASA of the heavy amino acid side chain is less than 10%.

[0055] In some embodiments, the relative SASA of the heavy amino acid side chain is greater than 30%, and the relative SASA of the light amino acid side chain is less than 15%, and the relative SASA of the light amino acid side chain is greater than 30%, and the relative SASA of the heavy amino acid side chain is less than 15%.

[0056] In some embodiments, the relative SASA of the heavy chain and the light chain amino acid side chains is less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, or less than 45%.

[0057] In one embodiment, the present disclosure relates to an antibody-drug conjugate comprising an antibody or antibody fragment described herein, covalently bound to a payload.

[0058] In one embodiment, this disclosure relates to nucleic acids encoding the light chain or heavy chain of an antibody or antibody fragment described herein.

[0059] In one embodiment, the present disclosure relates to a vector comprising a nucleic acid as described herein, wherein the nucleic acid is operably linked to a promoter.

[0060] In one aspect, this disclosure relates to cells comprising nucleic acids or vectors described herein.

[0061] In one embodiment, the present disclosure relates to a method for generating an antibody or an antibody fragment thereof, the method comprising culturing cells as described herein under conditions sufficient to cause the cells to generate an antibody or antibody fragment, and collecting the antibody or antibody fragment produced by the cells.

[0062] In one embodiment, the present disclosure relates to a method for preparing an antibody-drug conjugate (ADC), comprising the following steps: method, (a) Incubating the antibody or antibody fragment described herein with a reducing agent to reduce the natural interchain disulfide bonds in the antibody or antibody fragment to produce reduced thiol groups, (b) The process includes introducing an excess amount of a payload having a reactive group so as to react with the reduced thiol group generated in step (a).

[0063] In some embodiments, the method further comprises (c) adding an effective amount of N-acetylcysteine ​​to quench the excess payload, and then recovering the resulting antibody-drug conjugate.

[0064] In some embodiments, the payload includes maleimide groups, thiols, monobromomaleimide, dibromomaleimide, iodoacetamide, N-methyl-N-phenylvinylsulfonamide, divinylpyrimidine, and the like.

[0065] In some embodiments, the payload is ligated to an antibody or an antibody fragment via a thiol-based conjugation.

[0066] In some embodiments, at least 50%, 60%, 70%, 80%, or 90% of the resulting ADC products have a drug-to-antibody ratio (DAR) of approximately 4.

[0067] In some embodiments, at least 50%, 60%, 70%, 80%, or 90% of the resulting ADC products have a drug-to-antibody ratio (DAR) of about 6.

[0068] In one embodiment, the present disclosure relates to a method for producing a modified antibody or a fragment of that antibody, the method being (a) To provide an antibody or an antibody fragment thereof, wherein the antibody or antibody fragment does not have a disulfide bond between the amino acid residue at position 220 of the heavy chain constant region and the amino acid residue at position 214 of the light chain constant region, wherein the amino acid positions are based on EU numbering. (b) Identifying heavy chain amino acid residues in the heavy chain constant region (CH1) and light chain amino acid residues in the light chain constant region (CL), wherein (1) the sum of the relative solvent exposure area (SASA) of the heavy chain amino acid residue side chain and (2) the relative SASA of the light chain amino acid residue side chain is less than 65%, less than 60%, or less than 55%. (c) Substitution of each of the identified heavy chain amino acid residues and light chain amino acid residues with a cysteine ​​residue, wherein a non-natural disulfide bond is formed between the two cysteine ​​residues.

[0069] In some embodiments, the relative SASA of the heavy amino acid side chain is greater than 40% and the relative SASA of the light amino acid side chain is less than 10%, or the relative SASA of the light amino acid side chain is greater than 40% and the relative SASA of the heavy amino acid side chain is less than 10%.

[0070] In some embodiments, the relative SASA of the heavy amino acid side chain is greater than 30% and the relative SASA of the light amino acid side chain is less than 15%, or the relative SASA of the light amino acid side chain is greater than 30% and the relative SASA of the heavy amino acid side chain is less than 15%.

[0071] In some embodiments, the relative SASA of the heavy chain and the light chain amino acid side chains is less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, or less than 45%.

[0072] In some embodiments, the method further includes attaching a payload to a modified antibody or a fragment of that antibody.

[0073] In one embodiment, the disclosure relates to a method for treating a subject having cancer, the method comprising administering a therapeutically effective amount of a composition comprising the ADC described herein to the subject.

[0074] In some embodiments, the subject has a solid tumor carcinoma.

[0075] In some embodiments, cancer is breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial neoplasm, soft tissue sarcoma, esophageal cancer, or CNS tumor.

[0076] In one embodiment, the disclosure relates to a method for reducing the rate of tumor growth, the method comprising contacting tumor cells with an effective amount of a composition comprising an ADC or a pharmaceutically acceptable salt or solvate thereof as described herein.

[0077] In one embodiment, the disclosure relates to a method for killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising an ADC or a pharmaceutically acceptable salt or solvate thereof as described herein.

[0078] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising an ADC described herein or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

[0079] In some embodiments, the ADC comprises two pairs of heavy and light chains, where each heavy chain contains mutations F126C and C220S, and each light chain contains mutations Q124C and C214S.

[0080] In some embodiments, the ADC comprises two pairs of heavy and light chains, in which each heavy chain contains mutations H168C and C220S, and each light chain contains mutations T164C and C214S.

[0081] In some embodiments, the ADC comprises two pairs of heavy and light chains, in which each heavy chain comprises mutations F170C and C220S, and each light chain comprises T164C and C214S.

[0082] In some embodiments, the ADC comprises two pairs of heavy and light chains, where each heavy chain contains mutations F170C and C220S, and each light chain contains mutations S162C and C214S.

[0083] In some embodiments, the ADC comprises two pairs of heavy and light chains, in which each heavy chain contains mutations V173C and C220S, and each light chain contains mutations S162C and C214S.

[0084] In some embodiments, the ADC comprises two pairs of heavy and light chains, in which each heavy chain contains mutations S134C and C220S, and each light chain contains mutations F116C and C214S.

[0085] In some embodiments, the ADC comprises two pairs of heavy and light chains, in which case each heavy chain contains mutations K133C and C220S, and each light chain contains mutations F209C and C214S.

[0086] In some embodiments, the ADC comprises two pairs of heavy and light chains, in which case each heavy chain contains mutations K133C and C220S, and each light chain contains mutations I117C and C214S.

[0087] In some embodiments, the ADC comprises two pairs of heavy and light chains, where each heavy chain contains mutations P130C and C220S, and each light chain contains mutations F118C and C214S.

[0088] In some embodiments, the ADC comprises two pairs of heavy and light chains, in which each heavy chain contains mutations A141C and C220S, and each light chain contains mutations F116C and C214S.

[0089] As used herein, the terms “spontaneous” or “wild-type” refer to forms found in nature. For example, a spontaneously occurring or wild-type polypeptide or polynucleotide sequence is a sequence found in organisms that have not been intentionally modified by human intervention.

[0090] In this specification, the terms "first" and "second" are used simply to distinguish between two domains or two chains, but do not in any way indicate the location of the two domains or two chains.

[0091] As used herein, the terms “comprise” or “include” are intended to include, but not exclude, any other elements, integers, or steps described. When the terms “comprise” or “include” are used herein, unless otherwise indicated, they also include combinations of the described elements, integers, or steps. For example, when referring to an antibody variable region that “comprises” a particular sequence, it is also intended to include the antibody variable region consisting of that particular sequence.

[0092] As used herein, the term “modified” means a protein or polypeptide having an amino acid sequence that includes one or more amino acid substitutions, deletions, additions or insertions to the amino acid sequence of the original protein or polypeptide, or a protein or polypeptide having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with all or part of the amino acid sequence of the original protein or polypeptide. Sequence identity of amino acid sequences can be obtained by known methods of comparing amino acid sequences, for example, such comparisons can be performed using BLAST (Base Local Alignment Search Tool of the National Center for Biological Information) or similar tools with initial settings.

[0093] The terms "amino acid substitution" or "amino acid mutation" refer to the substitution of at least one amino acid residue in a given parent amino acid sequence with a different "substituting" amino acid residue. The substituted residue or group of residues may be "naturally occurring amino acid residues" (i.e., encoded by the genetic code) and are selected from the group consisting of alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Substitutions by one or more unnatural amino acid residues are also included within the definition of amino acid substitution as specified herein. "Naturally unexisting amino acid residues" refer to residues other than those naturally present in a polypeptide chain that can be covalently bonded to adjacent amino acid residues. Examples of naturally unexisting amino acid residues include norleucine, ornithine, norvaline, homoserine, Aib, and other amino acid residue analogs.

[0094] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, frequency, percentage, dimension, size, volume, weight, or length that varies by approximately 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a given quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length. In certain embodiments, the terms “about” or “approximately” when preceding a number indicate a value plus or minus a range of 15%, 10%, 5%, or 1%.

[0095] Furthermore, as used herein, the terms “higher than,” “lower than,” “more than,” or “less than” a given value encompass a range equal to that value.

[0096] An "antibody-drug conjugate" or ADC refers to a conjugate formed by covalently binding a drug to an antibody, directly or indirectly, via one or more suitable linkers. ADCs are generally a form of antibody-linker-drug conjugate. Antibody-drug conjugates combine the ideal properties of both antibodies and cytotoxic agents by targeting the potent cytotoxic agent to antigen-expressing tumor cells, thereby enhancing their antitumor activity.

[0097] As used herein, the term “drug” as used in this disclosure refers to any therapeutic or diagnostic molecule. In some embodiments, the therapeutic molecule is a cytotoxic agent. In some embodiments, the therapeutic molecule has an antitumor effect. In some embodiments, the therapeutic molecule is at least one substituent or substructure that enables binding to a linker structure. In some embodiments, the drug may kill cancer cells and / or inhibit the growth, proliferation, or metastasis of cancer cells, thereby reducing, alleviating, or eliminating one or more symptoms of a disease or disorder.

[0098] As used herein, the term “antibody” as used in this disclosure includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent) antibody that binds to a particular antigen. Naturally occurring intact antibodies consist of two heavy chains and two light chains. Each heavy chain has a variable region as well as first, second, and third constant regions (CH1, CH2, and CH3), while each light chain has a variable region and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains are classified as λ or κ. The variable region generally contains three highly variable loops called complementarity-determining regions (CDRs) (light chain (L)CDRs containing LCDR1, LCDR2, and LCDR3, and heavy chain (H)CDRs containing HCDR1, HCDR2, and HCDR3). The CDR boundary of an antibody is defined or specified according to the guidelines of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. Dec 5;186(3):651-63 (1985), Chothia, C. and Lesk, AM, J. Mol. Biol., 196,901 (1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83 (1989); Kabat EA et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are sandwiched between adjacent stretches known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold for supporting the hypervariable loop. Each variable region contains four FRs, and the CDRs and FRs are arranged from the amino terminus to the carboxyl terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain.The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In relation to antibodies, a "heavy-light chain pair" refers to one heavy chain and one light chain that form a pair to create an antigen-binding site.

[0099] The term "CH1 region" refers to the portion of an antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 region is the CH1 region of human IgG1, IgG2, IgG3, or IgG4.

[0100] In one embodiment, the CH1 region contains the amino acid sequence ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO: 90).

[0101] The terms “Fc domain” or “Fc region” are used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin “Fc domain” contains two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in native antibodies, the immunoglobulin Fc domain contains second and third constant domains (CH2 and CH3 domains) derived from two heavy chains of IgG, IgA, and IgD antibodies, or second, third, and fourth constant domains (CH2, CH3, and CH4 domains) derived from two heavy chains of IgM and IgE antibodies. Unless otherwise specified herein, amino acid residue numbers in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interests, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimeric Fc, and two different Fc regions can heterodimerize to form a heterodimeric Fc. As used herein, the terms “Fc region,” “Fc portion,” and “dimeric Fc (e.g., heterodimeric Fc)” do not include the heavy chain variable region VH and light chain variable region VL, as well as the heavy chain constant region CH1 and light chain constant region CL of immunoglobulins, although in some cases the configuration may include a hinge region at the N-terminus of the heavy chain constant region. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, or from Cys226, or from Asp231 to the carboxyl terminus of the heavy chain.

[0102] In one embodiment, the Fc region is derived from a human Fc region. The antibody's Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the human IgG4 subtype. In one embodiment, the Fc region belongs to the human IgG1 subtype.

[0103] Amino acid mutations are indicated as (original amino acid, amino acid position, mutated amino acid). If the mutation site is located in the C region, "Q124C" means that glutamine at position 124 of EU is substituted with cysteine ​​(C). When referring to a combination of mutations, the mutations in the combination are connected by " / ". "F126C / C220S" means that both mutations F126C and C220S are present. The possibility of multiple mutations at a particular position is represented herein by the symbol " / ". For example, the mutation "C220A / V / S" means that residue C at position 220 may be substituted with residues A, V, or S.

[0104] Where an amino acid position is referred to in this invention, unless otherwise specified, it refers to an amino acid position numbered based on the IgG1 heavy chain or kappa light chain, that is, an amino acid position numbered based on the IgG1 heavy chain or kappa light chain, and covers the amino acid positions of other heavy chains or light chains corresponding to the aforementioned amino acid positions. For example, when referring to C220, it includes the amino acid at position 220 under EU numbering in the IgG1 heavy chain, as well as the corresponding amino acid in other heavy chains, such as the amino acid at position 131 in IgG2, IgG3, or IgG4.

[0105] When describing mutations, it should be noted that the original amino acid at a particular position may be the amino acid described, or it may be a different amino acid at the corresponding position. For example, when describing the mutation S162C, it includes the mutation of serine to cysteine ​​at position 162 of CH1, and also includes the mutation of the corresponding amino acid at position 162 to cysteine ​​when the amino acid at the corresponding position CH162 is not serine, but it should be noted that this is conditional on the original amino acid corresponding to position 162 of the constant region.

[0106] As used herein, “antibody fragment” refers to a portion of a full-length antibody, generally including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4):315-323), fragments produced by Fab expression libraries, anti-idiotype (anti-Id) antibodies, CDRs (complementarity-determining regions), and any epitope-binding fragments described herein that bind immunospecifically to cancer cell antigens, viral antigens, or microbial antigens, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen fragment is an antigen-binding fragment.

[0107] As used herein, “disulfide bond” refers to a covalent bond with the structure RSS-R'. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond with a second thiol group, for example, from another cysteine ​​residue. Disulfide bonds are formed between the thiol groups of two cysteine ​​residues present on two polypeptide chains, thereby forming interchain bridges or interchain bonds. As used herein, the term “unnatural disulfide bond” refers to a disulfide bond that is not naturally present in wild-type proteins. In some embodiments, the unnatural disulfide bond is formed by two cysteine ​​residues, where at least one of them is a mutation. In some embodiments, two of them are mutations. In some embodiments, at least one or two cysteine ​​residues are introduced by substitution. In contrast, “natural disulfide bond” refers to a disulfide bond that is naturally present in wild-type proteins.

[0108] As used herein, the terms “EU numbering” or “EU numbering system” refer to the EU numbering conventions for constant regions of antibodies described in Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 5th Edition, 1991, each of which is incorporated herein by reference in its entirety.

[0109] As used herein, the term “solvent exposed area (SASA)” refers to a value calculated by representing each atom as a set of grid points distributed on a sphere whose radius is the sum of the atom’s van der Waals radius and probe radius (1.4 Å). Grid points embedded within another atomic sphere contribute to the embedded surface area. Grid points not embedded within another atomic sphere contribute to the exposed surface area. SASA can be calculated using Discovery Studio software version 2022.

[0110] As used herein, the term relative solvent exposure area (SASA) in percent is calculated by the following formula: residue SASA divided by 100 by the residue SASA of the fully exposed amino acid residue calculated using the extended Ala-X-Ala tripeptide, where X is the residue of interest.

[0111] As used herein, antibody-drug conjugates (ADCs) refer to compounds obtained by linking an antibody to a (small molecule) drug via a linker.

[0112] The term "linker" refers to a structural fragment that links a drug (e.g., a small molecule drug) to an antibody portion. It is understood that, prior to binding to the antibody or its antigen-binding fragment, the linker has functional groups that can form a bond with the functional groups of the antibody or its antigen-binding fragment.

[0113] The term "linker-payload" refers to a compound formed by linking a payload, such as a drug (e.g., a small molecule drug), to a linker.

[0114] As used herein, the term "alkyl" refers to a fully saturated branched or unbranched hydrocarbon group. Preferably, alkyl groups contain 1 to 16 carbon atoms, for example, 1 to 12 carbon atoms, 1 to 10 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. Typical examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.

[0115] The term "alkylene" refers to the alkyl group as defined above, but is divalent, meaning it has two single bonds linked to two other groups. Non-restrictive examples of alkylene groups include -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH(-CH2CH3)-, or -CH2CH(-CH3)-.

[0116] The term "alkenyl" refers to a straight-chain or branched-chain hydrocarbon group containing 2 to 16 carbon atoms, containing at least one double bond, and not containing a triple bond. Alkenyl groups preferably contain 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Typical examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.

[0117] The term "alkynyl" refers to a linear or branched hydrocarbon group containing 2 to 16 carbon atoms and at least one triple bond. Preferably, the alkynyl group contains 2 to 12 carbon atoms, 2 to 10 carbon atoms, 2 to 8 carbon atoms, 2 to 6 carbon atoms, or 2 to 4 carbon atoms. Typical examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0118] The terms "halogen" or "halo" refer to fluoro(-F), chloro(-Cl), bromo(-Br), and iodine(-I).

[0119] The term "haloalkyl" refers to an alkyl group as defined herein, substituted with one or more halo groups as defined herein. Haloalkyl groups may preferably be monohaloalkyl groups, dihaloalkyl groups, or polyhaloalkyl groups (including perhaloalkyl groups). Monohaloalkyl groups may contain one iodine, bromo, chloro, or fluoro atom in the alkyl group. Dihaloalkyl groups and polyhaloalkyl groups may contain two or more of the same halogen atoms in the alkyl group or in combination with different halo groups. Polyhaloalkyl groups preferably contain up to 12, 10, or 8, or 6, or 4, or 3, or 2 halo groups. Non-limiting examples of haloalkyl groups include fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl, and dichloropropyl. Perhaloalkyl groups refer to alkyl groups in which all hydrogen atoms are replaced by halogen atoms.

[0120] The term "haloalkenyl" refers to an alkenyl group as defined herein that is substituted with one or more halo groups as defined herein. The term "haloalkynyl" refers to an alkynyl group as defined herein that is substituted with one or more halo groups as defined herein. The meaning of "halo" as defined in "haloalkyl" applies to "haloalkenyl" and "haloalkynyl".

[0121] The term "amino acid" refers to both natural and synthetic amino acids. Amino acids may be either L-isomers or D-isomers. Conventional amino acids as referred to herein are described according to conventional approaches. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Furthermore, in this disclosure, amino acids are generally represented by one- and three-letter abbreviations commonly known in the art. For example, the amino acids may be selected from the group consisting of phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), and glutamine (Gln).

[0122] The terms "optional" or "at will" mean whether or not the event or state described thereafter is present, and that the description includes both cases where the event or state is present and cases where it is not. For example, if a base or structure is "optionally substituted," the base or structure may or may not be substituted.

[0123] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effect of the ADC conjugate of the present invention and is not a salt that is biologically or otherwise undesirable. The ADC conjugates of the present invention may exist in the form of their pharmaceutically acceptable salts, including acid addition salts and base addition salts. In the present invention, a pharmaceutically acceptable acid addition salt refers to a salt formed by the ADC conjugate of the present invention with an organic or inorganic acid, including but not limited to the following: hydrochloric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, nitric acid, perchloric acid, acetic acid, oxalic acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid, lactic acid, propionic acid, benzoic acid, p-toluenesulfonic acid, malic acid, etc. A pharmaceutically acceptable base addition salt means a salt formed by the ADC conjugate of the present invention with an organic or inorganic base, including but not limited to the following: alkali metal salts such as lithium salts, sodium salts, or potassium salts; alkaline earth metal salts such as calcium salts or magnesium salts; or organic base salts, such as ammonium salts, formed with an N-group-containing organic base.

[0124] The term "solvate" refers to an associated complex of one or more solvent molecules having the ADC antibody-drug conjugate of the present invention. Examples of solvents that form solvates include, but are not limited to, water, methanol, ethanol, isopropanol, ethyl acetate, tetrahydrofuran, N-dimethylformamide, and dimethyl sulfoxide.

[0125] "Pharmacologically acceptable" and "pharmaceutically useful" are used interchangeably in this specification, unless otherwise specified in context.

[0126] The term “drug-to-antibody ratio” or “DAR” refers to the ratio of the drug moiety (D) conjugated with the Ab moiety as described herein to the Ab moiety. In some embodiments described herein, the DAR may be determined by p of formula I. For example, the DAR may be 1–16, e.g., 2–16, 4–16, 5–12, 6–10, 2–8, 3–8, 2–6, 4–6, 6–10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Alternatively, the DAR may be calculated as the average DAR of the molecular population in the product, i.e., the overall ratio of the drug moiety (D) conjugated with the Ab moiety as described herein to the Ab moiety, measured in the product by a detection method (e.g., conventional methods such as chromatography, mass spectrometry, ELISA assay, electrophoresis, and / or HPLC), for example, the DAR is referred herein as the average DAR or the measured DAR. In some embodiments, the average DAR value of the conjugate of the present invention is 1 to 16, for example 2 to 16, 4 to 16, 5 to 12, 6 to 10, 2 to 8, 3 to 8, 2 to 6, 4 to 6, 6 to 10, for example 1.0 to 8.0, 2.0 to 6.0, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4 0.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 The range is 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, and has two of these values ​​as its endpoints.When the average DAR value is mentioned, it should be understood that the ADC according to the present invention refers to a collection of ADC molecules, or a mixture of ADC molecules containing ADC molecules having the same and / or different DARs.

[0127] As used herein, the term “therapeutic agent” includes any substance effective in preventing or treating tumors (such as cancer), including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (such as immunosuppressants).

[0128] As used in this invention, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function, and / or a substance that causes cell death or destruction.

[0129] "Chemotherapy agents" include compounds useful for treating cancer or immune system disorders.

[0130] The term "prodrug" refers to a chemically modified active or inactive compound that, upon administration to a subject, undergoes physiological effects (e.g., hydrolysis, metabolism) in vivo to become an active drug. The techniques for producing and using prodrugs are well known to those skilled in the art.

[0131] The term "small molecule drug" refers to drugs with a low molecular weight. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, generally less than 2 kD, preferably less than 1 kD, and more preferably less than 500 kD. Examples of small molecules include, but are not limited to, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetic compounds, and antibody mimetic compounds. As therapeutic agents, small molecules can penetrate cells more easily than macromolecules, are less susceptible to degradation, and have a lower tendency to induce immune responses.

[0132] "Anticumonochemicals" are pharmaceutically active compounds that are effective against tumors, and include, but are not limited to, cytotoxic or chemotherapeutic agents, particularly small molecule cytotoxic or chemotherapeutic agents including topoisomerase I inhibitors, small molecule tubulin inhibitors such as camptothecin, exatecan (a topoisomerase I inhibitor), Dxd (an exatecan derivative as a novel topoisomerase I inhibitor), as disclosed in WO2021 / 173773, WO2022180581, CN102574866, etc., or maytansinoids such as monomethyl auristatin E (MMAE), MMAF, or small molecule tubulin inhibitors DM1 and DM4. It will be understood that antitumor compounds may be substituted with isotopes, including but not limited to deuterium and tritium. For example, after substitution with deuterium, i.e., after replacing a carbon-hydrogen bond with a carbon-deuterium bond, the former is more stable than the latter. Therefore, this substitution directly affects certain properties of the drug, such as absorption, distribution, metabolism, and excretion, thereby improving the drug's efficacy, safety, and tolerability. Accordingly, the “antitumor compounds” of this application may include compounds substituted with deuterium.

[0133] "Deuterium-substituted" means that hydrogen atoms in a molecule are replaced by deuterium, for example, one or more hydrogen atoms, such as 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), are replaced by deuterium.

[0134] "Camptothecin" refers to a compound that possesses the core structure of camptothecin (for example, the condensed penta-ring structure or its condensed tetra-ring structure) and exhibits antitumor activity. "Camptothecin" is a commonly used term in medicinal chemistry. Based on the structure of a compound, those skilled in the art can easily determine whether a compound belongs to the camptothecin family.

[0135] "Auristatin" refers to a compound that has the core structure of auristatin and possesses antitumor activity.

[0136] "Meitansinoids" refer to compounds that have a meitansinoid core structure and possess antitumor activity.

[0137] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. While methods and materials for use in the present invention are described herein, other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to limit the scope of use. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated in their entirety by reference. In case of any inconsistency, this specification, including definitions, shall prevail.

