Compositions for use in treating MHC-I diseases - Patent application

Abstract

The present disclosure provides a method for treating a subject having an immune disease by administering to the subject a therapeutically effective amount of an endoplasmic reticulum aminopeptidase 2 (ERAP2) inhibitor in combination with an HLA-A29 or HLA-B27 inhibitory nucleic acid molecule.

Description

[Technical field]

[0001] Sequence Listing Reference This application contains a Sequence Listing that has been submitted electronically as a text file with file name 18923810702SEQ, created on April 6, 2022, with a size of 1027 kilobytes. This Sequence Listing is incorporated herein by reference.

[0002] The present disclosure relates in part to a method of treating a subject having an immune disorder by administering to the subject a therapeutically effective amount of an endoplasmic reticulum aminopeptidase 2 (ERAP2) inhibitor in combination with an HLA-A29 or HLA-B27 inhibitory nucleic acid molecule. [Background technology]

[0003] The cellular immune response in humans depends at least in part on the presentation of small peptides of 8-10 amino acids in length that are binding proteins of the major histocompatibility complex (MHC) (i.e. class I MHC molecules). These small peptides are derived from the proteolytic degradation of proteins (foreign and autoantigens). One source of these antigens arises from infected or malignantly transformed cells that express specific protein molecules that, upon degradation, result in distinct antigenic peptides that are presented on the cell surface complexed with MHC class I molecules (MHCI). Cytotoxic T cells can recognize these complexes of MHC molecules and degraded protein antigens and induce apoptotic cell death. The aberrant generation of antigenic peptides can lead to immune system evasion or autoimmune reactions.

[0004] Most antigenic peptides are initially generated by the proteasome, many of which are larger than the final antigen epitope and contain one or more additional amino acids at the N-terminus. These antigenic peptide precursors are transported into the endoplasmic reticulum (ER), where they are further degraded by at least two different aminopeptidases (ERAP1 and ERAP2) to generate mature antigenic peptides for complex formation with MHC class I molecules. Thus, the activity of ERAP1 and ERAP2 can directly affect the presentation of antigenic peptides complexed with specific MHC molecules in a beneficial or detrimental manner, thereby altering the immune response. Thus, there remains a need for the identification of subjects with specific MHC-I diseases associated with ERAP2 activity and their treatment.

[0005] Shotgun chorioretinopathy (BSCR) is a rare autoimmune uveitis that preferentially affects individuals over 50 years of age of European descent and is treated with immunomodulatory therapy. The disease presents with vitreous inflammation and progressive loss in visual acuity due to inflammatory lesions and atrophy of the choroid and retina. T cells have been identified in the retinal and choroidal tissues and in the vitreous of affected BSCR eyes. Summary of the Invention

[0006] The present disclosure provides a method for treating a subject having an immune disease, the method comprising administering an ERAP2 inhibitor and i) an HLA-A29 inhibitor and / or ii) HLA-B27 to the subject.

[0007] The present disclosure relates to a method for treating a subject having MHC-I disease, comprising: performing an assay on a biological sample derived from the subject to determine whether the subject contains i) HLA-A29 allele and / or HLA-B27, and ii) a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, or performing such an assay already; administering a therapeutically effective amount of E to a subject having a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, and having an HLA-A29 allele; administering an RAP2 inhibitor and an HLA-A29 inhibitor, or administering therapeutically effective amounts of an ERAP2 inhibitor and an HLA-B27 inhibitor to a subject having a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, and having an HLA-B27 allele, wherein the presence of both i) HLA-A29 and / or HLA-B27 allele, and ii) a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein indicates that the subject is a candidate for treatment of MHC-I disease by inhibiting ERAP2.

[0008] The present disclosure provides a combination of an ERAP2 inhibitor and an HLA-A29 inhibitor for use in the treatment of an immune disease. The present disclosure provides a combination of an ERAP2 inhibitor and an HLA-B27 inhibitor for use in the treatment of an immune disease.

[0009] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0010] [Figure 1]Table showing HLA-A second allele frequency in the French cohort compared to European A29 carriers in UKB and GHS. Alleles belonging to the Aw19 broad antigen group that increase risk are A29, A30, A31 and A33 (red), while A32 shows protection (green). Fisher's exact test combining all Aw19 risk alleles provides the strongest enrichment in all comparisons. Only alleles with 3 or more case carriers are presented. Table is filtered by p-value when comparing case frequencies against A29 controls in UKB. [Diagram 2] Enrichment of Aw19 in shotcretinopathy cases is shown. Odds ratios for BSCR comparing the frequency of 14 HLA-A alleles present in 3 or more cases (>1%, x-axis) in 286 UParis cases compared to 108 UParis controls (blue), GHS control cohort #1 (n=4,014, dark green), GHS control cohort #2 (n=2,829, light green), and UKB controls (n=38,543, yellow). The Aw19 allele shows the highest odds ratio reproduced by the large A29 control cohort (red box), with the exception of A32, which has a reduced number of cases (green box). *p<0.01. [Diagram 3] Table showing HLA-A second allele frequency in the French cohort compared to UKB and GHS European A29 carriers. Alleles belonging to the Aw19 broad antigen group that increase risk are A29, A30, A31 and A33 (red), A32 shows protection (green). Logistic regression test of covariates including sex and 6 principal components calculated on gene array data of each analysis set. Results are presented for all HLA-A alleles with 3 or more case carriers. Alleles are sorted as in Figure 1. *3 principal components. [Figure 4]A table of top SNPs in the ERAP2 region is shown. When analyzed together with previous results (125 cases and 670 controls (Kuiper 2014)), the ERAP2 variant is genome-wide significant. rs10044354 was highly associated at the ERAP1-ERAP2 locus in previous genome-wide association studies of Dutch and Spanish cohorts, while rs27432 was highly associated at the region in this French cohort. The LD between the two loci is also shown. *The reference A allele is the minor allele, and the risk is the G allele. [Diagram 5] It is shown that ERAP2 splice region variant is protective for BSCR. The common ERAP2 splice region variant rs2248374 that disrupts ERAP2 expression is protective in the present BSCR cohort as well as previous Spanish and Dutch cohorts. [Figure 6] Showing the combined risk of ERAP2 and two copies of Aw19. 286 shotgun chorioretinopathy cases and 4,014 controls from GHS cohort #1 are used to calculate the additive risk of combining risk factors of ERAP2 and Aw19. Additive genotype model of ERAP2 risk signal tagged by rs10044354 and single (A29 / -) or double (A29 / Aw19) copies of Aw19 compared to the lowest risk combination of rs10044354-CC and one copy of Aw19 allele (A29). [Figure 7] A table of the combined risk of ERAP2 and Aw19 is shown. Using 286 shotgun chorioretinopathy cases and 4,014 controls from GHS cohort #1, the additive risk of combining risk factors of ERAP2 and Aw19 is calculated. Additive genotype model of ERAP2 risk signal tagged by rs10044354 and single (A29 / -) or double (A29 / Aw19) copies of Aw19 compared to the lowest risk combination of rs10044354-CC and one copy of Aw19 allele (A29). [Figure 8]Differences between the risk Aw19 allele and A32 are shown. Panel A) Sequence differences between the risk Aw19 allele (red) and the protective A32 allele (green). A32 exhibits an F at position 9 as in the reference A:01:01 allele, whereas the risk allele is either a T or an S at that position. The Bw4 epitope sequence is found only at positions 79-83 or A32. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Various terms relating to aspects of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0012] Unless expressly stated otherwise, it is not intended that any method or aspect described herein be interpreted as requiring that its steps be performed in a particular order. Thus, unless a method claim specifically states in the claims or description that the steps are to be limited to a particular order, no order is intended to be implied in any respect. This is true for all potentially unclear criteria for interpretation, including questions of logic regarding the arrangement of steps or operational flow, the plain meaning derived from grammatical construction or punctuation, or the number or type of aspects described herein.

[0013] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the term "about" means that the recited numerical values ​​are approximate and that small variations would not significantly affect the practice of the disclosed embodiments. When numerical values ​​are used, unless otherwise indicated by the context, the term "about" means that the numerical values ​​can vary by ±10% and remain within the scope of the disclosed embodiments.

[0014] As used herein, the term "comprising" may in certain embodiments be replaced with "consisting of" or "consisting essentially of," as desired.

[0015] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "polynucleotide," or "oligonucleotide" include polymeric forms of nucleotides of any length, including DNA and / or RNA, and can be single-stranded, double-stranded, or multistranded. A strand of a nucleic acid also refers to its complement.

[0016] As used herein, the term "subject" includes any animal, including mammals. Mammals include, but are not limited to, farm animals (e.g., horses, cows, pigs, etc.), pet animals (e.g., dogs, cats, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), and non-human primates (e.g., apes and monkeys, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a patient under the care of a physician.

[0017] The present disclosure provides a method for treating a subject having an immune disease, comprising administering an ERAP2 inhibitor to the subject. In some embodiments, the immune disease is MHC-I disease (MHC-I-opathy). In some embodiments, the immune disease is MHC-II disease (MHC-II-opathy). In some embodiments, the MHC-I disease is BSCR, ankylosing spondylitis (AS), psoriasis with uveitis, or juvenile idiopathic arthritis (JIA). In some embodiments, the MHC-I disease is BSCR. In some embodiments, the MHC-I disease is AS. In some embodiments, the MHC-I disease is psoriasis with uveitis. In some embodiments, the MHC-I disease is JIA.

[0018] In some embodiments, the MHC-I disease is BSCR. In some embodiments, the method further comprises detecting the presence or absence of an HLA-Aw19 allele in a biological sample obtained from the subject. In some embodiments, the subject is + In some embodiments, the subject is HLA-A29 + , HLA-A30 + , HLA-A31 + , or HLA-A33 + In some embodiments, the subject is or is suspected of having one copy of the HLA-Aw19 allele, or any combination thereof. In some embodiments, the method further comprises determining whether the subject has one copy of the HLA-Aw19 allele or two copies of the HLA-Aw19 allele. In some embodiments, the subject has a single copy of HLA-Aw19. In some embodiments, the subject has two copies of HLA-Aw19. In some embodiments, the subject has an HLA-A29 + / HLA-A30 + In some embodiments, the subject is HLA-A29 + / HLA-A31 + In some embodiments, the subject is HLA-A29 + / HLA-A33 + It is.

