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

Background: Certain phosphorylated peptides are differentially presented by major histocompatibility complex (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 non-phosphorylated counterparts. This stability is thought to contribute to enhanced immunogenicity.

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

Background: Certain 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.

Dominant epitopes presented by prevalent HLA alleles permit wide use of banked CMVpp65 T cells in adoptive therapy.

We established and characterized a bank of 138 CMVpp65 peptide-specific T-cell (CMVpp65CTLs) lines from healthy marrow transplant donors who consented to their use for treatment of individuals other than their transplant recipient. CMVpp65CTL lines included 131 containing predominantly CD8+ T cells and 7 CD4+ T cells. CD8+ CMVpp65CTLs were specific for 1 to 3 epitopes each presented by one of only 34 of the 148 class I alleles in the bank.

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

Exploring the repertoire of peptides presented on major histocompatibility complexes (MHCs) helps identify targets for immunotherapy in many hematologic malignancies. However, there is a paucity of such data for diffuse large B-cell lymphomas (DLBCLs), which might be explained by the profound downregulation of MHC expression in many DLBCLs, and in particular in the enhancer of zeste homolog 2 (EZH2)-mutated subgroup. Epigenetic drug treatment, especially in the context of interferon-γ (IFN-γ), restored MHC expression in DLBCL.

Immunogenicity and therapeutic targeting of a public neoantigen derived from mutated PIK3CA.

Public neoantigens (NeoAgs) represent an elite class of shared cancer-specific epitopes derived from recurrently mutated driver genes. Here we describe a high-throughput platform combining single-cell transcriptomic and T cell receptor (TCR) sequencing to establish whether mutant PIK3CA, among the most frequently genomically altered driver oncogenes, generates an immunogenic public NeoAg. Using this strategy, we developed a panel of TCRs that recognize an endogenously processed neopeptide encompassing a common PIK3CA hotspot mutation restricted by the prevalent human leukocyte antigen (HLA)-A*03:01 allele.

A TCR mimic CAR T cell specific for NDC80 is broadly reactive with solid tumors and hematologic malignancies.

Target identification for chimeric antigen receptor (CAR) T-cell therapies remains challenging due to the limited repertoire of tumor-specific surface proteins. Intracellular proteins presented in the context of cell surface HLA provide a wide pool of potential antigens targetable through T-cell receptor mimic antibodies. Mass spectrometry (MS) of HLA ligands from 8 hematologic and nonhematologic cancer cell lines identified a shared, non-immunogenic, HLA-A*02-restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene.

A TCR mimic monoclonal antibody for the HPV-16 E7-epitope p11-19/HLA-A*02:01 complex.

More effective treatments are needed for human papilloma virus (HPV)-induced cancers despite HPV virus vaccination. The oncogenic HPV protein targets are currently undruggable and intracellular and therefore there are no antibodies to these targets. Here we report the discovery of TCR mimic monoclonal antibodies (TCRm mAb) specific for the HPV E7 protein p11-19, YMLDLQPET, when presented on the cell surface in the context of HLA-A*02:01 by use of human phage display libraries.

A TCR mimic monoclonal antibody reactive with the "public" phospho-neoantigen pIRS2/HLA-A*02:01 complex.

Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed "public" cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope's presentation by HLA-A*02:01 was confirmed by mass spectrometry.

Low-dose CDK4/6 inhibitors induce presentation of pathway specific MHC ligands as potential targets for cancer immunotherapy.

Cyclin dependent kinase 4/6 inhibitors (CDK4/6i) lead to cell-cycle arrest but also trigger T cell-mediated immunity, which might be mediated by changes in human leukocyte antigen (HLA) ligands. We investigated the effects of CDK4/6i, abemaciclib and palbociclib, on the immunopeptidome at nontoxic levels in breast cancer cell lines by biochemical identification of HLA ligands followed by network analyses. This treatment led to upregulation of HLA and revealed hundreds of induced HLA ligands in breast cancer cell lines.

Empirical and Rational Design of T Cell Receptor-Based Immunotherapies.

The use of T cells reactive with intracellular tumor-associated or tumor-specific antigens has been a promising strategy for cancer immunotherapies in the past three decades, but the approach has been constrained by a limited understanding of the T cell receptor's (TCR) complex functions and specificities. Newer TCR and T cell-based approaches are in development, including engineered adoptive T cells with enhanced TCR affinities, TCR mimic antibodies, and T cell-redirecting bispecific agents. These new therapeutic modalities are exciting opportunities by which TCR recognition can be further exploited for therapeutic benefit.

