InVivoMAb anti-human pan MHC Class II (HLA II)

Neoantigen-reactive CD4⁺ T cells play a key role in the anti-tumor immune response. However, the majority of epithelial tumors are negative for HLA class II (HLA-II) surface expression, and less is known about the processing of HLA-II antigens. Here, we directly identified naturally presented HLA-II neoantigens in HLA-II negative colorectal cancer (CRC) tissue using a proteogenomic approach. The neoantigens were immunogenic and induced patient CD4⁺ T cells with a Th1-like memory phenotype that produced IFN-γ, IL2 and TNF-α. Multiplex immunohistochemistry (IHC) demonstrated an interaction between Th cells and HLA-II-positive antigen-presenting cells (APCs) at the invasive margin and within the tertiary lymphoid structures (TLS). In our CRC cohort, the density of stromal APCs was associated with HLA-II antigen presentation in the tumor microenvironment (TME), and the number of TLS was positively correlated with the number of somatic mutations in the tumors. These results demonstrate the presence of neoantigen-specific CD4⁺ surveillance in HLA-II-negative CRC and suggest a potential role for macrophages and dendritic cells (DCs) at the invasive margin and in TLS for antigen presentation. Stromal APCs in the TME can potentially be used as a source for HLA-II neoantigen identification.

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) poses a major challenge in hemophilia A (HA) treatment. The formation of FVIII inhibitors is a CD4+ T-cell dependent mechanism which includes antigen presenting cells (APCs), B- and T-helper lymphocytes. APCs present FVIII derived peptides on major histocompatibility complex class II (MHC-II) to CD4+ Tcells. We previously established a mass spectrometry based approach to delineate the FVIII repertoire presented on HLA-DR and HLA-DQ. In this study, specific attention was directed towards the identification of FVIII peptides presented on HLA-DP. A data-set of naturally processed FVIII peptides was generated by incubating human FVIII with immature monocytes-derived dendritic cells (moDCs) from HLA-typed healthy donors. Using this method, we identified 176 to 1352 different HLA-DP presented peptides per donor, including 26 different FVIII derived peptides. The most frequently presented peptides derived from the A3, and C2 domains of FVIII. Comparison of the FVIII repertoire presented on HLA-DP with that presented on HLA-DR revealed considerable overlap but also suggested preferential presentation of specific peptides on either HLA-DR or HLA-DP. Fourteen FVIII peptides presented on HLA-DP were synthesized and evaluated for their binding ability to the commonly expressed HLA-DP4 molecule which is highly prevalent in the Caucasian population. Peptide binding studies showed that 7 of 14 peptides competed with a reference peptide to HLADP4. Interestingly, an A3 domain derived peptide bound with high affinity to HLA-DP4 positioning this peptide as a prime candidate for the development of novel peptide-based tolerogenic strategies for FVIII inhibitors.

Neoantigens arising from somatic mutations are tumor specific and induce antitumor host T cell responses. However, their sequences are individual specific and need to be identified for each patient for therapeutic applications. Here, we present a proteogenomic approach for neoantigen identification, named Neoantigen Selection using a Surrogate Immunopeptidome (NESSIE). This approach uses an autologous wild-type immunopeptidome as a surrogate for the tumor immunopeptidome and allows human leukocyte antigen (HLA)-agnostic identification of both HLA class I (HLA-I) and HLA class II (HLA-II) neoantigens. We demonstrate the direct identification of highly immunogenic HLA-I and HLA-II neoantigens using NESSIE in patients with colorectal cancer and endometrial cancer. Fresh or frozen tumor samples are not required for analysis, making it applicable to many patients in clinical settings. We also demonstrate tumor prevention by vaccination with selected neoantigens in a preclinical mouse model. This approach may benefit personalized T cell-mediated immunotherapies.

Acral lentiginous melanoma (ALM) is the most common melanoma subtype in non-Caucasians. Despite advances in cancer immunotherapy, current immune checkpoint inhibitors remain unsatisfactory for ALM. Hence, we conducted comprehensive immune profiling using single-cell phenotyping with reactivity screening of the T cell receptors of tumor-infiltrating T lymphocytes (TILs) in ALM. Compared with cutaneous melanoma, ALM showed a lower frequency of tumor-reactive CD8 clusters and an enrichment of regulatory T cells with direct tumor recognition ability, suggesting a suppressive immune microenvironment in ALM. Tumor-reactive CD8 TILs showed heterogeneous expression of coinhibitory molecules, including KLRC1 (NKG2A), in subpopulations with therapeutic implications. Overall, our study provides a foundation for enhancing the efficacy of immunotherapy in ALM.