Modified conjugation polypeptides for optimized drug conjugation

Abstract

We provide engineered antibodies that are stable and conjugate to ligands or drugs in ligand / drug-versus-antibody ratios greater than 3, making these conjugates suitable for the treatment of various indications. [Solution] Antibody heavy chain constant (C H The domain contains a dual-operated cysteine-reactive amino acid residue at a position selected from the group consisting of K274C and A339C, K274C and K360C, K274C and N384C, K274C and G385C, K274C and V422C, and K274C and S440C, according to the numbering of the Kabat EU index, and an antigen-binding domain and antibody heavy chain constant (C H An antigen-binding protein or its antigen-binding fragment containing a ) domain is provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 982,943, filed on 28 February 2020, the entirety of which is incorporated herein by reference.

[0002] This disclosure relates to compositions and methods for preparing dual-engineered antigen-binding proteins for ligand conjugation. [Background technology]

[0003] The use of specific antigen-binding proteins or antibodies to treat humans and other animals is a powerful tool and is highly effective in treating many conditions and disorders. However, there is a greater need for more effective targeted therapies, particularly targeted therapies with higher efficacy and a wider therapeutic window. One of these targeted therapies utilizes antibody-ligand conjugates, in which a specific antibody targets the desired treatment site and delivers a targeted ligand to that site. This ligand may be a drug, for example, a biologically active cytotoxic payload. The advantage of using antibody-ligand conjugate molecules is that they are designed to distinguish between healthy and affected tissue. Indeed, such molecules exhibit improved therapeutic indicators, namely higher efficacy and / or lower toxicity profiles than untargeted antibodies in a clinical setting. However, the development of such therapeutic agents is challenging, as many factors (including the stability of the antibody itself and its conjugation) can significantly affect the specificity of the disease target (e.g., tumor), thereby reducing efficacy. Antibody-ligand conjugates, which have been shown to exhibit high levels of nonspecific binding and low stability in circulation, are eliminated during their migration through normal tissue, even before reaching their target site. Furthermore, antibody-ligand conjugates with significant subpopulations of high drug load may generate aggregates that are removed by macrophages, resulting in shorter half-lives. Therefore, the importance of process control and improvement of antibody-ligand conjugates, as well as the need to prevent troublesome problems such as product aggregation and nonspecific toxicity, is increasing.

[0004] A small number of antibody-ligand conjugates specific to particular indications have been approved in the United States. Examples include: brentuximab vedotin for relapsed or refractory Hodgkin lymphoma and systemic anaplastic large cell lymphoma (ALCL); trastuzumab emtansine for HER2+ breast cancer; inotuzumab ozogamicin for acute lymphoblastic leukemia (ALL); and gemtuzumab ozogamicin for relapsed acute myeloid leukemia (AML). However, there remains a need for antibody-ligand conjugates that are stable, can deliver high payloads (i.e., high ligand / drug vs. antibody ratios), and can be used for a variety of indications. [Overview of the project] [Problems that the invention aims to solve]

[0005] In this specification, the inventors disclose engineered antibodies that are stable and conjugate to ligands or drugs in ligand / drug-versus-antibody ratios greater than 3, making these conjugates suitable for the treatment of various indications. [Means for solving the problem]

[0006] This disclosure provides an antigen-binding protein or a fragment thereof.

[0007] In one embodiment, the present disclosure relates to an antibody heavy chain constant (C) comprising a reactive amino acid residue manipulated at a first position and a reactive amino acid residue manipulated at a second position. H The system provides an antigen-binding protein or fragment thereof, including a domain; where, according to the numbering of the EU index of Kabat, the first position is 274, and the second position is selected from the group consisting of 339, 360, 384, 385, 422, 440, and any combination thereof.

[0008] In certain embodiments, according to the numbering of the Kabat EU index, the first position is 274th and the second position is 339th. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 274th and the second position is 360th. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 274th and the second position is 384th. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 274th and the second position is 385th. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 274th and the second position is 422nd. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 274th and the second position is 440th.

[0009] In certain embodiments, the antigen-binding protein or its fragment contains a K274C amino acid substitution at the first position and an A339C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains a K274C amino acid substitution at the first position and a K360C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains a K274C amino acid substitution at the first position and an N384C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains a K274C amino acid substitution at the first position and a G385C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains a K274C amino acid substitution at the first position and a V422C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains a K274C amino acid substitution at the first position and an S440C amino acid substitution at the second position, according to the numbering of the Kabat EU index.

[0010] In another embodiment, the present disclosure relates to an antibody heavy chain constant (C) comprising a reactive amino acid residue manipulated at a first position and a reactive amino acid residue manipulated at a second position. H The system provides an antigen-binding protein or fragment thereof, including a domain; where, according to the numbering of the EU index of Kabat, the first position is 339, and the second position is selected from the group consisting of 290, 360, 384, 385, 422, 440, and any combination thereof.

[0011] In certain embodiments, according to the numbering of the Kabat EU index, the first position is 339th and the second position is 290. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 339th and the second position is 360. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 339th and the second position is 384. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 339th and the second position is 385. In certain embodiments, according to the numbering of the Kabat EU index According to the U-index numbering, the first position is 339th and the second position is 422nd. In a particular embodiment, according to the Kabat EU-index numbering, the first position is 339th and the second position is 440th.

[0012] In certain embodiments, the antigen-binding protein or its fragment contains an A339C amino acid substitution at the first position and a K360C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an A339C amino acid substitution at the first position and a N384C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an A339C amino acid substitution at the first position and a G385C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an A339C amino acid substitution at the first position and a V422C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an A339C amino acid substitution at the first position and a S440C amino acid substitution at the second position, according to the Kabat EU index numbering.

[0013] In another embodiment, the present disclosure relates to an antibody heavy chain constant (C) comprising a reactive amino acid residue manipulated at a first position and a reactive amino acid residue manipulated at a second position. H The system provides an antigen-binding protein or fragment thereof, including a domain; where, according to the numbering of the EU index of Kabat, the first position is 118, and the second position is selected from the group consisting of 274, 339, 384, 385, 422, 440, and any combination thereof.

[0014] In certain embodiments, according to the numbering of the Kabat EU index, the first position is 118th and the second position is 274. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 118th and the second position is 339. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 118th and the second position is 384. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 118th and the second position is 385. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 118th and the second position is 422. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 118th and the second position is 440.

[0015] In certain embodiments, the antigen-binding protein or its fragment contains an A118C amino acid substitution at the first position and a K274C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an A118C amino acid substitution at the first position and an A339C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an A118C amino acid substitution at the first position and an N384C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an A118C amino acid substitution at the first position and a G385C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an A118C amino acid substitution at the first position and a V422C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment is the numbering of the EU index of Kabat. According to the formula, it includes an A118C amino acid substitution at the first position and an S440C amino acid substitution at the second position.

[0016] In another embodiment, the present disclosure relates to an antibody heavy chain constant (C) comprising a reactive amino acid residue manipulated at a first position and a reactive amino acid residue manipulated at a second position. H The system provides an antigen-binding protein or fragment thereof, including a domain; where, according to the numbering of the EU index of Kabat, the first position is 384, and the second position is selected from the group consisting of 118, 274, 290, 339, and any combination thereof.

[0017] In certain embodiments, according to the numbering of the Kabat EU index, the first position is 384th and the second position is 118th. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 384th and the second position is 274th. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 384th and the second position is 290th. In certain embodiments, according to the numbering of the Kabat EU index, the first position is 339th and the second position is 118th.

[0018] In certain embodiments, the antigen-binding protein or its fragment contains an N384C amino acid substitution at the first position and an A118C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an N384C amino acid substitution at the first position and a K274C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an N384C amino acid substitution at the first position and a K290C amino acid substitution at the second position, according to the Kabat EU index numbering. In certain embodiments, the antigen-binding protein or its fragment contains an N384C amino acid substitution at the first position and an A339C amino acid substitution at the second position, according to the Kabat EU index numbering.

[0019] In certain embodiments, the manipulated reactive amino acid residue is selected from the group consisting of cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, and arginine. In certain embodiments, the manipulated reactive amino acid residue is cysteine. In certain embodiments, the manipulated reactive amino acid residue is lysine.

[0020] In certain embodiments, the manipulated reactive amino acid residue is conjugated to a ligand via a reactive moiety. In certain embodiments, the antigen-binding protein or a fragment thereof further comprises a linker that conjugates the manipulated reactive amino acid residue to a ligand. In certain embodiments, the linker is cleavable. In certain embodiments, the linker is not cleavable.

[0021] In certain embodiments, the antigen-binding protein or its fragment contains a ligand-to-antibody ratio (LAR) of at least 3.0. In certain embodiments, the LAR is at least 3.4.

[0022] In certain embodiments, the ligand is a detection probe. In certain embodiments, the detection probe is selected from the group consisting of biotin, polyethylene glycol (PEG), fluorescent tags, visualization peptides, and combinations thereof. In certain embodiments, the detection probe is PEG.

[0023] In certain embodiments, the ligand is the targeting moiety. In certain embodiments, the targeting moiety is selected from the group consisting of proteins, nucleic acids, lipids, carbohydrates, and combinations thereof.

[0024] In certain embodiments, the ligand is a drug.

[0025] In certain embodiments, the antigen-binding protein or fragment contains a drug-to-antibody ratio (DAR) of at least 3.0. In certain embodiments, the DAR is at least 3.4.

[0026] In certain embodiments, the drug is a prodrug selected from the group consisting of: phosphate-containing prodrugs, amino acid-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, β-lactam-containing prodrugs, phenoxyacetamide-containing prodrugs, phenylacetamide-containing prodrugs, 5-fluorocytosine prodrugs, 5-fluorouridine prodrugs, and any combination thereof.

[0027] In certain embodiments, the drug is selected from the group consisting of: anticancer agents, anti-inflammatory agents, anti-infective agents, anesthetic agents, cytotoxic agents, radionuclides, immunomodulators, cell signaling peptides, growth factors, enzymes, oligonucleotides, photoactive agents, and any combination thereof.

[0028] In certain embodiments, the anticancer agent is selected from the group consisting of: cell division inhibitors, cytotoxic nucleosides, tubulin binders, hormones and hormone antagonists, anti-angiogenic agents, enzyme inhibitors, gene regulatory factors, proteasome inhibitors, pteridines, diinene, podophyllotoxin, auristatin, geldanamycin, calicheamicin, gramicidin D, maytansinoids, neocartinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracinedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansin derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptonigrin derivatives, auristatin derivatives, duocalmycin derivatives, and any combination thereof.

[0029] In certain embodiments, the cell division inhibitor is selected from the group consisting of: anthracine, DNA synthesis inhibitors, DNA intercalators, DNA-RNA transcription regulators, anthamycin benzoquinone, quinonoid derivatives, busulfan, ifosfamide, mechloretamine, triaziquane, diaziquane, carbazylquinone, indolequinone E09, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, nitrosourea compounds, and any combination thereof.

[0030] In certain embodiments, the cytotoxic nucleoside is selected from the group consisting of: adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, furoxiuridine, futraful, 6-mercaptopurine, and any combination thereof.

[0031] In certain embodiments, the tubulin binder is selected from the group consisting of: taxoids, nocodazole, rhizoxin, dorastatin, colchicine, colchicinoids, combretastatin, vinca alkaloids, and any combination thereof.

[0032] In certain embodiments, hormones and hormone antagonists are: corticosteroids Selected from the group consisting of iodines, progestins, estrogens, anti-estrogens, androgens, aromatase inhibitors, 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefelzin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide, luteinizing hormone-releasing hormone, pifislin-a, rapamycin, sex hormone-binding globulin, thapsigardin, and any combination thereof.

[0033] In certain embodiments, the anti-angiogenic agent is selected from the group consisting of: angiostatin Kl-3, DL-α-difluoromethyl-ornithine, endostatin, fumagiline, genistein, minocycline, staurosporine, (+)-thalidomide, and any combination thereof.

[0034] In certain embodiments, the enzyme inhibitor is selected from the group consisting of: S(+)-camptothecin, curcumin, (-)-deguerin, 5,6-dichlorobenzo-imidazole I-β-D-ribofuranoside, etoposide, formestan, fosaliesin, hispidin, 2-imino-1-imidazolidinedacetate, mebinolin, trichostatin A, tilhostin AG34, tilhostin AG879, and any combination thereof.

[0035] In certain embodiments, the gene regulatory factor is: 5-aza-2'-deoxycytidine, 5 - Selected from the group consisting of azacitidine, cholecalciferol, 4-hydroxytamoxifen, melatonin, mifepristone, raloxifen, trans-retinal, retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol, tamoxifen, troglitazone, and any combination thereof.

[0036] In certain embodiments, the antigen-binding protein or its fragment is an antibody heavy chain variable (V H ) Includes further domains. In certain embodiments, the antigen-binding protein or its fragment is an antibody light chain variable (V L ) Includes further domains.

[0037] In certain embodiments, the antigen-binding protein is a chimeric or humanized antibody. In certain embodiments, the antigen-binding protein is a human antibody. In certain embodiments, the antigen-binding protein is a monoclonal antibody.

[0038] In certain embodiments, the antigen-binding protein comprises one or more full-length antibody heavy chains including an Fc region. In certain embodiments, the Fc region is a human IgG1 Fc region.

[0039] In another embodiment, the Disclosure relates to a method for producing an antigen-binding protein or fragment thereof comprising a reactive amino acid residue manipulated at a first position and a reactive amino acid residue manipulated at a second position, comprising: (a) producing a first library of manipulated parent antigen-binding proteins or fragments thereof, wherein each parent antigen-binding protein or fragment comprises a first manipulated reactive amino acid residue; (b) producing a second library of ligand-conjugate manipulated parent antigen-binding proteins or fragments thereof by conjugating a ligand to the first manipulated reactive amino acid residue of each manipulated parent antigen-binding protein or fragment in the first library; (c) producing a third library of manipulated positions by screening the second library for a ligand-to-antibody ratio (LAR) greater than 1.7, wherein the third library of manipulated positions comprises positions where manipulated parent antigen-binding proteins or fragments having an LAR greater than 1.7 have the manipulated reactive amino acid residue; and (d) the antigen-binding protein or fragment The present invention provides a method for producing a fourth library of antigen-binding proteins or fragments, wherein each antigen-binding protein or fragment comprises a reactive amino acid residue manipulated at a first position selected from a third library of manipulated positions and a reactive amino acid residue manipulated at a second position selected from a third library of manipulated positions; (e) producing a fifth library of ligand-conjugate type dual-manipulated antigen-binding proteins or fragments by conjugating a ligand to the reactive amino acid residue manipulated at the first position and the reactive amino acid residue manipulated at the second position; and (f) producing a sixth library of dual-manipulated antigen-binding proteins or fragments by screening the fifth library for LARs exceeding 3.4.

[0040] In a particular embodiment, a method for producing an antigen-binding protein or fragment thereof further includes the step of producing a third library of the manipulated position by screening a second library for 60% or more conjugations of one ligand per single manipulated parent antigen-binding protein or fragment thereof, 20% or less conjugations of multiple ligands per single manipulated parent antigen-binding protein or fragment thereof, and 20% or less conjugations of zero ligands per single manipulated parent antigen-binding protein or fragment thereof.

[0041] In a particular embodiment, a method for producing an antigen-binding protein or fragment thereof further includes the step of producing a sixth library of dual-engineered antigen-binding proteins or fragments by screening a fifth library for conjugations of 80% or more of one or two ligands per dual-engineered antigen-binding protein or fragment thereof, 10% or less of multiple ligands per dual-engineered antigen-binding protein or fragment thereof, and 5% or less of zero ligands per dual-engineered antigen-binding protein or fragment thereof.

[0042] In certain embodiments, a method for producing an antigen-binding protein or fragment thereof further comprises the step of conjugating a ligand to an engineered reactive amino acid residue of a dual-engineered antigen-binding protein or fragment thereof, comprising a sixth library.

[0043] In certain embodiments, the manipulated reactive amino acid residue is selected from the group consisting of cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, and arginine. In certain embodiments, the manipulated reactive amino acid residue is cysteine. In certain embodiments, the manipulated reactive amino acid residue is lysine. In certain embodiments, the manipulated reactive amino acid residue is conjugated to a ligand via a reactive moiety.

[0044] In certain embodiments, a method for producing an antigen-binding protein or a fragment thereof further comprises a linker that conjugates a manipulated reactive amino acid residue to a ligand. In certain embodiments, the linker is cleavable. In certain embodiments, the linker is not cleavable.

[0045] In certain embodiments, LAR is at least 3.0. In certain embodiments, LAR is at least 3.4. In certain embodiments, the ligand is a detection probe. In certain embodiments, the detection probe is: biotin, polyethylene glycol (PEG), fluorescent tag, visualization PEG Tides and combinations thereof. In certain embodiments, the detection probe is selected from the group consisting of PEG.

[0046] In certain embodiments, the ligand is the targeting moiety. In certain embodiments, the targeting moiety is selected from the group consisting of proteins, nucleic acids, lipids, carbohydrates, and combinations thereof. In certain embodiments, the ligand is a drug. In certain embodiments, the method for producing an antigen-binding protein or a fragment thereof includes a drug-to-antibody ratio (DAR) of at least 3.0. In certain embodiments, the DAR is at least 3.4.

[0047] In certain embodiments, the drug is a prodrug selected from the group consisting of: phosphate-containing prodrugs, amino acid-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, β-lactam-containing prodrugs, phenoxyacetamide-containing prodrugs, phenylacetamide-containing prodrugs, 5-fluorocytosine prodrugs, 5-fluorouridine prodrugs, and combinations thereof.

[0048] In certain embodiments, the drug is selected from the group consisting of: anticancer agents, anti-inflammatory agents, anti-infective agents, anesthetic agents, cytotoxic agents, radionuclides, immunomodulators, cell signaling peptides, growth factors, enzymes, oligonucleotides, photoactive agents, and combinations thereof.

[0049] In certain embodiments, the anticancer agent is selected from the group consisting of: cell division inhibitors, cytotoxic nucleosides, tubulin binders, hormones and hormone antagonists, anti-angiogenic agents, enzyme inhibitors, gene regulatory factors, proteasome inhibitors, pteridines, diinene, podophyllotoxin, auristatin, geldanamycin, calicheamicin, gramicidin D, maytansinoids, neocartinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracinedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansin derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptonigrin derivatives, auristatin derivatives, duocalmycin derivatives, and any combination thereof.

[0050] In certain embodiments, the cell division inhibitor is selected from the group consisting of: anthracine, DNA synthesis inhibitors, DNA intercalators, DNA-RNA transcription regulators, anthamycin benzoquinone, quinonoid derivatives, busulfan, ifosfamide, mechloretamine, triaziquane, diaziquane, carbazylquinone, indolequinone E09, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, nitrosourea compounds, and any combination thereof.

[0051] In certain embodiments, the cytotoxic nucleoside is selected from the group consisting of: adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, furoxiuridine, futraful, 6-mercaptopurine, and any combination thereof.

[0052] In certain embodiments, the tubulin binder is selected from the group consisting of: taxoids, nocodazole, rhizoxin, dorastatin, colchicine, colchicinoids, combretastatin, vinca alkaloids, and any combination thereof.

[0053] In certain embodiments, hormones and hormone antagonists are: corticosteroids Selected from the group consisting of iodines, progestins, estrogens, anti-estrogens, androgens, aromatase inhibitors, 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefelzin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide, luteinizing hormone-releasing hormone, pifislin-a, rapamycin, sex hormone-binding globulin, thapsigardin, and any combination thereof.

[0054] In certain embodiments, the anti-angiogenic agent is selected from the group consisting of: angiostatin Kl-3, DL-α-difluoromethyl-ornithine, endostatin, fumagiline, genistein, minocycline, staurosporine, (+)-thalidomide, and any combination thereof.

[0055] In certain embodiments, the enzyme inhibitor is selected from the group consisting of: S(+)-camptothecin, curcumin, (-)-deguerin, 5,6-dichlorobenzo-imidazole I-β-D-ribofuranoside, etoposide, formestan, fosaliesin, hispidin, 2-imino-1-imidazolidinedacetate, mebinolin, trichostatin A, tilhostin AG34, tilhostin AG879, and any combination thereof.

[0056] In certain embodiments, the gene regulatory factor is: 5-aza-2'-deoxycytidine, 5 - Selected from the group consisting of azacitidine, cholecalciferol, 4-hydroxytamoxifen, melatonin, mifepristone, raloxifen, trans-retinal, retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol, tamoxifen, troglitazone, and any combination thereof.

[0057] In a particular embodiment, a method for producing an antigen-binding protein or a fragment thereof is used, which involves antibody heavy chain variable (V H ) further comprises a domain. In a particular embodiment, a method for producing an antigen-binding protein or a fragment thereof is an antibody light chain variable (V L ) Includes further domains.

[0058] In certain embodiments, the antigen-binding protein is a chimeric or humanized antibody. In certain embodiments, the antigen-binding protein is a human antibody. In certain embodiments, the antigen-binding protein is a monoclonal antibody.

[0059] In certain embodiments, the antigen-binding protein comprises one or more full-length antibody heavy chains including an Fc region. In certain embodiments, the Fc region is a human IgG1 Fc region.

[0060] In another embodiment, the present disclosure provides a pharmaceutical composition comprising any antigen-binding protein or fragment thereof disclosed herein, further comprising a pharmaceutically acceptable carrier.

[0061] In another embodiment, the Disclosure provides a method for treating a disease or disorder in a subject, comprising the step of administering to a subject in need of such treatment any antigen-binding protein or fragment thereof, or any pharmaceutical composition disclosed herein.

[0062] In another embodiment, the present disclosure provides isolated nucleic acid molecules encoding any antigen-binding protein or fragment thereof as disclosed herein.

[0063] In another embodiment, the present disclosure provides an expression vector comprising nucleic acid molecules disclosed herein.

[0064] In another embodiment, the present disclosure provides a host cell comprising an expression vector disclosed herein.