[0019] In some embodiments, the subject with BSCR is HLA-A29 + isn't it. In some embodiments, the subject with BSCR has at least any two copies of HLA-A29, HLA-A30, HLA-A31, or HLA-A33.In some embodiments, the subject with BSCR has at least any three copies of HLA-A29, HLA-A30, HLA-A31, or HLA-A33.In some embodiments, the subject with BSCR has all copies of HLA-A29, HLA-A30, HLA-A31, or HLA-A33.

[0020] In some embodiments, a subject with a BSCR has one copy of each of HLA-A29 and HLA-A30. In some embodiments, a subject with a BSCR has one copy of each of HLA-A29 and HLA-A31. In some embodiments, a subject with a BSCR has one copy of each of HLA-A29 and HLA-A33. In some embodiments, a subject with a BSCR has one copy of each of HLA-A30 and HLA-A31. In some embodiments, a subject with a BSCR has one copy of each of HLA-A30 and HLA-A33. In some embodiments, a subject with a BSCR has one copy of each of HLA-A31 and HLA-A33.

[0021] In some embodiments, a subject with a BSCR has one copy of HLA-A29 and two copies of HLA-A30. In some embodiments, a subject with a BSCR has one copy of HLA-A29 and two copies of HLA-A31. In some embodiments, a subject with a BSCR has one copy of HLA-A29 and two copies of HLA-A33. In some embodiments, a subject with a BSCR has one copy of HLA-A30 and two copies of HLA-A31. In some embodiments, a subject with a BSCR has one copy of HLA-A30 and two copies of HLA-A33. In some embodiments, a subject with a BSCR has one copy of HLA-A31 and two copies of HLA-A33.

[0022] In some embodiments, a subject with a BSCR has two copies of HLA-A29 and one copy of HLA-A30. In some embodiments, a subject with a BSCR has two copies of HLA-A29 and one copy of HLA-A31. In some embodiments, a subject with a BSCR has two copies of HLA-A29 and one copy of HLA-A33. In some embodiments, a subject with a BSCR has two copies of HLA-A30 and one copy of HLA-A31. In some embodiments, a subject with a BSCR has two copies of HLA-A30 and one copy of HLA-A33. In some embodiments, a subject with a BSCR has two copies of HLA-A31 and one copy of HLA-A33.

[0023] In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A29 and two copies of HLA-A30. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A29 and two copies of HLA-A31. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A29 and two copies of HLA-A33. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A30 and two copies of HLA-A31. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A30 and two copies of HLA-A33. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A31 and two copies of HLA-A33.

[0024] In some embodiments, the method further comprises administering an HLA-Aw19 inhibitor to the subject. In some embodiments, the HLA-Aw19 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-A29 antibody. In some embodiments, the HLA-Aw19 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes to HLA-Aw19. In some embodiments, the HLA-Aw19 is HLA-A29.

[0025] In some embodiments, the MHC-I disease is AS. In some embodiments, the method further comprises detecting the presence or absence of HLA-B27 or HLA-B40 in a biological sample obtained from the subject. In some embodiments, the subject is HLA-B27 + In some embodiments, the subject is HLA-B40 +In some embodiments, the method further comprises determining whether the subject has one copy of HLA-B27 or HLA-B40 or two copies of HLA-B27 or HLA-B40. In some embodiments, the subject has a single copy of HLA-B27 or HLA-B40. In some embodiments, the subject has two copies of HLA-B27 or HLA-B40. In some embodiments, the method further comprises administering an HLA-B27 inhibitor or an HLA-B40 inhibitor to the subject. In some embodiments, the HLA-B27 inhibitor or an HLA-B40 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-B27 antibody or an anti-HLA-B40 antibody. In some embodiments, the HLA-B27 inhibitor or an HLA-B40 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27 or HLA-B40.

[0026] In some embodiments, the MHC-I disease is psoriasis with uveitis. In some embodiments, the uveitis is anterior uveitis. In some embodiments, the method further comprises detecting the presence or absence of HLA-B27 in a biological sample obtained from the subject. In some embodiments, the subject has HLA-B27 +In some embodiments, the method further comprises determining whether the subject has one copy of HLA-B27 or two copies of HLA-B27. In some embodiments, the subject has a single copy of HLA-B27. In some embodiments, the subject has two copies of HLA-B27. In some embodiments, the method further comprises administering an HLA-B27 inhibitor to the subject. In some embodiments, the HLA-B27 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-B27 antibody. In some embodiments, the HLA-B27 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27.

[0027] In some embodiments, the MHC-I disease is JIA. In some embodiments, the method further comprises detecting the presence or absence of HLA-B27 or DRB1 in a biological sample obtained from the subject. In some embodiments, the subject is HLA-B27 + and / or DRB1 +In some embodiments, the method further comprises determining whether the subject has one copy of HLA-B27 and / or DRB1 or two copies of HLA-B27 and / or DRB1. In some embodiments, the subject has a single copy of HLA-B27 and / or DRB1. In some embodiments, the subject has two copies of HLA-B27 and / or DRB1. In some embodiments, the method further comprises administering an HLA-B27 inhibitor and / or DRB1 inhibitor to the subject. In some embodiments, the HLA-B27 inhibitor and / or DRB1 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-B27 antibody or an anti-DRB1 antibody. In some embodiments, the HLA-B27 inhibitor or DRB1 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27 or DRB1.

[0028] In some embodiments, the ERAP2 inhibitor comprises a small molecule degrader, a chimera targeting protein degradation, an immunomodulator, or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to the mRNA of ERAP2. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule that hybridizes to the mRNA of ERAP2. In some embodiments, the inhibitory nucleic acid molecule comprises an siRNA that hybridizes to the mRNA of ERAP2. In some embodiments, the inhibitory nucleic acid molecule comprises an shRNA that hybridizes to the mRNA of ERAP2. In some embodiments, the ERAP2 inhibitor comprises an anti-ERAP2 antibody. In some embodiments, the ERAP2 inhibitor comprises a pseudopeptide. In some embodiments, the pseudopeptide is a phosphinic pseudopeptide. In some embodiments, the phosphinic acid pseudopeptide is DG002 or DG013 (see, for example, Zervoudi et al., Proc. Natl. Acad. Sci. USA, 2013, 110, 19890-19895). In some embodiments, the phosphinic acid pseudopeptide is DG002. In some embodiments, the phosphinic acid pseudopeptide is DG013. In some embodiments, the ERAP2 inhibitor comprises a small molecule. In some embodiments, the ERAP2 inhibitor is compound 4, compound 15, compound 16, compound 5, or an analog of compound 5, which are drug-like carboxylic acids and bioisosteres screened for enhanced selectivity of ERAP2 over ERAP1 (see Medve et al., European Journal of Medicinal Chemistry, 2021, 211, 113053). In some embodiments, the ERAP2 inhibitor is a phosphonic acid compound or a phosphinic acid compound that has a higher affinity for ERAP2 than for ERAP1 (see Weglarz-Tomczak et al., Bioorg. Med. Chem. Lett., 2016, 26, 4122-4126).Additional ERAP2 inhibitors are described, for example, in Georgiadis et al., Curr. Medic. Chem., 2019, 26, 2715-2729.

[0029] In any of the embodiments described herein, any of the inhibitors or other agents described herein may form a component of an antibody-drug conjugate (ADC).For example, an ERAP2 inhibitor may be conjugated to an antibody or an antigen-binding fragment thereof.The inhibitor may include a small molecule degrader, a chimera that targets protein degradation, an immunomodulator, or an inhibitory nucleic acid molecule.

[0030] The present disclosure also provides a method for treating a subject having an MHC-I disease. In some embodiments, the method includes performing an assay on a biological sample from the subject or performing an assay to determine whether the subject comprises i) an MHC-I disease-associated HLA genotype; and ii) a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein. In some embodiments, the method includes administering a therapeutically effective amount of an ERAP2 inhibitor to a subject, wherein the subject comprises both an MHC-I disease-associated HLA genotype and a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein. The presence of both an MHC-I disease-associated HLA genotype and a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein indicates that the subject is a candidate for treatment of MHC-I disease by inhibition of ERAP2.

[0031] In some embodiments, the MHC-I disease is BSCR and the MHC-I disease associated HLA genotype comprises an HLA-Aw19 allele. In some embodiments, the HLA-Aw19 allele comprises an HLA-A29 allele, an HLA-A30 allele, an HLA-A31 allele, or an HLA-A33 allele, or any combination thereof. In some embodiments, the subject has a single copy of an HLA-Aw19 allele. In some embodiments, the HLA-Aw19 allele comprises an HLA-A29 allele. In some embodiments, the HLA-Aw19 allele comprises an HLA-A30 allele. In some embodiments, the HLA-Aw19 allele comprises an HLA-A31 allele. In some embodiments, the HLA-Aw19 allele comprises an HLA-A33 allele. In some embodiments, the subject has two copies of an HLA-Aw19 allele. In some embodiments, the subject has a single copy of an HLA-Aw19 allele. In some embodiments, the subject has a single copy of an HLA-Aw19 allele. In some embodiments, the HLA-Aw19 allele comprises an HLA-A29 allele. In some embodiments, the HLA-Aw19 allele comprises an HLA-A30 allele. In some embodiments, the HLA-Aw19 allele comprises an HLA-A31 allele. In some embodiments, the HLA-Aw19 allele comprises an HLA-A33 allele. In some embodiments, the subject has a single copy of an HLA-Aw19 allele. In some embodiments, the subject has a single copy of an HLA-Aw19 allele. + / HLA-A30 + In some embodiments, the subject is HLA-A29 + / HLA-A31 + In some embodiments, the subject is HLA-A29 + / HLA-A33 + is, or is suspected to be,

[0032] In some embodiments, the subject with BSCR is HLA-A29 + isn't it. In some embodiments, the subject with BSCR has at least any two copies of HLA-A29, HLA-A30, HLA-A31, or HLA-A33.In some embodiments, the subject with BSCR has at least any three copies of HLA-A29, HLA-A30, HLA-A31, or HLA-A33.In some embodiments, the subject with BSCR has all copies of HLA-A29, HLA-A30, HLA-A31, or HLA-A33.

[0033] In some embodiments, a subject with a BSCR has one copy of each of HLA-A29 and HLA-A30. In some embodiments, a subject with a BSCR has one copy of each of HLA-A29 and HLA-A31. In some embodiments, a subject with a BSCR has one copy of each of HLA-A29 and HLA-A33. In some embodiments, a subject with a BSCR has one copy of each of HLA-A30 and HLA-A31. In some embodiments, a subject with a BSCR has one copy of each of HLA-A30 and HLA-A33. In some embodiments, a subject with a BSCR has one copy of each of HLA-A31 and HLA-A33.

