Identification and structural characterization of a mutant KRAS-G12V specific TCR restricted by HLA-A3.

Mutations in KRAS are some of the most common across multiple cancer types and are thus attractive targets for therapy. Recent studies demonstrated that mutant KRAS generates immunogenic neoantigens that are targetable by adoptive T-cell therapy in metastatic diseases. To expand mutant KRAS-specific immunotherapies, it is critical to identify additional HLA-I allotypes that can present KRAS neoantigens and their cognate T-cell receptors (TCR). Here, we identified a murine TCR specific to a KRAS-G12V neoantigen (7VVVGAVGVGK16) using a vaccination approach with transgenic mice expressing HLA-A*03:01 (HLA-A3). This TCR demonstrated exquisite specificity for mutant G12V and not WT KRAS peptides. To investigate the molecular basis for neoantigen recognition by this TCR, we determined its structure in complex with HLA-A3(G12V). G12V-TCR CDR3ß and CDR1ß formed a hydrophobic pocket to interact with p6 Val of the G12V but not the WT KRAS peptide. To improve the tumor sensitivity of this TCR, we designed rational substitutions to improve TCR:HLA-A3 contacts. Two substitutions exhibited modest improvements in TCR binding avidity to HLA-A3 (G12V) but did not sufficiently improve T-cell sensitivity for further clinical development. Our study provides mechanistic insight into how TCRs detect neoantigens and reveals the challenges in targeting KRAS-G12V mutations.

Iron loading in HFE p.C282Y homozygotes found by population screening: relationships to HLA-type and T-lymphocyte subsets.

Iron loading in p.C282Y homozygous HFE hemochromatosis subjects is highly variable, and it is unclear what factors cause this variability. Finding such factors could aid in predicting which patients are at highest risk and require closest follow-up. The degree of iron loading has previously been associated with certain HLA-types and with abnormally low CD8 + cell counts in peripheral blood. In 183 Norwegian, p.C282Y homozygotes (104 men, 79 women) originally found through population screening we determined HLA type and measured total T-lymphocytes, CD4 + and CD8 + cells, and compared this with data on iron loading. In p.C282Y homozygous men, but not in homozygous women, we found that the presence of two HLA-A*03 alleles increased the iron load on average by approximately 2-fold compared to p.C282Y homozygous men carrying zero or one A*03 allele. On the other hand, the presence of two HLA-A*01 alleles, in male subjects, apparently reduced the iron loading. In p.C282Y homozygous individuals, the iron loading was increased if the CD8 + cell number was below the 25 percentile or if the CD4 + cell number was above the 75 percentile. This effect appeared to be additive to the effect of the number of HLA-A*03 alleles. Our data indicate that homozygosity for the HLA-A*03 allele significantly increases the risk of excessive iron loading in Norwegian p.C282Y homozygous male patients. In addition, low CD8 + cell number or high CD4 + cell number further increases the risk of excessive iron loading.

A new HLA-A*03 null allele - A*03:162N caused by an exon 4 insertion and discovered during an external quality assessment exercise.

HLA-A*03:162N differs from A*03:01:01:01 by an exon 4, 664-665insCATG causing E198A and A199X.

A new non-expressed allele HLA-A*03:168N, identified by serological and sequence-based typing in a voluntary Norwegian hematopoietic stem cell donor.

HLA-A*03:168N differs from HLA-A*03:01:01 by a single nucleotide substitution resulting in a coding change Y27X.

Identification of the novel HLA-A*03:453 allele in a Chinese blood donor.

HLA-A*03:453 differs from HLA-A*03:02:01:01 by one single nucleotide substitution at position 376 G > A.

Identification of the novel HLA-A allele, HLA-A*03:01:120, by next-generation sequencing.

The novel HLA-A*03:01:120 allele was characterized using next generation sequencing technology.

The novel HLA-A allele, HLA-A*03:478, identified in a Korean individual.

One nucleotide substitution in codon 320 of A*03:01:01:01 results in the novel allele, HLA-A*03:478.