[0065] The aforementioned and other features and advantages of the present invention will be better understood from the following detailed description of exemplary embodiments in conjunction with the accompanying drawings. The patent or application file contains at least one drawing prepared in color. A copy of this patent or patent application publication containing the color drawing will be provided by the Patent Office upon request and payment of the required fees. [Brief explanation of the drawing]

[0066] [Figure 1] This figure shows the single-modified parental antibody purified by SDS-PAGE. A single-modified parental antibody (mutant) with a cysteine ​​mutation at position 1 was added at 4 μg protein / row to a 4-12% non-reduced tris-glycine SDS-PAGE gel. [Figure 2]This figure shows nanoDSF measurements of single-modified parent antibodies. NanoDSF (differential scanning fluorescence) measurements were performed to assess the stability of single-modified parent antibodies (mutants) with a cysteine ​​mutation at position 1. Two melting points (Tm1 and Tm2) corresponding to the melting temperature at which 50% of the two spherical regions of the antibody unfolded were measured. Modified antibodies with a Tm1 more than 5°C lower than the unmodified antibody control are shown in bold. All modified antibodies were comparable to or slightly more stable than the control in terms of Tm2. [Figure 3] This figure shows the screening of single-modified parental antibodies using DTT. Single-modified parental antibodies (mutants) with a cysteine ​​mutation at position 1 were screened with a DTT reducing agent by adding them to a PEG-stained SDS-PAGE containing 64 equivalents of DTT for cap removal. Each modified antibody shown was screened using both reducing and non-reducing conditions. [Figure 4] This figure shows a comparison of single-modified parental antibodies using a heatmap. Single-modified parental antibodies (mutants) with a cysteine ​​mutation at position 1 were analyzed by Coomassi staining based on four criteria: % monoPEGylation, % multiPEGylation, PAR (ratio of PEG to antibody), and % unPEGylation. The shades (columns) for each criterion are as follows: Mono-PEGylation: >60% to <100% - light gray, >30% to <59.9% - no color, >5% to <29.9% - dark gray; Multi-PEGylation: >0% to <15% - light gray, >15.1% to <20% - no color, >20.1% to <100% - dark gray; PAR: >1.7 to <2.5 - light gray, >1 to <1.69 - no color, >0 to <0.99 - dark gray; UnPEGylation: >0% to <15% - light gray, >15.1% to <20% - no color, >20.1% to <100% - dark gray. [Figure 5] This figure shows the selected top single-modified parental antibodies. The selection of single-modified parental antibodies (mutants) with a cysteine ​​mutation at position 1 was based on four criteria measured by Coomersie staining: ≥60% monoPEGylation, ≤20% multiPEGylation, PAR (ratio of PEG to antibody) ≥1.7, and ≤20% unPEGylation. [Figure 6] This figure shows the top 10 single-operated parental antibodies selected. Antibodies at primary positions K274C, K290C, A339C, and K360C are shown in bold, while those at primary positions N384C, G385C, Q418C, V422C, S440C, and S442C are shown in italics. [Figure 7] This figure shows dual-mutated antibodies purified by SDS-PAGE. Dual-mutated antibodies (mutants) having a cysteine ​​mutation at the first position and a cysteine ​​mutation at the second position were added to an SDS-PAGE gel under reducing and non-reducing conditions. [Figure 8] This figure shows nanoDSF measurements of double-modified antibodies. NanoDSF (differential scanning fluorescence) measurements were performed to measure the stability of double-modified antibodies (mutants) having cysteine ​​mutations at the first and second positions. Two melting points (Tm1 and Tm2) corresponding to the melting temperature at which 50% of the two spherical regions of the antibody were unfolded were measured. Modified antibodies with Tm1 and Tm2 more than 2°C lower (bold) or more than 2°C higher (underlined and italic) than the unmodified antibody control are shown. [Figure 9] This figure shows the PEGylation screening of dual-engineered antibodies. Dual-engineered antibodies (mutants) having a cysteine ​​mutation at the first position and a cysteine ​​mutation at the second position were conjugated with PEG and then screened with a DTT reducing agent by adding them to a PEG-stained SDS-PAGE containing 64 equivalents of DTT for cap removal. Each shown mutant antibody was screened using reducing and non-reducing conditions to determine its PEG-to-antibody ratio (PAR). [Figure 10] This figure shows the PEGylation efficiency / selectivity of double-engineered antibodies. Double-engineered antibodies (mutants) with a cysteine ​​mutation at the first position and a cysteine ​​mutation at the second position were evaluated by Coomassi staining for four criteria: PAR, % mono- and di-PEGylation, % multi-PEGylation, and % unPEGylation. [Figure 11]This figure shows the top 19 dual-engineered antibodies selected. The selection of dual-engineered antibodies (mutants) having a cysteine ​​mutation at the first position and a cysteine ​​mutation at the second position was based on four criteria measured by Coomersie staining: ≥80% mono- and di-PEGylation, ≤10% multi-PEGylation, PAR ≥3.4, and ≤5% unPEGylation. [Figure 12] This figure shows the selected superior double-manipulated antibody with the first position being K274C. [Figure 13] This figure shows the selected superior double-operated antibody whose first position is A339C. [Figure 14] This figure shows the selected superior double-operated antibody whose first position is A118C. [Figure 15] This figure shows the screening of single-modified parental antibodies using the reducing agent tris(2-carboxyethyl)phosphine (TCEP). Single-modified parental antibodies (mutants) with a cysteine ​​mutation at position 1 were screened with TCEP by adding them to PEG-stained SDS-PAGE. Each modified antibody shown was screened under both reducing and non-reducing conditions. [Figure 16-1] This figure shows a comparison of parent antibodies that were TCEP-treated and DTT-treated (single-process). The comparison was performed by measurement using Coomassi staining under four criteria: mono-PEGylated, multi-PEGylated, PAR (ratio of PEG to antibody), and unPEGylated. [Figure 16-2] Continuation of Figure 16-1. [Figure 17]This figure shows a comparison of single-modified parental antibodies using a heatmap of TCEP-treated antibodies. Single-modified parental antibodies (mutants) with a cysteine ​​mutation at position 1 were analyzed by Coomassi staining based on four criteria: % mono-PEGylation, % multi-PEGylation, PAR (ratio of PEG to antibody), and % unPEGylation. The shades (columns) for each criterion are as follows: Mono-PEGylation: >60% to <100% - light gray, >30% to <59.9% - no color, >5% to <29.9% - dark gray; Multi-PEGylation: >0% to <15% - light gray, >15.1% to <20% - no color, >20.1% to <100% - dark gray; PAR: >1.7 to <2.5 - light gray, >1 to <1.69 - no color, >0 to <0.99 - dark gray; UnPEGylation: >0% to <15% - light gray, >15.1% to <20% - no color, >20.1% to <100% - dark gray. [Figure 18] This figure shows three single-modified parent antibody sites used to test the effects on conjugation and FcγRIIIa binding. The sites tested were K290, Q295, and S442. [Figure 19] This figure shows dynamic sensorgrams demonstrating the binding of FcγRIIIa to each of the single manipulated parent antibody sites used for conjugation. The sites tested were K290, Q295, and S442. Each site was conjugated with either 2kDa maleimide, PEG, or biotin. [Figure 20] This figure shows SDS-PAGE and PEG staining of PEG-conjugated single-cysteine ​​manipulated parent antibodies. Twenty-seven PEGylated single-cysteine ​​manipulated parent antibodies (13 μg each) were analyzed using 4–12% Bis-Tris NuPAGE under reducing conditions. The gels were stained using PEG staining. Similarly PEGylated wild-type antibodies (WT) were used as a control. PageRuler-stained protein ladders were used as protein molecular weight standards (MWStd). [Figure 21]This figure shows a Western blot of a PEGylated antibody sample (0.1 μg) incubated in mouse plasma containing an anti-PEG antibody. As indicated by the arrows, the mono-PEGylated antibody band was analyzed using AlphaView software to determine the amount of PEG remaining after 96 hours of incubation in plasma. The diffused band above the mono-PEGylated antibody band is a multi-PEGylated species that reacts strongly to the anti-PEG antibody because it contains more PEG. [Figure 22] This figure shows the quantification of Western blots using anti-PEG antibodies against PEGylated monocysteine ​​mutants. PEGylated antibodies were incubated in mouse plasma for 0 and 96 hours, and then analyzed using Western blots with anti-PEG antibodies. The percentage at 0 hours represents the band area of ​​the monoPEGylated antibody band in the 96-hour sample divided by the band area of ​​the 0-hour band, multiplied by 100. [Figure 23] This figure shows SDS-PAGE and PEG staining of two monocysteine ​​mutants, N297C and S298C, after PEGylation. The two monocysteine ​​mutants, N297C and S298C, were PEGylated and analyzed using 4–12% Bis-Tris NuPAGE under non-reducing and reducing conditions. The gels were stained using PEG staining. Under non-reducing conditions, a significant amount of half-antibody conjugate was detected in the gel. PageRuler-stained protein ladders were used as protein molecular weight standards (MWStd). [Figure 24]This figure shows the screening of PEGylation of dual-engineered antibodies. Dual-engineered antibodies (mutants) with a cysteine ​​mutation at the first position and a cysteine ​​mutation at the second position were conjugated with PEG and then screened with a DTT reducing agent by adding them to PEG-stained SDS-PAGE containing 64 equivalents of DTT for cap removal. Each shown mutant antibody was screened using reducing and non-reducing conditions to determine the ratio of PEG to the antibody (PAR). One of the mutants, A118C+A339C (highlighted in bold and underlined), showed insufficient expression and a diffused band after PEGylation. Rearrangement revealed a sequence mismatch. As shown, the original mutant clone with the correct sequence was reexpressed and PEGylated. [Figure 25] This figure shows a heatmap of PEGylated double cysteine ​​mutants. Different colors represent the range of PAR, mono- and di-PEGylated, multi-PEGylated, and unPEGylated species. PAR: >3.4 green <4; >2.5 yellow <3.3; >0 red <2.4. Mono- and di-PEGylated: >85% green <100%; >30% yellow <84.9%; >5% red <29.9%. Multi-PEGylated: <10% green >0%; >10.1% yellow <20%; >20.1% red <100%. UnPEGylated: <6% green >0%; >6.1% yellow <10%; >10.1% red <100%. Selectivity: >0.7 green <1.0, >0.51 yellow <0.69, >0 red <0.5. [Modes for carrying out the invention]

[0067] The inventors disclose engineered antibodies that are stable and conjugate to ligands or drugs in ligand / drug-to-antibody ratios greater than 3, making these conjugates suitable for the treatment of various indications. Methods for producing these engineered antibodies are also provided.

[0068] In general, the nomenclature used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization is well known and commonly used in the art. The methods and techniques provided herein are usually carried out in accordance with conventional methods well known in the art, and unless otherwise indicated, they are based on various general methods referenced and considered throughout this specification. and as described in more specific references. Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications, as is commonly practiced in the art or as described herein. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, as well as their experimental procedures and techniques, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical formulation, prescription, and delivery, as well as for patient treatment.

[0069] Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. Where there is a potentially ambiguous matter, the definitions provided herein take precedence over dictionary or external definitions. Unless otherwise required by context, singular forms include plurals and plural forms include singulars. The use of "or" means "and / or" unless otherwise stated. This is not to limit the use of other forms of terms such as "including" and "includes" and "included".

[0070] Certain terms are defined first so that the present invention may be more easily understood.

[0071] As used herein, the terms “antigen-binding protein” or “antibody” refer to immunoglobulin molecules that specifically bind to an antigen or epitope, or are immunologically reactive, including both polyclonal and monoclonal antibodies, as well as functional antibody fragments, including but not limited to antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term “antibody” includes genetically engineered or other modified forms of immunoglobulins (e.g., intrabody, peptibody, chimeric antibody, fully human antibody, humanized antibody, medotope-capable antibody), heteroconjugate antibodies (e.g., bispecific antibody, diabody, triabody, tetrabody, tandem di-scFv, tandem tri-scFv), etc. Unless otherwise stated, the term “antibody” should be understood to encompass its functional antibody fragments.

[0072] As used herein, the terms “parent antigen-binding protein” or “unmanipulated antigen-binding protein” refer to an antibody that can be manipulated by altering one or more of its amino acid residues in order to produce a derived “manipulated antigen-binding protein” or “manipulated antibody.” Such a manipulated antigen-binding protein / antibody derived from a parent antigen-binding protein / antibody may be a “single-manipulated antigen-binding protein,” in which only one amino acid residue is replaced with a different manipulated amino acid residue. A manipulated antigen-binding protein / antibody derived from a parent antigen-binding protein / antibody may also be a “double-manipulated antigen-binding protein,” in which an amino acid residue at a first position is replaced with a different manipulated amino acid residue and an amino acid residue at a second position is replaced with a different manipulated amino acid residue.

[0073] As used herein, the term “single-engineered parent antigen-binding protein” refers to a parent antigen-binding protein that is modified at a first position with an engineered amino acid residue and can be further engineered by changing an amino acid residue at a second position in order to produce a derived “double-engineered antigen-binding protein.”

[0074] As used herein, the term "reactive amino acid residue" refers to an amino acid having a reactive side chain. Reactive amino acid residues include cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, and arginine. It can be done.

[0075] As used herein, the term "manipulated amino acid residue" refers to an amino acid residue in an antigen-binding protein that has been substituted with a different amino acid residue. As used herein, the term "manipulated reactive amino acid residue" refers to an amino acid residue in an antigen-binding protein that has been substituted with a different amino acid residue that is a reactive amino acid residue.

[0076] As used herein, the term "ligand" refers to a molecule or atom covalently linked to a reactive amino acid residue of an antigen-binding protein, either directly, through a linker, or through several other reactive moieties. Ligands include, but are not limited to, polypeptides, carbohydrates, radionuclides, lipids, nucleic acids, synthetic compounds, and small molecules.

[0077] As used herein, the terms “conjugate,” “conjugated,” and “conjugation” refer to a covalent bond between two or more chemical compounds.

[0078] As used herein, the terms “reactive moiety” and “reactive group” refer to specific substituents or parts within a molecule that cause characteristic chemical reactions. The same reactive moiety undergoes the same or similar chemical reactions regardless of the size of the molecules that make up part of it.

[0079] As used herein, the term “linker” refers to a chemical portion of a polypeptide that includes a chain of covalent bonds or atoms covalently attaches to a ligand, a drug, a ligand-to-ligand, or a ligand-to-drug. The linker may be a “cleavable linker” that facilitates the release of a cytotoxic agent or growth inhibitor in a cell. For example, acid-unstable linkers, peptidase-sensitive linkers, esterase-unstable linkers, photosensitive linkers, or disulfide-containing linkers (see, for example, U.S. Patent No. 5,208,020) may be used. The linker may also be a “non-cleavable linker” (e.g., an SMCC linker) which in some cases may result in good resistance.

[0080] As used herein, the terms "ligand-to-antibody ratio" or "LAR" refer to the stoichiometric ratio of the number of ligand molecules bound to one antibody. Similarly, the terms "drug-to-antibody ratio" or "DAR" refer to the stoichiometric ratio of the number of drug molecules bound to one antibody. The terms "PEG-to-antibody ratio" or "PAR" refer to the stoichiometric ratio of the number of PEG molecules bound to one antibody.

[0081] As used herein, the term "detection probe" refers to ligands, including but not limited to biotin, polyethylene glycol (PEG), fluorescent tags, visualization peptides, and combinations thereof.

[0082] As used herein, the terms "PEG" or "polyethylene glycol" refer to polyether compounds having a structure commonly represented as H-(O-CH2-CH2)n-OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight.

[0083] As used herein, the term “targeting moiety” refers to a ligand that can act to direct an antibody to be conjugated to a specific target. Examples of such targets include, but are not limited to, cell membranes and cancer cells.

[0084] As used herein, the term "drug" refers to a molecule that, when administered to an organism, causes a change in the organism's physiology or psychology. Drugs include: anti-inflammatory, anti-cancer, anti-infective (e.g., anti- These include, but are not limited to, fungal, antibacterial, antiparasitic, and antiviral agents, as well as anesthetic therapeutic agents. The drugs may also be anticancer or cytotoxic agents or prodrugs.

[0085] As used herein, the term “prodrug” refers to a pharmaceutically active agent in the form of a precursor or derivative that has less activity, reactivity, or side effects compared to the parent drug, and has the ability to be enzymatically activated in vivo or otherwise converted to a more active form.

[0086] As used herein, the terms “anti-cancer agent” or “anti-cancer agent” refer to molecules that are harmful to the growth and / or proliferation of neoplasms, tumors or cancer cells, and that may act to reduce, inhibit or destroy malignant tumors.

[0087] As used herein, the term "cell division inhibitor" refers to a molecule that inhibits cell growth and duplication.

[0088] As used herein, the term "cytotoxic nucleoside" refers to a nucleic acid base or nucleoside analog that exerts a cytotoxic effect by mimicking endogenous nucleosides.

[0089] As used herein, the term "tubulin binder" refers to a molecule that directly associates with a tubulin system.

[0090] As used herein, the term “hormone” refers to any member of the class of signaling molecules produced by glands in multicellular organisms, transported by the circulatory system, and targeting distant organs to regulate physiological and / or behavioral processes. “Hormone antagonist” is a specific type of receptor antagonist that acts on hormone receptors.

[0091] As used herein, the term "anti-angiogenic agent" refers to a molecule that inhibits the physiological process of angiogenesis, in which new blood vessels are formed from existing vessels.

[0092] As used herein, the term "enzyme inhibitor" refers to a molecule that inhibits the function of a specific enzyme.

[0093] As used herein, the term "gene regulatory factor" refers to a molecule that can positively or negatively influence gene transcription.

[0094] As used herein, the term "monoclonal antibody" refers to antibodies produced by identical immune cells, which are clones of a unique parent cell. Monoclonal antibodies can have monovalent affinity in that they bind to the same epitope.

[0095] As used herein, the term "library" refers to a defined set of members. When a library refers to an antigen-binding protein, it refers to a defined set of antigen-binding proteins. When a library refers to an amino acid position, it refers to a defined set of amino acid positions within an antigen-binding protein.

[0096] As used herein, the term “expression vector” refers to a vehicle through which a DNA or RNA sequence (e.g., an exogenous gene) is introduced into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. Such vectors may include regulatory elements, such as promoters, enhancers, and terminators, which, upon administration to a subject, induce or direct the expression of the polypeptide. Examples of promoters and enhancers used in expression vectors for animal cells include the early SV40 LTR promoters and enhancers of Moloney's mouse leukemia virus (Mizukami T. et al. 1987), LTR promoters and enhancers of Moloney's mouse leukemia virus (Kuwana Y et al. 1987), promoters of immunoglobulin H chains (Mason including, for example, J O et al. (1985) and enhancer (Gillies S D et al. 1983).

[0097] As used herein, the term "domain" generally refers to any region of a protein that is defined based on sequence homology and is often associated with a particular structural or functional entity.

[0098] As used herein, the phrase "chimeric antibody" in the broadest sense refers to an engineered antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies. In one embodiment, the chimeric antibody is the C of another antibody, in one embodiment a human antibody H domain and C L domain associated with the V of an antibody derived from a non-human animal H domain and VL domain. Any animal, such as a mouse, rat, hamster, rabbit, etc., can be used as the non-human animal. The chimeric antibody can also exhibit a multispecific antibody having specificity for at least two different antigens.

[0099] As used herein, the phrase "humanized antibody" refers to an antibody that is wholly or partially of non-human origin and is modified such that certain amino acids (e.g., the framework regions of the V H and V L domains) are replaced to avoid or minimize the immune response in humans. The constant domains of humanized antibodies are generally human C H and C L domains.

[0100] As used herein, the term "fragment" refers to an intact antibody, particularly a portion or domain of the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fv, Fab, F(ab′)2, Fab′, dsFv, (dsFv)2, scFv, sc(Fv)2, diabodies, bispecific and multispecific antibodies formed from antibody fragments. Fragments of conventional antibodies may also be single-domain antibodies, such as heavy-chain antibodies or VHHs.

[0101] As used herein, the term “cytotoxic therapeutic agent” refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. The term “cytotoxic agent” is intended to include chemotherapeutic agents, enzymes, antibiotics, and toxins, e.g., small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin (including their fragments and / or variants), and various antitumor or anticancer agents disclosed below. In some embodiments, cytotoxic agents are taxoids, vinca, mytansinoids or mytansinoid analogs, e.g., DM1 or DM4, small drugs, leptomycin derivatives, auristatin or drastatin analogs, prodrugs, topoisomerase II inhibitors, DNA alkylating agents, antitubulin agents, CC-1065 or CC-1065 analogs.

[0102] As used herein, the term “pharmaceutically acceptable carrier” refers to any physiologically compatible solvent, dispersion medium, coating agent, antimicrobial and antifungal agent, etc. Examples of suitable carriers, diluents and / or excipients include water, amino acids, saline, phosphate-buffered saline, phosphate buffer, acetate buffer, citrate buffer, succinate buffer; amino acids and derivatives, e.g., histidine, arginine, glycine, proline, glycylglycine; inorganic salts NaCl, calcium chloride; sugars or polyalcohols, e.g., dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants, e.g., polysorbate 80, polysorbate 20, poloxamer 188; and one or more combinations thereof. Often, isotonic agents, e.g., sugars, are included in the composition. The formulation may preferably contain a polyalcohol or sodium chloride, and may also contain an antioxidant, such as tryptamine, and a stabilizer, such as Tween 20.

[0103] As used herein, the term “to treat” means to reverse, alleviate, inhibit the progression of, or prevent a disease, disorder, or condition, or one or more symptoms of such disease, disorder, or condition. For example, treating cancer means inhibiting the growth of malignant cells of a tumor and / or the progression of metastases from said tumor. Such treatment can also result in regression of tumor growth, i.e., a reduction in the measurable size of the tumor. In particular, such treatment can result in the complete regression of the tumor or metastases.

[0104] A. Antigen-binding protein or antibody As will be discussed in more detail below, the collective term “antigen-binding protein” or “antibody” includes five distinct classes of antibodies that can be biochemically distinguished. While all five classes of antibodies are clearly within the scope of this disclosure, the following discussion will generally focus on immunoglobulin molecules of the IgG class. With respect to IgG, immunoglobulins consist of two identical light chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with molecular weights of 53,000 to 70,000. The four chains are joined by disulfide bonds in a “Y” configuration, with the light chains attached to the heavy chains, starting at the mouth portion of the “Y” and continuing through a variable region.

[0105] The light chains of immunoglobulins are classified as either kappa or lambda (κ, λ).

[0106] Each heavy chain class can be bound to either a kappa or lambda light chain. Generally, the light and heavy chains are covalently linked to each other, with the "tails" of the two heavy chains being linked to each other by covalent disulfide linkages or non-covalent linkages (when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells). In the heavy chain, the amino acid sequence extends from the N-terminus to the C-terminus at the bottom of each chain, with a fork-like Y-configuration. As is obvious to those skilled in the art, the heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) (with several subclasses within them (e.g., γ1~γ4)). The properties of this chain determine the "class" of the antibody, which is IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well-characterized and known to confer functional specialization. Modified versions of each of these classes and isotypes are readily recognizable to those skilled in the art in light of this disclosure and are therefore within the scope of this disclosure.

[0107] Both light and heavy chains are divided into regions of structural and functional homology. The term “region” refers to a part or portion of an immunoglobulin or antibody chain, including constant or variable regions, as well as several further distinct parts or portions of such regions. For example, a light chain variable region includes “complementarity-determining regions” or “CDRs” incorporated between “framework regions” or “FRs” as defined herein.

[0108] Regions of immunoglobulin heavy or light chains can be defined as "constant" (C) regions or "variable" (V) regions based on whether there is relatively little sequence variation within the regions of various class members in the case of "constant regions," or significant variation within the regions of various class members in the case of "variable regions." The terms "constant region" and "variable region" can also be used functionally. In this context, the variable region of an immunoglobulin or antibody naturally determines antigen recognition and specificity. Conversely, the constant region of an immunoglobulin or antibody confers important effector functions such as secretion, transplacental migration, Fc receptor binding, and complement binding. The subunit structure and three-dimensional arrangement of the constant region of various immunoglobulin classes are well known.

[0109] The constant and variable regions of immunoglobulin heavy and light chains fold to form domains. The term "domain" refers to a spherical region of a heavy or light chain that contains, for example, β-pleated sheets and / or peptide loops stabilized by intrachain disulfide bonds (e.g., containing 3-4 peptide loops). The constant region domain of an immunoglobulin light chain is interchangeably referred to as the "light chain constant region domain" or "C L "domain" or "C L These are called "domains." These are the constant domains of heavy chains (e.g., hinges, C). H 1. C H 2 or C H The 3 domains are interchangeable: "heavy chain constant region domain", "C H "domain" or "C H It is called a "domain". The variable domain of the light chain is interchangeable with the "light chain variable domain", "V L"domain" or "V L It is called a "domain".