[0138] Other features and advantages of the present invention will be apparent from the following detailed description and figures, as well as from the claims. [Brief explanation of the drawing]

[0139] [Figure 1] Figure 1 is a schematic diagram of the ADC structure. [Figure 2] Figure 2 shows the results of the RP-HPLC analysis. [Figure 3] Figure 3 shows the SEC analysis results. [Figure 4] Figure 4 shows the HIC analysis results. [Figure 5] Figure 5 is a schematic diagram of the ADC structure. [Figure 6] Figure 6 shows the RP-HPLC analysis results. [Figure 7] Figure 7 shows the SEC analysis results. [Figure 8] Figure 8 shows the HIC analysis results. [Figure 9] Figure 9 is a schematic diagram of the ADC structure. [Figure 10] Figure 10 shows the results of the RP-HPLC analysis. [Figure 11] Figure 11 shows the SEC analysis results. [Figure 12] Figure 12 shows the HIC analysis results. [Figure 13] Figure 13 is a schematic diagram of the ADC structure. [Figure 14] Figure 14 shows the results of the RP-HPLC analysis. [Figure 15] Figure 15 shows the SEC analysis results. [Figure 16] Figure 16 shows the HIC analysis results. [Figure 17] Figure 17 is a schematic diagram of the ADC structure. [Figure 18] Figure 18 shows the results of the RP-HPLC analysis. [Figure 19] Figure 19 shows the SEC analysis results. [Figure 20] Figure 20 shows the HIC analysis results. [Figure 21] Figure 21 is a schematic diagram of the ADC structure. [Figure 22] Figure 22 shows the RP-HPLC analysis results. [Figure 23] Figure 23 shows the SEC analysis results. [Figure 24] Figure 24 shows the HIC analysis results. [Figure 25] Figure 25 is a schematic diagram of the ADC structure. [Figure 26] Figure 26 shows the results of the RP-HPLC analysis. [Figure 27] Figure 27 shows the HIC analysis results. [Figure 28] Figure 28 is a schematic diagram of the ADC structure. [Figure 29] Figure 29 shows the results of the RP-HPLC analysis. [Figure 30] Figure 30 shows the SEC analysis results. [Figure 31] Figure 31 shows the HIC analysis results. [Figure 32] Figure 32 is a schematic diagram of the ADC structure. [Figure 33] Figure 33 shows the RP-HPLC analysis results. [Figure 34] Figure 34 shows the SEC analysis results. [Figure 35] Figure 35 is a schematic diagram of the ADC structure. [Figure 36] Figure 36 shows the results of the RP-HPLC analysis. [Figure 37] Figure 37 shows the SEC analysis results. [Figure 38] Figure 38 shows the results of the RP-HPLC analysis. [Figure 39] Figure 39 shows the results of the RP-HPLC analysis. [Figure 40] Figure 40 shows the results of the RP-HPLC analysis. [Figure 41] Figure 41 shows the results of the RP-HPLC analysis. [Figure 42] Figure 42 shows the results of the RP-HPLC analysis. [Figure 43] Figure 43 shows the RP-HPLC analysis results. [Figure 44] Figure 44 shows the results of the RP-HPLC analysis. [Figure 45] Figure 45 shows the results of the RP-HPLC analysis. [Figure 46] Figure 46 shows the results of the RP-HPLC analysis. [Figure 47] Figure 47 shows the results of the RP-HPLC analysis. [Figure 48] Figure 48 shows the results of the RP-HPLC analysis. [Figure 49] Figure 49 shows the results of the RP-HPLC analysis. [Figure 50] Figure 50 shows the HIC analysis results. [Figure 51] Figure 51 shows the results of the RP-HPLC analysis. [Figure 52] Figure 52 shows the HIC analysis results. [Figure 53] Figure 53 shows the results of the RP-HPLC analysis. [Figure 54] Figure 54 shows the HIC analysis results. [Figure 55] Figure 55 shows the results of the RP-HPLC analysis. [Figure 56] Figure 56 shows the HIC analysis results. [Figure 57]Figure 57 shows the results of the RP-HPLC analysis. [Figure 58] Figure 58 shows the HIC analysis results. [Figure 59] Figure 59 is a schematic diagram of the ADC structure. [Figure 60] Figure 60 shows the results of the RP-HPLC analysis. [Figure 61] Figure 61 shows the HIC analysis results. [Figure 62] Figure 62 is a schematic diagram of the ADC structure. [Figure 63] Figure 63 shows the results of the RP-HPLC analysis. [Figure 64] Figure 64 shows the HIC analysis results. [Figure 65] Figure 65 is a schematic diagram of the ADC structure. [Figure 66] Figure 66 shows the results of the RP-HPLC analysis. [Figure 67] Figure 67 shows the HIC analysis results. [Figure 68] Figure 68 is a schematic diagram of the ADC structure. [Figure 69] Figure 69 shows the results of the RP-HPLC analysis. [Figure 70] Figure 70 shows the HIC analysis results. [Figure 71] Figure 71 is a schematic diagram of the ADC structure. [Figure 72] Figure 72 shows the RP-HPLC analysis results. [Figure 73] Figure 73 shows the HIC analysis results. [Figure 74] Figure 74 is a schematic diagram of the ADC structure. [Figure 75] Figure 75 shows the linear relationship between DAR and its TCEP equivalent. [Figure 76] Figure 76 shows the HIC analysis results. [Figure 77] Figure 77 shows the results of the RP-HPLC analysis. [Figure 78] Figure 78 shows the SEC analysis results. [Figure 79] Figure 79 is a schematic diagram of the ADC structure. [Figure 80] Figure 80 shows the linear relationship between DAR and its TCEP equivalent. [Figure 81] Figure 81 shows the results of the RP-HPLC analysis. [Figure 82] Figure 82 shows the SEC analysis results. [Figure 83] Figure 83 shows the HIC analysis results. [Figure 84] Figures 84-90 show the results for plasma stability. [Figure 85] Figures 84-90 show the results for plasma stability. [Figure 86] Figures 84-90 show the results for plasma stability. [Figure 87] Figures 84-90 show the results for plasma stability. [Figure 88] Figures 84-90 show the results for plasma stability. [Figure 89] Figures 84-90 show the results for plasma stability. [Figure 90] Figures 84-90 show the results for plasma stability. [Figure 91] Figure 91 shows the results of the cell binding assay. The X-axis represents the concentration of the antibody or ADC. The Y-axis represents the fluorescence intensity. [Figure 92] Figure 92 shows the results of the cytotoxicity assay. The X-axis represents the concentration of the antibody or ADC. The Y-axis represents cell viability. [Figure 93] Figure 93 shows the mean tumor volume in different groups of CB17-SCID mice injected with NCI-N87 cells and treated with ADC. [Figure 94] Figure 94 shows the mean body weight changes in different groups of CB17-SCID mice injected with NCI-N87 cells and treated with ADC. [Figure 95] Figure 95 is a schematic diagram of the ADC structure. [Figure 96] Figure 96 shows the results of the RP-HPLC analysis. [Figure 97] Figure 97 shows the HIC analysis results. [Figure 98]Figure 98 is a schematic diagram of the ADC structure. [Figure 99] Figure 99 shows the results of the RP-HPLC analysis. [Figure 100] Figure 100 shows the HIC analysis results. [Figure 101-1] Figure 101 lists the relevant amino acid sequences. [Figure 101-2] Figure 101 lists the relevant amino acid sequences. [Figure 101-3] Figure 101 lists the relevant amino acid sequences. [Figure 101-4] Figure 101 lists the relevant amino acid sequences. [Figure 101-5] Figure 101 lists the relevant amino acid sequences. [Figure 101-6] Figure 101 lists the relevant amino acid sequences. [Figure 101-7] Figure 101 lists the relevant amino acid sequences. [Figure 101-8] Figure 101 lists the relevant amino acid sequences. [Figure 101-9] Figure 101 lists the relevant amino acid sequences. [Figure 101-10] Figure 101 lists the relevant amino acid sequences. [Figure 101-11] Figure 101 lists the relevant amino acid sequences. [Figure 101-12] Figure 101 lists the relevant amino acid sequences. [Figure 101-13] Figure 101 lists the relevant amino acid sequences. [Figure 101-14] Figure 101 lists the relevant amino acid sequences. [Figure 101-15] Figure 101 lists the relevant amino acid sequences. [Figure 101-16] Figure 101 lists the relevant amino acid sequences. [Figure 101-17] Figure 101 lists the relevant amino acid sequences. [Figure 101-18] Figure 101 lists the relevant amino acid sequences. [Figure 101-19]Figure 101 lists the relevant amino acid sequences. [Figure 101-20] Figure 101 lists the relevant amino acid sequences. [Figure 101-21] Figure 101 lists the relevant amino acid sequences. [Figure 101-22] Figure 101 lists the relevant amino acid sequences. [Figure 101-23] Figure 101 lists the relevant amino acid sequences. [Figure 101-24] Figure 101 lists the relevant amino acid sequences. [Figure 101-25] Figure 101 lists the relevant amino acid sequences. [Figure 101-26] Figure 101 lists the relevant amino acid sequences. [Modes for carrying out the invention]

[0140] Antibody-drug conjugates (ADCs) have gained attention as a key class of biologics for targeted cancer therapy. To date, 14 ADCs have been approved by the US FDA. ADCs are complex molecules consisting of antibodies linked to biologically active cytotoxic drugs. Conventional strategies for drug conjugation (i.e., lysine and interchain cysteine-based conjugations to achieve target-level mean drug-to-antibody ratios (DARs)) have been plagued by heterogeneity of conjugation sites and bound drugs. Progress in the development of site-directed conjugations, including thiomap, disulfide recrosslinking, incorporation of non-natural amino acids, and enzymatic bioconjugations, has enabled the preparation of homogeneous ADCs. However, most of these strategies require antibody sequence redesign, multi-step operations, and expensive substrates, thus limiting the range of industrial applications.

[0141] Boosted by the clinical success of Enhertu and Trodelvy, interchain cysteine-based conjugation has become a primary strategy for the development of novel ADCs. Apart from DAR8 ADCs, which open all interchain disulfide bonds for conjugation, tunable DARs, namely DAR4 and DAR6, are highly desirable when very potent drugs or dual drugs with different mechanisms are used as payloads. One strategy involves replacing some of the interchain cysteine ​​with serine, thus reducing the eight potential conjugation sites to four or six. While homogeneous DAR6 and DAR4 ADCs can be successfully produced, the removal of interchain disulfide bonds can destabilize the antibody and cause light chain dissociation. This disclosure provides a cost-effective and easy-to-handle cysteine-based conjugation method that provides site-specific ADCs with stable structures and defined DARs.

[0142] In one embodiment, the disclosure combined in silico analysis and experimental testing to define specific manipulated cysteine ​​pairs for the synthesis of stable and homogeneous DAR4 and DAR6 ADCs in a cost-effective and practical manner. In some embodiments, the manipulated cysteine ​​pairs can form stable disulfide bonds resistant to TCEP reduction. In some embodiments, the resulting DAR4 or DAR6 species accounted for approximately 90% of the product.

[0143] In some embodiments, this disclosure provides modified antibodies DAR4 and antibody fragments that can be used for the synthesis of site-specific monospecific ADCs of DAR4 and DAR6, and bispecific ADCs of DAR4 and DAR6. In some embodiments, modified antibodies and antibody fragments can be used to produce more homogeneous ADC products. In some embodiments, the payload may be selected from cytotoxic drugs, biologically active proteins, synthetic polymers, enzymes, nucleic acids, and fragments thereof, such as DNA or RNA.

[0144] Modified antibodies and their antibody fragments, as well as thiol-based conjugations To avoid side effects, drugs should be delivered specifically to cancer cells via binding to ligands that can specifically recognize cancer-related biomarkers such as antigens. Among targeted therapeutic ligands, antibodies are excellent candidates due to their specific recognition and high affinity. Today, antibody-drug conjugates (ADCs) are attracting considerable attention for targeted cancer therapy.

[0145] Antibody-drug conjugates are biological therapeutic agents that consist of an antibody, a potent drug, and a linker between them. The antibody guides the drug to target cancer cells. In some embodiments, the drug is a prodrug, which can be chemically or enzymatically converted into its active form. By conjugating cytotoxins to antibodies that specifically bind to tumor cell surface antigens, drugs can be targeted and delivered to cancer cells while normal cells remain unaffected. More importantly, many cytotoxic drugs that are too toxic for use in conventional chemotherapy can also be used to construct antibody-drug conjugates. The linker is also an essential component of the antibody-drug conjugate and contributes to stability in circulation, good pharmacokinetics, and efficient release of toxic drugs in tumor cells.

[0146] Using thiols at interchain cysteine ​​residues in monoclonal antibodies as drug molecule binding sites is one of the most commonly used conjugation methods. Human IgG1 has four interchain disulfide bonds that can be used as potential conjugation sites. These four interchain disulfide bonds can be reduced by tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT), which yields eight thiol groups available for drug molecule conjugates. Through this method, different drug-antibody ratio (DAR) conjugates can be obtained when targeting 2 to 6 typical DARs. Furthermore, the antibody-drug conjugate at each drug-antibody ratio has several isomers. Therefore, many different species exist within the antibody-drug conjugate.

[0147] Homogeneous antibody-drug conjugates can be formed via cysteine ​​residues when all interchain cysteines are bound to the drug. An exemplary ADC contains cAC10, an anti-CD30 monoclonal antibody, and monomethyl auristatin E (MMAE). This cAC10-vcMMAE conjugate contains eight drugs per antibody, which is the highest drug-to-antibody ratio (DAR) that can be obtained by using interchain cysteine ​​as the conjugate site. However, antibody-drug conjugates with four drugs per antibody generally show improved in vivo performance.

[0148] A detailed review of ADCs and thiol-based conjugations is provided in Yao et al., "Methods for Designing and Synthesizing Antibody-Drug Conjugates (ADCs)," International Journal of Molecular Sciences 17.2 (2016): 194, which is incorporated herein by reference in its entirety.

[0149] In one embodiment, the disclosure provides a modified antibody and antibody fragment thereof in which one or more of the natural heavy-chain-light-chain (interchain) disulfide bonds are rearranged to a region inaccessible to the solvent so that these disulfide bonds do not cleave under typical reducing conditions. As a result, the maleimide-derived linker-payload can be site-specifically conjugated with other interchain cysteine ​​residues to achieve a defined DAR. In some embodiments, both or one of the disulfide bonds between the heavy and light chains (heavy-chain disulfide bonds) were mutated and the linker-payload was conjugated at the hinge region to obtain DAR4 and DAR6 ADCs, respectively.

[0150] In some embodiments, amino acid pairs at the heavy-chain and light-chain binding interfaces are screened to identify a panel of engineered cysteine ​​pairs that tend to form disulfide bonds and are inaccessible to the solvent. This analysis is made possible by calculating the solvent exposure area (SASA) of the side chains of candidate amino acid pairs in the structure of the Fab fragment, and disulfide bond formation is confirmed by non-reducing SDS-PAGE. Site-specific conjugations can be tested with different engineered pairs, different antibodies, and different linker-payloads, and the in vitro and in vivo efficacy of the resulting ADCs can be validated in cell assays and animal models, respectively.

[0151] In one embodiment, the disclosure relates to various modified antibodies and antibody fragments thereof. In some embodiments, various modifications are made to the antibody fragment to modify the DAR of the ADC. In some embodiments, one or more of the Fab region disulfide bonds are moved to a region that cannot access the solvent.

[0152] In some embodiments, one or more natural heavy-light disulfide bonds between the heavy and light chains are absent in the modified antibody or its antibody fragment.

[0153] In some embodiments, the native heavy-light chain disulfide bond in the modified antibody or its antibody fragment is mutated into a non-disulfide bond.

[0154] In some embodiments, the heavy chain cysteine ​​C220 (containing C131S / A / V in the corresponding position of the IgG1 subtype, or IgG2, IgG3, or IgG4) is mutated to a non-cysteine ​​residue. In some embodiments, the heavy chain cysteine ​​C220 is mutated to A, R, N, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, for example, S, A, or V. In some embodiments, the modified antibody or antibody fragment contains a C220S mutation in the heavy chain CH1 region. In some embodiments, the modified antibody or antibody fragment contains two heavy chains, and the heavy chain cysteine ​​C220 is mutated to a non-cysteine ​​residue in only one of the two heavy chains. In some embodiments, the modified antibody or antibody fragment contains two heavy chains, each containing the C220S mutation. In some embodiments, the modified antibody or antibody fragment contains two heavy chains, with only one heavy chain containing the C220S mutation.

[0155] In some embodiments, the light chain cysteine ​​C214 is mutated to a non-cysteine ​​residue. In some embodiments, the light chain cysteine ​​C214 is mutated to A, R, N, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V, for example, S, A, or V. In some embodiments, the modified antibody or antibody fragment contains the C214S mutation in the light chain constant region CL. In some embodiments, the modified antibody or antibody fragment contains two light chains, and the light chain cysteine ​​C214 is mutated to a non-cysteine ​​residue in both light chains. In some embodiments, the modified antibody or antibody fragment contains two light chains, and the light chain cysteine ​​C214 is mutated to a non-cysteine ​​residue in only one of the two light chains. In some embodiments, the modified antibody or antibody fragment contains two light chains, each containing the C214S mutation. In some embodiments, the modified antibody or antibody fragment contains two light chains, with only one light chain containing the C214S mutation.

[0156] In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond. In some embodiments, the modified antibody or antibody fragment described herein has a non-natural disulfide bond at one of the positions in Table 1. In some embodiments, the non-natural disulfide bond is located at one of the following CH1 / CL positions: F126 / Q124, H168 / T164, F170 / T164, F170 / S162, V173 / S162, S134 / F116, K133 / F209, K133 / I117, P130 / F118, and A141 / F116. In some embodiments, the modified antibody or antibody fragment includes at least a non-natural disulfide bond in a region that does not have access to the solvent. In some embodiments, the modified antibody or antibody fragment contains two pairs of heavy and light chains, each pair containing one of the following CH1 / CL mutations: F126 / Q124, H168 / T164, F170 / T164, F170 / S162, V173 / S162, S134 / F116, K133 / F209, K133 / I117, P130 / F118, and A141 / F116. In some embodiments, the modified antibody or antibody fragment contains two pairs of heavy and light chains, with only one pair of heavy and light chains containing one of the following CH1 / CL mutations: F126 / Q124, H168 / T164, F170 / T164, F170 / S162, V173 / S162, S134 / F116, K133 / F209, K133 / I117, P130 / F118, and A141 / F116.

[0157] In some embodiments, one amino acid in the heavy chain constant region of the modified antibody or antibody fragment is mutated to cysteine. In some embodiments, the amino acid is selected from the group consisting of: F126, H168, F170, V173, S134, K133, P130, and A141. In some embodiments, the modified antibody or antibody fragment contains one or more of the following mutations in the heavy chain constant region: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C. In some embodiments, the modified antibody or antibody fragment contains two heavy chains, each containing one or more of the following mutations in the heavy chain constant region: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C. In some embodiments, the modified antibody or antibody fragment contains two heavy chains, and only one of the heavy chains contains one or more of the following mutations in the heavy chain constant region: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C.

[0158] In some embodiments, one amino acid in the light chain constant region of the modified antibody or antibody fragment is mutated to cysteine. In some embodiments, the amino acid is selected from the group consisting of: Q124, T164, S162, F116, F209, I117, and F118. In some embodiments, the modified antibody or antibody fragment contains one or more of the following mutations in the light chain constant region: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C. In some embodiments, the modified antibody or antibody fragment contains two light chains, each containing one or more of the following mutations in the light chain constant region: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C. In some embodiments, the modified antibody or antibody fragment contains two light chains, and only one light chain contains one or more of the following mutations in its constant region: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C.

[0159] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment has F126C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has Q124C mutations and C214S mutations in the light chain constant region. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: F126 / Q124. In some embodiments, the modified antibody or antibody fragment has two heavy chains, each having F126C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, each having Q124C mutations and C214S mutations in the light chain constant region.

[0160] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment has H168C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has T164C mutations and C214S mutations in the light chain constant region. In some embodiments, the modified antibody or antibody fragment has a non-native disulfide bond at the following CH1 / CL position: H168 / T164. In some embodiments, the modified antibody or antibody fragment has two heavy chains, each having H168C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, each having T164C mutations and C214S mutations in the light chain constant region.

[0161] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment has F170C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has T164C mutations and C214S mutations in the light chain constant region. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: F170 / T164. In some embodiments, the modified antibody or antibody fragment has two heavy chains, each having F170C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, each having T164C mutations and C214S mutations in the light chain constant region.

[0162] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment has F170C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has S162C mutations and C214S mutations in the light chain constant region. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: F170 / S162. In some embodiments, the modified antibody or antibody fragment has two heavy chains, each having F170C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, each having S162C mutations and C214S mutations in the light chain constant region.

[0163] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment has V173C mutation and C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has S162C mutation and C214S mutation in the light chain constant region. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: V173 / S162. In some embodiments, the modified antibody or antibody fragment has two heavy chains, each having V173C mutation and C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, each having S162C mutation and C214S mutation in the light chain constant region.

[0164] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment has S134C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has F116C mutations and C214S mutations in the light chain constant region. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: S134 / F116. In some embodiments, the modified antibody or antibody fragment has two heavy chains, each having S134C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, each having F116C mutations and C214S mutations in the light chain constant region.

[0165] In some embodiments, the modified antibody or antibody fragment thereof has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment thereof has a K133C mutation and a C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has an F209C mutation and a C214S mutation in the light chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has an unnatural disulfide bond at the following CH1 / CL positions: K133 / F209. In some embodiments, the modified antibody or antibody fragment thereof has two heavy chains, each of which has a K133C mutation and a C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has two light chains, each of which has an F209C mutation and a C214S mutation in the light chain constant region.

[0166] In some embodiments, the modified antibody or antibody fragment thereof has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment thereof has a K133C mutation and a C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has an I117C mutation and a C214S mutation in the light chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has an unnatural disulfide bond at the following CH1 / CL positions: K133 / I117. In some embodiments, the modified antibody or antibody fragment thereof has two heavy chains, each of which has a K133C mutation and a C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has two light chains, each of which has an I117C mutation and a C214S mutation in the light chain constant region.

[0167] In some embodiments, the modified antibody or antibody fragment thereof has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment thereof has a P130C mutation and a C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has an F118C mutation and a C214S mutation in the light chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has a non-natural disulfide bond at the following CH1 / CL positions: P130 / F118. In some embodiments, the modified antibody or antibody fragment thereof has two heavy chains, each of which has a P130C mutation and a C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has two light chains, each of which has an F118C mutation and a C214S mutation in the light chain constant region.

[0168] In some embodiments, the modified antibody or antibody fragment thereof has two heavy chain constant region mutations and two light chain constant region mutations. In some embodiments, the modified antibody or antibody fragment thereof has an A141C mutation and a C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has an F116C mutation and a C214S mutation in the light chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has a non-natural disulfide bond at the following CH1 / CL positions: A141 / F116. In some embodiments, the modified antibody or antibody fragment thereof has two heavy chains, each of which has an A141C mutation and a C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment thereof has two light chains, each of which has an F116C mutation and a C214S mutation in the light chain constant region.

[0169] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has F126C mutations and C220S mutations in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has Q124C mutations and C214S mutations in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: F126 / Q124. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains F126C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains Q124C mutations and C214S mutations in the light chain constant region.

[0170] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has K133C mutation and C220S mutation in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has F209C mutation and C214S mutation in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: K133 / F209. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains K133C mutation and C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains F209C mutation and C214S mutation in the light chain constant region.

[0171] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has K133C mutation and C220S mutation in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has I117C mutation and C214S mutation in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: K133 / I117. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains K133C mutation and C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains I117C mutation and C214S mutation in the light chain constant region.

[0172] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has P130C mutations and C220S mutations in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has F118C mutations and C214S mutations in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: P130 / F118. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains P130C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains F118C mutations and C214S mutations in the light chain constant region.

[0173] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has A141C mutation and C220S mutation in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has F116C mutation and C214S mutation in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: A141 / F116. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains A141C mutation and C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains F116C mutation and C214S mutation in the light chain constant region.

[0174] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has H168C mutation and C220S mutation in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has T164C mutation and C214S mutation in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: H168 / T164. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains H168C mutation and C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains T164C mutation and C214S mutation in the light chain constant region.

[0175] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has F170C mutations and C220S mutations in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has T164C mutations and C214S mutations in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: F170 / T164. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains F170C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains T164C mutations and C214S mutations in the light chain constant region.

[0176] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has F170C mutations and C220S mutations in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has S162C mutations and C214S mutations in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: F170 / S162. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains F170C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains S162C mutations and C214S mutations in the light chain constant region.

[0177] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has V173C mutation and C220S mutation in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has S162C mutation and C214S mutation in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: V173 / S162. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains V173C mutation and C220S mutation in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains S162C mutation and C214S mutation in the light chain constant region.