Characterization of the novel HLA-A*03:444 detected in a potential hematopoietic stem cell donor.

The novel HLA-A*03:444 allele was characterized using next generation sequencing technology.

The novel HLA-A*03 allele, HLA-A*03:440, identified in a potential hematopoietic stem cell donor.

The novel HLA-A*03:440 allele was characterized using next generation sequencing technology.

Two novel HLA-A alleles detected in a potential hematopoietic stem cell donors: HLA-A*03:418 and -A*26:220.

HLA-A*03:418 and -A*26:220, two novel alleles detected by next generation sequencing.

Genome-wide association studies of response and side effects to the BNT162b2 vaccine in Italian healthcare workers: Increased antibody levels and side effects in carriers of the HLA-A*03:01 allele.

The remarkable variability of response to vaccines against SARS-CoV-2 is apparent. The present study aims to estimate the extent to which the host genetic background contributes to this variability in terms of immune response and side effects following the administration of the BNT162b2 vaccine. We carried out a genome wide association study (GWAS) by genotyping 873 Italian healthcare workers who underwent anti-SARS-CoV-2 vaccination with the BNT162b2 vaccine and for whom information about anti-SARS-CoV-2 spike antibodies titers and vaccine side effects were available. The GWAS revealed a significant association between the HLA locus and the anti-SARS-CoV-2 Spike antibodies level at 2 months following the first dose of vaccine (SNP: rs1737060; p = 9.80 × 10-11 ). In particular, we observed a positive association between the antibody levels and the presence of the HLA-A*03:01 allele. The same allele was found associated with a 2-2.4-fold increased risk of experiencing specific side effects such as fever, chills and myalgia and a 1.5-1.8-fold increased risk of joint pain, nausea, fatigue, headache and asthenia, independently of age and sex. This study confirms that the heterogeneity in the immune response to the BNT162b2 vaccine and in its side effects are at least partially influenced by genetic variants. This information, integrated with individual biological and lifestyle-related correlates, could be of use in the definition of algorithms aimed at the identification of subjects in which the administration of additional vaccine doses would be particularly beneficial to maintain immunity against the virus.

Identification of two novel HLA alleles, HLA-A*03:344:02 and -DQB1*04:02:24 in Russian individuals.

The novel HLA alleles HLA-A*03:344:02 and -DQB1*04:02:24 have synonymous mutations.

A Lysine Residue at the C-Terminus of MHC Class I Ligands Correlates with Low C-Terminal Proteasomal Cleavage Probability.

The majority of peptides presented by MHC class I result from proteasomal protein turnover. The specialized immunoproteasome, which is induced during inflammation, plays a major role in antigenic peptide generation. However, other cellular proteases can, either alone or together with the proteasome, contribute peptides to MHC class I loading non-canonically. We used an immunopeptidomics workflow combined with prediction software for proteasomal cleavage probabilities to analyze how inflammatory conditions affect the proteasomal processing of immune epitopes presented by A549 cells. The treatment of A549 cells with IFNγ enhanced the proteasomal cleavage probability of MHC class I ligands for both the constitutive proteasome and the immunoproteasome. Furthermore, IFNγ alters the contribution of the different HLA allotypes to the immunopeptidome. When we calculated the HLA allotype-specific proteasomal cleavage probabilities for MHC class I ligands, the peptides presented by HLA-A*30:01 showed characteristics hinting at a reduced C-terminal proteasomal cleavage probability independently of the type of proteasome. This was confirmed by HLA-A*30:01 ligands from the immune epitope database, which also showed this effect. Furthermore, two additional HLA allotypes, namely, HLA-A*03:01 and HLA-A*11:01, presented peptides with a markedly reduced C-terminal proteasomal cleavage probability. The peptides eluted from all three HLA allotypes shared a peptide binding motif with a C-terminal lysine residue, suggesting that this lysine residue impairs proteasome-dependent HLA ligand production and might, in turn, favor peptide generation by other cellular proteases.

