Biology subjects

Klatt, M. G.

Publications and source records attributed to Klatt, M. G..

5 recordsLinked to original sources

bioRxiv2023

Identification of tumor-specific MHC ligands through improved biochemical isolation and incorporation of machine learning

Isolation of MHC ligands and subsequent analysis by mass spectrometry is considered the gold standard for defining targets for TCR-T immunotherapies. However, as many targets of high tumor-specificity are only presented at low abundance on the cell surface of tumor cells, the efficient isolation of these peptides is crucial for their successful detection. Here, we demonstrate how different isolation strategies, which consider hydrophobicity and post-translational modifications, can improve the detection of MHC ligands, including cysteinylated MHC ligands from cancer germline antigens or point-mutated neoepitopes. Furthermore, we developed a novel MHC class I ligand prediction algorithm (ARDisplay-I) that outperforms the current state-of-the-art and facilitates the assignment of peptides to the correct MHC allele. The model has other applications, such as the identification of additional MHC ligands not detected from mass spectrometry or determining whether the MHC ligands can be presented on the cell surface via MHC alleles not included in the study. The implementation of these strategies can augment the development of T cell receptor-based therapies (i.a. TIL1-derived T cells, genetically engineered T cells expressing tumor recognizing receptors or TCR-mimic antibodies) by facilitating the identification of novel immunotherapy targets and by enriching the resources available in the field of computational immunology. SignificanceThis study demonstrates how the isolation of different tumor-specific MHC ligands can be optimized when considering their hydrophobicity and post-translational modification status. Additionally, we developed a novel machine-learning model for the probability prediction of the MHC ligands presentation on the cell surface. The algorithm can assign these MHC ligands to their respective MHC alleles which is essential for the design of TCR-T immunotherapies.

immunology↗

bioRxiv2023

The landscape of MHC-presented phosphopeptides yields actionable shared tumor antigens for cancer immunotherapy across multiple HLA alleles

BackgroundCertain phosphorylated peptides are differentially presented by MHC molecules on cancer cells characterized by aberrant phosphorylation. Phosphopeptides presented in complex with the human leukocyte antigen HLA-A*02:01 provide a stability advantage over their nonphosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity. Whether tumor-associated phosphopeptides presented by other common alleles exhibit immunogenicity and structural characteristics similar to those presented by A*02:01 is unclear. Therefore, we determined the identity, structural features, and immunogenicity of phosphopeptides presented by the prevalent alleles HLA-A*03:01, -A*11:01, -C*07:01, and - C*07:02. MethodsWe isolated peptide-MHC complexes by immunoprecipitation from 10 healthy and neoplastic tissue samples using mass spectrometry, and then combined the resulting data with public immunopeptidomics datasets to assemble a curated set of phosphopeptides presented by 20 distinct healthy and neoplastic tissue types. We determined the biochemical features of selected phosphopeptides by in vitro binding assays and in silico docking, and their immunogenicity by analyzing healthy donor T cells for phosphopeptide-specific multimer binding and cytokine production. ResultsWe identified a subset of phosphopeptides presented by HLA-A*03:01, A*11:01, C*07:01 and C*07:02 on multiple tumor types, particularly lymphomas and leukemias, but not healthy tissues. These phosphopeptides are products of genes essential to lymphoma and leukemia survival. The presented phosphopeptides generally exhibited similar or worse binding to A*03:01 than their nonphosphorylated counterparts. HLA-C*07:01 generally presented phosphopeptides but not their unmodified counterparts. Phosphopeptide binding to HLA-C*07:01 was dependent on B- pocket interactions that were absent in HLA-C*07:02. While HLA-A*02:01 and -A*11:01 phosphopeptide-specific T cells could be readily detected in an autologous setting even when the nonphosphorylated peptide was co-presented, HLA-A*03:01 or -C*07:01 phosphopeptides were repeatedly nonimmunogenic, requiring use of allogeneic T cells to induce phosphopeptide- specific T cells. ConclusionsPhosphopeptides presented by multiple alleles that are differentially expressed on tumors constitute tumor-specific antigens that could be targeted for cancer immunotherapy, but the immunogenicity of such phosphopeptides is not a general feature. In particular, phosphopeptides presented by HLA-A*02:01 and A*11:01 exhibit consistent immunogenicity, while phosphopeptides presented by HLA-A*03:01 and C*07:01, although appropriately presented, are not immunogenic. Thus, to address an expanded patient population, phosphopeptide-targeted immunotherapies should be wary of allele-specific differences. What is already known on this topic - Phosphorylated peptides presented by the common HLA alleles A*02:01 and B*07:02 are differentially expressed by multiple tumor types, exhibit structural fitness due to phosphorylation, and are targets of healthy donor T cell surveillance, but it is not clear, however, whether such features apply to phosphopeptides presented by other common HLA alleles. What this study adds - We investigated the tumor presentation, binding, structural features, and immunogenicity of phosphopeptides to the prevalent alleles A*03:01, A*11:01, C*07:01, and C*07:02, selected on the basis of their presentation by malignant cells but not normal cells. We found tumor antigens derived from genetic dependencies in lymphomas and leukemias that bind HLA-A3, -A11, -C7 molecules. While we could detect circulating T cell responses in healthy individuals to A*02:01 and A*11:01 phosphopeptides, we did not find such responses to A*03:01 or C*07:01 phosphopeptides, except when utilizing allogeneic donor T cells, indicating that these phosphopeptides may not be immunogenic in an autologous setting but can still be targeted by other means. How this study might affect research, practice or policy - An expanded patient population expressing alleles other than A*02:01 can be addressed through the development of immunotherapies specific for phosphopeptides profiled in the present work, provided the nuances we describe between alleles are taken into consideration.