[0110] The heavy chain variable domain is interchangeable with the "heavy chain variable domain", "V H "domain" or "V H It is called a "domain".

[0111] By convention, the numbering of variable constant region domains increases as they are further away from the antigen-binding site or the amino terminus of the immunoglobulin or antibody. The N-terminus of each heavy and light immunoglobulin chain is the variable region, and the C-terminus is the constant region. H 3 and C L The domains actually contain the carboxyl termini of the heavy chain and light chain, respectively. Therefore, the domains of light chain immunoglobulins are V L -C L Although arranged in orientation, the heavy chain domain is VH-CH1-hinge-C H 2-C H They are arranged in a 3-way orientation.

[0112] C H 1. Hinge, C H 2, C H 3, and C L The amino acid positions in the heavy chain constant region, including the amino acid positions within the domain, may be numbered according to the Kabat index numbering system (see Kabat et al, in “Sequences of Proteins of Immunological Interest,” US Dept. Health and Human Services, 5th edition, 1991). Alternatively, the amino acid positions of the antibody may be numbered according to the EU index numbering system (see Kabat et al, ibid.). The term "V" as used herein H The term "domain" includes the amino-terminal variable domain of the immunoglobulin heavy chain, and the term "V" L The "domain" includes the amino-terminal variable domain of the immunoglobulin light chain.

[0113] The term "C" as used in this specification H "Domain 1" includes, for example, the first (most amino-terminal) constant region domain of the immunoglobulin heavy chain extending from approximately position 114-223 (EU position 118-215) in the Kabat numbering system. H 1 domain is V H It is adjacent to the domain, located at the amino terminus relative to the hinge region of the immunoglobulin heavy chain molecule, and does not form part of the Fc region of the immunoglobulin heavy chain.

[0114] As used herein, the term "hinge region" refers to C H 1 domain C H It contains a heavy chain molecule that connects the two domains. This hinge region contains approximately 25 residues and is mobile, thus allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle, and lower hinge domains (Roux et al. J. Immunol. 1998, 161:4083).

[0115] The term "C" as used in this specification H The "2 domains" include, for example, a portion of a heavy-chain immunoglobulin molecule that extends approximately from position 244 to 360 (EU position 231 to 340) in the Kabat numbering system. H The two domains are unique in that they are not closely paired with the other domain. Rather, the two N-linked branched carbohydrate chains are the two C-linked chains of an intact natural IgG molecule. H It is inserted between the two domains. In one embodiment, the antigen-binding protein of this disclosure is C derived from IgG1 molecule (e.g., human IgG1 molecule) H Includes 2 domains.

[0116] The term "C" as used in this specification H "3 domains" is C HThis includes a portion of the heavy-chain immunoglobulin molecule extending approximately 110 residues from the N-terminus of the two domains, for example, approximately positions 361-476 (EU positions 341-445) in the Kabat numbering system. H The three domains typically form the C-terminal portion of an antibody. However, in some immunoglobulins, an additional domain is present in the C-terminal region. H3 It can extend from the domain and form the C-terminal portion of the molecule (for example, the C in the μ-chain of IgM and the ε-chain of IgE). H (4 domains). In one embodiment, the antigen-binding protein of the present disclosure is derived from an IgG1 molecule (e.g., a human IgG1 molecule) C H Includes 3 domains. The term "C" is used herein. L The "domain" includes, for example, the constant region domain of the immunoglobulin light chain extending from approximately Kabat positions 107A to 216. L The domain is V L Adjacent to the domain. In one embodiment, the antigen-binding protein of the present disclosure is derived from a kappa light chain (e.g., human kappa light chain) C L Includes the domain.

[0117] As used herein, the term “Fc region” is defined as the portion of the heavy chain constant region that begins in the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 of IgG, with the first residue of the heavy chain constant region being 114) and ends at the C-terminus of the antibody. Thus, the complete Fc region is defined as at least the hinge domain, C H 2 domains, and C H Includes 3 domains.

[0118] As used herein, the term “natural Fc” refers to a molecule comprising a sequence of non-antigen-binding fragments, which may contain a hinge region, and which are produced by the digestion of an antibody or by other means, and which may be in monomeric or multimeric form. The original immunoglobulin source of natural Fc may be of human origin and may be any immunoglobulin, such as IgG1 or IgG2.

[0119] Natural Fc molecules consist of monomer polypeptides that can be linked by covalent (i.e., disulfide bonds) and non-covalent associations to form dimers or multimers. The number of intermolecular disulfide bonds between monomer subunits in natural Fc molecules ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of natural Fc is a disulfide-bonded dimer resulting from the papain digestion of IgG. As used herein, the term “natural Fc” is a general term encompassing monomer, dimer, and multimer forms.

[0120] As used herein, the term “Fc variant” refers to a molecule or sequence that has been modified from native Fc but still contains a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Examples of Fc variants and their interactions with salvage receptors are known in the art. Therefore, the term “Fc variant” may include molecules or sequences that have been humanized from non-human native Fc. Furthermore, native Fc contains regions that can be removed because they result in structural features or biological activity not required for antibody-like antigen-binding proteins. Therefore, the term “Fc variant” includes molecules or sequences lacking one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity in expression in selected host cells, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC), or molecules or sequences in which one or more Fc sites or residues have been modified. As used herein, the term “Fc domain” encompasses native Fc and Fc variants and sequences as defined above. As with Fc variants and native Fc molecules, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from the whole antibody or produced by other means.

[0121] As shown above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the V of the antibody L Domain and V H The domain is formed by the combination of variable regions (Fv) that define a three-dimensional antigen-binding site. This quaternary antibody structure forms antigen-binding sites located at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three complementarity-determining regions (CDRs) in each of the variable regions of the heavy and light chains. As used herein, the term “antigen-binding site” includes a site that specifically binds to (immunely reacts to) an antigen (e.g., a cell surface or soluble antigen). The antigen-binding site includes the variable regions of the immunoglobulin heavy and light chains, and the binding site formed by these variable regions determines the specificity of the antibody. The antigen-binding site is formed by variable regions that vary from antibody to antibody. The modified antibodies in this disclosure include at least one antigen-binding site.

[0122] In certain embodiments, the antigen-binding proteins of this disclosure include at least two antigen-binding domains that provide association of the antigen-binding protein with a selected antigen. The antigen-binding domains do not necessarily have to be derived from the same immunoglobulin molecule. In this context, the variable region may be derived from or originates from any type of animal that may be introduced to produce an immunoglobulin against a desired antigen and possess a humoral response. As such, the variable region of an antigen-binding protein may be of mammalian origin, e.g., human, mouse, rat, goat, sheep, non-human primates (e.g., cynomolgus monkey, macaque monkey, etc.), wolf (lupine), or camelid (e.g., camel, llama, and related species). In naturally occurring antibodies, assuming that the antibody takes its three-dimensional configuration in an aqueous environment, the six CDRs present in each monomer antibody are short, discontinuous sequences of amino acids specifically positioned to form an antigen-binding site. The remainder of the heavy and light chain variable domains exhibit lower intermolecular variability in the amino acid sequence and are referred to as the framework region. The framework regions largely adopt a β-sheet structure, and the CDRs connect these β-sheet structures, forming loops that in some cases form part of the β-sheet structure. Thus, these framework regions act to form a scaffold that provides the correct orientation for positioning the six CDRs through interchain non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines surface complementarity to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of antibodies to the immunoreactive antigen epitope.

[0123] Examples of antigen-binding proteins include antibody variants. As used herein, the term “antibody variant” includes synthetic and manipulated forms of antibodies that have been modified to not exist in nature, e.g., antibodies containing at least two heavy chain moieties but not two full heavy chains (e.g., domain deletion antibodies or minibodies); multispecific forms of antibodies modified to bind to two or more different antigens or to different epitopes on a single antigen (e.g., bispecific, tripspecific, etc.); and heavy chain molecules conjugated to scFv molecules, etc. Furthermore, the term “antibody variant” also includes multivalent forms of antibodies (e.g., trivalent, tetravalent, etc., antibodies that bind to three, four or more copies of the same antigen).

[0124] As used herein, the term “bindance” refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to one target molecule or a specific site on a target molecule. If a polypeptide contains more than one target binding site, each target binding site may specifically bind to the same or different molecules (e.g., different ligands or different antigens, or different epitopes on the same antigen). Antigen-binding proteins of the subject typically have at least one binding site specific to a human antigen molecule.

[0125] The term "specificity" refers to the ability to specifically bind to a given target antigen (e.g., a human target antigen) (e.g., to trigger an immune response). Antigen-binding proteins can be single-specific and target A polypeptide may contain one or more binding sites that bind specifically, or the polypeptide may be multispecific and may contain two or more binding sites that bind specifically to the same or different targets. In certain embodiments, an antigen-binding protein is specific to two different (e.g., non-overlapping) portions of the same target. In certain embodiments, an antigen-binding protein is specific to one or more targets. Antigen-binding proteins (e.g., antibodies) that include antigen-binding sites that bind to antigens expressed on tumor cells are known in the art, and one or more CDRs from such antibodies may be included in the antibodies described herein.

[0126] Sometimes, residues derived from non-hypervariable or framework regions (FRs) affect the overall domain structure, and therefore the binding site. Thus, complementarity-determining regions, or CDRs, refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. Each of the light and heavy chains of an immunoglobulin has three CDRs, named CDR1-L, CDR2-L, CDR3-L, and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, a conventional antibody-antigen binding site contains six CDRs, including sets of CDRs derived from each of the heavy and light chain V regions.

[0127] A.1. Methods for producing antibodies The antibodies of the present invention can be produced by any technique known in the art, for example, any chemical, biological, genetic, or enzymatic technique, either alone or in combination, without limitation.

[0128] If the amino acid sequence of the desired sequence is known, those skilled in the art can readily produce the antibody or immunoglobulin chain by standard techniques for polypeptide production. For example, those skilled in the art can synthesize it using commercially available peptide synthesizers (e.g., those made by Applied Biosystems, Foster City, Calif.) and known solid-phase methods according to the manufacturer's instructions. Alternatively, the antibodies and immunoglobulin chains of the present invention can be synthesized by recombinant DNA techniques known in the art. For example, these fragments can be obtained as DNA expression products after the incorporation of the DNA sequence encoding the desired (poly)peptide into an expression vector, and the introduction of such a vector into a suitable eukaryotic or prokaryotic host expressing the desired polypeptide, from which they can be subsequently isolated using known techniques.

[0129] The present invention further relates to a method for producing the antibody of the present invention, the method comprising the steps of: (i) culturing host cells transformed according to the present invention; (ii) expressing the antibody or polypeptide; and (iii) recovering the expressed antibody or polypeptide. The antibody of the present invention can be appropriately separated from the culture medium by conventional immunoglobulin purification procedures, such as protein A Sepharose, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0130] The humanized chimeric antibody of the present invention is, as previously described, humanized V L and V H Obtaining nucleic acid sequences encoding the domain, human chimeric antibody expression vectors, and human antibody C H and human antibody C L It can be constructed by inserting the gene encoding it into an expression vector for animal cells, and produced by introducing the expression vector into animal cells to express the coding sequence. Human chimeric antibodies C HThe domain may be any region belonging to the human immunoglobulin heavy chain, but one of the IgG class is appropriate, and any one of the subclasses belonging to the IgG class, such as IgG1, IgG2, IgG3, and IgG4, may be used. Furthermore, the CL of the human chimeric antibody may be any region belonging to the human immunoglobulin light chain, and one of the kappa class or lambda class may be used.

[0131] Methods for producing humanized or chimeric antibodies include conventional recombinant DNA and gene transfection techniques well known in the art (see Morrison S L. et al. (1984) and U.S. Patents 5,202,238 and 5,204,244). Methods for producing humanized antibodies based on conventional recombinant DNA and gene transfection techniques are well known in the art (see, for example, Riechmann L. et al. 1988; Neuberger M S. et al. 1985). Antibodies can be humanized using various techniques known in the art, including, for example, techniques disclosed in patent application WO2009 / 032661, CDR grafting (EP239,400; PCT publication WO91 / 09967; U.S. Patents 5,225,539; 5,530,101; and 5,585,089), veneerization or resurfacing (EP592,106; EP 519,596; Padlan EA (1991); Studnicka GM et al. (1994); Roguska M A. et al. (1994)), and strand shuffling (U.S. Patent 5,565,332). Common recombinant DNA techniques for formulations of such antibodies are also known (see European Patent Application EP125023 and International Patent Application WO 96 / 02576).

[0132] Numerous methods for humanizing antibody sequences are publicly known in the field; see, for example, the review by Almagro & Fransson (2008) Front Biosci. 13: 1619-1633. One commonly used method is CDR grafting or antibody reshaping, which involves grafting the CDR sequence of a donor antibody (generally a mouse antibody) onto a framework scaffold of a human antibody of different specificity. Since CDR grafting can reduce the binding specificity and affinity (and therefore bioactivity) of the CDR-grafted non-human antibody, a reverse mutation may be introduced at a selected location in the CDR-grafted antibody to preserve the binding specificity and affinity of the parent antibody. Identification of possible reverse mutation sites can be carried out using information available in the literature and antibody databases. Candidate amino acid residues for reverse mutations are typically located on the surface of the antibody molecule, but embedded residues with a low degree of surface exposure are usually not modified. An alternative humanization technique to CDR grafting and revertant mutation is resurfacing, in which non-human non-surface-exposed residues are retained, but surface residues are replaced with human residues. Another alternative technique is "Guided Selection" (Jespers Known as et al. (1994) Biotechnology 12, 899), it is used to derive from mouse antibodies and fully human antibodies that preserve the epitope and binding properties of the parent antibody.

[0133] With respect to chimeric antibodies, humanization typically involves modification of the framework region of the variable region sequence. Amino acid residues that are part of the CDR are typically not altered in connection with humanization, but in certain cases, it is desirable to alter the CDR amino acid residues of an individual, for example, by removing glycosylation sites, deamide sites, or undesirable cysteine ​​residues. N-linked glycosylation occurs by the attachment of oligosaccharide chains to asparagine residues in the tripeptide sequences Asn-X-Ser or Asn-X-Thr (where X is any amino acid other than Pro). Removal of N-glycosylation sites is achieved by mutating the Asn or Ser / Thr residue to a different residue, for example, by means of conservative substitution. Deamide of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly sensitive to deamide when present primarily in the sequence Asn-Gly and to a lesser extent in other dipeptide sequences (e.g., Asn-Ala). Therefore, if such a deamide site, for example Asn-Gly, is present in a CDR sequence, it would be desirable to remove the site by removing one of the residues that are typically related by a conservative substitution. Substitutions in the CDR sequence that remove one of the residues are also intended to be included in the present invention.

[0134] Common expression systems include E. coli host cells and plasmid vectors, insect host cells and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) and primary or established mammalian cell cultures (e.g., those produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells (in which the dihydrofolate reductase gene (hereinafter referred to as the "DHFR gene") is deleted (Urlaub G et al; 1980)), and rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereafter referred to as "YB2 / 0 cells"). In one embodiment, YB2 / 0 cells are used because the ADCC activity of the chimeric or humanized antibody is enhanced when expressed in these cells.

[0135] A.2. Modified antibodies Modifications and alterations may be made to the structure and encoding DNA sequence of the antibody of the present invention, still resulting in a functional antibody or polypeptide with desirable characteristics. In altering the amino sequence of a polypeptide, the hydrophobic-hydrophilic index of amino acids may be considered. The importance of the hydrophobic-hydrophilic amino acid index in conferring reciprocal biological functions in proteins is generally understood in the art. It is accepted that the relative hydrophobic-hydrophilic traits of amino acids contribute to the resulting secondary structure of the protein, which in turn governs the interaction between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc. Each amino acid is assigned a hydrophobic-hydrophilic index based on its hydrophobic and charge characteristics, which are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0136] A further challenge of the present invention also encompasses functionally conserving variants of the polypeptide. For example, a particular amino acid may be substituted with another amino acid in the protein structure without a noticeable loss of activity. Since the interactivity and properties of a protein determine its biological activity, a particular amino acid substitution may be made in the protein sequence (of course, in its DNA coding sequence), but nevertheless, a protein with similar properties can be obtained simultaneously. Therefore, it is intended that various changes may be made in the antibody sequence of the present invention or the corresponding DNA sequence encoding the polypeptide without a noticeable loss of their biological activity.

[0137] It is well known in the art that substituting a specific amino acid with another amino acid having a similar hydrophobicity or hydrophilicity index or score can produce a protein that still has similar biological activity, i.e., a protein that is still biologically and functionally equivalent. Using well-established techniques, such as the alanine scanning approach, it is also possible to identify all amino acids that can be substituted in the antibody or polypeptide of the present invention without significantly losing antigen-binding ability. Such residues may be considered neutral because they do not participate in antigen binding or the maintenance of antibody structure. One or more of these neutral positions can be substituted with alanine or another amino acid without altering the main characteristics of the antibody or polypeptide of the present invention.

[0138] Therefore, as outlined above, amino acid substitutions are generally based on the relative similarities of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Substitutions in which any of the aforementioned features are considered are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine.

[0139] A.2.a. FC modification In certain embodiments, the antigen-binding protein may include an antibody constant region (e.g., an IgG constant region, e.g., a human IgG constant region, e.g., a human IgG1 or IgG4 constant region) that mediates one or more effector functions. For example, binding of the C1-complex to the antibody constant region may activate the complement system. Complement system activation is important for opsonization and lysis of cellular pathogens. Complement system activation may also stimulate inflammatory responses and may be involved in autoimmune hypersensitivity. Furthermore, antibodies bind to various cell receptors via their Fc region (the Fc receptor binding site of the antibody Fc region binds to the cell's Fc receptor (FcR). There are several Fc receptors specific to different classes of antibodies, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). The binding of antibodies to Fc receptors on the cell surface triggers several important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, transplacental transfer, and regulation of immunoglobulin production. In some embodiments, the antigen-binding protein characterized in the present invention (e.g., its antibody or antigen-binding fragment) binds to the Fc-gamma receptor. In alternative embodiments, the antigen-binding protein may include a constant region lacking one or more effector functions (e.g., ADCC activity) and / or unable to bind to the Fc-gamma receptor with respect to IgG.

[0140] In certain embodiments, the antibody includes one in which at least one amino acid in one or more constant region domains is deleted or otherwise modified to provide additional desired biochemical features, such as reduced or enhanced effector function, non-covalent dimerization ability, increased ability to localize to tumor sites, reduced serum half-life, or increased serum half-life, when compared to an unmodified antibody of substantially the same immunogenicity, or to an antibody containing only the modifications characterized in the present invention. For example, certain antibodies for use in the diagnostic and treatment methods described herein are domain deletion antibodies that contain a polypeptide chain similar to an immunoglobulin heavy chain but lack at least a portion of one or more heavy chain domains. For example, in certain antibodies, one entire domain of the constant region of the modified antibody is deleted, e.g., C H All or part of the two domains are deleted.

[0141] In certain other embodiments, the antigen-binding protein includes constant regions derived from different antibody isotypes (e.g., constant regions derived from two or more human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the antigen-binding protein includes a chimeric hinge (i.e., derived from hinge domains of different antibody isotypes). The hinge includes a hinge portion, for example, an upper hinge domain and an IgG1 intermediate hinge domain derived from an IgG4 molecule. In one embodiment, the antigen-binding protein includes a human IgG4 molecule and an Fc region or portion derived from the Ser228Pro mutation (EU numbering) in the core hinge region of the molecule.

[0142] In certain embodiments, the Fc moiety may be mutated to increase or decrease effector function using techniques known in the art. For example, deletion or inactivation of the constant region domain (by point mutation or other means) reduces Fc receptor binding of the circulating modified antibody, thereby increasing tumor localization. In other cases, constant region modifications consistent with the present invention may suppress complement binding, and therefore reduce the serum half-life and nonspecific association of the conjugated cytotoxin. Furthermore, other modifications of the constant region may be used to modify the disulfide linkage or oligosaccharide moiety, thereby enabling enhanced localization due to increased antigen specificity or mobility. The resulting physiological profiles, bioavailability, and other biochemical effects, such as tumor localization, in vivo distribution, and serum half-life, can be readily measured and quantified using well-known immunological techniques without excessive experimentation.

[0143] The Fc domain using the antibody characterized in the present invention is an Fc variant. As used herein, the term "Fc variant" refers to an Fc domain having at least one amino acid substitution compared to the wild-type Fc domain from which the Fc domain is derived. For example, here the Fc domain is derived from a human IgG1 antibody, and the Fc variant of the human IgG1 Fc domain contains at least one amino acid substitution compared to the Fc domain.

[0144] In addition to the variants characterized in the present invention, amino acid substitutions of Fc variants are located at any position within the Fc domain (i.e., any amino acid position of any EU convention). In one embodiment, the Fc variant includes additional substitutions at an amino acid position located in or part of the hinge domain. In another embodiment, the Fc variant is C H The 2 domain or part thereof includes additional substitutions at amino acid positions. In another embodiment, the Fc variant is C H 3. Includes additional substitutions at amino acid positions located in the domain or part thereof. In another embodiment, the Fc variant is CH This includes additional substitutions at amino acid positions located in the 4 domain or part thereof.

[0145] The antigen-binding protein may use any Fc variant known in the art to confer improvement (e.g., reduction or enhancement) to effector function and / or FcR binding. The aforementioned Fc variants include, for example, the International PCT publications WO88 / 07089A1, W096 / 14339A1, WO98 / 05787A1, W098 / 23289A1, W099 / 51642A1, W099 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351 A2, and WO04 / 074455 A2, WO04 / 099249A2, WO05 / 040217A2, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2, and WO06 / 085967A2 or US 5,648,260; 5,739,277; 5,834,250; 5 Includes any one of the amino acid substitutions disclosed in ,869,046;6,096,871;6,121,022;6,194,551;6,242,195;6,277,375;6,528,624;6,538,124;6,737,056;6,821,505;6,998,253; and 7,083,784 (each of which is incorporated herein by reference in its entirety). This is also acceptable. In one exemplary embodiment, the antigen-binding protein may include an Fc variant containing an amino acid substitution at EU position 268 (e.g., H268D or H268E). In another exemplary embodiment, the antigen-binding protein may contain an amino acid substitution at EU position 239 (e.g., S239D or S239E) and / or EU position 332 (e.g., I332D or I332Q).

