Class ii hla with interchain disulfide bond

Abstract

The present disclosure provides modified HLA molecules, and methods of using the same to induce or enhance cytotoxicity, the molecules comprising an alpha chain and a beta chain, wherein both chains comprise one or more substituted cysteines that pair to form disulfide bonds, and wherein the modified HLA molecules are optionally further conjugated to an Fc domain at the 3' end.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Application Serial No. 63 / 613,880, filed December 22, 2023. The full disclosure of the application referred to in this paragraph is incorporated herein by reference. sequence list

[0002] This application contains references to amino acid sequences and / or nucleic acid sequences, which are submitted herein as a sequence listing XML file named “000002wopoa_SequenceListing.XML”, 38,105 bytes in size, created on December 20, 2024. Pursuant to 37 CFR §1.52(e)(5), the above sequence listing is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure generally relates to modified HLA molecules, wherein the α and β chains are engineered to include substituted cysteine ​​pairs; to compositions comprising modified HLA molecules; to compositions of modified HLA molecules for treating cancer, pathogen-driven diseases, and alloimmune responses; to methods for enhancing cytotoxicity; and to methods for producing modified HLA molecules. Background Technology

[0004] The major histocompatibility complex (MHC) encodes the human leukocyte antigen (HLA) gene. HLA is a key component of the immune system and is responsible for regulating immune responses by presenting antigens to T cells. HLA-DQ molecules are a subset of class II HLA molecules and are known to cause unintended alloimmune responses after solid organ transplantation, as well as various autoimmune diseases, including celiac disease. Biologics designed with HLA molecules as part of their formulation are emerging as a therapeutic option for these alloimmune and autoimmune disorders. However, the α and β chains of soluble class II HLA molecules cannot dimerize efficiently without their transmembrane domains, which hinders their production.

[0005] Serra et al. described fusing the α and β chains of class II MHC with mutant IgG1-Fc to form a knob-into-hole structure, which improved the stability and yield of MHC class II molecules. (Serra et al., “Increased yields and biological potency of knob-into-hole-based soluble MHC class II molecules,” Nature Communications, Vol. 10, No. 1, 4917, October 29, 2019).

[0006] WO 2011 / 101681A2 describes that the α and β chains of recombinant MHC class II molecules can be stabilized by disulfide bonds located between cysteine ​​residues at the following positions: Pro 96 α2 - Ser 119 β2 (Sort 1), Ser 95 α2 -Ser 121 β2 (Sort 2), Arg 94 α2 - Asn 151 β2 (Sorted 3), Phe 148 α2 - Gly 152 β2 (Ranked 4), Pro 96 α2 - Thr 101 β2 (Ranked 5), Pro 96 α2 - Ser 121 β2 (Sorted 6), Ile 106 α2 - Asn 151 β2 (Sorted 7), and Ser 95 α2 - Asp 122 β2 (Sorted 8).

[0007] WO 2024 / 006576A1 describes that MHC class II constructs can be stabilized by one or more disulfide bonds and one or more amino acid substitutions. Summary of the Invention

[0008] This section provides a general overview of this disclosure and is not a complete disclosure of the full scope or all features of this disclosure.

[0009] This disclosure provides a modified HLA molecule comprising a first peptide and a second peptide. The first peptide comprises a wild-type α chain and a signal peptide, wherein at least one amino acid of the wild-type α chain is substituted with a cysteine ​​residue. The second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein at least one amino acid of the wild-type β chain is substituted with a cysteine ​​residue. Each substituted cysteine ​​residue on the α chain is adjacent to a corresponding substituted cysteine ​​residue on the β chain, such that each substituted cysteine ​​residue on the α chain forms a disulfide bond with the substituted cysteine ​​residue on the β chain, thereby forming one or more interchain disulfide bonds. The substituted cysteine ​​residues are positioned such that, when the modified HLA molecule exhibits its quaternary structure, the substituted cysteine ​​residue on the α chain is adjacent to the substituted cysteine ​​residue on the β chain, and the pair of substituted cysteine ​​residues forms a disulfide bond. In embodiments, the modified HLA molecule is an HLA-DQ2.5, HLA-DQ7, HLA-DQ8, or HLA-DR molecule.

[0010] It is also disclosed that the first peptide has the amino acid sequence of SEQ ID No: 1, and the second peptide has the amino acid sequence of SEQ ID No: 2.

[0011] The nucleic acid sequence, which encodes a modified HLA-DQ molecule, was also disclosed.

[0012] It was also disclosed that the human IgG1 Fc fragment sequence linked to the 3' end of the β chain has at least one gain-of-function mutation.

[0013] Cells expressing modified HLA molecules or containing nucleic acid sequences encoding modified HLA molecules are also disclosed.

[0014] Compositions comprising modified HLA molecules present on the surface of cells without membranes or cells are also disclosed.

[0015] Compositions for treating cancer, pathogen-driven diseases, and immune responses, comprising modified HLA molecules, are also disclosed.

[0016] The invention also discloses a method for enhancing cytotoxicity, which includes targeting the disclosed modified HLA molecule, cells expressing the modified HLA molecule, or cells containing a nucleic acid sequence encoding the modified HLA molecule to target cells.

[0017] A method for producing modified HLA molecules is also disclosed, comprising: replacing one or more amino acids of the wild-type α chain with cysteine ​​in such a way that the α chain and β chain contain the same number of substituted cysteines; replacing one or more amino acids of the wild-type β chain with cysteine; and forming a disulfide bond between each substituted cysteine ​​on the α chain and each substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds. Attached Figure Description

[0018] The accompanying drawings described herein are for illustrative purposes only, and not for all possible implementations, and are not intended to limit the scope of this disclosure.

[0019] Figure 1 A describes the paired amino acid residues in HLA-DQ2 that are juxtaposed with each other and, if mutated to cysteine, are expected to form disulfide bonds. Figure 1 B classifies the disulfide bond formation potential of candidate pairings. Figure 1 C measures the expression level of modified HLA molecules using ELISA. Figure 1 D shows the expression of wild-type and mutant pDQ7-Fc protein by SDS-PAGE.

[0020] Figure 2 A depicts the α and β chains of HLA-DQ with potential amino acid pairs suitable for forming disulfide bonds.

[0021] Figure 2 B. Figure 2 C and Figure 2 D depicts the three-dimensional structures of YCDC, ACEC, and ACNC, as well as the mutated amino acid residue pairs in each molecule.

[0022] Figure 3 A depicts the analysis of modified HLA-DQs with different peptides by SDS-PAGE under reducing and non-reducing conditions.

[0023] Figure 3 B describes the quantitative expression levels of modified HLA molecules with different peptides.

[0024] Figure 4 A describes the binding properties of modified HLA molecules to anti-DQ7 as measured by ELISA.

[0025] Figure 4 B describes the binding characteristics of empty YCDC, YCDC with CLIP, and YCDC with Mem-36 as measured by ELISA.

[0026] Figure 4 C shows the protein imprint of the modified HLA molecule.

[0027] Figure 5 A provides a schematic diagram illustrating how the pDQ7-Fc protein can guide the killing of antibody-producing cells with specific B cell receptors.

[0028] Figure 5 B shows the binding of pDQ7-Fc (YCDC) to HB144 B cell hybridoma as measured by flow cytometry.

[0029] Figure 5 C shows the percentage of hybridoma cells that survived as the peptide concentration increased.

[0030] Figure 5 D shows the normalized survival percentage of hybridoma cells as peptide concentration increases.

[0031] Figure 6 The β chain and α chain are described.

[0032] Figure 7 The SDS-PAGE analysis of modified HLA molecules under reduction conditions was described.

[0033] Figure 8 An ELISA was used to depict pDQ2 expression in CHO cells.

[0034] Figure 9 The expression of mutant pDQ8-Fc (YCDC) under non-reducing conditions was depicted by SDS-PAGE.

[0035] Figure 10 The hierarchy of eight disulfide bond engineered candidate sites predicted by the MODIP program based on the HLA-DR4 model was depicted, and the expression of mutant pDR4-Fc (PCPC) under non-reducing conditions was depicted by SDS-PAGE. Detailed Implementation

[0036] The following description is merely illustrative in nature and is not intended to limit this disclosure, application, or use.

[0037] As used herein, an "HLA molecule" is a protein dimer comprising an α-chain and a β-chain, wherein the α-chain and β-chain are separate peptides that, when presenting their respective three-dimensional shapes, interact to form a protein with a quaternary structure. Human leukocyte antigen (HLA) is the human form of the major histocompatibility complex (MHC) found in all mammals. In several embodiments, HLA molecules are selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR. In one embodiment, the HLA molecule is HLA-DR. HLA-DQ molecules are a subset of HLA molecules. HLA-DQ molecules include HLA-DQ2, HLA-DQ3, HLA-DQ4, HLA-DQ5, HLA-DQ6, HLA-DQ7, HLA-DQ8, and HLA-DQ9. In one embodiment, HLA-DQ is HLA-DQ2.5. In one implementation, HLA-DQ is HLA-DQ7. In another implementation, HLA-DQ is HLA-DQ8.