[0178] In some embodiments, the modified antibody or antibody fragment has two heavy chain constant region mutations in the first heavy chain and two light chain constant region mutations in the first light chain. In some embodiments, the modified antibody or antibody fragment has S134C mutations and C220S mutations in the heavy chain constant region of the first heavy chain. In some embodiments, the modified antibody or antibody fragment has F116C mutations and C214S mutations in the light chain constant region of the first light chain. In some embodiments, the modified antibody or antibody fragment has a non-natural disulfide bond at the following CH1 / CL position: S134 / F116. In some embodiments, the modified antibody or antibody fragment has two heavy chains, and only one of the two heavy chains contains S134C mutations and C220S mutations in the heavy chain constant region. In some embodiments, the modified antibody or antibody fragment has two light chains, and only one of the two light chains contains F116C mutations and C214S mutations in the light chain constant region.

[0179] Therefore, in one embodiment of the present disclosure, the present disclosure relates to an antibody containing modified CH1 / CL, (i) CH1 contains the F126C mutation, and CL contains the Q124C mutation (EU numbering), (ii) CH1 contains the H168C mutation, and CL contains the T164C mutation (EU numbering), (iii) CH1 contains the F170C mutation, and CL contains the T164C mutation (EU numbering), (iv) CH1 contains the F170C mutation, and CL contains the S162C mutation (EU numbering), (v) CH1 contains the V173C mutation, and CL contains the S162C mutation (EU numbering), (vi) CH1 contains the S134C mutation, and CL contains the F116C mutation (EU numbering), (vii) CH1 contains the K133C mutation, and CL contains the F209C mutation (EU numbering), (viii) CH1 contains the K133C mutation, and CL contains the I117C mutation (EU numbering), (xi) CH1 contains the P130C mutation, and CL contains the F118C mutation (EU numbering), (x) CH1 contains the A141C mutation, and CL contains the F116C mutation (EU numbering), Optionally, CH1 further includes the C220S / A / V mutation (or the corresponding C131S / A / V), and CL further includes the C214S / A / V mutation (EU numbering), Preferably, CH1 further includes C220S (EU numbering), and CL further includes C214S (EU numbering).

[0180] In some embodiments, the antibody or antigen-binding fragment thereof comprises a pair of modified CH1 / CL, wherein the CH1 / CL are located on the heavy and light chains, respectively. In some embodiments, the antibody or antigen-binding fragment thereof comprises two pairs of modified CH1 / CL, wherein the CH1 / CL are located on the heavy and light chains, respectively.

[0181] In some embodiments, the antibody or antigen-binding fragment thereof comprises two heavy chains and two light chains, wherein the two heavy chains may be the same or different, and the two light chains may be the same or different. For example, the heavy and light chains contain the following combinations of mutations: [Table 1-1]

[0182] In some embodiments, when the antibody or antibody fragment thereof comprises a lambda light chain constant region, it may contain an S134C mutation (numbered according to heavy chain IgG1) in the heavy chain constant region and a T116C mutation (numbered according to lambda light chain) in the lambda light chain constant region.

[0183] In some embodiments, when the antibody or antibody fragment thereof comprises two heavy chains and two light chains, and both light chains comprise a lambda light chain constant region, each of the two heavy chains comprises an S134C mutation (numbered according to heavy chain IgG1) in the heavy chain constant region or CH1, and each of the two light chains comprises a T116C mutation (numbered according to lambda light chain) in the lambda light chain constant region.

[0184] In some embodiments, when the antibody or antibody fragment thereof comprises two heavy chains and two light chains, and one or both of the light chains comprise a lambda light chain constant region, one of the heavy chains contains the S134C mutation (numbered according to heavy chain IgG1) in the heavy chain constant region or CH1, and the light chain paired with the heavy chain containing the lambda constant region contains a T116C mutation (numbered according to lambda light chain) in the lambda light chain constant region.

[0185] In some embodiments, the modified antibody or antibody fragment has one or more additional amino acid mutations. In some embodiments, the modified antibody or antibody fragment may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 mutations. In some embodiments, the mutations may be deletions, insertions, or substitutions.

[0186] In some embodiments, the modified antibody or antibody fragment has an Fc region. In some embodiments, the Fc region is derived from human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the Fc region contains or consists of an amino acid sequence having at least 90% (e.g., 95%, 96%, 97%, 98%, 99%, or more) identity with the amino acid sequence of SEQ ID NO: 91 or 92.

[0187] In one embodiment, the Fc region is modified with an effector function of the Fc region (e.g., complement activation function of the Fc region). In one embodiment, the effector function is reduced or eliminated compared to the wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by a method selected from the group consisting of: the use of an Fc isotype in which the effector function is naturally reduced or eliminated, and Fc region modification.

[0188] In one preferred embodiment, the Fc region has reduced Fc region-mediated effector functions, such as reduced or eliminated ADCC, ADCP, or CDC effector functions, including mutations to achieve the above functions.

[0189] As will be understood by those skilled in the art, the antibodies or antibody fragments of the present disclosure may also be configured to include modifications in the Fc domain that alter the binding affinity to one or more Fc receptors, depending on the intended use of the antibody or antibody fragment of the present disclosure. In one embodiment, the Fc receptor is an Fc gamma receptor, in particular the human Fc gamma receptor. In some embodiments, the Fc region includes mutations that reduce binding to the Fc gamma receptor. In another preferred embodiment, the Fc region may be configured to have mutations that increase the serum half-life, for example, mutations that improve the binding of the Fc region to FcRn.

[0190] In some embodiments, the antibody is a human IgG1 antibody optionally having SI mutations, LALA mutations, N297A mutations, YTE mutations, and / or FLAA mutations in the Fc region. In some embodiments, the antibody is a human IgG4 antibody optionally having SI mutations, LALA mutations, N297A mutations, YTE mutations, and / or FLAA mutations. In some embodiments, the Fc region has LALA mutations (L234A and L235A mutations according to EU numbering) or LALA-PG mutations (L234A, L235A, P329G mutations according to EU numbering). In some embodiments, the Fc region has FLAA mutations (F234A and L235A according to EU numbering). In some embodiments, the Fc region has SI mutations (S239D and I332E mutations according to EU numbering). In some embodiments, the Fc region has the N297A mutation according to EU numbering. In some embodiments, Fc has YTE mutations (M252Y, S254T, and T256E according to EU numbering).

[0191] In some embodiments, the Fc region contained within the antibody or antibody fragment may contain mutations that promote heterodimerization between the first Fc region and the second Fc region. Preferably, a knob mutation and a hole mutation are introduced into the first and second Fc regions, respectively, based on the knob-in-hole technique. For this technique, see, for example, U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., Prot Eng 9,617-621 (1996), and Carter, J Immunol Meth 248,7-15 (2001) or WO96 / 27011. Alternatively, each mutation may be introduced into the first and second Fc regions based on the Innobody technique. For this technique, see, for example, PCT / CN2021 / 143141.

[0192] In some embodiments, antibody fragments, such as antibodies or antigen-binding fragments, do not have a functional Fc region. For example, the antibody or antigen-binding fragments are Fab, Fab', F(ab')2, and Fv fragments.

[0193] In some embodiments, antibody fragments, such as antibodies or antigen-binding fragments applicable to this disclosure, also include mutations in the FR of the variable region, for example, the Q38D mutation in the light chain variable region and the Q39K mutation in the heavy chain variable region.

[0194] Antibody and antigen-binding fragments Antibodies not limited to this disclosure may be intact four-unit immunoglobulin chain antibodies comprising two heavy chains and two light chains (also called “whole antibody” or “full-length antibody”). The heavy chains of the antibody may be any isotype, including IgM, IgG, IgE, IgA, or IgD, or a subisotype, including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chains may be kappa light chains or lambda light chains. The antibody may consist of two identical copies of the light chains and two identical copies of the heavy chains. Each heavy chain contains one variable domain (or variable region, VH) and multiple constant domains (or constant regions), which are bound to each other via disulfide bonds within their constant domains to form the “stem” of the antibody. Each light chain contains one variable domain (or variable region, VH). L ) and one constant domain (or constant region) are contained, each bonded to a heavy chain via a disulfide bond. The variable region of each light chain aligns with the variable region of the heavy chain to which it is bonded. The variable regions of both the light and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FRs).

[0195] These hypervariable regions, known as complementarity-determining regions (CDRs), form loops containing the antibody's primary antigen-binding surface. The four framework regions primarily employ a beta-sheet structure, with the CDRs forming loops connecting the beta-sheet structures and, in some cases, forming parts of them. The CDRs of each chain are held in close proximity by the framework regions, and the CDRs from the other chain contribute to the formation of the antigen-binding region.

[0196] CDRs are important for recognizing the epitopes of antigens. As used herein, “epitope” is the smallest portion of a target molecule that can be specifically bound by the antigen-binding domain of an antibody. The minimum size of an epitope may be about 3, 4, 5, 6, or 7 amino acids, but these amino acids do not need to be in a continuous linear sequence of the primary structure of the antigen, as the epitope may depend on the three-dimensional configuration of the antigen based on the secondary and tertiary structures of the antigen.

[0197] In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved and differ in their constant regions, particularly their hinges and upper CH2 domains. The sequences and differences of IgG subclasses are publicly known in the art, for example, as described in Vitarsson, et al., “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5 (2014); Irani, et al., “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2(2015): 171-182; and Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016, each of which is incorporated herein by reference in whole.

[0198] Antibodies may also be immunoglobulin molecules derived from any species (e.g., humans, rodents, mice, camelids). Antibodies disclosed herein also include, but are not limited to, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, polyspecific antibodies, and chimeric antibodies comprising an immunoglobulin-binding domain fused to another polypeptide.

[0199] In some embodiments, the antibody fragment is an antigen-binding fragment.

[0200] The terms “antigen-binding domain” or “antigen-binding fragment” refer to any part of an antibody that retains the specific binding activity of an intact antibody, i.e., any portion of the antibody that can specifically bind to an epitope on the target molecule of the intact antibody. This includes, for example, Fab, Fab', F(ab')2, and variants of these fragments. Thus, in some embodiments, the antibody or its antigen-binding fragment may be, for example, Fv, Fd, dAb, bispecific antibodies, diabodies, linear antibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments, and any polypeptide that is an antigen-binding domain or a binding domain homologous to an antigen-binding domain. Non-limiting examples of antigen-binding domains include, for example, the heavy and / or light chain CDRs of an intact antibody, the heavy and / or light chain variable regions of an intact antibody, the full-length heavy or light chain of an intact antibody, or individual CDRs from either the heavy or light chain of an intact antibody.

[0201] In some embodiments, the antibody is a full-length antibody.

[0202] Antibodies and their antibody fragments may also be antibodies or antibody fragments and antibody variants (including derivatives and conjugates) of multispecific (e.g., bispecific) antibodies or antibody fragments. Additional antibodies provided herein are polyclonal, monoclonal, multispecific (multimer, e.g., bispecific), human antibodies, chimeric antibodies (e.g., human-mouse chimeric), single-chain antibodies, intracellularly produced antibodies (i.e., in vivo), and their antigen-binding fragments. Antibodies or their antibody fragments may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In some embodiments, the antibody or its antigen-binding fragment is an IgG antibody or its antigen-binding fragment.

[0203] The Fab fragment contains a variable domain and a constant domain of the light chain, as well as a variable domain and a first constant domain (CH1) of the heavy chain. The F(ab')2 antibody fragment contains a pair of Fab fragments generally covalently bonded near the carboxyl terminus by a hinge cysteine ​​between them. Other chemical couplings of antibody fragments are also known in the art.

[0204] A diabody is a small antibody fragment having two antigen-binding sites, the fragment containing VH bound to VL in the same polypeptide chain (VH and VL). By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, creating two antigen-binding sites.

[0205] Linear antibodies contain a pair of serial Fd segments (VH-CH1-VH-CH1) that, together with a complementary light chain polypeptide, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0206] A one-armed antibody may have a heavy chain and a light chain, as well as a heavy chain fragment containing the CH2 and CH3 domains of IgG. In some embodiments, a one-armed antibody is an antibody that has only one of two antigen-binding arms in a typical antibody. In some embodiments, a one-armed antibody includes an antigen-binding arm (e.g., VH+CH1 and VL+CL) and Fc.

[0207] The antibodies and antibody fragments of this disclosure may be modified within the Fc region to provide a desired effector function or serum half-life. In some embodiments, the Fc region may be modified to silence or reduce complement-dependent cytotoxicity (CDC) or antibody-dependent cytotoxicity (ADCC).

[0208] Antibody multimerization can be achieved through innate antibody aggregation or through chemical or recombinant conjugation techniques known in the art. For example, a certain proportion of purified antibody preparations (e.g., purified IgG1 molecules) spontaneously form protein aggregates containing antibody homodimers and other higher-order antibody multimers.

[0209] Alternatively, antibody homodimers may be formed via chemical bonding techniques known in the art. For example, antibody polymers can be formed using heterobifunctional crosslinking agents, including but not limited to SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate) and SATA (N-succinimidyl S-acylthioacetate). An exemplary protocol for antibody homodimer formation is described by Ghetie et al. (Proc. Natl. Acad. Sci. USA 94: 7509-7514, 1997). Antibody homodimers can be converted to Fab'2 homodimers via digestion with pepsin. Another method for forming antibody homodimers is via the use of the autoaffinity T15 peptide described by Zhao et al. (J. Immunol. 25:396-404, 2002).

[0210] In some embodiments, multispecific antibodies are bispecific antibodies. Bispecific antibodies can be produced by manipulating the interface between a pair of antibody molecules to maximize the proportion of heterodimers recovered from recombinant cell culture. For example, the interface may contain at least a portion of the CH3 domain of the antibody's constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size as one or more large side chains are produced on the interface of the second antibody molecule by replacing the large amino acid side chains with smaller side chains (e.g., alanine or threonine). This provides a mechanism that increases the yield of heterodimers compared to other undesirable end products such as homodimers. This method is described, for example, in WO96 / 27011, which is incorporated in whole by reference.

[0211] In some embodiments, the antibodies of this disclosure are bispecific antibodies. The term “bispecific antibody” refers to an antibody comprising a first antigen-binding region and a second antigen-binding region, wherein the first antigen-binding region binds to one antigen or epitope, and the second antigen-binding region binds to another antigen or another epitope. Thus, the bispecific antibodies of this disclosure have specificity to two different antigens, or to two different epitopes of one antigen. The bispecific antibody format includes IgG-like antibodies (Fan et al. (2015) Journal of Oncology. 8:130). The most common type of IgG-like antibody comprises two Fab regions and two Fc regions, where the heavy and light chains of each Fab may be derived from separate monoclonal antibodies. The bispecific antibodies of this disclosure can be prepared using bispecific antibody formats or techniques known in the art. Specific exemplary bispecificity formats that may be used in the context of the present invention are described, for example, in Labrijn, et al. Bispecific antibodies: a mechanistic review of the pipeline. Nature Reviews Drug Discovery, 2019, 18(8):1-24.

[0212] In some embodiments, the antibody of the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region, wherein one or more heavy chains comprises a modified heavy chain constant region or CH1 of the present invention, and one or more light chains comprises a modified light chain constant region of the present invention. In some embodiments, the antibody of the present invention comprises two heavy chains and two light chains, wherein the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region, wherein one or more heavy chains comprises a modified heavy chain constant region or CH1 of the present invention, and one or more light chains comprises a modified light chain constant region of the present invention.

[0213] In some embodiments, the antibody of the present invention comprises two different heavy chains and / or two different light chains. In some embodiments, the two different light chains each comprise two different light chain constant regions, for example, one comprising a lambda light chain constant region and the other comprising a kappa light chain constant region. In some embodiments, the antibody of the present invention is a bispecific antibody comprising one pair of heavy chains and light chains that specifically bind to a first antigen, and another pair of heavy chains and light chains that specifically bind to a second antigen.

[0214] The antibodies of the present invention, such as full-length antibodies or multispecific antibodies, can bind to any target, such as an antigen or a ligand or receptor suitable for the antibody. In some embodiments, the antigen is a tumor-associated antigen (TAA). In some embodiments, the tumor-associated antigen is an immune checkpoint molecule. In some embodiments, the target or antigen is selected from FAP, CEA, p95 HER2, BCMA, EpCAM, MSLN, MCSP, HER-1, HER-2, HER-3, CD19, CD20, CD22, CD33, CD38, CD52Flt3, EpCAM, IGF-1R, FOLR1, Trop-2, CA-12-5, HLA-DR, MUC-1 (mucin), GD2, A33 antigen, PSMA, PSCA, transferrin receptor, TNC (tendinin), CA-IX, CD3, B7H3, EGFR, Hel, cMET, Axl, GPRC5D, PD-1, PD-L1, CD47, and immunoactivating molecules, such as 4-1BB, CD40, and OX40.

[0215] In some embodiments, the target or antigen is selected from TROP2, HER2, or B7H3. In some embodiments, the bispecific antibody of the present invention binds to two different epitopes of one antigen or to two antigens. In some specific embodiments, the bispecific antibody of the present invention binds to two epitopes of HER2.

[0216] In some embodiments, the antibody or antigen-binding fragment of the present invention is an antibody or antigen-binding fragment that specifically binds to HER2 and includes modified CH1 and modified CL of the present invention. In some embodiments, the anti-HER2 antibody or antigen-binding fragment includes six CDRs of an antibody known to specifically bind to HER2 (e.g., trastuzumab or pertuzumab). In some embodiments, the anti-HER2 antibody or antigen-binding fragment includes three heavy chain variable region CDRs and / or three light chain variable region CDRs of an antibody known to specifically bind to HER2 (e.g., trastuzumab or pertuzumab). In some embodiments, the anti-HER2 antibody or antigen-binding fragment includes heavy chain variable regions and / or light chain variable regions of an antibody known to specifically bind to HER2 (e.g., trastuzumab or pertuzumab). In some embodiments, the HER2 antibody is a full-length antibody. In some embodiments, the HER2 antibody is trastuzumab or pertuzumab and includes modified CH1 and modified CL of the present invention.

[0217] In some embodiments, the anti-HER2 antibody is a bispecific antibody that specifically binds to two epitopes of HER2 and comprises two heavy chains and two light chains, where one heavy chain and one light chain comprises the modified CH1 and modified CL of the present invention, and / or the other heavy chain and the other light chain comprises the modified CH1 and modified CL of the present invention. In some embodiments, one heavy chain and one light chain of the anti-HER2 bispecific antibody each comprises three heavy chain variable regions CDRs and three light chain variable regions CDRs of trastuzumab, and the other heavy chain and the other light chain each comprises three heavy chain variable regions CDRs and three light chain variable regions CDRs of pertuzumab. In some embodiments, one heavy chain and one light chain of the anti-HER2 bispecific antibody each comprises a heavy chain variable region and a light chain variable region of trastuzumab, and the other heavy chain and one light chain each comprises a heavy chain variable region and a light chain variable region of pertuzumab.

[0218] In some embodiments, the heavy chain variable region of trastuzumab includes or comprises the amino acid sequence described in SEQ ID NO: 72 or 74, and the light chain variable region of trastuzumab includes or comprises the amino acid sequence described in SEQ ID NO: 73 or 75. In some embodiments, the heavy chain variable region of pertuzumab includes or comprises the amino acid sequence described in SEQ ID NO: 76, and the light chain variable region of pertuzumab includes or comprises the amino acid sequence described in SEQ ID NO: 77.

[0219] In some embodiments, the antibody or antigen-binding fragment of the present invention is an antibody or antigen-binding fragment that specifically binds to TROP2 and includes modified CH1 and modified CL of the present invention. In some embodiments, the TROP2 antibody or antigen-binding fragment includes three CDRs of the heavy chain variable region described in SEQ ID NO: 78 and / or three CDRs of the light chain variable region described in SEQ ID NO: 79. In some embodiments, the TROP2 antibody or antigen-binding fragment includes or consists of a heavy chain variable region and a light chain variable region, where the heavy chain variable region includes or consists of the amino acid sequence described in SEQ ID NO: 78 and / or the light chain variable region includes or consists of the amino acid sequence described in SEQ ID NO: 79. In some embodiments, the TROP2 antibody is a full-length antibody.

[0220] In some embodiments, the antibody or antigen-binding fragment of the present invention is an antibody or antigen-binding fragment that specifically binds to B7H3 and comprises modified CH1 and modified CL of the present invention. In some embodiments, the B7H3 antibody or antigen-binding fragment comprises three heavy chain variable region CDRs of the heavy chain variable region described in SEQ ID NO: 80 and / or three light chain variable region CDRs of the light chain variable region described in SEQ ID NO: 81. In some embodiments, the B7H3 antibody or antigen-binding fragment comprises or consists of a heavy chain variable region and a light chain variable region, where the heavy chain variable region comprises or consists of the amino acid sequence described in SEQ ID NO: 80 and / or the light chain variable region comprises or consists of the amino acid sequence described in SEQ ID NO: 81. In some embodiments, the B7H3 antibody is a full-length antibody.

[0221] Any antibody or antibody fragment described herein may be conjugated with a stabilizing molecule (e.g., a molecule that increases the half-life of the antibody or its antigen-binding fragment in a subject or solution). Non-limiting examples of stabilizing molecules include polymers (e.g., polyethylene glycol) or proteins (e.g., serum albumin, such as human serum albumin). Conjugation with a stabilizing molecule can increase the half-life of the antibody or antigen-binding fragment or extend its biological activity in vitro (e.g., in tissue culture or when stored as a pharmaceutical composition) or in vivo (e.g., in humans).

[0222] In some embodiments, the antibodies or antibody fragments described herein may be conjugated to a therapeutic agent. The antibody-drug conjugate, comprising an antibody or its antigen-binding fragment, may be covalently or non-covalently bonded to the therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cell proliferation inhibitor (e.g., cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine, meitansinoids such as DM-1 and DM-4, ​​dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and its analogues).

[0223] Antibody-drug conjugate (ADC) Antibodies or antibody fragments described herein may be conjugated to a payload (e.g., a therapeutic agent or drug). In some embodiments, the payload may be selected from cytotoxic agents, biologically active proteins, synthetic polymers, enzymes, nucleic acids, and fragments thereof such as DNA or RNA. The payload may be covalently or noncovalently bonded to the antibody or antigen-binding fragment or antigen-binding protein construct (e.g., a bispecific antibody). In some embodiments, the payload may be a small molecule (e.g., cytotoxic, radioisotope, proteolytic targeted chimera), nucleic acid (e.g., siRNA, antisense oligonucleotide), protein or peptide (e.g., toxin, cytokine), or lysosome-targeted chimera (LYTAC).

[0224] In some embodiments, the payload is a cytotoxic agent or a cell growth inhibitor (e.g., monomethyl auristatin E, monomethyl auristatin F, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracine, meitansinoids such as DM-1 and DM-4, ​​dione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamides and their analogues). Useful classes of cytotoxic agents, cell growth inhibitors, or immunomodulators include, for example, antitubulins, DNA minor groove binders, DNA replication inhibitors, and alkylating agents.

[0225] In some embodiments, the payload may include, but is not limited to, cytotoxic reagents such as chemotherapeutic agents and immunotherapeutic agents, antiviral agents, or antibacterial agents. In some embodiments, the conjugated payload may be selected from, but is not limited to, MMAE (monomethyl auristatin E), MMAD (monomethyl auristatin D), or MMAF (monomethyl auristatin F).

[0226] In some embodiments, the payload is an auristatin, such as auristatin E (also known in the art as a derivative of dorastatin-10) or a derivative thereof. The auristatin may be, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include AFP, MMAF, and MMAE. The synthesis and structure of exemplary auristatin are described in U.S. Patent Publication No. 2003-0083263, International Patent Publication No. WO04 / 010957, International Patent Publication No. WO02 / 088172, and U.S. Patents No. 7,498,298, 6,884,869, 6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483. These are described in Nos. 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414, each incorporated herein by reference in its entirety for all purposes.

[0227] Auristatin has been shown to interfere with microtubule dynamics as well as nuclear and cell division, and to possess anticancer activity. Auristatin can bind to tubulin and exert cytotoxic or cell proliferation inhibitory effects on cancer cells. There are many different assays known in the art that can be used to determine whether auristatin or the resulting antibody-drug conjugate exerts cell proliferation inhibitory or cytotoxic effects on desired cells.

