Pocket Engineering of HLA Alleles for Treating Autoimmunity

Abstract

Methods of preventing or treating autoimmune disease are disclosed. In some cases, subjects with having or at risk of developing autoimmune disease are identified as possessing one or more autoimmunity-susceptibility HLA alleles at one or more HLA loci. In many cases, the HLA loci are selected from Class I and Class II loci, for example Class I A, B, and C, and Class II DQ, DR, and DP. In many cases, subjects suffering from or at risk of developing an autoimmune disease may be administered a plurality engineered autologous HSCs modified to carry and express a variant susceptibility allele having at least one mutation in the antigen binding cleft that alters antigen binding and/or specificity of that variant HLA molecule. In many embodiments, the engineered HSCs are CD34+ immune cells that express one or more modified HLA proteins.

1 . A variant HLA-DRB protein comprising:
 an amino acid sequence at least 95% identical to a sequence encoded by a native allele of HLA-DRB1, HLA-DRB3, HLA-DRB4, or HLA-DRB5; and   at least one amino acid residue at an amino acid position of the amino acid sequence being different than an amino acid residue at the amino acid position of the sequence encoded by the native HLA allele, wherein the amino acid position is at or near Pocket 1 of an antigen binding cleft and the variant HLA-DRB protein has altered binding affinity for at least one peptide antigen compared to a protein encoded by the native HLA allele.   
     
     
         2 . The variant HLA-DRB protein of  claim 1 , wherein the allele is HLA-DRB1. 
     
     
         3 . The variant HLA-DRB protein of  claim 1 , wherein the allele is HLA-DRB3. 
     
     
         4 . The variant HLA-DRB protein of  claim 1 , wherein the allele is HLA-DRB4. 
     
     
         5 . The variant HLA-DRB protein of  claim 1 , wherein the allele is HLA-DRB5. 
     
     
         6 . The variant HLA-DRB protein of  claim 2 , wherein the amino acid position is position 86 of the amino acid sequence. 
     
     
         7 . The variant HLA-DRB protein of  claim 6 , wherein the at least one amino acid residue at an amino acid position of the amino acid sequence is methionine or leucine. 
     
     
         8 . The variant HLA-DRB protein of  claim 7 , wherein the at least one amino acid residue is methionine. 
     
     
         9 . The variant HLA-DRB protein of  claim 7 , wherein the at least one amino acid residue is leucine. 
     
     
         10 . The variant HLA-DRB protein of  claim 7 , wherein the amino acid sequence is selected from SEQ ID NO:74 and 75. 
     
     
         11 . The variant HLA-DRB protein of  claim 10  for use in treating a patient suffering from an autoimmune disorder selected from rheumatoid arthritis (RA), celiac disease, diabetes mellitus type 1, systemic lupus erythematosus (SLE), multiple sclerosis (MS), myelin oligodendrocyte glycoprotein antibody disorders (MOGAD), myasthenic syndromes and neuromyelitis optica (NMO), ankylosing spondylitis, Behcet's syndrome, Birdshot uveitis, narcolepsy, narcolepsy type 1 (NT1; previously termed narcolepsy with cataplexy), Kawasaki disease, Crohn's disease, psoriasis, dermatomyositis (DM), Addison's disease, irritable-bowl syndrome (IBS), Graves' disease, Henoch-Schönlein purpura (HSP), sarcoidosis, Sjögren's syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's disease, idiopathic thrombocytopenic purpura, polymyositis (PM), paraneoplastic neurological syndromes (PNS), autoimmune encephalitis, lupus nephritis (LN), myasthenia gravis (MG), psoriatic arthritis, graft rejection, graft-versus-host disease (GVHD), an unwanted delayed-type hypersensitivity reaction, T-cell mediated pulmonary disease, neuritis, vitiligo, autoimmune pancreatitis, inflammatory bowel diseases, ulcerative colitis, glomerulonephritis, scleroderma, autoimmune thyroid diseases, asthma, autoimmune uveoretinitis, pemphigus vulgaris, pulmonary fibrosis or idiopathic pulmonary fibrosis, primary biliary cirrhosis, and pernicious anemia. 
     