[0038] As used herein, the “modified HLA-DQ molecule” is an HLA-DQ molecule in which the amino acid sequences of the wild-type α chain and the wild-type β chain each contain at least one cysteine ​​substitution. Each substituted cysteine ​​on the α chain is adjacent to the corresponding substituted cysteine ​​on the β chain, such that each substituted cysteine ​​on the α chain forms a disulfide bond with the substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

[0039] As used herein, “modified HLA-DR molecule” refers to an HLA-DR molecule in which the amino acid sequences of the wild-type α chain and the wild-type β chain each contain at least one cysteine ​​substitution. Each substituted cysteine ​​on the α chain is adjacent to the corresponding substituted cysteine ​​on the β chain, such that each substituted cysteine ​​on the α chain forms a disulfide bond with the substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

[0040] As used herein, “wildtype” refers to an amino acid or nucleic acid sequence commonly found in natural populations. For example, the wild-type α chain refers to the presumed HLA α chain sequence commonly found in human populations. Similarly, the wild-type β chain refers to the presumed HLA β chain sequence commonly found in human populations. Wild-type sequences serve as the basis for cysteine ​​substitutions in the amino acid sequences encoded by nucleic acid sequences. For example, a substitution at position 16 of an amino acid sequence refers to altering the amino acid at position 16, which is naturally present in the general human population, and replacing it with another amino acid that is not commonly found at position 16.

[0041] As used in this article, “pharmaceutical acceptable” means that a pharmaceutical preparation is generally considered safe for such use, has been formally approved for such use by a national or state regulatory agency, or is listed in the United States Pharmacopeia or other recognized pharmacopoeia for use in animals, and especially in humans.

[0042] As used herein, "pharmaceuticalally acceptable carrier" means a diluent, adjuvant, excipient, or carrier, other component, or combination of components that provide a carrier or medium, either alone or together, wherein one or more compounds of the present invention are formulated and / or administered with such carrier or medium, and wherein each component or carrier as a whole is pharmaceutically acceptable.

[0043] As used herein, “pharmaceuticalally acceptable excipients” refers to any substance other than the active pharmaceutical ingredient (API) incorporated into a pharmaceutical formulation to facilitate the manufacturing process, enhance stability, improve bioavailability, or promote drug administration. These excipients are pharmacologically inactive and are selected based on their compatibility with the active pharmaceutical ingredient and their ability to perform specific functions such as binding, filling, disintegration, or preservation.

[0044] As used in this article, "cancer" refers to benign or malignant tumors, including but not limited to fibromas, lipomas, adenomas, hemangiomas, leiomyomas, myxomas, chondromas, osteomas, fibrous histiocytomas, hemangiopericytomas, lymphangiomas, plasmacytosis, rhabdomyomas, papillomas, hepatic adenomas, renal tubular adenomas, bile duct adenomas, transitional cell papillomas, hydatidiform moles, meningiomas, schwannomas, neurofibromas, carcinomas, sarcomas, leukemia, lymphomas, myeloma, gliomas, glioblastomas, and neuroblastomas. Malignant medulloblastoma, malignant meningioma, malignant Schwann cell tumor, neurofibrosarcoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, hemangioendothelioma, angiosarcoma, lymphangiosarcoma, mesothelioma, plasmacytoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, adenocarcinoma, hepatocellular carcinoma, renal cell carcinoma, bile duct carcinoma, seminoma, embryonal cell carcinoma, as well as various brain cancers and central nervous system cancers.

[0045] As used in this article, "cancer cells" refers to cancer cells.

[0046] As used herein, "pathogen" refers to pathogens including, but not limited to, the following: Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, Salmonella enterica, Clostridium botulinum, Bacillus anthracis, Vibrio cholerae, Helicobacter pylori, influenza virus, HIV, SARS-CoV-2, hepatitis B virus, Ebola virus, Zika virus, rabies virus, herpes simplex virus, Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides immitis. Plasmodium falciparum, Giardia lamblia, Toxoplasma gondii, Trypanosoma brevicornu, Leishmania donovani, Schistosoma mansoni, Trichinella spiralis, Onchocerca salina, prions causing Creutzfeldt-Jakob disease, prions causing kuru, prions causing fatal familial insomnia, Acinetobacter baumannii, Actinobacillus isatis, Propionibacterium propionate, Adenoviridae, Trypanosoma brevicornu, Entamoeba histolytica, Anaplasma, Strongyloides angiostrongylus, Anisakis, Cryptococcus hemolyticus, Junin virus, Ascaridium lumbricoides. Insects, Astroviridae, Babesia, Bacillus cereus, Borrelia burgdorferi, Brucella, Campylobacter jejuni, Chlamydia trachomatis, Corynebacterium diphtheriae, Coxsella brevis, Cryptosporidium microsporum, Cyclospora cayetana, Dengue virus, Echinococcus granulosus, Enterococcus faecalis, Tulafrancsis, Haemophilus influenzae, Hantavirus, Hendra virus, Human papillomavirus, Klebsiella pneumoniae, Lassa virus, Legionella pneumophila, Leptospira question mark, Listeria monocytogenes Bacteria, Marburg virus, measles virus, mumps virus, Neisseria gonorrhoeae, Neisseria meningitidis, norovirus, parvovirus B19, Pneumocystis jirovecii, Pseudomonas aeruginosa, respiratory syncytial virus, Rickettsia rickettsiae, Rift Valley fever virus, rotavirus, rubella virus, Shigella dysenteriae, Streptococcus pneumoniae, Streptococcus pyogenes, Taenia solium, Treponema pallidum, Trichomonas vaginalis, varicella-zoster virus, Vibrio vulnificus, West Nile virus, Yersinia pestis, and Zygomycosis.

[0047] As used in this article, "pathogen-driven disease" refers to a disease caused by a pathogen.

[0048] As used herein, “immune response” refers to both autoimmune and alloimmune responses, and includes, but is not limited to, diseases such as: rheumatoid arthritis, systemic lupus erythematosus (lupus), myositis, polymyalgia rheumatica, psoriasis, psoriatic arthritis, dermatomyositis, scleroderma, vitiligo, bullous pemphigoid, lichen planus, and alopecia areata. In addition, autoimmune diseases affecting the digestive system include Crohn's disease, celiac disease, ulcerative colitis, and autoimmune hepatitis. Endocrine disorders such as type 1 diabetes, Addison's disease, Hashimoto's thyroiditis, and Graves' disease are also included. The nervous system can be affected by multiple sclerosis (MS), myasthenia gravis (MG), Guillain-Barré syndrome, and chronic inflammatory demyelinating polyneuropathy (CIDP). Blood and vascular disorders include autoimmune hemolytic anemia, vasculitis, and antiphospholipid syndrome. Other autoimmune diseases include Sjögren's syndrome, autoimmune uveitis, Goodpasture syndrome, Wegener's granulomatosis, autoimmune gastritis, autoimmune pancreatitis, autoimmune encephalitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune neutropenia, autoimmune thrombocytopenia, autoimmune retinopathy, autoimmune ganglionosis, autoimmune enteropathy, autoimmune hepatitis, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune polyendocrine syndrome (APS), autoimmune progesterone dermatitis, autoimmune pulmonary alveolar proteinosis, autoimmune thyroiditis, autoimmune vascular disease, autoimmune vitiligo, and autoimmune vulvovaginitis.

[0049] As used in this article, “unwanted cells” refers to the following types of cells, including but not limited to cancer cells, infected cells, senescent cells, autoimmune cells, damaged cells, and excess cells.

[0050] Modified HLA molecules

[0051] This disclosure provides a modified HLA molecule in which the wild-type sequences of the α and β chains are modified such that they contain paired substituted cysteine ​​residues forming disulfide bonds between the α and β chains. In one embodiment, the α chain contains an amino acid sequence in which one or more amino acids of the wild-type α chain are substituted with cysteine ​​residues; and the β chain contains an amino acid sequence in which one or more amino acids of the wild-type β chain are substituted with cysteine ​​residues. The α and β chains contain the same number of substituted cysteine ​​residues, and when the modified HLA molecule exhibits its quaternary structure, the substituted cysteine ​​residues on each chain are adjacent to each other, and each substituted cysteine ​​residue on the α chain forms a disulfide bond with the corresponding substituted cysteine ​​residue on the β chain. The mutant residues in the α and β chains have limited solvent exposure. The modified HLA molecule can be further linked to additional domains, such as an Fc domain, a histidine tag, or a streptavidin tag. This additional domain can be used to assist purification or to induce complement-dependent or cell-dependent cytotoxicity, such as antibody-dependent cell-mediated cytotoxicity. The Fc domain may attach to the 3' end of either the α or β chain. In a specific embodiment, the Fc domain is attached to the 3' end of the β chain. In a specific embodiment, the Fc domain is an Fc fragment. In a specific embodiment, the Fc domain is an IgG Fc fragment. In a specific embodiment, the Fc domain is a human IgG1 Fc fragment. The Fc domain may contain gain-of-function mutations to enhance the potency of complement-dependent or cell-dependent cytotoxicity.