[0146] In certain embodiments, the antigen-binding protein may include Fc variants containing amino acid substitutions that alter the antigen-independent effector function of the antibody, particularly the circulating half-life of the antigen-binding protein. Such antigen-binding proteins may exhibit either increased or decreased binding to FcRn compared to antigen-binding proteins lacking these substitutions, and therefore have increased or decreased half-lives, respectively. Fc variants with improved affinity for FcRn are expected to have a longer serum half-life, and such molecules have useful applications in methods of treating mammals, for example, to treat chronic diseases or disorders, where a longer half-life of the administered antibody is desired. In contrast, Fc variants with reduced FcRn binding affinity are expected to have a shorter half-life, and such molecules are also useful for administration to mammals, for example, for in vivo diagnostic imaging, or in situations where toxic side effects are present if the starting antibody remains in circulation for a long period, where a shortened circulating time may be advantageous. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta and are therefore useful in the treatment of diseases or disorders in pregnant women. Furthermore, other applications where reduced FcRn binding affinity may be desired include applications localized to the brain, kidneys, and / or liver. In one exemplary embodiment, the modified antigen-binding protein (e.g., an antibody or its antigen-binding fragment) exhibits reduced transport from vascular structures to the epithelium of the renal glomeruli. In another embodiment, the modified antigen-binding protein (e.g., an antibody or its antigen-binding fragment) exhibits reduced transport across the blood-brain barrier (BBB) ​​from the brain to the vascular space. In one embodiment, an antibody with modified FcRn binding contains an Fc domain having one or more amino acid substitutions within the “FcRn binding loop” of the Fc domain. The FcRn binding loop consists of amino acid residues 280–299 (according to EU numbering).

[0147] Examples of amino acid substitutions that alter FcRn binding activity are disclosed in International PCT Publication WO05 / 047327 (which is incorporated herein by reference in its entirety). In specific exemplary embodiments, the antigen-binding protein (e.g., an antibody or its antigen-binding fragment) comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering). In yet another embodiment, the binding molecule comprises a human Fc domain having the double mutation H433K / N434F (see, for example, U.S. Patent No. 8,163,881). In other embodiments, antigen-binding proteins for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgG1 or IgG4 heavy chain constant region, that has been modified to reduce or eliminate glycosylation. For example, an antigen-binding protein (e.g., an antibody or its antigen-binding fragment) may also include an Fc variant that includes an amino acid substitution that modifies the glycosylation of the antibody Fc. For example, the Fc variant may have reduced glycosylation (e.g., N- or O-linked glycosylation). In an exemplary embodiment, the Fc variant includes reduced glycosylation of an N-linked glycan commonly found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, e.g., an N-linked glycosylation motif, containing the amino acid sequence NXT or NXS. In a particular embodiment, the antibody includes an Fc variant having the amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody is a constant IgG1 or IgG4 antibody containing the S228P and T299A mutations (EU numbering). Includes the region.

[0148] Double cysteine-operated Fc-containing binding polypeptide In one embodiment, the disclosure provides an isolated Fc domain variant comprising or compounded with (e.g., fused with) at least one binding domain (e.g., at least one binding polypeptide).

[0149] In certain embodiments, the Fc domain variant comprises a reactive amino acid residue manipulated at a first position and a reactive amino acid residue manipulated at a second position in the antibody heavy chain constant (C H ) including the domain; here, according to the numbering of the Kabat EU index, the first position is 274th, and the second position is selected from the group consisting of 339, 360, 384, 385, 422, 440, and any combination thereof.

[0150] In certain embodiments, the Fc domain variant comprises a reactive amino acid residue manipulated at a first position and a reactive amino acid residue manipulated at a second position in the antibody heavy chain constant (C H ) including the domain; here, according to the numbering of the Kabat EU index, the first position is 339th, and the second position is selected from the group consisting of 290, 360, 384, 385, 422, 440, and any combination thereof.

[0151] In certain embodiments, the Fc domain variant comprises a reactive amino acid residue manipulated at a first position and a reactive amino acid residue manipulated at a second position in the antibody heavy chain constant (C H ) including the domain; here, according to the numbering of the Kabat EU index, the first position is 384th, and the second position is selected from the group consisting of 274, 290, 339, and any combination thereof.

[0152] In certain embodiments, the Fc domain variant further comprises a CH1 domain containing a reactive amino acid residue manipulated to the second position of 118 in the CH1 domain, according to the numbering of the Kabat EU index.

[0153] In certain embodiments, the Fc domain variant further comprises a CH1 domain containing a reactive amino acid residue manipulated at a first position of 118 in the CH1 domain according to the numbering of the Kabat EU index, and a reactive amino acid residue manipulated at a second position selected from the group consisting of 274, 339, 384, 385, 422, 440, and any combination thereof, according to the numbering of the Kabat EU index.

[0154] In certain embodiments, the manipulated reactive amino acid residue is selected from the group consisting of cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, and arginine. In certain embodiments, the manipulated reactive amino acid residue is cysteine.

[0155] In certain embodiments, the binding domain comprises one or more antigen-binding domains. The antigen-binding domain does not necessarily have to be derived from the same molecule as the parent Fc domain (i.e., the Fc domain that does not contain the first and second manipulated reactive amino acid residues). In certain embodiments, the Fc domain variant is present in the antibody.

[0156] In one embodiment, the Fc domain variant is present in or conjugated with an antibody. Antibodies from any source or species may be used with the Fc domain variants disclosed herein. Suitable antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, or human antibodies. Suitable antibodies include, but are not limited to, full-length antibodies, monoclonal antibodies, polyclonal antibodies, or single-domain antibodies, such as VHH antibodies. Born.

[0157] In specific exemplary embodiments, the Fc domain variant may be bound to or compounded with an antigen-binding fragment of an antibody. The term “antigen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which it is derived) for antigen binding (i.e., specific binding). Antigen-binding fragments may be produced by recombinant or biochemical methods well known in the art. Examples of antigen-binding fragments include Fv, Fab, Fab', and (Fab')2. In specific exemplary embodiments, the binding polypeptide of this disclosure comprises an antigen-binding fragment and an Fc domain variant.

[0158] In some embodiments, the binding polypeptide comprises a single-chain variable region sequence (ScFv). The single-chain variable region sequence comprises a single polypeptide having one or more antigen-binding sites, e.g., a VL domain linked to a VH domain by a mobile linker. The ScFv molecule may be constructed in a VH-linker-VL orientation or a VL-linker-VH orientation. The mobile hinge linking the VL and VH domains constituting the antigen-binding site comprises about 10 to about 50 amino acid residues. The linking of peptides is known in the art. The binding polypeptide may comprise at least one scFv and / or at least one constant region. In one embodiment, the binding polypeptide of the present disclosure may comprise at least one scFv linked to or fused to an Fc domain variant.

[0159] In some embodiments, the binding polypeptide of the Disclosure is a polyvalent (e.g., tetravalent) antibody produced by fusing a DNA sequence encoding an antibody having an ScFv molecule (e.g., a modified ScFv molecule). For example, in one embodiment, these sequences are combined such that the ScFv molecule (e.g., a modified ScFv molecule) is linked to an Fc domain variant via a mobile linker (e.g., a gly / ser linker) at its N-terminus or C-terminus. In another embodiment, the tetravalent antibody of the Disclosure may be made by fusing an ScFv molecule to a binding peptide, which is then fusing this peptide to an Fc domain variant to construct an ScFv-Fab tetravalent molecule.

[0160] In another embodiment, the binding polypeptide of the present disclosure is a modified minibody. The modified minibody of the present disclosure is a dimer molecule composed of two polypeptide chains, each containing an ScFv molecule, which is fused to an Fc domain variant via a linking peptide. The minibody can be made by constructing the ScFv component and linking the peptide component using the methods described in the art (see, for example, U.S. Patent No. 5,837,821 or WO94 / 09817Al). In another embodiment, a tetravalent minibody can be constructed. The tetravalent minibody can be constructed in the same manner as the minibody, except that two ScFv molecules are linked using a movable linker. The linked scFv-scFv construct is then conjugated to an Fc domain variant.

[0161] In another embodiment, the binding polypeptide of the present disclosure comprises a diabody. The diabody is a tetravalent molecule of a dimer, each having a polypeptide similar to an scFv molecule, such that the VL and VH domains of the same polypeptide chain cannot interact, but having a typically short (less than 10, e.g., about 1 to about 5) amino acid residue linker connecting both variable domains. Instead, the VL and VH domains of one polypeptide chain interact with the (respectively) VH and VL domains of a second polypeptide chain (see, e.g., WO02 / 02781). The diabody of the present disclosure comprises an scFv-like molecule fused to an Fc domain variant.

[0162] In other embodiments, the binding polypeptide is attached to the same polypeptide chain one or more times This includes multispecific or multivalent antibodies containing more than one variable domain, such as tandem variable domain (TVD) polypeptides. An example of a TVD polypeptide is the “double-headed” or “dual-Fv” configuration described in U.S. Patent No. 5,989,830. In the dual-Fv configuration, the variable domains of two different antibodies are expressed in tandem orientation on two separate chains (one heavy chain and one light chain), where one polypeptide chain has two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of a complementary VL domain consecutively linked by a peptide linker (VL1-linker-VL2). In a crossover double-head configuration, the variable domains of two different antibodies are expressed in tandem orientation on two separate chains (one heavy chain and one light chain), where one polypeptide chain has two consecutive VH domains separated by a peptide linker (VH1-linker-VH2), and the other polypeptide chain consists of a complementary VL domain consecutively linked by the peptide linker in the opposite orientation (VL2-linker-VL1). Additional antibody variants based on the "dual-Fv" format include dual-variable-domain IgG (DVD-IgG) bispecific antibodies (see U.S. Patent No. 7,612,181) and the TBTI format (see U.S.2010 / 0226923A1). In some embodiments, the conjugated polypeptide includes a multispecific or multivalent antibody containing one or more consecutive variable domains on the same polypeptide fused to the Fc domain variant.

[0163] In another embodiment, the conjugated polypeptide comprises a crossover bivariable domain IgG (CODV-IgG) bispecific antibody based on a “double-head” configuration (see US20120251541 A1, which is incorporated herein by reference in its entirety).

[0164] In other embodiments, the conjugated polypeptide includes a CrossMab or CrossMab-Fab multispecific format (see WO2009080253 and Schaefer, et al., PNAS (2011), 108: 11187-1191). Antibody variants based on the CrossMab format have a crossover of antibody domains within one arm of a bispecific IgG antibody, enabling correct chain association.

[0165] In other embodiments, the glycosylated effector-competent polypeptide contains a multispecific antibody in a T-cell engager format. “T-cell engager” refers to a binding protein that targets the host immune system, more specifically the cytotoxic activity of T cells, and tumor target proteins. In some embodiments, the isolated effector-competent polypeptide contains a multispecific antibody in an NK-cell engager format. “NK-cell engager” refers to a binding protein containing a monoclonal antibody fragment that targets the activation of the NK cell receptor, antigen-specific targeting region, and Fc region (Gauthier, et al. Cell (2019), 177: 1701-13).

[0166] The conjugated polypeptides of this disclosure, including the Fc domain variants described herein, may include the CDR sequence or variable domain sequence of a known “parent” antibody. In some embodiments, the parent antibody and the antibody of this disclosure may be similar or share the same sequence, except for modifications to the Fc domain disclosed herein.

[0167] B. Antibody-ligand conjugate Antibody-ligand conjugates can be used for a wide range of applications, including targeted therapeutics. In target-specific treatment, the antigen-binding portion of the antibody targets the treatment site. The ligand conjugated to the antibody can then be delivered to that site. This ligand may be a drug, for example, a biologically active cytotoxic payload. The advantage of using antibody-ligand-conjugate molecules is that they are designed to distinguish between healthy and affected tissue. In fact, such molecules exhibit improved therapeutic indicators, namely higher efficacy and / or lower toxicity profiles than untargeted antibodies in a clinical setting.

[0168] Conventional methods for attaching ligands to antibodies utilize covalent linkage of the ligand to a reactive amino acid (e.g., lysine) already present in the antibody. This results in a heterogeneous mixture of antibodies, with ligands attached to several sites on the antibody. Depending on the reaction state, the heterogeneous mixture can contain a distribution of antibodies with attached ligands ranging from 0 to 10 or more. Analytical and preparation methods are insufficient to separate these different antibody-ligand conjugates, resulting in non-uniform or poorly defined mixtures that are therefore unpredictable in treatment regimens.

[0169] Antibodies are large, complex, and structurally diverse biomolecules, often possessing numerous reactive groups. Their reactivity with linker reagents and ligand-linker intermediates depends on factors such as ligand concentration, pH, salt concentration, and the characteristics of the cosolvent. Furthermore, multi-step conjugation processes involving the existing reactive groups of antibodies are often irreproducible due to the difficulty in controlling the reaction state and characterizing the reactants and intermediates.

[0170] B.1. Antibodies having ligands conjugated to manipulated reactive amino acid residues By introducing engineered reactive amino acid residues onto the surface of an antibody (the residues are available for conjugation to a ligand but do not disrupt the antibody's folding and assembly or alter its antigen-binding and effector functions), engineered antigen-binding proteins or engineered antibodies can be produced and used to prepare a defined antibody-ligand conjugate population.

[0171] In certain embodiments of the present invention disclosed herein, the manipulated amino acid residue is an amino acid residue in an antigen-binding protein that is substituted with a different amino acid residue. In certain embodiments, the manipulated reactive amino acid residue is an amino acid residue in an antigen-binding protein that is substituted with a different amino acid residue that is a reactive amino acid residue. In certain embodiments, the manipulated reactive amino acid residue is cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, or arginine. In certain embodiments, the manipulated reactive amino acid residue is lysine. In certain embodiments, the manipulated reactive amino acid residue is cysteine.

[0172] In certain embodiments of the present invention disclosed herein, a parent antigen-binding protein is engineered by altering one or more of its amino acid residues to produce a derived engineered antigen-binding protein or engineered antibody. Such an engineered antigen-binding protein / antibody derived from a parent antigen-binding protein / antibody may be a single engineered antigen-binding protein in which only one amino acid residue is substituted with a different engineered amino acid residue. The engineered antigen-binding protein / antibody derived from a parent antigen-binding protein / antibody may also be a double engineered antigen-binding protein in which an amino acid residue at a first position is substituted with a different engineered amino acid residue and an amino acid residue at a second position is substituted with a different engineered amino acid residue.

[0173] In certain embodiments, a single-operated parent antigen-binding protein is a derived double-operated protein. An antigen-binding protein having an amino acid residue manipulated at a first position and which can be further manipulated by changing an amino acid residue at a second position in order to produce an antigen-binding protein.

[0174] In certain embodiments, an engineered antigen-binding protein is produced in which an engineered reactive amino acid residue is introduced at one to five positions within the antigen-binding protein. In certain embodiments, an engineered antigen-binding protein is produced in which an engineered reactive amino acid residue is introduced at a first position within the antigen-binding protein, resulting in a single engineered antigen-binding protein. In certain embodiments, an engineered antigen-binding protein is produced in which an engineered reactive amino acid residue is introduced at a first position within the antigen-binding protein, and an engineered reactive amino acid residue is introduced at a second position, resulting in a double engineered antigen-binding protein. In certain embodiments, the engineered reactive amino acid is lysine. In certain embodiments, the engineered reactive amino acid is cysteine.

[0175] In one embodiment of a dual-engineered antigen-binding protein or fragment disclosed herein, the dual-engineered antigen-binding protein or fragment comprises a reactive amino acid residue engineered at a first position and a reactive amino acid residue engineered at a second position in an antibody heavy chain constant (C H) containing a domain; where, according to the numbering of the Kabat EU index, the first position is 274, and the second position is selected from the group consisting of 339, 360, 384, 385, 422, 440, and any combination thereof. In certain embodiments, the manipulated reactive amino acid residue is cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, or arginine. In certain embodiments, the manipulated reactive amino acid residue is lysine. In certain embodiments, the manipulated reactive amino acid residue is cysteine.

[0176] In another embodiment of the dual-engineered antigen-binding protein or fragment disclosed herein, the dual-engineered antigen-binding protein or fragment comprises a reactive amino acid residue engineered at a first position and a reactive amino acid residue engineered at a second position in an antibody heavy chain constant (C H ) containing a domain; where, according to the numbering of the Kabat EU index, the first position is 339, and the second position is selected from the group consisting of 290, 360, 384, 385, 422, 440, and any combination thereof. In certain embodiments, the manipulated reactive amino acid residue is cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, or arginine. In certain embodiments, the manipulated reactive amino acid residue is lysine. In certain embodiments, the manipulated reactive amino acid residue is cysteine.

[0177] In another embodiment of the dual-engineered antigen-binding protein or fragment disclosed herein, the dual-engineered antigen-binding protein or fragment comprises a reactive amino acid residue engineered at a first position and a reactive amino acid residue engineered at a second position in an antibody heavy chain constant (C H) containing a domain; where, according to the numbering of the Kabat EU index, the first position is 118, and the second position is selected from the group consisting of 274, 339, 384, 385, 422, 440, and any combination thereof. In certain embodiments, the manipulated reactive amino acid residue is cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, or arginine. In certain embodiments, the manipulated reactive amino acid residue is lysine. In certain embodiments, the manipulated reactive amino acid residue is cysteine.

[0178] In another embodiment of the dual-engineered antigen-binding protein or fragment disclosed herein, the dual-engineered antigen-binding protein or fragment comprises a reactive amino acid residue engineered at a first position and a reactive amino acid residue engineered at a second position in an antibody heavy chain constant (C H ) containing a domain; where, according to the numbering of the Kabat EU index, the first position is position 384, and the second position is selected from the group consisting of 118, 274, 290, 339, and any combination thereof. In certain embodiments, the manipulated reactive amino acid residue is cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, or arginine. In certain embodiments, the manipulated reactive amino acid residue is lysine. In certain embodiments, the manipulated reactive amino acid residue is cysteine.

[0179] In certain embodiments, the dual-engineered antigen-binding protein or fragment disclosed herein comprises an engineered reactive amino acid residue conjugated to a ligand via a reactive moiety. In certain embodiments, the engineered reactive amino acid residue is conjugated to the ligand by a linker. In certain embodiments, the linker is cleavable. In certain embodiments, the linker is not cleavable. In certain embodiments, the ligand-to-antibody ratio (LAR) is at least 3.0. In certain embodiments, the LAR is at least 3.1. In certain embodiments, the LAR is at least 3.2. In certain embodiments, the LAR is at least 3.3. In certain embodiments, the LAR is at least 3.4. In certain embodiments, the ligand is PEG and the PEG-to-antibody ratio (PAR) is at least 3.0. In certain embodiments, the PAR is at least 3.1. In certain embodiments, the PAR is at least 3.2. In certain embodiments, the PAR is at least 3.3. In certain embodiments, the PAR is at least 3.4. In certain embodiments, the ligand is a drug and the drug-to-antibody ratio (DAR) is at least 3.0. In certain embodiments, DAR is at least 3.1. In certain embodiments, DAR is at least 3.2. In certain embodiments, DAR is at least 3.3. In certain embodiments, DAR is at least 3.4.

[0180] In certain embodiments, the single-operated antigen-binding protein or fragment disclosed herein comprises an operated reactive amino acid residue conjugated to a ligand via a reactive moiety. In certain embodiments, the operated reactive amino acid residue is conjugated to the ligand by a linker. In certain embodiments, the linker is cleavable. In certain embodiments, the linker is not cleavable. In certain embodiments, the ligand-to-antibody ratio (LAR) is at least 1.5. In certain embodiments, the LAR is at least 1.6. In certain embodiments, the LAR is at least 1.7. In certain embodiments, the LAR is at least 1.8. In certain embodiments, the LAR is at least 1.9. In certain embodiments, the ligand is PEG and the PEG-to-antibody ratio (PAR) is at least 1.5. In certain embodiments, the PAR is at least 1.6. In certain embodiments, the PAR is at least 1.7. In certain embodiments, the PAR is at least 1.8. In certain embodiments, the PAR is at least 1.9. In certain embodiments, the ligand is a drug and the drug-to-antibody ratio (DAR) is at least 1.5. In certain embodiments, DAR is at least 1.6. In certain embodiments, DAR is at least 1.7. In certain embodiments, DAR is at least 1.8. In certain embodiments, DAR is at least 1.9.

[0181] In certain embodiments of the manipulated antigen-binding proteins or fragments disclosed herein, the manipulated antigen-binding proteins or fragments are manipulated The constant state of the antibody heavy chain containing reactive amino acid residues (C H ) domains and antibody heavy chain variable (V H ) further comprises a domain. In certain embodiments, the manipulated antigen-binding protein or fragment thereof is an antibody light chain variable (V L) further includes a domain. In certain embodiments, the engineered antigen-binding protein or fragment is a chimeric antibody. In certain embodiments, the engineered antigen-binding protein or fragment is a humanized antibody. In certain embodiments, the engineered antigen-binding protein or fragment is a human antibody. In certain embodiments, the engineered antigen-binding protein or fragment is a monoclonal antibody. In certain embodiments, the engineered antigen-binding protein or fragment comprises one or more full-length antibody heavy chains including an Fc region. In certain embodiments, the Fc region is human IgG1 This is the Fc region.

[0182] In one embodiment of the method disclosed herein, a method for producing a dual-operated antigen-binding protein or fragment comprising a reactive amino acid residue operated at a first position and a reactive amino acid residue operated at a second position is: (a) producing a first library of single-operated parent antigen-binding proteins or fragments, each parent antigen-binding protein or fragment comprising a single-operated reactive amino acid residue; (b) producing a second library of ligand-conjugate single-operated parent antigen-binding proteins or fragments by conjugating ligands to the single-operated reactive amino acid residues of each single-operated parent antigen-binding protein or fragment comprising the first library; (c) producing a third library of operated positions by screening the second library for a ligand-to-antibody ratio (LAR) greater than 1.7, wherein the single-operated parent antigen-binding proteins have an LAR greater than 1.7 (d) a step of producing a fourth library of dual-engineered antigen-binding proteins or fragments, wherein the position having the manipulated reactive amino acid residue of the protein or fragment comprises a third library of manipulated positions; (e) a step of producing a fifth library of ligand-conjugate dual-engineered antigen-binding proteins or fragments by conjugating a ligand to the reactive amino acid residue at the first position and the reactive amino acid residue at the second position selected from the third library of manipulated positions; and (f) a step of producing a sixth library of dual-engineered antigen-binding proteins or fragments by screening the fifth library for LARs greater than 3.4.

[0183] In a particular embodiment, a method for producing a dual-engineered antigen-binding protein or fragment further comprises the step of producing a third library of the engineered position by screening a second library with respect to 60% or more conjugations of one ligand per single-engineered parent antigen-binding protein or fragment, 20% or less conjugations of multiple ligands per single-engineered parent antigen-binding protein or fragment, and 20% or less conjugations of zero ligands per single-engineered parent antigen-binding protein or fragment. The method further includes the step of producing a library of 3. In a particular embodiment, the method for producing a dual-engineered antigen-binding protein or fragment thereof further includes the step of producing a third library of the engineered position by screening the second library for 60% to 90% conjugations of one ligand per single-engineered parent antigen-binding protein or fragment thereof, 10% to 20% conjugations of multiple ligands per single-engineered parent antigen-binding protein or fragment thereof, and 10% to 20% conjugations of zero ligands per single-engineered parent antigen-binding protein or fragment thereof. In a particular embodiment, a method for producing a dual-engineered antigen-binding protein or fragment thereof further includes the step of producing a third library of the engineered position by screening a second library for 60% to 85% conjugations of one ligand per single-engineered parent antigen-binding protein or fragment thereof, 15% to 20% conjugations of multiple ligands per single-engineered parent antigen-binding protein or fragment thereof, and 15% to 20% conjugations of zero ligands per single-engineered parent antigen-binding protein or fragment thereof.