[0228] In some embodiments, the payload is a chemotherapeutic agent. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®), alkyl sulfonates such as busulfan, improsulfan and piposulfan, aziridines such as benzodepa, carbocon, meturedopa and uredopa, ethyleneimines and methylamelamamines such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide and trimethyloromelamamine, chlorambucil, chlornafadin, etc. Lophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, fenestrine, prednimustine, trophosphamide, nitrogenous mustards such as uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, and other nitrosoureas, such as acrasinomycin, actinomycin, autoramycin, azaserin, bleomycin, kakutinomycin, calicheamicin, carabicin. Antibiotics such as carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogaramycin, olibomycin, peplomycin, potophyllomycin, puromycin, keramycin, ronorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin, methotrexa Antimetabolites such as folate and 5-fluorouracil (5-FU), folate analogs such as denopterin, methotrexate, pteropterin, and trimethrexate, purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine, for example, pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmoflu, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, and 5-FU, carsterone, dromostanolone propionate, epithiostanol, mepitiostane,Androgens such as testactone, anti-adrenal drugs such as aminoglutethimide, mitotane, and trilostane, folic acid supplements such as floric acid, and acegraton, aldofamide glycoside, aminolevulinic acid, amsacrin, bestrabusil, bisantren, edatraxate, dehofamine, demecolsin, diazicon, elfornithine, eriptinium acetate, etogluside, gallium nitrate, hydroxyurea, lentinan, ronidamin, mitogluzone, mitoxantrone, mopidamol, nitracrin, pen Tostatin, fenamet, pirarubicin, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK7, razoxane, schizophyllan, spirogermanium, tenuazonic acid, triadicone, 2',2',2'-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitobronitol, mitobronitol, pipobromane, gasitosine, arabinoside (Ara-C), cyclophosphamide, e.g., paclitaxel (TAXOL®, Bristol-Myers Examples include taxanes such as Squibb Oncology (Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony France), platinum analogs such as chlorambucil, gemcitabine, 6-thioguanine, cisplatin and carboplatin; vinblastone, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeroda, ibandronate, CPT-11; topoisomerase inhibitors RFS2000, difluoromethylornithine (DMFO), retinoic acid, esperamicin, capecitabine, and any pharmaceutically acceptable salts, acids or derivatives of any of the above. Furthermore, this definition includes, for example, anti-estrogens such as tamoxifen, raloxifen, 4(5)-imidazole-inhibiting aromatase, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and toremifene (Fareston), as well as anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin.This also includes anti-hormone agents that act to modulate or inhibit the hormonal effects on tumors, such as any pharmaceutically acceptable salts, acids, or derivatives of any of the above. A detailed description of chemotherapeutic agents can be found, for example, in US20180193477A1, which is incorporated in its entirety by reference.

[0229] In some embodiments, the antigen-binding construct is bound to the payload via a cleavable linker, such as an SPBD linker or a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (VC) linker. In some embodiments, the antigen-binding construct is bound to the drug via a non-cleavable linker, such as an MCC linker formed using SMCC or sulfo-SMCC. The selection of an appropriate linker for a given ADC can be easily made by a person skilled in the art, taking into account relevant factors such as the binding site to the antigen-binding construct, any structural constraints of the drug, and the hydrophobicity of the drug (see, for example, Nolting, Chapter 5, Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer).

[0230] Examples of linkers suitable for use in the present invention include cathepsin-degradable linkers such as Val-Cit linkers (e.g., vc-PAB), cBu-Cit linkers, and CX linkers; non-cleavable linkers such as SMCC linkers or MD linkers; acid-sensitive linkers; silicone-structured linkers; disulfide-carbamate linkers; MC-GGFG linkers; TRX linkers; galactoside-containing linkers; pyrophosphate linkers; near-infrared-sensitive linkers; and UV-sensitive linkers such as PC4AP (Antibody-drug conjugates: Recent advances in linker chemistry, Su, Z., Xiao, D., Xie, F., Liu, L., Wang, Y., Fan, S., … Li, S. (2021). Antibody-drug conjugates: Recent advances in linker chemistry. Acta Pharmaceutica Sinica B.). A linker suitable for use in the present invention may also be a combination of one or more linkers; for example, a cathepsin-degrading linker may be combined with other types of linkers to form a new linker. Therefore, as used herein, the term "linker" encompasses a single type of linker or a combination of different types of linkers, provided that it can bind the antibody of the present invention to a drug. In one embodiment, a linker suitable for use in the present invention is a maleimide-containing linker. Therefore, in one embodiment, a linker suitable for use in the present invention is mc-VC-PAB or mc-GGFG.

[0231] Several specific linker-toxin combinations are described and may be used with the antigen-binding constructs described herein to prepare ADCs in certain embodiments. Examples include, but are not limited to, cleavable peptide linkers having auristatins such as MMAE and MMAF; camptothecines such as SN-38, duocalmycin, and PDB dimers; non-cleavable MC linkers having auristatins MMAF and MMAE; acid-unstable hydrazone linkers having calitiamycin and doxorubicin; disulfide linkers having mayansinoids such as DM1 and DM4; and bis-maleimide-trioxyethylene glycol (BMPEO) linkers having mayansinoid DM1. Some payloads and linkers are described, for example, in Peters & Brown, (2015) Biosci. Rep. e00225, Dosio et al., (2014) Recent Patents on Anti-Cancer Drug Discovery 9:35-65, U.S. Patent Publication US2015 / 0374847, and U.S. Patent Publication US20180193477A1, which are incorporated herein by reference in their entirety.

[0232] Depending on the desired drug and the selected linker, those skilled in the art can choose a suitable method for linking them together. For example, several conventional coupling methods, such as amine coupling methods, can be used to form the desired drug-linker complex that still contains a reactive group for conjugation to the antibody via covalent bonding. In some embodiments, drug-maleimide complexes (i.e., maleimide-bound drugs) may be used as the payload-supporting reactive group of this disclosure. Maleimide is the most common reactive group that can be bound to a thiol group in an ADC preparation. Furthermore, organobromids and iodides are also frequently used.

[0233] ADCs can be prepared by one of several routes known in the art using organic chemical reactions, conditions, and reagents known to those skilled in the art (see, for example, Bioconjugate Techniques (G. Thermanson, 2013, Academic Press)). For example, conjugation is achieved by (1) the reaction of a nucleophile or electrophile of an antibody with a divalent linker reagent to form an antibody linker intermediate Ab-L via covalent bonding before reaction with the active drug moiety D, or (2) the reaction of a nucleophile or electrophile of a drug moiety with a linker reagent to form a drug linker intermediate DL via covalent bonding before reaction with the nucleophile or electrophile of an antibody. Conjugation methods (1) and (2) can be used with various antibodies, drug moieties, and linkers to prepare the ADCs described herein. Various prepared linkers, linker components, and toxins are commercially available or can be prepared using standard synthetic organic chemistry techniques. These methods are described, for example, in March's Advanced Organic Chemistry (Smith & These are described in March, 2006, Sixth Ed., Wiley; Toki et al., (2002) J. Org. Chem. 67:1866-1872; Frisch et al., (1997) Bioconj. Chem. 7:180-186; Bioconjugate Techniques (G. Thermanson, 2013, Academic Press), US20210379193A1, and US20180193477A1, the entirety of which is incorporated herein by reference. Furthermore, several pre-formed drug linkers suitable for reaction with selected antigen-binding constructs are also commercially available, for example, linker toxins containing DM1, DM4, MMAE, MMAF, or ducalmycin SA are available from Creative BioLabs (Shirley, NY).

[0234] It is understood that the pharmaceutically acceptable salt or solvate forms of ADC are also applicable to the various embodiments described herein.

[0235] In some embodiments, the present invention relates to an antibody-drug conjugate of formula (I):

number

[0236] It should be understood that the S in equation (I) is sulfur from the antibody Ab.

[0237] In some embodiments, D in formula (I) of the present invention may be any antitumor compound and is not particularly limited as long as it has an antitumor effect and a portion that can be linked to the linker structure. The antitumor compound may be a pharmaceutically active compound that acts on tumors. For ADCs, it is preferable that part or all of the linker is cleaved within tumor cells to release the antitumor compound, thereby exhibiting an antitumor effect. When the linker is cleaved at the site where it links to the drug, the antitumor compound is released in an unmodified form and exhibits its original antitumor effect. For example, D may be a payload as defined herein.

[0238] In some embodiments, the antitumor compound is, for example, a cytotoxic agent, a topoisomerase I inhibitor, a small molecule tubulin inhibitor such as camptothecin, auristatin, or a maytansinoid such as exatecan, Dxd, MMAE, MMAF, DM1, or DM4.

[0239] In some embodiments, -L- has the following structure:

number

[0240] In the above-mentioned -Z-L1-L2-L3-, it will be understood that Z is connected to S of Ab, and L3 is connected to D.

[0241] In some embodiments, Z is [ka] Selected from the following, where m is 1, 2, 3, 4, 5, 6, 7, or 8.

[0242] In some embodiments, Z is [ka] Selected from.

[0243] In some embodiments, L1 is absent or [ka] The formula is chosen from, where each n1 is an integer selected from 0 to 12, such as 1, 2, 3, 4, 5, 6, 7, or 8, independently.

[0244] In some embodiments, L2 is a peptide residue consisting of 2, 3, 4, 5, 6, or 7 amino acids.

[0245] The amino acids constituting L2 are not particularly limited, and are, for example, L- or D-amino acids, preferably L-amino acids. In addition to α-amino acids, they may also be amino acids having structures such as β-alanine, ε-aminocaproic acid, and γ-aminobutyric acid. Furthermore, they may also be unnatural amino acids such as N-methylated amino acids.

[0246] The amino acids that make up L2 can be independently selected from phenylalanine (Phe; F), tyrosine (Tyr; Y), leucine (Leu; L), glycine (Gly; G), alanine (Ala; A), valine (Val; V), lysine (Lys; K), citrulline (Cit), serine (Ser; S), glutamic acid (Glu; E), aspartic acid (Asp; D), asparagine (Asn), isoleucine (Ile), arginine (Arg), proline (Pro), glutamine (Gln), and others.

[0247] In some embodiments, L2 is selected from -Val-Ala-, -Val-Cit-, -Ala-Ala-, -Ala-Cit-, -Ala-Lys-, -Ala-Val-, -Asn-Cit-, -Asp-Cit-, -Asn-Lys-, -Asp-Val-, -Cit-Ala-, -Cit-Asn-, -Cit-Asp-, -Cit-Cit-, -Cit-Lys-, -Cit-Ser-, -Cit-Val-, -Glu-Val-, -Glu-Gly-, -Ile-Cit-, -Ile-Pro-, -Ile-Val-, -Leu-Cit-, -Lys-Cit-, -Phe-Arg-, -Phe-Cit-, -Phe-Lys-, -Pro-Lys-, -Ser-Cit-, -Trp-Cit-, -Ala-Val-, -Val-Asp-, -Cit-Val-, -Val-Glu-, -Val-Lys-, -Gly-Gly-Gly-, -Gly-Gly-Arg-, -Phe-Lys-Gly-, -Leu-Lys-Gly-, -Leu-Leu-Gly-, -Glu-Val-Cit-, -Cit-Ala-Glu-, -Val-Lys-Gly-, -Val-Lys-Ala-, -Val-Gly-Gly-, -Val-Cit-Gly-, -Val-Gln-Gly-, -Val-Glu-Gly-, -Val-Lys-Gly-, -Val-Lys-Leu-, -Ala-Ala-Ala-, -Asn-Ala-Ala-, -Gly-Gly-Phe-Gly-, -Gly-Gly-Gly-Gly-, -Gly-Gly-Leu-Gly-, -Gly-Phe-Leu-Gly-, -Gly-Val-Lys-Gly-, -A1a-Leu-A1a-Leu-, -Gly-Phe-Leu-Gly-, -Ala-Leu-Ala-Leu-, -Gly-Phe-Gly-Gly-, -Val-Lys-Gly-Gly-, and -Pro-Leu-Gly-Leu-Ala-Gly-.

[0248] In some embodiments, L2 is selected from -Val-Ala-, -Gly-Gly-Phe-Gly-, and -Val-Cit-.

[0249] L2 is linked to L1 or Z via the amino group of the amino acid on its left side, and to L3 via the carbonyl group of the amino acid on its right side, which will be understood to be consistent with the following explanation.

[0250] In some embodiments, L3 is [ka] Selected from, in the formula, R 1c n2 is selected from H and C1-C6 alkyl groups, n2 is 1, 2, 3, or 4, and n3 and n4 are independently selected from 1, 2, 3, 4, 5, or 6.

[0251] In some embodiments, L3 is absent or [ka] Selected from.

[0252] L3 is linked to L2 through the amino group on its left side and to D through groups such as the carbonyl group on its right side, which should be understood to be consistent with the following explanation.

[0253] In some embodiments, -Z-L1-L2-L3- is selected from the following structures. [ka]

[0254] In some embodiments, D has a structure represented by formula (D-1a) or formula (D-1b). [ka]

[0255] In the formula, R 1a These are selected from H and C1-C6 alkyl groups. R2a is H, halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR5a and -SR 5a selected from, R 3a is H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl, and -OR 5a selected from, or R 2a and R 3a together form -O(CR 6a R 7a ) n O-, where n is 1, 2, 3, or 4, R 4a and R 5a are independently selected from H and C1-C4 alkyl, R 6a and R 7a are independently selected from H and halogen, either X is absent or is selected from -O(CH2) m C(O)-, m is 1, 2, 3, 4, 5, or 6, and the carbonyl on the right side of the group is bonded to the nitrogen in formula (D-1a),

Chemical formula

[0256] In some embodiments, R 1a H is R 2a It is a C1-C6 alkyl group, and R 3a is a halogen, preferably -F, and R 4a The compound is a C1-C4 alkyl group, preferably ethyl.

[0257] In some embodiments, R 1b , R 4b , and R 8b Each is independent of C 1-2 Selected from alkyl, preferably methyl, R 2b , R 3b , and R 5b Each is independent of C 3-4 Selected from alkyl groups, R 6b and R 7b However, each is independent of C 1-2 Selected from alkoxy, R 9b However, C 1-4 Selected from alkyl, R 10b However, is it OH, or R 9b However, it is COOH, and R 10b However, it is H.

[0258] In some embodiments, D has a structure represented by formula (D-2a) or formula (D-2b): [ka] In the formula, R 1a , R 2a , R 3a , R 4a , and X are as defined above, or [ka] And in the formula, R 1b , R 2b , R 3b , R4b , R 5b , R 6b , R 7b , R 8b , R 9b , and R 10b This is defined as described above.

[0259] In some embodiments, D has a structure represented by formula (D-3a) or formula (D-3b). [ka]

[0260] Unless otherwise specified and unless otherwise inconsistent with the context, it should be understood that, for the ADCs of the present invention, the bond on the left side of the divalent group as shown herein is to an Ab or a group near the Ab terminus, and the bond on the right side of the divalent group is to a D or a group near the D terminus. For example, L2 is [ka] In this case, the amino group on the left is bonded to L1, and the carbonyl group on the right is bonded to L3; In some embodiments, the antibody-drug conjugate has an average DAR of 2-10, 6-10, 4-8, 7-9, or 2-4.

[0261] In some embodiments, the antibody-drug conjugate is [ka] Selected from the formula, where Ab is the antibody or antigen-binding fragment of the present invention, preferably p is as defined above, and preferably the antibody-drug conjugate has an average DAR of, for example, about 4 or 6.

[0262] It will be understood that the sulfur atom bonded to Ab in the above ADC originates from Ab. The disulfide bond of Ab cleaves under the action of a reducing agent such as TCEP to produce a thiol (-SH), which then bonds to the terminal functional group of the linker, such as the maleimide moiety.

[0263] Several specific examples of methods for preparing ADCs are known in the art and are described in U.S. Patent No. 8,624,003 (Pot Method), U.S. Patent No. 8,163,888 (One-Step Method), and U.S. Patent No. 5,208,020 (Two-Step Method), and U.S. 20180193477A1, which are incorporated herein by reference in their entirety. Other methods are known in the art and include those described in Antibody-Drug Conjugates: Methods in Molecular Biology, 2013, Ducry (Ed.), Springer.

[0264] The drug load is expressed by the number of drug moieties per antibody in the ADC molecule. For some antibody-drug conjugates, the drug load may be limited by the number of binding sites on the antibody. For example, if the binding is to cysteinethiol, as in certain exemplary embodiments described herein, the drug load may range from 0 to 8 drug moieties per antibody. In certain embodiments, a higher drug load, e.g., p ≥ 5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of a particular antibody-drug conjugate. In certain embodiments, the average drug load of an antibody-drug conjugate is in the range of 1 to about 8, about 2 to about 6, or about 3 to about 5. In practice, it has been shown that for a particular antibody-drug conjugate, the optimal ratio of drug moieties per antibody may be about 4. In some embodiments, the drug-to-antibody ratio (DAR) is about or at least 1, 2, 3, 4, 5, 6, 7, or 8. In some embodiments, the average DAR in the composition is about 1 to about 2, about 2 to about 3, about 3 to about 4, about 3 to about 5, about 4 to about 5, about 5 to about 6, about 6 to about 7, or about 7 to about 8.

[0265] In some embodiments, an excess amount of reducing agent is added to the antibody or antibody fragment to reduce the hinge region disulfide bond. In some embodiments, the concentration of the antibody or antibody fragment is greater than 0.1 mg / ml, greater than 0.2 mg / ml, greater than 0.3 mg / ml, greater than 0.4 mg / ml, greater than 0.5 mg / ml, greater than 1 mg / ml, greater than 2 mg / ml, greater than 3 mg / ml, greater than 4 mg / ml, greater than 5 mg / ml, greater than 10 mg / ml, greater than 20 mg / ml, greater than 30 mg / ml, greater than 40 mg / ml, or greater than 50 mg / ml. In some embodiments, the antibody concentration is less than 0.1 mg / ml, less than 0.2 mg / ml, less than 0.3 mg / ml, less than 0.4 mg / ml, less than 0.5 mg / ml, less than 1 mg / ml, less than 2 mg / ml, less than 3 mg / ml, less than 4 mg / ml, less than 5 mg / ml, less than 10 mg / ml, less than 20 mg / ml, less than 30 mg / ml, less than 40 mg / ml, or less than 50 mg / ml. In some embodiments, the concentration of the antibody or antibody fragment is 0.1 to 50 mg / ml, 1 to 50 mg / ml, 1 to 10 mg / ml, or 1 to 5 mg / ml. In some embodiments, the reduction potential of the reducing agent is about -0.29 V. In some embodiments, the reduction potential of the reducing agent is about -0.24 V to -0.32 V. In some embodiments, the reduction potential of the reducing agent is approximately -0.24V, approximately -0.25V, approximately -0.26V, approximately -0.27V, approximately -0.28V, approximately -0.29V, approximately -0.30V, approximately -0.31V, or approximately -0.32V. In some embodiments, the reducing agent is added in amounts greater than 2 equivalents, 3 equivalents, 4 equivalents, 5 equivalents, 6 equivalents, 7 equivalents, 8 equivalents, 9 equivalents, 10 equivalents, 15 equivalents, 20 equivalents, 25 equivalents, 30 equivalents, 40 equivalents, or 50 equivalents per single antibody molecule. In some embodiments, the reducing agent is added in amounts less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 equivalents per single antibody molecule. In some embodiments, the reducing agent is added in amounts of 4 to 50 equivalents or 8 to 30 equivalents per single antibody molecule. In some embodiments, the reducing agent is added in amounts of 10 to 30 equivalents per single antibody molecule.In some embodiments, the reducing agent is added in amounts of 10, 11, 12, 13, 14, 15, 20, 25, or 30 equivalents per single antibody molecule. In some embodiments, the reduction reaction takes place over a period of more than 15 minutes, more than 30 minutes, more than 1 hour, more than 1.5 hours, more than 2 hours, more than 3 hours, more than 4 hours, more than 6 hours, more than 8 hours, more than 12 hours, or more than 24 hours. In some embodiments, the reduction reaction takes place over a period of less than 15 minutes, less than 30 minutes, less than 1 hour, less than 1.5 hours, less than 2 hours, less than 3 hours, less than 4 hours, less than 6 hours, less than 8 hours, less than 12 hours, or less than 24 hours.

[0266] In some embodiments, the reducing agent is tris(2-carboxyethyl)phosphine hydrochloride (TCEP), beta-mercaptoethanol (β-ME), dithiothreitol (DTT), dithioerythritol (DTE), tris-(hydroxymethyl)phosphine (THMP), tris(3-hydroxypropyl)phosphine (THPP), 2-mercaptoethylamine·HCl (2-MEA), or glutathione (GSH). In some embodiments, the reducing agent is selected from TCEP, THMP, and THPP. In some embodiments, the reducing agent is TCEP.

[0267] In some embodiments, an excess of TCEP (10 to 30 equivalents, e.g., 10, 11, 12, 13, 14, 15, 20, 25, or 30 equivalents per single antibody molecule) is added to the antibody or antibody fragment to reduce the hinge region disulfide bonds (e.g., at 2 mg / ml). In some embodiments, the reduction reaction takes place over a period of more than 15 minutes, more than 30 minutes, more than 1 hour, more than 1.5 hours, more than 2 hours, more than 3 hours, more than 4 hours, more than 6 hours, more than 8 hours, more than 12 hours, or more than 24 hours. In some embodiments, the reduction reaction takes place over a period of less than 15 minutes, less than 30 minutes, less than 1 hour, less than 1.5 hours, less than 2 hours, less than 3 hours, less than 4 hours, less than 6 hours, less than 8 hours, less than 12 hours, or less than 24 hours. In some embodiments, the reduction reaction takes place at room temperature. In some embodiments, the reduction reaction takes place in phosphate-buffered saline (PBS) buffer. In some embodiments, the reduction reaction takes place in histidine buffer (e.g., 20 mM, pH=6.5) at 37°C for 2 hours. In some embodiments, the disulfide bonds in the hinge region were completely reduced. In some embodiments, more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the disulfide bonds in the hinge region were reduced. In some embodiments, the reduction reaction is carried out in a buffer system selected from hepes, histidine buffer, PBS, and MES. In some embodiments, the pH of the buffer system is 5.5 to 8. In some embodiments, the reduction reaction is carried out at a temperature of 0°C to 37°C.

[0268] In some embodiments, an excess linker payload (e.g., 12 equivalents per single antibody molecule) was added to the reduced antibody or antibody fragment to form an ADC. In some embodiments, the linker payload was added in amounts greater than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 equivalents per single antibody molecule. In some embodiments, the linker payload is added in amounts less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, or 50 equivalents per single antibody molecule. In some embodiments, the linker payload is added in amounts of 5 to 20 equivalents or 10 to 15 equivalents per single antibody molecule. In some embodiments, the linker payload is added in amounts of 10, 11, 12, 13, 14, 15, or 20 equivalents per single antibody molecule. In some embodiments, the coupling reaction takes place over a period of more than 15 minutes, more than 30 minutes, more than 1 hour, more than 1.5 hours, more than 2 hours, more than 3 hours, more than 4 hours, more than 6 hours, more than 8 hours, more than 12 hours, or more than 24 hours. In some embodiments, the coupling reaction takes place over a period of less than 15 minutes, less than 30 minutes, less than 1 hour, less than 1.5 hours, less than 2 hours, less than 3 hours, less than 4 hours, less than 6 hours, less than 8 hours, less than 12 hours, or less than 24 hours. In some embodiments, the coupling reaction takes place at room temperature. In some embodiments, the coupling reaction takes place in PBS buffer. In some embodiments, the coupling reaction takes place in 10% dimethyl sulfoxide (DMSO) at 25°C for 1.5 hours. In some embodiments, the linker is a maleimide linker. In some embodiments, the linker is a maleimide caproyl linker. In some embodiments, the payload is a drug. In some embodiments, the payload is a therapeutic agent. In some embodiments, the payload is a cytotoxic agent. In some embodiments, the coupling reaction is carried out in a buffer system selected from hepes, histidine buffer, PBS, and MES.In some embodiments, the pH of the buffer system is 5.5 to 8. In some embodiments, the linker-payload contains a maleimide moiety, bromide, or iodide. In some embodiments, the coupling reaction is carried out at a temperature of 0°C to 37°C. In some embodiments, the payload contains a diagnostic agent, therapeutic agent, or labeling agent.

[0269] In some embodiments, the unreacted linker-payload was quenched by adding an excess of N-acetylcysteine ​​(e.g., 30 equivalents per single antibody molecule). In some embodiments, the quench reaction was carried out for more than 15 minutes, more than 30 minutes, more than 1 hour, more than 1.5 hours, more than 2 hours, more than 3 hours, more than 4 hours, more than 6 hours, more than 8 hours, more than 12 hours, or more than 24 hours. In some embodiments, the quench reaction was carried out for less than 15 minutes, less than 30 minutes, less than 1 hour, less than 1.5 hours, less than 2 hours, less than 3 hours, less than 4 hours, less than 6 hours, less than 8 hours, less than 12 hours, or less than 24 hours. In some embodiments, the quench reaction occurred at room temperature. In some embodiments, the quench reaction occurred in PBS buffer. In some embodiments, the quench reaction was carried out for 20 minutes. In some embodiments, the quench reaction was carried out in a buffer system selected from hepes, histidine buffer, PBS, and MES. In some embodiments, the pH value of the buffer system was 5.5 to 8. In some embodiments, the quench reaction is carried out at a temperature of 0°C to 37°C.

