Methods of making diabodies

Abstract

The present disclosure relates to dimeric immunotherapeutics comprising two IgGs crosslinked by a disulfide bond. The two IgGs can be a chimera composed of two different heavy chains, wherein one heavy chain comprises a cysteine mutation forming the disulfide bond, and the other heavy chain does not comprise the cysteine mutation. By the presence of the cysteine mutation only on one of the two heavy chains of the IgG, the formation of two disulfide bonds between the two IgGs is avoided, increasing the spatial orientations that can be adopted between the two crosslinked IgGs, and further avoiding the formation of trimers and higher order oligomers.

Description

Cross-references to related applications

[0001] This international patent application claims priority and benefit to U.S. Patent Application No. 18 / 396,466, filed on December 26, 2023, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0002] This disclosure relates to antibodies that form dimers by cross-linking via disulfide bonds and methods for manufacturing such dimers. sequence list

[0003] This disclosure includes a sequence list named "sequence_listing_1200590028.xml", created on September 5, 2024, with a file size of 10,459 bytes, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0004] Therapeutic antibodies have contributed to significant improvements in cancer outcomes and, for at least a subset of patients, have offered new opportunities to cure cancer. The first antibody-based cancer immunotherapy, Rituxan®, was approved in the United States in 1997 for the treatment of B-cell non-Hodgkin's lymphoma, and its lifetime sales have exceeded $100 billion. Despite widespread competition, Rituxan® still generates over $1 billion in annual sales. For example, the competitive antibody Zevalin® was approved in the United States in 2002. Both Rituxan® and Zevalin® target CD20, a B-cell antigen, and both immunotherapies work by depleting B cells. When Rituxan® binds to CD20, it triggers antibody-dependent cytotoxicity and leukocyte-mediated cell death, while Zevalin® is chemically modified to chelate a radioactive isotope, thereby enabling additional radiation-induced cell death. Therapeutic antibodies can also be conjugated to cytotoxic drugs via cleavable linkers, allowing the antibody-drug conjugate to release its cytotoxic payload upon antigen binding. There are many other antibody-based cancer treatment strategies.

[0005] In the late 20th century, dimer antibodies were evaluated as potential immunotherapeutic agents. Dimeric antibodies can be prepared through chemical cross-linking or by using engineered disulfide bonds. Chemical cross-linking typically modifies the lysine amino acids commonly found in antibodies, thus producing a heterogeneous product population composed of various different dimers exhibiting different pharmacological effects. Engineered disulfide bonds reduce heterogeneity, but currently, no chemically cross-linked dimers or any disulfide-linked dimers have been approved for marketing to treat human health conditions.

[0006] While therapeutic antibodies have revolutionized the medical field, progress has been incremental. Innovative strategies to improve upon existing antibody technologies are still needed. Summary of the Invention

[0007] Various aspects of this disclosure relate to the development of improved methods for preparing disulfide-linked dimer antibodies. In short, it involves introducing cysteine ​​mutations into first IgG antibodies. For example... Figure 1 As shown, the first IgG antibody and the second IgG antibody are then mixed under mild reducing conditions to reduce the disulfide bonds that crosslink the two different heavy chains of the two IgGs. Subsequently, the IgGs separate and reform a chimeric antibody, which comprises one heavy chain and one light chain from each of the first and second IgGs, respectively. The chimeric antibody is then subjected to mild oxidative conditions to form a disulfide-linked dimer of the chimeric antibody. The disulfide-linked dimer in this specification is referred to as a "dimeric immunotherapeutic agent".

[0008] The term “chimeric antibody” is used in a manner different from its usual use in the field of immunology. In this disclosure, the term “chimeric antibody” refers to an IgG antibody having one or both of the following: (1) two heavy chains comprising two different amino acid sequences; and (2) two light chains comprising two different amino acid sequences. For example, methods for preparing such chimeric antibodies are described in U.S. Patent Nos. 9,862,769 B2 and 10,344,050 B2, which are incorporated herein by reference in their entirety. These methods are branded using the DuoBody® platform (Genmab, Denmark) for the manufacture of bispecific antibodies that bind to two different antigens. While the chimeric antibodies of this disclosure may optionally bind to two different antigens, the novelty and advantage of this disclosure arise from a cysteine ​​mutation present only on one chain of the chimeric antibody, which causes the chimeric antibody to form a single disulfide bond.

[0009] The heavy or light chain of a chimeric antibody has two different amino acid sequences, at least because either of the two heavy chains or either of the two light chains contains a cysteine ​​mutation to enable the formation of the single disulfide bond. U.S. Patents 9,862,769B2 and 10,344,050B2 also describe other mutations that favor the segregation of the IgG heavy chain, such as the F405L and K409R mutations in IgG1. However, these other mutations are not necessary for the construction of chimeric antibodies, but merely improve the relative yield. The amino acid sequence may also be changed, for example, to prepare a bispecific antibody dimer, as an artificial product of cloning, or for other reasons that do not limit this disclosure.

[0010] The inventors discovered a defect in conventional disulfide-linked dimer antibodies, which the presently disclosed dimer immunotherapeutic agent addresses. Antibodies contain multiple copies of the same amino acid sequence; therefore, engineering the introduction of cysteine ​​residues into an antibody can result in more cysteine ​​residues than are required to form a single disulfide bond. For example, IgG contains two copies of each amino acid sequence. These extra cysteine ​​residues can still participate in undesirable reactions, such as by forming additional intramolecular disulfide bonds, potentially limiting the conformational possibilities of the dimer, or causing the dimer to crosslink with other antibodies or entirely different molecules. The presently disclosed dimer immunotherapeutic agent comprises two engineered antibodies, each containing only one cysteine ​​residue. Therefore, by avoiding the introduction of additional cysteine ​​residues that could have detrimental effects, the presently disclosed dimer immunotherapeutic agent overcomes this previously unrecognized problem.

[0011] Although this disclosure focuses on IgG1 antibodies, the innovations of this disclosure are generally compatible with any IgG antibody. For example, IgG4 antibodies readily chimerize in the presence of 2-mercaptoethanol, molecular cysteine, and glutathione, as described in van der NeutKolfschoten, M. et al., “Anti-inflammatory activity of human IgG4 antibodies by dynamic Fab arm exchange,” Science, September 14, 2007; 317(5844):1554-57.

[0012] The foregoing background and summary sections are merely a brief introduction to the subject matter and a summary of some of the technical improvements and advantages it provides. The background and summary should not be construed as identifying essential aspects of the subject matter, nor should they be construed as limiting this specification or any patent claims formed based on this specification.

[0013] Brief description of the attached figures

[0014] This specification can be better understood by referring to the following figures. The figures are for illustrative purposes only, and neither this specification nor any patent claims formed based on this specification should be construed as limiting the scope of the figures.

[0015] Figure 1 This is a schematic diagram illustrating a general method for preparing a dimer immunotherapeutic agent, wherein a single disulfide bond crosslinks two distinct IgGs.

[0016] Figure 2AThis is a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image of the first IgG used to prepare the chimeric antibody of this disclosure, wherein lane 1 is loaded with 1 microgram of the first IgG under non-reducing conditions, lane 7 is loaded with 1 microgram of control IgG under non-reducing conditions, lane 8 is loaded with a molecular weight standard, lane 9 is loaded with 2 micrograms of the first IgG under reducing conditions, and lane 15 is loaded with 2 micrograms of the control IgG under reducing conditions.

[0017] Figure 2B The chromatogram of the first IgG obtained by liquid chromatography analysis is shown, in which the detection wavelength is 280 nm, indicating that the purity of the first IgG is about 91.96%.

[0018] Figure 3A This is an SDS-PAGE gel image of the second IgG used to prepare the chimeric antibody of this disclosure, wherein lane 6 is loaded with 1 microgram of the second IgG under non-reducing conditions, lane 7 is loaded with 1 microgram of control IgG under non-reducing conditions, lane 8 is loaded with a molecular weight standard, lane 14 is loaded with 2 micrograms of the second IgG under reducing conditions, and lane 15 is loaded with 2 micrograms of the control IgG under reducing conditions.

[0019] Figure 3B The chromatogram of the second IgG obtained by liquid chromatography analysis is shown, in which the detection wavelength is 280 nm, indicating that the purity of the second IgG is approximately 92.39%.

[0020] Figure 4A These are SDS-PAGE gel images of the monomers and dimers of chimeric IgG disclosed herein, wherein lane 3 was loaded with 1 microgram of the chimeric IgG under non-reducing conditions, lane 7 was loaded with 1 microgram of control IgG under non-reducing conditions, lane 8 was loaded with a molecular weight standard, lane 11 was loaded with 2 micrograms of the chimeric IgG under reducing conditions, and lane 15 was loaded with 2 micrograms of the control IgG under reducing conditions. Arrows indicate the chimeric IgG dimers displayed on the gel.

[0021] Figure 4B A chromatogram of the reaction mixture used to prepare the dimer immunotherapeutic agent of this disclosure is shown by liquid chromatography analysis, wherein the detection wavelength is 280 nm. The chromatogram indicates that the reaction mixture contains approximately 28.16% by weight of the dimer immunotherapeutic agent. Detailed Implementation

[0022] Various aspects of this disclosure relate to methods for preparing dimeric immunotherapeutic agents. Figure 1The method of this disclosure is illustrated. A first IgG 1 is provided, comprising two hemimolecules 2, each hemimolecule 2 comprising a heavy chain and a light chain. The amino acid sequence of the heavy chain of each hemimolecule 2 is identical, and the amino acid sequence of the light chain of each hemimolecule 2 is identical. The two hemimolecules 2 are cross-linked by disulfide bonds 3 located in the hinge region of the first IgG 1. Each hemimolecule 2 contains a mutation of the native amino acid to cysteine ​​4, which may occur in either the heavy chain or the light chain. The first IgG 1 contains two such cysteine ​​mutations 4, shown in the figure as present in the heavy chain.

[0023] A second IgG 5 is also provided, comprising two hemimolecules 6, each hemimolecule 6 containing one heavy chain and one light chain. The heavy chain amino acid sequence is identical in each hemimolecule 6, and the light chain amino acid sequence is also identical in each hemimolecule 6. The two hemimolecules 6 are cross-linked by disulfide bonds 3 located in the hinge region of the second IgG 5. Each hemimolecule 6 does not contain the mutation that changes the native amino acid to cysteine.

[0024] Under mild reducing conditions, such as in the presence of cysteine, first IgG1 and second IgG5 are mixed to reduce disulfide bond 3 without reducing other disulfide bonds in first IgG1 and second IgG5. These other disulfide bonds include disulfide bonds that covalently link the light chain to the heavy chain and disulfide bonds that maintain tertiary structure. Non-limiting examples of suitable reducing agents include cysteine.

[0025] Following the reduction of disulfide bond 3, half-molecule 2 of the first IgG1 can dissociate, and half-molecule 6 of the second IgG5 can dissociate, subsequently allowing half-molecule 2 of the first IgG1 to pair with half-molecule 6 of the second IgG5. Various mutations such as F405L and K409R (as described herein, when the first IgG1 and second IgG5 are IgG1) can promote one or both of the following: the dissociation of the two half-molecules 2 and 6 of the first IgG1 and / or the second IgG5, and the pairing of half-molecule 2 of the first IgG1 with half-molecule 6 of the second IgG5.

[0026] First IgG1 and second IgG5 are incubated under reducing conditions, causing their half-molecules 2 and 6 to dissociate and re-pair. Then, the paired half-molecule 2 of first IgG1 and half-molecule 6 of second IgG5 are oxidized to form disulfide bonds 8, thereby crosslinking the half-molecule 2 of first IgG1 with the half-molecule 6 of second IgG5, yielding a chimeric immunotherapeutic agent 7. The chimeric immunotherapeutic agent 7 contains a single half-molecule 2 of first IgG1, and therefore contains a single mutation site that mutates the natural amino acid to cysteine ​​4.

[0027] Incubating the first IgG1 and the second IgG5 under reducing conditions causes the half-molecules 2 and 6 of both to dissociate and re-pair. Then, cysteine ​​4 on the two different chimeric immunotherapeutic agents 7 is oxidized to form disulfide bonds 10, thereby crosslinking the two different chimeric immunotherapeutic agents 7 to form homodimeric antibodies 9.

[0028] Figure 1 The disulfide bonds 8 of the chimeric immunotherapeutic agent 7 are shown to form before the disulfide bonds 10 of the two different chimeric immunotherapeutic agents 7 are crosslinked to form the homodimeric antibody 9, and these disulfide bonds 8 are likely formed before the disulfide bonds 10 of the two different chimeric immunotherapeutic agents 7 are crosslinked, for example, due to the spatial proximity maintained by the non-covalent interaction between the half-molecules 2 and 6 of the first IgG 1 and the second IgG 5. Nevertheless, Figure 1 The order shown should not limit this disclosure or any patent claims formed based on this disclosure, for example, because the disulfide bond 10 of crosslinking two different chimeric immunotherapeutic agents 7 can be formed simultaneously with the disulfide bond 8 of the half-molecules 2, 6 of the first IgG 1 and the second IgG 5, or even before these disulfide bonds 8 are formed.

[0029] In some embodiments, the method includes providing a first immunotherapeutic agent and a second immunotherapeutic agent. In some specific embodiments, the first immunotherapeutic agent comprises a first IgG, and the second immunotherapeutic agent comprises a second IgG. In some very specific embodiments, the first immunotherapeutic agent is the first IgG, and the second immunotherapeutic agent is the second IgG. Nevertheless, the first immunotherapeutic agent may comprise the first IgG and a covalently linked linker for coupling a drug load or chelating agent of the first immunotherapeutic agent to the first IgG; and / or, the second immunotherapeutic agent may comprise the second IgG and a covalently linked linker for coupling the same or different drug loads or the same or different chelating agents of the second immunotherapeutic agent to the second IgG.