A novel HLA allele, HLA-A*03:420, identified by next-generation sequencing.

HLA-A*03:420 differs from HLA-A*03:01:01:01 by a single nucleotide in exon 1, codon -22.

Characterization of the novel HLA-A*03:425 allele by two next-generation sequencing methods.

The novel HLA-A*03:425 allele was characterized using two next-generation sequencing technologies.

Characterization of the novel HLA-A*03:436 allele by sequencing-based typing.

HLA-A*03:436 differs from HLA-A*03:01:01:05 by one nucleotide substitution in codon 26 in exon 2.

In-silico design of peptides for inhibition of HLA-A*03-KLIETYFSK complex as a new drug design for treatment of multiples sclerosis disease.

Multiple sclerosis is recognized as a chronic inflammatory disease. Human leukocyte antigen (HLA) plays an important role in initiating adaptive immune responses. HLA class I is present in almost all nucleated cells and presents the cleaved endogenous peptide antigens to cytotoxic T cells. HLA-A*03 is one of the HLA class I alleles, which is reported as substantially related HLA to MS disease. In 2011, the structure of the HLA-A*03 in complex was identified with an immunodominant proteolipid protein (PLP) epitope (KLIETYFSK). This complex has been reported as an important autoantigen-presenting complex in MS pathogenesis. In this study, new peptides were designed to bind to this complex that may prevent specific pathogenic cytotoxic T cell binding to this autoantigen-presenting complex and CNS demyelination. Herein, 14 new helical peptides containing 19 amino acids were designed and their structures were predicted using the PEP-FOLD server. The binding of each designed peptide to the mentioned complex was then performed. A mutation approach was used by the BeAtMuSiC server to improve the binding affinity of the designed peptide. In each position, amino acid substitutions leading to an increase in the binding affinity of the peptide to the mentioned complex were determined. Finally, the resulting complexes were simulated for 40 ns using AMBER18 software. The results revealed that out of 14 designed peptides, "WRYWWKDWAKQFRQFYRWF" peptide exhibited the highest affinity for binding to the mentioned complex. This peptide can be considered as a potential drug to control multiple sclerosis disease in patients carrying the HLA-A*03 allele.

The genomic full-length sequence of the HLA-A*03:30 allele, identified by full-length group-specific sequencing.

The genomic full-length sequence of HLA-A*03:30 was identified by a group-specific sequencing approach from China.

The Association Between HLA-A*03:01 and HLA-B*07:02 Alleles and Oxcarbazepine-induced Maculopapular Eruption in the Uighur Chinese Population.

PURPOSE: To determine genetic associations between oxcarbazepine (OXC)-induced maculopapular eruption (MPE) and human leukocyte antigen (HLA) variants in the Uighur Chinese population. METHODS: A total of 208 patients were enrolled in this study, including 20 subjects with OXC-induced MPE (case group) and 188 OXC-tolerant patients (control group). We detected the whole sequence of the HLA alleles in the 208 Uighur patients using next-generation sequencing (NGS). RESULTS: The carried rate of HLA-A*03:01 was significantly different between the OXC-induced MPE group and the tolerant group (7/20, 35% versus 4/188, 2.13%; P = 0.000, odds ratio [OR] = 24.77, 95% confidence interval [CI] = 6.41-95.65). Additionally, the carried rate of HLA-B*07:02 was significantly different between the OXC-induced MPE group and the tolerant group (3/20, 15% versus 2/188, 0.011%; P = 0.000, odds ratio [OR] = 16.41, 95% confidence interval (CI) = 2.56-105.09). CONCLUSIONS: HLA-A*03:01 and HLA-B*07:02 may be the risk alleles for MPE in the Uighur Chinese population.

The novel HLA-A*03 variant, HLA-A*03:08:01:02, detected by sequencing-based typing.

One nucleotide change in position 2606 of HLA-A*03:08:01:01 results in the novel allele, HLA-A*03:08:01:02.