[0184] In a particular embodiment, a method for producing a dual-engineered antigen-binding protein or fragment further includes the step of producing a sixth library of dual-engineered antigen-binding proteins or fragments by screening a fifth library for conjugations of 80% or more of one or two ligands per dual-engineered antigen-binding protein or fragment, 10% or less of multiple ligands per dual-engineered antigen-binding protein or fragment, and 5% or less of zero ligands per dual-engineered antigen-binding protein or fragment. In a particular embodiment, a method for producing a dual-engineered antigen-binding protein or fragment further includes the step of producing a sixth library of dual-engineered antigen-binding proteins or fragments by screening a fifth library for conjugations of 80% to 99% of one or two ligands per dual-engineered antigen-binding protein or fragment, 1% to 10% of multiple ligands per dual-engineered antigen-binding protein or fragment, and 1% to 5% of zero ligands per dual-engineered antigen-binding protein or fragment. In a particular embodiment, a method for producing a dual-engineered antigen-binding protein or fragment thereof further includes the step of producing a sixth library of dual-engineered antigen-binding proteins or fragments by screening a fifth library for 80% to 95% conjugations of one or two ligands per dual-engineered antigen-binding protein or fragment thereof, 2% to 10% conjugations of multiple ligands per dual-engineered antigen-binding protein or fragment thereof, and 2% to 5% conjugations of zero ligands per dual-engineered antigen-binding protein or fragment thereof.In a particular embodiment, a method for producing a dual-engineered antigen-binding protein or fragment thereof further includes the step of producing a sixth library of dual-engineered antigen-binding proteins or fragments by screening a fifth library for 80% to 90% conjugations of one or two ligands per dual-engineered antigen-binding protein or fragment thereof, 5% to 10% conjugations of multiple ligands per dual-engineered antigen-binding protein or fragment thereof, and 3% to 5% conjugations of zero ligands per dual-engineered antigen-binding protein or fragment thereof.

[0185] In a particular embodiment, a method for producing a dual-engineered antigen-binding protein or fragment thereof involves using a ligand to produce a dual-engineered antigen-binding protein comprising a sixth library. The process further includes conjugating a protein or fragment of a protein to an engineered reactive amino acid residue. In certain embodiments, the engineered reactive amino acid residue is cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, or arginine. In certain embodiments, the engineered reactive amino acid residue is cysteine. In certain embodiments, the engineered reactive amino acid residue is lysine. In certain embodiments, the engineered reactive amino acid residue is conjugated to a ligand via a reactive moiety. In certain embodiments, the engineered reactive amino acid residue is conjugated to a ligand by a linker. In certain embodiments, the linker is cleavable. In certain embodiments, the linker is not cleavable. In certain embodiments, the linker is not cleavable. In certain embodiments, the LAR is at least 3.0. In certain embodiments, the linker is not cleavable. In certain embodiments, the LAR is at least 3.1. In certain embodiments, the linker is not cleavable. In certain embodiments, the LAR is at least 3.2. In certain embodiments, the linker is inseparable. In certain embodiments, the LAR is at least 3.3. In certain embodiments, the linker is inseparable. In certain embodiments, the LAR is at least 3.4.

[0186] In a particular embodiment, a method for producing a dual-engineered antigen-binding protein or fragment thereof further includes the step of screening a first library of single-engineered parent antigen-binding proteins or fragments thereof for thermal stability equivalent to that of unengineered antigen-binding proteins or fragments thereof.

[0187] In a particular embodiment of a method for producing an engineered antigen-binding protein or fragment thereof, the engineered antigen-binding protein or fragment thereof comprises an antibody heavy chain containing an engineered reactive amino acid residue (C H ) domains and antibody heavy chain variable (VH ) further comprises a domain. In certain embodiments, the manipulated antigen-binding protein or fragment thereof is an antibody light chain variable (V L ) further includes a domain. In certain embodiments, the engineered antigen-binding protein or fragment is a chimeric antibody. In certain embodiments, the engineered antigen-binding protein or fragment is a humanized antibody. In certain embodiments, the engineered antigen-binding protein or fragment is a human antibody. In certain embodiments, the engineered antigen-binding protein or fragment is a monoclonal antibody. In certain embodiments, the engineered antigen-binding protein or fragment comprises one or more full-length antibody heavy chains including an Fc region. In certain embodiments, the Fc region is a human IgG1 Fc region.

[0188] B.1.a. Ligand In certain embodiments, the ligand is an agent having biological or other functional activity (e.g., a protein, nucleic acid, lipid, carbohydrate, glycopeptide, or a fragment thereof). In certain embodiments, a modified antigen-binding protein containing a ligand conjugated to an antigen-binding protein has at least one additional function or property compared to an unconjugated antibody.

[0189] In certain embodiments, the ligand is conjugated to an antigen-binding protein or antibody to serve a variety of purposes and / or functions, including but not limited to serving as a targeting moiety, serving as a diagnostic agent, serving as a drug substitute, and / or serving as a drug. In certain embodiments, the ligand is the targeting moiety, which may be a protein, nucleic acid, lipid, carbohydrate, and / or a combination thereof.

[0190] In certain embodiments, the ligand includes a targeting moiety, which is one or more It specifically binds to the target molecule. Any type of targeting moiety can be used, without limitation, including proteins, nucleic acids, lipids, carbohydrates (e.g., glycans), and combinations thereof (e.g., glycoproteins, glycopeptides, and glycolipids). In certain embodiments, the targeting moiety is a carbohydrate or glycopeptide. In one embodiment, it is a trivalent glycopeptide (e.g., a trivalent GalNAc glycan-containing glycopeptide or a trivalent galactose-containing glycopeptide). In certain embodiments, the trivalent galactose-containing polypeptide is lactose3-Cys3Gly4. In certain embodiments, the targeting moiety is a glycan. The targeting moiety may be a naturally occurring or non-naturally occurring molecule. Targeting moieties suitable for conjugation may include those containing an aminooxylinker.

[0191] The targeting moieties described in the present invention may bind to any type of cell, including, but not limited to, animal (e.g., mammalian), plant, or insect cells, either in vitro or in vivo. The cells may be of endoderm, mesoderm, or ectoderm origin, and may include any cell type. In certain embodiments, the targeting moiety binds to cells, for example, mammalian cells, and facilitates the delivery of antigen-binding proteins to targeted cells, thereby improving, for example, cell targeting and / or uptake. Examples of target cells include, but are not limited to, immune cells (e.g., lymphocytes, e.g., B cells, T cells, natural killer (NK) cells, basophils, macrophages, or dendritic cells), liver cells (e.g., hepatocytes or non-parenchymal cells, e.g., hepatic sinusoidal endothelial cells, Kupffer cells, or hepatic astrocytic cells), tumor cells (e.g., any malignant or benign cells, including hepatocellular carcinoma cells, lung cancer cells, sarcoma cells, leukemia cells, or lymphoma cells), vascular cells (e.g., aortic endothelial cells or pulmonary artery endothelial cells), epithelial cells (e.g., simple squamous epithelial cells, simple columnar epithelial cells, pseudostratified columnar epithelial cells, or stratified squamous epithelial cells), or mesenchymal cells (e.g., lymphatic and circulatory, bone, and chondrocyte cells).

[0192] In one embodiment, the antigen-binding protein is internally translocated by the cell. In another embodiment, the amount of antigen-binding protein internally translocated by the cell is greater than the amount of reference antigen-binding protein lacking a targeting region that is internally translocated by the cell.

[0193] In one embodiment, the targeting moiety binds to a receptor on the target cell. For example, the targeting moiety may include a mannose-6-phosphate moiety that binds to the cell's mannose-6-phosphate receptor. In another exemplary embodiment, the targeting moiety binds to a Siglec on the target cell. Example Siglecs include sialoadhesine (Siglec-1), CD22 (Siglec-2), CD33 (Siglec-3), MAG (Siglec-4), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12, Siglec-14, or Siglec-15. In yet another embodiment, the targeting moiety includes an α2,3-, α2,6-, or α2,8-linked sialic acid residue. In yet another embodiment, the targeting moiety includes an α2,3-sialyl lactose moiety or an α2,6-sialyl lactose moiety. Other example receptors include lectin receptors, which include, but are not limited to, C-type lectin receptors, galectins, and L-type lectin receptors. Examples of lectin receptors include: TDEC-205, macrophage mannose receptor (MMR), Dectin-1, Dectin-2, macrophage-inducible C-type lectin (Mincle), dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN, CD209), DC NK lectin group receptor-1 (DNGR-1), Langerin (CD207), CD169, lectican, asialoglycoprotein receptor, DCIR, MGL, DC receptor, collectin, selectin, NK-cell receptor, multi-CTLD endocytosis receptor, Reg group (type VII) lectin, and chondrolectin. ) include tetranectin, polycystin, attractin (ATRN), eosinophil major basic protein (EMBP), DGCR2, thrombomodulin, Bimlec, SEEC, and CB CP / Frem 1 / QBRICK.

[0194] In certain embodiments, the antigen-binding protein of the present disclosure is conjugated to a ligand that includes a diagnostic agent. In one embodiment, the diagnostic agent is a detectable small molecule label, such as a fluorophore, chromophore, spin resonance probe, imaging agent, or radioactive label. Example fluorophores include fluorescent dyes (e.g., fluorescein, rhodamine, etc.) and other luminescent molecules (e.g., luminol). Fluorophores are sensitive to the environment, and their fluorescence changes upon binding to a substrate (e.g., dansyl probe) when located near one or more residues in the modified antigen-binding protein, undergoing a structural change. Example radioactive labels include small molecules containing atoms with one or more low-sensitivity nuclei. The radionuclide has a half-life appropriate to enable activity or detection after the elapsed time between administration and localization at the imaging site, e.g., a gamma, photon, or positron-emitting radionuclide.

[0195] In one embodiment, the diagnostic agent is a polypeptide. Example diagnostic polypeptides include enzymes having fluorescence-generating or chromogenic activity, e.g., the ability to cleave a substrate that forms a fluorophore or chromophore as a product (i.e., a reporter protein, e.g., luciferase). Other diagnostic proteins may have intrinsic fluorescence-generating or chromogenic activity (e.g., green, red, and yellow fluorescent bioluminescent aequorin proteins from bioluminescent marine organisms) or they have one or more low-energy radioactive nuclei ( 13 C, 15 N, 2 H, 125 I, 124 I, 123 I, 99 Tc, 43 K, 52 Fe,64 Cu, 68 Ga, 111 In, etc.) may be included.

[0196] In connection with the use of radiolabeled conjugates in conjunction with the present disclosure, the antigen-binding proteins of the present disclosure may be directly labeled (e.g., by iodination) or indirectly labeled by the use of a chelating agent. Both the phrases "indirect labeling" and "indirect labeling approach" as used herein mean that the chelating agent is covalently attached to the antigen-binding protein and at least one radionuclide associates with the chelating agent. Such chelating agents are typically referred to as bifunctional chelating agents such that they bind to both the polypeptide and the radioisotope. Exemplary chelating agents include 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid ("MX-DTPA") and cyclohexyldiethylenetriaminepentaacetic acid ("CHX-DTPA") derivatives. Other chelating agents include P-DOTA and EDTA derivatives. Radionuclides for particularly indirect labeling are, 111 In and 90 Y. Most imaging studies utilize 5 mCi 111 In-labeled antibodies, both because this dose is safe and increases imaging efficiency compared to lower doses, and optimal imaging occurs 3 to 6 days after antibody administration. See, e.g., Murray, (1985), J. Nuc. Med. 26: 3328 and Carraguillo et al, (1985), J. Nuc. Med. 26: 67. Radionuclides for direct labeling may be, for example, 131 I. It will be apparent to those skilled in the art that non-radiolabeled conjugates may also be assembled depending on the selected agent to be conjugated.

[0197] In certain embodiments, the diagnostic agent is a FRET (fluorescence resonance energy transfer) probe. FRET is used for a variety of diagnostic applications, including cancer diagnosis. The FRET probe may include a cleavable linker (enzyme-sensitive or pH linker) connecting the donor and acceptor portions of the FRET probe, where cleavage produces enhanced fluorescence (including near-infrared) (e.g., A. Cobos-Correa et al.). Membrane-bound FRET probe visualizes MM P12 activity in pulmonary inflammation, Nature Chemical Biology (2009), 5(9), 628-63; S. Gehrig et.al. Spatially Resolved Monitoring of Neutrophil Elastase Activity See Ratiometric Fluorescent Reporters (2012) Angew. Chem. Int. Ed., 51, 6258-6261.

[0198] In certain embodiments, the ligand is functionalized to contain additional groups. For example, the ligand may contain a cleavable linker that releases the ligand from the antigen-binding protein under specific conditions. In exemplary embodiments, the ligand may contain a linker that can be cleaved by a cellular enzyme and / or be pH-sensitive. Furthermore, or alternatively, the ligand may contain a disulfide bond that is cleaved by intracellular glutathione upon uptake into the cell.

[0199] In yet another embodiment, the ligand may include hydrophilic and biocompatible moieties, such as poly(glycine), poly(oxazoline), or PEG moieties.

[0200] In other embodiments, the ligand is a moiety that includes poly(ethylene glycol) (PEG, PEO, or POE). PEG is an oligomer or polymer of ethylene oxide and has the chemical structure Η-(O-CΗ2-CH2)n-ΟΗ (where the elements in parentheses are repeated). PEGylation (or pegylation) is the process by which a PEG polymer chain attaches to another molecule (e.g., an antigen-binding protein), which is then described as being PEGylated (or pegylated). PEGylation serves to reduce immunogenicity and antigenicity and increase the hydrodynamic size (size in solution) of the molecule to which it attaches, reduce renal clearance, and extend the circulation time. PEGylation can also create a more water-soluble molecule. In one embodiment of the invention, the PEG moiety can include mono-PEG, bi-PEG, or tri-PEG. In another embodiment, the PEG moiety includes from 3 to 3.5 PEG.

[0201] In certain embodiments, the ligand contains an aminooxy group, which facilitates conjugation to an antigen-binding protein via a stable oxime linkage.

[0202] In other embodiments, the ligand contains a hydrazide and / or N-alkylated hydrazine group to facilitate conjugation to an antigen-binding protein via a stable hydrazone linkage.

[0203] The antigen-binding proteins of the present invention can be used to drive toxic compounds and harmful substances into the liver in a plurality of diseases by targeting carbohydrate receptors (e.g., mannose 6-phosphate receptor, mannose receptor, and asialoglycoprotein receptor). Ganesan, L.P. et al: Rapid and Efficient Clearance of Blood-borne Virus by Liver See Sinusoidal Endothelium. PLoS Pathogens 2011, 9: 1; and Monnier, V.M. et al: Glucosepane: a poorly understood advanced glycation end product of growing importance for diabetes and its complications. Clin Chem Lab Med 2014; 52: 21.

[0204] The antigen-binding proteins of the present invention can also target different cell receptors, including, but not limited to, carbohydrate receptors, asialoglycoprotein receptors, and Siglecs, and can be used to target tumor cells by targeting these receptors. See Chen, W.C. et al: In vivo targeting of B-cell lymphoma with glycan ligands of CD22. Blood 2010, 115: 4778; Chen, W.C. et al: Targeting B lymphoma with nanoparticles bearing glycan ligands of CD22. Leuk Lymphoma 2012, 53: 208; Hatakeyama, S. et al: Targeted drug delivery to tumor vasculature by a carbohydrate mimetic peptide. PNAS, 2011, 108: 19587; Hong, F. et al: β-Glucan Functions as an Adjuvant for Monoclonal Antibody Immunotherapy by Recruiting Tumoricidal Granulocytes as Killer Cells. Cancer Res. 2003, 23: 9023; Kawasakia, N. et al: Targeted delivery of lipid antigen to macrophages via the CD169 / sialoadhesin endocytic See also: pathway induces robust invariant natural killer T cell activation. PNAS 2013, 110: 7826; and Medina, SH et al: N-acetylgalactosamine-functionalized dendrimers as hepatic cancer cell-targeted carriers. Biomaterials 2011, 32: 4118.

[0205] The conjugated peptides of the present invention can be used to control immune responses by a variety of receptors, including, but not limited to, carbohydrate receptors, DC-SIGN, or Siglec.

[0206] B.1.b. Drug substitutes In certain embodiments, the ligand is a detection probe, which may be biotin, polyethylene glycol (PEG), a fluorescent tag, a visualization peptide, and / or a combination thereof. In certain embodiments, a detection probe conjugated to an antigen-binding protein or antibody can serve as a drug substitute, partly because the ligand is comparable in size to the drug. In certain embodiments, the detection probe is biotin. In certain embodiments, the detection probe is PEG. In certain embodiments, PEG has a molecular weight of 15,000 to 20,000 g / mol or 15 to 20 kDa. In certain embodiments, PEG has a molecular weight of 10,000 to 15,000 g / mol or 10 to 15 kDa. In certain embodiments, PEG has a molecular weight of 5,000 to 10,000 g / mol or 5 to 10 kDa. In certain embodiments, PEG has a molecular weight of 4,000 to 5,000 g / mol or 4 to 5 kDa. In certain embodiments, PEG has a molecular weight of 3,000 to 4,000 g / mol or 3 to 4 kDa. In certain embodiments, PEG has a molecular weight of 2,000 to 3,000 g / mol or 2 to 3 kDa. In certain embodiments, PEG has a molecular weight of 1,000 to 2,000 g / mol or 1 to 2 kDa. In certain embodiments, PEG has a molecular weight of 900 to 1,000 g / mol or 0.9 to 1 kDa. In certain embodiments, PEG has a molecular weight of 800 to 900 g / mol or 0.8 to 0.9 kDa. In certain embodiments, PEG has a molecular weight of 700 to 800 g / mol or 0.7 to 0.8 kDa. In certain embodiments, PEG has a molecular weight of 600 to 700 g / mol or 0.6 to 0.7 kDa. In certain embodiments, PEG has a molecular weight of 500 to 600 g / mol It has a molecular weight of 0.5 to 0.6 kDa. In certain embodiments, PEG has a molecular weight of 400 to 500 g / mol or 0.4 to 0.5 kDa. In certain embodiments, PEG has a molecular weight of 300 to 400 g / mol or 0.3 to 0.4 kDa.

[0207] Using ligands or detection probes as drug substitutes can allow for screening antibody-ligand conjugates for desirable properties, including but not limited to thermal stability, structural integrity, antigen-binding ability, and / or conjugation efficiency. One measure of conjugation efficiency is the ligand-to-antibody ratio (LAR), PEG-to-antibody ratio (PAR), and / or drug-to-antibody ratio (DAR).

[0208] B.1.c. Drugs In certain embodiments, the ligand is a drug.

[0209] In certain embodiments, conjugation of an antibody with a cytotoxic drug ligand results in the formation of an antibody having drug-mediated cytotoxicity as a second function (i.e., in addition to antigen binding). In certain embodiments, conjugation of a second antibody with the antibody may confer additional binding properties. In certain embodiments, if the ligand is a genetically encoded therapeutic or diagnostic protein or nucleic acid, the ligand may be synthesized or expressed by either peptide synthesis or recombinant DNA methods known in the art. In certain embodiments, if the ligand is a non-genetically encoded peptide or drug, the ligand may be artificially synthesized or purified from natural sources.

[0210] In certain embodiments, the drug is a prodrug. Prodrugs include, but are not limited to, phosphate-containing prodrugs, amino acid-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine, and other 5-fluorouridine prodrugs that can be converted to more active non-cytotoxic drugs. Those skilled in the art may make chemical modifications to a desired drug or its prodrug to facilitate the reaction of the compound for the purpose of preparing the modified antigen-binding proteins of this disclosure. Drugs also include derivatives, pharmaceutically acceptable salts, esters, amides, and ethers of the drugs described herein. Derivatives include modifications to the drugs identified herein that can improve or not significantly reduce the desired therapeutic activity of a particular drug.

[0211] In certain embodiments, the drug may be an anti-cancer agent, an anti-inflammatory agent, an anti-infective agent, an anesthetic agent, a cytotoxic agent, a radionuclide, an immunomodulator, a cell signaling peptide, a growth factor, an enzyme, an oligonucleotide, a photoactive agent, and / or a combination thereof.

[0212] In certain embodiments, the drug is an anticancer therapeutic agent or anticancer agent. Examples of such agents include cell division inhibitors, cytotoxic nucleosides, tubulin binders, hormones and hormone antagonists, anti-angiogenic agents, enzyme inhibitors, gene regulatory factors, proteasome inhibitors, pteridines, diinene, podophyllotoxin, auristatin, geldanamycin, calicheamicin, gramicidin D, maytansinoids, neocarcinosin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracinedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansin derivatives, and This includes, but is not limited to, tracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptonigrin derivatives, auristatin derivatives, duocalmycin derivatives, and / or any combination thereof.

[0213] In certain embodiments, the drug may be anthracine, a DNA synthesis inhibitor, a DNA intercalator, a DNA-RNA transcription regulator, ansamycin benzoquinone, a quinonoid derivative, busulfan, ifosfamide, mechloretamine, triaziquane, diaziquane, carbazylquinone, indolequinone E09, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, a nitrosourea compound, and / or any combination thereof, a cell division inhibitor.

[0214] Examples of cell division-inhibiting anticancer drugs include alkylating agents, such as drugs of the anthracycline family (e.g., adriamycin, carminomycin, cyclosporine-A, chloroquine, metopterin, mitramycin, porphyromycin, streptonigrin, porphyromycin, anthracendione, and aziridine). Other cell division-inhibiting anticancer agents include DNA synthesis inhibitors (e.g., methotrexate and dichloromethotrexate, 3-amino-l,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D, and mitomycin C), DNA intercalators or cross-linkers (e.g., bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diamineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin), and DNA-RNA transcription regulators (e.g., actinomycin D, daunorubicin, doxorubicin, homohalingtonin, and idarubicin). Other examples of cell division inhibitors that conform to this disclosure include ansamycin benzoquinone, quinonoid derivatives (e.g., quinolones, genistein, bactacyclines), busulfan, ifosfamide, mechloretamine, triaziquane, diaziquane, carbazylquinone, indolequinone E09, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0215] In certain embodiments, the drug is a cytotoxic nucleoside, which may be adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, furoxiuridine, futraful, 6-mercaptopurine, and / or any combination thereof.

[0216] In certain embodiments, the drug is a tubulin binder. Examples of tubulin binders include, but are not limited to, taxoids (e.g., paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, drastatin (e.g., drastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD6126), combretastatin (e.g., combretastatin A-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)).

[0217] In certain embodiments, the drugs are hormones and hormone antagonists. Examples of anti-cancer hormones and hormone antagonists include: corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone or medroprogesterone), estrogens (e.g., diethylstilbestrol), anti-estrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminoglutethimide), and 17-(allylamino)-17-demethoxyl. Examples of anticancer and anti-angiogenic compounds include, but are not limited to, geldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefelzin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifislin-a, rapamycin, sex hormone-binding globulin, and thapsigardin. Examples of anticancer and anti-angiogenic compounds include, but are not limited to, angiostatin Kl-3, DL-α-difluoromethyl-ornithine, endostatin, fumagiline, genistein, minocycline, staurosporine, and (+)-thalidomide.

[0218] In certain embodiments, the drug is an anti-angiogenic agent, which may be angiostatin Kl-3, DL-α-difluoromethyl-ornithine, endostatin, fumagiline, genistein, minocycline, staurosporine, (+)-thalidomide, and / or any combination thereof.