[0030] The properties of the first IgG and the second IgG are not limited. For example, the first IgG may be selected from IgG1, IgG2, IgG3, and IgG4, and the second IgG may be selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the first IgG and the second IgG are each one of IgG1, IgG2, IgG3, or IgG4, such that the first IgG and the second IgG are of the same type of IgG.

[0031] The first IgG may be selected from human / animal chimeric antibodies (e.g., human / mouse chimeric antibodies), humanized antibodies, and fully human antibodies, and the second IgG may be selected from human / animal chimeric antibodies (e.g., human / mouse chimeric antibodies), humanized antibodies, and fully human antibodies. In some embodiments, the first IgG and the second IgG are each one of human / animal chimeric antibodies, humanized antibodies, and fully human antibodies, such that the first IgG and the second IgG are of the same type of IgG.

[0032] The word “chimeric” in the term “human / animal chimeric antibody” takes its conventional meaning when used with the word “antibody”. Therefore, for the purposes of this disclosure, the term “human / animal chimeric antibody” is different from the term “chimeric antibody”.

[0033] In some embodiments, the first IgG includes a human heavy chain constant domain 3 (CH3 region). In some embodiments, the second IgG includes a human CH3 region. In some specific embodiments, both the first IgG and the second IgG include human CH3 regions.

[0034] The first IgG is typically a monoclonal antibody. In some embodiments, both the first IgG and the second IgG are monoclonal antibodies.

[0035] In some embodiments, the first IgG has at least 95% amino acid sequence identity with a therapeutic antibody selected from: 3F8 monoclonal antibody (3F8), abagovomab, abituzumab, adecatumumab, amatuximab, andecaliximab, anrukinzumab, apolizumab, ascrinvacumab, and atezolizumab. tezolizumab, avelumab, basiliximab, bavituximab, belimumab, bemarituzumab, bermekimab, bevacizumab, bivatuzumab, bleselumab, blontuvetmab, brontictuzumab b) Cabiralizumab, Camrelizumab, Capromab, Carotuximab, Cimiprimab, Cetrelimab, Cetuximab, Cibisatamab, Cirmtuzumab, Cixutumumab, Clazakizumab, Colotuximab The following are listed: codrituzumab, conatumumab, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, daratumumab, dectrekumab, demcizumab, detumomab, dinutuximab, dinutuximab beta, dostarlimab, drozitumab, duligotuzumab, durvalumab, and dusigitumab.Ecomeximab, edrecolomab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enoblituzumab, enoticumab, encipituximab, epratuzumab zumab, etaracizumab, etigilimab, faricimab, farletuzumab, fibatuzumab, ficlatuzumab, figitumumab, flanvotumab, fresolimumab, futuximab, galiximab, ganitumab, gatipotuzumab zumab, gevokizumab, gilvetmab, gimsilumab, girentuximab, gomiliximab, icrucumab, ifabotuzumab, imalumab, imaprelimab, imgatuzumab, inebilizumab, inolizumab ab), ipilimumab, iratumumab, isatuximab, iscalimab, isistiratumab, labetuzumab, lacnotuzumab, lebrikizumab, lenzilumab, leronlimab, lexatumumab, lintuzumab, lirilumab.Lucarumumab, Lulizumab Pegol, Lumiliximab, Luretuzumab, Lutikizumab, Mapatumumab, Margetuximab, Matuzumab, Milatuzumab, Mitumomab, Modotuximab, Mogamulizumab, Monalizumab, Namilumab, Namilumab Monoclonal antibodies (narnatumab), navicixizumab, naxitamab, necitumumab, nesvacumab, nimotuzumab, nivolumab, obinutuzumab, ocaratuzumab, ocrelizumab, ofatumumab, olaratumab, oleclum ab), olokizumab, omburtamab, ontuxizumab, oregovomab, otlertuzumab, panitumumab, pankomab, parsatuzumab, pastoxuxizumab, patritumab, pembrolizumab, pemtumomab, pertuzumab ( pertuzumab, pidilizumab, pritumumab, prolgolimab, racotumomab, radretumab, ramucirumab, ravagalimab, relatlimab, retifanlimab, rilotumumab, rinucumab, rituximabRobatumumab, Romilkimab, Rosmantuzumab, Samalizumab, Sarilumab, Selicrelumab, Seribantumab, Sibrotuzumab, Siltuximab, Sintilimab, Sirukumab, Spartalizumab, Taba Tabalumab, Tafasitamab, Talacotuzumab, Tarextumab, Tavolimab, Telisotuzumab, Tenatumomab, Teneliximab, Tepoditamab, Teprotumumab, Theralizumab, Tigatuzumab, Telmitor Timigutuzumab, tiragotumab, tislelizumab, tomouzotuximab, tositumomab, tovetumab, tralokinumab, trastuzumab, tremelimumab, ublituximab, ulocuplumab, urelumab, utoluximab Utomilumab, Vanalilumab, Vantictumab, Vanucizumab, Varisacumab, Varlilumab, Veltuzumab, Volociximab, Vonlerolizumab, Votumumab, Xentuzumab, Zalutumumab, ZatuximabZenocutuzumab and zolbetuximab. In some specific embodiments, the first IgG has at least 98% amino acid sequence identity with the therapeutic antibody. In some very specific embodiments, the first IgG has at least 99% amino acid sequence identity with the therapeutic antibody. The first IgG does not have 100% amino acid sequence identity with the therapeutic antibody because the first IgG contains a mutation in the natural amino acid to cysteine ​​(e.g., one of S444C or S119C) to form a disulfide bond, thereby cross-linking the chimeric IgG of the dimer immunotherapeutic agent. The first IgG may also not have 100% amino acid sequence identity with the therapeutic antibody, for example, by introducing one or more mutations (e.g., one of F405L or K409R) that favor the half-molecule dissociation of the first IgG. The first IgG may also not have 100% amino acid sequence identity with the therapeutic antibody, for example, due to removal or alteration of glycosylation sites, regulation of effector function, regulation of in vivo half-life, improvement of stability, reduction of in vivo antigenicity, as a result of cloning, and / or any number of other reasons.

[0036] In some embodiments, the first IgG has at least 95% amino acid sequence similarity to the therapeutic antibody described above, and the second IgG has at least 95% amino acid sequence similarity to the same therapeutic antibody. In some specific embodiments, the first IgG has at least 98% amino acid sequence similarity to the therapeutic antibody described above, and the second IgG has at least 98% amino acid sequence similarity to the same therapeutic antibody. In some more specific embodiments, the first IgG has at least 99% amino acid sequence similarity to the therapeutic antibody described above, and the second IgG has at least 99% amino acid sequence similarity to the same therapeutic antibody.

[0037] In some embodiments, the first IgG has at least 95% of its amino acid sequence similar to that of the therapeutic antibody; the first IgG specifically binds to the antigen; and the second IgG is a different therapeutic antibody that binds to a different epitope of the same antigen. In some specific embodiments, the first IgG has at least 98% of its amino acid sequence similar to that of the therapeutic antibody. In some very specific embodiments, the first IgG has at least 99% of its amino acid sequence similar to that of the therapeutic antibody.

[0038] In some embodiments, the first IgG has at least 95% of its amino acid sequence similar to that of the therapeutic antibody; the first IgG specifically binds to an antigen; and the second IgG is a different therapeutic antibody that binds to a different antigen. In some specific embodiments, the first IgG has at least 98% of its amino acid sequence similar to that of the therapeutic antibody. In some very specific embodiments, the first IgG has at least 99% of its amino acid sequence similar to that of the therapeutic antibody.

[0039] In some embodiments, the first immunotherapy agent is a variant selected from the following parental immunotherapy agents: 3F8 monoclonal antibody (3F8), abagovomab, abituzumab, adecatumumab, amatuximab, andecaliximab, anrukinzumab, apolizumab, ascrinvacumab, and atezolizumab. ab), avelumab, basiliximab, bavituximab, belimumab, bemarituzumab, bermekimab, bevacizumab, bivatuzumab, bleselumab, blontuvetmab, brontictuzumab, carbirap Cabiralizumab, Camrelizumab, Capromab, Carotuximab, Cimiprilimab, Cetrelimab, Cetuximab, Cibisatamab, Cirmtuzumab, Cixutumumab, Clazakizumab, Cod The following are listed: rituzumab, conatumumab, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, daratumumab, dectrekumab, demcizumab, detumomab, dinutuximab, dinutuximab beta, dostarlimab, drozitumab, duligotuzumab, durvalumab, dusigitumab, and ecoromeximab.Edrecolomab, eldelumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emibetuzumab, enavatuzumab, enoblituzumab, enoticumab, ensituximab, epratuzumab, etarac izumab), etigilimab, faricimab, farletuzumab, fibatuzumab, ficlatuzumab, figitumumab, flanvotumab, fresolimumab, futuximab, galiximab, gatipotuzumab, gatipotuzumab, gevoki zumab, gilvetmab, gimsilumab, girentuximab, gomiliximab, icrucumab, ifabotuzumab, imalumab, imaprelimab, imgatuzumab, inebilizumab, inotetumumab, ipilimumab b) Iratumumab, Isatuximab, Iscalimab, Istiratumab, Labetuzumab, Lacnotuzumab, Lebrikizumab, Lenzilumab, Leronlimab, Lexatumumab, Lintuzumab, Lirilumab, LucatumumabLulizumab, Lumiliximab, Luretuzumab, Lutikizumab, Mapatumumab, Margetuximab, Matuzumab, Milatuzumab, Mitumomab, Modotuximab, Mogamulizumab, Monalizumab, Namilumab, and Nanatuzumab are all names of traditional Chinese medicines. Arnatumab, Navicixizumab, Naxitamab, Necitumumab, Nesvacumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Ofatumumab, Olaratumab, Oleclumab, Oloccizumab Olokizumab, omburtamab, ontuxizumab, oregovomab, otlertuzumab, panitumumab, pankomab, parsatuzumab, pastoxuxizumab, patritumab, pembrolizumab, pemtumomab, pertuzumab, pilduzumab Pidilizumab, pritumumab, prolgolimab, racotumomab, radretumab, ramucirumab, ravagalimab, relatlimab, retifanlimab, rilotumumab, rinucumab, rituximab, robatumumab.Romilkimab, Rosmantuzumab, Samalizumab, Sarilumab, Selicrelumab, Seribantumab, Sibrotuzumab, Siltuximab, Sintilimab, Sirukumab, Spartalizumab, Tabalumab, Tafasitam ab), talacotuzumab, tarextumab, tabolimab, telisotuzumab, tenatumomab, teneliximab, tepoditamab, teprotumumab, theralizumab, tigatuzumab, timigutuzumab, tiragotumab, tiragotumab Tislelizumab, tomouzotuximab, tositumomab, tovetumab, tralokinumab, trastuzumab, tremelimumab, ublituximab, ulocuplumab, urelumab, utomilumab, vanalimab, vantictumab, and more. Vanucizumab, varisacumab, varlilumab, veltuzumab, volociximab, vonlerolizumab, votumumab, xentuzumab, zalutumumab, zatuximab, zenocutuzumab, zolbetuximab, and anetumab ravtansine.Aprutumab ixadotin, azintuxizumab vedotin, belantamab mafodotin, brentuximab vedotin, camidanlumab tesirine, cantuzumab mertansine, cantuzumab ravtansine, CBR96-doxorubicin immunoconjugate, cergutuzumab amunaleukin, cofetuzumab pelidotin, and coltuximab lavanten. The following are listed: ravtansine, denintuzumab mafodotin, depatuxizumab mafodotin, derbyotuximab biotin, enapotamab vedotin, enfortumab vedotin, gemtuzumab ozogamicin, glembatumumab vedotin, iladatuzumab vedotin, indatuximab ravtansine, indusatumab vedotin, and inotuzumab ozogamicin. ozogamicin, ladiratuzumab vedotin, lapituximab emtansine, lidostuzumab vedotin, loncastuximab tesirine, lorvotuzumab mertansine, losatuxizumab vedotin, mirvetuximab soravtansine, and moxetumomab pasudotox.Naratuximab emtansine, pinatuzumab vedotin, polotuzumab vedotin, rovalpituzumab tesirine, sacituzumab govitecan, samolatamab vedotin, sirtratumab vedotin, sofituzumab vedotin, tapelitumomab paptox, telisotuzumab vedotin, tisotumab vedotin, and trastuzumab derutecan. deruxtecan), trastuzumab duocarmazine, trastuzumab emtansine, tucotuzumab celmoleukin, vadastuximab talirine, vandortuzumab vedotin, vorsetuzumab mafodotin, clivatuzumab tetraxetan, ibritumomab tiuxetan, lilotomab satetraxetan, tacatuzumab Tetraxetan and tositumomab, wherein the first immunotherapy agent is a variant of the parental immunotherapy agent because the amino acid sequence of the first IgG of the first immunotherapy agent contains one or more mutations relative to the amino acid sequence of the parental immunotherapy agent, the one or more mutations including mutations in the natural amino acid to cysteine ​​(e.g., one of S444C or S119C), and optionally including one or more other mutations, such as one or more mutations that favor the dissociation of the first IgG half-molecule under reducing conditions (e.g., F405L or K409R).

[0040] In some embodiments, the first immunotherapy agent is a variant of a parental immunotherapy agent; the second immunotherapy agent is one of the preceding second parental immunotherapy agent or a variant of the preceding second parental immunotherapy agent; the first immunotherapy agent is a variant of the parental immunotherapy agent because the amino acid sequence of the first IgG of the first immunotherapy agent contains one or more mutations relative to the amino acid sequence of the parental immunotherapy agent, said one or more mutations including mutations from the natural amino acid to cysteine ​​(e.g., S444C or S119C), and optionally includes one or more other mutations, such as one or more mutations that favor the dissociation of the first IgG half-molecule under reducing conditions (e.g., F405L or K409R); and the second immunotherapy agent is optionally a variant of the second parental immunotherapy agent because the amino acid sequence of the second IgG of the second immunotherapy agent contains one or more mutations relative to the amino acid sequence of the second parental immunotherapy agent, such as one or more mutations that favor the dissociation of the second IgG half-molecule under reducing conditions (e.g., F405L or K409R). The parental immunotherapy agent and the second parental immunotherapy agent are optionally the same as or different from the preceding parental immunotherapy agent. In some specific embodiments, the parental immunotherapy agent and the second parental immunotherapy agent are the same parental immunotherapy agents as the first one.