Impact of tumor heterogeneity and microenvironment in identifying neoantigens in a patient with ovarian cancer.

Identification of neoepitopes as tumor-specific targets remains challenging, especially for cancers with low mutational burden, such as ovarian cancer. To identify mutated human leukocyte antigen (HLA) ligands as potential targets for immunotherapy in ovarian cancer, we combined mass spectrometry analysis of the major histocompatibility complex (MHC) class I peptidomes of ovarian cancer cells with parallel sequencing of whole exome and RNA in a patient with high-grade serous ovarian cancer. Four of six predicted mutated epitopes capable of binding to HLA-A*02:01 induced peptide-specific T cell responses in blood from healthy donors.

Solving an MHC allele-specific bias in the reported immunopeptidome.

Identification of MHC class I-bound peptides by immunopurification of MHC complexes and subsequent analysis by mass spectrometry is crucial for understanding T cell immunology and immunotherapy. Investigation of the steps for the MHC ligand isolation process revealed biases in widely used isolation techniques toward peptides of lower hydrophobicity. As MHC ligand hydrophobicity correlates positively with immunogenicity, identification of more hydrophobic MHC ligands could potentially lead to more effective isolation of immunogenic peptides as targets for immunotherapies.

An input-controlled model system for identification of MHC bound peptides enabling laboratory comparisons of immunopeptidome experiments.

Characterization of MHC-bound peptides by mass spectrometry (MS) is an essential technique for immunologic studies. Many efforts have been made to quantify the number of MHC-presented ligands by MS and to define the limits of detection of a specific MHC ligand. However, these experiments are often complex and comparisons across different laboratories are challenging.

Identification of the Targets of T-cell Receptor Therapeutic Agents and Cells by Use of a High-Throughput Genetic Platform.

T-cell receptor (TCR)-based therapeutic cells and agents have emerged as a new class of effective cancer therapies. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on MHC receptors. However, cross-reactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events.

ALK and RET Inhibitors Promote HLA Class I Antigen Presentation and Unmask New Antigens within the Tumor Immunopeptidome.

T-cell immunotherapies are often thwarted by the limited presentation of tumor-specific antigens abetted by the downregulation of human leukocyte antigen (HLA). We showed that drugs inhibiting ALK and RET produced dose-related increases in cell-surface HLA in tumor cells bearing these mutated kinases and , as well as elevated transcript and protein expression of HLA and other antigen-processing machinery. Subsequent analysis of HLA-presented peptides after ALK and RET inhibitor treatment identified large changes in the immunopeptidome with the appearance of hundreds of new antigens, including T-cell epitopes associated with impaired peptide processing (TEIPP) peptides.

Depleting T regulatory cells by targeting intracellular Foxp3 with a TCR mimic antibody.

Depletion of T regulatory cells (Tregs) in the tumor microenvironment is a promising cancer immunotherapy strategy. Current approaches for depleting Tregs are limited by lack of specificity and concurrent depletion of anti-tumor effector T cells. The transcription factor forkhead box p3 (Foxp3) plays a central role in the development and function of Tregs and is an ideal target in Tregs, but Foxp3 is an intracellular, undruggable protein to date.

Carcinogenesis of renal cell carcinoma reflected in HLA ligands: A novel approach for synergistic peptide vaccination design.

Despite recent advances in immunotherapy of renal cell carcinoma (RCC), peptide vaccination strategies still lack an approach for personalized peptide vaccination that takes intra- and inter-tumoral heterogeneity and biological characteristics into account. In this study, we use an immunoprecipitation and mass spectrometry-based approach supplemented by network analysis of HLA ligands to target this goal. By analyzing HLA-presented peptides for HLA class I and II of 11 malignant and 6 non-malignant kidney tissue samples, more than 2,700 peptides and 1,600 corresponding source proteins were identified.

The transcription factor BATF operates as an essential differentiation checkpoint in early effector CD8+ T cells.

The transcription factor BATF is required for the differentiation of interleukin 17 (IL-17)-producing helper T cells (TH17 cells) and follicular helper T cells (TFH cells). Here we identified a fundamental role for BATF in regulating the differentiation of effector of CD8(+) T cells. BATF-deficient CD8(+) T cells showed profound defects in effector population expansion and underwent proliferative and metabolic catastrophe early after encountering antigen.