[0270] In some embodiments, the ADC is purified by a desalting column. In some embodiments, the ADC is purified by a Zeba® Spin Desalting column (e.g., 10 ml). In some embodiments, the remaining ADC is concentrated to obtain a solution of the corresponding ADC.

[0271] In some embodiments, the resulting ADCs are classified as D0, D2, D4, D6, and D8 based on the ratio of antibody to payload molecules in the ADC, where D0 refers to an antibody molecule that is not bound to any payload molecules, D2 refers to an ADC in which two payload molecules are bound to the antibody molecule, D4 ​​refers to an ADC in which four payload molecules are bound to the antibody molecule, D6 refers to an ADC in which six payload molecules are bound to the antibody molecule, and D8 refers to an ADC in which eight payload molecules are bound to the antibody molecule.

[0272] In some embodiments, the modified antibodies or antibody fragments described herein can be used to produce site-directed DAR4 ADCs.

[0273] In some embodiments, the ADCs described herein have a mean drug-to-antibody ratio (DAR) greater than 3, greater than 3.2, greater than 3.4, greater than 3.6, greater than 3.8, greater than 4, greater than 4.2, greater than 4.4, or greater than 4.6, as determined by HPLC. In some embodiments, the ADCs described herein have a mean DAR less than 3, less than 3.2, less than 3.4, less than 3.6, less than 3.8, less than 4, less than 4.2, less than 4.4, or less than 4.6, as determined by HPLC. In some embodiments, the ADCs described herein have a mean DAR of 3-5, 3.5-5, 3.5-4.5, 3.7-4.3, or 3.8-4.2. In some embodiments, the ADCs described herein have a mean DAR of 3.74-4 or 3.74-3.95. In some embodiments, the ADCs described in this disclosure have a mean DAR of 5.70-6 or 5.70-5.85.

[0274] In some embodiments, DAR4 constitutes more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the different DAR types in the ADC described herein. In some embodiments, DAR6 constitutes more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the different DAR types in the ADC described herein. In some embodiments, at least 90% of the resulting ADC product has about 4 DARs. In some embodiments, at least 90% of the resulting ADC product has about 6 DARs.

[0275] In some embodiments, the antibodies or antigen-binding fragments described herein, or ADCs derived therefrom, have a purity of over 90%, over 91%, over 92%, over 93%, over 94%, over 95%, over 96%, over 97%, or over 98% as determined by size exclusion chromatography (SEC). In some embodiments, the antibodies or antibody fragments described herein, or ADCs derived therefrom, have hydrophobic interaction chromatography (HIC) retention times of over 2 minutes, over 2.5 minutes, over 3 minutes, over 3.5 minutes, over 4 minutes, or over 4.5 minutes.

[0276] In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, are stable in plasma. In some embodiments, plasma stability can be measured by an in vitro plasma stability assay. In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, are stable in plasma for at least 1 day, at least 2 days, at least 3 days, at least 4 days, or at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, or at least 18 days.

[0277] In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, can bind to tumor cells. In some embodiments, cell binding can be measured by an in vitro cell binding assay. In some embodiments, cell binding can be measured by the half-effect concentration (EC50). In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, have a cell-binding EC50 of less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, or less than 0.15 nM.

[0278] In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, can kill tumor cells. In some embodiments, the cytotoxicity can be measured by an in vitro cytotoxicity assay. In some embodiments, cytotoxicity can be measured by the half-effective concentration (EC50). In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, have a cytotoxicity EC50 of less than 3 nM, less than 2.5 nM, less than 2 nM, less than 1.5 nM, less than 1 nM, less than 0.75 nM, less than 0.5 nM, less than 0.25 nM, or less than 0.15 nM.

[0279] In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, have a tumor growth inhibition rate (TGI%) of more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 110%, more than 120%, more than 130%, more than 140%, more than 150%, more than 160%, more than 170%, more than 180%, more than 190%, or more than 200%. In some embodiments, the antibodies or antigen-binding fragments thereof described herein, or ADCs derived therefrom, have tumor growth inhibition rates of less than 60%, less than 70%, less than 80%, less than 90%, less than 100%, less than 110%, less than 120%, less than 130%, less than 140%, less than 150%, less than 160%, less than 170%, less than 180%, less than 190%, or less than 200%. The TGI% can be determined, for example, at 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. When used herein, tumor growth inhibition rate (TGI%) is calculated using the following formula:

number

[0280] Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume of the treatment group on day 0. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day zero.

[0281] Method for producing a modified antibody or an antibody fragment thereof. Variants of antibodies or antibody fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the human, humanized, or chimeric antibodies or their antigen-binding fragments described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues in the amino acid sequence constituting the antigen-binding site of the antibody or antigen-binding domain. In a population of such variants, some antibodies or antibody fragments will have increased affinity for the target protein. Any combination of deletions, insertions, and / or combinations can be made to obtain antibodies or antigen-binding fragments with increased binding affinity to the target. Amino acid changes introduced into antibodies or antigen-binding fragments can also alter or introduce new post-translational modifications to the antibody or antigen-binding fragment, such as changing the number of glycosylation sites (e.g., increasing or decreasing), changing the type of glycosylation site (e.g., changing the amino acid sequence so that different sugars are bound by enzymes present in the cell), or introducing new glycosylation sites.

[0282] The antibodies disclosed herein may originate from any animal species, including mammals. Non-limiting examples of natural antibodies include antibodies derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

[0283] The antibodies produced by the mouse have a fully human VH, a fully human VL, and a mouse constant region. In some embodiments, the human VH and human VL are linked to a human IgG constant region (e.g., IgG1, IgG2, IgG3, and IgG4).

[0284] Human antibodies and humanized antibodies include antibodies having a variable region and a constant region derived from (or having the same amino acid sequence as) a human germline immunoglobulin sequence. Human antibodies may also include, for example, amino acid residues not encoded by a human germline immunoglobulin sequence in a CDR (mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo).

[0285] Humanized antibodies typically have a human framework (FR) into which a non-human CDR has been transplanted. Therefore, humanized antibodies have one or more amino acid sequences introduced from a non-human source. These non-human amino acid residues are typically often referred to as “import” residues and usually originate from “import” variable domains. Humanization can essentially be carried out, for example, by substituting a rodent CDR or CDR sequence with the corresponding sequence in a human antibody. These methods are described, for example, in Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); and Verhoeyen et al., Science, 239:1534-1536 (1988), each of which is incorporated herein by reference in its entirety. Thus, a “humanized” antibody is a chimeric antibody in which substantially fewer parts of the intact human V domain are replaced with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically mouse antibodies in which some CDR residues and some FR residues are replaced with residues from similar sites in human antibodies.

[0286] The selection of human VH and VL domains used in the production of humanized antibodies is crucial for reducing immunogenicity. According to the so-called best match method, the V domain sequence of a mouse antibody is screened against an entire library of known human domain sequences. The human sequence most closely resembling the mouse sequence is then accepted as the human FR for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).

[0287] It is even more important that antibodies are humanized while retaining high specificity and affinity for antigens, as well as other desirable biological properties. To achieve this objective, humanized antibodies can be prepared by analytical processes of the parent sequence and various conceptual humanized products using three-dimensional models of the parent sequence and the humanized sequence. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the putative three-dimensional structure of selected candidate immunoglobulin sequences. Examination of these displays allows for the analysis of the potential role of residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient sequence and import sequence so as to achieve desired antibody properties, such as increased affinity for one or more target antigens.

[0288] Typically, amino acid sequence variants of human antibodies, humanized antibodies, or chimeric antibodies contain amino acid sequences that have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the sequences present in the light or heavy chain of the original antibody.

[0289] Sequence identity or homology is typically the percentage of amino acid residues present in a candidate sequence that is identical to a sequence present in a human, humanized, or chimeric antibody or fragment, after the sequences have been aligned to achieve the maximum possible sequence identity and gaps have been introduced as necessary, without considering any conservative substitutions as part of the sequence identity.

[0290] Further modifications can be made to the antibody or antibody fragment. For example, one or more cysteine ​​residues can be introduced into the Fc region, thereby enabling the formation of interchain disulfide bonds in this region. The homodimer antibody thus produced may have any increase in half-life in vitro and / or in vivo. Homodimer antibodies with increased half-life in vitro and / or in vivo can also be prepared using heterobifunctional crosslinkers, for example, as shown by Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, the antibody can be manipulated to have a double Fc region (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

[0291] In some embodiments, covalent modifications can be performed on antibodies or their antigen-binding fragments. These covalent modifications can be prepared by chemical synthesis or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of antibodies or antibody fragments are introduced intramolecularly by reacting target amino acid residues of the antibody or fragment with an organic derivatizing agent that can react with selected side chains or N-terminal or C-terminal residues.

[0292] In some embodiments, antibody variants are provided having carbohydrate structures lacking fucose (directly or indirectly) attached to the Fc region. For example, the amount of fucose in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycans at Asn297 relative to the sum of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, as measured by MALDI-TOF mass spectrometry, as described, for example, in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (Eu numbering of the Fc region residue, or position 314 in Kabat numbering), although slight sequence changes in the antibody may cause Asn297 to be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region of the antibody can be further manipulated to replace the asparagine at position 297 with alanine (N297A).

[0293] In some embodiments, the Fc region of the antibody was further manipulated to replace the serine at position 228 (EU numbering) of IgG4 with proline (S228P) to enhance production efficiency by avoiding Fab-arm exchange. A detailed description of the S228 mutation is, for example, found in Silva et al., "The S228P mutation prevents in vivo and in vitro IgG4 Fab-arm exchange as demonstrated using a combination of novel quantitative immunoassays and physiological matrix preparation," Journal of Biological Chemistry 290.9(2015):5462-5469, which is incorporated in its entirety by reference.

[0294] In one aspect, the present disclosure provides a method for manufacturing an ADC, the method being (1) To produce a nucleic acid sequence encoding the heavy chain of an antibody or antibody fragment, wherein the cysteine ​​residue at position 220 is substituted with a non-cysteine ​​amino acid, and / or to produce one or more of the following mutations: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C, and / or to produce a nucleic acid sequence encoding the light chain of an antibody or antibody fragment by substituting the cysteine ​​at position 214 with a non-cysteine ​​amino acid, and / or to produce one or more of the following mutations: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C, (2) Expressing the antibody or an antibody fragment, (3) Isolating the antibody or an antibody fragment, (4) Reacting the antibody or an antibody fragment with a reducing agent, (5) The antibody or an antibody fragment thereof is reacted with a payload.

[0295] Recombinant vectors This disclosure also provides a recombinant vector (e.g., an expression vector) comprising an isolated polynucleotide (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell into which the recombinant vector is introduced (i.e., the host cell contains the polynucleotide and / or the vector containing the polynucleotide), and the production of a recombinant antibody polypeptide or a fragment thereof by recombinant technology.

[0296] As used herein, “vector” is any construct capable of delivering one or more target polynucleotides to a host cell when the vector is introduced into the host cell. “Expression vector” can be delivered and expressed in the host cell into which the expression vector has been introduced as a polypeptide encoding one or more target polynucleotides. Thus, in an expression vector, the target polynucleotide is positioned for expression in the vector by being operably ligated with regulatory elements such as promoters, enhancers, and / or poly-A tails either within the vector or near or adjacent to the integration site of the target polynucleotide in the host cell genome, and as a result the target polynucleotide is translated in the host cell into which it has been introduced with the expression vector.

[0297] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., by recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which may be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors associated with cationic condensants.

[0298] In some implementations, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of non-pathogenic (defective), reproducible viruses or replication-deficient viruses. In the latter case, viral replication generally occurs only in complementary viral packaging cells.

[0299] For expression, DNA inserts containing polynucleotides encoding antibodies or polypeptides disclosed herein may be operably ligated to suitable promoters (e.g., heterologous promoters), such as, to name a few, the phage-lambda PL promoter, the E. coli lac promoter, the trp promoter, and the tac promoter, the early and late promoters of SV40, and the promoter of retroviral LTRs. Other suitable promoters are known to those skilled in the art. The expression construct may further include sites for transcription initiation and termination, and a ribosome-binding site for translation within the transcription region. The coding portion of the mature transcript expressed by the construct may initially include a transcription initiation site and a stop codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide being translated.

[0300] As shown, the expression vector may be configured to include at least one selection marker. Such markers include dihydrofolate reductase or neomycin resistance to eukaryotic cell culture, as well as tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella tifrium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HK293 cells, and plant cells. Suitable culture media and conditions for the host cells described herein are known in the art.

[0301] The introduction of constructs into host cells can be achieved by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in numerous standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986), which are incorporated herein by reference in their entirety.

[0302] Transcription of the DNA encoding the antibodies of this disclosure by higher eukaryotes can be increased by inserting enhancer sequences into the vector. Enhancers are cis-acting elements of DNA, typically about 10–300 bp in length, that act to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located post-origin at base pairs 100–270, the cytomegalovirus early promoter enhancer, the post-origin polyoma enhancer, and the adenovirus enhancer.

[0303] Appropriate secretory signals may be incorporated into the expressed polypeptide for the secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment. The signal may be endogenous to the polypeptide or a heterologous signal.

[0304] Polypeptides (e.g., antibodies) can be expressed in modified forms, such as fusion proteins (e.g., GST fusions), or with histidine tags, and may include not only secretory signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, may be added to the N-terminus of the polypeptide to improve stability and persistence in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. These regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides for secretion or excretion to improve stability and facilitate purification is a well-known and common technique, particularly in the art.

[0305] Treatment method The antibodies or antibody fragments thereof, and ADCs derived therefrom, or pharmaceutically acceptable salts or solvates thereof, can be used for a variety of therapeutic purposes.

[0306] In one embodiment, the disclosure provides a method for treating cancer in a subject, a method for reducing the rate of tumor volume increase in the subject over time, a method for reducing the risk of metastasis, or a method for reducing the risk of additional metastasis in the subject. In some embodiments, the treatment can stop, delay, suppress, or inhibit the progression of cancer. In some embodiments, the treatment can result in a reduction in the number, severity, and / or duration of one or more cancer symptoms in the subject.

[0307] In one embodiment, the Disclosure relates to a method for administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof disclosed herein, or an ADC derived therefrom, to a subject in need (e.g., a subject identified or diagnosed with cancer), such as breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, urinary tract cancer, or hematological malignancy. In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-resistant prostate cancer. In some embodiments, the cancer is NSCLC, ovarian cancer, melanoma, colorectal cancer, breast cancer, hematological malignancies, head and neck cancer, gastrointestinal cancer, bladder cancer, or bone cancer. In some embodiments, the subjects have solid tumors. In some embodiments, the cancer is head and neck squamous cell carcinoma (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the subjects have Hodgkin lymphoma. In some embodiments, the subjects have triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, hematological malignancies, particularly non-Hodgkin lymphoma, lymphoma, chronic lymphocytic leukemia, or progressive solid tumors. In some embodiments, cancer is breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, bladder cancer, gastroesophageal junction cancer, gastric cancer, non-small cell lung cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, non-Hodgkin lymphoma, solid tumor, lymphoma, epithelial tumor, soft tissue sarcoma, esophageal cancer, or CNS tumor.

[0308] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Patients with cancer can be identified by various methods known in the art.

[0309] As used herein, “effective dose” means an amount or dose sufficient to produce a beneficial or desired outcome, including stopping, delaying, suppressing, or inhibiting the progression of a disease, such as an autoimmune disease or cancer. The effective dose varies depending on the age and weight of the subject to whom the antibody, antigen-binding fragment, ADC, polynucleotide encoding the antibody, a vector containing a polynucleotide, and / or a composition thereof is administered, the severity of the symptoms, and the route of administration, and therefore the dose may be determined on an individual basis.

[0310] An effective dose can be administered in one or more doses. For example, an effective dose of an antibody or antigen-binding fragment, or an ADC derived therefrom, is sufficient to improve, halt, stabilize, reverse, inhibit, delay, and / or slow the progression of an autoimmune disease or cancer in a patient, or sufficient to improve, halt, stabilize, reverse, delay, and / or slow the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, an effective dose of an antibody, antigen-binding fragment, or an ADC derived therefrom may vary depending, among other factors, such as the patient's medical history and the type (and / or dosage) of the antibody or ADC used.

[0311] The effective doses and schedules for administering the antibodies, antibody fragments, ADCs derived therefrom, polynucleotides encoding the antibodies, and / or compositions disclosed herein may be determined empirically, and such determinations are within the scope of the art. Those skilled in the art will understand that the dose to be administered will vary depending on, for example, the mammal receiving the antibodies, antibody fragments, ADCs derived therefrom, polynucleotides encoding the antibodies, and / or compositions disclosed herein, the route of administration used, the specific type of antibody, ADC, polynucleotide encoding the antibody, antigen-binding fragment, and / or compositions disclosed herein, and other agents administered to the mammal. Examples of daily doses of effective amounts of antibodies, antibody fragments, or ADCs derived therefrom range from 0.01 mg / kg to 100 mg / kg. In some embodiments, the dose may be less than 100 mg / kg, less than 10 mg / kg, less than 9 mg / kg, less than 8 mg / kg, less than 7 mg / kg, less than 6 mg / kg, less than 5 mg / kg, less than 4 mg / kg, less than 3 mg / kg, less than 2 mg / kg, less than 1 mg / kg, less than 0.5 mg / kg, or less than 0.1 mg / kg. In some embodiments, the dose may be greater than 10 mg / kg, greater than 9 mg / kg, greater than 8 mg / kg, greater than 7 mg / kg, greater than 6 mg / kg, greater than 5 mg / kg, greater than 4 mg / kg, greater than 3 mg / kg, greater than 2 mg / kg, greater than 1 mg / kg, greater than 0.5 mg / kg, greater than 0.1 mg / kg, greater than 0.05 mg / kg, or greater than 0.01 mg / kg. In some embodiments, the dosage is approximately 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.9 mg / kg, 0.8 mg / kg, 0.7 mg / kg, 0.6 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, or 0.1 mg / kg.

[0312] In any of the methods described herein, at least one antibody, its antigen-binding fragment, an ADC derived therefrom, or a pharmaceutical composition (e.g., any of the antibodies, an antigen-binding fragment, an ADC derived therefrom, or a pharmaceutical composition described herein), and optionally at least one additional therapeutic agent, can be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day). In some embodiments, at least two different antibodies, antibody fragments, or ADCs derived therefrom are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody, an antigen-binding fragment, or an ADC derived therefrom, and at least one additional therapeutic agent are administered in the same composition (e.g., a liquid composition). In some embodiments, at least one antibody, an antigen-binding fragment, or an ADC derived therefrom, and at least one additional therapeutic agent are administered in two different compositions (e.g., a liquid composition containing at least one antibody, an antigen-binding fragment, or an ADC derived therefrom, and a solid oral composition containing at least one additional therapeutic agent). In some embodiments, at least one additional therapeutic agent is administered as a tablet, capsule, or cereal. In some embodiments, at least one additional therapeutic agent is administered as a sustained-release oral formulation.

[0313] In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of at least one antibody, antigen-binding antibody fragment, ADC derived therefrom, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, ADCs derived therefrom, or pharmaceutical compositions described herein). In some embodiments, the one or more additional therapeutic agents and the at least one antibody, antigen-binding antibody fragment, ADC derived therefrom, or pharmaceutical composition (e.g., any of the antibodies, antigen-binding antibody fragments, ADCs derived therefrom, or pharmaceutical compositions described herein) are administered to the subject such that the activity times of the one or more additional therapeutic agents and the at least one antibody or antigen-binding fragment (e.g., any of the antibody fragments or ADCs derived therefrom described herein) overlap in the subject.

[0314] In some embodiments, one or more additional therapeutic agents may be administered to the target. The additional therapeutic agents may comprise one or more inhibitors selected from the group consisting of B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K / mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat) inhibitor.

[0315] In some embodiments, the additional therapeutic agent may comprise one or more inhibitors selected from the group consisting of OX40 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of the activated Hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

[0316] In some embodiments, additional therapeutic agents include trabectedin, nab-paclitaxel, trevananib, pazopanib, cedilanib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, riolisin, alimta, zicasia, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, and AGS-003. The composition may include one or more therapeutic agents selected from the group consisting of cabozantinib, vinflunin, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastone, taromib, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, lenalidomide, bortezomib, albicin, carfilzomib, pralatrexate, and enzastaurin.

[0317] In some embodiments, the additional therapeutic agent may comprise one or more therapeutic agents selected from the group consisting of adjuvants, TLR agonists, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1-targeted therapies, CXCL9-targeted therapies, CXCL10-targeted therapies, CCL5-targeted therapies, LFA-1 agonists, ICAM1 agonists, HER2 agonists, and OX40 agonists.

[0318] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the target patient.

[0319] In some embodiments, additional therapeutic agents include anti-OX40 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, anti-ICOS antibody, anti-CD27 antibody, anti-4-1BB antibody, and / or anti-GITR antibody.

[0320] Pharmaceutical composition and route of administration Furthermore, this specification provides pharmaceutical compositions containing at least one (e.g., one, two, three, or four) of the antibodies, antibody fragments, or ADCs derived therefrom, or pharmaceutically acceptable salts or solvates thereof described herein. Two or more (e.g., two, three, or four) of the antibodies, antibody fragments, or ADCs derived therefrom, or pharmaceutically acceptable salts or solvates thereof described herein may be present in any combination in the pharmaceutical composition. The pharmaceutical composition may be formulated in any way known in the art.

[0321] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions may include sterile diluents (e.g., sterile water or saline), non-volatile oils, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antimicrobial or antifungal agents (e.g., benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.), antioxidants (e.g., ascorbic acid or sodium bisulfite), chelating agents (e.g., ethylenediaminetetraacetic acid), buffers (e.g., acetates, citrates, or phosphates), isotonic agents (e.g., sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers (see, for example, U.S. Patent No. 4,522,811). The preparation of the composition may be in a form that is formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. If necessary (e.g., as in injectable formulations), appropriate fluidity may be maintained by the use of a coating, such as lecithin or a surfactant. The absorption of antibodies, their antigen-binding fragments, or ADCs derived therefrom can be prolonged by including absorption-delaying agents (e.g., aluminum monostearate and gelatin). Alternatively, controlled release may be achieved by implantable and microencapsulated delivery systems that may include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, Alza Corporation and Nova Pharmaceutical, Inc.).

[0322] Compositions containing one or more of the antibodies, antibody fragments, or ADCs derived therefrom described herein can be formulated in unit dosage forms (i.e., physically distinct units containing a predetermined amount of the active compound for ease of administration and uniformity of drug delivery) for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0323] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions may be provided in unit dosage forms (i.e., doses for single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, antibodies, antibody fragments, or ADCs derived therefrom may be formulated in aqueous solution, preferably in a physiologically compatible buffer, to reduce injection site discomfort. The solution may contain formulations such as suspensions, stabilizers, and / or dispersants. Alternatively, antibodies, antibody fragments, or ADCs derived therefrom may be in lyophilized form for formulation with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

[0324] The toxicity and therapeutic efficacy of a composition can be measured by standard medical procedures in cell cultures or experimental animals (e.g., monkeys). For example, the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population) can be measured, and the therapeutic index is the ratio of LD50:ED50. Drugs exhibiting high therapeutic indices are preferred. If a drug exhibits undesirable side effects, care must be taken to minimize potential damage (i.e., reduce the undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard medical procedures.

[0325] Data obtained from cell culture assays and animal studies can be used to formulate appropriate doses of any given drug for use in subjects (e.g., humans). A therapeutically effective dose of one or more (e.g., one, two, three, or four) antibodies, antibody fragments thereof, or ADCs derived therefrom (e.g., any of the antibodies, antibody fragments, or ADCs derived therefrom described herein) is an amount that treats the disease (e.g., kills cancer cells) in a subject (e.g., a human subject identified as having cancer) or a subject identified as being at risk of developing the disease (e.g., a subject who has previously had cancer but is now cured), and reduces the severity, frequency, and / or duration of one or more symptoms of the disease in the subject (e.g., humans). The efficacy and administration of any of the antibodies, antibody fragments, or ADCs derived therefrom described herein can be determined by a healthcare professional or veterinarian using methods known in the art, and by observation of one or more symptoms of the disease in a subject (e.g., humans). Certain factors may influence the dosage and timing required to effectively treat the subject (e.g., severity of the disease or disability, previous treatment, the subject's general health and / or age, and the presence of other diseases).