[0041] When the parental immunotherapy agent is a radioimmunoconjugate, such as clivatuzumab tetraxetan, ibritumomab tiuxetan, lilotomabsatetraxetan, tacatuzumab tetraxetan, or tositumomab, the first immunotherapy agent (or second immunotherapy agent) may be a variant of the parental immunotherapy agent. This is because, unlike the parental immunotherapy agent, the chelating agent contained in the first immunotherapy agent (or second immunotherapy agent) typically does not form a chelate with the radioisotope before the dimeric immunotherapy agent is actually used, but rather chelates the radioisotope when the dimeric immunotherapy agent is used, for example, for cancer imaging or treatment.

[0042] The first IgG contains two heavy chains having a first amino acid sequence, and the second IgG contains two heavy chains having a second amino acid sequence. The first immunotherapeutic agent contains two heavy chains because the first IgG contains two heavy chains, and the second immunotherapeutic agent contains two heavy chains because the second IgG contains two heavy chains. In other words, the two heavy chains of the first IgG and the first immunotherapeutic agent are the same heavy chains, and the two heavy chains of the second IgG and the second first immunotherapeutic agent are the same heavy chains.

[0043] The first IgG comprises two light chains having a first light chain amino acid sequence, and the second IgG comprises two light chains having a second light chain amino acid sequence. The first immunotherapeutic agent comprises two light chains because the first IgG comprises two light chains, and the second immunotherapeutic agent comprises two light chains because the second IgG comprises two light chains. In other words, the two light chains of the first IgG and the first immunotherapeutic agent are identical light chains, and the two light chains of the second IgG and the second first immunotherapeutic agent are identical light chains.

[0044] In some embodiments, the first amino acid sequence includes a mutation from a natural amino acid to cysteine, and the second amino acid sequence does not have this mutation. In some specific embodiments, the first amino acid sequence includes a mutation from a natural amino acid to cysteine; the second amino acid sequence does not have this mutation; the first light chain amino acid sequence does not have this mutation; and the second light chain amino acid sequence does not have this mutation. In other words, the first amino acid sequence of the heavy chain of the first IgG contains this mutation.

[0045] This disclosure covers "mutations of natural amino acids to cysteine," said mutations occurring on either the heavy or light chain of the first IgG. To explicitly support both configurations, this disclosure is generally arranged as follows: first, a combination of a technical feature and a mutation of a natural amino acid to cysteine ​​occurring on the heavy chain is disclosed in one paragraph; subsequently, where applicable, a similar technical feature and a combination of a mutation of a natural amino acid to cysteine ​​occurring on the light chain is disclosed in another paragraph.

[0046] In some embodiments, the first light chain amino acid sequence includes a mutation from a natural amino acid to cysteine, and the second light chain amino acid sequence does not have said mutation. In some specific embodiments, the first light chain amino acid sequence includes a mutation from a natural amino acid to cysteine; the second light chain amino acid sequence does not have said mutation; the first amino acid sequence does not have said mutation; and the second amino acid sequence does not have said mutation. In other words, the first light chain amino acid sequence of the light chain of the first IgG contains said mutation.

[0047] In some embodiments, the two heavy chains of the first IgG and the two heavy chains of the second IgG are different heavy chains; therefore, the two heavy chains of the first immunotherapeutic agent and the two heavy chains of the second immunotherapeutic agent are also different heavy chains. In some specific embodiments, the two heavy chains of the first IgG and the two heavy chains of the second IgG are different heavy chains; therefore, the two heavy chains of the first immunotherapeutic agent and the two heavy chains of the second immunotherapeutic agent are also different heavy chains; the two heavy chains of the first IgG contain a mutation from the natural amino acid to cysteine; and the two heavy chains of the second IgG do not have said mutation. In some very specific embodiments, the two heavy chains of the first IgG and the two heavy chains of the second IgG are different heavy chains; therefore, the two heavy chains of the first immunotherapeutic agent and the two heavy chains of the second immunotherapeutic agent are also different heavy chains; the two heavy chains of the first IgG contain a mutation from the natural amino acid to cysteine; the two heavy chains of the second IgG do not have said mutation; the two light chains of the first IgG do not have said mutation; and the two light chains of the second IgG do not have said mutation. In some embodiments, the two light chains of the first IgG each have the same amino acid sequence, and the two light chains of the second IgG each have the same amino acid sequence (in contrast, the two heavy chains of the first IgG are different from each other, and the two heavy chains of the second IgG are also different from each other).

[0048] In some embodiments, the two light chains of the first IgG and the two light chains of the second IgG are different light chains; therefore, the two light chains of the first immunotherapeutic agent and the two light chains of the second immunotherapeutic agent are also different light chains. In some specific embodiments, the two light chains of the first IgG and the two light chains of the second IgG are different light chains; therefore, the two light chains of the first immunotherapeutic agent and the two light chains of the second immunotherapeutic agent are also different light chains; the two light chains of the first IgG contain a mutation from the natural amino acid to cysteine; and the two light chains of the second IgG do not have said mutation. In some very specific embodiments, the two light chains of the first IgG and the two light chains of the second IgG are different light chains; therefore, the two light chains of the first immunotherapeutic agent and the two light chains of the second immunotherapeutic agent are also different light chains; the two light chains of the first IgG contain a mutation from the natural amino acid to cysteine; the two light chains of the second IgG do not have said mutation; the two heavy chains of the first IgG do not have said mutation; and the two heavy chains of the second IgG do not have said mutation. In some embodiments, the two heavy chains of the first immunotherapeutic agent and the two heavy chains of the second immunotherapeutic agent each have the same amino acid sequence (in contrast, the two light chains of the first IgG are different from each other, and the two light chains of the second IgG are also different from each other).

[0049] In some embodiments, the two heavy chains of the first immunotherapeutic agent are covalently cross-linked via one or more disulfide bonds of a first group, and the two heavy chains of the second immunotherapeutic agent are covalently cross-linked via one or more disulfide bonds of a second group. In some specific embodiments, the two heavy chains of the first immunotherapeutic agent are covalently cross-linked via two disulfide bonds of a first group, and the two heavy chains of the second immunotherapeutic agent are covalently cross-linked via two disulfide bonds of a second group. The disulfide bonds that covalently cross-link the two heavy chains are typically disulfide bonds in the hinge region of IgG. The two heavy chains of the first immunotherapeutic agent and / or the two heavy chains of the second immunotherapeutic agent are not necessarily cross-linked via one or more disulfide bonds of a first group and one or more disulfide bonds of a second group, for example, because the first immunotherapeutic agent and / or the second immunotherapeutic agent may be provided under mild reducing conditions.

[0050] In some embodiments, (a) the first immunotherapeutic agent comprises a first IgG having two heavy chains having a first amino acid sequence; (b) the second immunotherapeutic agent comprises a second IgG having two heavy chains having a second amino acid sequence; (c) the first amino acid sequence includes a mutation from a natural amino acid to cysteine; (d) the second amino acid sequence does not have the mutation; (e) the two heavy chains of the first immunotherapeutic agent are covalently cross-linked by one or more disulfide bonds of a first group; and (f) the two heavy chains of the second immunotherapeutic agent are covalently cross-linked by one or more disulfide bonds of a second group.

[0051] In some embodiments, (a) the first immunotherapeutic agent comprises a first IgG having two light chains having a first light chain amino acid sequence; (b) the second immunotherapeutic agent comprises a second IgG having two light chains having a second light chain amino acid sequence; (c) the first light chain amino acid sequence includes a mutation from a natural amino acid to cysteine; (d) the second light chain amino acid sequence does not have the mutation; (e) the two heavy chains of the first immunotherapeutic agent are covalently cross-linked by one or more disulfide bonds of a first group; and (f) the two heavy chains of the second immunotherapeutic agent are covalently cross-linked by one or more disulfide bonds of a second group.

[0052] In some embodiments, the method includes incubating a solution containing a first immunotherapeutic agent and a second immunotherapeutic agent under reducing conditions to reduce one or more disulfide bonds in the first group and one or more disulfide bonds in the second group. In some specific embodiments, the reducing conditions include the presence of one or more selected from β-mercaptoethanol, molecular forms of cysteine, cysteamine, and glutathione in the solution. In some more specific embodiments, the reducing conditions include the presence of cysteamine in the solution.

[0053] In some embodiments, the method includes incubating the solution under reducing conditions such that: (a) the two heavy chains of the first immunotherapeutic agent dissociate from each other to form a half-molecule of the first immunotherapeutic agent; and (b) the two heavy chains of the second immunotherapeutic agent dissociate from each other to form a half-molecule of the second immunotherapeutic agent. In some specific embodiments, the method includes incubating the solution under reducing conditions such that: (a) the two heavy chains of the first immunotherapeutic agent dissociate from each other to form a half-molecule of the first immunotherapeutic agent; and (b) the two heavy chains of the second immunotherapeutic agent dissociate from each other to form a half-molecule of the second immunotherapeutic agent, wherein "first immunotherapeutic agent" refers to a single molecule of the first immunotherapeutic agent, and "second immunotherapeutic agent" refers to a single molecule of the second immunotherapeutic agent. The solution may optionally contain additional molecules of one or both of the first and second immunotherapeutic agents, which may also optionally dissociate; and in practice, the solution typically contains additional molecules of the first and second immunotherapeutic agents, including first and second immunotherapeutic agent molecules that also dissociate.

[0054] When the two heavy chains of the first immunotherapeutic agent dissociate from each other, the two light chains of the first immunotherapeutic agent are typically each covalently linked to one heavy chain and dissociate along with their respective linked heavy chains. Each light chain of the first immunotherapeutic agent is typically covalently linked to one heavy chain, and the reduction conditions of this disclosure are advantageously mild enough to maintain this covalent link.

[0055] When the two heavy chains of the second immunotherapeutic agent dissociate from each other, the two light chains of the second immunotherapeutic agent are typically each covalently linked to one heavy chain and dissociate along with their respective linked heavy chains. Each light chain of the second immunotherapeutic agent is typically covalently linked to one heavy chain, and the reduction conditions of this disclosure are advantageously mild enough to maintain this covalent link.

[0056] In some embodiments, the method includes incubating a solution under reducing conditions such that (a) at least a portion of the two heavy chains of a first immunotherapeutic agent dissociates to produce a half-molecule of the first immunotherapeutic agent; and (b) at least a portion of the two heavy chains of a second immunotherapeutic agent dissociates to produce a half-molecule of the second immunotherapeutic agent. In some specific embodiments, the method includes incubating a solution under reducing conditions such that (a) at least a portion of the two heavy chains of a first immunotherapeutic agent dissociates to produce a half-molecule of the first immunotherapeutic agent; and (b) at least a portion of the two heavy chains of a second immunotherapeutic agent dissociates to produce a half-molecule of the second immunotherapeutic agent, wherein the first immunotherapeutic agent refers to a plurality of molecules of the first immunotherapeutic agent such that at least a portion of the plurality of molecules of the first immunotherapeutic agent has two heavy chains that have dissociated; and the second immunotherapeutic agent refers to a plurality of molecules of the second immunotherapeutic agent such that at least a portion of the plurality of molecules of the second immunotherapeutic agent has two heavy chains that have dissociated.

[0057] In some embodiments, the method includes incubating a solution under reducing conditions such that a half-molecule of a first immunotherapeutic agent recombines with a half-molecule of a second immunotherapeutic agent to produce a chimeric immunotherapeutic agent that is chimeric at least because it contains both of the following: (i) a heavy chain from a first IgG containing the cysteine; and (ii) a heavy chain from a second IgG that does not contain the cysteine. In some specific embodiments, the method includes incubating a solution under reducing conditions such that a half-molecule of a first immunotherapeutic agent recombines with a half-molecule of a second immunotherapeutic agent to produce a chimeric immunotherapeutic agent, which is chimeric at least because it simultaneously contains: (i) a heavy chain containing the cysteine ​​from a first IgG; and (ii) a heavy chain without the cysteine ​​from a second IgG, wherein the first immunotherapeutic agent refers to a single molecule of the first immunotherapeutic agent; the second immunotherapeutic agent refers to a single molecule of the second immunotherapeutic agent; a half-molecule of the first immunotherapeutic agent refers to a single half-molecule of the first immunotherapeutic agent; a half-molecule of the second immunotherapeutic agent refers to a single half-molecule of the second immunotherapeutic agent; and the chimeric immunotherapeutic agent refers to a single molecule of the chimeric immunotherapeutic agent. The term "cysteine" refers to cysteine ​​in the context of "a mutation from a natural amino acid to cysteine". Chimeric antibodies typically further comprise: (i) a light chain from a first IgG, covalently bound to a heavy chain from the first IgG; and (ii) a light chain from a second IgG, covalently bound to a heavy chain from the second IgG. Various methods of this disclosure require at least two chimeric immunotherapeutic agents, for example, for forming a dimer immunotherapeutic agent, and in practice, the incubation step of this paragraph typically produces a plurality of chimeric immunotherapeutic agent molecules comprising said chimeric immunotherapeutic agent.

