Biology subjects

Scheinberg, D. A.

Publications and source records attributed to Scheinberg, D. A..

6 recordsLinked to original sources

bioRxiv2023

Host-cell Interactions of Engineered T cell Micropharmacies

Genetically engineered, cytotoxic, adoptive T cells localize to antigen positive cancer cells inside patients, but tumor heterogeneity and multiple immune escape mechanisms have prevented the eradication of most solid tumor types. More effective, multifunctional engineered T cells are in development to overcome the barriers to the treatment of solid tumors, but the interactions of these highly modified cells with the host are poorly understood. We previously engineered prodrug-activating enzymatic functions into chimeric antigen receptor (CAR) T cells, endowing them with an orthogonal killing mechanism to conventional T-cell cytotoxicity. These drug-delivering cells, termed Synthetic Enzyme-Armed KillER (SEAKER) cells, demonstrated efficacy in mouse lymphoma xenograft models. However, the interactions of an immunocompromised xenograft with such complex engineered T cells are distinct from those in an immunocompetent host, precluding an understanding of how these physiologic processes may affect the therapy. Here, we also expand the repertoire of SEAKER cells to target solid-tumor melanomas in syngeneic mouse models using specific targeting with TCR-engineered T cells. We demonstrate that SEAKER cells localize specifically to tumors, and activate bioactive prodrugs, despite host immune responses. We additionally show that TCR-engineered SEAKER cells are efficacious in immunocompetent hosts, demonstrating that the SEAKER platform is applicable to many adoptive cell therapies.

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↗

bioRxiv2020

Low-Dose CDK4/6 Inhibitors Induce Presentation of Pathway Specific MHC ligands as Targets for Cancer Immunotherapy

PurposeCyclin dependent kinase 4/6 inhibitors (CDK4/6i) lead to cell-cycle arrest but also demonstrate antineoplastic activity through triggering T cell-mediated immunity. One of the potential mechanisms responsible for this immunological effect might be qualitative and quantitative changes in human leukocyte antigen (HLA) ligands on the cell surface after treatment with CDK4/6i. These changes may increase the immunogenicity of breast cancer cells offering potential synergies for combinations with cancer immunotherapies. Experimental DesignWe investigated the ability of two CDK4/6 inhibitors (CDK4/6i), Abemaciclib and Palbociclib, to alter the immunopeptidome at subclinical, non-toxic, levels in different breast cancer cell lines. Biochemical isolation of HLA ligands, identification by mass spectrometry and subsequent network analysis after drug treatment were used to characterize the changes in the immunopeptidome. The mechanisms for altered CDK4/6 presentation were explored. ResultsLow-dose treatment with 100nM of Abemaciclib and Palbociclib led to upregulation of cell surface HLA levels and induced hundreds of HLA ligands in breast cancer cell lines. These new ligands were significantly and most strongly enriched for peptides derived from proteins involved in the "G1/S phase transition of cell cycle" pathway and included among others, HLA ligands from CDK4, CDK6, Cyclin D1 and Cyclin E1. An increase in transcript, protein, and subsequent ubiquitination for Cyclin D1, which could lead to enhanced degradation of the target protein, was identified as a potential mechanism for the altered presentation of peptides. ConclusionsCDK4/6i treatment gave rise to drug-induced antigens through cell cycle disruption and increased antigen presentation. Interestingly, these induced HLA ligands are often sourced from the proteins of the CDK4/CDK6/CCND1 complex or more downstream interaction partners, providing evidence that inhibition of a distinct cellular pathway leads to increased presentation of the proteins involved. These findings suggested CDK4/6i provided a tool for highly selective induction of HLA ligands that may be targeted by T cell-based immunotherapeutics. Translational RelevanceThese data demonstrated that low-dose treatment of breast cancer cells with CDK4/6 inhibitors, Abemaciclib and Palbociclib, induced marked changes in presentation of HLA ligands, especially from proteins involved in the G1/S phase transition, the phase in which these drugs arrest the cells. Enhanced ubiquitination and degradation was identified as a mechanism for the altered presentation for one of the relevant proteins. The induced HLA ligands may provide ideal specific targets for combination immunotherapies. The data show for the first time that selective inhibition of a distinct pathway can lead to specific presentation of HLA ligands in breast cancer cells. This work supports the rationale for testing the combination of low-dose CDK4/6i with immunotherapeutic agents, such as immune checkpoint blockade antibodies or T-cell-based approaches specifically directed against one of the induced HLA ligands.

cancer biology↗

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↗