[0052] The modified HLA molecule can be a class I HLA molecule (e.g., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G) or a class II HLA molecule (e.g., HLA-DP, HLA-DQ, or HLA-DR). In several embodiments, the modified HLA molecule is HLA-DQ, selected from the group consisting of HLA-DQ2, HLA-DQ3, HLA-DQ4, HLA-DQ5, HLA-DQ6, HLA-DQ7, HLA-DQ8, and HLA-DQ9. In a specific embodiment, HLA-DQ is HLA-DQ2.5. In a specific embodiment, HLA-DQ is HLA-DQ7. In a specific embodiment, HLA-DQ is HLA-DQ8. The modified HLA molecule can be a class I HLA molecule coupled to Fc or a class II HLA molecule coupled to Fc. In a specific embodiment, the modified HLA molecule is a class II HLA-Fc conjugate. When the modified HLA molecule is an HLA-DQ molecule, the substituted cysteine ​​residue can be located at position 19, position 83, position 84, or a combination thereof on the α chain. When the modified HLA molecule is an HLA-DQ molecule, the substituted cysteine ​​residue can be located at position 5, position 6, position 33, or a combination thereof on the β chain. When the modified HLA molecule is an HLA-DR molecule, the substituted cysteine ​​residue can be located at positions 83 and 84, or a combination thereof on the α chain. When the modified HLA molecule is an HLA-DR molecule, the substituted cysteine ​​residue can be located at positions 5 and 33, or a combination thereof on the β chain.

[0053] In one embodiment, the modified HLA molecule is HLA-DQ7, which comprises a first peptide and a second peptide. The first peptide comprises a wild-type α chain and a signal peptide, wherein at least one amino acid of the wild-type α chain is substituted with a cysteine ​​residue. The second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein at least one amino acid of the wild-type β chain is substituted with a cysteine ​​residue. Each substituted cysteine ​​residue on the α chain is adjacent to a corresponding substituted cysteine ​​residue on the β chain, such that each substituted cysteine ​​residue on the α chain forms a disulfide bond with the substituted cysteine ​​residue on the β chain, thereby forming one or more interchain disulfide bonds. In one embodiment, the signal peptide of the first peptide is derived from azuril proprotein. In one embodiment, the placeholder peptide of the second peptide is a CLIP placeholder peptide. In one embodiment, the linker of the second peptide is a polyglycine linker. In one embodiment, the Fc fragment sequence is a human IgG1 Fc fragment sequence. In one embodiment, the first peptide has the amino acid sequence of SEQ ID No: 1, and the second peptide has the amino acid sequence of SEQ ID No: 2. The substituted cysteine ​​on the α chain may be located at multiple amino acid positions, including positions selected from the group consisting of positions 19, 83, 84, and combinations thereof. The substituted cysteine ​​on the β chain may be located at multiple amino acid positions, including positions selected from the group consisting of positions 6, 5, 33, and combinations thereof. In one embodiment, the substituted cysteine ​​on the α chain is located at amino acid position 19, and the substituted cysteine ​​on the β chain is located at amino acid position 6; wherein the substituted cysteine ​​on the α chain is located at amino acid position 83, and the substituted cysteine ​​on the β chain is located at amino acid position 5; and wherein the substituted cysteine ​​on the α chain is located at amino acid position 84, and the substituted cysteine ​​on the β chain is located at amino acid position 33, or combinations thereof. In another embodiment, the substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C; wherein, the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C; wherein, the substituted cysteine ​​on the α chain is A84C, and the substituted cysteine ​​on the β chain is N33C, or combinations thereof. In yet another embodiment, the substituted cysteine ​​on the α chain is located at amino acid position 19, and the substituted cysteine ​​on the β chain is located at amino acid position 6; and wherein, the substituted cysteine ​​on the α chain is located at amino acid position 83, and the substituted cysteine ​​on the β chain is located at amino acid position 5. In yet another embodiment, the substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C; and wherein, the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C.

[0054] In yet another embodiment, the modified HLA molecule is Figure 2 Any of the sequences depicted. In yet another embodiment, the human IgG1 Fc fragment sequence linked to the 3' end of the β chain has at least one gain-of-function mutation. In yet another embodiment, the gain-of-function mutation is E345R and / or E430G. In a specific embodiment, the modified HLA-DQ7 molecule comprises: a first peptide having the amino acid sequence of SEQ ID No: 1 and a second peptide having the amino acid sequence of SEQ ID No: 2, the first peptide comprising a wild-type α chain and a signal peptide, wherein cysteine ​​substitutions on the wild-type α chain are located at amino acid positions 19, 83, and 84; the second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein cysteine ​​substitutions on the wild-type β chain are located at amino acid positions 5, 6, and 33; wherein the substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C. Wherein, the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C; wherein, the substituted cysteine ​​on the α chain is A84C, and the substituted cysteine ​​on the β chain is N33C, or a combination thereof; wherein each substituted cysteine ​​on the α chain is adjacent to the corresponding substituted cysteine ​​on the β chain, such that each substituted cysteine ​​on the α chain can form a disulfide bond with the substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

[0055] In a specific embodiment, the modified HLA-DQ7 molecule comprises a first peptide and a second peptide; the first peptide has the following amino acid sequence, which has at least 85% sequence identity with SEQ ID No: 1, and comprises a wild-type α chain and a signal peptide, wherein the cysteine ​​substitution on the wild-type α chain is located at amino acid positions 19, 83, and / or 84; the second peptide has the following amino acid sequence, which has at least 85% sequence identity with SEQ ID No: 2, and comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein the cysteine ​​substitution on the wild-type β chain is located at amino acid positions 5, 6, and / or 33. The substituted cysteine ​​residue on the α chain is Y19C, and the substituted cysteine ​​residue on the β chain is D6C; the substituted cysteine ​​residue on the α chain is A83C, and the substituted cysteine ​​residue on the β chain is E5C; the substituted cysteine ​​residue on the α chain is A84C, and the substituted cysteine ​​residue on the β chain is N33C, or combinations thereof. Each substituted cysteine ​​residue on the α chain is adjacent to a corresponding substituted cysteine ​​residue on the β chain, such that each substituted cysteine ​​residue on the α chain can form a disulfide bond with a substituted cysteine ​​residue on the β chain, thereby forming one or more interchain disulfide bonds.

[0056] In one embodiment, the modified HLA molecule comprises a first peptide and a second peptide, wherein the first peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1; and the second peptide has at least 85%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2. In one embodiment, the modified HLA-DQ molecule comprises a first peptide and a second peptide, wherein the first peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1; and the second peptide has at least 85%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2. In one embodiment, the modified HLA-DQ7 molecule comprises a first peptide and a second peptide, wherein the first peptide has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 1; and the second peptide has at least 85%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2.

[0057] In an alternative embodiment, the modified HLA molecule is an HLA-DR molecule. The modified HLA-DR molecule may have the same substitutions as those described for the modified HLA-DQ molecule. In a specific embodiment, the HLA-DR molecule comprises a first peptide and a second peptide, the first peptide comprising a wild-type α chain and a signal peptide, wherein at least one amino acid of the wild-type α chain is substituted with a cysteine ​​residue; the second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein at least one amino acid of the wild-type β chain is substituted with a cysteine ​​residue. Each substituted cysteine ​​residue on the α chain is adjacent to a corresponding substituted cysteine ​​residue on the β chain, such that each substituted cysteine ​​residue on the α chain forms a disulfide bond with the substituted cysteine ​​residue on the β chain, thereby forming one or more interchain disulfide bonds. In one embodiment, the signal peptide of the first peptide is derived from azuril proprotein. In one embodiment, the placeholder peptide of the second peptide is a CLIP placeholder peptide. In one embodiment, the linker of the second peptide is a polyglycine linker. In one embodiment, the Fc fragment sequence is a human IgG1 Fc fragment sequence. In one embodiment, the first peptide has the amino acid sequence of SEQ ID No: 9, and the second peptide has the amino acid sequence of SEQ ID No: 10. The substituted cysteine ​​on the α-chain can be located at various amino acid positions, including positions selected from the group consisting of positions 83 and 84, and combinations thereof. The substituted cysteine ​​on the β-chain can be located at various amino acid positions, including positions selected from the group consisting of positions 5 and 33, and combinations thereof. In one embodiment, the substituted cysteine ​​on the α-chain is located at amino acid position 83, and the substituted cysteine ​​on the β-chain is located at amino acid position 5; the substituted cysteine ​​on the α-chain is located at amino acid position 84, and the substituted cysteine ​​on the β-chain is located at amino acid position 33, or combinations thereof. In another embodiment, the substituted cysteine ​​on the α-chain is P83C, and the substituted cysteine ​​on the β-chain is P5C; the substituted cysteine ​​on the α-chain is I84C, and the substituted cysteine ​​on the β-chain is H33C, or combinations thereof. In another embodiment, the substituted cysteine ​​on the α chain is located at amino acid position 83, and the substituted cysteine ​​on the β chain is located at amino acid position 5. In another embodiment, the substituted cysteine ​​on the α chain is located at amino acid position 84, and the substituted cysteine ​​on the β chain is located at amino acid position 33. In yet another embodiment, the substituted cysteine ​​on the α chain is P83C, and the substituted cysteine ​​on the β chain is P5C. In yet another embodiment, the substituted cysteine ​​on the α chain is located at amino acid position I84C, and the substituted cysteine ​​on the β chain is located at amino acid position H33C.