[0034] In some embodiments, a subject with a BSCR has one copy of HLA-A29 and two copies of HLA-A30. In some embodiments, a subject with a BSCR has one copy of HLA-A29 and two copies of HLA-A31. In some embodiments, a subject with a BSCR has one copy of HLA-A29 and two copies of HLA-A33. In some embodiments, a subject with a BSCR has one copy of HLA-A30 and two copies of HLA-A31. In some embodiments, a subject with a BSCR has one copy of HLA-A30 and two copies of HLA-A33. In some embodiments, a subject with a BSCR has one copy of HLA-A31 and two copies of HLA-A33.

[0035] In some embodiments, a subject with a BSCR has two copies of HLA-A29 and one copy of HLA-A30. In some embodiments, a subject with a BSCR has two copies of HLA-A29 and one copy of HLA-A31. In some embodiments, a subject with a BSCR has two copies of HLA-A29 and one copy of HLA-A33. In some embodiments, a subject with a BSCR has two copies of HLA-A30 and one copy of HLA-A31. In some embodiments, a subject with a BSCR has two copies of HLA-A30 and one copy of HLA-A33. In some embodiments, a subject with a BSCR has two copies of HLA-A31 and one copy of HLA-A33.

[0036] In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A29 and two copies of HLA-A30. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A29 and two copies of HLA-A31. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A29 and two copies of HLA-A33. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A30 and two copies of HLA-A31. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A30 and two copies of HLA-A33. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A31 and two copies of HLA-A33.

[0037] In some embodiments, the method further comprises administering an HLA-Aw19 inhibitor to the subject. In some embodiments, the HLA-Aw19 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-A29 antibody. In some embodiments, the HLA-Aw19 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-Aw19. In some embodiments, the HLA-Aw19 is HLA-A29.

[0038] In some embodiments, the MHC-I disease is AS, and the MHC-I disease-associated HLA genotype comprises an HLA-B27 allele or an HLA-B40 allele. In some embodiments, the subject has a single copy of HLA-B27 or HLA-B40. In some embodiments, the subject has two copies of HLA-B27 or HLA-B40. In some embodiments, the method further comprises administering to the subject an HLA-B27 inhibitor or an HLA-B40 inhibitor. In some embodiments, the HLA-B27 inhibitor or an HLA-B40 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-B27 antibody or an anti-HLA-B40 antibody. In some embodiments, the HLA-B27 inhibitor or an HLA-B40 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27 or HLA-B40.

[0039] In some embodiments, the MHC-I disease is psoriasis with uveitis, and the MHC-I disease-associated HLA genotype comprises an HLA-B27 allele. In some embodiments, the uveitis is anterior uveitis. In some embodiments, the subject has a single copy of HLA-B27. In some embodiments, the subject has two copies of HLA-B27. In some embodiments, the method further comprises administering an HLA-B27 inhibitor to the subject. In some embodiments, the HLA-B27 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-B27 antibody. In some embodiments, the HLA-B27 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27.

[0040] In some embodiments, the MHC-I disease is JIA and the MHC-I disease associated HLA genotype comprises HLA-B27 and / or DRB1. In some embodiments, the subject has a single copy of HLA-B27 and / or DRB1. In some embodiments, the subject has two copies of HLA-B27 and / or. In some embodiments, the method further comprises administering an HLA-B27 inhibitor and / or DRB1 inhibitor to the subject. In some embodiments, the HLA-B27 inhibitor and / or DRB1 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-B27 antibody and / or DRB1 antibody. In some embodiments, the HLA-B27 inhibitor or DRB1 inhibitor comprises a small molecule degrader or inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27 or DRB1.

[0041] In any of the embodiments described herein, the nucleic acid molecule comprises genomic DNA, mRNA, or cDNA obtained from mRNA. In some embodiments, the nucleic acid molecule comprises genomic DNA. In some embodiments, the nucleic acid molecule comprises mRNA. In some embodiments, the nucleic acid molecule comprises cDNA obtained from mRNA.

[0042] In any of the embodiments described herein, the ERAP2 inhibitor comprises a small molecule degrader, a chimera targeting protein degradation, an immunomodulator, or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to the mRNA of ERAP2. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule that hybridizes to the mRNA of ERAP2. In some embodiments, the inhibitory nucleic acid molecule comprises an siRNA that hybridizes to the mRNA of ERAP2. In some embodiments, the inhibitory nucleic acid molecule comprises an shRNA that hybridizes to the mRNA of ERAP2. In some embodiments, the ERAP2 inhibitor comprises an anti-ERAP2 antibody. In some embodiments, the ERAP2 inhibitor comprises a pseudopeptide. In some embodiments, the pseudopeptide is a phosphinic acid pseudopeptide. In some embodiments, the phosphinic acid pseudopeptide is DG002 or DG013. In some embodiments, the ERAP2 inhibitor comprises a small molecule.

[0043] It has been reported that HLA class I antibodies can be produced by a number of methodologies to achieve different degrees of antigen / allele specificity and are used for in vitro assays. HLA-B27 antibodies can be produced by a number of methodologies. In addition, three commercially available antibodies for flow cytometric screening of HLA-B27 include monoclonal mouse anti-human ABC-m3, FD705, and GS145.2, each of which has been shown to have different levels of cross-reactivity to other HLA-B antigens / alleles (Levering et al., Cytometry B Clin. Cytom., 2003, 54, 28-38).

[0044] In some embodiments, the assay for determining whether a subject contains MHC-I disease-related and / or MHC-II disease-related HLA genotype and functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein is a genotyping assay or a sequence assay. In some embodiments, the nucleic acid molecule encoding a functional ERAP2 protein includes genomic DNA, mRNA, or cDNA obtained from mRNA. By comparing the nucleotide or protein sequence of the ERAP2 protein in a sample from a subject with the wild-type sequence of the ERAP2 protein or nucleic acid molecule, or with the published sequence of a variant ERAP2 protein or nucleic acid molecule with reduced or no activity, it can be determined whether a subject contains a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein. In addition, although an individual ERAP2 protein may have biological activity, the overall function of the ERAP2 protein may not be functional due to a reduced level of expression. Thus, as used herein, an ERAP2 protein may be determined to be non-functional because the ERAP2 protein lacks or reduces biological activity, or because the expression level is reduced.

[0045] Determining whether a subject has an MHC-I disease-associated and / or MHC-II disease-associated HLA genotype, and / or a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein in a biological sample from the subject can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a biological sample obtained from the subject.

[0046] Biological samples can be derived from any cell, tissue, or biological fluid from a subject. Biological samples can include any clinically significant tissue, such as bone marrow samples, tumor biopsies, fine needle aspirates, or samples of bodily fluids (such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine). In some cases, samples include buccal swabs. The biological samples used in the methods disclosed herein can vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. Depending on the assay being used, biological samples can be processed differently. For example, when detecting any specific nucleic acid molecule, preliminary processing designed to isolate or enrich the biological sample for the specific nucleic acid molecule can be used. A variety of techniques can be used for this purpose. A variety of methods can be used to detect the presence or level of mRNA molecules, or the presence of specific genomic DNA loci.

[0047] In some embodiments, the biological sample comprises a cell or cell lysate. Such a method may further comprise obtaining a biological sample from a subject, comprising, for example, a genomic nucleic acid molecule or an mRNA molecule, and, if mRNA, optionally reverse transcribing the mRNA into cDNA. In some embodiments, the method is an in vitro method. In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay also comprises reverse transcribing the mRNA into cDNA, such as by reverse transcriptase polymerase chain reaction (RT-PCR).

[0048] Illustrative examples of techniques for sequencing nucleic acids include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Other methods include nucleic acid hybridization methods other than sequencing, including using labeled primers or probes on purified DNA, amplified DNA, and fixed cell preparations (fluorescence in situ hybridization (FISH)). In some methods, the target nucleic acid molecule can be amplified prior to or simultaneously with detection. Illustrative examples of nucleic acid amplification techniques include, but are not limited to, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and nucleic acid sequence-based amplification (NASBA). Other methods include, but are not limited to, ligase chain reaction, strand displacement amplification, and thermophilic SDA (tSDA).

[0049] The administration of any of the therapeutic agents described herein (including ERAP2 inhibitors and / or HLA inhibitors) may be a therapeutically effective amount determined by a health care professional. The administration of any of the therapeutic agents may be repeated, for example, after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, or 3 months. The repeated administration may be the same dose or a different dose. The administration may be repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. For example, according to a certain dosing regimen, a subject may receive therapy for an extended period of time (such as 6 months, 1 year, or more).

[0050] Administration of any of the therapeutic agents may be by any suitable route, including, but not limited to, parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, intraarticular, intravitreal, intracameral, subretinal, suprachoroidal, or intramuscular.

[0051] Pharmaceutical compositions for administration are desirably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions may be provided in unit dosage form (i.e., dosage for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically and pharma- ceutical acceptable carriers, diluents, excipients, or adjuvants. Formulations depend on the chosen route of administration. The term "pharmaceutical acceptable" means that the carrier, diluent, excipient, or adjuvant is compatible with the other ingredients of the formulation and is not substantially deleterious to the recipient thereof.

[0052] The terms "treat", "treating", and "treatment" as well as "prevent", "preventing", and "prevention", as used herein, refer to eliciting a desired biological response (such as a therapeutic effect and a prophylactic effect), respectively. In some embodiments, a therapeutic effect is a reduction / reduction in MHC-I disease and / or MHC-II disease, a reduction / reduction in the severity of MHC-I disease and / or MHC-II disease (such as reducing or inhibiting the onset of MHC-I disease and / or MHC-II disease), a reduction / reduction in symptoms and MHC-I disease-related effects and / or MHC-II disease-related effects, a delay in the onset of symptoms and MHC-I disease-related effects and / or MHC-II disease-related effects, a reduction in the severity of symptoms of MHC-I disease-related effects and / or MHC-II disease-related effects, a reduction in the severity of acute episodes, a reduction in symptoms and ... The prophylactic effect includes one or more of the following: reduction in the number of MHC-I and / or MHC-II disease-related effects, reduction in the latency of symptoms and MHC-I and / or MHC-II disease-related effects, amelioration of symptoms and MHC-I and / or MHC-II disease-related effects, reduction in secondary symptoms, reduction in secondary infections, prevention of recurrence of MHC-I and / or MHC-II disease, reduction in the number or frequency of recurrent episodes, increase in the latency between symptomatic episodes, increase in the time to sustained progression, rapid recovery, or increase in the efficacy of or decrease in resistance to alternative treatments and / or increase in the survival time of the subject after administration of the agent or composition constituting the agent. The prophylactic effect may include complete or partial avoidance / inhibition or delay (e.g., complete or partial avoidance / inhibition or delay, etc.) of the onset / progression of MHC-I and / or MHC-II disease after administration of the treatment protocol, as well as increase in the survival time of the affected subject. Treatment of MHC-I Disease and / or MHC-II Disease encompasses treatment of subjects already diagnosed with any form of MHC-I Disease and / or MHC-II Disease at any clinical stage or appearance, delaying the onset or development or progression or worsening of symptoms or signs of MHC-I Disease and / or MHC-II Disease, and / or preventing and / or reducing the severity of MHC-I Disease and / or MHC-II Disease.