     
         12 . An engineered hematopoietic cell comprising the variant HLA-DRB protein of  claim 10 . 
     
     
         13 . A nucleic acid coding for a variant HLA-DRB protein according to  claim 10 . 
     
     
         14 . An engineered hematopoietic cell comprising the nucleic acid of  claim 13 . 
     
     
         15 . (canceled) 
     
     
         16 . A method of modification of an HLA allele, comprising:
 identifying an HLA allele associated with one or more autoimmune diseases;   identifying one or more susceptible HLA alleles, wherein the susceptible HLA allele is associated with susceptibility to the one or more autoimmune diseases;   identifying a target sequence corresponding to an amino acid position at or near a pocket of an antigen binding cleft, the pocket being a set of primary anchor amino acid residues for antigen binding within the antigen binding cleft;   modifying the target sequence to cause substitution of a target amino acid at the amino acid position with a second amino acid having a side chain larger than the target amino acid to create an occlusion in the pocket, wherein the side chain of the second amino acid affects the antigen binding in the pocket of the antigen binding cleft; and thereby   occluding the pocket of the HLA allele.   
     
     
         17 . The method of  claim 16 , wherein the HLA allele is selected from HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5. 
     
     
         18 . The method of  claim 17 , wherein the pocket is pocket 1. 
     
     
         19 . The method of  claim 18 , wherein the amino acid position is 86 of an amino acid sequence encoded by the HLA allele. 
     
     
         20 . The method of  claim 19 , wherein the second amino acid is selected from methionine and leucine. 
     
     
         21 . The method of  claim 16 , wherein the HLA protein with the occlusion in the pocket possess lower binding affinity for at least one self-peptide associated with an autoimmune disease, optionally wherein the at least one self-peptide is deiminated, and further optionally wherein the target amino acid is not at the T-cell receptor binding interface. 
     
     
         22 . A method of treating a subject suffering from or at risk of developing an autoimmune disease, comprising:
 identifying a susceptible HLA allele of an HLA gene within an HLA complex of the subject, wherein the susceptible HLA allele is associated with susceptibility to the autoimmune disease;   isolating a plurality of CD34+ immune cells from the subject;   modifying the HLA complex of the CD34+ immune cells to create engineered CD34+ immune cells, wherein the engineered CD34+ immune cells do not express the susceptible HLA allele, and express an engineered HLA allele, wherein a protein encoded for by the engineered HLA allele differs from a protein encoded for by the susceptible HLA allele by presence of an occlusion within a pocket of an antigen binding cleft of the protein encoded for by the engineered HLA allele, and alters binding affinity for at least one self-peptide as compared to a protein coded for by the susceptible HLA allele;   isolating a plurality of the engineered CD34+ immune cells;   administering the isolated, engineered CD34+ immune cells to the subject; and thereby   treating the subject suffering from or at risk of developing the autoimmune disease.   
     
     
         23 . The method of  claim 22 , wherein the HLA allele is selected from HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5. 
     
     
         24 . The method of  claim 23 , wherein the pocket is pocket 1. 
     
     
         25 . The method of  claim 24 , wherein the occlusion within the pocket of the antigen binding cleft results from an amino acid substitution at position 86 of the protein encoded for by the engineered HLA allele as compared to the protein encoded for by the susceptible HLA allele. 
     
     
         26 . The method of  claim 25 , wherein the amino acid substitution replaces the amino acid at position 86 with an amino acid selected from methionine and leucine. 
     
     
         27 . The method of  claim 26 , wherein the protein encoded by the engineered HLA allele possess lower binding affinity for the at least one self-peptide associated with the autoimmune disease. 
     
     
         28 . The method of  claim 27 , the self-peptide is selected from a peptide comprising at least one deiminated residue. 
     
     
         29 . The method of  claim 21 , wherein the engineered HLA allele has an amino acid sequence of SEQ ID NO:74 or 75. 
     
     
         30 . The method of  claim 27 , wherein the engineered HLA allele has an amino acid sequence of SEQ ID NO:74 or 75. 
     
     
         31 . The method of  claim 22 , wherein administering the isolated, engineered CD34+ immune cells to the subject does not elicit a rejection or graft-versus-host disease in the subject.