[0058] In an alternative embodiment, the modified HLA molecule is an HLA-DQ2.5 molecule. The modified HLA-DQ2.5 molecule may have the same substitutions as those described in the modified HLA-DQ molecule. The modified HLA-DQ2.5 comprises a first peptide and a second peptide, the first peptide comprising a wild-type α chain and a signal peptide, wherein at least one amino acid of the wild-type α chain is substituted with a cysteine ​​residue; the second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein at least one amino acid of the wild-type β chain is substituted with a cysteine ​​residue. Each substituted cysteine ​​residue on the α chain is adjacent to a corresponding substituted cysteine ​​residue on the β chain, such that each substituted cysteine ​​residue on the α chain forms a disulfide bond with the substituted cysteine ​​residue on the β chain, thereby forming one or more interchain disulfide bonds. In one embodiment, the signal peptide of the first peptide is derived from azuril proprotein. In one embodiment, the placeholder peptide of the second peptide is a glia-α1a placeholder peptide. In one embodiment, the linker of the second peptide is a polyglycine linker. In one embodiment, the Fc fragment sequence is the human IgG1 Fc fragment sequence. In one embodiment, the first peptide has the amino acid sequence of SEQ ID No: 1, and the second peptide has the amino acid sequence of SEQ ID No: 6. Substituted cysteine ​​residues on the α chain can be located at various amino acid positions, including positions selected from the group consisting of positions 19, 83, 84, and combinations thereof. Substituted cysteine ​​residues on the β chain can be located at various amino acid positions, including positions selected from the group consisting of positions 6, 5, 33, and combinations thereof. In one embodiment, the substituted cysteine ​​residue on the α chain is located at amino acid position 19, and the substituted cysteine ​​residue on the β chain is located at amino acid position 6; wherein the substituted cysteine ​​residue on the α chain is located at amino acid position 83, and the substituted cysteine ​​residue on the β chain is located at amino acid position 5; and wherein the substituted cysteine ​​residue on the α chain is located at amino acid position 84, and the substituted cysteine ​​residue on the β chain is located at amino acid position 33, or combinations thereof. In another embodiment, the substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C; wherein the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C; wherein the substituted cysteine ​​on the α chain is A84C, and the substituted cysteine ​​on the β chain is N33C, or combinations thereof. In yet another embodiment, the substituted cysteine ​​on the α chain is located at amino acid position 19, and the substituted cysteine ​​on the β chain is located at amino acid position 6; and wherein the substituted cysteine ​​on the α chain is located at amino acid position 83, and the substituted cysteine ​​on the β chain is located at amino acid position 5.In yet another embodiment, the substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C; and wherein the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C.

[0059] In an alternative embodiment, the modified HLA molecule is an HLA-DQ8 molecule. The modified HLA-DQ8 molecule may have the same substitutions as those described in the modified HLA-DQ molecule. The modified HLA-DQ8 comprises a first peptide and a second peptide, the first peptide comprising a wild-type α chain and a signal peptide, wherein at least one amino acid of the wild-type α chain is substituted with a cysteine ​​residue; the second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein at least one amino acid of the wild-type β chain is substituted with a cysteine ​​residue. Each substituted cysteine ​​residue on the α chain is adjacent to a corresponding substituted cysteine ​​residue on the β chain, such that each substituted cysteine ​​residue on the α chain forms a disulfide bond with the substituted cysteine ​​residue on the β chain, thereby forming one or more interchain disulfide bonds. In one embodiment, the signal peptide of the first peptide is derived from azuril proprotein. In one embodiment, the placeholder peptide of the second peptide is a CLIP placeholder peptide. In one embodiment, the linker of the second peptide is a polyglycine linker. In one embodiment, the Fc fragment sequence is a human IgG1 Fc fragment sequence. In one embodiment, the first peptide has the amino acid sequence of SEQ ID No: 8, and the second peptide has the amino acid sequence of SEQ ID No: 7. The substituted cysteine ​​on the α-chain can be located at various amino acid positions, including positions selected from the group consisting of positions 19, 83, 84, and combinations thereof. The substituted cysteine ​​on the β-chain can be located at various amino acid positions, including positions selected from the group consisting of positions 6, 5, 33, and combinations thereof. In one embodiment, the substituted cysteine ​​on the α-chain is located at amino acid position 19, and the substituted cysteine ​​on the β-chain is located at amino acid position 6; wherein the substituted cysteine ​​on the α-chain is located at amino acid position 83, and the substituted cysteine ​​on the β-chain is located at amino acid position 5; and wherein the substituted cysteine ​​on the α-chain is located at amino acid position 84, and the substituted cysteine ​​on the β-chain is located at amino acid position 33; or combinations thereof. In another embodiment, the substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C; wherein the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C; wherein the substituted cysteine ​​on the α chain is A84C, and the substituted cysteine ​​on the β chain is N33C; or combinations thereof. In yet another embodiment, the substituted cysteine ​​on the α chain is located at amino acid position 19, and the substituted cysteine ​​on the β chain is located at amino acid position 6; and wherein the substituted cysteine ​​on the α chain is located at amino acid position 83, and the substituted cysteine ​​on the β chain is located at amino acid position 5. In yet another embodiment, the substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C; and wherein the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C.

[0060] In one embodiment, the first peptide of the modified HLA-DQ7 molecule has a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with SEQ ID NO: 11. The second peptide of the modified HLA-DQ7 molecule has a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with SEQ ID NO: 12.

[0061] In one embodiment, the second peptide of the modified HLA-DQ2.5 molecule has a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with SEQ ID NO: 16.

[0062] In one embodiment, the occupant sequence is Mem_36, and the HLA molecule has a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with SEQ ID NO: 13.

[0063] In one embodiment, the gain-of-function mutation is E345R, and the modified HLA molecule has a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with SEQ ID NO: 14.

[0064] In one embodiment, the gain-of-function mutation is E430G, and the modified HLA molecule has a nucleotide sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with SEQ ID NO: 15.

[0065] In one embodiment, the first peptide of the modified HLA-DQ8 molecule has a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 17. The second peptide of the modified HLA-DQ8 molecule has a nucleotide sequence having at least 80%, 85%, 90%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 18.

[0066] In one embodiment, the first peptide of the modified HLA-DR molecule has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19. The second peptide of the modified HLA-DR molecule has a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20.

[0067] In specific implementation schemes, the positional combinations of cysteine ​​residues substituted on the α and β chains are shown in Table 1.

[0068] Table 1 alpha chain β chain YCDC Y19C D6C ACEC A83C E5C ACNC A84C N33C

[0069] Preparation of modified HLA molecules and nucleotide sequences

[0070] The modified HLA molecules described herein can be prepared using conventional methods known in the art. The amino acid sequence of the modified HLA molecule can be encoded by a nucleotide sequence (e.g., sequence I) and provided to cells (e.g., hybridomas, bacteria, yeast, mammalian cells, etc.), which translate the nucleotide sequence into the modified HLA molecule. The nucleotide sequence described herein can be integrated into vectors (e.g., viral vectors, plasmids, etc.) for insertion into cells (e.g., transformation, transfection, etc.) for subsequent protein production.

[0071] In any embodiment, this document provides a nucleotide sequence comprising sequence I encoding an α chain and / or a β chain. For example, the nucleotide sequence may encode an α chain comprising substituted cysteine ​​residues at positions 19, 83, and / or 84, including specific substitutions of Y19C, A83C, and / or A84C. The nucleotide sequence may also encode a β chain comprising substituted cysteine ​​residues at positions 5, 6, and / or 33, including specific substitutions of E5C, D6C, and / or N33C.

[0072] The nucleotide sequence may also encode an Fc domain attached to the 3' end of the α or β chain. In a specific embodiment, the nucleotide sequence encodes an Fc domain attached to the 3' end of the β chain. The Fc domain encoded by this nucleotide sequence may contain a gain-of-function mutation that enhances antigen-specific killing efficacy.

[0073] Cells, nucleic acids and compositions

[0074] This disclosure provides cells expressing the modified HLA-DQ molecule described herein, and nucleic acids encoding the modified HLA-DQ molecule.

[0075] This disclosure also provides a composition comprising: a modified HLA-DQ molecule present on the surface of a cell-free membrane or a cell, and a pharmaceutically acceptable carrier. The composition may also comprise one or more pharmaceutically acceptable excipients. In one embodiment, the cell is an engineered cell. In a specific embodiment, the engineered cell is selected from the group consisting of monocytes, leukocytes, macrophages, and antigen-presenting cells. In one embodiment, the cell-free membrane is an exosome. In various embodiments, the composition is used to treat a variety of diseases and symptoms, including, for example, cancer, pathogen-driven diseases, and immune diseases.