[0058] In some embodiments, the method includes incubating a solution under reducing conditions such that a half-molecule of a first immunotherapeutic agent recombines with a half-molecule of a second immunotherapeutic agent to produce a chimeric immunotherapeutic agent that is chimeric at least because it contains both of the following: (i) a light chain of a first IgG containing the cysteine; and (ii) a light chain of a second IgG not containing the cysteine. In some specific embodiments, the method includes incubating a solution under reducing conditions to recombine a half-molecule of a first immunotherapeutic agent with a half-molecule of a second immunotherapeutic agent to produce a chimeric immunotherapeutic agent that is chimeric at least because it simultaneously contains: (i) a light chain of a first IgG containing the cysteine; and (ii) a light chain of a second IgG not containing the cysteine, wherein the first immunotherapeutic agent refers to a single molecule of the first immunotherapeutic agent; the second immunotherapeutic agent refers to a single molecule of the second immunotherapeutic agent; a half-molecule of the first immunotherapeutic agent refers to a single half-molecule of the first immunotherapeutic agent; a half-molecule of the second immunotherapeutic agent refers to a single half-molecule of the second immunotherapeutic agent; and the chimeric immunotherapeutic agent refers to a single molecule of the chimeric immunotherapeutic agent. The term "cysteine" refers to cysteine ​​in "a mutation from the natural amino acid to cysteine". The chimeric antibody further comprises: (i) a heavy chain from the first IgG covalently bound to a light chain from the first IgG; and (ii) a heavy chain from the second IgG covalently bound to a light chain from the second IgG. The various methods disclosed herein require at least two chimeric immunotherapeutic agents, for example, to form a dimer immunotherapeutic agent, and in practice, the incubation step of this section typically produces multiple chimeric immunotherapeutic agent molecules comprising the chimeric immunotherapeutic agent.

[0059] In some embodiments, the method includes incubating a solution under reducing conditions such that at least a portion of a half-molecule of a first immunotherapeutic agent recombines with at least a portion of a half-molecule of a second immunotherapeutic agent to produce a chimeric immunotherapeutic agent that is chimeric at least because it simultaneously contains: (i) a heavy chain from a first IgG containing the cysteine; and (ii) a heavy chain from a second IgG that does not contain the cysteine. In some specific embodiments, the method includes incubating a solution under reducing conditions such that at least a portion of a half-molecule of a first immunotherapeutic agent recombines with at least a portion of a half-molecule of a second immunotherapeutic agent to produce a chimeric immunotherapeutic agent that is chimeric at least because it simultaneously contains: (i) a heavy chain containing the cysteine ​​from a first IgG; and (ii) a heavy chain without the cysteine ​​from a second IgG, wherein the first immunotherapeutic agent refers to a plurality of molecules of the first immunotherapeutic agent; the second immunotherapeutic agent refers to a plurality of molecules of the second immunotherapeutic agent; a half-molecule of the first immunotherapeutic agent refers to a plurality of half-molecules of the first immunotherapeutic agent; a half-molecule of the second immunotherapeutic agent refers to a plurality of half-molecules of the second immunotherapeutic agent; and the chimeric immunotherapeutic agent refers to a plurality of molecules of the chimeric immunotherapeutic agent. The term "cysteine" refers to cysteine ​​in "a mutation from a natural amino acid to cysteine". Chimeric antibodies typically also include: (i) a light chain from a first IgG, which is covalently bound to a heavy chain from the first IgG; and (ii) a light chain from a second IgG, which is covalently bound to a heavy chain from the second IgG.

[0060] In some embodiments, the method includes incubating a solution under reducing conditions such that at least a portion of a half-molecule of a first immunotherapeutic agent recombines with at least a portion of a half-molecule of a second immunotherapeutic agent to produce a chimeric immunotherapeutic agent that is chimeric at least because it contains both of the following: (i) a light chain of a first IgG containing the cysteine; and (ii) a light chain of a second IgG not containing the cysteine. In some specific embodiments, the method includes incubating a solution under reducing conditions such that at least a portion of a half-molecule of a first immunotherapeutic agent recombines with at least a portion of a half-molecule of a second immunotherapeutic agent to produce a chimeric immunotherapeutic agent that is chimeric at least because it simultaneously contains: (i) a light chain of a first IgG containing the cysteine; and (ii) a light chain of a second IgG not containing the cysteine, wherein the first immunotherapeutic agent refers to a plurality of molecules of the first immunotherapeutic agent; the second immunotherapeutic agent refers to a plurality of molecules of the second immunotherapeutic agent; a half-molecule of the first immunotherapeutic agent refers to a plurality of half-molecules of the first immunotherapeutic agent; a half-molecule of the second immunotherapeutic agent refers to a plurality of half-molecules of the second immunotherapeutic agent; and the chimeric immunotherapeutic agent refers to a plurality of molecules of the chimeric immunotherapeutic agent. The term "cysteine" refers to cysteine ​​in "a mutation from a natural amino acid to cysteine". The chimeric antibody further comprises: (i) a heavy chain from a first IgG, which is covalently bound to a light chain of the first IgG; and (ii) a heavy chain from a second IgG, which is covalently bound to a light chain of the second IgG.

[0061] In some embodiments, the method includes incubating a solution containing a first immunotherapeutic agent and a second immunotherapeutic agent under reducing conditions to reduce one or more disulfide bonds of the first group and one or more disulfide bonds of the second group, such that: (a) the two heavy chains of the first immunotherapeutic agent dissociate to produce a half-molecule of the first immunotherapeutic agent; (b) the two heavy chains of the second immunotherapeutic agent dissociate to produce a half-molecule of the second immunotherapeutic agent; and (c) a half-molecule of the first immunotherapeutic agent recombines with a half-molecule of the second immunotherapeutic agent to produce a chimeric immunotherapeutic agent, which is chimeric at least because it contains both: (i) a heavy chain containing the cysteine ​​from the first IgG; and (ii) a heavy chain from the second IgG that does not contain the cysteine.

[0062] In some embodiments, the method includes incubating a solution containing a first immunotherapeutic agent and a second immunotherapeutic agent under reducing conditions to reduce one or more disulfide bonds of the first group and one or more disulfide bonds of the second group, such that: (a) the two heavy chains of the first immunotherapeutic agent dissociate to produce a half-molecule of the first immunotherapeutic agent; (b) the two heavy chains of the second immunotherapeutic agent dissociate to produce a half-molecule of the second immunotherapeutic agent; and (c) a half-molecule of the first immunotherapeutic agent recombines with a half-molecule of the second immunotherapeutic agent to produce a chimeric immunotherapeutic agent, which is chimeric at least because it contains both of the following: (i) a light chain of the first IgG containing the cysteine; and (ii) a light chain of the second IgG not containing the cysteine.

[0063] In some embodiments, the method includes incubating a solution containing a first immunotherapeutic agent and a second immunotherapeutic agent under reducing conditions to reduce one or more disulfide bonds of the first group and one or more disulfide bonds of the second group, such that: (a) at least a portion of the two heavy chains of the first immunotherapeutic agent dissociates to produce a half-molecule of the first immunotherapeutic agent; (b) at least a portion of the two heavy chains of the second immunotherapeutic agent dissociates to produce a half-molecule of the second immunotherapeutic agent; and (c) at least a portion of the half-molecule of the first immunotherapeutic agent recombines with at least a portion of the half-molecule of the second immunotherapeutic agent to produce a chimeric immunotherapeutic agent, which is chimeric at least because it contains both of the following: (i) a heavy chain containing the cysteine ​​from the first IgG; and (ii) a heavy chain from the second IgG that does not contain the cysteine.

[0064] In some embodiments, the method includes incubating a solution containing a first immunotherapeutic agent and a second immunotherapeutic agent under reducing conditions to reduce one or more disulfide bonds of the first group and one or more disulfide bonds of the second group, such that: (a) at least a portion of the two heavy chains of the first immunotherapeutic agent dissociates to produce a half-molecule of the first immunotherapeutic agent; (b) at least a portion of the two heavy chains of the second immunotherapeutic agent dissociates to produce a half-molecule of the second immunotherapeutic agent; and (c) at least a portion of the half-molecule of the first immunotherapeutic agent recombines with at least a portion of the half-molecule of the second immunotherapeutic agent to produce a chimeric immunotherapeutic agent, which is chimeric at least because it contains both of the following: (i) a light chain of the first IgG containing the cysteine; and (ii) a light chain of the second IgG not containing the cysteine.

[0065] In some embodiments, the method includes incubating the chimeric immunotherapeutic agent under oxidative conditions, such that one or more cysteine ​​residues from the heavy chain of a first IgG in the chimeric immunotherapeutic agent form one or more disulfide bonds with one or more cysteine ​​residues from the heavy chain of a second IgG; wherein the one or more cysteine ​​residues from the heavy chain of the first IgG originally participated in forming one or more disulfide bonds of a first group, while the one or more cysteine ​​residues from the heavy chain of the second IgG originally participated in forming one or more disulfide bonds of a second group.

[0066] In this disclosure, the term "cysteine" in "one or more cysteines" (as described above) does not include "the cysteine" in "mutations of natural amino acids to cysteine," because "cysteine" in "one or more cysteines" refers to cysteine ​​that crosslinks two half-molecules of IgG, while "the cysteine" in "mutations of natural amino acids to cysteine" crosslinks two chimeric IgGs of a dimer immunotherapeutic agent. "Molecular cysteine" does not constitute part of any amino acid sequence but is soluble in solution, such that it exists, for example, as a zwitterion containing an ammonium group and a carboxyl group.

[0067] In some embodiments, the method includes incubating the chimeric immunotherapeutic agent under oxidative conditions to form a disulfide bond between (a) the cysteine ​​residue of a first molecule of the chimeric immunotherapeutic agent from the heavy chain of a first IgG; and (b) the cysteine ​​residue of a second molecule of the chimeric immunotherapeutic agent from the heavy chain of the first IgG, thereby producing a dimer immunotherapeutic agent.

[0068] In this disclosure, the plural terms “one or more disulfide bonds” and “disulfide bond” do not include the singular form of said disulfide bond (such as “one disulfide bond” in the preceding paragraph), because the disulfide bond in “one or more disulfide bonds” and “disulfide bond” refers to a disulfide bond that crosslinks two half-molecules of IgG, while “said disulfide bond” (such as “one disulfide bond” in the preceding paragraph) crosslinks two chimeric IgGs of a dimer immunotherapeutic agent.

[0069] In some embodiments, the method includes incubating the chimeric immunotherapeutic agent under oxidative conditions to form a disulfide bond between (a) the cysteine ​​residue of a first molecule of the chimeric immunotherapeutic agent from the light chain of a first IgG; and (b) the cysteine ​​residue of a second molecule of the chimeric immunotherapeutic agent from the light chain of the first IgG, thereby producing a dimer immunotherapeutic agent.

[0070] In some embodiments, the following two operations constitute the same incubation process: (1) incubating the chimeric immunotherapeutic agent under oxidative conditions to form one or more disulfide bonds between one or more cysteine ​​residues derived from the heavy chain of the first IgG and one or more cysteine ​​residues derived from the heavy chain of the second IgG; and (2) incubating the chimeric immunotherapeutic agent under oxidative conditions to obtain the dimer immunotherapeutic agent. For example, in the same incubation process, the formation of the one or more disulfide bonds and the preparation of the dimer immunotherapeutic agent can be performed in any order, simultaneously, or in a sequence that is difficult to distinguish clearly.

[0071] In some embodiments, the first IgG specifically binds to antigens selected from the following: 4-1BB, 5′-nucleotidase, 5T4, activin receptor-like kinase 1, alpha-fetoprotein, angiopoietin 2, AXL, B7-H3, B cell activating factor (BAFF), B cell maturation antigen (BCMA), B cell receptor (BCR), c-Met, C242, CA-125, CanAg, carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen, CCR4, CCR5, CD3, CD4, CD19, CD20, CD22, CD23, CD25, CD27, CD28, CD30, CD33, CD37, CD38, CD40, CD44, CD51, CD56, CD70, CD74, CD79B, CD80, CD123, CD134, CD152, CD200, CD276, CD319, CEACAM5, and claudin. 18. Coagulation factor III, connective tissue growth factor (CTGF), colony-stimulating factor 1 (CSF1), colony-stimulating factor 1 receptor (CSF1R), colony-stimulating factor 2 (CSF2), CTLA-4, CXCR4, dendritic cell-associated lectin 2, DLL3, DLL4, DR5, EGFL7, EGFR, endothelial glycoprotein, EpCAM, liver glycoside receptor A3 (EPHA3), epidermal growth factor receptor (EGFR), ERBB3 (HER3), ERB4, fibroblast activation protein α (FAP), FGFR2, fibronectin extradomain B, folate hydrolase, folate receptor 1, Frizzled receptor, GD2 ganglioside, GD3 ganglioside, gelatinase B, glycoprotein 100 (gp100), phosphatidylinositol proteoglycan 3, GPNMB, G protein-coupled receptor 5D (GPRC5D), GUCY2C, hepatocyte growth factor (HGF), HER1, HER2, HGFR, histone complex, HLA-DR, human scattering factor receptor kinase, IGF-1 receptor (IGF-1R);CD221), IGF-2, Interleukin 1α, Interleukin-2, Interleukin-6, Interleukin-13, Integrin alph5beta1, Integrin alphaVbeta3, KIR2D, LAG3, Lewis-Y antigen, LIV-1, LRRC15, Macrophage migration inhibitory factor (MIF), MCP-1, Melanoma cell adhesion molecule (MCAM), Mesothelin, MUC1, MUC5AC, Nectin-4, NGNA gangliosides, Notch 1, Notch receptor, NRP1, PCDC1, PD-1, PD-L1, PDGFRA, Sodium phosphate cotransporter, Phosphatidylserine, PTK7, Root plate-specific spondin 3, ROR1, SDC1, SLAMF7, SLITRK6, Sp17, STEAP1, Syndecan 1. TEM1, tendinin C, TGF-β, TIGIT, TRAIL-R1, TRAIL-R2, tumor-associated calcium signaling transducer 2 (TROP-2), tumor antigen CTAA16.88, tumor-specific glycosylated MUC1, tumor-associated glycoprotein 72 (TAG-72), TWEAK receptor, TYRP1, VEGF-A, VEGFR-1, VEGFR-2, and vimentin. In some specific embodiments, the first IgG binds to Sp17 (human spermin 17).

[0072] In some implementations, the second IgG specifically binds to the antigen described in the preceding segment.

[0073] The first IgG and the second IgG may bind to the same or different antigens. In some specific embodiments, the first IgG and the second IgG bind to the same antigen.