[0219] In certain embodiments, the drug is an enzyme inhibitor. Examples of anticancer enzyme inhibitors include, but are not limited to, S(+)-camptothecin, curcumin, (-)-deguerin, 5,6-dichlorobenzo-imidazole I-β-D-ribofuranoside, etoposide, formestan, fosaliesin, hispidin, 2-imino-1-imidazolidinedacetate (cyclocreatine), mebinoline, trichostatin A, tilhostin AG34, and tilhostin AG879.

[0220] In certain embodiments, the drug is a gene regulator. Examples of anti-cancer gene regulators include, but are not limited to, 5-aza-2'-deoxycytidine, 5-azacitidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifen, trans-retinal (vitamin A aldehyde), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0221] Other classes of anticancer agents include, but are not limited to, drugs of the pteridine family, diinen, and podophyllotoxins. Particularly useful members of these classes include, for example, metopterin, podophyllotoxin, or podophyllotoxin derivatives, such as etoposide or etoposide phosphate, leulosidine, vindesine, and leulosine. Further anticancer agents suitable for the teachings herein include auristatins (e.g., auristatin E and monomethyl auristane E), geldanamycin, calicheamicin, gramicidin D, maytansinoids (e.g., maytansine), neocarutinostatin, topotecan, taxane, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracinedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and their analogs or homologues. Further anticancer agents that conform to the teachings herein include maytansine derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptonigrin derivatives, auristatin derivatives, and duocalmycin derivatives.

[0222] Another class of suitable anticancer agents that can be used as drugs are radiosensitizers that can efficiently target tumor or immunoreactive cells. Such drug portions enhance sensitivity to ionizing radiation, thereby increasing the efficacy of radiotherapy. Though not limited by theory, antibodies modified with radiosensitizers and internalized by tumor cells will deliver the radiosensitizer closer to the nucleus where radiosensitization will be maximized. Antibodies that lose their radiosensitizer portion are rapidly eliminated from the blood, and the remaining radiosensitizer localizes in the target tumor, with minimal uptake into normal tissue. After clearance from the blood, adjuvant radiotherapy may involve external beam radiation that specifically targets the tumor, or radioactive materials directly implanted in the tumor. Or may be administered by systemic radioimmunotherapy with the same modified antibody. In one embodiment, the therapeutic agent comprises a radionuclide or radiolabel having high-energy ionizing radiation capable of causing multiple strand breaks in nuclear DNA and resulting in cell death. Exemplary high-energy radionuclides include 90 Y, 125 I, 131 I, 123 I, m In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re and 188 Re. These isotopes typically generate high-energy α or β particles with short path lengths. Such radionuclides kill cells in close proximity to them, such as neoplastic cells to which the conjugate is attached or has entered. They have little or no effect on non-localized cells and are essentially non-immunogenic. Alternatively, high-energy isotopes can be generated by thermal irradiation of another stable isotope, such as in boron neutron capture therapy (Guan et al., PNAS, 95: 13206-10, 1998)(Guan et al., PNAS, 95: 13206-10, 1998).

[0223] In certain embodiments, the drug is a radioisotope. Examples of radioisotopes include, but are not limited to, radioisotopes suitable for the treatment of cancer, such as At 211 , Bi 212 , Er 169 , I 131 , I 125 , Y 90 , In 111 , R 32 , Re 186 , Re 188 , Sm 153 , Sr 89This includes radioactive isotopes of , and Lu. Such radioactive isotopes generally emit primarily beta radiation. In one embodiment, the radioactive isotope is the alpha-emitter isotope, more precisely thorium-227, which emits alpha radiation.

[0224] B.1.d. Proteolytic-Targeted Chimeric Ligands (PROTACs) The terms “protein degradation-targeting chimera” or “PROTAC” as defined herein refer to a bifunctional molecule comprising a protein of interest (POI) ligand (i.e., a ligand that binds to the protein of interest) and an E3 ubiquitin ligase (E3) recruiting ligand linked by a linker. PROTAC initiates a degradation cascade by causing the POI and E3 to form a ternary complex, bringing the ubiquitin-binding mechanism closer for subsequent POI ubiquitin binding. The polyubiquitinated POI is then recognized and degraded by the 26S proteasome. The 26S proteasome is part of the ubiquitin-proteasome system (UPS), which is the primary mechanism used by eukaryotic cells to control protein levels. Therefore, PROTAC is a useful ligand for the targeting and degradation of intracellular proteins. Additional PROTAC disclosures and example PROTACs are described in Pettersson et al. (Drug Discov Today Technol. 2019. 31: 15-27) and Maneiro et al. (ACS Chem Biol. 2020. 15(6): 1306-1312), each of which is incorporated herein by reference.

[0225] In certain embodiments, the antigen-binding protein or fragment thereof of the present disclosure comprises an engineered reactive amino acid residue that is conjugated to a PROTAC via a reactive moiety. In further embodiments, a linker conjugates the engineered reactive amino acid residue to the PROTAC.

[0226] B.1.e. Lysosome-targeted chimeric (LYTAC) ligands The terms “lysosome-targeted chimera” or “LYTAC” as defined herein refer to a bifunctional molecule comprising a region capable of binding a cell surface lysosomal-targeted receptor and a region capable of binding the extracellular domain of a target protein, including, but not limited to, secreted extracellular proteins and extracellular domains of membrane-bound proteins. Therefore, LYTACs are a useful alternative to the above-mentioned PROTACs when the target protein of interest is not intracellular. Further LYTAC disclosures and examples of LYTACs are found in Banik et al. (ChemRxiv. 2019), Banik The findings are described in et al. (Nature. 2020. 584: 291-297), WO2015 / 143091, and WO2020 / 132100, each of which is incorporated herein by reference. The portions of LYTAC used herein that have the ability to bind to the extracellular domain of a target protein correspond to the antigen-binding proteins or fragments thereof of this disclosure.

[0227] In certain embodiments, the antigen-binding protein or fragment thereof of the present disclosure comprises an engineered reactive amino acid residue that is conjugated to LYTAC via a reactive moiety. In further embodiments, a linker conjugates the engineered reactive amino acid residue to LYTAC.

[0228] In certain embodiments, the region of LYTAC capable of binding to cell surface lysosomal targeting receptors includes mannose-6-phosphate (M6P) or its derivatives, GalNAc (e.g., trivalent GalNAc), and glycopeptides. In certain embodiments, the cell surface lysosomal targeting receptors include asialoglycoprotein receptors (ASGPR), mannose-6-phosphate receptors (M6PR) (including, but not limited to, cation-independent M6PR), and sialic acid-binding immunoglobulin-type lectins (Siglec).

[0229] B.1.f. Ligand-to-antibody ratio (LAR) As defined herein, the terms “ligand-to-antibody ratio” or “LAR” refer to the stoichiometric ratio of the number of ligand molecules bound to one antibody. When the ligand is a PEG molecule, which can act as a drug substitute, the terms “PEG-to-antibody ratio” or “PAR” refer to the stoichiometric ratio of the number of PEG molecules bound to one antibody. When the ligand is a drug, the terms “drug-to-antibody ratio” or “DAR” refer to the stoichiometric ratio of the number of drug molecules bound to one antibody.

[0230] In one embodiment, the conjugate according to the present invention is characterized by a LAR / PAR / DAR in the range of 1 to 10, for example, 2 to 5, and particularly 3 to 4. This is generally the case for conjugates containing mytansinoid molecules. This LAR / PAR / DAR number may vary depending on the properties of the antibody and drug (i.e., growth inhibitor) used in accordance with the experimental conditions used for conjugation (if any, the ratio of growth inhibitor / antibody, reaction time, solvent properties and co-solvent properties, etc.). Thus, contact between the antibody and the growth inhibitor results in a mixture containing several conjugates that differ from each other by different ligand / PEG / drug versus antibody ratios; sometimes naked antibodies; and sometimes aggregates. The LAR / PAR / DAR determined is therefore an average value.

[0231] The method used to determine LAR / PAR / DAR consists of spectrophotometrically measuring the ratio of the absorbance at λD to 280 nm in a substantially purified conjugate solution. 280 nm is a wavelength commonly used to measure protein concentrations, e.g., antibody concentrations. The wavelength λD is chosen to allow for the differentiation of the drug from the antibody; that is, as is known to those skilled in the art, λD is the wavelength at which the drug has high absorption, and λD is far enough away from 280 nm to avoid substantial overlap of the absorbance peaks of the drug and the antibody. In the case of mytansinoid molecules, λD may be chosen to be 252 nm. The method for calculating LAR / PAR / DAR may be derived from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science.

[0232] LAR / PAR / DAR are calculated as follows: The conjugate generally contains 1 to 10 mytansinoid molecules that covalently attach to the antibody (LAR or DAR). This number can vary depending on the properties of the antibody and mytansinoid used in accordance with the experimental conditions used for conjugation (if any, mytansinoid / antibody ratio, reaction time, solvent properties and co-solvent properties, etc.). Thus, contact between antibody and mytansinoid results in a mixture containing several conjugates that differ from each other by different ligand / PEG / drug versus antibody ratios; sometimes naked antibody; and sometimes aggregates. The determined LAR / PAR / DAR is therefore an average value.

[0233] The method used herein to determine the LAR consists of spectrophotometrically measuring the ratio of absorbances at 252 nm and 280 nm of a substantially purified conjugate solution. In particular, the LAR is the ratio of the extinction coefficients (ε) measured at 280 and 252 nm with respect to the antibody and mytansinoid, respectively. D250 = 5,180M -1 cm -1and ε D252 =26,159M -1 cm -1 This can be determined spectrophotometrically by using ). The calculation method is derived from Antony S. Dimitrov (ed), LLC, 2009, Therapeutic Antibodies and Protocols, vol 525, 445, Springer Science, and is described in more detail below:

[0234] λ D (A λD ) and 280nm(A 280 The absorbance for the conjugate in ) is measured either by the monomer peak of size exclusion chromatography (SEC) analysis (which allows for the calculation of the "LAR(SEC)" parameter) or by using a classical spectrophotometer (which allows for the calculation of the "LAR(UV)" parameter). The absorbance can be expressed as follows: A λD =(c D ×ε DλD )+(c A ×ε AλD ) A 280 =(c D ×ε D280 )+(c A ×ε A280 ) During the ceremony ·c D and c A These are the concentrations of the ligand / drug and antibody in solution, respectively. ·ε DλD and ε D280 These are λ, respectively. D and the molar extinction coefficient of the ligand / drug at 280 nm. ·ε AλD and ε A280 These are λ, respectively. D And the molar extinction coefficient of the antibody at 280 nm. The solution to these two equations with two unknowns yields the following equation: c D =[(ε A280 ×(ε AλD ×A280 )] / [(ε DλD ×ε A280 )-(ε AλD ×ε D280 )] c A =[A 280 -(c D ×ε D280 )] / ε A280

[0235] The average LAR is then calculated from the ratio of drug concentration to antibody concentration: LAR = c D / c A .

[0236] B.2. Method for producing antibody-ligand conjugates As used herein, the term “reactive moiety” refers to a moiety comprising one or more atoms and one or more bonds, or the entire functional group containing them, that are responsible for a characteristic chemical reaction. In the example embodiments, the reactive moiety includes, but is not limited to, aldehyde moieties, alkynes, aminooxy moieties, azides, hydrazines, keto moieties, and thiols. In some embodiments, the reactive moiety is a terminal reactive moiety. In the reaction step, a first reactive moiety reacts with a second reactive moiety to form a ligand-conjugate antigen-binding protein. As used herein, the “aldehyde” moiety refers to a formyl functional group, represented by the following structural formula: [ka]

[0237] For example, CMP sialic acid derivatives containing terminal aldehyde moieties include, but are not limited to, the following structural formulas: [ka]

[0238] As used herein, the term "alkyne" refers to a carbon-carbon triple bond.

[0239] As used herein, the “aminooxy” portion refers to a nitrogen-oxygen single bond and is represented by the following structural formula: [ka]

[0240] As used herein, the "azide" portion refers to the RN3 portion, which can be represented by the following structural formula: [ka]

[0241] As used herein, the “hydrazine” portion refers to at least one nitrogen-nitrogen single bond and is represented by the following structural formula: [ka]

[0242] As used herein, the “imine” portion refers to a carbon-nitrogen double bond and is represented by the following structural formula: [ka]

[0243] In some embodiments, the targeted or ligand-conjugate type antigen-binding protein includes an imine. For example, one type of imine is an aldimine, a hydroxyl This includes, but is not limited to, silamines, hydrazones, ketamines, or oximes. As used herein, the “hydrazone” portion refers to one type of imine, represented by the following structural formula: [ka]

[0244] In some embodiments, the hydrazone may be a terminal hydrazone. In some embodiments, the hydrazone linkage includes an additional functional group, such as a linker or a portion of the linkage, along with the hydrazone moiety.

[0245] As used herein, the "keto" or "ketone" portion refers to a carbonyl functional group, represented by the following structural formula: [ka]

[0246] The term "maleimide" as used herein includes an unsaturated imide and is represented by the following structural formula: [ka]

[0247] The "oxime" portion is a type of imine, represented by the following structural formula: [ka]

[0248] "Thiol" refers to a portion containing an -SH functional group, which may also be called a sulfhydryl group. In some embodiments, the thiol contains a carbon-bonded sulfhydryl group.

[0249] When referring to a reactive moiety, the term “terminal” as used herein describes a group bonded to the end of a linear or branched moiety. In some embodiments, the terminal reactive moiety is a substituent of a functional group.

[0250] The term "oxidizing agent" refers to a compound or reagent that accepts or gains electrons from another compound or reagent, thereby undergoing reduction, while the other compound or reagent is oxidized. Examples of oxidizing agents include, but are not limited to, sodium periodate, periodate oxidase, galactose oxidase, hydrogen peroxide, and copper compounds (e.g., copper(II) sulfate).

[0251] As used herein, the term “ambient temperature” is equivalent to the term “room temperature,” and refers to an average temperature of approximately 23°C (73°F) within the temperature range of 20°C to 26°C (equivalent to 68°F to 79°F).

[0252] Antibody-ligand conjugates may be prepared by in vitro methods known in the art. Linkers or linking groups are used to link a ligand, such as a drug or prodrug, to an antibody. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid-unstable groups, photosensitive groups, peptidase-unstable groups, and esterase-unstable groups. The conjugation of the antibody of the present invention with a cytotoxic agent or growth inhibitor includes N-succinimidylpyridyl dithiobutyrate (SPDB), 4-[(5-nitro-2-pyridinyl)dithio]-2,5-dioxo-1-pyrrolidinyl ester butanoate (nitro-SPDB), 4-(pyridine-2-yldisulfanyl)-2-sulfobutyrate (sulfo-SPDB), N-succinimidyl (2-pyridyldithio)propionate (SPDP), succinimidyl (N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), and a bifunctional derivative of an imide ester (e.g., dimethyl adipate). This can be done using a variety of bifunctional protein coupling agents, including, but not limited to, HCl, active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)-hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, lysine immunotoxins can be prepared as described in Vitetta et al (1987). Carbon-labeled 1-isothiocyanatobenzylmethyldiethylenetriaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for the conjugation of radioactive nucleotides to antibodies (WO94 / 11026).

[0253] The antibodies of the present invention may also be used in Dependent Enzyme-Mediated Prodrug Therapy by conjugating polypeptides to prodrug-activating enzymes that convert prodrugs (e.g., peptidyl chemotherapeutic agents, see WO81 / 01145) into active anticancer drugs (e.g., see WO88 / 07378 and U.S. Patent No. 4,975,278). Components of immunoconjugates useful for ADEPT include any enzyme capable of acting on the prodrug in a manner that converts it into a more active cytotoxic form. Enzymes useful in the method of the present invention include alkaline phosphatases useful for converting phosphate-containing prodrugs into free drugs; aryl sulfatases useful for converting sulfate-containing prodrugs into free drugs; cytosine deaminases useful for converting non-toxic fluorocytosine into the anticancer drug 5-fluorouracil; proteases useful for converting peptide-containing prodrugs into free drugs, such as serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L); and enzymes containing D-amino acid substituents. The enzymes include, but are not limited to, D-alanyl carboxypeptidases useful for converting prodrugs; carbohydrate-cleaving enzymes useful for converting glycosylated prodrugs into free drugs, such as O-galactosidases and iraminidases; P-lactamases useful for converting drugs derivatized with P-lactams into free drugs; and penicillin amidases useful for converting drugs whose amine nitrogen is derivatized with a phenoxyacetyl or phenylacetyl group into free drugs, respectively, such as penicillin V amidase or penicillin G amidase. The enzymes may be covalently bonded to the polypeptides of the present invention by techniques well known in the art, such as the use of heterobifunctional crosslinking reagents discussed above.

[0254] Generally, a conjugate can be obtained by a method comprising: (i) optionally contacting a buffered aqueous solution of a cell-binding agent (e.g., an antibody according to the present invention) with a solution of a linker and a cytotoxic compound; and (ii) optionally separating the conjugate formed in (i) from the unreacted cell-binding agent.

[0255] The aqueous solution of the cell binder can be buffered with a buffer, such as potassium phosphate, acetate, citrate, or N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes buffer). The buffer depends on the properties of the cell binder. The cytotoxic compound is in solution in an organic polar solvent, such as dimethyl sulfoxide (DMSO) or dimethylacetamide (DMA). The reaction temperature is usually set between 20 and 40°C. The reaction time can vary from 1 to 24 hours. The reaction between the cell binder and the cytotoxic agent can be monitored by size exclusion chromatography (SEC) with a refractive index and / or UV detector. If the yield of the conjugate is too low, the reaction time may be extended.

[0256] Several different chromatographic methods are used by those skilled in the art to carry out the separation in step (ii): the conjugate is purified by, for example, SEC, adsorption chromatography (e.g., ion exchange chromatography, IEC), hydrophobic interaction chromatography (HIC), affinity chromatography, mixed support chromatography (e.g., hydroxyapatite chromatography), or high-performance liquid chromatography (HPLC). Purification by dialysis or diafiltration may also be used.

[0257] After step (i) or (ii), the conjugate-containing solution may be subjected to an additional step (iii) of chromatography, ultrafiltration, and / or diafiltration. The conjugate is recovered in aqueous solution at the end of these steps.

[0258] C. Expression of antigen-binding proteins In one embodiment, nucleic acid molecules encoding antigen-binding proteins disclosed herein are provided. Methods for producing antigen-binding proteins, including expressing these nucleic acid molecules or polynucleotides, are also provided.

[0259] Nucleic acid molecules or polynucleotides encoding antigen-binding proteins disclosed herein are inserted into expression vectors for introduction into host cells which can be used to produce a desired amount of the claimed antibody or fragment thereof. Accordingly, in certain embodiments, this disclosure provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0260] An expression vector is a vehicle for introducing and expressing a desired gene into cells. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, a vector suitable for the present invention includes a selection marker and a suitable restriction site for facilitating the cloning of the desired gene, as well as the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0261] Numerous expression vector systems can be used for the purposes of the present invention. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others include the use of polycistronic systems having an internal ribosome binding site. Furthermore, cells into which DNA has been incorporated into their chromosomes can be transfected host cells. Selection can be achieved by introducing one or more markers that enable selection. The markers may provide prototrophicity to a trophic-requiring host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene may be directly ligated to the DNA sequence to be expressed, or introduced into the same cell by co-transformation. Additional elements may also be necessary for optimal mRNA synthesis. These elements may include signal sequences, splice signals, as well as transcription promoters, enhancers, and termination signals. In some embodiments, the cloned variable region gene is inserted into an expression vector along with synthesized heavy and light chain constant region genes (e.g., human constant region genes) as discussed above.

[0262] In other embodiments, antigen-binding proteins may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as the heavy and light chains of antibodies, may be produced from a single polycistronic construct. These systems advantageously utilize internal ribosome entry sites (IRESs) to deliver relatively high levels of polypeptides to eukaryotic host cells. A suitable IRES sequence is disclosed in U.S. Patent No. 6,193,980 (which, for all purposes, is incorporated herein by reference in its entirety). As will be obvious to those skilled in the art, such expression systems can be used to efficiently produce the entire range of polypeptides disclosed in this application.

[0263] More generally, once a vector or DNA sequence encoding an antibody or a fragment thereof is prepared, the expression vector can be introduced into a suitable host cell; that is, the host cell can be transformed. Plasmid introduction into host cells can be achieved by various techniques well known to those skilled in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion using enveloped DNA, microinjection, and infection using intact viruses. See Ridgway, AAG “Mammalian Expression Vectors” Chapter 24.2, pp. 470-472; Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Plasmid introduction into the host may also be by electroporation. Transformed cells are grown under conditions suitable for light and heavy chain production and assayed for heavy and / or light chain protein synthesis. Examples of assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorter analysis (FACS), and immunohistochemistry.

[0264] As used herein, the term "transformation" is used in a broad sense to refer to the introduction of DNA into a recipient host cell that alters the genotype and consequently causes changes in the recipient cell.

[0265] Similarly, “host cells” refer to cells constructed using recombinant DNA technology and transformed with a vector encoding at least one heterologous gene. In describing the process for isolating polypeptides from recombinant hosts, the terms “cells” and “cell culture” are used interchangeably to indicate the source of antibodies unless otherwise explicitly specified. In other words, the recovery of polypeptides from “cells” may mean from whole cells centrifuged or from cell cultures containing both culture medium and suspended cells.

[0266] In one embodiment, the host cell line used for antibody expression is of mammalian origin. Those skilled in the art can determine the specific host cell line best suited to the desired gene product to be expressed therein. Examples of host cell lines include DG44 and DUXB11. Includes, but is not limited to, (Chinese hamster ovary strain, DHFR-negative), HELA (human cervical cancer), CV-1 (monkey kidney strain), COS (CV-1 derived cells with SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney strain), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). In one embodiment, the cell lines result in modified glycosylation of antibodies expressed therefrom, e.g., afucosylation (e.g., PER.C6® (Crucell) or FUT8-knockout CHO cell line (Potelligent® cells) (Biowa, Princeton, NJ)). In one embodiment, NS0 cells may be used. CHO cells are particularly useful. Host cell lines are typically available through commercial services, such as the American Tissue Culture Collection or from published literature.

[0267] In vitro production allows for scale-up to obtain large quantities of the desired bound polypeptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in airlift reactors or continuous agitation reactors, or cell cultures immobilized or captured in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. Where necessary and / or desired, polypeptide solutions may be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography in DEAE-cellulose, and / or (immuno-)affinity chromatography.

[0268] The genes encoding antigen-binding proteins characterized in this invention can also be expressed in non-mammalian cells such as bacteria, yeast, or plant cells. In this context, it is natural that various unicellular non-mammalian microorganisms, such as bacteria, can also be transformed, i.e., they can be grown by culture or fermentation. Bacteria that are susceptible to transformation include members of the Enterobacteriaceae family, such as Escherichia coli or Salmonella lineages; Bacillaceae family, such as Bacillus subtilis; Streptococcus pneumoniae; Streptococcus, and Haemophilus influenzae. Furthermore, it is natural that when expressed in bacteria, polypeptides can become part of inclusion bodies. Polypeptides must be isolated, purified, and then assembled into functional molecules.