[0076] Methods to enhance cytotoxicity

[0077] In various embodiments, the modified HLA molecule is used to enhance the cytotoxicity of target cells. In one embodiment, the modified HLA molecule can be targeted to target cells, such as cancer cells (benign or malignant), pathogens, or other unwanted cells. The modified HLA molecule can be administered as part of a cell (e.g., engineered monocytes, leukocytes, macrophages, antigen-presenting cells) or administered in a cell-membrane-free environment (e.g., exosomes).

[0078] Engineered cells or cell-free membranes expressing the modified HLA molecules are administered to patients requiring this treatment. Administration can be performed via various routes, including intravenous injection, direct tissue injection, and intraperitoneal injection. The dosage and frequency of administration are determined based on the patient's condition, disease severity, and desired treatment outcome.

[0079] Modified HLA molecules in engineered cells or cell-free membranes can be used in a variety of therapeutic applications, including but not limited to autoimmune diseases, transplantation, cancer immunotherapy, and infectious diseases. In autoimmune diseases, the modified HLA molecules can help modulate the immune response. In transplantation, the engineered cells or cell-free membranes can be used to improve graft acceptance and reduce the risk of rejection in organ and tissue transplantation. In cancer immunotherapy, the modified HLA molecules can enhance the presentation of tumor antigens, thereby enhancing the immune system's ability to recognize and destroy cancer cells. In infectious diseases, the engineered cells or cell-free membranes can be used to enhance the immune response against infection. Example

[0080] Example 1: Disulfide bond engineering of soluble peptide-HLA-DQ7-Fc (pDQ7-Fc) fusion protein

[0081] To identify the paired amino acid residues of the interchain disulfide bonds introduced through mutagenesis in HLA-DQ7, existing structural models of HLA-DQ2 and HLA-DQ8 molecules (PDB IDs: 1S9V and 1JK8, respectively) were analyzed visually and using the Protein Disulfide Bond Modeling (MODIP) program. Numerous residue pairs from the α1 / β1 domain of the heterodimer were juxtaposed at the bottom or sides of the peptide-binding groove, and additional sites were also identified between the α2 / β1 and α2 / β2 domains. Figure 1A). The MODIP program grades candidate sites based on whether the atomic distance can accommodate a disulfide bond and whether there is any significant steric strain. Grade A indicates that the modeled disulfide bond distance and dihedral angle are within acceptable ranges; Grade B indicates that the bond is geometrically suitable but has some spatial distortion; Grade C indicates that the site is too close to the disulfide bond in space; Grade D indicates that the sulfur atom cannot be geometrically fixed at that location and is therefore unsuitable for disulfide bonding. Most candidate pairings are poorly rated and there are frequent inconsistencies between the structural models of HLA-DQ2 and HLA-DQ8. Figure 1 B).

[0082] DNA encoding the paired α and β strands of wild-type and mutant HLA-DQ7 was transfected into Chinese hamster ovary (CHO) cell lines, and the expression of these recombinant proteins in the supernatant was measured. In addition to introducing the pairing mutations for disulfide bond formation, a signal peptide sequence, a CLIP occupant peptide sequence (PVSKMRMATPLLMQA), and a flexible polyglycine linker (GGGSG)2 were fused to the 5' end of the β strand sequence. The 3' end of the β strand sequence was fused to the human IgG1 Fc fragment sequence (…). Figure 6 The design was intended to generate a peptide-HLA-DQ7 (pDQ7) complex, which could further dimerize via the Fc fragment to form a divalent pDQ7-Fc fusion protein within the immunoglobulin-like backbone. As measured by ELISA, extremely low expression of wild-type pDQ7-Fc was observed in the supernatant; however, for the YCDC, ACEC, and ACNC mutants, expression levels were significantly increased, up to 165-fold compared to wild-type. Figure 1 C). Compared to the wild type, the other mutants did not show significantly increased expression levels.

[0083] Wild-type and mutant pDQ7-Fc proteins were purified and evaluated by SDS-PAGE analysis. Figure 1 D). Under non-reducing conditions, the YCDC, ACEC, and ACNC mutants showed a clear band of approximately 150 kDa, consistent with the expected molecular weight of the pDQ7-Fc molecule. However, the wild-type and the remaining mutants did not exhibit this band, and a smaller band of approximately 100 kDa was observed, which had the same size as the β-Fc dimer in the absence of paired α chains. Under reducing conditions that disrupted inter- and intra-chain disulfide bonds, a β-chain of approximately 50 kDa was observed in all mutants; for some mutants, an α-chain of approximately 25 kDa with varying intensities was also observed. Figure 7 These data together demonstrate that stable pDQ7-Fc mutants can be generated with increased expression levels and yields through site-directed disulfide bond engineering.

[0084] Example 2: Characteristics of amino acid residues successfully engineered for disulfide bonds through mutation

[0085] HLA molecules exhibit high polymorphism in the human population, enabling the presentation of various exogenous peptides or neoantigens to initiate adaptive immunity. In 337 HLA-DQA1 and 1516 HLA-DQB1 proteins (IPD-IMGT / HLA database version 3.54, released October 2023) (Barker et al., Nucleic Acids Res, 2023), three pairs of amino acid residues successfully mutated for disulfide bond engineering were all conserved. Furthermore, cysteine ​​residues have not been found at these positions in humans. Figure 2 A).

[0086] Solvent accessibility of the mutated residues was visualized. Solvent accessibility can indicate the likelihood of generating new epitopes after mutation. Figure 2 B- Figure 2 (D) In ​​the HLA-DQ7 model created using the pHLA3D procedure, all three pairs of mutated residues in YCDC, ACEC, and ACNC were visible on the model surface. From the side view, the 83A / 5E pair appeared more exposed than the Y19 / D6 and A84 / N33 pairs. From the top of the molecule, these three pairs, especially the latter two, were largely obscured by the α-helix of the α-chain and therefore unlikely to be recognized by the T-cell receptor (TCR). Regarding potential recognition by the B-cell receptor (BCR), these three pairs of mutated residues are conserved in humans and are not known to be immunogenic. All 27 antibody-validated HLA-DQ eplets in the HLA epitope registry were re-examined. Whether located on the α-chain or β-chain, these eplets did not contact the three pairs of residues mutated for disulfide bond engineering. These data suggest the potentially low immunogenicity of these mutations, necessitating further investigation in alloimmune models.

[0087] Example 3: Effect of peptides on pDQ7-Fc(YCDC) fusion protein

[0088] The stability of pDQ7-Fc(YCDC) was investigated to determine whether it was peptide-dependent. Without CLIP, the DQ7-Fc(YCDC) construct failed to express any detectable protein of the expected size. Figure 3 A), indicating that endogenous peptides in cultured cells are insufficient to stabilize the soluble protein. A search of the Immunological Epitope Database (IEDB) (Vita et al., Nucleic Acids Res., 2019) identified three SARS-CoV-2-derived peptides that bind to DQ7 with high affinity, defined as the half-maximum inhibitory concentration (IC50) in a radioligand competitive binding assay.50 <1000 nM (Heide et al., PloS Pathol, 2021). By replacing CLIP in the pDQ7-Fc(YCDC) protein with the identified SARS-CoV-2 peptide Mem_36 (SEQ ID NO:3) (IC 50 :48 nM), Mem_34 (IC 50 :649 nM) and Ncl_54 (IC 50 Variants were generated using a method involving 794 nM. PAGE analysis showed that variants containing the Mem_36 peptide expressed significantly more than those containing CLIP. In contrast, variants containing the other two SARS-CoV-2 peptides were undetectable. Figure 3 A). Quantification of these variant proteins in the supernatant by ELISA showed that the expression level of the Mem_36 variant was 8 times higher than that of the CLIP variant, while the expression of variants containing Mem_34 and Ncl_54 was extremely low. Figure 3 B). These data support the view that stable expression of disulfide-engineered pDQ7-Fc is peptide-dependent. The presence of higher-affinity peptides is associated with higher yields of the fusion protein.

[0089] Example 4: Binding characteristics of the pDQ7-Fc fusion protein

[0090] The proper folding and intact surface epitopes of the pDQ7-Fc protein were investigated. pDQ7-Fc (knob-in-hole) was produced as a reference (pDQ7(Ref)) by co-expressing the α-chain linked with Fc(knob) and the β-chain linked with Fc(hole) in CHO cells (Serra et al., Nat. Commun., 2019). Heterodimerization of Fc(knob) and Fc(hole) (typically used for the production of bispecific antibodies) promoted the formation of the pDQ7 complex in this case. As measured by ELISA, serially diluted YCDC, ACEC, and ACNC variants of pDQ7-Fc showed similar binding curves to the anti-DQ7 antibody (HB144). Figure 4 A). All three variants exhibited higher affinity than the reference protein. In contrast, the wild-type and pDQ7-Fc ICGC variants showed extremely low binding. The YCDC variants carrying CLIP and Mem_36 peptides, respectively, also showed similar binding profiles. Both bound the anti-DQ7 antibody with higher affinity than the variants without the peptide. Figure 4 B).