[0053] In some embodiments, the ERAP2 antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences shown in SEQ ID NOs: 1 to 858. In some embodiments, the ERAP2 siRNA molecule comprises or consists of any of the nucleotide sequences shown in SEQ ID NOs: 859 to 2748 (sense strand and antisense strand shown in order) (for example, the sense strand is, for example, SEQ ID NO: 859, and the corresponding antisense strand is, for example, SEQ ID NO: 860; the sense strand is, for example, SEQ ID NO: 861, and the corresponding antisense strand is, for example, SEQ ID NO: 862; the sense strand is, for example, SEQ ID NO: 2747, and the corresponding antisense strand is, for example, SEQ ID NO: 2748, and so on).

[0054] In some embodiments, the HLA-A antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 2749-2763. In some embodiments, the HLA-A siRNA molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 2764-2777 (sense and antisense strands set forth sequentially) (e.g., the sense strand is, e.g., SEQ ID NO: 2764 and the corresponding antisense strand is, e.g., SEQ ID NO: 2765; the sense strand is, e.g., SEQ ID NO: 2766 and the corresponding antisense strand is, e.g., SEQ ID NO: 2767; the sense strand is, e.g., SEQ ID NO: 2776 and the corresponding antisense strand is, e.g., SEQ ID NO: 2777, etc.).

[0055] In some embodiments, the HLA-B antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 2778 and 2779. In some embodiments, the HLA-B siRNA molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 2780-2783 (sense and antisense strands set forth sequentially) (e.g., the sense strand is, e.g., SEQ ID NO: 2780 and the corresponding antisense strand is, e.g., SEQ ID NO: 2781; the sense strand is, e.g., SEQ ID NO: 2783 and the corresponding antisense strand is, e.g., SEQ ID NO: 2783, etc.).

[0056] In some embodiments, the HLA-C antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 2784 and 2785. In some embodiments, the HLA-C siRNA molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 2786 and 2787 (sense and antisense strands set forth sequentially).

[0057] In some embodiments, the B27 antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 2788-3132. In some embodiments, the B27 siRNA molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 3133-3870 (sense and antisense strands set forth sequentially) (e.g., the sense strand is, e.g., SEQ ID NO: 3133 and the corresponding antisense strand is, e.g., SEQ ID NO: 3134; the sense strand is, e.g., SEQ ID NO: 3135 and the corresponding antisense strand is, e.g., SEQ ID NO: 3136; the sense strand is, e.g., SEQ ID NO: 3869 and the corresponding antisense strand is, e.g., SEQ ID NO: 3870, etc.).

[0058] In some embodiments, the A29 antisense nucleic acid molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 3871-4145. In some embodiments, the A29 siRNA molecule comprises or consists of any of the nucleotide sequences set forth in SEQ ID NOs: 4146-4755 (sense and antisense strands set forth sequentially) (e.g., the sense strand is, e.g., SEQ ID NO: 4146 and the corresponding antisense strand is, e.g., SEQ ID NO: 4147; the sense strand is, e.g., SEQ ID NO: 4148 and the corresponding antisense strand is, e.g., SEQ ID NO: 4149; the sense strand is, e.g., SEQ ID NO: 4754 and the corresponding antisense strand is, e.g., SEQ ID NO: 4755, etc.).

[0059] The inhibitory nucleic acid molecules disclosed herein may comprise RNA, DNA, or both RNA and DNA. The inhibitory nucleic acid molecules may also be linked or fused to a heterologous nucleic acid sequence (such as in a vector) or a heterologous label. For example, the inhibitory nucleic acid molecules disclosed herein may be in a vector that comprises the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence, or as an exogenous donor sequence that comprises the inhibitory nucleic acid molecule and a heterologous nucleic acid sequence. The inhibitory nucleic acid molecules may also be linked or fused to a heterologous label. The label may be directly detectable (such as a fluorophore) or indirectly detectable (such as a hapten, enzyme, or fluorophore quencher). Such labels may be detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Such labels include, for example, radioisotope labels, dyes, dyes, chromogens, spin labels, and fluorescent labels. The label may also be, for example, a chemiluminescent substance; a metal-containing substance; or an enzyme (where enzyme-dependent secondary generation of a signal occurs). The term "label" may also refer to a "tag" or hapten that can selectively bind to the conjugated molecule such that the conjugated molecule is subsequently added with a substrate and used to generate a detectable signal. For example, biotin can be used as a tag to bind to the tag with an avidin or streptavidin conjugate of horseradish peroxidase (HRP), and a colorimetric substrate (such as tetramethylbenzidine (TMB)) or a fluorogenic substrate can be used to detect the presence of HRP. Exemplary labels that can be used as tags to facilitate purification include, but are not limited to, myc, HA, FLAG or 3xFLAG, 6xHis or polyhistidine, glutathione-S-transferase (GST), maltose binding protein, epitope tags, or the Fc portion of an immunoglobulin. Numerous labels include, for example, particles, fluorophores, haptens, enzymes, and their colorimetric, fluorogenic, and chemiluminescent substrates, as well as other labels.

[0060] The disclosed inhibitory nucleic acid molecules can include, for example, nucleotides, or non-natural or modified nucleotides, such as nucleotide analogs or nucleotide substitutes. Such nucleotides include nucleotides that contain modified bases, sugars, or phosphate groups, or nucleotides that incorporate non-natural moieties into their structure. Examples of non-natural nucleotides include, but are not limited to, dideoxynucleotides, biotinylated nucleotides, aminated nucleotides, deaminated nucleotides, alkylated nucleotides, benzylated nucleotides, and fluorophore-labeled nucleotides.

[0061] The inhibitory nucleic acid molecules disclosed herein may also include one or more nucleotide analogs or substitutions. A nucleotide analog is a nucleotide that contains a modification to either the base, sugar, or phosphate moiety. Modifications to the base moiety include, but are not limited to, natural and synthetic modifications of A, C, G, and T / U, and different purine or pyrimidine bases, such as pseudouridine, uracil-5-yl, hypoxanthine-9-yl (I), and 2-aminoadenine-9-yl. Modified bases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (cytosine), and thymine. These include, but are not limited to, 8-substituted adenines and guanines, 5-halo (such as 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine.

[0062] Nucleotide analogs can also include modifications to the sugar moiety, including, but not limited to, natural and synthetic modifications of ribose and deoxyribose. Sugar modifications include the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C 1-10 Alkyl or C 2-10 Alkenyl, and C 2-10 Exemplary 2' sugar modifications include, but are not limited to, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -ONH2 and -O(CH2) n ON [(CH2) n Other modifications at the 2' position include, but are not limited to, C 1-10These include, but are not limited to, alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of the 5' terminal nucleotide. Modified sugars can also include those containing modifications at the bridging ring oxygen (such as CH2 and S). Nucleotide sugar analogs can also have sugar mimetics (such as cyclobutyl moieties) in place of the pentofuranosyl sugar.

[0063] Nucleotide analogs can also be modified at the phosphate moiety, such as when the linkage between the two nucleotides is a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methylphosphonate, and other alkylphosphonates. These phosphate linkages or modified phosphate linkages between two nucleotides can be via 3'-5' linkages or 2'-5' linkages, and the linkages can contain reverse polarity (such as 3'-5' to 5'-3' or 2'-5' to 5'-2'). Also included are various salts, mixed salts, and free acid forms. Nucleotide substitutes also include peptide nucleic acids (PNAs).

[0064] In some embodiments, the antisense nucleic acid molecule is a gapmer, whereby the first 1-7 nucleotides at the 5' and 3' ends each have a 2'-methoxyethyl (2'-MOE) modification. In some embodiments, the first five nucleotides at the 5' and 3' ends each have a 2'-MOE modification. In some embodiments, the first 1-7 nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, the first five nucleotides at the 5' and 3' ends are RNA nucleotides. In some embodiments, each of the internucleotide backbone linkages is a phosphorothioate linkage.

[0065] In some embodiments, the siRNA molecule has a terminal modification. In some embodiments, the 5' end of the antisense strand is phosphorylated. A 5'-phosphate analog that cannot be hydrolyzed is used, such as a 5'-(E)-vinyl-phosphonate.

[0066] In some embodiments, the siRNA molecule has a backbone modification. In some embodiments, modified phosphodiester groups linking consecutive ribose nucleosides have been shown to promote siRNA stability and in vivo bioavailability. The non-ester group (-OH, =O) of the phosphodiester bond can be replaced by sulfur, boron, or acetate to obtain phosphorothioate linkages, boranophosphate linkages, and phosphonoacetate linkages. In addition, the replacement of phosphodiester groups with phosphotriesters can facilitate cellular uptake of siRNA and retention on serum components by eliminating their negative charge. In some embodiments, the siRNA molecule has a sugar modification. In some embodiments, the sugar is deprotonated (a reaction catalyzed by exonucleases and endonucleases), in which case the 2'-hydroxyl can act as a nucleophile and attack the adjacent phosphorus in the phosphodiester bond. Such alternatives include 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro modifications.

[0067] In some embodiments, the siRNA molecule has base modifications, in some embodiments, the bases can be replaced with modified bases, such as pseudouridine, 5'-methylcytidine, N6-methyladenosine, inosine, and N7-methylguanosine.

[0068] In some embodiments, siRNA molecule is conjugated to lipid.Lipid can be conjugated to 5'-end or 3'-end of siRNA to improve in vivo bioavailability by enabling association with serum lipoprotein.Representative lipid includes, but is not limited to, cholesterol and vitamin E, and fatty acid (such as palmitate and tocopherol).