[0074] The first IgG and the second IgG can bind to the same or different epitopes of the antigen. In some specific embodiments, the first IgG and the second IgG bind to the same epitope.

[0075] In some embodiments, (1) the first IgG has the amino acid sequence of the first light chain variable domain complementarity-determining region 1 (VL CDR1), the first VL CDR2, the first VL CDR3, the first heavy chain variable domain CDR1 (VH CDR1), the first VH CDR2, and the first VH CDR3; (2) the second IgG has the amino acid sequence of the second VL CDR1, the second VL CDR2, the second VL CDR3, the second VHCDR1, the second VH CDR2, and the second VH CDR3; (3) the first VL CDR1 amino acid sequence and the second VL CDR1 amino acid sequence are identical; (4) the first VL CDR2 amino acid sequence and the second VL CDR2 amino acid sequence are identical; (5) the first VL CDR3 amino acid sequence and the second VL CDR3 amino acid sequence are identical; (6) the first VH CDR1 amino acid sequence and the second VH CDR1 amino acid sequence are identical; (7) the first VH CDR2 amino acid sequence and the second VH CDR3 amino acid sequence are identical. The CDR2 amino acid sequence is the same; and (8) the first VH CDR3 amino acid sequence and the second VH CDR3 amino acid sequence are the same.

[0076] In some embodiments, (1) the first IgG has a first light chain variable domain amino acid sequence and a first heavy chain variable domain amino acid sequence; (2) the second IgG has a second light chain variable domain amino acid sequence and a second heavy chain variable domain amino acid sequence; (3) the first light chain variable domain amino acid sequence and the second light chain variable domain amino acid sequence are identical; and (4) the first heavy chain variable domain amino acid sequence and the second heavy chain variable domain amino acid sequence are identical.

[0077] In some embodiments, (1) the reducing agent, the first immunotherapy agent, and the second immunotherapy agent in the solution each have a corresponding molar concentration; (2) the solution has a total molar concentration of the first immunotherapy agent and the second immunotherapy agent, the total molar concentration being equal to the sum of the molar concentrations of the first immunotherapy agent and the second immunotherapy agent; and (3) the molar concentration of the reducing agent is greater than the total molar concentration. In some specific embodiments, the molar concentration of the reducing agent is at least twice the total molar concentration (e.g., the total molar concentration is 7.5 mmol / L, and the molar concentration of the reducing agent is at least 15 mmol / L). In some more specific embodiments, the molar concentration of the reducing agent is at least four times the total molar concentration (e.g., the total molar concentration is 7.5 mmol / L, and the molar concentration of the reducing agent is at least 30 mmol / L).

[0078] In some embodiments, the reducing agent is selected from β-mercaptoethanol, molecular cysteine, cysteamine, and glutathione. In some specific embodiments, the reducing agent is cysteamine.

[0079] In some embodiments, the concentration of the reducing agent is at least 5 mmol and no more than or greater than 750 mmol. In some specific embodiments, the concentration of the reducing agent is at least 10 mmol and no more than 500 mmol. In some very specific embodiments, the concentration of the reducing agent is at least 25 mmol and no more than 250 mmol.

[0080] In some embodiments, the method includes purifying the chimeric immunotherapeutic agent, wherein (1) the solution contains a reducing agent; and (2) the purification separates the chimeric immunotherapeutic agent from the reducing agent. In some specific embodiments, the method includes purifying the chimeric immunotherapeutic agent, wherein (1) the solution contains a reducing agent; (2) the purification separates the chimeric immunotherapeutic agent from the reducing agent; and (3) incubating the chimeric immunotherapeutic agent under oxidative conditions includes the purification.

[0081] The purification method is not limited. In some embodiments, the purification is selected from dialysis, protein A purification, protein G purification, protein A / G purification, protein L purification, ammonium sulfate precipitation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, thiophilic adsorption, and immobilized metal chelation chromatography.

[0082] In some embodiments, the purification of the chimeric immunotherapeutic agent is performed simultaneously with incubation of the chimeric immunotherapeutic agent under oxidative conditions. For example, the purification may involve exposing the chimeric immunotherapeutic agent to oxidative conditions.

[0083] In some embodiments, incubating the chimeric immunotherapeutic agent under oxidative conditions includes incubating the chimeric immunotherapeutic agent at a pH of at least 7.0. In some specific embodiments, incubating the chimeric immunotherapeutic agent under oxidative conditions includes incubating the chimeric immunotherapeutic agent at a pH of at least 7.0 and not exceeding 8.0. In some very specific embodiments, incubating the chimeric immunotherapeutic agent under oxidative conditions includes incubating the chimeric immunotherapeutic agent at a pH of at least 7.2 and not exceeding 7.8. For example, incubating the chimeric immunotherapeutic agent at a pH of at least 7.0 in the presence of dissolved oxygen may be sufficient to constitute oxidative conditions because such conditions deprotonate cysteine.

[0084] In some embodiments, the first IgG is human IgG1, with the natural amino acid S444, and mutated to S444C. In some specific embodiments, the first IgG is human IgG1; with the natural amino acid S444; mutated to S444C; and optionally contains one or more other substitutions, deletions, and / or insertions.

[0085] In this disclosure, the amino acid positions of IgG are defined according to the EU numbering system proposed by Kabat, EA et al., in “Sequences of proteins of immunological interest.”, 5th edition (US Department of Health and Human Services, NIH Publication No. 91-3242, 1991) (hereinafter referred to as “Kabat”), the entire contents of which are incorporated herein by reference.

[0086] In some embodiments, the first IgG is human IgG1, with the natural amino acid S119, and mutated to S119C. In some specific embodiments, the first IgG is human IgG1; with the natural amino acid S119; mutated to S119C; and optionally contains one or more other substitutions, deletions, and / or insertions.

[0087] S119C and S444C are representative mutations that convert natural amino acid residues to cysteine, which are known to enable efficient cross-linking of IgG1 antibodies. Other suitable natural amino acid residues for conversion to cysteine ​​can be identified, for example, by analyzing the crystal structure of IgG to identify natural amino acid residues with a solvent-accessible surface area greater than that of the embedded natural amino acid residues. Other suitable natural amino acid residues may include, for example, asparagine residues that would otherwise be glycosylated. Other suitable natural amino acid residues may also include, for example: (1) alanine residues with a solvent-accessible surface area greater than that of other alanine residues in IgG; (2) serine residues; (3) threonine residues; (4) aspartic acid residues; (5) glutamic acid residues; (6) asparagine residues; (7) glutamine residues; (8) histidine residues; (9) arginine residues; (10) lysine residues; (11) methionine residues; and (12) tyrosine residues. However, suitable natural amino acid residues for mutation sites are not limited to the residues mentioned above; even hydrophobic amino acid residues can be suitable natural amino acid residues as long as they have a significant solvent-accessible surface area.

[0088] In some embodiments, the first IgG is human IgG1, and the first IgG contains a mutation at F405, and also contains one or more other substitutions, deletions, and / or insertions, including mutations from a native amino acid to the cysteine ​​residue. In some specific embodiments, the mutation is selected from F405A, F405D, F405E, F405G, F405H, F405I, F405K, F405L, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In some very specific embodiments, the mutation is F405L.

[0089] In some embodiments, the first IgG is human IgG1, and the first IgG comprises one, two, three, or four mutations in one, two, three, or each of L368, D399, F405, and Y407, and also comprises one or more other substitutions, deletions, and / or insertions, including mutations in the natural amino acid to the cysteine. In some specific embodiments, the mutation is selected from L368A, L368D, L368E, L368G, L368H, L368I, L368N, L368Q, L368R, L368S, L368T, L368V, L368W, D399A, D399F, D399H, D399K, D399R, D399Y, F405A, F405D, F405E, F405G, F405H, F405I, F405K, F405L, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, F405Y, Y407G, Y407L, Y407M, and Y407W.

[0090] In some embodiments, the second IgG is human IgG1, and the second IgG contains a mutation at K409, and optionally also contains one or more other substitutions, deletions, and / or insertions. In some specific embodiments, the mutation is selected from K409A, K409C, K409D, K409E, K409F, K409G, K409H, K409I, K409N, K409P, K409Q, K409R, K409S, K409T, K409V, K409W, and K409Y. In some very specific embodiments, the mutation is K409R.

[0091] In some embodiments, the first IgG is human IgG1; the first IgG contains a mutation at F405 and also contains one or more other substitutions, deletions and / or insertions, including mutations from a natural amino acid to the cysteine; the second IgG is human IgG1; and the second IgG contains a mutation at K409 and optionally also contains one or more other substitutions, deletions and / or insertions. In some specific embodiments, the mutation at F405 is selected from F405A, F405D, F405E, F405G, F405H, F405I, F405K, F405L, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y; and the mutation at K409 is selected from K409A, K409C, K409D, K409E, K409F, K409G, K409H, K409I, K409N, K409P, K409Q, K409R, K409S, K409T, K409V, K409W, and K409Y. In some very specific implementations, the mutation at F405 is F405L, and the mutation at K409 is K409R.

[0092] In some embodiments, the first IgG is human IgG1; the first IgG comprises one, two, three, or four mutations in one, two, three, or each of L368, D399, F405, and Y407, and also comprises one or more other substitutions, deletions, and / or insertions, including mutations in the natural amino acid to the cysteine; the second IgG is human IgG1; and the second IgG comprises a mutation at K409, and optionally also comprises one or more other substitutions, deletions, and / or insertions. In some specific embodiments, the mutations in one, two, three, or each of L368, D399, F405, and Y407 are selected from L368A, L368D, L368E, L368G, L368H, L368I, L368N, L368Q, L368R, L368S, L368T, L368V, L368W, D399A, D399F, D399H, D399K, D399R, D399Y, F405A, F405D, F405E, F405G, F405H, and F405. I, F405K, F405L, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, F405Y, Y407G, Y407L, Y407M, and Y407W; and the mutation at K409 is selected from K409A, K409C, K409D, K409E, K409F, K409G, K409H, K409I, K409N, K409P, K409Q, K409R, K409S, K409T, K409V, K409W, and K409Y. In some very specific implementations, the mutations for one, two, three, or each of L368, D399, F405, and Y407 include F405L, and the mutation at K409 is K409R.

[0093] In some embodiments, the first IgG is human IgG1, and the first IgG comprises a mutation at K409, and also comprises one or more other substitutions, deletions, and / or insertions, including mutations in the natural amino acid to the cysteine ​​residue. In some specific embodiments, the mutation is selected from K409A, K409C, K409D, K409E, K409F, K409G, K409H, K409I, K409N, K409P, K409Q, K409R, K409S, K409T, K409V, K409W, and K409Y. In some very specific embodiments, the mutation is K409R.

[0094] In some embodiments, the second IgG is human IgG1, and the second IgG contains a mutation at F405, and optionally also contains one or more other substitutions, deletions, and / or insertions. In some specific embodiments, the mutation is selected from F405A, F405D, F405E, F405G, F405H, F405I, F405K, F405L, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In some very specific embodiments, the mutation is F405L.

[0095] In some embodiments, the second IgG is human IgG1, and the second IgG contains one, two, three, or four mutations in one, two, three, or each of L368, D399, F405, and Y407, and optionally also contains one or more other substitutions, deletions, and / or insertions. In some specific embodiments, the mutation is selected from L368A, L368D, L368E, L368G, L368H, L368I, L368N, L368Q, L368R, L368S, L368T, L368V, L368W, D399A, D399F, D399H, D399K, D399R, D399Y, F405A, F405D, F405E, F405G, F405H, F405I, F405K, F405L, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, F405Y, Y407G, Y407L, Y407M, and Y407W.

[0096] In some embodiments, the first IgG is human IgG1; the first IgG contains a mutation at K409 and also contains one or more other substitutions, deletions and / or insertions, including mutations from a natural amino acid to the cysteine; the second IgG is human IgG1; and the second IgG contains a mutation at F405 and optionally also contains one or more other substitutions, deletions and / or insertions. In some specific embodiments, the mutation at K409 is selected from K409A, K409C, K409D, K409E, K409F, K409G, K409H, K409I, K409N, K409P, K409Q, K409R, K409S, K409T, K409V, K409W, and K409Y; and the mutation at F405 is selected from F405A, F405D, F405E, F405G, F405H, F405I, F405K, F405L, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, and F405Y. In some very specific implementations, the mutation at K409 is K409R, and the mutation at F405 is F405L.

[0097] In some embodiments, the first IgG is human IgG1; the first IgG contains a mutation at K409 and also contains one or more other substitutions, deletions and / or insertions, including mutations from a natural amino acid to the cysteine; the second IgG is human IgG1; and the second IgG contains one, two, three or each of one, two, three or four mutations in L368, D399, F405 and Y407, and optionally also contains one or more other substitutions, deletions and / or insertions. In some specific embodiments, the mutation at K409 is selected from K409A, K409C, K409D, K409E, K409F, K409G, K409H, K409I, K409N, K409P, K409Q, K409R, K409S, K409T, K409V, K409W, and K409Y; and the mutations for one, two, three, or each of L368, D399, F405, and Y407 are selected from L368A, L368D, L368E, L368G, L368H, L368... I, L368N, L368Q, L368R, L368S, L368T, L368V, L368W, D399A, D399F, D399H, D399K, D399R, D399Y, F405A, F405D, F405E, F4 05G, F405H, F405I, F405K, F405L, F405M, F405N, F405Q, F405S, F405T, F405V, F405W, F405Y, Y407G, Y407L, Y407M and Y407W. In some very specific implementations, the mutation at K409 is K409R, and the mutations for one, two, three, or each of L368, D399, F405, and Y407 include F405L.

[0098] In some embodiments, the 409th amino acid of the first IgG is lysine, and the 409th amino acid of the second IgG is arginine.

[0099] In some embodiments, the 405th amino acid of the first IgG is phenylalanine, and the 405th amino acid of the second IgG is leucine.