[0326] Exemplary doses include milligrams or micrograms of any of the antibodies, antibody fragments, or ADCs derived therefrom described herein per kilogram of the subject's body weight (e.g., approximately 1 μg / kg to approximately 500 mg / kg, approximately 100 μg / kg to approximately 500 mg / kg, approximately 100 μg / kg to approximately 50 mg / kg, approximately 10 μg / kg to approximately 5 mg / kg, approximately 10 μg / kg to approximately 0.5 mg / kg, or approximately 1 μg / kg to approximately 50 μg / kg). While these doses cover a wide range, those skilled in the art will understand that therapeutic agents containing antibodies, antibody fragments, or ADCs derived therefrom vary in their potency, and that effective doses can be determined by methods known in the art. Typically, a relatively low dose is administered initially, and the healthcare professional or veterinary expert (in the case of therapeutic use) or researcher (if still working in the development stage) may then gradually increase the dose until an appropriate response is obtained. Furthermore, it is understood that a specific dose level for any particular subject depends on various factors, including the activity of the specific compound used, the subject's age, weight, overall health, sex, and diet, administration time, route of administration, excretion rate, and the half-life of the antibody, antibody fragment, or ADC derived therefrom in vivo.

[0327] The pharmaceutical composition may be contained in a container, pack, or dispenser, along with instructions for administration. This disclosure also provides methods for producing antibodies, their antigen-binding fragments, or ADCs derived therefrom for the various uses described herein.

[0328] Specific Embodiments This disclosure further refers to the following embodiments: 1. An antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof comprising a payload and an antibody or an antibody fragment thereof (e.g., an antigen-binding fragment), e.g., an antigen-binding fragment, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises unnatural disulfide bonds formed at the positions of heavy-chain amino acid residues and light-chain amino acid residues, and the sum of (1) the relative solvent exposure area (SASA) of the heavy-chain amino acid residue side chain and (2) the relative SASA of the light-chain amino acid residue side chain is less than 65%, less than 60%, or less than 55%, and the antibody or antibody fragment does not have a disulfide bond between the amino acid residue at position 220 of the heavy-chain constant region and the amino acid residue at position 214 of the light-chain constant region, and the amino acid positions are based on EU numbering.

[0329] 2. The ADC described in Embodiment 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the relative SASA of the heavy chain amino acid side chain is greater than 30% and the relative SASA of the light chain amino acid side chain is less than 15%, or the relative SASA of the light chain amino acid side chain is greater than 30% and the relative SASA of the heavy chain amino acid side chain is less than 15%.

[0330] 3. An ADC according to Embodiment 1 or Embodiment 2, or a pharmaceutically acceptable salt or solvate thereof, wherein the relative SASA of the heavy chain amino acid side chain is greater than 40% and the relative SASA of the light chain amino acid side chain is less than 10%, or the relative SASA of the light chain amino acid side chain is greater than 40% and the relative SASA of the heavy chain amino acid side chain is less than 10%.

[0331] 4. An ADC according to any one of Embodiments 1 to 3, or a pharmaceutically acceptable salt or solvate thereof, wherein the relative SASA of the amino acid side chains of the heavy chain and the light chain, respectively, is less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, or less than 45%.

[0332] 5. An ADC according to any one of Embodiments 1 to 4, or a pharmaceutically acceptable salt or solvate thereof, wherein (1) the amino acid residue at position 220 of the heavy chain constant region is a non-cysteine ​​residue, or (2) the amino acid residue at position 214 of the light chain constant region is a non-cysteine ​​residue, and the amino acid position is based on EU numbering.

[0333] 6. An ADC or a pharmaceutically acceptable salt or solvate thereof according to any one of Embodiments 1 to 5, wherein the non-natural disulfide bond is resistant to reduction with tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and / or dithiothreitol (DTT).

[0334] 7. An ADC according to any one of Embodiments 1 to 6, or a pharmaceutically acceptable salt or solvate thereof, wherein when the antibody or antibody fragment is reduced with 10 to 30 equivalents of TCEP per single antibody molecule, more than 90% or more than 95% of the unnatural disulfide bonds remain intact.

[0335] 8. The ADC according to Embodiment 7 or a pharmaceutically acceptable salt or solvate thereof, wherein the concentration of the antibody or antibody fragment is 0.1 to 50 mg / ml during the reduction reaction.

[0336] 9. An ADC or a pharmaceutically acceptable salt or solvate thereof according to any one of Embodiments 1 to 8, wherein the non-natural disulfide bond remains stable under reducing conditions that can reduce more than 90% of the hinge region disulfide bonds of the antibody or antibody fragment.

[0337] 10. An ADC according to any one of Embodiments 1 to 9, or a pharmaceutically acceptable salt or solvate thereof, wherein at least one non-cysteine ​​amino acid residue of the heavy chain is mutated to a cysteine ​​residue.

[0338] 11. The ADC according to Embodiment 10 or a pharmaceutically acceptable salt or solvate thereof, wherein the non-cysteine ​​amino acid residue is located at a position selected from the group consisting of 126, 168, 170, 173, 134, 133, 130, and 141, and the amino acid position conforms to EU numbering.

[0339] 12. A pharmaceutically acceptable salt or solvate thereof according to any one of Embodiments 1 to 11, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment contains one or more of the following mutations in the heavy chain constant region: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C.

[0340] 13. A pharmaceutically acceptable salt or solvate thereof of the ADC or a pharmaceutically acceptable salt or solvate thereof, as described in any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two heavy chains, each of which contains one or more of the following mutations in the heavy chain constant region: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C.

[0341] 14. A pharmaceutically acceptable salt or solvate thereof according to any one of Embodiments 1 to 12, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two heavy chains, and only one heavy chain contains one or more of the following mutations in the heavy chain constant region: F126C, H168C, F170C, V173C, S134C, K133C, P130C, and A141C.

[0342] 15. An ADC according to any one of Embodiments 1 to 14, or a pharmaceutically acceptable salt or solvate thereof, wherein at least one non-cysteine ​​amino acid in the light chain constant region of the antibody or antibody fragment is mutated to a cysteine ​​residue.

[0343] 16. The ADC according to Embodiment 15 or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the at least one non-cysteine ​​amino acid is located at a position selected from the group consisting of 124, 164, 162, 116, 209, 117, and 118.

[0344] 17. A pharmaceutically acceptable salt or solvate thereof of the ADC or any pharmaceutically acceptable salt or solvate thereof, as described in any one of Embodiments 1 to 16, wherein the antibody or antibody fragment contains one or more of the following mutations in the light chain constant region: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C.

[0345] 18. A pharmaceutically acceptable salt or solvate thereof of the ADC or any pharmaceutically acceptable salt or solvate thereof, as described in any one of Embodiments 1 to 17, wherein the antibody or antibody fragment comprises two light chains, each of which contains one or more of the following mutations in the constant region of the light chain: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C.

[0346] 19. A pharmaceutically acceptable salt or solvate thereof according to any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two light chains, and only one light chain contains one or more of the following mutations in the light chain constant region: Q124C, T164C, S162C, F116C, F209C, I117C, and F118C.

[0347] 20. An ADC according to any one of Embodiments 1 to 19, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises the following set of mutations: (1) F126C in the heavy chain and Q124C in the light chain, (2) In the heavy chain, H168C, and in the light chain, T164C, (3) F170C in the heavy chain and T164C in the light chain, (4) F170C in the heavy chain and S162C in the light chain, (5) V173C in the heavy chain and S162C in the light chain, (6) S134C in the heavy chain and F116C in the light chain, (7) K133C in the heavy chain and F209C in the light chain, (8) K133C in the heavy chain and I117C in the light chain, (9) P130C in the heavy chain, and F118C in the light chain, (10) A pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable salt or solvate thereof, comprising one or more of A141C in the heavy chain and F116C in the light chain.

[0348] 21. An ADC or a pharmaceutically acceptable salt or solvate thereof as described in any one of Embodiments 1 to 20, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy and light chains, and only one pair of heavy and light chains comprises the following set of mutations: (1) F126C in the heavy chain and Q124C in the light chain, (2) In the heavy chain, H168C, and in the light chain, T164C, (3) F170C in the heavy chain and T164C in the light chain, (4) F170C in the heavy chain and S162C in the light chain, (5) V173C in the heavy chain and S162C in the light chain, (6) S134C in the heavy chain and F116C in the light chain, (7) K133C in the heavy chain and F209C in the light chain, (8) K133C in the heavy chain and I117C in the light chain, (9) P130C in the heavy chain and F118C in the light chain, (10) ADC or a pharmaceutically acceptable salt or solvate thereof, comprising A141C in the heavy chain and one of F116C in the light chain.

[0349] 22. An ADC or a pharmaceutically acceptable salt or solvate thereof as described in any one of Embodiments 1 to 20, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, and each pair of heavy chains and light chains comprises the following set of mutations: (1) F126C in the heavy chain and Q124C in the light chain, (2) In the heavy chain, H168C, and in the light chain, T164C, (3) F170C in the heavy chain and T164C in the light chain, (4) F170C in the heavy chain and S162C in the light chain, (5) V173C in the heavy chain and S162C in the light chain, (6) S134C in the heavy chain and F116C in the light chain, (7) K133C in the heavy chain and F209C in the light chain, (8) K133C in the heavy chain and I117C in the light chain, (9) P130C in the heavy chain and F118C in the light chain, (10) A pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the heavy chain contains one or more of A141C and the light chain contains one or more of F116C.

[0350] 23. The ADC according to Embodiment 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising F126C and each light chain comprising Q124C.

[0351] 24. The ADC according to Embodiment 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising H168C and each light chain comprising T164C.

[0352] 25. The ADC according to Embodiment 22 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising F170C and each light chain comprising T164C.

[0353] 26. The ADC according to Embodiment 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising F170C and each light chain comprising S162C.

[0354] 27. The ADC according to Embodiment 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising V173C and each light chain comprising S162C.

[0355] 28. The ADC according to Embodiment 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising S134C and each light chain comprising F116C.

[0356] 29. The ADC according to Embodiment 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising K133C and each light chain comprising F209C.

[0357] 30. The ADC according to Embodiment 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising K133C and each light chain comprising I117C.

[0358] 31. The ADC according to Embodiment 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising P130C and each light chain comprising F118C.

[0359] 32. The ADC according to Embodiment 22, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises two pairs of heavy chains and light chains, each heavy chain comprising A141C and each light chain comprising F116C.

[0360] 33. A pharmaceutically acceptable salt or solvate thereof comprising an ADC and an antibody or an antibody fragment thereof, wherein the antibody or antibody fragment comprises a non-natural disulfide bond formed at the positions of a heavy chain amino acid residue and a light chain amino acid residue, the antibody or antibody fragment does not have a disulfide bond between the amino acid residue at position 220 of the heavy chain constant region and the amino acid residue at position 214 of the light chain constant region, and when the antibody or antibody fragment is exposed to reducing conditions that reduce more than 90% or more of the hinged disulfide bond, more than 90% or more of the non-natural disulfide bond remains intact.

[0361] 34. An ADC according to any one of Embodiments 1 to 33, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises the following set of heavy chain mutations: (1) F126C and C220S, (2) H168C and C220S, (3) F170C and C220S, (4) V173C and C220S, (5) S134C and C220S, (6) K133C and C220S, (7) P130C and C220S, and (8) ADC or a pharmaceutically acceptable salt or solvate thereof, comprising one or more of A141C and C220S.

[0362] 35. An ADC according to any one of Embodiments 1 to 34, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises the following set of light chain mutations: (1) Q124C and C214S; (2) T164C and C214S, (3) S162C and C214S, (4) F116C and C214S, (5) F209C and C214S, (6) I117C and C214S, and (7) ADC or a pharmaceutically acceptable salt or solvate thereof, comprising one or more of F118C and C214S.

[0363] 36. An ADC according to any one of Embodiments 1 to 35, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment comprises the following set of mutations: (1) F126C and C220S in the heavy chain, and Q124C and C214S in the light chain, (2) In the heavy chain, H168C and C220S, and in the light chain, T164C and C214S, (3) F170C and C220S in the heavy chain, and T164C and C214S in the light chain, (4) F170C and C220S in the heavy chain, and S162C and C214S in the light chain, (5) In the heavy chain, V173C and C220S, and in the light chain, S162C and C214S, (6) S134C and C220S in the heavy chain, and F116C and C214S in the light chain, (7) In the heavy chain, K133C and C220S, and in the light chain, F209C and C214S, (8) In the heavy chain, K133C and C220S, and in the light chain, I117C and C214S, (9) P130C and C220S in the heavy chain, and F118C and C214S in the light chain, (10) A pharmaceutically acceptable salt or solvate thereof, or a pharmaceutically acceptable salt or solvate thereof, comprising one or more of A141C and C220S in the heavy chain and F116C and C214S in the light chain.

[0364] 37. An ADC according to any one of Embodiments 1 to 36, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment is a human or humanized antibody or antibody fragment thereof, a one-armed antibody, and / or a multispecific antibody (e.g., a bispecific antibody).

[0365] 38. An ADC according to any one of Embodiments 1 to 37, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment is a human IgG1, IgG1, IgG2, or IgG4 antibody or antibody fragment thereof.

[0366] 39. An ADC according to any one of Embodiments 1 to 38, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antibody fragment includes a fragment crystallizable region (Fc region).

[0367] 40. An ADC or a pharmaceutically acceptable salt or solvate thereof according to any one of Embodiments 1 to 39, wherein the payload is a cytotoxic agent, a cell proliferation inhibitor, a bioactive protein, a synthetic polymer, an enzyme, a nucleic acid (e.g., DNA or RNA), or a fragment thereof.

[0368] 41. An ADC according to any one of Embodiments 1 to 40, or a pharmaceutically acceptable salt or solvate thereof, having a drug-to-antibody ratio (DAR) of 3.74 to 4.

[0369] 42. An ADC according to any one of Embodiments 1 to 40, or a pharmaceutically acceptable salt or solvate thereof, having a drug-to-antibody ratio (DAR) of 5.70 to 6.

[0370] 43. An ADC according to any one of Embodiments 1 to 42, or a pharmaceutically acceptable salt or solvate thereof, wherein the payload is covalently bound to an antibody or an antibody fragment via a thiol-based conjugation.

[0371] 44. An antibody or antibody fragment comprising unnatural disulfide bonds formed at the positions of heavy chain amino acid residues and light chain amino acid residues, wherein (1) the sum of the relative solvent exposure area (SASA) of the heavy chain amino acid residue side chain and (2) the relative SASA of the light chain amino acid residue side chain is less than 65%, less than 60%, or less than 55%, and the antibody or antibody fragment does not have a disulfide bond between the amino acid residue at position 220 of the heavy chain constant region and the amino acid residue at position 214 of the light chain constant region, and the amino acid positions are based on EU numbering.

[0372] 45. For example, an antibody or antibody fragment (e.g., antigen-binding fragment) according to Embodiment 44, wherein the relative SASA of the heavy amino acid side chain is greater than 30% and the relative SASA of the light amino acid side chain is less than 15%, or the relative SASA of the light amino acid side chain is greater than 30% and the relative SASA of the heavy amino acid side chain is less than 15%.

[0373] 46. ​​The antibody or antibody fragment according to Embodiment 44 or Embodiment 45, wherein the relative SASA of the heavy amino acid side chain is greater than 40% and the relative SASA of the light amino acid side chain is less than 10%, or the relative SASA of the light amino acid side chain is greater than 40% and the relative SASA of the heavy amino acid side chain is less than 10%.

[0374] 47. The antibody or antibody fragment according to any one of Embodiments 44 to 46, wherein the relative SASA of the amino acid side chains of the heavy chain and the light chain, respectively, is less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, or less than 45%.

[0375] 48. An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprising an antibody or antibody fragment according to any one of embodiments 44 to 47, covalently bound to a payload.

[0376] 49. A nucleic acid encoding the light chain or heavy chain of the antibody or antibody fragment described in any one of Embodiments 1 to 48.

[0377] 50. A vector comprising nucleic acid according to Embodiment 49, wherein the nucleic acid is operably linked to a promoter.

[0378] 51. A cell comprising the nucleic acid of Embodiment 49 or the vector of Embodiment 50.

[0379] 52. A method for producing an antibody or an antibody fragment thereof, (a) Culturing the cells according to Embodiment 51 under conditions sufficient for the cells to produce the antibody or the antibody fragment, (b) A method comprising collecting the antibody or antibody fragment produced by the cells.

[0380] 53. A method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof, (a) Incubating an antibody or antibody fragment described in any one of Embodiments 1 to 47 with a reducing agent to reduce the natural disulfide bond in the antibody or antibody fragment and generate a reduced thiol group, A method comprising (b) introducing an excess amount of a payload having a reactive group so as to react with the reduced thiol group generated in step (a).

[0381] 54.(c) The method according to Embodiment 53, further comprising adding an effective amount of N-acetylcysteine ​​to quench an excess payload, and then recovering the resulting antibody-drug conjugate.

[0382] 55. The method according to Embodiment 53 or Embodiment 54, wherein the payload contains a maleimide group.

[0383] 56. The method according to any one of embodiments 53 to 55, wherein the payload is linked to an antibody or an antibody fragment via a thiol-based conjugation.

[0384] 57. The method according to any one of Embodiments 53 to 56, wherein at least 50%, 60%, 70%, 80%, or 90% of the resulting ADC product has a drug-to-antibody ratio (DAR) of about 4.

[0385] 58. The method according to any one of Embodiments 53 to 56, wherein at least 50%, 60%, 70%, 80%, or 90% of the resulting ADC products have a drug-to-antibody ratio (DAR) of about 6.

[0386] 59. A method for producing a modified antibody or an antibody fragment thereof, (a) To provide an antibody or an antibody fragment thereof, wherein the antibody or antibody fragment has no disulfide bond between the amino acid residue at position 220 of the heavy chain constant region and the amino acid residue at position 214 of the light chain constant region, and the amino acid positions are based on EU numbering. (b) Identifying the heavy chain amino acid residues of the heavy chain constant region (CH1) and the light chain amino acid residues of the light chain constant region (CL), wherein the sum of (1) the relative solvent exposure area (SASA) of the heavy chain amino acid residue side chain and (2) the relative SASA of the light chain amino acid residue side chain is less than 65%, less than 60%, or less than 55%. (c) A method comprising substituting each of the identified heavy chain amino acid residues and light chain amino acid residues with a cysteine ​​residue such that a non-natural disulfide bond is formed between the two cysteine ​​residues.

[0387] 60. The method according to Embodiment 59, wherein the relative SASA of the heavy chain amino acid side chain is greater than 30% and the relative SASA of the light chain amino acid side chain is less than 15%, or the relative SASA of the light chain amino acid side chain is greater than 30% and the relative SASA of the heavy chain amino acid side chain is less than 15%.

[0388] 61. The method according to Embodiment 59 or Embodiment 60, wherein the relative SASA of the heavy chain amino acid side chain is greater than 40% and the relative SASA of the light chain amino acid side chain is less than 10%, or the relative SASA of the light chain amino acid side chain is greater than 40% and the relative SASA of the heavy chain amino acid side chain is less than 10%.

[0389] 62. The method according to any one of Embodiments 59 to 61, wherein the relative SASA of the amino acid side chains of the heavy chain and the light chain is less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, or less than 45%.

[0390] 63. The method according to any one of embodiments 59 to 62, further comprising adding a payload to the modified antibody or an antibody fragment thereof.

[0391] 64. A method for treating a subject having cancer, comprising administering to the subject a therapeutically effective amount of an ADC or a pharmaceutically acceptable salt or solvate thereof described in any one of Embodiments 1 to 43 and 48, or a composition comprising a pharmaceutically acceptable salt or solvate thereof.

[0392] 65. The method according to Embodiment 64, wherein the subject has a solid tumor carcinoma.

[0393] 66. The method according to Embodiment 64, wherein the cancer is breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma, lymphoma, epithelial neoplasm, soft tissue sarcoma, esophageal cancer, or CNS tumor.

[0394] 67. A method for reducing the rate of tumor growth, the method comprising contacting tumor cells with an effective amount of an ADC or a pharmaceutically acceptable salt or solvate thereof described in any one of embodiments 1 to 43 and 48, or a composition comprising a pharmaceutically acceptable salt or solvate thereof.

[0395] 68. A method for killing tumor cells, the method comprising contacting tumor cells with an effective amount of an ADC described in any one of embodiments 1 to 43 and 48, or a pharmaceutically acceptable salt or solvate thereof, or a composition comprising a pharmaceutically acceptable salt or solvate thereof.

[0396] 69. A pharmaceutical composition comprising an ADC or a pharmaceutically acceptable salt or solvate thereof as described in any one of Embodiments 1 to 43 and 48, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier. [Examples]

[0397] The present invention is further described in the following embodiments, but this does not limit the scope of the invention as described in the claims.

[0398] The following materials were used in the following examples.

[0399] [Table 1-2] [Table 1-3] [Table 1-4]

[0400] Example 1. Design of a disulfide bond that cannot access the solvent. Firstly, the cysteine ​​residue corresponding to the natural heavy-chain-light-chain disulfide is mutated to a non-cysteine ​​residue, thereby eliminating the natural heavy-chain-light-chain disulfide.

[0401] Secondly, appropriate amino acid pairs are selected at the interface between the heavy and light chains and mutated with cysteine ​​residues to form new disulfide bonds. Amino acid pairs at the interface between the heavy and light chains are screened, and pairs with an α-carbon distance of less than 10 Å, preferably less than 8 Å, and more preferably less than 6 Å are selected, with the side chains facing each other. The selected amino acid pairs are shown in the table below as "candidate positions for unnatural disulfide bonds (CH1 / CL)" (EU numbering).

[0402] To achieve the DAR4 objective, the new disulfide bond should be hidden within the protein structural domain in a region inaccessible to the solvent, and as a result, the new disulfide bond will not be cleaved by reducing agents at normal reducing concentrations. The relative solvent exposure area (SASA) of the side chains of candidate amino acid pairs in the structure of the Fab fragment was calculated. The results are shown in the table below as relative solvent exposure area (%).

[0403] SASA was calculated by representing each atom as a set of grid points distributed on a sphere whose radius is the sum of the atom's van der Waals radius and probe radius (1.4 Å). Grid points embedded within another atomic sphere contribute to the embedded surface area. Grid points not embedded within another atomic sphere contribute to the exposed surface area. These results were calculated using Discovery Studio software version 2022.

[0404] The relative residue SASA (percentage) was calculated using the following formula: 100 times the residue SASA obtained by dividing the residue SASA of the fully exposed amino acid residue calculated using the extended Ala-X-Ala tripeptide, where X is the residue in question.

[0405] [Table 1-5]

[0406] In the following examples, modified antibodies were constructed based on the candidate positions in the table above. The modified antibodies were tested for disulfide bond formation using gel electrophoresis.

[0407] Conjugation experiments were performed to produce site-directed ADCs, and it was tested whether the modified antibody or its antibody fragment met the requirements for constructing DAR4 ADC or DAR6 ADC. The proportions of DAR4 ADC and DAR6 ADC are shown in the table above.

[0408] Example 2: Antibody expression and purification Nucleic acids encoding each strand of a modified antibody or its antibody fragment were synthesized and constructed on a pCDNA3.1 template plasmid.

[0409] The plasmid was filtered through a 0.22 μm filter for transient expression in cells. Expi293 cells (Invitrogen) were cultured to the desired transfection dose, and the cell density was set to 3 × 10⁶ the day before transfection. 6The cell density was adjusted to cells / mL. On the day of transfection, the cell density was again adjusted to 3 × 10⁶. 6 The volume was adjusted to cells / mL. Opti-MEM medium (Gibco catalog number 31985-070) at a final volume of 1 / 10 (v / v) was used as the transfection buffer. For monoclonal antibodies, the expression plasmids of (1) the heavy chain and (2) the light chain were mixed in a 1:1 or other more appropriate ratio. For bispecific antibodies, the expression plasmids of (1) the first heavy chain, (2) the first light chain, (3) the second heavy chain, and (4) the second light chain were mixed in a 1:1:1:1 or other more appropriate ratio.

[0410] An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid from the previous step (the mass ratio of plasmid to PEI was 1:3), mixed until homogeneous, and incubated at room temperature for 10 minutes to obtain the DNA / PEI mixture. The DNA / PEI mixture was gently poured into HEK293 cells, mixed well, and incubated at 37°C under 8% CO2 for 24 hours. After 24 hours, the culture was supplemented with VPA (Sigma, catalog no. P4543-100G) to a final VPA concentration of 2 mM, supplemented with 2% (v / v) feed (1 G / L Phyton Peptone + 1 G / L DIFFCO Selective Phyton), and the culture was continued for 6 days. Cells were collected and centrifuged at 4000 rpm for 30 minutes. The cell supernatant was filtered through a 0.45 μM filter and purified by protein A affinity chromatography. A sample was further purified using ion exchange chromatography, KappaSelect affinity chromatography, and LambdaFabSelect affinity chromatography.

[0411] For protein A affinity chromatography purification, samples were purified using a pre-packed Hitrap Mabselect Sure column (GE, 11-0034-95). The specific steps were: (1) equilibrating the packed column with 5 column volumes of equilibration solution (20 mM Tris, 150 mM NaCl, pH 7.2) before purification; (2) passing the collected sample through the column; (3) washing the packed column with 10 column volumes of equilibration solution to remove nonspecific binding proteins; (4) eluting with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5); and (5) collecting the eluate. 2 M Tris was added to neutralize the pH to 6.5. The purified antibody was analyzed by liquid chromatography-mass spectrometry (LC-MS) to evaluate the ratio.