[0069] In some embodiments, an exemplary siRNA has the formula: Sense: mN*mN* / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / *mN* / 32FN / Antisense: / 52FN / * / i2FN / *mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN / i2FN / mN*N*N, where "N" is a base; "2F" is a 2'-F modification; "m" is a 2'-O methyl modification, "I" is an internal base; and "*" is a phosphorothioate backbone linkage.

[0070] The present disclosure also provides a vector comprising any one or more of the inhibitory nucleic acid molecules disclosed herein. In some embodiments, the vector comprises any one or more of the inhibitory nucleic acid molecules disclosed herein and a heterologous nucleic acid. The vector can be a viral vector or a non-viral vector capable of transporting a nucleic acid molecule. In some embodiments, the vector is a plasmid or a cosmid (such as a circular double-stranded DNA to which additional DNA segments can be ligated). In some embodiments, the vector is a viral vector to which additional DNA segments can be ligated into the viral genome. Expression vectors include, but are not limited to, plasmids, cosmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses (such as cauliflower mosaic virus and tobacco mosaic virus), yeast artificial chromosomes (YACs), Epstein-Barr (EBV) derived episomes, and other expression vectors known in the art.

[0071] The present disclosure also provides compositions comprising any one or more of the inhibitory nucleic acid molecules disclosed herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a carrier and / or excipient. Examples of carriers include, but are not limited to, poly(lactic acid) (PLA) microspheres, poly(D,L-lactic-coglycolic acid) (PLGA) microspheres, liposomes, micelles, reverse micelles, lipid cochleates, and lipid microtubules. Carriers may include buffered salt solutions, PBS, HBSS, etc.

[0072] The present disclosure also provides a method for identifying a subject having an increased risk for developing MHC-I disease and / or MHC-II disease. The method includes performing an assay on a biological sample from the subject or performing an assay, and determining whether the subject comprises i) an MHC-I disease-associated HLA genotype and / or an MHC-II disease-associated HLA genotype; and ii) a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein. If the subject has both an MHC-I disease-associated HLA genotype and / or an MHC-II disease-associated HLA genotype, and a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, then the subject has an increased risk for developing MHC-I disease and / or MHC-II disease. If the subject lacks MHC-I disease-associated HLA genotype and / or MHC-II disease-associated HLA genotype, or lacks functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, or lacks both, then the subject has a reduced risk of developing MHC-I disease and / or MHC-II disease. In some embodiments, the method further comprises determining whether the subject has a single copy of MHC-I disease-associated HLA genotype and / or MHC-II disease-associated HLA genotype or two copies of MHC-I disease-associated HLA genotype and / or MHC-II disease-associated HLA genotype. If the subject includes two copies of MHC-I disease-associated HLA genotype and / or MHC-II disease-associated HLA genotype, then the subject has an increased risk of developing MHC-I disease and / or MHC-II disease compared to those who include a single copy of MHC-I disease-associated HLA genotype and / or MHC-II disease-associated HLA genotype.

[0073] In some embodiments, the MHC-I disease is BSCR. + In some embodiments, the subject is HLA-A29 + , HLA-A30 + , HLA-A31 + , or HLA-A33+ In some embodiments, the subject has or is suspected of having HLA-Aw19, HLA-Aw29, HLA-Aw39, HLA-Aw40, HLA-Aw50, HLA-Aw60, HLA-Aw70, HLA-Aw80, HLA-Aw90, HLA-Aw10, HLA-Aw11, HLA-Aw22, HLA-Aw30, HLA-Aw40, HLA-Aw50, HLA-Aw60, HLA-Aw70, HLA-Aw80, HLA-Aw90, HLA-Aw10, HLA-Aw11, HLA-Aw12, HLA-Aw13, HLA-Aw14, HLA-Aw15, HLA-Aw16, HLA-Aw17, HLA-Aw18, HLA-Aw19, HLA-Aw29, HLA + / HLA-A30 + In some embodiments, the subject is HLA-A29 + / HLA-A31 + In some embodiments, the subject is HLA-A29 + / HLA-A33 + It is.

[0074] In some embodiments, the subject with BSCR is HLA-A29 + isn't it. In some embodiments, the subject with BSCR has at least any two copies of HLA-A29, HLA-A30, HLA-A31, or HLA-A33.In some embodiments, the subject with BSCR has at least any three copies of HLA-A29, HLA-A30, HLA-A31, or HLA-A33.In some embodiments, the subject with BSCR has all copies of HLA-A29, HLA-A30, HLA-A31, or HLA-A33.

[0075] In some embodiments, a subject with a BSCR has one copy of each of HLA-A29 and HLA-A30. In some embodiments, a subject with a BSCR has one copy of each of HLA-A29 and HLA-A31. In some embodiments, a subject with a BSCR has one copy of each of HLA-A29 and HLA-A33. In some embodiments, a subject with a BSCR has one copy of each of HLA-A30 and HLA-A31. In some embodiments, a subject with a BSCR has one copy of each of HLA-A30 and HLA-A33. In some embodiments, a subject with a BSCR has one copy of each of HLA-A31 and HLA-A33.

[0076] In some embodiments, a subject with a BSCR has one copy of HLA-A29 and two copies of HLA-A30. In some embodiments, a subject with a BSCR has one copy of HLA-A29 and two copies of HLA-A31. In some embodiments, a subject with a BSCR has one copy of HLA-A29 and two copies of HLA-A33. In some embodiments, a subject with a BSCR has one copy of HLA-A30 and two copies of HLA-A31. In some embodiments, a subject with a BSCR has one copy of HLA-A30 and two copies of HLA-A33. In some embodiments, a subject with a BSCR has one copy of HLA-A31 and two copies of HLA-A33.

[0077] In some embodiments, a subject with a BSCR has two copies of HLA-A29 and one copy of HLA-A30. In some embodiments, a subject with a BSCR has two copies of HLA-A29 and one copy of HLA-A31. In some embodiments, a subject with a BSCR has two copies of HLA-A29 and one copy of HLA-A33. In some embodiments, a subject with a BSCR has two copies of HLA-A30 and one copy of HLA-A31. In some embodiments, a subject with a BSCR has two copies of HLA-A30 and one copy of HLA-A33. In some embodiments, a subject with a BSCR has two copies of HLA-A31 and one copy of HLA-A33.

[0078] In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A29 and two copies of HLA-A30. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A29 and two copies of HLA-A31. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A29 and two copies of HLA-A33. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A30 and two copies of HLA-A31. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A30 and two copies of HLA-A33. In some embodiments, a subject having or suspected of having a BSCR has two copies of HLA-A31 and two copies of HLA-A33.

[0079] In some embodiments, the method further comprises administering an HLA-Aw19 inhibitor to the subject. In some embodiments, the HLA-Aw19 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-A29 antibody. In some embodiments, the HLA-Aw19 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA) that hybridizes to HLA-Aw19. In some embodiments, the HLA-Aw19 is HLA-A29.

[0080] In some embodiments, the MHC-I disease is AS. In some embodiments, the subject is HLA-B27 + / HLA-B40 + In some embodiments, the subject has a single copy of HLA-B27 or HLA-B40. In some embodiments, the subject has two copies of HLA-B27 or HLA-B40. In some embodiments, the method further comprises administering an HLA-B27 inhibitor or an HLA-B40 inhibitor to the subject. In some embodiments, the HLA-B27 inhibitor or an HLA-B40 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-B27 antibody or an anti-HLA-B40 antibody. In some embodiments, the HLA-B27 inhibitor or an HLA-B40 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27 or HLA-B40.

[0081] In some embodiments, the MHC-I disease is psoriasis with uveitis. In some embodiments, the uveitis is anterior uveitis. In some embodiments, the subject is HLA-B27 +In some embodiments, the subject has or is suspected to have HLA-B27. In some embodiments, the subject has a single copy of HLA-B27. In some embodiments, the subject has two copies of HLA-B27. In some embodiments, the method further comprises administering an HLA-B27 inhibitor to the subject. In some embodiments, the HLA-B27 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-B27 antibody. In some embodiments, the HLA-B27 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27.

[0082] In some embodiments, the MHC-I disease is JIA. + and / or DRB1 + In some embodiments, the subject has or is suspected to have a HLA-B27 and / or DRB1. In some embodiments, the subject has two copies of HLA-B27 and / or DRB1. In some embodiments, the method further comprises administering an HLA-B27 inhibitor and / or DRB1 inhibitor to the subject. In some embodiments, the HLA-B27 inhibitor and / or DRB1 inhibitor comprises an antibody. In some embodiments, the antibody comprises an anti-HLA-B27 antibody or an anti-DRB1 antibody. In some embodiments, the HLA-B27 inhibitor or DRB1 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to HLA-B27 or DRB1.

[0083] In any of the embodiments described herein, the method may further include administering an ERAP2 inhibitor to a subject having an MHC-I disease-associated HLA genotype and / or an increased risk of developing an MHC-II disease. In some embodiments, the ERAP2 inhibitor comprises a small molecule degrader, a chimera targeting protein degradation, an immunomodulator, or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, siRNA, or shRNA that hybridizes to the mRNA of ERAP2. In some embodiments, the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule that hybridizes to the mRNA of ERAP2. In some embodiments, the inhibitory nucleic acid molecule comprises an siRNA that hybridizes to the mRNA of ERAP2. In some embodiments, the inhibitory nucleic acid molecule comprises an shRNA that hybridizes to the mRNA of ERAP2. In some embodiments, the ERAP2 inhibitor comprises an anti-ERAP2 antibody. In some embodiments, the ERAP2 inhibitor comprises a pseudopeptide. In some embodiments, the pseudopeptide is a phosphinic acid pseudopeptide. In some embodiments, the phosphinic acid pseudopeptide is DG002 or DG013. In some embodiments, the phosphinic acid pseudopeptide is DG002. In some embodiments, the phosphinic acid pseudopeptide is DG013. In some embodiments, the ERAP2 inhibitor comprises a small molecule.

[0084] In some embodiments, the assay for determining whether a subject contains MHC-I disease-related and / or MHC-II disease-related HLA genotype and functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein is a genotyping assay or a sequence assay. In some embodiments, the nucleic acid molecule encoding a functional ERAP2 protein includes genomic DNA, mRNA, or cDNA obtained from mRNA. By comparing the nucleotide or protein sequence of the ERAP2 protein in a sample from a subject with the wild-type sequence of the ERAP2 protein or nucleic acid molecule, or with the published sequence of a variant ERAP2 protein or nucleic acid molecule with reduced or no activity, it can be determined whether a subject contains a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein. In addition, although an individual ERAP2 protein may have biological activity, the overall function of the ERAP2 protein may not be functional due to a reduced level of expression. Thus, as used herein, an ERAP2 protein may be determined to be non-functional because the ERAP2 protein lacks or reduces biological activity, or because the expression level is reduced.