[0100] In some embodiments, the 409th amino acid of the first IgG is lysine and the 405th amino acid is phenylalanine, and the 409th amino acid of the second IgG is arginine and the 405th amino acid is leucine.

[0101] In some embodiments, the 409th amino acid of the first IgG is arginine, and the 409th amino acid of the second IgG is lysine.

[0102] In some embodiments, the 405th amino acid of the first IgG is leucine, and the 405th amino acid of the second IgG is phenylalanine.

[0103] In some embodiments, the 409th amino acid of the first IgG is arginine and the 405th amino acid is leucine, and the 409th amino acid of the second IgG is lysine and the 405th amino acid is phenylalanine.

[0104] In some embodiments, the first IgG is human IgG1; the second IgG is human IgG1; and satisfies one of the following (a) or (b): (a)(i) the first IgG contains the F405L mutation and (ii) the second IgG contains the K409R mutation; or (b)(i) the first IgG contains the K409R mutation and (ii) the second IgG contains the F405L mutation.

[0105] Other non-limiting mutation embodiments that may be performed on one or both of the first IgG and the second IgG are described, for example, in U.S. Patent No. 5,731,168 (Genentech), U.S. Patent No. 8,592,562 (Amgen), U.S. Patent No. 9,505,848 (Merck), U.S. Patent No. 10,011,858 (Chugai), and U.S. Patent No. 10,597,464 (Genmab), all of which are incorporated herein by reference in their entirety.

[0106] Various aspects of this disclosure relate to dimer immunotherapeutic agents prepared according to the methods described herein. Such dimer immunotherapeutic agents comprise two chimeric immunotherapeutic agents described herein, said two chimeric immunotherapeutic agents being cross-linked via disulfide bonds.

[0107] Various aspects of this disclosure relate to chimeric immunotherapeutic agents prepared according to the methods described herein, which include mutations in the natural amino acid cysteine.

[0108] Various aspects of this disclosure relate to a dimeric immunotherapeutic agent comprising a first chimeric immunotherapeutic agent and a second chimeric immunotherapeutic agent. In some embodiments, the first chimeric immunotherapeutic agent and the second chimeric immunotherapeutic agent have the same amino acid sequence. However, differences may still exist between the first chimeric immunotherapeutic agent and the second chimeric immunotherapeutic agent. For example, such differences may be caused by post-translational modifications (such as heterogeneity in glycosylation patterns); when the chimeric immunotherapeutic agent is an immunoconjugate, heterogeneity in chemical conjugation may also lead to such differences. The first chimeric immunotherapeutic agent and the second chimeric immunotherapeutic agent are "chimeric" because they are prepared from "half-molecules" of two different immunotherapeutic agents (e.g., the first immunotherapeutic agent and the second immunotherapeutic agent) described herein.

[0109] In some embodiments, (1) the first chimeric immunotherapy agent comprises a first chimeric IgG, the first chimeric IgG comprising a first light chain, a first heavy chain, a second light chain, and a second heavy chain each having an amino acid sequence; (2) the second chimeric immunotherapy agent comprises a second chimeric IgG, the second chimeric IgG comprising a first light chain, a first heavy chain, a second light chain, and a second heavy chain each having an amino acid sequence; (3) the first light chain of the first chimeric IgG and the first light chain of the second chimeric IgG have the same amino acid sequence; (4) the second light chain of the first chimeric IgG and the second light chain of the second chimeric IgG have the same amino acid sequence; (5) the first heavy chain of the first chimeric IgG and the first heavy chain of the second chimeric IgG have the same amino acid sequence; (6) the second heavy chain of the first chimeric IgG and the second heavy chain of the second chimeric IgG have the same amino acid sequence; (7) the first heavy chain of the first chimeric IgG and the first heavy chain of the second chimeric IgG each have an amino acid sequence; (8) A mutation comprising natural amino acids up to cysteine; (9) Cysteine ​​in the amino acid sequence of the first heavy chain of the first chimeric IgG and cysteine ​​in the amino acid sequence of the first heavy chain of the second chimeric IgG forming a disulfide bond with each other; (10) The amino acid sequences of the second heavy chain of the first chimeric IgG and the second heavy chain of the second chimeric IgG each do not have the mutation; (11) The first chimeric immunotherapy is chimeric at least for the following reasons: the amino acid sequence of the first heavy chain of the first chimeric IgG contains the mutation, while the amino acid sequence of the second heavy chain of the first chimeric IgG does not have the mutation, such that the first chimeric IgG comprises two different heavy chains; and (12) The second chimeric immunotherapy is chimeric at least for the following reasons: the amino acid sequence of the first heavy chain of the second chimeric IgG contains the mutation, while the amino acid sequence of the second heavy chain of the second chimeric IgG does not have the mutation, such that the second chimeric IgG comprises two different heavy chains.

[0110] In some embodiments, (1) the first chimeric immunotherapy agent comprises a first chimeric IgG, the first chimeric IgG comprising a first light chain, a first heavy chain, a second light chain, and a second heavy chain, each having an amino acid sequence; (2) the second chimeric immunotherapy agent comprises a second chimeric IgG, the second chimeric IgG comprising a first light chain, a first heavy chain, a second light chain, and a second heavy chain, each having an amino acid sequence; (3) the first light chain of the first chimeric IgG and the first light chain of the second chimeric IgG have the same amino acid sequence; (4) the second light chain of the first chimeric IgG and the second light chain of the second chimeric IgG have the same amino acid sequence; (5) the first heavy chain of the first chimeric IgG and the first heavy chain of the second chimeric IgG have the same amino acid sequence; (6) the second heavy chain of the first chimeric IgG and the second heavy chain of the second chimeric IgG have the same amino acid sequence; (7) the first light chain of the first chimeric IgG and the first light chain of the second chimeric IgG each have an amino acid sequence. (8) A mutation from natural amino acids to cysteine; (9) Cysteine ​​in the amino acid sequence of the first light chain of the first chimeric IgG and cysteine ​​in the amino acid sequence of the first light chain of the second chimeric IgG form a disulfide bond; (10) The amino acid sequences of the second light chain of the first chimeric IgG and the second light chain of the second chimeric IgG do not each have the mutation; (11) The first chimeric immunotherapy is chimeric at least because the amino acid sequence of the first light chain of the first chimeric IgG contains the mutation, while the amino acid sequence of the second light chain of the first chimeric IgG does not have the mutation, such that the first chimeric IgG contains two different light chains; and (12) The second chimeric immunotherapy is chimeric at least because the amino acid sequence of the first light chain of the second chimeric IgG contains the mutation, while the amino acid sequence of the second light chain of the second chimeric IgG does not have the mutation, such that the second chimeric IgG contains two different light chains.

[0111] In some embodiments, both the first chimeric IgG and the second chimeric IgG are chimeric human IgG1; the natural amino acid is S444; and they are mutated to S444C.

[0112] In some embodiments, both the first chimeric IgG and the second chimeric IgG are chimeric human IgG1; the natural amino acid is S119; and they are mutated to S119C.

[0113] In some embodiments, the first heavy chain of the first chimeric IgG and the second chimeric IgG each includes an F405L mutation. In some embodiments, the amino acid sequences of the second heavy chain of the first chimeric IgG and the second heavy chain of the second chimeric IgG each include a K409R mutation. In some specific embodiments, the amino acid sequences of the first heavy chain of the first chimeric IgG and the first heavy chain of the second chimeric IgG each include an F405L mutation; and the amino acid sequences of the second heavy chain of the first chimeric IgG and the second heavy chain of the second chimeric IgG each include a K409R mutation.

[0114] In some embodiments, the amino acid sequences of the first heavy chain of the first chimeric IgG and the first heavy chain of the second chimeric IgG each include a K409R mutation. In some embodiments, the amino acid sequences of the second heavy chain of the first chimeric IgG and the second heavy chain of the second chimeric IgG each include an F405L mutation. In some specific embodiments, both the first chimeric IgG and the second chimeric IgG are chimeric human IgG1; the amino acid sequences of the first heavy chain of the first chimeric IgG and the first heavy chain of the second chimeric IgG each include a K409R mutation; and the amino acid sequences of the second heavy chain of the first chimeric IgG and the second heavy chain of the second chimeric IgG each include an F405L mutation.

[0115] In some embodiments, the first heavy chain of the first chimeric IgG, the second heavy chain of the first chimeric IgG, the first heavy chain of the second chimeric IgG, and the second heavy chain of the second chimeric IgG each include a heavy chain variable region having the same amino acid sequence; and the first light chain of the first chimeric IgG, the second light chain of the first chimeric IgG, the first light chain of the second chimeric IgG, and the second light chain of the second chimeric IgG each include a light chain variable region having the same amino acid sequence.

[0116] In some embodiments, both the first chimeric IgG and the second chimeric IgG are chimeric human IgG1; the natural amino acid is S444; the mutation is S444C; the amino acid sequences of the first heavy chain of the first chimeric IgG and the first heavy chain of the second chimeric IgG each include the F405L mutation; the amino acid sequences of the second heavy chain of the first chimeric IgG and the second heavy chain of the second chimeric IgG each include the K409R mutation; the first heavy chain of the first chimeric IgG, the second heavy chain of the first chimeric IgG, the first heavy chain of the second chimeric IgG and the second heavy chain of the second chimeric IgG each contain a heavy chain variable region having the same amino acid sequence; and the first light chain of the first chimeric IgG, the second light chain of the first chimeric IgG, the first light chain of the second chimeric IgG and the second light chain of the second chimeric IgG each contain a light chain variable region having the same amino acid sequence.

[0117] In some embodiments, the first light chain of the first chimeric immunotherapy agent, the second light chain of the first chimeric immunotherapy agent, the first light chain of the second chimeric immunotherapy agent, and the second light chain of the second chimeric immunotherapy agent have the same amino acid sequence.

[0118] In some embodiments, the first heavy chain of the first chimeric immunotherapy agent, the second heavy chain of the first chimeric immunotherapy agent, the first heavy chain of the second chimeric immunotherapy agent, and the second heavy chain of the second chimeric immunotherapy agent have the same amino acid sequence.

[0119] In some embodiments, the first heavy chain of the first chimeric IgG, the second heavy chain of the first chimeric IgG, the first heavy chain of the second chimeric IgG, and the second heavy chain of the second chimeric IgG each include a heavy chain variable region having the same amino acid sequence; and the first light chain of the first chimeric IgG, the second light chain of the first chimeric IgG, the first light chain of the second chimeric IgG, and the second light chain of the second chimeric IgG each include a light chain variable region having the same amino acid sequence.

[0120] In some embodiments, the first heavy chain of the first chimeric IgG, the second heavy chain of the first chimeric IgG, the first heavy chain of the second chimeric IgG, and the second heavy chain of the second chimeric IgG each comprise: (i) a VH CDR1 region comprising the same amino acid sequence as SEQ ID NO:5; (ii) a VH CDR2 region comprising the same amino acid sequence as SEQ ID NO:6; and (iii) a VH CDR3 region comprising the same amino acid sequence as SEQ ID NO:7; and the first light chain of the first chimeric IgG, the second light chain of the first chimeric IgG, the first light chain of the second chimeric IgG, and the second light chain of the second chimeric IgG each comprise: (i) a VL CDR1 region comprising the same amino acid sequence as SEQ ID NO:8; (ii) a VL CDR2 region comprising the same amino acid sequence as SEQ ID NO:9; and (iii) a VL CDR3 region comprising the same amino acid sequence as SEQ ID NO:10.

[0121] In some embodiments, the first heavy chain of the first chimeric IgG, the second heavy chain of the first chimeric IgG, the first heavy chain of the second chimeric IgG, and the second heavy chain of the second chimeric IgG each include a heavy chain variable region having at least 90% sequence identity with SEQ ID NO:3; and the first light chain of the first chimeric IgG, the second light chain of the first chimeric IgG, the first light chain of the second chimeric IgG, and the second light chain of the second chimeric IgG each include a light chain variable region having at least 90% sequence identity with SEQ ID NO:4. In some specific embodiments, the first heavy chain of the first chimeric IgG, the second heavy chain of the first chimeric IgG, the first heavy chain of the second chimeric IgG, and the second heavy chain of the second chimeric IgG each include a heavy chain variable region having at least 95% sequence identity with SEQ ID NO:3; and the first light chain of the first chimeric IgG, the second light chain of the first chimeric IgG, the first light chain of the second chimeric IgG, and the second light chain of the second chimeric IgG each include a light chain variable region having at least 95% sequence identity with SEQ ID NO:4. In some very specific embodiments, the first heavy chain of the first chimeric IgG, the second heavy chain of the first chimeric IgG, the first heavy chain of the second chimeric IgG, and the second heavy chain of the second chimeric IgG each include a heavy chain variable region having SEQ ID NO:3; and the first light chain of the first chimeric IgG, the second light chain of the first chimeric IgG, the first light chain of the second chimeric IgG, and the second light chain of the second chimeric IgG each include a light chain variable region having SEQ ID NO:4.

[0122] Following the general and specific description of various features of this disclosure in the foregoing detailed embodiments, the following examples provide specific examples of preparing the dimer immunotherapeutic agents described herein. Through these examples and within the context of the foregoing detailed embodiments, those skilled in the art will immediately recognize variations in the methods described in these examples (e.g., selection of different first IgG and / or different second IgG). The following examples are illustrative only and should not limit this disclosure or any patent claims formed by this disclosure. Any patent claim formed by this disclosure should be limited only to the features expressly recited in the context of its dependent relation and interpreted in accordance with the conventional claim interpretation principles applicable to this disclosure.

[0123] Example Example. Method for preparing a dimer immunotherapeutic agent.