[0269] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae or common baker's yeast are the most commonly used among eukaryotic microorganisms, but several other strains are also commonly available. For expression in Saccharomyces, plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used, for example. This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow in tryptophan (e.g., ATCC number 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of trpl damage as a feature of the yeast host cell genome then provides a suitable environment for detecting transformation by growth in the absence of tryptophan.

[0270] D. Method of administering antigen-binding proteins Methods for preparing and administering antigen-binding proteins (e.g., the antigen-binding proteins disclosed herein) are well known to those skilled in the art or readily determined by those skilled in the art. The routes of administration of the antigen-binding proteins disclosed herein may be oral, parenteral, inhaled, or topical. As used herein, "parenteral" includes intravenous, intra-arterial, intraperitoneal, and intramuscular administration. This includes internal, subcutaneous, rectal, or vaginal administration. All of these dosage forms are expressly intended to be within the scope of this disclosure, but the dosage form will be an injectable solution, particularly for intravenous or intra-arterial injection or infusion. Typically, pharmaceutical compositions suitable for injection may include buffers (e.g., acetic acid, phosphoric acid, or citrate buffer), surfactants (e.g., polysorbate), and possibly stabilizers (e.g., human albumin). However, in other ways compatible with the teachings herein, modified antibodies may be delivered directly to the site of harmful cell populations, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0271] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including physiological saline and buffering media. Pharmaceutically acceptable carriers in the compositions and methods of this disclosure include, but are not limited to, 0.01–0.1 M or 0.05 M phosphate buffer, or 0.8% saline. Other common parenteral vehicles include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate solution, or fixative oils. Intravenous vehicles include fluids and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextrose. Preservatives and other additives such as antibacterial agents, antioxidants, chelating agents, and inert gases may also be present. More specifically, pharmaceutical compositions suitable for injectable applications include sterile aqueous solutions (if water-soluble) or dispersants, and sterile powders for the immediate preparation of sterile injectable solutions or dispersants. In such cases, the composition must be sterile and fluid enough to allow for easy injection. It must be stable under manufacturing and storage conditions and protected from microbial contamination such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or a suitable mixture thereof. Adequate fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersibility, and by the use of a surfactant.

[0272] Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. Isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, may also be included in the composition. Extended absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0273] In any case, sterile injectable solutions may be prepared by incorporating the required amount of the active compound (e.g., a modified antigen-binding protein, either by itself or in combination with other active agents) into a suitable solvent along with one or a combination of the components listed herein, and subsequently by sterile filtration as required. Generally, dispersants are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injectable solutions, typical preparation methods include vacuum drying and freeze-drying, which yield a powder of the active component from the solution pre-sterilically filtered and any additional desired components. Formulations for injection are processed according to methods known in the art, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under sterile conditions. Furthermore, formulations may be packaged and sold in kit form (e.g., those described in the concurrently pending USSN 09 / 259,337 and USSN 09 / 259,338, each of which is incorporated by reference herein). Such products include the relevant composition The label or accompanying information may include a statement indicating that the product is useful for treating subjects who have an autoimmune or neoplastic disorder, or who are susceptible to autoimmune or neoplastic disorders.

[0274] The effective dose of the compositions of this disclosure for treating the above conditions will vary depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, may also be treated. The treatment dose may be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0275] With regard to passive immunity with antigen-binding proteins, the dosage may range from approximately 0.0001 to 100 mg / kg of host body weight, and more typically from 0.01 to 5 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.). For example, the dosage may be in the range of 1 mg / kg body weight or 10 mg / kg body weight or 1 to 10 mg / kg, for example, at least 1 mg / kg. Intermediate doses within the above ranges are also intended to be within the scope of this disclosure. Such doses may be administered to subjects daily, every other day, weekly, or according to any other schedule determined by empirical analysis. Exemplary treatments necessarily involve administration of multiple dosages over an extended period, for example, for at least 6 months. Additional exemplary treatment regimens necessarily involve administration every 2 weeks, once a month, or once every 3 to 6 months. Example dosage schedules include 1–10 mg / kg or 15 mg / kg daily, 30 mg / kg every other day, or 60 mg / kg weekly. In some cases, two or more antigen-binding proteins with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered is within the indicated range.

[0276] The antigen-binding proteins described herein may be administered in many cases. The interval between single doses may be weekly, monthly, or yearly. The interval may be irregular as indicated by measuring the blood levels of the antigen-binding protein or antigen in the patient. In some methods, the dosage is adjusted to achieve plasma concentrations of 1–1000 μg / ml of modified antigen-binding protein, and in some methods, plasma concentrations of approximately 25–300 μg / ml of modified antigen-binding protein are achieved. Alternatively, the antigen-binding protein may be administered as a sustained-release formulation, in which case the required frequency of administration is lower. For antibodies, the dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit the longest half-lives, followed by chimeric antibodies and non-human antibodies.

[0277] The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, the composition or cocktail containing this antibody is administered to patients who are not yet diseased to enhance their resistance. Such amounts are defined as the “effective prophylactic dose.” In this use, the exact amount, again, depends on the patient’s health and systemic immunity, but generally ranges from 0.1 to 25 mg per dose, particularly from 0.5 to 2.5 mg per dose. Relatively low doses are administered over a long period at relatively infrequent intervals. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses (e.g., approximately 1 to 400 mg / kg of antibody per dose, 5 to 25 mg for radioactive immune complexes, and higher doses more commonly used for cytotoxic-drug modified antibodies) may be required at relatively short intervals until disease progression is reduced or terminated, or until the patient shows partial or complete remission of disease symptoms. Subsequently, the patient may be administered a prophylactic regimen.

[0278] The antigen-binding proteins described herein may be administered in combination with other agents that are effective in treating disorders or conditions requiring treatment (e.g., prophylactic or therapeutic). 90The effective single-treatment dose (i.e., therapeutically effective dose) of the Y-labeled modified antibody of this disclosure ranges between about 5 and about 75 mCi, for example, between about 10 and about 40 mCi. 131 The effective single-dose non-myeloablative dose of I-modified antibody ranges between approximately 5 and 70 mCi, for example, between approximately 5 and 40 mCi. 131 The effective single-treatment depletion dose of I-labeled antibody (i.e., the dose that may require autologous bone marrow transplantation) ranges between approximately 30 and approximately 600 mCi, for example, between approximately 50 and less than approximately 500 mCi. Combined with chimeric antibodies, due to their longer circulating half-life compared to mouse antibodies, the effective single-treatment non-bone marrow apheresis dose of 131-labeled chimeric antibody ranges between approximately 5 and approximately 40 mCi, such as less than approximately 30 mCi. For example, 111 The imaging threshold for In-labeled labels is typically less than approximately 5 mCi.

[0279] While antigen-binding proteins may be administered as described immediately above, it should be emphasized that in other embodiments, antigen-binding proteins may be administered in a different manner to healthy patients as a first-line treatment. In such embodiments, antigen-binding proteins may be administered to patients with normal or average red bone marrow reserve and / or patients who have not received and are not receiving one or more other treatments. The administration of modified antibodies or fragments thereof in conjunction with or in combination with adjuvant therapy as used herein means the administration or application of the treatment and the disclosed antibody in a sequential, simultaneous, identical, synchronous, concomitant, or concurrent manner. As is obvious to those skilled in the art, the administration or application of various components of a combined treatment regimen may be timed to enhance the overall efficacy of the treatment. Those skilled in the art (e.g., experienced oncologists) will be able to readily recognize effective combined treatment regimens based on the selected adjuvant therapy and the teachings herein, without excessive experimentation.

[0280] As previously discussed, the antigen-binding proteins, immunoreactive fragments, or recombinants thereof of this disclosure may be administered in pharmaceutically effective amounts for the in vivo treatment of mammalian disorders. In this context, it is natural that the disclosed antigen-binding proteins are formulated to facilitate administration and enhance the stability of the activator.

[0281] Pharmaceutical compositions relating to this disclosure typically include a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, a non-toxic buffer, or a preservative. For the purposes of this application, a pharmaceutically effective amount of a modified antigen-binding protein, immunoreactive fragment, or recombinant thereof, conjugated or unconjugated to a therapeutic agent, is considered to mean a sufficient amount to achieve effective binding to an antigen and to achieve a benefit, for example, to alleviate the symptoms of a disease or disorder, or to detect a substance or cell. In the case of tumor cells, the modified antigen-binding protein typically has the ability to interact with a selected immunoreactive antigen on neoplastic or immunoreactive cells, resulting in increased cell death. Naturally, the pharmaceutical compositions relating to this disclosure may be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified antigen-binding protein.

[0282] In accordance with the scope of this disclosure, the antigen-binding proteins of the disclosure may be administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect, in accordance with the treatment methods described above. The antigen-binding proteins of the disclosure may be administered to such humans or other animals in a conventional dosing form prepared by combining the antibodies of the disclosure with conventional pharmaceutically acceptable carriers or diluents in accordance with known techniques. It will be recognized by those skilled in the art that the form and characteristics of a pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient to be mixed, the route of administration, and other well-known variables. Furthermore, it will be obvious to those skilled in the art that the disclosure A cocktail containing one or more of the antigen-binding proteins described may prove particularly effective.

[0283] Pharmaceutical composition The antibodies or immunoconjugates of the present invention can be combined with pharmaceutically acceptable excipients and, optionally, with a persistent matrix, such as a biodegradable polymer, to form therapeutic compositions.

[0284] The form of the pharmaceutical composition, the route of administration, the dosage, and the regimen will inevitably depend on the condition being treated, the severity of the disease, the patient's age, weight, and sex, etc. The pharmaceutical composition of the present invention can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc.

[0285] In one embodiment, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for an injectable formulation. These may be an isotonic sterile saline solution (such as monosodium or disodium phosphate, sodium chloride, potassium, calcium or magnesium, or a mixture of such salts), or a dry, particularly lyophilized, composition to which sterile water or saline solution is optionally added to constitute an injectable solution.

[0286] The pharmaceutical composition may be administered by a drug combination device.

[0287] The dose used for administration may be adapted as a function of various parameters, for example, the mode of administration used (for the relevant medical condition), or alternatively, as a function of the desired duration of treatment.

[0288] For the preparation of a pharmaceutical composition, an effective amount of the antibody or immunoconjugate of the present invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0289] Suitable pharmaceutical forms for injectable applications include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injectable solutions or dispersants. In all cases, the form must be sterile and injectable in a suitable device or system for delivery without degradation. It must be stable under manufacturing and storage conditions and protected from microbial contamination such as bacteria and fungi.

[0290] Solutions of the active compound, either as a free base or a pharmacologically acceptable salt, may be prepared in water appropriately mixed with a surfactant. Dispersions can also be prepared with glycerol, liquid polyethylene glycol, and mixtures thereof, and oils. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth.

[0291] The polypeptides, antibodies, or immunoconjugates of the present invention can be formulated into compositions in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may also be derived from inorganic bases such as sodium hydroxide, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, glycine, histidine, or procaine.

[0292] The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, or polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol). These may be suitable mixtures thereof, and vegetable oils. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Extended absorption of the injectable composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0293] Sterile injectable solutions are prepared by incorporating the required amount of the active compound in a suitable solvent into any other components listed above, and subsequently by sterile filtration, if necessary. Generally, dispersants are prepared by incorporating various sterile active ingredients into a sterile vehicle, which contains a basic dispersion medium and other necessary components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, which yield a powder of the active ingredient from the solution that has been sterile filtered beforehand and any additional desired components.

[0294] Formulations of more concentrated or highly concentrated solutions for direct injection are also being considered, with the use of DMSO as the solvent expected to result in extremely rapid penetration and delivery of high concentrations of the active ingredient to small tumor areas.

[0295] During formulation, the solvent is administered in a form compatible with the administered formulation and in a therapeutically effective amount. The formulation can be easily administered in various dosage forms, such as the injectable solution type described above, but drug-releasing capsules may also be used.

[0296] For parenteral administration in aqueous solutions, for example, the solution should be appropriately buffered as needed, and the liquid diluent should first be isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used are known to those skilled in the art from the perspective of this disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to either 1000 ml of subcutaneous infusion fluid or injection at the proposed infusion site (see, e.g., “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will inevitably occur depending on the condition of the person being treated. In any case, the person administering the drug will determine the appropriate dose for each individual patient.

[0297] The antibody or immunoconjugate of the present invention can be formulated within a therapeutic mixture to contain approximately 0.01 to 100 milligrams per dose.

[0298] In addition to parenteral administration, such as antibodies or immunoconjugates formulated for intravenous or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; sustained-release capsules; and any other forms currently in use.

[0299] In certain embodiments, the use of liposomes and / or nanoparticles is intended for the introduction of polypeptides into host cells. The formation and use of liposomes and / or nanoparticles are known to those skilled in the art.

[0300] Nanocapsules can generally encapsulate compounds in a stable and regenerative manner. To avoid side effects from intracellular polymer overload, such ultrafine particles (approximately 0.1 μm in size) are generally designed using polymers that can be degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles, or biodegradable polylactic acid or polylactic acid-co-glycolide nanoparticles that meet these requirements are intended for use in the present invention, and such particles can be easily manufactured.

[0301] Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilayer concentric bilayer vesicles (also called multilayer vesicles (MLVs)). MLVs generally have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small monolayer vesicles (SUVs) with a diameter ranging from 200 to 500 Å, containing an aqueous solution in the core. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0302] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. While specific embodiments have been described in detail, these will be better understood by referring to the following examples, which are included for illustrative purposes only and are not intended to limit the scope of the embodiments disclosed herein. [Examples]

[0303] Preparation of a single-modified parental cysteine ​​antibody Twenty-nine heavy-chain antibody variants were generated using QuickChange Lightning mutagenesis or the Q5 site-directed mutagenesis kit according to the recommended procedure. Antibodies were expressed in human embryonic kidney (HEK) cell-derived Expi293 cells when generating sequences encoding specific, single-manipulated parental cysteine ​​variant antibodies. Protein expression was performed in a 96-well plate format, with 0.5 mL of culture medium per well and 3 or 5 mL per sample in the 96-well plate. Culture medium was collected 4 days after transfection. Antibody expression was performed according to the established Expi293 cell procedure.

[0304] The antibodies were purified using a protein A-based purification method. Sample 3 or 5 ml was used at Hamilton Microlab STAR Liquid Handling. The protein was added to a 20 or 80 μl PhyNexus tip of the System. The protein was captured, washed, and eluted with a dedicated PhyNexus buffer (Figure 1 and Table 1).

[0305] [Table 1-1] [Table 1-2]

[0306] The thermal stability of single-modified parental cysteine ​​mutants was measured and compared to that of unmodified antibodies using nanoDSF (Figure 2).

[0307] nanoDSF is a modified differential scanning fluorescence (DSF) method for determining protein stability using intrinsic tryptophan or tyrosine fluorescence. Protein stability was typically investigated by thermal or chemical evacuation experiments. Thermal evacuation experiments utilized a linear temperature gradient to unfold the protein, while chemical evacuation experiments used increasing concentrations of denaturants. The thermal stability of a protein is usually expressed as its "melting temperature" or "Tm," at which 50% of the protein population unfolds, corresponding to the midpoint of the transition from folding to unfolding. In contrast to conventional DSF methods, nanoDSF uses tryptophan or tyrosine fluorescence to monitor protein unfolding. Both fluorescence intensity and maximum fluorescence intensity were strongly dependent on the vicinity of tryptophan. Therefore, the ratio of fluorescence intensity at 350 nm and 330 nm was suitable, for example, for detecting changes in protein structure due to protein unfolding. Applications of this method include antibody engineering, membrane protein research, quality control, and formulation development. [Examples]

[0308] Preparation of conjugated, single-modified parental cysteine ​​antibodies The conjugation capabilities of various antibody cysteine ​​variants were investigated using the THIOMAB approach with maleimide PEG (5 kDa). The THIOMAB approach involves the specific manipulation of cysteine ​​residues in the constant region of the antibody. This approach is described in more detail in Sochaj et al. (Biotechnology Advances. 2015. 33(6):775~784), which is incorporated herein by reference. The conjugation method used to obtain the bolded residues in Figure 6 (K274C, K290C, A339C, K360C) was selected after comparing various methods, including partial reduction with TCEP and cysteine. PEGylation was detected using both Coomasie and PEG staining of SDS-PAGE gels. However, only Coomasie staining was used for conjugation ranking. Variants were screened under non-reducible and reduced conditions. Cap removal was performed with 64 equivalents of DTT (Figures 3, 4, 5, and 6).

[0309] Twenty-seven antibody variants containing unpaired cysteine ​​residues were expressed. The variants were ranked according to the degree and selectivity of PEGylation, which serves as a substitute for drug-based conjugation based on PEG molecule size. The selection criteria are listed below: ≥60% mono PEG conversion ≤20% multi-PEG conversion PEG:antibody ratio (PAR)≧1.7 ≦20% PEG-free

[0310] Based on the above selection criteria, 14 single-modified cysteine ​​variants were identified. Of the 14 selected mutants, the following 10 were considered preferable: A339C, S440C, K290C, S442C, K274C, V422C, N384C, G385C, Q418C, and K360C.

[0311] The method used to identify desirable mutations was based on the following formula: F モノ -F マルチ -F無 F モノ =Mono-PEG fraction, F マルチ =Multi-PEGylated fraction, F 無 = UnPEGylated fraction • Mutants that did not meet the above criteria or that contained significantly high levels of free HL (free heavy chain as indicated by SDS-PAGE) were excluded.

[0312] PEG staining confirmed that the high molecular weight bands were due to PEGylation rather than aggregation. In other words, these high molecular weight bands corresponded to a higher degree of PEGylation, rather than PEGylation of high molecular weight species. [Examples]

[0313] Preparation of a double-processed cysteine ​​antibody Based on the screening results of the single-processed cysteine ​​mutants described above, expression was performed in 5 or 10 ml scales in 50 ml bioreactor tubes, and double mutants were generated in the same manner as described in Example 1, except that a HiTrap Protein A column was used for antibody purification using Protein Maker (Protein BioSolutions). PEG conjugation and PEGylation screening were performed as detailed in Example 2 above.

[0314] The proposed double cysteine ​​mutants are listed in Table 2 below.

[0315] [Table 2]

[0316] The double cysteine ​​mutants were prepared as follows. Mutagenesis was performed using the Q5 or QuickChange Lightning SDM kit. Expression was performed using Expi293 expression in a 10 ml bioreactor tube. Purification was performed using protein A-based purification with 1 ml MabSelect Sure on Protein Maker. Subsequently, the buffer was replaced with Amicon-15 (cutoff 10 kD). Representative purifications under non-reducing and reducing conditions are shown in Figure 7, while the purification data for all double cysteine ​​mutants are shown in Table 3. Thermal stability analysis using nanoDSF is shown in Figure 8.

[0317] [Table 3] [Examples]

[0318] PEGylation screening of dual-processed cysteine ​​antibodies As in Example 2, the conjugation ability of antibodies with dual-engineered cysteine ​​variants was investigated using the THIOMAB approach with maleimide PEG (5 kDa).

[0319] Double cysteine ​​variants were ranked according to the degree and selectivity of PEGylation, which allows them to function as substitutes for drug-based conjugations based on PEG molecule size (Figures 9 and 10). The selection criteria are listed below: ≥80% mono and diPEGylation ≤10% multi-PEGylation PEG:antibody ratio (PAR)≧3.4 ≦5% PEG-free

[0320] Based on the above selection criteria, 19 dual-engineered cysteine ​​variants were identified (Figures 11, 12, 13, and 14). [Examples]

[0321] Preparation of a single, manipulated parental cysteine ​​antibody conjugated with another reducing agent, TCEP. In addition to the aforementioned DTT reduction for removing the cap of the manipulated single cysteine ​​residue, TCEP reduction was also investigated. The conjugation ability of 27 diverse antibody-cysteine ​​variants was examined using a THIOMAB approach with maleimide PEG (5 kDa) and TCEP. PEGylation was detected using both Coomasie and PEG staining on SDS-PAGE gels. However, only Coomasie staining was used for conjugation ranking. Variants were screened under both non-reducible and reduced conditions (Figure 15).

[0322] As in previous screenings using DTT, variants were ranked using various indicators, including conjugation efficiency (PAR) and selectivity (% monoPEGylated, % unPEGylated, and % multiPEGylated heavy chains) (Figures 16 and 17).

[0323] Mutations K326C, T299C, A339C, K274C, G385C, Q386C, Y300C, K414C, S440C, and S415C were identified as the top 10 variants in the TCEP screening. Compared to the original DTT screening, four variants (A339C, K274C, S440C, and G385C) were among the top 10 in conjugations using either TCEP or DTT cap removal.

[0324] The data showed that conjugation using TCEP resulted in reduced multi-PEGylation and PAR. The difference in conjugation efficiency between DTT and TCEP cap removal may have been due to local hydrophobicity. [Examples]

[0325] Conjugation efficiency observed in single-cysteine ​​mutants The conjugation capabilities of these variants were further investigated using PEGylation. Antibody variants were partially reduced with DTT and then reoxidized with dehydroascorbic acid (dHAA). The antibodies were then PEGylated and analyzed using SDS-PAGE under non-reducing and reducing conditions, with gel staining using Coomassie blue (Figure 3). PEGylation of the same samples was confirmed on reduced SDS-PAGE gels stained with PEG (Figure 20). MonoPEGylated, unPEGylated, and multi-PEGylated protein bands were detected on reduced Coomassie blue-stained gels scanned using ProteinSimple. Percentages for each were determined using AlphaView software.

[0326] Various single-cysteine ​​mutants were PEGylated with varying efficiencies (ratio of PAR or PEG to antibody) and with varying selectivity, calculated by subtracting unPEGylated and multi-PEGylated bands (undesired species, where more than two PEGs were conjugated to each antibody heavy chain) from monoPEGylated bands (desired species) (Figure 3). Interestingly, As shown by non-reducing SDS-PAGE, most conjugates showed only slight protein aggregates, although some exhibited a certain level of aggregation even before conjugation, as previously shown (Figure 1). Under non-reducing SDS-PAGE, a considerable number of semi-antibody conjugate species were detected using mutants N297C and N298C. This was confirmed by PEG staining (Figure 23).

[0327] Based on high conjugation efficiency (PAR > 1.7) and selectivity (>60% mono-PEGylation, <20% multi-PEGylation, and <20% unPEGylation), the top 10 single-cysteine ​​variants were selected from these 27 variants: A339C, S440C, K290C, S442C, K274C, V422C, N384C, G385C, Q418C, and K360C. Some of the other variants showed high PAR, but were excluded from the top list due to low selectivity or the presence of significant amounts of semi-antibody conjugates. [Examples]

[0328] Influence of conjugation on FcγRIIIa binding Three cysteine ​​variants were PEGylated with 2kDa maleimide PEG or conjugated with the PEG2-biotin linker using DTT cap removal of unpaired cysteine. Specific variants K290C, Q295C, and S442C were selected (Figure 18). During conjugation, antibodies were analyzed for FcγRIIIa binding using Biacore. Table 4 below shows the number of conjugates per antibody at each site.

[0329] [Table 4]

[0330] To test FcγRIIIa binding, an anti-HPC4 tagged antibody was immobilized on a CM5 sensor chip. Subsequently, HPC4-tagged FcγRIIIa was captured by the immobilized anti-HPC4 antibody (Ca 2+ (In the presence of [substance name]). Next, each conjugated single mutant antibody was injected three times at a constant concentration. Dynamic studies were performed in 180-second binding and 180-second dissociation phases. For analysis, the mean of the steady-state binding response for each variant was calculated.