[0085] Determining whether a subject has an MHC-I disease-associated and / or MHC-II disease-associated HLA genotype, and / or a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein in a biological sample from the subject can be performed by any of the methods described herein. In some embodiments, these methods can be performed in vitro. In some embodiments, these methods can be performed in situ. In some embodiments, these methods can be performed in vivo. In any of these embodiments, the nucleic acid molecule can be present in a biological sample obtained from the subject.

[0086] Biological samples can be derived from any cell, tissue, or biological fluid from a subject. Biological samples can include any clinically significant tissue, such as bone marrow samples, tumor biopsies, fine needle aspirates, or samples of bodily fluids (such as blood, gingival crevicular fluid, plasma, serum, lymph, ascites, cyst fluid, or urine). In some cases, samples include buccal swabs. The biological samples used in the methods disclosed herein can vary based on the assay format, the nature of the detection method, and the tissue, cell, or extract used as the sample. Depending on the assay being used, biological samples can be processed differently. For example, when detecting any specific nucleic acid molecule, preliminary processing designed to isolate or enrich the biological sample for the specific nucleic acid molecule can be used. A variety of techniques can be used for this purpose. A variety of methods can be used to detect the presence or level of mRNA molecules, or the presence of specific genomic DNA loci.

[0087] In some embodiments, the biological sample comprises a cell or cell lysate. Such a method may further comprise obtaining a biological sample from a subject, comprising, for example, a genomic nucleic acid molecule or an mRNA molecule, and, if mRNA, optionally reverse transcribing the mRNA into cDNA. In some embodiments, the method is an in vitro method. In some embodiments, the assay comprises RNA sequencing (RNA-Seq). In some embodiments, the assay also comprises reverse transcribing the mRNA into cDNA, such as by reverse transcriptase polymerase chain reaction (RT-PCR).

[0088] Detection of the presence or absence of any particular HLA allele can be performed by a number of techniques. Detection of HLA-A alleles can be performed at two and four-digit resolution. For example, assays targeting the HLA region at high resolution (all class I and class II genes) can be used. In some embodiments, the assay amplifies the complete HLA gene (HLA-A in this case) from 5'UTR to 3'UTR, providing gene variants across the complete amplicon (the DNA that is the product of this amplification of the gene). Methods that call HLA-A alleles with high accuracy can then be used (e.g. PHLAT2; Bai et al., Methods Mol. Biol., 2018, 1802, 193-201). The output of PHLAT2 provides four-digit allele data of HLA-A for each sample, which can be used for analysis to identify other Aw19 alleles enriched in scattershot chorioretinopathy cases. In addition, commercial sources of HLA typing are available.

[0089] Detection of the presence or absence of functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein can be performed by a number of techniques. For example, detection of the presence or absence of ERAP2 protein and related nucleotide sequences can be performed as described in Andres et al., PLoS Genetics, 2010, 6, 1-13. For example, a subject having an intronic variant of ERAP2 designated as rs2248374-A has a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, and has an increased risk of developing MHC-I disease. A subject having an ERAP2 variant designated as rs10044354, HapA has a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, and has an increased risk of developing MHC-I disease. A subject having the ERAP2 splice variant designated as rs2248374-G does not have a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, and has a reduced risk of developing MHC-I disease.

[0090] Representative embodiments of the present disclosure include, but are not limited to, the following. The present disclosure provides a method for treating a subject having an immune disease, comprising administering to the subject an ERAP2 inhibitor and i) an HLA-A29 inhibitor and / or ii) HLA-B27. In some embodiments, the immune disease is an MHC-I-opathy.

[0091] The present disclosure also relates to a method for treating a subject having MHC-I disease (MHC-I-opathy), comprising: performing an assay on a biological sample derived from the subject to determine whether the subject contains i) HLA-A29 allele and / or HLA-B27, and ii) a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, or having already performed such an assay; and administering a therapeutically effective amount of ... Also provided is a method comprising administering an ERAP2 inhibitor and an HLA-A29 inhibitor, or administering therapeutically effective amounts of an ERAP2 inhibitor and an HLA-B27 inhibitor to a subject having a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein, and having an HLA-B27 allele, wherein the presence of both i) HLA-A29 and / or HLA-B27 allele, and ii) a functional ERAP2 protein or a nucleic acid molecule encoding a functional ERAP2 protein indicates that the subject is a candidate for treatment of MHC-I disease by inhibiting ERAP2.

[0092] The present disclosure also provides a combination of an ERAP2 inhibitor and an HLA-A29 inhibitor for use in treating an immune disease. The present disclosure also provides a combination of an ERAP2 inhibitor and an HLA-A29 inhibitor for use in preparing a medicament for treating an immune disease. In some embodiments, the immune disease is an MHC-I-opathy.

[0093] In some embodiments, the MHC-I disease is shotcretinopathy (BSCR), and the method includes administering an ERAP2 inhibitor and an HLA-A29 inhibitor to the subject. In some embodiments, the method further includes detecting the presence or absence of an HLA-A29 allele in a biological sample obtained from the subject. In some embodiments, the method further includes determining whether the subject has one copy of the HLA-A29 allele or two copies of the HLA-A29 allele. In some embodiments, the HLA-29 inhibitor includes an antibody. In some embodiments, the HLA-A29 inhibitor includes a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or short hairpin RNA that hybridizes to HLA-A29. In some embodiments, the antisense nucleic acid molecule includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3871-4145. In some embodiments, the siRNA molecule includes an antisense strand corresponding to a sense strand selected from the group consisting of SEQ ID NOs: 4146-4755.

[0094] In some embodiments, the MHC-I disease is AS, and the method includes administering an ERAP2 inhibitor and an HLA-B27 inhibitor to the subject. In some embodiments, the method further includes detecting the presence or absence of an HLA-B27 allele in a biological sample obtained from the subject. In some embodiments, the method further includes determining whether the subject has one copy of the HLA-B27 allele or two copies of the HLA-B27 allele. In some embodiments, the HLA-B27 inhibitor includes an antibody. In some embodiments, the HLA-B27 inhibitor includes a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or short hairpin RNA that hybridizes to HLA-B27. In some embodiments, the antisense nucleic acid molecule includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2788-3132. In some embodiments, the siRNA molecule includes an antisense strand corresponding to a sense strand selected from the group consisting of SEQ ID NOs: 3133-3870.

[0095] In some embodiments, the MHC-I disease is psoriasis, and the subject also has uveitis, comprising administering to the subject an ERAP2 inhibitor and an HLA-B27 inhibitor. In some embodiments, the uveitis is anterior uveitis. In some embodiments, the method further comprises detecting the presence or absence of an HLA-B27 allele in a biological sample obtained from the subject. In some embodiments, the method further comprises determining whether the subject has one copy of the HLA-B27 allele or has two copies of the HLA-B27 allele. In some embodiments, the HLA-B27 inhibitor comprises an antibody. In some embodiments, the HLA-B27 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or short hairpin RNA that hybridizes to HLA-B27. In some embodiments, the antisense nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2788-3132. In some embodiments, the siRNA molecule comprises a sense strand and a corresponding antisense strand selected from the group consisting of SEQ ID NOs: 3133-3870.

[0096] In some embodiments, the MHC-I disease is JIA, and the method includes administering an ERAP2 inhibitor and an HLA-B27 inhibitor to the subject. In some embodiments, the method further includes detecting the presence or absence of an HLA-B27 allele in a biological sample obtained from the subject. In some embodiments, the method further includes determining whether the subject has one copy of the HLA-B27 allele or two copies of the HLA-B27 allele. In some embodiments, the HLA-B27 inhibitor includes an antibody. In some embodiments, the HLA-B27 inhibitor includes a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or short hairpin RNA that hybridizes to HLA-B27. In some embodiments, the antisense nucleic acid molecule includes a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2788-3132. In some embodiments, the siRNA molecule includes an antisense strand corresponding to a sense strand selected from the group consisting of SEQ ID NOs: 3133-3870.

[0097] In some embodiments, the ERAP2 inhibitor comprises a small molecule degrader or an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA, or short hairpin RNA that hybridizes to the mRNA of ERAP2. In some embodiments, the antisense nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 858. In some embodiments, the siRNA molecule comprises an antisense strand corresponding to a sense strand selected from the group consisting of SEQ ID NOs: 859 to 2748. In some embodiments, the ERAP2 inhibitor comprises an anti-ERAP2 antibody. In some embodiments, the ERAP2 inhibitor comprises a pseudopeptide. In some embodiments, the pseudopeptide is a phosphinic acid pseudopeptide. In some embodiments, the phosphinic acid pseudopeptide is DG002 or DG013. In some embodiments, the ERAP2 inhibitor comprises a small molecule.

[0098] The present disclosure also provides a pharmaceutical composition comprising a combination of one or more ERAP2 inhibitors and one or more HLA-A29 inhibitors.The present disclosure also provides a pharmaceutical composition comprising a combination of one or more ERAP2 inhibitors and one or more HLA-B27 inhibitors.The present disclosure also provides a pharmaceutical composition comprising a combination of one or more ERAP2 inhibitors and one or more HLA-A29 inhibitors and one or more HLA-B27 inhibitors.

[0099] In order to more effectively understand the subject matter disclosed herein, the following examples are provided. It should be understood that these examples are for illustrative purposes only and cannot be construed as limiting the claimed subject matter in any manner. Throughout these examples, molecular cloning reactions and other standard recombinant DNA techniques were carried out according to the methods described in Maniatis et al., Molecular Cloning - A Laboratory Manual, 2nd ed., Cold Spring Harbor Press (1989), using commercially available reagents, unless otherwise indicated.