[0091] The binding of pDQ7-Fc variants to three anti-DQ antibodies (HB144, SPV-L3, and 1A3) was examined by Western blotting. These antibodies exhibited unique binding specificities: HB144 was specific for the DQ3 parent type (including the DQ7 subtype); SPV-L3 was specific for the DQ α chain; and 1A3 was specific for the DQ β chain. pDQ7 (Ref) and the four pDQ7-Fc variants tested (i.e., YCDC (CLIP), ACEC (CLIP), ACNC (CLIP), and YCDC (Mem_36)) were detected by all three antibodies and showed the expected size ( Figure 4 C). Neither the wild-type nor the ICGC variant protein was detectable by any antibody. pDR3-Fc (knob-in-hole) (pDR3(Ref)) was produced as another negative control and was not detected by any DQ-specific antibody. Figure 4 C). The above results indicate that various disulfide-engineered pDQ7-Fc proteins possess intact surface epitopes that are recognized by antibodies with known specificity.

[0092] Example 5: Complement-dependent cytotoxic effects of pDQ7-Fc on homologous B-cell hybridomas

[0093] The ability of disulfide-engineered pDQ7-Fc protein to mediate antigen-specific killing of homologous antibody-producing cells (e.g., HB144 B-cell hybridomas) was investigated. The pDQ7 complex guides the fusion protein to specific B-cell receptors; simultaneously, the Fc moiety activates the complement cascade and triggers cytotoxic effects. Figure 5 A). In fact, significant binding of pDQ7-Fc(YCDC) to HB144 B cell hybridomas was detected by flow cytometry. In contrast, wild-type and ICGC variants were not detected on the cells. Figure 5 B). Flow cytometry complement-dependent cytotoxicity assays (Webber et al., Blood, 2022) showed that the YCDC and ACEC variants of pDQ7-Fc significantly killed HB144 cells in a dose-dependent manner. However, the killing effect appeared less potent than previously reported class I HLA-Fc proteins (Id.). To further enhance complement-dependent killing, gain-of-function mutations were introduced into the Fc portion of the fusion protein. Through mutations in E345R (SEQ ID NO:4) and E430G (SEQ ID NO:5), the half-maximal cytotoxic concentration (CMC) of pDQ7-Fc (YCDC) was significantly increased. 50The concentrations decreased from 0.003 mg / mL to 0.00048 mg / mL and 0.00071 mg / mL, respectively. These results demonstrate that the dimerized, disulfide-engineered pDQ7-Fc protein functions as a targeting agent and has the potential to kill antigen-specific cells that generate homologous antibodies.

[0094] Example 6: Disulfide bond engineering of soluble peptide-HLA-DQ2-Fc (pDQ2-Fc) fusion protein

[0095] Two DNA constructs encoding the two strands of pDQ2-Fc: (i) the extracellular domain of the DQ2α strand (SEQ ID NO: 1), including an N-terminal signal peptide; and (ii) the extracellular domain of the DQ2β strand (SEQ ID NO: 6), with the signal peptide and glutenin peptide glia-α1a (QLQPFPQPELPY) attached to its N-terminus, and human IgG1 Fc attached to its C-terminus. Figure 8 ).

[0096] DNA encoding paired α and β strands of wild-type and variant HLA-DQ2 was transfected into CHO cell lines, and the expression of these recombinant proteins in the supernatant was measured. In addition to the paired mutations introduced to form disulfide bonds, sequences of the signal peptide, glia-α1a (QLQPFPQPELPY) locator peptide, and flexible polyglycine linker (GGGSG)2 were fused to the 5' end of the β strand sequence. The 3' end of the β strand sequence was fused to a human IgG1 Fc fragment sequence. This design was intended to generate a peptide-HLA-DQ2 (pDQ2) complex, which would further dimerize via the Fc fragment, forming a divalent pDQ2-Fc fusion protein within the immunoglobulin-like backbone. As measured by ELISA (coated with SPV-L3 antibody at 0.25 μg / mL), extremely low expression of wild-type pDQ2-Fc was observed in the supernatant (data not shown), while expression levels were significantly increased in the YCDC and ACEC mutants. Compared with the wild type, the remaining mutants did not show significantly increased expression levels. Figure 8 ).

[0097] Example 7: Disulfide bond engineering of soluble peptide-HLA-DQ8-Fc (pDQ8-Fc) fusion protein

[0098] Two DNA constructs encoding two strands of pDQ8-Fc (YCDC): (i) the extracellular domain of the DQ8α strand (SEQ ID NO: 8), including an N-terminal signal peptide with a Y19C mutation; and (ii) the extracellular domain of the DQ8β strand (SEQ ID NO: 7), with the signal peptide and CLIP peptide (PVSKMRMATPLLMQA) attached to its N-terminus, and human IgG1 Fc attached to its C-terminus. Figure 9 The mutant was purified by protein A and evaluated by SDS-PAGE analysis. Under non-reducing conditions, the YCDC-pDQ8 variant showed a clear band of approximately 150 kDa, consistent with the expected molecular weight of the pDQ8-Fc molecule.

[0099] Example 8: Disulfide bond engineering of soluble peptide-HLA-DR4-Fc (pDR4-Fc) fusion protein

[0100] Based on the crystal structures of DR4 with different peptides (including 5LAX (pep 26), 4IS6 (gl100), 5JLZ (Cit_pep 26), and 2SEB (COL2)), candidate positions for introducing cysteine ​​residues for disulfide bond formation in DR4 (SEQ ID NOs: 9-10) were predicted using the MODIP program. Based on these structures, eight pairs of potential mutations capable of forming disulfide bonds were predicted. Figure 10 Interestingly, the P83C-P5C pair (corresponding to the pDQ7 lead pair) showed high ratings in all four models. The P83C-P5C pair was mutated with a cysteine ​​residue and used CLIP as a placeholder peptide for protein production assays targeting pDQ7, as described above. The β-chain was side-linked to both the lead peptide and the CLIP peptide at the N-terminus and to human IgG1 Fc at the C-terminus. A clear band of approximately 150 kDa (marked with an asterisk) was observed in non-reducing SDS-PAGE, consistent with the expected molecular weight of the dimeric pDR4-Fc molecule, at a yield of 16 mg / L cell culture.

[0101] The foregoing description of the embodiments is provided for illustrative and descriptive purposes. It is not intended to be exhaustive or limiting of this disclosure. Individual elements or features of a particular embodiment are generally not limited to that particular embodiment, but are interchangeable and applicable to selected embodiments where applicable, even if not specifically shown or described. Similarly, they may vary in various ways. Such variations are not considered to depart from this disclosure, and all such modifications are intended to be included within the scope of this disclosure.

[0102] method

[0103] Cell culture

[0104] CHO-S cells (Invitrogen) were suspended and grown in CHOgro expression medium (Mirus) at 37°C in a shaker until transfection for class II HLA protein production. IVD12 B hybridoma cells were obtained from ATCC and cultured at 37°C in DMEM supplemented with glucose (4.5 g / L), 1 mM sodium pyruvate, 15% heat-inactivated fetal bovine serum, and antibiotics.

[0105] plasmid

[0106] The extracellular domain coding sequences of the HLA-DQ7 β chain (DQB1*03:01) and α chain (DQA1*05:01) were codon-optimized for mammalian cells (GenScript) and cloned into the pcDNA3.4 vector. The natural signal peptide sequence (MILNKALMLGALALTTVMSPCGG) of the α chain was replaced with the signal peptide sequence (MTRLTVLALLAGLLASSRA) from the azuril progenitor protein to enhance protein secretion into mammalian cell culture supernatant. For the β chain, its natural signal peptide sequence was retained. A (GGGSG)2 flexible linker and a placeholder peptide CLIP (PVSKMRMATPLLMQA) or other test peptide were inserted between the signal peptide and the β chain sequence; the β chain sequence was followed by a human IgG1 Fc fragment. All sequences encoding wild-type and variant DQ7Fc proteins were synthesized using GenScript.

[0107] Protein expression and purification

[0108] First, the dimeric DQ7Fc recombinant protein was transiently expressed in CHO-S cells (Invitrogen). Following the manufacturer's recommendations, transfection with TransIT-PRO (Mirus) was performed in Optimum Growth Flasks (Thompson) at a viability >97% and a concentration of approximately 4.0 × 10⁻⁶. 6Healthy CHO-S cells / mL were used. TransIT-PRO was premixed with plasmids of the DQ7Fc β and α chains at a molar ratio of 1:2 to a final concentration of 1 mg total DNA per liter of culture. Immediately after transfection, the recommended volume of transfection kit enhancer was added. Cultures were collected after 7 days at 32°C. The supernatant of the cell culture was collected by centrifugation at 5000g for 10 min at 4°C, followed by filtration through a 0.22 μm regenerated cellulose membrane. The secreted protein was purified by affinity chromatography using a MabSelect PrismA Protein A column (Cytia). The Fc fusion protein was eluted with 0.1 M sodium citrate (pH 3.0) and immediately neutralized with 1 M Tris-HCl (pH 9.0). The purified protein was then transferred to 1× PBS.