immunology↗

bioRxiv2021

Unmasking the cryptic immunopeptidome of EZH2 mutated diffuse large B-cell lymphomas through combination drug treatment

Exploring the repertoire of peptides presented on major histocompatibility complexes (MHC) has been utilized to identify targets for immunotherapy in many hematological malignancies. However, such data have not been described systematically for diffuse large B-cell lymphomas (DLBCL), which might be explained by the profound downregulation of MHC expression in many DLBCLs, and in particular in the EZH2-mutated subgroup. Epigenetic drug treatment, especially in the context of interferon gamma (IFNg), restored MHC expression in DLBCL. DLBCL MHC-presented peptides were identified via mass spectrometry following tazemetostat or decitabine treatments alone, or in combination with IFNg. Such treatment synergistically increased MHC class I surface protein expression up to 50-fold and class II expression up to 3-fold. Peptides presented on MHC complexes increased to a similar extent for MHC class I and remained constant for class II. Overall, these treatments restored the diversity of the immunopeptidome to levels described in healthy B cells and allowed the systematic search for new targets for immunotherapy. Consequently, we identified multiple MHC ligands from regulator of G protein signaling 13 (RGS13) and E2F transcription factor 8 (E2F8) on different MHC alleles, none of which have been described in healthy tissues and therefore represent tumor-specific MHC ligands, which are unmasked only after drug treatment. Overall, our results show that EZH2 inhibition in combination with decitabine and IFNg can expand the repertoire of MHC ligands presented on DLBCLs by revealing cryptic epitopes, thus allowing the systematic analysis and identification of new potential immunotherapy targets. Key pointsO_LICombination therapy of interferon gamma with epigenetic regulators leads to large increases in the immunopeptidome of DLBCL. C_LIO_LIHLA ligands from proteins RGS13 and E2F8 may provide DLBCL-specific targets for immunotherapy. C_LI

immunology↗

bioRxiv2021

Immunogenicity of a public neoantigen derived from mutated PIK3CA

Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrent mutations in driver genes that are restricted by prevalent HLA alleles. Here, we report on a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant (Mut) PIK3CA, among the most common genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this method, we developed a library of TCRs that recognize an endogenously processed neoepitope containing a common PIK3CA hotspot mutation that is restricted by HLA-A*03:01. Mechanistically, immunogenicity to this public NeoAg arises primarily from enhanced stability of the neopeptide/HLA complex caused by a preferred HLA anchor substitution. Structural studies indicated that the HLA-bound neopeptide presents a relatively "featureless" surface dominated by the peptides backbone. To overcome the challenge of binding such an epitope with high specificity and affinity, we discovered that a lead TCR clinical candidate engages the neopeptide through an extended interface aided by an unusually long {beta}-chain complementarity-determining region 3 (CDR3{beta}) loop. In a pan-cancer cohort of patients with diverse malignancies that express the PIK3CA public NeoAg, we observed spontaneous immunogenicity, NeoAg clonal conservation, and in a limited number of cases, evidence of targeted immune escape. Together, these results establish the immunogenic potential of Mut PIK3CA, creating a framework for off-the-shelf immunotherapies targeting this public NeoAg.

immunology↗

bioRxiv2018

Prospective identification of cross-reactive human peptide-MHC ligands for T cell receptor based therapies

T cell receptor (TCR)-based therapeutic cells and agents have emerged as a new class of effective cancer therapeutics. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on major histocompatibility complex receptors. However, cross-reactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events. We have developed a high throughput genetic platform (termed \"PresentER\") that encodes MHC-I peptide minigenes for functional immunological assays as well as for determining the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this report, we demonstrate that PresentER can be used to identify the on-and-off targets of T cells and TCR mimic antibodies using in vitro co-culture assays or binding assays. We find dozens of MHC-I ligands that are cross-reactive with two TCR mimic antibodies and two native TCRs and that are not easily predictable by other methods.

cancer biology↗