[0100] Working Example Example 1: Methods Study subjects and samples Genomic DNA samples from 286 patients with BSCR and 108 unrelated healthy local French volunteers who showed HLA tissue typing common in the French population were included in this study. Patients were recruited at the Hôpital Cochin, Paris, France. All patients fulfilled the criteria for the diagnosis of BSCR as defined by both the International Consensus Conference held in 2002 and the Standardization of Uveitis Nomenclature (SUN) Working Group. Briefly, all patients had posterior bilateral uveitis with multifocal cream-colored or yellow-orange ovoid or round choroidal lesions ("shotgun-like spots"). Although the presence of the HLA-A29 allele was not a requirement for the diagnosis of BSCR according to international criteria, all patients included in the current study carried the HLA-A29 allele. Control DNA samples were collected from volunteer donors recruited by the Hematopoietic Stem Cell Donor Center of Rheims for the France Greffe de Moelle Registry and from healthy individuals from the local controls of the registry. DNA samples were isolated from peripheral blood samples using standard salting out methods or the QIAamp Blood Kit (Qiagen, Chatsworth, CA, USA). DNA quality and quantity were determined by UV spectrophotometry and the concentration was adjusted to 100ng / ml. Signed informed consent documents were obtained from all participants and all studies adhered to the principles outlined in the Declaration of Helsinki. All study-related data acquisition was approved by the Paris Cochin Institutional Review Board.

[0101] Genetic Data For the exome sequence and all samples, an inclusive approach was adopted for both genotyping to enable the identification of common variants and rare variants filtered based on high-quality calls. DNA from participants was genotyped on the Illumina Global Screening Array (GSA) and imputed to the HRC reference panel. Prior to imputation, variants with MAF >= 0.1%, missingness < 1%, and HWE p-value > 10 -15 were retained. Imputation using the HRC reference panel yielded 8,385,561 variants with imputation INFO > 0.3 and MAF > 0.5%.

[0102] Exome sequencing was performed to an average depth of 31x, followed by variant calling and quality control as previously reported (Van Hout et al., Nature, 2020, 586, 749-756), yielding 238,942 variants. When integrated, this generated an overall dataset of 8,459,907 variants [65.5% common (MAF > 5%), 34.5% low-frequency (0.5% < MAF < 5%), and 0.01% rare (MAF < 0.5%)].

[0103] HLA genotyping HLA class I genes (HLA-A, HLA-B, and HLA-C) were amplified in multiplex PCR reactions with primers covering the complete genomic locus for each target. The resulting amplicons were enzymatically fragmented to an average size of 250 base pairs and prepared for Illumina sequencing (New England Biolabs, Ipswich, MA). Libraries were sequenced on an Illumina HiSeq 2500 platform on a rapid run flow cell using paired-end 125 base pair reads with dual 10 base pair indexes. Upon completion of sequencing, raw data from each Illumina HiSeq run was collected in local buffer storage and uploaded to the DNAnexus platform (Reid et al., BMC Bioinformatics, 2014, 15, 30) for automated analysis. FASTQ format reads were converted from BCL files and assigned to samples identified by specific barcodes using bcl2fastq conversion software (Illumina Inc., San Diego, CA). All reads in sample-specific FASTQ files were subjected to HLA typing analysis using an updated version of the PHLAT program (Bai et al., BMC Genomics, 2014, 15, 325) with reference sequences consisting of the GRCh38 genome sequence and HLA type reference sequences in the IPD-IMGT / HLA database v3.30.0 (Robinson et al., Hum. Immunol., 2016, 77, 233-237).

[0104] In addition, HLA allele imputation was performed with the T1DGC HLA allele reference panel (Rich et al., Ann. NY Acad. Sci., 2006, 1079, 1-8) after SNP2HLA (Jia et al., PLoS One, 2013, 8, e64683). HRC imputed genotypes in the extended major histocompatibility complex (MHC) region (chr6:25-35Mb) were filtered for high INFO score (>0.9) and confidence (max GP>0.8 for all genotypes) to increase overlap with the T1DGC reference panel, rephased together with chromosome 6 array genotypes using SHAPEIT4 (Delaneau et al., Nat. Commun., 2019, 10, 5436) and imputed using Minimac4 (Das et al., Nat. Genet., 2016, 48, 1284-1287). The quality of HLA allele imputation was assessed by examining the INFO score vs. MAF and the imputation vs. reference panel MAF.

[0105] Genetic association analysis Association analysis in each study was performed using the genome-wide Firth logistic regression test implemented in SAIGE (Mbatchou et al., bioRxiv, 2020, 2020.2006.2019.162354, doi:10.1101 / 2020.06.19.162354; and Zhou et al., Nat. Genet., 2018, 50, 1335-1341). In this implementation, the Firth approach is applied when the p-value from the standard logistic regression score test is less than 0.05. Minor allele frequency (MAF) > 1%, < 10% missing, Hardy-Weinberg equilibrium test p-value > 10 -15 , and linkage disequilibrium (LD) pruning (1000 variant window, 100 variant sliding window, and r 2Directly genotyped variants with a mean mean of 1.0 (<0.1) were included for GRM on SAIGE. Association models included the covariate sex and the first 10 ancestry-informative principal components (PCs) from the GRM dataset. Haplotype analysis was performed using PLINK 1.0 (Purcell et al., Am. J. Hum. Genet., 2007, 81, 559-575) --chap, --hap-assoc, and --hap-logistic, and in R. High haplotype imputation and phasing quality was indicated by PLINK --hap-phase, where all of the posterior probabilities of the most likely haplotype genotypes are equal to 1.

[0106] HLA-A allele association analysis HLA-A allele association was performed as follows: For each sample, both HLA-A alleles were typed as described above. Following HLA allele typing, relevant samples were removed. For the remaining cohort of 282 cases and 106 controls, one HLA-A allele (the "second" allele) that was not A29 was then obtained. Samples carrying two copies of A29 were determined to have A29 as the second allele. The cohort was then subjected to Fisher's exact test to test the association of each allele identified in 3 or more BSCR cases with case status-control status. To answer the question of whether the A19 allele group was also associated with case status-control status, samples were combined and tested together for the carriage of all Aw19 alleles (A29, A30, A31, A32, and A33) in two different ways. Because A32 is biologically distinct from other Aw19 alleles in the peptide-binding domain, we also constructed and tested a group made up of samples carrying all Aw19 alleles except A32. The final odds ratios and p-values ​​are presented in the table in Figure 1.

[0107] Example 2: Wide range of antigen serotype alleles of HLA-Aw19 and BSCR risk The HLA-A29 controlled cohort allows for the examination of the HLA region, and thus the detection of possible additional association signals in the HLA region, while controlling for the strong association of HLA-A29 with BSCR.

[0108] First, we asked whether rare variants on an HLA-A29 background were enriched in BSCR cases.No significant enrichment of rare single or aggregated variants was identified within or outside the MHC region.

[0109] Second, we wondered whether other HLA-A alleles in addition to the HLA-A29 allele might increase BSCR risk. We constructed an assay to type HLA-A alleles in this cohort (see Methods) and tested a second HLA-A allele (other than the known first HLA-A29) for association with BSCR. Additional HLA-A alleles were found to be associated with BSCR, with the largest effectors belonging to the same HLA-Aw19 broad antigen serotype group: HLA-A29:02, A30:02, A31:01, and A33:01 (Figure 1). As a group, the HLA-Aw19 allele was significantly enriched in second alleles in BSCR patients (odds ratio = 4.44, p = 2.2e-03, Figure 2, blue bars). This result suggests that, for example, individuals carrying two copies of HLA-A29 may be at higher risk of developing BSCR compared to those carrying one copy. This also suggests that other Aw19 alleles may play a role in BSCR co-susceptibility or pathogenesis in cooperation with A29. The only exception within the HLA-Aw19 serogroup is HLA-A32, which has been reported not to share the defined binding domain of Aw19 (McKenzie et al., Genes Immun., 1999, 1, 120-129). HLA-A32 is reduced in BSCR cases and therefore appears to be protective against BSCR (odds ratio = 0.28, p = 0.1).

[0110] The above results raised two issues due to the small number of controls in UParis (n=108): 1) the allele frequency may not represent the frequency of HLA-A alleles in the general European population; and 2) although the high odds ratio is reproduced in multiple HLA-Aw19 controls, the number is not sufficient to support a significant association. To address these issues, we investigated the frequency of HLA-A alleles in three other large control populations of European (EUR) ancestry (two cohorts from the Geisinger Health System [GHS cohort #1, n=77,198, and GHS cohort #2, n=59,072], and the UK Biobank (UKB, n=463,315)]. We selected European samples carrying at least one HLA-A29 allele in all three datasets and matched the BSCR cohort to 4,014 A29 carriers from GHS cohort #1 (5.2% of all European subjects), 2,829 A29 carriers from GHS cohort #2 (4.8% of all European), and 38,543 A29 carriers from UKB (8.3% of all European). The frequency of the second HLA-A allele in these cohorts was compared to that observed in the BSCR cohort (Figure 2, Figure 1). Results supported enrichment of four of the five HLA-Aw19 alleles in BSCR cases, with the highest increased risk for HLA-A30:02 (GHS cohort #1 odds ratio = 4.31, GHS cohort #2 odds ratio = 6.6, UKB odds ratio = 4.6) and HLA-A33 (GHS cohort #1 odds ratio = 3.4, GHS cohort #2 odds ratio = 2.8, UKB odds ratio = 4.9). When samples carrying the four co-susceptibility alleles A29, A30, A31, and A33 were combined, highly significant enrichment was found in BSCR cases compared to the larger control cohort (GHS cohort #1, p-value = 1.29E-06; GHS cohort #2, p-value = 1.07E-06; UKB, p-value = 9.62E-07; Figure 1, top). This analysis excludes A32 due to biological differences in the sequence of the peptide binding domain, as previously reported.Additional analysis with all Aw19 alleles, including A32, showed that enrichment of cases was reduced when A32 was included (Figure 1, bottom).

[0111] To test whether these associations were influenced by measurable confounding factors, logistic regression tests were performed to evaluate the effect of a second HLA-A allele in HLA-A29 carriers in UParis BSCR cases compared to each control cohort, calculated based on the genetic array data of each analysis set, with covariates including sex and principal components (Figure 3). Results were consistent with an increased risk for HLA-Aw19 co-susceptibility alleles (A29, A30, A31, and A33).

[0112] Example 3: HLA-A32 shows protection from BSCR in HLA-A29 positive cohorts HLA-A32 was less prevalent in BSCR cases (3 / 286, approximately 1%) compared to A29 carrier controls (4 / 108, 3.7%), corresponding to a nominally significant protection from risk (odds ratio = 0.28, p = 0.1; Figure 1). When compared to a larger control cohort, the trend of protection is maintained both by UKB controls (3.4%, odds ratio = 0.3, p = 0.02) and by GHS controls (cohort #1: 3.8%, odds ratio = 0.27, p = 0.01; cohort #2: 3.7%, odds ratio = 0.27, p = 0.02). Although nominally significant, this result does not exceed the threshold for multiple testing correction (p = 3.57e-03) and will need to be further validated by additional case cohorts.