[0331] As shown in Figure 19, all three variants exhibited a similar FcγRIIIa binding response to the WT antibody. PEGylation reduced binding for all three variants, while biotin conjugation had little effect on the binding of K290C or S442C. [Examples]

[0332] Plasma stability of PEGylated monocysteine ​​mutants Seven single-cysteine ​​mutants (A339C, S440C, S442C, K274C, V422C, N384C, and G385C) were incubated in mouse plasma (0.02 mg / mL, close to the initial plasma concentration of mice injected at 1 mg / kg in vivo). The samples were incubated in a CO2 incubator at 37°C for 0 hours and 96 hours. After incubation, the samples were analyzed using Western blotting. Western blotting was performed with recombinant rabbit anti-PEG antibody. Mono-PEGylated bands were analyzed. In the Western blot, the diffused band at the top was a multi-PEGylated species that reacted strongly with the anti-PEG antibody due to the presence of more PEG. The Western blot is shown in Figure 21. The mono-PEGylated bands of the Western blot were analyzed and plotted as shown in Figure 22. The results showed that the PEGylated variants were stable, with at least 75% of the PEGylation remaining in the protein after 96 hours of incubation, compared to samples that were not incubated in plasma. [Examples]

[0333] Further preparation of double-engineered cysteine ​​antibodies Thirty-eight dual-cysteine ​​mutants were designed based on previous results of PEGylation screening of single-cysteine ​​mutants. The top ten single-cysteine ​​mutants, excluding Q418C which was PEGylated by DTT reduction as described above, were combined with each other or with the previously reported A118C (U.S. Patent No. 7,521,541). These were generated using site-directed mutagenesis and expressed from Expi293 cells. Most of the manipulated dual-cysteine ​​mutants showed comparable expression titers, with the exception of the dual-cysteine ​​mutant K360C+K290C, which showed a titer reduction of at least one-quarter (Table 5). SDS-PAGE results showed high aggregation in mutant S440C+N384C (Figure 7), and SEC-HPLC analysis also detected many aggregates in this dual-cysteine ​​mutant and A339C+S440C. The thermal stability of these mutants also showed a thermal transition temperature (Tm1) at least 2 degrees lower than that of the wild-type antibody, compared to the wild-type antibody and most of the double cysteine ​​mutants containing the A339C mutation, including A339C+N384C, A339C+G385C, A339C+V422C, A339C+S442C, A118C+A339C, and K274C+A339C (Table 5).

[0334] [Table 5]

[0335] The double cysteine ​​variants listed above were screened for conjugation efficiency and selectivity using PEGylation. First, DTT, dHAA, and PEG were screened. After investigating optimal conditions for various quantities, the PEGylation procedure was applied for screening (data not shown). Mutants were partially reduced using DTT to remove the caps of the two manipulated cysteine ​​residues. After reoxidation, the antibody mutants were conjugated with PEG. PEGylated mutants were analyzed using SDS-PAGE and stained with Coomassie blue (Figures 9 and 24). Most mutants showed low aggregation after PEGylation when analyzed under non-reducing conditions. Furthermore, there was a double-cysteine ​​mutant, S442C+V422C, which did not show conjugation similar to the wild-type antibody. PEGylation of this mutant repeated similar results. As shown in Figure 10, the double-cysteine ​​mutants showed varying conjugation efficiencies (PAR). Conjugation selectivity was calculated by subtracting the unPEGylated and multi-PEGylated bands (more than 3 PEGs conjugated per antibody heavy chain) (undesired species) from the mono- and diPEGylated bands (desired species), and variability was observed among mutants. Of the 38 mutants, 17 showed a PAR greater than 3.4 after conjugation. Furthermore, good selectivity (>0.7) was demonstrated for over 85% mono- and di-PEGylated proteins, less than 10% multi-PEGylated species, and less than 6% unPEGylated species (Figures 11 and 25, heatmaps). This selection criterion differed from that of Example 4. The top-performing double-cysteine ​​mutants included A118C paired with A339C, G385C, K274C, N384C, S440C, or V422C; A339C paired with G385C, K290C, N384C, S440C, or V422C; K274C paired with A339C, G385C, N384C, S440C, or V422C; and K290C paired with N384C. [Examples]

[0336] Conjugation of double cysteine ​​mutants with PROTAC linker As described regarding PEGylation, the top three double-cysteine ​​mutants were reduced with DTT (120 equivalents) and reoxidized with dHAA (30 equivalents). Next, the partially reduced and reoxidized antibody mutants were conjugated with 20 equivalents of BCN-PEG3-maleimide linker, and then with 20 equivalents of PROTAC linker, PROTAC BRD4 Degrader-5-CO-PEG3-N3, as described by Manerio et al. (ACS Chem Biol. 2020.15(6):1306~1312). The conjugates were analyzed using MALDI-TOF MS (linear positive mode) for intact protein analysis. DAR was calculated by dividing the difference in average mass between the conjugated and unconjugated mutants by the masses of the two linkers. The results are shown in Table 6 below. The DAR values ​​were lower than the PAR values ​​of the same double-cysteine ​​mutants listed above. The PROTAC linker used in this study was of low quality, which contributed to the decrease in DAR values. Repeated analyses of these dual cysteine ​​mutations due to PEGylation confirmed that the decrease in DAR values ​​was not a result of loss of activity at the manipulated cysteine ​​site.

[0337] [Table 6]

Claims

1. A constant state of the antibody heavy chain (C) containing a reactive amino acid residue manipulated at the first position and a reactive amino acid residue manipulated at the second position. Ｈ An antigen-binding protein or fragment thereof comprising a domain; wherein, according to the numbering of the EU index of Kabat, the first position is at position 274, and the second position is selected from the group consisting of 339, 360, 384, 385, 422, 440, and any combination thereof.

2. A constant state of the antibody heavy chain (C) containing a reactive amino acid residue manipulated at the first position and a reactive amino acid residue manipulated at the second position. Ｈ An antigen-binding protein or fragment thereof comprising a domain; wherein, according to the numbering of the EU index of Kabat, the first position is at position 339, and the second position is selected from the group consisting of 290, 360, 384, 385, 422, 440, and any combination thereof.

3. A constant state of the antibody heavy chain (C) containing a reactive amino acid residue manipulated at the first position and a reactive amino acid residue manipulated at the second position. Ｈ An antigen-binding protein or fragment thereof comprising a domain; wherein, according to the numbering of the EU index of Kabat, the first position is at position 118, and the second position is selected from the group consisting of 274, 339, 384, 385, 422, 440, and any combination thereof.

4. A constant state of the antibody heavy chain (C) containing a reactive amino acid residue manipulated at the first position and a reactive amino acid residue manipulated at the second position. Ｈ An antigen-binding protein or fragment thereof comprising a domain; wherein, according to the numbering of the EU index of Kabat, the first position is at position 384, and the second position is selected from the group consisting of 118, 274, 290, 339, and any combination thereof.

5. The antigen-binding protein or fragment thereof according to any one of claims 1 to 4, wherein the manipulated reactive amino acid residue is selected from the group consisting of cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, and arginine.

6. The antigen-binding protein or fragment thereof according to claim 5, wherein the manipulated reactive amino acid residue is cysteine.

7. The antigen-binding protein or fragment thereof according to claim 5, wherein the manipulated reactive amino acid residue is lysine.

8. The antigen-binding protein or fragment thereof according to any one of claims 1 to 7, wherein the manipulated reactive amino acid residue is conjugated to a ligand via the reactive moiety.

9. The antigen-binding protein or fragment thereof according to claim 8, further comprising a linker that conjugates a manipulated reactive amino acid residue to a ligand.

10. The antigen-binding protein or fragment thereof according to claim 9, wherein the linker is cleavable.

11. The linker is indestructible, antigen-binding protein or flag according to claim 9. Ment.

12. An antigen-binding protein or fragment thereof according to any one of claims 8 to 11, comprising a ligand-to-antibody ratio (LAR) of at least 3.

0.

13. An antigen-binding protein or fragment thereof according to any one of claims 8 to 11, comprising at least 3.4 LARs.

14. The antigen-binding protein or fragment thereof according to any one of claims 8 to 13, wherein the ligand is a detection probe.

15. The antigen-binding protein or fragment thereof according to claim 14, wherein the detection probe is selected from the group consisting of biotin, polyethylene glycol (PEG), a fluorescent tag, a visualization peptide, and combinations thereof.

16. The antigen-binding protein or fragment thereof according to claim 15, wherein the detection probe is PEG.

17. The antigen-binding protein or fragment thereof according to any one of claims 8 to 13, wherein the ligand is the targeting portion.

18. The antigen-binding protein or fragment thereof according to claim 17, wherein the targeting portion is selected from the group consisting of proteins, nucleic acids, lipids, carbohydrates, and combinations thereof.

19. The antigen-binding protein or fragment thereof according to any one of claims 8 to 13, wherein the ligand is a drug.

20. The antigen-binding protein or fragment thereof according to claim 19, comprising a drug-to-antibody ratio (DAR) of at least 3.

0.

21. The antigen-binding protein or fragment thereof according to claim 19, comprising at least 3.4 DARs.

22. The drug is a prodrug selected from the group consisting of: a phosphate-containing prodrug, an amino acid-containing prodrug, a thiophosphate-containing prodrug, a sulfate-containing prodrug, a peptide-containing prodrug, a β-lactam-containing prodrug, a phenoxyacetamide-containing prodrug, a phenylacetamide-containing prodrug, a 5-fluorocytosine prodrug, a 5-fluorouridine prodrug, and any combination thereof, the antigen-binding protein or fragment thereof according to any one of claims 19 to 21.

23. The drug is an antigen-binding protein or fragment thereof according to any one of claims 19 to 21, selected from the group consisting of: anticancer agents, anti-inflammatory agents, anti-infective agents, anesthetic agents, cytotoxic agents, radionuclides, immunomodulators, cell signaling peptides, growth factors, enzymes, oligonucleotides, photoactive agents, and any combination thereof.

24. Anticancer drugs include: cell division inhibitors, cytotoxic nucleosides, tubulin binders, hormones and hormone antagonists, anti-angiogenic agents, enzyme inhibitors, gene regulators, proteasome inhibitors, pteridine, diinen, podophyllotoxin, auristatin, geldanamycin, calicheamicin, gramicidin D, meitansinoids, neocartinostatin, topotecan, taxane, cytochalasin B, ethidium bromide, emetine, and teno An antigen-binding protein or fragment thereof according to claim 23, selected from the group consisting of poside, colchicine, dihydroxyanthracinedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, meitansine derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptonigrin derivatives, auristatin derivatives, duocalmycin derivatives, and any combination thereof.

25. The cell division inhibitor is selected from the group consisting of anthracine, DNA synthesis inhibitors, DNA intercalators, DNA-RNA transcription regulators, anthamycin benzoquinone, quinonoid derivatives, busulfan, ifosfamide, mechloretamine, triaziquane, diaziquane, carbazylquinone, indolequinone E09, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, nitrosourea compounds, and any combination thereof, according to claim 24, which is an antigen-binding protein or fragment thereof.

26. The antigen-binding protein or fragment thereof according to claim 24, wherein the cytotoxic nucleoside is selected from the group consisting of: adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, furoxiuridine, futraful, 6-mercaptopurine, and any combination thereof.

27. The antigen-binding protein or fragment thereof according to claim 24, wherein the tubulin binder is selected from the group consisting of taxoids, nocodazole, rhizoxin, dorastatin, colchicine, colchicinoids, combretastatin, vinca alkaloids, and any combination thereof.

28. The antigen-binding protein or fragment thereof according to claim 24, wherein the hormone and hormone antagonist is selected from the group consisting of: corticosteroids, progestins, estrogens, antiestrogens, androgens, aromatase inhibitors, 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefelzin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide, luteinizing hormone-releasing hormone, pifislin-a, rapamycin, sex hormone-binding globulin, thapsigardin, and any combination thereof.

29. The anti-angiogenic agent is selected from the group consisting of angiostatin Kl-3, DL-α-difluoromethyl-ornithine, endostatin, fumagiline, genistein, minocycline, staurosporine, (+)-thalidomide, and any combination thereof, according to claim 24, which is an antigen-binding protein or fragment thereof.

30. The enzyme inhibitor is selected from the group consisting of: S(+)-camptothecin, curcumin, (-)-deguerin, 5,6-dichlorobenzoimidazole I-β-D-ribofuranoside, etoposide, formestan, fosaliesin, hispidin, 2-imino-1-imidazolidinedioacetic acid, mebinolin, trichostatin A, tilhostin AG34, tilhostin AG879, and any combination thereof, the antigen-binding protein or fragment thereof according to claim 24.

31. The antigen-binding protein or fragment thereof according to claim 24, wherein the gene regulatory factor is selected from the group consisting of: 5-aza-2'-deoxycytidine, 5-azacitidine, cholecalciferol, 4-hydroxytamoxifen, melatonin, mifepristone, raloxifen, trans-retinal, retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol, tamoxifen, troglitazone, and any combination thereof.

32. Antibody heavy chain variable (V Ｈ An antigen-binding protein or fragment thereof according to any one of claims 1 to 31, further comprising a domain.

33. Antibody light chain variable (V Ｌ An antigen-binding protein or fragment thereof according to any one of claims 1 to 32, further comprising a domain.

34. The antigen-binding protein or a fragment thereof according to any one of claims 1 to 33, wherein the antigen-binding protein is a chimeric or humanized antibody.

35. The antigen-binding protein or a fragment thereof according to any one of claims 1 to 33, wherein the antigen-binding protein is a human antibody.

36. The antigen-binding protein or a fragment thereof according to any one of claims 1 to 35, wherein the antigen-binding protein is a monoclonal antibody.

37. The antigen-binding protein or a fragment thereof according to any one of claims 1 to 36, wherein the antigen-binding protein comprises one or more full-length antibody heavy chains including an Fc region.

38. The antigen-binding protein or fragment thereof according to claim 37, wherein the Fc region is a human IgG1 Fc region.

39. A method for producing an antigen-binding protein or fragment thereof comprising a reactive amino acid residue manipulated at a first position and a reactive amino acid residue manipulated at a second position: (a) producing a first library of manipulated parent antigen-binding proteins or fragment thereof, wherein each parent antigen-binding protein or fragment comprises the first manipulated reactive amino acid residue; (b) Producing a second library of ligand-conjugate type manipulated parent antigen-binding proteins or fragments by conjugating ligands to the first manipulated reactive amino acid residues of each manipulated parent antigen-binding protein or fragment in the first library; (c) A step of producing a third library of the manipulated position by screening a second library for a ligand-to-antibody ratio (LAR) greater than 1.7, wherein the position where the manipulated parent antigen-binding protein or fragment having a LAR greater than 1.7 has a manipulated reactive amino acid residue is included in the third library of the manipulated position; (d) A step of producing a fourth library of antigen-binding proteins or fragments thereof, wherein each antigen-binding protein or fragment thereof comprises a reactive amino acid residue manipulated at a first position selected from a third library at the manipulated position and a reactive amino acid residue manipulated at a second position selected from the third library at the manipulated position; (e) producing a fifth library of ligand-conjugate dual-engineered antigen-binding proteins or fragments thereof by conjugating a ligand to a reactive amino acid residue manipulated at the first position and a reactive amino acid residue manipulated at the second position; and (f) A step of producing a sixth library of dual-engineered antigen-binding proteins or fragments by screening the fifth library for LARs greater than 3.

4. Methods that include...

40. The method according to claim 39, further comprising the step of producing a third library of the manipulated position by screening a second library for 60% or more conjugations of one ligand per single manipulated parent antigen-binding protein or fragment thereof, 20% or less conjugations of multiple ligands per single manipulated parent antigen-binding protein or fragment thereof, and 20% or less conjugations of zero ligands per single manipulated parent antigen-binding protein or fragment thereof.

41. The method according to claim 39 or 40, further comprising the step of producing a sixth library of dual-engineered antigen-binding proteins or fragments by screening a fifth library for conjugations of 80% or more of one or two ligands per dual-engineered antigen-binding protein or fragment, 10% or less of multiple ligands per dual-engineered antigen-binding protein or fragment, and 5% or less of zero ligands per dual-engineered antigen-binding protein or fragment.

42. The method according to any one of claims 39 to 41, further comprising the step of conjugating a ligand to an engineered reactive amino acid residue of a dual-engineered antigen-binding protein or fragment thereof, comprising a sixth library.

43. The method according to any one of claims 39 to 42, wherein the manipulated reactive amino acid residue is selected from the group consisting of cysteine, lysine, histidine, serine, methionine, tryptophan, tyrosine, aspartic acid, glutamic acid, and arginine.

44. The method according to claim 43, wherein the manipulated reactive amino acid residue is cysteine.

45. The method according to claim 43, wherein the manipulated reactive amino acid residue is lysine.

46. The method according to any one of claims 39 to 45, wherein the manipulated reactive amino acid residue is conjugated to a ligand via the reactive moiety.

47. The method according to claim 46, further comprising a linker that conjugates a manipulated reactive amino acid residue to a ligand.

48. The method according to claim 47, wherein the linker is cuttable.

49. The method according to claim 47, wherein the linker is not cut.

50. The method according to any one of claims 46 to 49, comprising at least 3.0 LAR.

51. The method according to any one of claims 46 to 49, comprising at least 3.4 LARs.

52. The method according to any one of claims 46 to 51, wherein the ligand is a detection probe.

53. The method according to claim 52, wherein the detection probe is selected from the group consisting of biotin, polyethylene glycol (PEG), fluorescent tags, visualization peptides, and combinations thereof.

54. The method according to claim 53, wherein the detection probe is PEG.

55. The method according to any one of claims 46 to 51, wherein the ligand is the targeting portion.

56. The method according to claim 55, wherein the targeted portion is selected from the group consisting of proteins, nucleic acids, lipids, carbohydrates, and combinations thereof.

57. The method according to any one of claims 46 to 51, wherein the ligand is a drug.

58. The method according to claim 57, comprising a drug-to-antibody ratio (DAR) of at least 3.

0.

59. The method according to claim 57, comprising at least 3.4 DARs.

60. The method according to any one of claims 57 to 59, wherein the drug is a prodrug selected from the group consisting of: phosphate-containing prodrugs, amino acid-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, β-lactam-containing prodrugs, phenoxyacetamide-containing prodrugs, phenylacetamide-containing prodrugs, 5-fluorocytosine prodrugs, 5-fluorouridine prodrugs, and combinations thereof.

61. The method according to any one of claims 57 to 59, wherein the drug is selected from the group consisting of: anticancer agents, anti-inflammatory agents, anti-infective agents, anesthetic agents, cytotoxic agents, radionuclides, immunomodulators, cell signaling peptides, growth factors, enzymes, oligonucleotides, photoactive therapeutic agents, and combinations thereof.

62. The anticancer treatment agent is selected from the group consisting of: cell division inhibitors, cytotoxic nucleosides, tubulin binders, hormones and hormone antagonists, anti-angiogenic agents, enzyme inhibitors, gene regulatory factors, proteasome inhibitors, pteridine, diinen, podophyllotoxin, auristatin, geldanamycin, calicheamicin, gramicidin D, maytansinoids, neocarlutinostatin, topotecan, taxane, cytochalasin B, ethidium bromide, emetine, tenoposide, colchicine, dihydroxyanthracinedione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansin derivatives, anthracycline derivatives, bisphosphonate derivatives, leptomycin derivatives, streptonigrin derivatives, auristatin derivatives, duocalmycin derivatives, and any combination thereof, according to claim 61.

63. The method according to claim 62, wherein the cell division inhibitor is selected from the group consisting of: anthracine, DNA synthesis inhibitors, DNA intercalators, DNA-RNA transcription regulators, anthamycin benzoquinone, quinonoid derivatives, busulfan, ifosfamide, mechloretamine, triaziquane, diaziquane, carbazylquinone, indolequinone E09, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, nitrosourea compounds, and any combination thereof.

64. The method according to claim 62, wherein the cytotoxic nucleoside is selected from the group consisting of: adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, furoxiuridine, futraful, 6-mercaptopurine, and any combination thereof.

65. The method according to claim 62, wherein the tubulin binder is selected from the group consisting of taxoids, nocodazole, rhizoxin, dorastatin, colchicine, colchicinoids, combretastatin, vinca alkaloids, and any combination thereof.

66. The method according to claim 62, wherein the hormone and hormone antagonist are selected from the group consisting of: corticosteroids, progestins, estrogens, antiestrogens, androgens, aromatase inhibitors, 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefelzin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide, luteinizing hormone-releasing hormone, pifislin-a, rapamycin, sex hormone-binding globulin, thapsigardin, and any combination thereof.

67. The method according to claim 62, wherein the anti-angiogenic agent is selected from the group consisting of angiostatin Kl-3, DL-α-difluoromethyl-ornithine, endostatin, fumagiline, genistein, minocycline, staurosporine, (+)-thalidomide, and any combination thereof.

68. The method according to claim 62, wherein the enzyme inhibitor is selected from the group consisting of: S(+)-camptothecin, curcumin, (-)-deguerin, 5,6-dichlorobenzoimidazole I-β-D-ribofuranoside, etoposide, formestan, fosaliesin, hispidin, 2-imino-1-imidazolidinedioacetic acid, mebinolin, trichostatin A, tilhostin AG34, tilhostin AG879, and any combination thereof.

69. The method according to claim 62, wherein the gene regulatory factor is selected from the group consisting of: 5-aza-2'-deoxycytidine, 5-azacitidine, cholecalciferol, 4-hydroxytamoxifen, melatonin, mifepristone, raloxifen, trans-retinal, retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol, tamoxifen, troglitazone, and any combination thereof.

70. Antibody heavy chain variable (V Ｈ The method according to any one of claims 39 to 69, further comprising a domain.

71. Antibody light chain variable (V Ｌ The method according to any one of claims 39 to 70, further comprising a domain.

72. The method according to any one of claims 39 to 71, wherein the antigen-binding protein is a chimeric or humanized antibody.

73. The method according to any one of claims 39 to 71, wherein the antigen-binding protein is a human antibody.

74. The method according to any one of claims 39 to 73, wherein the antigen-binding protein is a monoclonal antibody.

75. The method according to any one of claims 39 to 74, wherein the antigen-binding protein comprises one or more full-length antibody heavy chains including an Fc region.

76. The method according to claim 75, wherein the Fc region is a human IgG1 Fc region.

77. The method according to any one of claims 39 to 76, wherein a first library of single-operated parent antigen-binding proteins or fragments thereof is screened for thermal stability equivalent to that of unoperated parent antigen-binding proteins or fragments thereof.

78. The antigen-binding protein or fragment thereof according to any one of claims 1 to 77 A pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

79. A method for treating a disease or disorder in a subject, comprising the step of administering to a subject in need of such treatment an antigen-binding protein or a fragment thereof according to any one of claims 1 to 77 or a pharmaceutical composition according to claim 78.

80. An isolated nucleic acid molecule encoding an antigen-binding protein or a fragment thereof, as described in any one of claims 1 to 79.

81. An expression vector comprising the nucleic acid molecule described in claim 80.

82. A host cell comprising the expression vector described in claim 81.

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