[0124] First IgG1 containing the F405L and S444C mutations was cloned, expressed, and purified. The positions of F405L and S444C were defined according to the EU numbering described in Kabat. SDS-PAGE analysis of the first IgG1 is shown in [Figure / Reference]. Figure 2ALane 1 corresponds to 1 microgram of first IgG1 loaded under non-reducing conditions, lane 7 corresponds to 1 microgram of IgG standard loaded under non-reducing conditions, lane 8 corresponds to molecular weight standard, lane 9 corresponds to 2 micrograms of first IgG1 loaded under reducing conditions, and lane 15 corresponds to 2 micrograms of IgG standard loaded under reducing conditions. Figure 2B As shown, the purity of the first IgG1 was approximately 91.96% as determined by liquid chromatography.

[0125] The first IgG1 specifically binds to Sp17. The nucleotide sequences encoding the heavy chain variable region and light chain variable region of the first IgG1 antibody are shown in SEQ ID NO:1 and 2, respectively. The amino acid sequences encoded by these nucleotide sequences are shown in SEQ ID NO:3 and 4, respectively. SEQ ID NO:5 contains the VH CDR1 region of the first IgG1. SEQ ID NO:6 contains the VH CDR2 region of the first IgG1. SEQ ID NO:7 contains the VH CDR3 region of the first IgG1. SEQ ID NO:8 contains the VL CDR1 region of the first IgG1. SEQ ID NO:9 contains the VL CDR2 region of the first IgG1. SEQ ID NO:10 contains the VL CDR3 region of the first IgG1.

[0126] Table 1. Nucleotide sequences of the VH and VL regions of exemplary IgG

[0127]

[0128] Table 2. Amino acid sequences of the VH and VL regions of exemplary IgG

[0129]

[0130] Table 3. Amino acid sequences of CDRs containing the VH and VL regions of exemplary IgG.

[0131]

[0132] A second IgG1 containing the K409R mutation was cloned, expressed, and purified. The position of K409R was defined according to the EU numbering described in Kabat. SDS-PAGE analysis of the second IgG1 is shown in [Figure / Reference]. Figure 3A Lane 6 corresponds to 1 microgram of second IgG1 loaded under non-reducing conditions, lane 7 corresponds to 1 microgram of IgG standard loaded under non-reducing conditions, lane 8 corresponds to molecular weight standard, lane 14 corresponds to 2 micrograms of second IgG1 loaded under reducing conditions, and lane 15 corresponds to 2 micrograms of IgG standard loaded under reducing conditions. Figure 3BAs shown, the purity of the second IgG1 was approximately 92.39% as determined by liquid chromatography.

[0133] Except for the F405L and S444C mutations in the first IgG1 and the K409R mutation in the second IgG1, the amino acid sequences of the first IgG1 and the second IgG1 are identical.

[0134] The second IgG1 specifically binds to Sp17 and contains the same variable region as the first IgG1. The nucleotide sequences encoding the heavy chain and light chain variable regions of the second IgG1 antibody are shown in SEQ ID NO:1 and 2. These nucleotide sequences encode the amino acid sequences shown in SEQ ID NO:3 and 4, respectively. SEQ ID NO:5 contains the VH CDR1 region of the second IgG1. SEQ ID NO:6 contains the VH CDR2 region of the second IgG1. SEQ ID NO:7 contains the VH CDR3 region of the second IgG1. SEQ ID NO:8 contains the VL CDR1 region of the second IgG1. SEQ ID NO:9 contains the VL CDR2 region of the second IgG1. SEQ ID NO:10 contains the VL CDR3 region of the second IgG1.

[0135] 0.5 mg of first IgG1 in 193.8 μL was mixed with 0.5 mg of second IgG1 in 204.9 μL, and then 44.3 μL of 750 mmol / L cysteine ​​was added to obtain a reaction mixture with a total volume of 443 μL, containing 0.5 mg of first IgG1 (approximately 7.5 mmol / L), 0.5 mg of second IgG1 (approximately 7.5 mmol / L), and 75 mmol / L cysteine. The reaction mixture was incubated at 31 °C for 5 hours to selectively reduce the disulfide bonds in the hinge region of IgG1, thereby dissociating IgG1 into half-molecules and allowing the half-molecules of first IgG1 and second IgG1 to recombine to form chimeric IgG1. The F405L and K409R mutations favor the dissociation of the first IgG1 and second IgG1 half-molecules, respectively, and allow these half-molecules to recombine to form chimeric IgG1, thus shifting the equilibrium towards the formation of chimeric IgG1 from the first IgG1 and second IgG1 half-molecules, rather than the reformation of the original first IgG1 and second IgG1.

[0136] Five hours later, the reaction mixture was transferred to a dialysis bag with a molecular weight cutoff of 10 kDa (MWCO) and then to 5 liters of phosphate-buffered saline (PBS) adjusted to pH 7.4. The first round of dialysis was performed for four hours. The dialysis bag was then transferred to 5 liters of fresh PBS (pH 7.4) and dialyzed overnight at 4°C. At the weakly alkaline pH of 7.4, the following equilibrium was formed: some cysteine ​​residues of chimeric IgG1 were deprotonated, resulting in the spontaneous oxidation of the following substances: (1) cysteine ​​residues in the hinge region of chimeric IgG1 formed disulfide bonds that crosslinked the first and second IgG1 molecules in the chimeric IgG1; and (2) S444C cysteine ​​residues of different chimeric IgG1s formed a dimer immunotherapeutic agent.

[0137] After dialysis, the reaction mixture was transferred to a microcentrifuge tube, and 100 μL of 100 mmol / L cysteine ​​was added to block any remaining free cysteine. The blocked reaction mixture was then transferred back to a dialysis bag with a molecular weight of 10 kDa MWCO, and the dialysis bag was transferred to 5 L of 20 mmol / L phosphate buffer (PB) adjusted to pH 6.0. The first round of dialysis was performed for four hours, and then the dialysis bag was transferred to fresh 5 L of PB (pH 6.0) for a second round of dialysis for four hours.

[0138] SDS-PAGE analysis of the reaction mixture is shown in Figure 4A Lane 3 corresponds to 1 microgram of protein from the reaction mixture loaded under non-reducing conditions, lane 7 corresponds to 1 microgram of IgG standard loaded under non-reducing conditions, lane 8 corresponds to molecular weight standard, lane 11 corresponds to 2 micrograms of protein from the reaction mixture loaded under reducing conditions, and lane 15 corresponds to 2 micrograms of IgG standard loaded under reducing conditions. Figure 4A The arrows indicate protein bands showing molecular weights corresponding to the dimer immunotherapeutic agent. Liquid chromatography analysis showed that approximately 28.16% of the proteins in the reaction mixture had molecular weights consistent with the dimer immunotherapeutic agent.

[0139] Any patent claim formed based on this disclosure should not be construed as requiring any feature of the foregoing embodiments. Any method described in the claims or specification, unless expressly stated otherwise, should not be construed as requiring the steps to be performed in a particular order. Unless expressly stated otherwise, the method should be construed as supporting the steps to be performed in any order.

[0140] Certain features described in the context of different implementations may also be combined and implemented in a single implementation. Conversely, various features described in the context of a single implementation may also be implemented separately in multiple implementations or in any suitable sub-combination. Furthermore, although features may be described above as certain combinations, or even initially claimed in this way, in some cases one or more features in the claimed combination may be removed from that combination, and the claimed combination may involve sub-combinations or variations of sub-combinations.

[0141] The exemplary configurations described herein do not represent all examples that are implementable or fall within the scope of the claims. The term "example" should be interpreted as "used as an example, illustration, or description," rather than "preferred" or "superior to other examples."

[0142] Articles such as “the,” “a,” and “an” can indicate singular or plural. When the word “or” is not preceded by “any one” (or other similar wording that clearly indicates the exclusive meaning of “or,” such as only one of x or y), “or” should be interpreted as inclusive (e.g., “x or y” means one or both of x and y).

[0143] The term “and / or” should also be interpreted as inclusive (e.g., “x and / or y” means one or both of x and y). Where “and / or” or “or” is used as a conjunction connecting three or more items, the group of items should be interpreted as including a single item, all items together, or any combination or number of said items.

[0144] The terms “has,” “contain(s),” and “include(s)” should be interpreted as synonymous with the term “comprise(s)” and are inclusive or open-ended, thus not excluding other unlisted subjects. Narrower alternative implementations are also disclosed and supported using the foregoing four terms, wherein these terms are replaced by “consisting” or “consisting essentially of,” the latter two being closed-ended, thereby excluding other unlisted subjects.

[0145] Unless otherwise stated, all numbers or expressions used in the specification (other than the claims), such as those indicating concentration, ratio, quantity, etc., are to be understood as being modified by the term "about" in all cases. At least, and not in an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter modified by the term "about" in the specification or claims shall be interpreted in conjunction with the number of significant figures listed and using conventional rounding methods. All disclosed scopes shall be understood to encompass and support any subscopes included in each scope, as well as any and all claims containing individual numerical values. For example, the scope of "at least 90%" shall be interpreted to include support for at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, and at least 99.9%.

[0146] Terms recited in the claims shall be given their ordinary and customary meaning, determined by reference to relevant entries in widely used general dictionaries, relevant technical references, and meanings commonly understood by those skilled in the art. It should be understood that the broadest meaning given by any one or a combination of these sources shall be given to the claim terms (e.g., two or more relevant references shall be combined to provide the broadest meaning of that combination), subject only to the following two exceptions: (a) when a term is used in a manner broader than its ordinary and customary meaning, it shall be given its ordinary and customary meaning as well as any additional extended meaning; and (b) when a term is explicitly defined with a different meaning by using "in this disclosure" or similar language simultaneously with its definition, the term shall be limited to that definition (e.g., the use of the term "chimera" in relation to antibodies differs from its common usage in the relevant technical field; therefore, "chimera antibody" and similar terms such as "chimera IgG" shall be limited to the scope defined in this disclosure). References to specific examples shall not trigger the foregoing exception (b) nor otherwise limit the scope of the claim terms. Except as provided in the foregoing exception (b), nothing contained herein shall be construed as a disclaimer or waiver of the scope of the claims.

[0147] The subject matter recited in the claims differs from the scope of any embodiment, feature, or combination of features described or illustrated herein, and should not be construed as being identical to its scope. This conclusion applies even if only a single embodiment of a feature or combination of features is shown and described.

[0148] The entire contents of each of the following documents are incorporated herein by reference (collectively, the “Incorporated Documents”). Where the same term is used both herein and in one or more of the Incorporated Documents, it shall be interpreted as having the broadest meaning given to it by any of those sources, or a combination thereof, unless it has been expressly defined herein as having a different meaning. In the event of any incorporation inconsistent with this document, this document shall prevail. The incorporated subject matter shall not be used to limit or narrow the scope of the subject matter expressly recorded or described.

[0149] - U.S. Patent No. 5,731,168 A, entitled "Method for Manufacturing Heteromeric Polypeptides", was granted on March 24, 1998; - U.S. Patent No. 8,592,562 B2, entitled "Method for manufacturing antibody Fc heterodimer molecules using electrostatic guiding effect", was granted on November 26, 2013; - U.S. Patent No. 9,505,848 B2, entitled "Engineered Heterodimeric Protein Domain", was granted on November 29, 2016; - U.S. Patent No. 9,862,769 B2, entitled "Anti-HER2 Monoclonal Antibody", was granted on January 9, 2018; - U.S. Patent No. 10,011,858 B2, entitled "Method for generating polypeptides by regulating polypeptide association", granted on July 3, 2018; - U.S. Patent No. 10,344,050 B2, entitled "Generation of Heterodimeric Proteins", was granted on July 9, 2019; - U.S. Patent No. 10,597,464 B2, entitled "Heterodimeric antibody protein containing Fc and method for generating the same thereto", was granted on March 24, 2020; - Kabat, EA et al., Immunology-associated protein sequences, 5th edition—NIH Publication No. 91-3242 (1991). - Shopes, B., "Genetically engineered human IgG mutants with enhanced cytolytic activity", THE JOURNAL OF IMMUNOLOGY, May 1, 1992; 148(9):2918-22; - Shopes, B., “Genetically engineered human IgG with limited flexibility and full activation of complement-mediated cytolysis”, MOLECULAR IMMUNOLOGY, April 1993; 30(6):603-9; and - van der Neut Kolfschoten, M. et al., “Anti-inflammatory activity of human IgG4 antibody achieved through dynamic Fab arm exchange”, SCIENCE, 14 Sep 2007; 317(5844):1554-7.

Claims

1. A method for manufacturing a dimer immunotherapeutic agent, comprising: A first immunotherapeutic agent and a second immunotherapeutic agent are provided, wherein: (a) the first immunotherapeutic agent comprises a first IgG having two heavy chains having a first amino acid sequence; (b) the second immunotherapeutic agent comprises a second IgG having two heavy chains having a second amino acid sequence; (c) the first amino acid sequence includes a mutation that mutates a natural amino acid to cysteine; (d) the second amino acid sequence does not have said mutation; (e) the two heavy chains of the first immunotherapeutic agent are covalently cross-linked by one or more disulfide bonds of a first group; and (f) the two heavy chains of the second immunotherapeutic agent are covalently cross-linked by one or more disulfide bonds of a second group; A solution containing the first immunotherapeutic agent and the second immunotherapeutic agent is incubated under reducing conditions to reduce one or more disulfide bonds in the first group and one or more disulfide bonds in the second group, such that: (a) the two heavy chains of the first immunotherapeutic agent dissociate, thereby producing a half-molecule of the first immunotherapeutic agent; (b) the two heavy chains of the second immunotherapeutic agent dissociate, thereby producing a half-molecule of the second immunotherapeutic agent; and (c) the half-molecule of the first immunotherapeutic agent recombines with the half-molecule of the second immunotherapeutic agent, thereby producing a chimeric immunotherapeutic agent, said chimeric immunotherapeutic agent being chimeric at least by simultaneously containing: (i) a heavy chain containing the cysteine ​​from the first IgG, and (ii) a heavy chain not containing the cysteine ​​from the second IgG; and The chimeric immunotherapeutic agent is incubated under oxidative conditions to form a disulfide bond between (a) a cysteine ​​residue from the heavy chain of the first IgG in a first molecule of the chimeric immunotherapeutic agent, and (b) a cysteine ​​residue from the heavy chain of the first IgG in a second molecule of the chimeric immunotherapeutic agent, thereby producing the dimer immunotherapeutic agent, wherein: The first IgG has a first VL CDR1 amino acid sequence, a first VL CDR2 amino acid sequence, a first VL CDR3 amino acid sequence, a first VH CDR1 amino acid sequence, a first VH CDR2 amino acid sequence, and a first VH CDR3 amino acid sequence; The second IgG has a second VL CDR1 amino acid sequence, a second VL CDR2 amino acid sequence, a second VL CDR3 amino acid sequence, a second VH CDR1 amino acid sequence, a second VH CDR2 amino acid sequence, and a second VH CDR3 amino acid sequence; The first VL CDR1 amino acid sequence is identical to the second VL CDR1 amino acid sequence; The first VL CDR2 amino acid sequence is identical to the second VL CDR2 amino acid sequence; The first VL CDR3 amino acid sequence is identical to the second VL CDR3 amino acid sequence; The first VH CDR1 amino acid sequence is identical to the second VH CDR1 amino acid sequence; The first VH CDR2 amino acid sequence is identical to the second VH CDR2 amino acid sequence; The first VH CDR3 amino acid sequence is identical to the second VH CDR3 amino acid sequence; The reducing agent is cysteamine; The first IgG is human IgG1; The natural amino acid is S444; The mutation is S444C; The first IgG contains the F405L mutation; The second IgG is human IgG1; and The second IgG contains the K409R mutation.