[0412] For ion exchange chromatography purification, the sample was purified using Capto HiRes S 10 / 100 (GE, catalog no. 29275879). The specific procedure was as follows: (1) replace the sample with low-salt PB buffer using an ultrafiltration concentrator tube (MILLIPORE, catalog no. UFC901096); (2) equilibrate the packed column with 5 column volumes of pH 6.0 low-salt PB buffer before purification; (3) pass the liquid-exchanged sample through the column; (4) wash the packed column with 10 column volumes of pH 6.0 low-salt PB buffer to remove nonspecific binding proteins; and (5) wash with pH 6.0 high-salt PB buffer (10 mM phosphate, pH 6.0, 1 M). The procedure includes: (6) eluting by linearly increasing the concentration of NaCl (linear gradient: increasing the ratio of high-salt PB buffer at pH 6.0 from 30% to 100% over 30 column volumes); (7) collecting the eluted main peak; and (8) replacing the eluted material with PBS buffer (Gibco, catalog no. 70011-044) using an ultrafiltration concentrator (MILLIPORE, catalog no. UFC901096). The purified antibody was analyzed by liquid chromatography-mass spectrometry (LC-MS) to evaluate the ratio.

[0413] For KappaSelect affinity chromatography purification, samples were purified using a pre-packed HiTrap KappaSelect column (GE, 17-5458-11). The specific procedure was as follows: (1) Before purification, the packed column was equilibrated with 5 column volumes of equilibration solution (PBS, pH 7.4); (2) The sample was passed through the column; (3) The packed column was then washed with 10 column volumes of equilibration solution to remove nonspecific binding proteins; (4) The sample was eluted with 5 column volumes of elution buffer (0.1 M glycine buffer, pH 2.5); and (5) The eluted product was collected. 2 M Tris was added to neutralize the pH to 6.5. The purified antibody was analyzed by liquid chromatography-mass spectrometry (LC-MS) to evaluate the ratio.

[0414] For LambdaFabSelect affinity chromatography purification, samples were purified using a pre-packed Hitrap LambdaFabSelect column (GE, 17-5482-11). The specific steps were: (1) equilibrating the packed column with 5 column volumes of equilibration solution (PBS, pH 7.4) before purification; (2) passing the sample through the column; (3) washing the packed column with 10 column volumes of equilibration solution to remove nonspecific binding proteins; (4) eluting with 5 column volumes of elution buffer (0.1 M acetate buffer, pH 3.5); and (5) collecting the eluted product. 2 M Tris was added to neutralize the pH to 6.5. The purified antibody was analyzed by liquid chromatography-mass spectrometry (LC-MS) to evaluate the ratio.

[0415] Example 3: Production of site-specific ADCs and common analytical procedures

[0416] Purified antibodies were conjugated to drugs according to the methods of Examples 4-6 below to obtain various ADCs. General procedures A-D were performed to characterize these ADCs.

[0417] General Procedure A: DTT treatment for complete reduction of all interchain disulfide bonds. An antibody-drug conjugate solution (approximately 1 mg / mL, 50 μL) was mixed with an aqueous solution of dithiothreitol (DTT) (1 M, 1.0 μL). The mixture was adjusted to pH 7.5 with Tris buffer (1 M, pH 8.5) and incubated at 37°C for 30 minutes to prepare a sample readily usable for HPLC analysis.

[0418] General Procedure B: Measurement of drug-to-antibody ratio (DAR) of ADCs using RP-HPLC analysis. RP-HPLC analysis was performed under the measurement conditions shown in the table below. Signals were detected using dual wavelengths of 280 nm / 360 nm for ADCs carrying Dxd and 280 nm / 248 nm for ADCs carrying MMAE. Either a 45-minute or 30-minute method may be used.

[0419] [Table 2]

[0420] General Procedure C: Measurement of ADC purity using SEC analysis. SEC analysis was performed under the measurement conditions shown in the table below. The signal was detected using dual wavelengths of 280 nm / 360 nm for ADCs supporting Dxd, and 280 nm / 248 nm for ADCs supporting MMAE.

[0421] [Table 3]

[0422] General Procedure D: Measurement of drug distribution in ADCs using HIC analysis. HIC analysis was performed under the measurement conditions shown in the table below. Signals were detected using dual wavelengths of 280 nm / 360 nm for ADCs carrying Dxd and 280 nm / 248 nm for ADCs carrying MMAE.

[0423] [Table 4]

[0424] Example 4: Production and analysis of site-specific DAR4 ADCs The purified antibody was conjugated to the drug according to the following method to obtain an ADC.

[0425] 1. HC_F126C / C220S, LC_Q124C / C214S (both arms) An aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (179 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of humanized anti-HER2 antibody (trastuzumab (TZB)) containing disulfide bond mutations, with both arms (HC_F126C / C220S, LC_Q124C / C214S) (13.0 mg) in histidine buffer (5.2 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0426] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 111.3 μL, 10 mg / mL in DMSO, 12 equivalents per single antibody molecule) was added, and additional DMSO was added to reach a final concentration of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (54 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the mixture was stirred for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin desalting column (10 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (12.05 mg, 2.51 mg / mL, 4.8 mL).

[0427] The resulting ADC will be named 126C_124C, TZB(126C_124C)-DXd, or TZB-126-124-DXd.

[0428] Figure 1 is a schematic diagram of the ADC structure.

[0429] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.84 (Figure 2).

[0430] SEC analysis performed according to general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 0.51% and 99.49%, respectively (Figure 3).

[0431] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D2, 12.75%; D4, 87.55% (Figure 4).

[0432] 2. HC_H168C / C220S, LC_T164C / C214S (both arms) An aqueous solution of TCEP (82.8 μL, 10 mM in H2O, 30 equivalents per single antibody molecule) was added to a solution of humanized anti-HER2 antibody (trastuzumab) containing disulfide bond mutations in both arms (HC_H168C / C220S, LC_T164C / C214S) (4.0 mg) in histidine buffer (2.0 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0433] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 28.5 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was further incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (16.6 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin desalting column (5 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (3.43 mg, 2.45 mg / mL, 1.4 mL).

[0434] The resulting ADC will be named 168C_164C, or TZB(168C_164C)-DXd, or TZB-168-164-DXd.

[0435] Figure 5 is a schematic diagram of the ADC structure.

[0436] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.81 (Figure 6).

[0437] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 1.57% and 98.43%, respectively (Figure 7).

[0438] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D2, 11.53%; D4, 88.47% (Figure 8).

[0439] 3. HC_F170C / C220S, LC_T164C / C214S (both arms) An aqueous solution of TCEP (82.8 μL, 10 mM in H2O, 30 equivalents per single antibody molecule) was added to a solution of humanized anti-HER2 antibody (trastuzumab) containing disulfide bond mutations in both arms (HC_F170C / C220S, LC_T164C / C214S) (4.0 mg) in histidine buffer (2.0 mL, 20 mM, pH=6.5). The resulting mixture was then incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0440] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 28.5 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was further incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (16.6 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting Column (5 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (3.48 mg, 2.90 mg / mL, 1.2 mL).

[0441] The resulting ADC will be named 170C_164C, TZB(170C_164C)-DXd, or TZB-170-164-DXd.

[0442] Figure 9 is a schematic diagram of the ADC structure.

[0443] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.83 (Figure 10).

[0444] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 0.25% and 99.75%, respectively (Figure 11).

[0445] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D2, 12.85%; D4, 87.15% (Figure 12).

[0446] 4. HC_F170C / C220S, LC_S162C / C214S (both arms) An aqueous solution of TCEP (82.8 μL, 10 mM in H2O, 30 equivalents per single antibody molecule) was added to a solution of humanized anti-HER2 antibody (trastuzumab) containing disulfide bond mutations in both arms (HC_F170C / C220S, LC_S162C / C214S) (4.0 mg) in histidine buffer (2.0 mL, 20 mM, pH=6.5). The resulting mixture was then incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0447] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 28.5 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was further incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (16.6 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting Column (5 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (3.48 mg, 3.37 mg / mL, 1.0 mL).

[0448] The resulting ADC will be named 170C_162C, or TZB(170C_162C)-Dxd, or TZB-170-162-Dxd.

[0449] Figure 13 is a schematic diagram of the ADC structure.

[0450] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.83 (Figure 14).

[0451] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 0.39% and 99.61%, respectively (Figure 15).

[0452] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 0.23%; D2, 12.14%; D4, 87.63% (Figure 16).

[0453] 5. HC_V173C / C220S, LC_S162C / C214S (both arms) A TCEP aqueous solution (13.8 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of humanized anti-HER2 antibody (trastuzumab) containing disulfide bond mutations in both arms (HC_V173C / C220S, LC_S162C / C214S) (1.0 mg) in histidine buffer (0.67 mL, 20 mM, pH=6.5). The resulting mixture was then incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0454] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 7.2 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was further incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (4.2 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting column (2 mL), and the remaining solution was concentrated to obtain the corresponding ADC solution (0.78 mg, 2.55 mg / mL, 0.3 mL).

[0455] The resulting ADC will be named 173C_162C or TZB(173C_162C)-DXd.

[0456] Figure 17 is a schematic diagram of the ADC structure.

[0457] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.79 (Figure 18).

[0458] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 0.13% and 99.87%, respectively (Figure 19).

[0459] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 0.72%; D2, 9.28%; D4, 90.00% (Figure 20).

[0460] 6. HC_S134C / C220S, LCF116C / C214S (both arms) A TCEP aqueous solution (310 μL, 10 mM in H2O, 30 equivalents per single antibody molecule) was added to a solution of humanized anti-HER2 antibody (trastuzumab) containing disulfide bond mutations in both arms (HC_S134C / C220S, LCK_F116C / C214S) (15.0 mg) in histidine buffer (6.0 mL, 20 mM, pH=6.5). The resulting mixture was then incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0461] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 128.4 μL, 10 mg / mL in DMSO, 12 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated further at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (62 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin desalting column (10 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (13.35 mg, 2.67 mg / mL, 5.0 mL).

[0462] The resulting ADC is named 134C_116C, TZB(134C_116C)-DXd, or TZB-134-116-DXd.

[0463] Figure 21 is a schematic diagram of the ADC structure.

[0464] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.82 (Figure 22).

[0465] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 0.76% and 99.24%, respectively (Figure 23).

[0466] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D2, 9.69%; D4, 90.31% (Figure 24).

[0467] 7. HC_F126C / C220S, LC_Q124C / C214S (both arms) An aqueous solution of TCEP (5.1 μL, 10 mM in H2O, 25 equivalents per single antibody molecule) was added to a solution of anti-B7H3 antibody (HC_F126C / C220S, LC_Q124C / C214S) (0.3 mg) with disulfide bond mutations in both arms in histidine buffer (0.2 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0468] After cooling to room temperature, the linker-payload solution (mc-VC-PAB-MMAE, 4.05 μL, 10 mg / mL in DMSO, 15 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (1.2 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0469] Figure 25 is a schematic diagram of the ADC structure.

[0470] The obtained ADC product was first treated with DTT according to general procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.85 (Figure 26).

[0471] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 2.86%; D2, 6.17%; D4, 90.97% (Figure 27).

[0472] 8. HC_F126C / C220S, LC_Q124C / C214S (both arms) A TCEP aqueous solution (6.9 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of anti-Trop22 antibody containing disulfide bond mutations in both arms (HC_F126C / C220S, LC_Q124C / C214S) (0.5 mg) in histidine buffer (0.33 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0473] After cooling to room temperature, the linker-payload solution (mc-VC-PAB-MMAE, 5.44 μL, 10 mg / mL in DMSO, 12 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated further at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (2.1 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting column (2 mL), and the remaining solution was concentrated to obtain the corresponding ADC solution (0.3 mg, 2.96 mg / mL, 0.1 mL).

[0474] Figure 28 is a schematic diagram of the ADC structure.

[0475] The obtained ADC product was first treated with DTT according to general procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.86 (Figure 29).

[0476] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 1.50% and 98.50%, respectively (Figure 30).

[0477] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 5.40%; D2, 4.47%; D4, 90.13% (Figure 31).

[0478] 9. HC_F126C / C220S, LC_Q124C / C214S (single arm) A TCEP aqueous solution (3.4 μL, 10 mM in H2O, 10 equivalents per single antibody molecule) was added to a solution of anti-B7H3 antibody (HC_F126C / C220S, LC_Q124C / C214S) (0.5 mg) containing a disulfide bond mutation in only one Fab arm in histidine buffer (0.33 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours.

[0479] After cooling to room temperature, the linker-payload solution (mc-VC-PAB-MMAE, 5.31 μL, 10 mg / mL in DMSO, 15 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was further incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (2.1 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting Column (2 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (0.3 mg, 3.15 mg / mL, 0.1 mL).

[0480] Figure 32 is a schematic diagram of the ADC structure.

[0481] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 5.77 (Figure 33).

[0482] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 1.89% and 98.11%, respectively (Figure 34).

[0483] 10. HC_F126C / C220S, LC_Q124C / C214S (single arm) A TCEP aqueous solution (13.8 μL, 10 mM in H2O, 10 equivalents per single antibody molecule) was added to a solution of a bispecific antibody (trastuzumab-pertuzumab, or TZB-PZB) with a disulfide bond mutation (PZB arm (HC_F126C / C220S, LC_Q124C / C214S) (2.0 mg)) in histidine buffer (0.8 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours.

[0484] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 17.2 μL, 10 mg / mL in DMSO, 12 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was further incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (8.3 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting Column (10 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (1.46 mg, 2.43 mg / mL, 0.6 mL).

[0485] Figure 35 is a schematic diagram of the ADC structure.

[0486] The obtained ADC product was first treated with DTT according to general procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 5.70 (Figure 36).

[0487] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS), monomers, and low molecular weight species (LMWS) were 0.87%, 92.19%, and 6.93%, respectively (Figure 37).

[0488] 11. HC_132C / C220S, LC_119C / C214S (both arms) An aqueous solution of TCEP (2.76 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB) containing disulfide bond mutations (HC_132C / C220S, LC_119C / C214S) (0.2 mg) in histidine buffer (80 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours.

[0489] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 1.42 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.8 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0490] RP-HPLC analysis after standard procedure B showed that the modified antibody was unable to produce DAR4 ADC (Figure 38).

[0491] Similar experiments were conducted with TZB-L128C_F118C, TZB-S134C_F118C, TZB-F170C_S174C, TZB-Q175C_T180C, TZB-S181C_T178C, and TZB-S183C_T178C, and none of the modified antibodies were able to produce DAR4 ADC.

[0492] 12. HC_T139C / C220S, LCF116C / C214S (both arms) An aqueous solution of TCEP (2.76 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB) containing disulfide bond mutations (HC_139C / C220S, LC_116C / C214S) (0.2 mg) in histidine buffer (80 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours.

[0493] RP-HPLC analysis of the reducing antibody after standard procedure B showed that more than 10% of the disulfide bonds between the light and heavy chains were cleaved, and therefore the modified antibody was unable to produce DAR4 ADC (Figure 39).

[0494] 13. HC_P171C / C220S, LC_S162C / C214S (both arms) An aqueous solution of TCEP (2.76 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB) containing disulfide bond mutations (HC_171C / C220S, LC_162C / C214S) (0.2 mg) in histidine buffer (80 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours.

[0495] RP-HPLC analysis of the reducing antibody after standard procedure B showed that more than 10% of the disulfide bonds between the light and heavy chains were cleaved, and therefore the modified antibody was unable to produce DAR4 ADC (Figure 40).

[0496] 14. HC_V173C / C220S, LC_Q160C / C214S (both arms) An aqueous solution of TCEP (2.76 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB) with a disulfide bond mutation (HC_173C / C220S, LC_160C / C214S) (0.2 mg) in histidine buffer (80 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours.

[0497] RP-HPLC analysis of the reducing antibody after standard procedure B showed that the disulfide bond between the light and heavy chains was cleaved, and therefore the modified antibody was unable to produce DAR4 ADC (Figure 41).

[0498] 15. HC_S136C / C220S, LC_S114C / C214S (both arms) An aqueous solution of TCEP (2.76 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB) containing disulfide bond mutations (HC_136C / C220S, LC_114C / C214S) (0.2 mg) in histidine buffer (80 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours.

[0499] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 1.42 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.8 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0500] The obtained ADC product was first treated with DTT according to general procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed that the modified antibody was unable to produce DAR4 ADC (Figure 42).

[0501] 16. TZB-S131C_P119C An aqueous solution of TCEP (2.76 μL, 5 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB), an anti-HER2 antibody containing disulfide bond mutations in both arms (HC_S131C / C220S, LCK_P119C / C214S) (0.1 mg) in histidine buffer (50 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0502] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 0.71 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.4 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0503] RP-HPLC analysis after standard procedure B showed that the modified antibody was unable to produce DAR4 ADC (Figure 43).

[0504] 17. TZB-S132C_F209C An aqueous solution of TCEP (2.76 μL, 5 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of the anti-HER2 antibody trastuzumab (TZB) containing disulfide bond mutations in both arms (HC_S132C / C220S, LCK_F209C / C214S) (0.1 mg) in histidine buffer (50 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0505] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 0.71 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.4 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0506] RP-HPLC analysis after standard procedure B showed that the modified antibody was unable to produce DAR4 ADC (Figure 44).

[0507] 18. TZB-K133C_I117C (Double Arm) An aqueous solution of TCEP (2.76 μL, 5 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of the anti-HER2 antibody trastuzumab (TZB) containing disulfide bond mutations in both arms (HC_K133C / C220S, LC_I117C / C214S) (0.1 mg) in histidine buffer (50 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0508] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 0.71 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.4 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0509] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.77 (Figure 45).

[0510] 19. TZB-K133C_K207C An aqueous solution of TCEP (2.76 μL, 5 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB), an anti-HER2 antibody containing disulfide bond mutations in both arms (HC_K133C / C220S, LCK_K207C / C214S) (0.1 mg) in histidine buffer (50 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0511] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 0.71 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.4 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0512] RP-HPLC analysis after standard procedure B showed that the modified antibody was unable to produce DAR4 ADC (Figure 46).

[0513] 20. TZB-K133C_F209C (Double Arm) An aqueous solution of TCEP (2.76 μL, 5 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB), an anti-HER2 antibody containing disulfide bond mutations in both arms (HC_K133C / C220S, LCK_F209C / C214S) (0.1 mg) in histidine buffer (50 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0514] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 0.71 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.4 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0515] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.80 (Figure 47).

[0516] 21. TZB-A141C_F116C (Double Arm) An aqueous solution of TCEP (2.76 μL, 5 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB), an anti-HER2 antibody containing disulfide bond mutations in both arms (HC_A141C / C220S, LCK_F116C / C214S) (0.1 mg) in histidine buffer (50 μL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0517] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 0.71 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.4 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0518] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.74 (Figure 48).

[0519] 22. TZB-K133C_I117C (Double Arm) An aqueous solution of TCEP (2.76 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB), an anti-HER2 antibody containing disulfide bond mutations in both arms (HC_K133C / C220S, LC_I117C / C214S) (0.2 mg) in histidine buffer (0.1 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0520] After cooling to room temperature, the linker-payload solution (mc-VC-PAB-MMAE, 1.82 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.8 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0521] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.76 (Figure 49).

[0522] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 4.89%; D2, 2.85%; D4, 92.25% (Figure 50).

[0523] 23. TZB-K133C_F209C (Double Arm) An aqueous solution of TCEP (2.76 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB), an anti-HER2 antibody containing disulfide bond mutations in both arms (HC_K133C / C220S, LCK_F209C / C214S) (0.2 mg) in histidine buffer (0.1 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0524] After cooling to room temperature, the linker-payload solution (mc-VC-PAB-MMAE, 1.82 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.8 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0525] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.89 (Figure 51).

[0526] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 1.58%; D2, 4.26%; D4, 94.16% (Figure 52).

[0527] 24. TZB-A141C_F116C (Double Arm) An aqueous solution of TCEP (2.76 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB), an anti-HER2 antibody containing disulfide bond mutations in both arms (HC_A141C / C220S, LCK_F116C / C214S) (0.2 mg) in histidine buffer (0.1 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0528] After cooling to room temperature, the linker-payload solution (mc-VC-PAB-MMAE, 1.82 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.8 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0529] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.74 (Figure 53).

[0530] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 3.59%; D2, 6.55%; D4, 89.77% (Figure 54).

[0531] 25. TZB-Hel-F126C_Q124C (Single Arm) An aqueous solution of TCEP (2.08 μL, 10 mM in H2O, 15 equivalents per single antibody molecule) was added to a solution of anti-HER22 bispecific antibodies (trastuzumab-Hel or TZB-Hel) with disulfide bond mutations in the TZB arm (HC_F126C / C220S, LC_Q124C / C214S) (0.2 mg) in histidine buffer (0.1 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0532] After cooling to room temperature, the linker-payload solution (mc-VC-PAB-MMAE, 2.74 μL, 10 mg / mL in DMSO, 15 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.8 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0533] The obtained ADC product was first treated with DTT according to general procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 5.85 (Figure 55).

[0534] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 0%; D2, 2.96%; D4, 4.70%; D6, 92.34% (Figure 56).

[0535] 26. TZB-P130C_F118C (Double Arm) An aqueous solution of TCEP (2.76 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of trastuzumab (TZB), an anti-HER2 antibody containing disulfide bond mutations in both arms (HC_P130C / C220S, LCK_F118C / C214S) (0.2 mg) in histidine buffer (0.1 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0536] After cooling to room temperature, the linker-payload solution (mc-VC-PAB-MMAE, 1.82 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.8 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without purification.

[0537] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.95 (Figure 57).

[0538] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 0.54%; D2, 0.84%; D4, 98.62% (Figure 58).

[0539] 27. HC_F170C / C220S, LC_T164C / C214S (single arm) An aqueous solution of TCEP (8.23 μL, 10 mM in H2O, 12 equivalents per single antibody molecule) was added to a solution of a bispecific antibody (trastuzumab-pertuzumab, or TZB-PZB) with a disulfide bond mutation in the PZB arm (HC_F170C / C220S, LC_T164C / C214S) (1.0 mg) in histidine buffer (0.33 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 25°C for 2 hours.

[0540] After cooling to room temperature, the linker-payload solution (mc-vc-PAB-MMAE, 10.8 μL, 10 mg / mL in DMSO, 12 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated further at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (4.2 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting column (2 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (0.83 mg, 16.6 mg / mL, 50 μL).

[0541] Figure 59 is a schematic diagram of the ADC structure.

[0542] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 5.80 (Figure 60).

[0543] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D2, 1.17%; D4, 4.48%; D6, 94.35% (Figure 61).

[0544] 28. HC_F170C / C220S, LC_S162C / C214S (Single-arm) A TCEP aqueous solution (4.11 μL, 10 mM in H2O, 12 equivalents per single antibody molecule) was added to a solution of a bispecific antibody (trastuzumab-pertuzumab, or TZB-PZB) with a disulfide bond mutation (PZB arm (HC_F170C / C220S, LC_S162C / C214S) (0.5 mg)) in histidine buffer (0.17 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 25°C for 2 hours.

[0545] After cooling to room temperature, the linker-payload solution (mc-vc-PAB-MMAE, 5.4 μL, 10 mg / mL in DMSO, 12 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was further incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (2.1 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin desalting column (0.5 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (0.42 mg, 8.3 mg / mL, 50 μL).

[0546] Figure 62 is a schematic diagram of the ADC structure.

[0547] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 5.76 (Figure 63).

[0548] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D2, 1.67%; D4, 7.35%; D6, 90.98% (Figure 64).

[0549] 29. HC_S134C / C220S, LCF116C / C214S (single arm) An aqueous solution of TCEP (8.23 μL, 10 mM in H2O, 12 equivalents per single antibody molecule) was added to a solution of a bispecific antibody (trastuzumab-pertuzumab, or TZB-PZB) with a disulfide bond mutation in the PZB arm (HC_S134C / C220S, LCK_F116C / C214S) (1.0 mg) in histidine buffer (0.33 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 25°C for 2 hours.

[0550] After cooling to room temperature, the linker-payload solution (mc-vc-PAB-MMAE, 10.8 μL, 10 mg / mL in DMSO, 12 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was further incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (4.2 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting Column (2 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (0.82 mg, 16.3 mg / mL, 50 μL).

[0551] Figure 65 is a schematic diagram of the ADC structure.

[0552] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 5.73 (Figure 66).

[0553] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D2, 1.32%; D4, 6.01%; D6, 92.68% (Figure 67).