[0113] Example 4: ERAP2 is independently associated with BSCR All variants and gene burdens were tested for association with case-control status using generalized linear mixed models (SAIGE), controlling for gender and 10 principal components. The expected strong HLA-A signal, due to the fact that both cases and controls were A29 allele carriers, was at least partially controlled, as evidenced by the strongest HLA p-value = 8.98E-07, compared with p = 6.6e-74 for 125 cases in the previous BSCR report (Kuiper et al., Hum. Mol. Genet., 2014, 23, 6081-6087). Overall, no loci exceeded the genome-wide significance threshold (p < 5e-8). Other than the residual signal of HLA-A, only the ERAP1 / ERAP2-LNPEP locus on chromosome 5 showed association with disease at p < 1e-6 (Figure 2).

[0114] The previously reported top association for BSCR at this locus tags a common variant near ERAP2 / LNPEP, rs10044354. This reported risk allele is in strong linkage disequilibrium (D' = 0.99, R 2 = 0.76). The results show a nominal association of rs10044354 with an increased risk of scattershot chorioretinopathy (odds ratio (95% confidence interval) = 1.55 (1.13-2.11), p = 5.8e-3). Furthermore, no significant evidence was found for an interaction between rs10044354 and the rs27432-rs2287987 haplotype (conditional haplotype test p = 0.46).

[0115] We then performed a meta-analysis of the results using published results from Kuiper et al., which yielded a genome-wide significant association with BSCR for the ERAP2 locus (rs10044354, odds ratio (95% confidence interval) = 1.95 (1.55-2.44), p = 6.2e-09) (Figure 1). The previous study and the current study showed a consistent direction for ERAP2 separated over 201,222 bp, with low linkage disequilibrium (LD) in the current cohort (R2 = 0.18, D' = 0.79).

[0116] It has been previously reported that a common splice region variant (rs2248374, AF=0.53) disrupts the expression of ERAP2, which causes missplicing of intron 10 and the resulting transcript degradation via nonsense-mediated decay (Andres et al., PLoS Genet., 2010, 6, e1001157; and Coulombe-Huntington et al., PLoS Genet., 2009, 5, e1000766), and is in high LD with rs10044354 (R2=0.8, D'=1). Therefore, it is estimated that about 25% of most ancestral populations (including Europe, AF=0.53; Africa, AF=0.57, and South Asia, AF=0.58) lack active ERAP2 protein. Both data sets were investigated for rs2248374 association, and it was found to be nominally significantly protective for BSCR in both data sets (Figure 4). Furthermore, ERAP2-rs2248374, which interferes with ERAP2 expression, is protective (odds ratio 0.56; 95% confidence interval [0.45-0.70]; p = 2.39e-07; Figure 5). In summary, higher expression of ERAP2 protein increases the risk for BSCR, and lower expression is protective.

[0117] Example 5: Cumulative effect of HLA-Aw19 alleles and ERAP2 haplotypes on BSCR risk Possible interactions between ERAP2-related signals and between HLA-Aw19 and ERAP2 signals were examined by calculating the cumulative effect of HLA-Aw19 and ERAP2 genotypes on BSCR risk using 286 cases and 4,014 A29 carriers from GHS cohort #1. First, we performed an analysis of the ERAP2-rs10044354 risk haplotype, which is the top non-MHC signal shown in Kuiper et al., and stratified it by single (A29 / -) and double (A29 / AW19) Aw19 backgrounds. A trend toward increased risk was observed for the additional ERAP2-rs10044354-T variant allele, especially in the double A29 / AW19 background (Figure 6). The combination of rs10044354-TT and two copies of Aw19 in 12 cases and 34 controls was found to be at highest risk (odds ratio = 9.9 [4.4 to 21.2], p = 1.66e-07, Figure 7).

[0118] Example 6: Discussion Sequencing of a new large BSCR patient cohort and HLA-A29 controls confirmed the importance of ERAP2 polymorphisms in increasing the risk of developing BSCR. The association of ERAP2 haplotypes is consistent with a mechanism by which increased expression of ERAP2 contributes to disease risk. Multiple studies have reported that ERAP2 haplotypes affect their expression and the resulting peptidome (Kuiper et al., Hum. Mol. Genet., 2018, 27, 4333-4343; Paladini et al., Sci. Rep., 2018, 8, 10398; and Sanz-Bravo et al., Mol. Cell Proteomics, 2018, 17, 1564-1577).

[0119] This study found that in addition to the HLA-A29 risk allele, multiple other HLA-Aw19 family alleles (HLA-A29, A30, A31, A33) confer additional risk as second HLA-A alleles. HLA-Aw19 family alleles have similar antigen-binding sequences and therefore are thought to bind similar peptide motifs. Thus, enrichment of Aw19 alleles in cases supports the speculated mechanism underlying the activation of immune responses in BSCR. Having two copies of these alleles may increase cell surface presentation of specific types of peptides in BSCR cases compared to HLA-A29 positive controls. Furthermore, it was found that the HLA-A32 allele within the Aw19 family may be protective.

[0120] HLA-A32 was the only HLA-Aw19 member found in a lower percentage of BSCR patients compared to controls, suggesting that it may be protective. The HLA-Aw19 serotype was initially identified by antibodies binding to related family members, which identify HLA-A proteins based on structures outside the peptide-binding groove. Since then, the serofamily has been reanalyzed by sequences of the entire peptide-binding region (McKenzie et al., Genes Immun. 1999, 1, 120-129). Comparison of sequences in the peptide-binding region revealed that HLA-A32 is more distantly related than the other Aw19 alleles identified as novel risk factors in this study: HLA-A29, A30, A31, and A33. When examining the differences in the sequences between these Aw19 alleles, two main differences are evident: at position 9, which is part of the peptide binding domain, and at a stretch of amino acids at positions 79-83 (Figure 8) that is found only in HLA-A32 and not in other Aw19 alleles. Theoretically, it is conceivable that the peptide pool bound by HLA-A32 does not activate the same subset of responding CD8 T cells as the remaining members of the Aw19 family. This adds further evidence supporting the hypothesis of a threshold requirement for increased concentration of the driving autoantigen peptide pool presented on high-risk HLA-A proteins as a driving component for the development of BSCR uveitis.

[0121] In addition to those described herein, various modifications of the described subject matter will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, genebank accession numbers, and the like) is hereby incorporated by reference in its entirety.

Claims

1. 1. A combination pharmaceutical for treating a subject with an immune disease, comprising: an endoplasmic reticulum aminopeptidase 2 (ERAP2) inhibitor; i) an HLA-A29 inhibitor and / or ii) an HLA-B27 inhibitor, A combination medicine comprising:

2. The pharmaceutical combination according to claim 1, wherein the immune disease is an MHC-I disease.

3. the MHC-I disease is shotcretinopathy (BSCR); The combination pharmaceutical according to claim 2, wherein the combination pharmaceutical comprises the ERAP2 inhibitor and the HLA-A29 inhibitor.

4. The pharmaceutical combination of claim 3, wherein the HLA-29 inhibitor comprises an antibody, a small molecule degrader, or an inhibitory nucleic acid molecule.

5. The pharmaceutical combination according to claim 4, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA that hybridizes to HLA-A29.

6. the antisense nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3871-4145; The pharmaceutical combination according to claim 5, wherein the siRNA molecule comprises a sense strand and a corresponding antisense strand selected from the group consisting of SEQ ID NOs: 4146 to 4755.

7. the MHC-I disease is ankylosing spondylitis (AS); The combination pharmaceutical according to claim 2, wherein the combination pharmaceutical comprises the ERAP2 inhibitor and the HLA-B27 inhibitor.

8. The pharmaceutical combination of claim 7, wherein the HLA-B27 inhibitor comprises an antibody, a small molecule degrader, or an inhibitory nucleic acid molecule.

9. The pharmaceutical combination of claim 8, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule that hybridizes to HLA-B27, a small interfering RNA (siRNA), or a short hairpin RNA.

10. the antisense nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2788-3132; The pharmaceutical combination according to claim 9, wherein the siRNA molecule comprises a sense strand and a corresponding antisense strand selected from the group consisting of SEQ ID NOs: 3133 to 3870.

11. the MHC-I disease is psoriasis and the subject also has uveitis; The combination pharmaceutical according to claim 2, wherein the combination pharmaceutical comprises the ERAP2 inhibitor and the HLA-B27 inhibitor.

12. The pharmaceutical combination of claim 11, wherein the HLA-B27 inhibitor comprises an antibody, a small molecule degrader, or an inhibitory nucleic acid molecule.

13. The pharmaceutical combination of claim 12, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule that hybridizes to HLA-B27, a small interfering RNA (siRNA), or a short hairpin RNA.

14. the antisense nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2788-3132; The pharmaceutical combination according to claim 13, wherein the siRNA molecule comprises a sense strand and a corresponding antisense strand selected from the group consisting of SEQ ID NOs: 3133 to 3870.

15. the MHC-I disease is juvenile idiopathic arthritis (JIA); The combination pharmaceutical according to claim 2, wherein the combination pharmaceutical comprises the ERAP2 inhibitor and the HLA-B27 inhibitor.

16. The pharmaceutical combination of claim 15, wherein the HLA-B27 inhibitor comprises an antibody, a small molecule degrader, or an inhibitory nucleic acid molecule.

17. The pharmaceutical combination of claim 16, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule that hybridizes to HLA-B27, a small interfering RNA (siRNA), or a short hairpin RNA.

18. the antisense nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2788-3132; The pharmaceutical combination according to claim 17, wherein the siRNA molecule comprises a sense strand and a corresponding antisense strand selected from the group consisting of SEQ ID NOs: 3133 to 3870.

19. The pharmaceutical combination according to any one of claims 1 to 18, wherein the ERAP2 inhibitor comprises an anti-ERAP2 antibody, a pseudopeptide, a small molecule, a small molecule degrader, or an inhibitory nucleic acid molecule.

20. The pharmaceutical combination according to claim 19, wherein the inhibitory nucleic acid molecule comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA that hybridizes to the mRNA of ERAP2.

21. the antisense nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-858; The pharmaceutical combination according to claim 20, wherein the siRNA molecule comprises a sense strand and a corresponding antisense strand selected from the group consisting of SEQ ID NOs: 859 to 2748.

22. The pharmaceutical combination according to claim 19, wherein the peptidomimetic is a phosphinic acid peptidomimetic.

23. The pharmaceutical combination according to claim 22, wherein the phosphinic acid pseudopeptide is DG002 or DG013.

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