2. A method for manufacturing a dimer immunotherapeutic agent, comprising: A first immunotherapeutic agent and a second immunotherapeutic agent are provided, wherein: (a) the first immunotherapeutic agent comprises a first IgG having two heavy chains having a first amino acid sequence; (b) the second immunotherapeutic agent comprises a second IgG having two heavy chains having a second amino acid sequence; (c) the first amino acid sequence includes a mutation that mutates a natural amino acid to cysteine; (d) the second amino acid sequence does not have said mutation; (e) the two heavy chains of the first immunotherapeutic agent are covalently cross-linked by one or more disulfide bonds of a first group; and (f) the two heavy chains of the second immunotherapeutic agent are covalently cross-linked by one or more disulfide bonds of a second group; A solution containing the first immunotherapeutic agent and the second immunotherapeutic agent is incubated under reducing conditions to reduce one or more disulfide bonds in the first group and one or more disulfide bonds in the second group, such that: (a) the two heavy chains of the first immunotherapeutic agent dissociate, thereby producing a half-molecule of the first immunotherapeutic agent; (b) the two heavy chains of the second immunotherapeutic agent dissociate, thereby producing a half-molecule of the second immunotherapeutic agent; and (c) the half-molecule of the first immunotherapeutic agent recombines with the half-molecule of the second immunotherapeutic agent, thereby producing a chimeric immunotherapeutic agent, said chimeric immunotherapeutic agent being chimeric at least by simultaneously containing: (i) a heavy chain containing the cysteine ​​from the first IgG, and (ii) a heavy chain not containing the cysteine ​​from the second IgG; and The chimeric immunotherapeutic agent is incubated under oxidative conditions to form a disulfide bond between (a) the cysteine ​​residue from the heavy chain of the first IgG in a first molecule of the chimeric immunotherapeutic agent and (b) the cysteine ​​residue from the heavy chain of the first IgG in a second molecule of the chimeric immunotherapeutic agent, thereby producing the dimer immunotherapeutic agent.

3. The method according to claim 2, wherein: The first IgG has a first VL CDR1 amino acid sequence, a first VL CDR2 amino acid sequence, a first VL CDR3 amino acid sequence, a first VH CDR1 amino acid sequence, a first VH CDR2 amino acid sequence, and a first VH CDR3 amino acid sequence; The second IgG has a second VL CDR1 amino acid sequence, a second VL CDR2 amino acid sequence, a second VL CDR3 amino acid sequence, a second VH CDR1 amino acid sequence, a second VH CDR2 amino acid sequence, and a second VH CDR3 amino acid sequence; The first VL CDR1 amino acid sequence is identical to the second VL CDR1 amino acid sequence; The first VL CDR2 amino acid sequence is identical to the second VL CDR2 amino acid sequence; The first VL CDR3 amino acid sequence is identical to the second VL CDR3 amino acid sequence; The first VH CDR1 amino acid sequence is identical to the second VH CDR1 amino acid sequence; The first VH CDR2 amino acid sequence is identical to the second VH CDR2 amino acid sequence; The first VH CDR3 amino acid sequence is identical to the second VH CDR3 amino acid sequence; The reducing agent is cysteamine; The first IgG is human IgG1; The natural amino acid is S444; The mutation is S444C; The first IgG contains the F405L mutation; The second IgG is human IgG1; and The second IgG contains the K409R mutation.

4. The method according to claim 2, wherein: The first IgG has a first VL CDR1 amino acid sequence, a first VL CDR2 amino acid sequence, a first VL CDR3 amino acid sequence, a first VH CDR1 amino acid sequence, a first VH CDR2 amino acid sequence, and a first VH CDR3 amino acid sequence; The second IgG has a second VL CDR1 amino acid sequence, a second VL CDR2 amino acid sequence, a second VL CDR3 amino acid sequence, a second VH CDR1 amino acid sequence, a second VH CDR2 amino acid sequence, and a second VH CDR3 amino acid sequence; The amino acid sequences of the first VL CDR1 and the second VL CDR1 are identical to those of SEQ ID NO: 8; The first VL CDR2 amino acid sequence and the second VL CDR2 amino acid sequence are identical to SEQ ID NO: 9; The first VL CDR3 amino acid sequence and the second VL CDR3 amino acid sequence are identical to those in SEQ ID NO: 10; The first VH CDR1 amino acid sequence and the second VH CDR1 amino acid sequence are identical to SEQ ID NO: 5; The first VH CDR2 amino acid sequence and the second VH CDR2 amino acid sequence are identical to SEQ ID NO: 6; and The first VH CDR3 amino acid sequence and the second VH CDR3 amino acid sequence are identical to SEQ ID NO:

7.

5. The method according to claim 2, wherein: The first amino acid sequence has at least 90% sequence identity with SEQ ID NO: 3; The second amino acid sequence has at least 90% sequence identity with SEQ ID NO: 3; The first IgG comprises two light chains, the two light chains having a first light chain amino acid sequence that is at least 90% sequence identical to SEQ ID NO: 4; and The second IgG comprises two light chains having a second light chain amino acid sequence that is at least 90% identical to that of SEQ ID NO:

4.

6. The method according to claim 2, wherein: The first amino acid sequence has at least 95% sequence identity with SEQ ID NO: 3; The second amino acid sequence has at least 98% sequence identity with SEQ ID NO: 3; The first IgG comprises two light chains, the two light chains having an amino acid sequence comprising the first light chain of SEQ ID NO: 4; and The second IgG comprises two light chains having a second light chain amino acid sequence including SEQ ID NO:

4.

7. The method according to claim 2, wherein: The first IgG comprises two light chains having a first light chain amino acid sequence; The second IgG comprises two light chains having a second light chain amino acid sequence; The first light chain amino acid sequence is identical to the second light chain amino acid sequence; and The first IgG and the second IgG bind to the same antigen.

8. The method of claim 2, further comprising purifying the chimeric immunotherapeutic agent, wherein: The solution comprises the reducing agent, the first immunotherapeutic agent, and the second immunotherapeutic agent, wherein the reducing agent, the first immunotherapeutic agent, and the second immunotherapeutic agent each have a molar concentration in the solution; The solution contains a combined molar concentration of the first immunotherapy agent and the second immunotherapy agent, the combined molar concentration being equal to the sum of the molar concentrations of the first immunotherapy agent and the second immunotherapy agent; The molar concentration of the reducing agent is at least four times the combined molar concentration; The purification process separates the chimeric immunotherapy agent from the reducing agent; The chimeric immunotherapeutic agent is incubated under oxidative conditions, including the purification process. The reducing agent is cysteamine; and Incubating the chimeric immunotherapeutic agent under oxidative conditions includes incubating the chimeric immunotherapeutic agent at a pH of at least 7.

0.

9. The method according to claim 2, wherein: The first IgG is human IgG4; The first IgG has lysine at amino acid position 409 and phenylalanine at amino acid position 405; The second IgG is human IgG4; and The second IgG has arginine at amino acid position 409 and leucine at amino acid position 405.

10. The method according to claim 2, wherein: The first IgG is human IgG4; The first IgG has arginine at amino acid position 409 and leucine at amino acid position 405; The second IgG is human IgG4; and The second IgG has lysine at amino acid position 409 and phenylalanine at amino acid position 405.

11. The method of claim 2, further comprising purifying the chimeric immunotherapeutic agent, wherein: The solution contains a reducing agent; The purification process separates the chimeric immunotherapy agent from the reducing agent; and Incubation of the chimeric immunotherapeutic agent under oxidative conditions includes the purification process; and Incubating the chimeric immunotherapeutic agent under oxidative conditions includes incubating the chimeric immunotherapeutic agent at a pH of at least 7.

0.

12. The method according to claim 2, wherein: The first IgG has a first VL CDR1 amino acid sequence, a first VL CDR2 amino acid sequence, a first VL CDR3 amino acid sequence, a first VH CDR1 amino acid sequence, a first VH CDR2 amino acid sequence, and a first VH CDR3 amino acid sequence; The second IgG has a second VL CDR1 amino acid sequence, a second VL CDR2 amino acid sequence, a second VL CDR3 amino acid sequence, a second VH CDR1 amino acid sequence, a second VH CDR2 amino acid sequence, and a second VH CDR3 amino acid sequence; The first VL CDR1 amino acid sequence is identical to the second VL CDR1 amino acid sequence; The first VL CDR2 amino acid sequence is identical to the second VL CDR2 amino acid sequence; The first VL CDR3 amino acid sequence is identical to the second VL CDR3 amino acid sequence; The first VH CDR1 amino acid sequence is identical to the second VH CDR1 amino acid sequence; The first VH CDR2 amino acid sequence is identical to the second VH CDR2 amino acid sequence; The first VH CDR3 amino acid sequence is identical to the second VH CDR3 amino acid sequence; The reducing agent is cysteamine; The first IgG is human IgG1; The natural amino acid is S444; and The mutation is S444C.

13. The method according to claim 2, wherein: The first IgG has a first VL CDR1 amino acid sequence, a first VL CDR2 amino acid sequence, a first VL CDR3 amino acid sequence, a first VH CDR1 amino acid sequence, a first VH CDR2 amino acid sequence, and a first VH CDR3 amino acid sequence; The second IgG has a second VL CDR1 amino acid sequence, a second VL CDR2 amino acid sequence, a second VL CDR3 amino acid sequence, a second VH CDR1 amino acid sequence, a second VH CDR2 amino acid sequence, and a second VH CDR3 amino acid sequence; The first VL CDR1 amino acid sequence is identical to the second VL CDR1 amino acid sequence; The first VL CDR2 amino acid sequence is identical to the second VL CDR2 amino acid sequence; The first VL CDR3 amino acid sequence is identical to the second VL CDR3 amino acid sequence; The first VH CDR1 amino acid sequence is identical to the second VH CDR1 amino acid sequence; The first VH CDR2 amino acid sequence is identical to the second VH CDR2 amino acid sequence; The first VH CDR3 amino acid sequence is identical to the second VH CDR3 amino acid sequence; The first IgG is human IgG1; The natural amino acid is S444; and The mutation is S444C.

14. The method according to claim 2, wherein: The first IgG has a first light chain variable domain amino acid sequence and a first heavy chain variable domain amino acid sequence; The second IgG has a second light chain variable domain amino acid sequence and a second heavy chain variable domain amino acid sequence; The amino acid sequence of the first light chain variable domain is identical to the amino acid sequence of the second light chain variable domain; and The amino acid sequence of the first heavy chain variable domain is the same as that of the second heavy chain variable domain.

15. The method according to claim 2, wherein: The first IgG has a first VL CDR1 amino acid sequence, a first VL CDR2 amino acid sequence, a first VL CDR3 amino acid sequence, a first VH CDR1 amino acid sequence, a first VH CDR2 amino acid sequence, and a first VH CDR3 amino acid sequence; The second IgG has a second VL CDR1 amino acid sequence, a second VL CDR2 amino acid sequence, a second VL CDR3 amino acid sequence, a second VH CDR1 amino acid sequence, a second VH CDR2 amino acid sequence, and a second VH CDR3 amino acid sequence; The first VL CDR1 amino acid sequence is identical to the second VL CDR1 amino acid sequence; The first VL CDR2 amino acid sequence is identical to the second VL CDR2 amino acid sequence; The first VL CDR3 amino acid sequence is identical to the second VL CDR3 amino acid sequence; The first VH CDR1 amino acid sequence is identical to the second VH CDR1 amino acid sequence; The first VH CDR2 amino acid sequence is identical to the second VH CDR2 amino acid sequence; The first VH CDR3 amino acid sequence is identical to the second VH CDR3 amino acid sequence.

16. The method of claim 2, wherein the first IgG binds to the same antigen as the second IgG.

17. The method according to claim 2, wherein: The first IgG is human IgG1; The second IgG is human IgG1; and it is either one of the following: (i) the first IgG contains the F405L mutation and (ii) the second IgG contains the K409R mutation; or (i) the first IgG contains the K409R mutation and (ii) the second IgG contains the F405L mutation.

18. The method according to claim 2, wherein: The first IgG is human IgG1; The natural amino acid is S444; and The mutation is S444C.

19. The method according to claim 2, wherein: The first IgG has lysine at amino acid position 409 and phenylalanine at amino acid position 405; and The second IgG has arginine at amino acid position 409 and leucine at amino acid position 405.

20. The method according to claim 2, wherein: The first IgG contains arginine at amino acid position 409 and leucine at amino acid position 405; and The second IgG has lysine at amino acid position 409 and phenylalanine at amino acid position 405.

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