[0109] ACECs were further purified using ion-exchange chromatography. The major dimer DQ7Fc was separated from minor aggregates and β-Fc dimers using Q Sepharose fast flow resin (Cytia #17051001). The DQ7Fc dimer was eluted with 250 mM NaCl in 20 mM phosphate buffer, while the aggregates and β-Fc dimers were eluted with a higher NaCl concentration. The purity of the eluted fractions was analyzed by non-reducing SDS-PAGE and analytical size exclusion chromatography (SEC; TSKgel G3000SWXL using 1×PBS as the elution buffer).

[0110] The reference protein DQ7Fc(KIH) was produced based on the work of Serra et al. (31). In short, the DNA sequences encoding the α strand (DQA1*05:01) and β strand (DQB1*03:01) of DQ7 were codon-optimized for mammalian cells and cloned into the pcDNA3.4 vector, respectively, with the downstream human IgG1 Fc_knob (S350C / T362W) and Fc_hole (Y377C / T394S / L369A / Y435V) sequences within the frame. The endotoxin-free plasmids encoding the paired α and β strands were co-transfected into CHO cells at a 1:1 ratio using TransIT-PRO (Mirus), and the cells were then cultured at 32°C for 7 days before collection. The DQ7FcKIH in the culture supernatant was purified by protein A chromatography and replaced with phosphate-buffered saline (PBS) as described above.

[0111] ELISA for protein quantification

[0112] Anti-DQ7 antibody purified from IVD12 B hybridoma cells was used to pre-coat 96-well polystyrene microplates (Nunc Maxisorp, Thermo Fisher) at a final concentration of 0.3 μg / mL. The wells were washed and blocked with 5% bovine serum albumin (0.1%) in PBS / T. Supernatant from day 7 of transfected CHO-S cell culture or purified protein was then added, and the plates were incubated at room temperature for 1 hour. After washing the plates again, horseradish peroxidase-conjugated goat anti-human IgG antibody (Jackson Immuno Research) was added. After incubation at room temperature for 1 hour followed by washing, a chromogenic substrate solution (3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide) was added. The reaction was terminated with sulfuric acid, and the optical density was measured at 450 nm using a Molecular Devices VersaMax microplate reader.

[0113] Protein electrophoresis

[0114] Protein samples (5 μg) were electrophoresed on 4%–20% SDS-PAGE gels (Bio-Rad). For reducing conditions, samples were diluted 1:4 with 4× Laemmli sample buffer (Bio-Rad) supplemented with 10% 2-mercaptoethanol (final concentration 2.5%) and then boiled at 100 °C for 5 min. Non-reducing conditions were evaluated by diluting samples 1:4 with 4× sample buffer (100 mM Tris-HCl, pH 6.8, 40% glycerol, and bromophenol blue) without boiling and loading them directly onto the gel. Gel images were captured using a ChemiDoc MP imaging system (Bio-Rad). The gel images were then analyzed using ImageLab software (Bio-Rad) for protein band quantification and characterization.

[0115] Protein blot

[0116] For Western blot analysis, proteins were transferred to nitrocellulose membranes (Bio-Rad) after SDS-PAGE. The membranes were blocked with 5% BSA in PBS / T (0.1%) for 1 hour, then incubated with one of the following antibodies: 1) IVD12 (self-purified, 0.3 mg / mL) diluted 1:200, 2) SPV-L3 (0.2 mg / mL; Novus Biologicals) diluted 1:250, and 3) 1A3 (10 mg / mL; Leinco Technologies) diluted 1:10,000. After washing with PBS / T, the membranes were incubated with IRDye 800CW goat anti-mouse (Licor) diluted 1:5000 for 1 hour. Finally, the membranes were washed again with PBS / T and analyzed using Licor ODYSSEY.

[0117] Bioinformatics Analysis

[0118] The structures of HLA-DQ2 (PDB ID: IS9V) and HLA-DQ8 (PDB ID: 1JK8) were visualized using the Mol* 3D viewer (60) on rcsb.org. The sites for disulfide bridge insertion were selected using the MODIP server (caps.ncbs.res.in / iws / modip.html). To analyze the mutation sites of disulfide bond engineering, multiple sequence alignment files of the HLA-DQA1 and HLA-DQB1 protein sequences were downloaded from the IPD-IMGT / HLA database (version 3.54, released October 2023) and processed using an internal script to generate a text document file listing each allele and its protein sequence on each line. A graphical representation of the HLA-DQA1 and HLA-DQB1 alignments was generated using weblogo (61) with default parameters. The surface exposure of mutated residues in HLA-DQ7 was visualized using pHLA3D (38).

[0119] FlowPRA assay

[0120] To verify the anti-DQ7 specificity of the antibody produced by IVD12 cells, FlowPRA assays were performed on an AttuneNxT instrument (Invitrogen) using the FlowPRA Single Antigen Class II HLA-4 Antibody Detection Kit (One Lambda, West Hills, CA. #FL2HD04) according to the manufacturer's instructions.

[0121] Cell binding assay

[0122] IVD12 hybridoma cells (ATCC) were washed in DPBS and seeded at 50,000 cells per well in V-bottom 96-well plates. After blocking with TruStain FcX (BioLegend) at a 1:200 dilution in staining buffer (1× PBS with 0.5% BSA and 2 mM EDTA), an increased amount of DQ7Fc protein was added to the buffer as shown, or PBS was added as a negative control. After incubation at 4°C for 20 min, the cells were washed three times in buffer and stained with FITC-conjugated anti-human IgG at a 1:100 dilution. After a second incubation at 4°C for 30 min, the cells were washed three times, resuspended in 200 μL buffer, and analyzed by flow cytometry on an Attune NxT instrument (Invitrogen).

[0123] Flow cytometry complement-dependent cytotoxicity (CDC) assay

[0124] DQ7-specific B-cell hybridoma cells (IVD12, ATCC) were washed in DPBS and seeded at 50,000 cells per well in 96-well V-bottom plates. Cells were pelleted by centrifugation at 1400 rpm and 4°C for 4 min. Freshly thawed rabbit serum (One Lambda, #CDR-50) was diluted 1:8 in DPBS. DQ7 Fc protein was added to the diluted rabbit serum at a concentration of 0.01 mg / mL, followed by serial dilutions at 1:2 to titers up to 256. Rabbit serum containing and without HLA-Fc was added at 45 μL / well to the pelleted hybridoma cells and incubated at room temperature in the dark for 3 h. Cells were washed in 150 μL, followed by 200 μL, of staining buffer, then resuspended in 100 μL of buffer containing 7-AAD diluted 1:50, and analyzed by flow cytometry on an Attune NxT instrument (Invitrogen).

[0125] Data analysis and statistics

[0126] All data were derived from at least three independent experiments. For protein expression levels measured by ELISA, the 95% confidence intervals and interpolated concentrations of the standard curves were reported from representative experiments. For antibodies binding to titrated DQ7Fc protein, the mean and standard deviation (SD) of OD values ​​were plotted against protein concentration and fitted with a four-parameter logistic (4PL) regression model. For CDC assays, the mean and SD of the percentage of viable treated cells compared to untreated cells (7AAD-) were plotted against protein concentration; the CC50 for each wild-type or variant protein was determined by nonlinear regression using a variable slope model. All data analyses were performed using Prism version 9.2.0 (GraphPad Software, LLC).

Claims

1. A modified HLA-DQ molecule, said molecule comprising: A first peptide comprising a wild-type α chain and a signal peptide, wherein at least one amino acid of the wild-type α chain is substituted with a cysteine ​​residue; and The second peptide comprises a wild-type β chain, a signal peptide, a placebo peptide, a linker, and an Fc fragment sequence, wherein at least one amino acid of the wild-type β chain is substituted with a cysteine ​​residue. in, Each substituted cysteine ​​on the α chain is adjacent to a corresponding substituted cysteine ​​on the β chain, such that each substituted cysteine ​​on the α chain forms a disulfide bond with the substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

2. The modified HLA-DQ molecule according to claim 1, wherein, The HLA-DQ molecule is HLA-DQ2.5, HLA-DQ7, or HLA-DQ8.

3. The modified HLA-DQ molecule according to claim 1, wherein, The signal peptide of the first peptide is derived from the azoxystrobin precursor protein.

4. The modified HLA-DQ molecule according to claim 1, wherein, The second peptide is either a CLIP peptide or a glia-α1a peptide.

5. The modified HLA-DQ molecule according to claim 1, wherein, The linker for the second peptide is a polyglycine linker.

6. The modified HLA-DQ molecule according to claim 1, wherein, The Fc fragment sequence is the human IgG1 Fc fragment sequence.

7. The modified HLA-DQ molecule according to claim 2, wherein, The first peptide has the following amino acid sequence, which has at least 85% sequence identity with SEQ ID No: 1, and the second peptide has the following amino acid sequence, which has at least 85% sequence identity with SEQ ID No:

2.

8. The modified HLA-DQ molecule according to claim 7, wherein, The substituted cysteine ​​on the α chain is located at an amino acid position selected from the group consisting of the 19th, 83rd, 84th positions and combinations thereof.

9. The modified HLA-DQ molecule according to claim 8, wherein, The substituted cysteine ​​on the β chain is located at an amino acid position selected from the group consisting of the 6th, 5th, 33rd positions and combinations thereof.