[0554] 30. HC_P130C / C220S, LCF118C / C214S (single arm) An aqueous solution of TCEP (8.23 μL, 10 mM in H2O, 12 equivalents per single antibody molecule) was added to a solution of a bispecific antibody (trastuzumab-pertuzumab, or TZB-PZB) with a disulfide bond mutation in the PZB arm (HC_P130C / C220S, LCK_F118C / C214S) (1.0 mg) in histidine buffer (0.33 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 25°C for 2 hours.

[0555] After cooling to room temperature, the linker-payload solution (mc-vc-PAB-MMAE, 10.8 μL, 10 mg / mL in DMSO, 12 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated further at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (4.2 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting Column (2 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (0.78 mg, 15.5 mg / mL, 50 μL).

[0556] Figure 68 is a schematic diagram of the ADC structure.

[0557] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 5.83 (Figure 69).

[0558] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D2, 1.03%; D4, 4.48%; D6, 94.49% (Figure 70).

[0559] 31. HC_S134C / C220S, LLC_T116C / C214S (both arms) An aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (3.43 μL, 10 mM in H2O, 10 equivalents per single antibody molecule) was added to a solution of humanized anti-HER2 antibody (trastuzumab (TZB)) containing disulfide bond mutations, with both arms (HC_S134C / C220S, LLC_T116C / C214S) (0.5 mg) in histidine buffer (0.2 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 25°C for 2 hours, resulting in complete reduction of the disulfide bond in the hinge region.

[0560] After cooling to room temperature, the linker-payload solution (mc-vc-PAB-MMAE, 4.5 μL, 10 mg / mL in DMSO, 10 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final concentration of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (2.1 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the mixture was stirred for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin desalting column (0.5 mL), and the remaining solution was concentrated to obtain the corresponding ADC solution (0.4 mg, 3.3 mg / mL, 0.12 mL).

[0561] Figure 71 is a schematic diagram of the ADC structure.

[0562] RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.84 (Figure 72).

[0563] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D2, 4.71%; D4, 92.20%; D6, 3.10% (Figure 73).

[0564] Example 5: Generation and Analysis of Random DAR4 ADCs The purified antibody was conjugated to the drug according to the following method to obtain an ADC.

[0565] a) Preliminary tests to determine the TCEP ratio A TCEP aqueous solution (2.76 μL, 1 mM in H2O, 2 equivalents per single antibody molecule) was added to a solution of anti-HER2 antibody (trastuzumab) (0.2 mg) in histidine buffer (0.10 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours.

[0566] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 1.14 μL, 10 mg / mL in DMSO, 8 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (0.83 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without further purification.

[0567] Following the procedure described above, two other TCEP ratios (4.14 μL, 1 mM in H2O, 3 equivalents per single antibody molecule; 5.52 μL, 1 mM in H2O, 4 equivalents per single antibody molecule) were tested, and the corresponding ADC products were obtained.

[0568] Figure 74 is a schematic diagram of the ADC structure.

[0569] The obtained ADC products were first treated with DTT according to general procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed that the drug-to-antibody ratios were 1.52, 2.41, and 3.14 for 2, 3, and 4 equivalents of TCEP, respectively (Figure 75).

[0570] b) Preparation of random DAR4 ADCs using the determined TCEP ratio Based on preliminary test results, a TCEP ratio of 5.1 should be used to generate the DAR4 ADC.

[0571] A TCEP aqueous solution (492.4 μL, 1 mM in H2O, 5.1 equivalents per single antibody molecule) was added to a solution of anti-HER2 antibody (trastuzumab) (14.0 mg) in histidine buffer (7.0 mL, 20 mM, pH=6.5). The resulting mixture was then incubated at 37°C for 2 hours.

[0572] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 79.9 μL, 10 mg / mL in DMSO, 8 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 10% (DMSO / buffer, v / v). The reaction mixture was further incubated at 25°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (58 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at the same temperature for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin desalting column (10 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (12.7 mg, 2.55 mg / mL, 5.0 mL).

[0573] The obtained ADC product (also called aveDAR4, TZB-Dxd, or TZB-DXd aveDAR4) was first treated with DTT according to general procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 4.14 (Figure 77).

[0574] HIC analysis following general procedure D showed the following distribution of the number of conjugated drugs: D0, 3.42%; D2, 23.52%; D4, 42.44%; D6, 19.31%; D8, 11.30% (Figure 76).

[0575] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 3.14% and 96.86%, respectively (Figure 78).

[0576] c) Preparation of control IgG1-DXd DAR4 A similar procedure was followed to prepare IgG-Dxd (control IgG1-DXd DAR4).

[0577] Figure 95 is a schematic diagram of the ADC structure.

[0578] The obtained ADC product was first treated with DTT according to general procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 4.14 (Figure 96).

[0579] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 0.25% and 99.75%, respectively (Figure 97).

[0580] d) Preparation of TZB-Dxd DAR8 An aqueous solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (200 μL, 10 mM in H2O, 20 equivalents per single antibody molecule) was added to a solution of humanized anti-HER2 antibody (trastuzumab (TZB)) (15 mg) in histidine buffer (6.0 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 37°C for 2 hours.

[0581] After cooling to room temperature, the linker-payload solution (mc-GGFG-Dxd, 139 μL, 10 mg / mL in DMSO, 13 equivalents per single antibody molecule) was added, and additional DMSO was added to reach a final concentration of 10% (DMSO / buffer, v / v). The reaction mixture was incubated at 25°C for 1.5 hours. Subsequently, a 50 mM aqueous solution of N-acetylcysteine ​​(63 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the mixture was stirred for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin desalting column (10 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (14.3 mg, 3.3 mg / mL, 4.3 mL).

[0582] Figure 98 is a schematic diagram of the ADC structure.

[0583] RP-HPLC analysis following general procedure B showed a drug-to-antibody ratio of 7.67 (Figure 99).

[0584] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 2.86% and 97.14%, respectively (Figure 100).

[0585] Example 6: Production of DAR4 ADC using a concentration method The purified antibody was conjugated to the drug according to the following method to obtain an ADC.

[0586] a) Preliminary tests to determine the TCEP ratio An aqueous solution of TCEP (1.025 μL, 5 mM in H2O, 2.5 equivalents per single antibody molecule) was added to a solution of anti-B7H3 antibody (M30) (0.3 mg) in histidine buffer (0.06 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 0°C for 6 hours.

[0587] A solution of the linker-payload (mc-GGFG-Dxd, 1.7 μL, 10 mg / mL in DMSO, 8 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 15% (DMSO / buffer, v / v). The reaction mixture was incubated at 0°C for 1.0 hour. Subsequently, 50 mM aqueous solution of N-acetylcysteine ​​(1.23 μL, pH=8.0 per single antibody molecule, 30 equivalents) was added, and the resulting mixture was stirred at 25°C for a further 20 minutes to quench the unreacted linker-payload. The mixture was then subjected to analysis without further purification.

[0588] Following the procedure described above, two other TCEP ratios (1.435 μL, 5 mM in H2O, 3.5 equivalents per single antibody molecule; 1.845 μL, 5 mM in H2O, 4.5 equivalents per single antibody molecule) were tested, and the corresponding ADC products were obtained.

[0589] Figure 79 is a schematic diagram of the ADC structure.

[0590] The obtained ADC products were first treated with DTT according to general procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed that the drug-to-antibody ratios were 3.08, 3.79, and 4.26 for TCEP ratios of 2.5, 3.5, and 4.5 equivalents, respectively (Figure 80).

[0591] b) Preparation of random DAR4 ADCs using the determined TCEP ratio According to preliminary test results, a TCEP ratio of 4.0 is required to generate the DAR4 ADC.

[0592] A TCEP aqueous solution (43.73 μL, 5 mM in H2O, 4.0 equivalents per single antibody molecule) was added to a solution of anti-B7H3 antibody (M30) (8.0 mg) in histidine buffer (1.6 mL, 20 mM, pH=6.5). The resulting mixture was incubated at 0°C for 6 hours.

[0593] A linker-payload solution (mc-GGFG-Dxd, 45.2 μL, 10 mg / mL in DMSO, 8 equivalents per single antibody molecule) was added, and additional DMSO was added to achieve a final ratio of 15% (DMSO / buffer, v / v). The reaction mixture was incubated at 0°C for 1.5 hours. Subsequently, a 50 mM N-acetylcysteine ​​aqueous solution (32.8 μL, pH=8.0, 30 equivalents per single antibody molecule) was added, and the resulting mixture was stirred at 25°C for a further 20 minutes to quench the unreacted linker-payload. The mixture was then desalted using a Zeba® Spin Desalting Column (10 mL), and the remaining solution was concentrated to obtain a solution of the corresponding ADC (7.2 mg, 2.25 mg / mL, 3.2 mL).

[0594] The obtained ADC product was first treated with DTT according to common procedure A, and then analyzed by RP-HPLC. RP-HPLC analysis after general procedure B showed a drug-to-antibody ratio of 3.80 (Figure 81).

[0595] SEC analysis following general procedure C showed that the proportions of high molecular weight species (HMWS) and monomers were 0.45% and 99.55%, respectively (Figure 82).

[0596] HIC analysis after general procedure D showed the following percentages of ADCs with different amounts of conjugated drugs: D0, 1.84%; D2, 23.47%; D4, 55.20%; D6, 17.59%; D8, 1.90% (Figure 83).

[0597] Example 7: Stability of ADCs in mouse plasma The stability of ADCs in mouse plasma was tested according to the steps shown below.

[0598] Each ADC sample (2 mg / ml) was added to mouse plasma to obtain a final concentration of 0.2 mg / mL (20 μL of ADC + 180 μL of plasma, n=5). The ADC-plasma mixture was incubated at 37°C. One sample was taken at each time point (days 0, 1, 3, 5, and 7) and stored below -40°C until analysis.

[0599] Total human antibodies were recovered from plasma by immunoprecipitation using Fc-specific anti-human IgG-agarose resin (Sigma, #A3316). 40 μL of the resin slurry was centrifuged to remove the solvent. The resin was rinsed twice with 200 μL of PBS (pH 7.4). ADC-containing plasma samples were then gently mixed with the resin at room temperature for 30 minutes. The resin was recovered by centrifugation, washed twice with PBS at pH 7.4, and then washed again with PBS at pH 6.0. The resin pellet was resuspended in 50 μL of citrate buffer at pH 3.0 and incubated further at room temperature for 5–10 minutes. The resin was removed by centrifugation. The supernatant was collected and adjusted to pH 8.0 with 1 M Tris.

[0600] The ADC concentration was measured using nanodrops.

[0601] The sample was reduced with 20 equivalents of TCEP and then analyzed by RP-HPLC according to general procedure B. The results are shown in Figures 84-90.

[0602] Example 8: Cell Binding Assay SK-BR-3 cells (ATCC, HTB-30) were resuspended in FACS buffer (2 mL, 2% FBS, 1 XPBS) and 1.5-2.0 × 10⁶ cells were extracted. 6 The solution was adjusted to 50 μL / mL, seeded into a 96-well plate (50 μL / well), and incubated at 4°C until use.

[0603] Trastuzumab and various ADC samples were diluted to 180 μg / mL with FACS buffer, and then serially diluted (4-fold) with the same buffer. The resulting solutions were added to plates (50 μL per well), and the plates were incubated at 4°C for 30 minutes. After centrifugation, the supernatant was discarded, and the cells were washed with FACS buffer. Next, PE goat anti-human IgG (purchased from Biolegend) was added, the cells were resuspended, and incubated in the dark at 4°C for 30 minutes. The cells were centrifuged, washed with FACS buffer, and analyzed by flow cytometry.

[0604] The results are shown in Figure 91.

[0605] Example 9; In vitro cytotoxicity assay: SK-BR-3 cells were seeded into 96-well plates at a rate of 2,000 cells per well. These plates were incubated overnight at 37°C with 5% CO2.

[0606] Starting at 40 nM, serial 3-fold dilutions were applied to the ADC samples using the corresponding media. The ADC samples were added to cells (2 wells for each concentration gradient), and the cells were cultured at 37°C and 5% CO2 for 6 days, after which CellTiter-Glo® Luminescent (100 μL per well) was added. The plates were gently shaken for 2 minutes and incubated for 10 minutes before measurement at 450 nm using a spectrometer. EC 50 The values ​​and cell viability curves were calculated using GraphPad Prism software.

[0607] The results are shown in Figure 92.

[0608] Example 10: In vivo efficacy study of the NCI-N87 model in CB17-SCID mice 4 x 10 6 Human gastric cancer cells (NCI-N87, purchased from ATCC) were suspended in physiological saline. The resulting solution was then subcutaneously transplanted into CB17-SCID mice (Charles, Charles, Nordisk) on day 0. The mice were randomly divided into groups on day 11.

[0609] After grouping was complete, mice were intravenously (iv) administered a single dose of 5 mg / kg each of anti-HER2 ADC produced using the concentration method (TZB-Dxd, Example 5) and site-specific ADC produced according to Example 4. Tumor size and weight were measured every 3 days. Tumor growth inhibition was monitored every 3 days.

[0610] Tumor inhibition (TGI%) was calculated as follows: TGI% = 100% * (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group initial tumor volume).

[0611] Tumor volume measurement: The longest axis (L) and widest axis (W) of the tumor were measured using calipers. The tumor volume was calculated using the following formula: V = L * W 2 / 2

[0612] The results are shown in Figures 93 and 94 and summarized in the table below.

[0613] [Table 5]

[0614] The ADC composition produced by the production method of the present invention has been demonstrated to have the same therapeutic efficacy as the ADC composition produced by the conventional production method (random DAR4).

[0615] Other Embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to illustrate, and not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the following claims.

Claims

1. An antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region or its CH1 region and a light chain constant region, and comprises unnatural disulfide bonds formed at the positions of heavy chain amino acid residues in the heavy chain constant region or its CH1 region and at the positions of light chain amino acid residues in the light chain constant region, wherein (1) the sum of the relative solvent exposure area (SASA) of the heavy chain amino acid residue side chain and (2) the relative SASA of the light chain amino acid residue side chain is less than 65%, less than 60%, or less than 55%, and the antibody or antigen-binding fragment does not have a disulfide bond between the amino acid residue at position 220 of the heavy chain constant region and the amino acid residue at position 214 of the light chain constant region, and the amino acid positions are based on EU numbering.

2. ADC according to claim 1 or a pharmaceutically acceptable salt or solvate thereof, wherein the relative SASA of the heavy chain amino acid side chain is greater than 30% and the relative SASA of the light chain amino acid side chain is less than 15%, or the relative SASA of the light chain amino acid side chain is greater than 30% and the relative SASA of the heavy chain amino acid side chain is less than 15%, preferably the relative SASA of the heavy chain amino acid side chain is greater than 40% and the relative SASA of the light chain amino acid side chain is less than 10%, or the relative SASA of the light chain amino acid side chain is greater than 40% and the relative SASA of the heavy chain amino acid side chain is less than 10%, more preferably the relative SASA of each of the heavy chain and light chain amino acid side chains is less than 20%, less than 25%, less than 30%, less than 35%, less than 40%, or less than 45%. The position of the heavy chain constant region is determined according to the amino acid positions of the IgG1 heavy chain numbering, and the position of the light chain constant region is determined according to the amino acid positions of the kappa light chain numbering or lambda light chain numbering. The aforementioned amino acid positions are based on EU numbering, and the ADC or a pharmaceutically acceptable salt or solvate thereof.

3. ADC according to claim 1 or 2 or a pharmaceutically acceptable salt or solvate thereof, wherein the non-natural disulfide bond is resistant to reduction by tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and / or dithiothreitol (DTT), for example, When the antibody or its antigen-binding fragment is reduced by 10 to 30 equivalents of TCEP per single antibody molecule, more than 90% or more than 95% of the non-natural disulfide bonds remain intact, for example, The concentration of the antibody or its antigen-binding fragment is 0.1 to 50 mg / ml during the reduction reaction. Optional, ADC or a pharmaceutically acceptable salt or solvate thereof, wherein the non-natural disulfide bond remains stable under reducing conditions that can reduce more than 90% or more of the hinge region disulfide bond of the antibody or antigen-binding fragment, for example, in which more than 90% or more of the non-natural disulfide bond remains intact.

4. An ADC according to any one of claims 1 to 3, wherein the cysteine ​​at position 220 of the heavy chain constant region and the cysteine ​​at position 214 of the light chain constant region of the antibody or its antigen-binding fragment are substituted with non-cysteine, for example, the antibody or its antigen-binding fragment contains C220S / A / V in the heavy chain constant region and C214S / A / V in the light chain constant region, or a pharmaceutically acceptable salt or solvate thereof.

5. ADC according to any one of claims 1 to 4, wherein the heavy chain amino acid residue of the heavy chain constant region or its CH1, and the light chain amino acid residue of the light chain constant region are mutated to cysteine ​​residues, forming a non-natural disulfide bond, or a pharmaceutically acceptable salt or solvate thereof.

6. A pharmaceutically acceptable salt or solvate of the ADC or the ADC according to claim 5, wherein the mutant heavy chain amino acid residue is located at one or more positions in the heavy chain constant region selected from the group consisting of 126, 168, 170, 173, 134, 133, 130, and 141, the amino acid position being based on EU numbering, and / or the mutant light chain amino acid residue is located at one or more positions in the light chain constant region selected from the group consisting of 124, 164, 162, 116, 209, 117, and 118, the position of the heavy chain constant region being determined according to the amino acid position of the IgG1 heavy chain numbering, and the position of the light chain constant region being determined according to the amino acid position of kappa light chain numbering or lambda light chain numbering.

7. ADC according to claim 6 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or its antigen-binding fragment is a combination of the following mutations: 126C in the heavy chain, 124C in the light chain, 168C in the heavy chain, 164C in the light chain, 170C in the heavy chain, 164C in the light chain, 170C for heavy chains, 162C for light chains. 173C in the heavy chain, 162C in the light chain, 134C in the heavy chain, 116C in the light chain, 133C in the heavy chain, 209C in the light chain, 133C in the heavy chain, 117C in the light chain, 130C in the heavy chain, 118C in the light chain, or ADC or a pharmaceutically acceptable salt or solvate thereof, comprising one or more of 141C in the heavy chain and 116C in the light chain.

8. ADC according to claim 7 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or its antigen-binding fragment comprises two pairs of heavy and light chains, and only one pair of heavy and light chains comprises the following combination of mutations: In the heavy chain, F126C, and in the light chain, Q124C, In the heavy chain, H168C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, S162C, In the heavy chain, V173C, and in the light chain, S162C, In the heavy chain, S134C, and in the light chain, F116C, In the heavy chain, K133C, and in the light chain, F209C, In the heavy chain, K133C, and in the light chain, I117C, In the heavy chain, P130C, and in the light chain, F118C, The heavy chain contains one of A141C, and the light chain contains one of F116C. ADC or a pharmaceutically acceptable salt or solvate thereof, wherein the heavy chain and light chain optionally further comprise C220S in the heavy chain and C214S in the light chain.

9. ADC according to claim 6 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or its antigen-binding fragment comprises two pairs of heavy chains and light chains, each pair of heavy chains and light chains having the following combination of mutations: In the heavy chain, F126C, and in the light chain, Q124C, In the heavy chain, H168C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, T164C, In the heavy chain, F170C, and in the light chain, S162C, In the heavy chain, V173C, and in the light chain, S162C, In the heavy chain, S134C, and in the light chain, F116C, In the heavy chain, K133C, and in the light chain, F209C, In the heavy chain, K133C, and in the light chain, I117C, In the heavy chain, P130C, and in the light chain, F118C, The heavy chain contains one or more A141C, and the light chain contains one or more F116C. ADC or a pharmaceutically acceptable salt or solvate thereof, wherein the heavy chain and light chain optionally further comprise C220S in the heavy chain and C214S in the light chain.

10. ADC according to claim 9 or a pharmaceutically acceptable salt or solvate thereof, wherein the antibody or its antigen-binding fragment comprises two pairs of heavy chains and light chains. Each heavy chain contains F126C, and each light chain contains Q124C. Each heavy chain contains H168C, and each light chain contains T164C. Each heavy chain contains F170C, and each light chain contains T164C. Each heavy chain contains F170C, and each light chain contains S162C. Each heavy chain contains V173C, and each light chain contains S162C. Each heavy chain contains S134C, and each light chain contains F116C. Each heavy chain contains K133C, and each light chain contains F209C. Each heavy chain contains K133C, and each light chain contains I117C. Each heavy chain contains P130C, and each light chain contains F118C, or Each heavy chain contains A141C, and each light chain contains F116C. ADC or a pharmaceutically acceptable salt or solvate thereof, optionally comprising each heavy chain further containing C220S and each light chain further containing C214S.

11. A DC according to any one of claims 1 to 10, wherein the antibody or its antigen-binding fragment comprises two pairs of heavy chains and light chains, the two heavy chains may be the same or different, and the two light chains may be the same or different, for example, the heavy chains and light chains contain the following combination of mutations. Table 1

12. An ADC according to any one of claims 1 to 11, or a pharmaceutically acceptable salt or solvate thereof, wherein the heavy chain steady region is the steady region of human IgG1, IgG2, IgG3, or IgG4, for example, the steady region of IgG1. The light chain steady region is either a kappa or lambda steady region, or CH1 is the CH1 region of human IgG1, IgG2, IgG3, or IgG4, and is an ADC or a pharmaceutically acceptable salt or solvate thereof.

13. A pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein the antibody or its antigen-binding fragment is a human or humanized or chimeric antibody or its antigen-binding fragment, a monoclonal antibody, a Fab fragment, a single-arm antibody and / or a multispecific antibody (e.g., a bispecific antibody), and for example, the antibody or its antigen-binding fragment specifically binds to HER2, B7H3 and / or TROP2.

14. A pharmaceutically acceptable salt or solvate of the ADC or the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment comprises a fragment crystallizable region (Fc region), and optionally the Fc region is the Fc region of human IgG1, IgG2, IgG3, or IgG4 having, for example, an SI mutation, a LALA mutation, an N297A mutation, a YTE mutation, and / or a FLAA mutation.

15. An ADC or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 14, wherein the ADC comprises a payload linked to an antibody or an antigen-binding fragment via a linker, the payload being covalently bound to the antibody or an antigen-binding fragment via a thiol-based conjugation, for example, the payload comprising a cytotoxic agent (e.g., Dxd or MMAE), a cell proliferation inhibitor, a biologically active protein, a synthetic polymer, an enzyme, a nucleic acid (e.g., DNA or RNA) and fragments thereof.

16. ADC according to any one of claims 1 to 14, wherein the ADC comprises a payload linked to an antibody or an antigen-binding fragment via a linker, the linker being linked to the antibody via an open disulfide bond thiol-SH, for example, the linker containing a maleimide moiety.

17. A pharmaceutically acceptable salt or solvate thereof according to any one of claims 1 to 14, wherein the ADC comprises a payload linked to an antibody or an antigen-binding fragment via a linker, and the linker-payload is selected from mc-GGFG-DXD or mc-VC-PAB-MMAE.

18. The ADC according to any one of claims 1 to 17, wherein the drug-to-antibody ratio (DAR) is 3.74 to 4, or the DAR is 5.70 to 6.

19. A method for preparing an antibody-drug conjugate (ADC) or a pharmaceutically acceptable salt or solvate thereof, comprising the following steps: (a) Incubating an antibody or antigen-binding fragment according to any one of claims 1 to 14 with a reducing agent to reduce the natural disulfide bond in the antibody or antigen-binding fragment to produce a reduced thiol group, (b) Introducing an excess amount of linker-payload having a reactive group so as to react with the reduced thiol group generated in step (a), The method further optionally includes (c) adding an effective amount of N-acetylcysteine ​​to quench the excess payload, and then recovering the resulting antibody-drug conjugate, Optionally, the linker-payload contains a maleimide group. A method wherein the linker-payload is optionally linked to the antibody or its antigen-binding fragment via a thiol-based conjugation.

20. The method according to claim 19, wherein at least 50%, 60%, 70%, 80%, or 90% of the resulting ADC products have a drug-to-antibody ratio (DAR) of about 4, or at least 50%, 60%, 70%, 80%, or 90% of the resulting ADC products have a drug-to-antibody ratio (DAR) of about 6.

21. A method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising ADC according to any one of claims 15 to 18, or a pharmaceutically acceptable salt or solvate thereof, for example, the subject having a solid tumor cancer, for example, the cancer being breast cancer, lung cancer, pancreatic cancer, melanoma, oral cancer, mesothelioma, ovarian cancer, colorectal cancer, gastric cancer, cervical cancer, brain cancer, skin cancer, multiple myeloma lymphoma, epithelial neoplasm, soft tissue sarcoma, esophageal cancer, or CNS tumor.

22. A method for reducing the rate of tumor growth, comprising contacting tumor cells with an effective amount of a composition comprising an ADC or a pharmaceutically acceptable salt or solvate thereof as described in any one of claims 15 to 18.

23. A pharmaceutical composition comprising an ADC according to any one of claims 1 to 18 or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier.

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