10. The modified HLA-DQ molecule according to claim 9, wherein, The substituted cysteine ​​on the α chain is located at the 19th amino acid position, and the substituted cysteine ​​on the β chain is located at the 6th amino acid position. Wherein, the substituted cysteine ​​on the α chain is located at the 83rd amino acid position, and the substituted cysteine ​​on the β chain is located at the 5th amino acid position; Wherein, the substituted cysteine ​​on the α chain is located at the 84th amino acid position, and the substituted cysteine ​​on the β chain is located at the 33rd amino acid position; or a combination thereof.

11. The modified HLA-DQ molecule according to claim 9, wherein, The substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C. Wherein, the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C; Wherein, the substituted cysteine ​​on the α chain is A84C, and the substituted cysteine ​​on the β chain is N33C; or a combination thereof.

12. The modified HLA-DQ molecule according to claim 10, wherein, The substituted cysteine ​​on the α chain is located at amino acid position 19, and the substituted cysteine ​​on the β chain is located at amino acid position 6; and / or The substituted cysteine ​​on the α chain is located at position 83 of the amino acid, and the substituted cysteine ​​on the β chain is located at position 5 of the amino acid.

13. The modified HLA-DQ molecule according to claim 11, wherein, The substituted cysteine ​​residue on the α chain is Y19C, and the substituted cysteine ​​residue on the β chain is D6C; and / or Wherein, the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C.

14. The modified HLA-DQ molecule according to claim 1, wherein the molecule is any one of those depicted in Figure 2.

15. The modified HLA-DQ molecule according to claim 1, wherein, The Fc fragment sequence attached to the 3' end of the β chain has at least one gain-of-function mutation.

16. The modified HLA-DQ molecule according to claim 15, wherein, The gain-of-function mutations are E345R and / or E430G.

17. A cell expressing the modified HLA-DQ molecule according to any one of claims 1 to 16.

18. A nucleic acid encoding the modified HLA-DQ molecule according to any one of claims 1 to 16.

19. A composition comprising, on the surface of a cell-membrane-free or cellular organism, any of the modified HLA-DQ molecules of claims 1 to 16, and a pharmaceutically acceptable carrier.

20. The composition according to claim 19, wherein, The cells are engineered cells.

21. The composition according to claim 20, wherein, The engineered cells are at least monocytes, leukocytes, macrophages, or antigen-presenting cells.

22. The composition according to claim 19, wherein, The cell-membrane-free form is at least an exosome.

23. The composition according to claim 19, wherein, The composition is used to treat cancer, pathogen-driven diseases, and immune responses.

24. A method for enhancing cytotoxicity, the method comprising: The modified HLA-DQ molecule of any one of claims 1 to 16, the cell expressing the modified HLA-DQ molecule, or the cell containing the nucleic acid sequence encoding the modified HLA-DQ molecule is targeted to the target cell.

25. The method according to claim 24, wherein, The cytotoxicity is complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity.

26. The method according to claim 24, wherein, The target cells are cancer cells, pathogens, and unwanted cells.

27. A method for producing modified HLA-DQ molecules, the method comprising: One or more amino acids in the wild-type α chain are replaced with cysteine; In order to make the α chain and β chain contain the same number of substituted cysteines, one or more amino acids of the wild-type β chain are replaced with cysteines. as well as A disulfide bond is formed between each substituted cysteine ​​on the α chain and each substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

28. A modified HLA-DQ7 molecule, said molecule comprising: A first peptide having the amino acid sequence of SEQ ID No: 1, comprising a wild-type α chain and a signal peptide, wherein a cysteine ​​residue on the wild-type α chain is substituted at amino acid positions 19, 83, 84, or a combination thereof; and The second peptide has the amino acid sequence of SEQ ID No: 2, and comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein the cysteine ​​residue on the wild-type β chain is substituted at amino acid positions 6, 5, 33, or a combination thereof. in, The substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C. Wherein, the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C; Wherein, the substituted cysteine ​​on the α chain is A84C, and the substituted cysteine ​​on the β chain is N33C; or a combination thereof. Each substituted cysteine ​​on the α chain is adjacent to a corresponding substituted cysteine ​​on the β chain, such that each substituted cysteine ​​on the α chain forms a disulfide bond with the substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

29. The modified HLA-DQ7 molecule according to claim 28, wherein, The signal peptide of the first peptide is derived from the azoxystrobin precursor protein.

30. The modified HLA-DQ7 molecule according to claim 28, wherein, The second peptide's placeholder peptide is the CLIP placeholder peptide.

31. The modified HLA-DQ7 molecule according to claim 28, wherein, The linker for the second peptide is a polyglycine linker.

32. The modified HLA-DQ7 molecule according to claim 28, wherein, The Fc fragment sequence is the human IgG1 Fc fragment sequence.

33. The modified HLA-DQ7 molecule according to claim 28, wherein, The human IgG1 Fc fragment sequence attached to the 3' end of the β chain has at least one gain-of-function mutation.

34. The modified HLA-DQ7 molecule according to claim 28, wherein, The gain-of-function mutations are E345R and / or E430G.

35. A modified HLA-DQ7 molecule, said molecule comprising: A first peptide having an amino acid sequence having at least 85% sequence identity with SEQ ID No: 1, comprising a wild-type α chain and a signal peptide, wherein cysteine ​​residues on the wild-type α chain are substituted at amino acid positions 19, 83, 84, or a combination thereof; and The second peptide has the following amino acid sequence, which has at least 85% sequence identity with SEQ ID No:

2. The second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein the cysteine ​​residue on the wild-type β chain is substituted at the amino acid positions 5, 33, or a combination thereof. Wherein, the substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C; Wherein, the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C; Wherein, the substituted cysteine ​​on the α chain is A84C, and the substituted cysteine ​​on the β chain is N33C; or a combination thereof. Each substituted cysteine ​​on the α chain is adjacent to a corresponding substituted cysteine ​​on the β chain, such that each substituted cysteine ​​on the α chain forms a disulfide bond with the substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

36. A modified HLA-DR molecule, said molecule comprising: A first peptide having an amino acid sequence having at least 85% sequence identity with SEQ ID No: 9, comprising a wild-type α chain and a signal peptide, wherein a cysteine ​​residue on the wild-type α chain replaces an amino acid at position 83 and / or 84; and The second peptide has the following amino acid sequence, which has at least 85% sequence identity with SEQ ID No:

10. The second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein the cysteine ​​residue on the wild-type β chain replaces the amino acid position 5 and / or 33. in, The substituted cysteine ​​residue on the α chain is P83C, and the substituted cysteine ​​residue on the β chain is P5C; and / or Wherein, the substituted cysteine ​​on the α chain is I84C, and the substituted cysteine ​​on the β chain is H33C. Each substituted cysteine ​​on the α chain is adjacent to a corresponding substituted cysteine ​​on the β chain, such that each substituted cysteine ​​on the α chain forms a disulfide bond with the substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

37. A modified HLA-DQ2.5 molecule, said molecule comprising: A first peptide having an amino acid sequence having at least 85% sequence identity with SEQ ID No: 1, comprising a wild-type α chain and a signal peptide, wherein cysteine ​​residues on the wild-type α chain are substituted at amino acid positions 19, 83, 84, or a combination thereof; and The second peptide has the following amino acid sequence, which has at least 85% sequence identity with SEQ ID No:

6. The second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein the cysteine ​​residue on the wild-type β chain is substituted at amino acid positions 6, 5, 33, or a combination thereof. in, The substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C. Wherein, the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C; Wherein, the substituted cysteine ​​on the α chain is A84C, and the substituted cysteine ​​on the β chain is N33C; or a combination thereof. Each substituted cysteine ​​on the α chain is adjacent to a corresponding substituted cysteine ​​on the β chain, such that each substituted cysteine ​​on the α chain forms a disulfide bond with the substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

38. A modified HLA-DQ8 molecule, said molecule comprising: A first peptide having an amino acid sequence having at least 85% sequence identity with SEQ ID No: 8, comprising a wild-type α chain and a signal peptide, wherein cysteine ​​residues on the wild-type α chain are substituted at amino acid positions 19, 83, 84, or a combination thereof; and The second peptide has the following amino acid sequence, which has at least 85% sequence identity with SEQ ID No:

7. The second peptide comprises a wild-type β chain, a signal peptide, a placeholder peptide, a linker, and an Fc fragment sequence, wherein the cysteine ​​residue on the wild-type β chain is substituted at amino acid positions 6, 5, 33, or a combination thereof. in, The substituted cysteine ​​on the α chain is Y19C, and the substituted cysteine ​​on the β chain is D6C. Wherein, the substituted cysteine ​​on the α chain is A83C, and the substituted cysteine ​​on the β chain is E5C; Wherein, the substituted cysteine ​​on the α chain is A84C, and the substituted cysteine ​​on the β chain is N33C; or a combination thereof. Each substituted cysteine ​​on the α chain is adjacent to a corresponding substituted cysteine ​​on the β chain, such that each substituted cysteine ​​on the α chain forms a disulfide bond with the substituted cysteine ​​on the β chain, thereby forming one or more interchain disulfide bonds.

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