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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. CAR T cells directed against the ALNEQIARL:HLA-A*02 complex exhibited high sensitivity and specificity for recognition and killing of multiple cancer types, especially those of hematologic origin, and were efficacious in mouse models against a human leukemia and a solid tumor. In contrast, no toxicities toward resting or activated healthy leukocytes as well as hematopoietic stem cells were observed. This shows how MS can inform the design of broadly reactive therapeutic T-cell receptor mimic CAR T-cell therapies that can target multiple cancer types currently not druggable by small molecules, conventional CAR T cells, T cells, or antibodies.

Epithelial ovarian cancer is the most lethal of gynecological cancers. The therapeutic efficacy of chimeric antigen receptor (CAR) T cell directed against single antigens is limited by the heterogeneous target antigen expression in epithelial ovarian tumors. To overcome this limitation, we describe an engineered cell with both dual targeting and orthogonal cytotoxic modalities directed against two tumor antigens that are highly expressed on ovarian cancer cells: cell surface Muc16 and intracellular WT1. Muc16-specific CAR-T cells (4H11) were engineered to secrete a bispecific T cell engager (BiTE) constructed from a TCR mimic antibody (ESK1) reactive with the WT1-derived epitope RMFPNAPYL (RMF) presented by HLA-A2 molecules. The secreted ESK1 BiTE recruited and redirected other T cells to WT1 on the tumor cells. We show that ESK1 BiTE-secreting 4H11 CAR-T cells exhibited enhanced anticancer activity against cancer cells with low Muc16 expression, compared to 4H11 CAR-T cells alone, both in vitro and in mouse tumor models. Dual orthogonal cytotoxic modalities with different specificities targeting both surface and intracellular tumor-associated antigens present a promising strategy to overcome resistance to CAR-T cell therapy in epithelial ovarian cancer and other cancers.

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. In DLBCL, peptides presented on MHCs were identified via mass spectrometry after treatment with tazemetostat or decitabine alone or in combination with IFN-γ. Such treatment synergistically increased the expression of MHC class I surface proteins up to 50-fold and the expression of class II surface proteins up to threefold. Peptides presented on MHCs increased to a similar extent for both class I and class II MHCs. Overall, these treatments restored the diversity of the immunopeptidome to levels described in healthy B cells for 2 of 3 cell lines and allowed the systematic search for new targets for immunotherapy. Consequently, we identified multiple MHC ligands from the 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 that are unmasked only after drug treatment. Overall, our results show that EZH2 inhibition in combination with decitabine and IFN-γ can expand the repertoire of MHC ligands presented on DLBCLs by revealing suppressed epitopes, thus allowing the systematic analysis and identification of new potential immunotherapy targets.

Supplementary Tables and Figures

Supplemental Figures and Table

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. We have developed a high-throughput genetic platform (termed “PresentER”) that encodes MHC-I peptide minigenes for functional immunologic assays and determines the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this article, we demonstrated that PresentER could be used to identify the on-and-off targets of T cells and TCR-mimic (TCRm) antibodies using in vitro coculture assays or binding assays. We found dozens of MHC-I ligands that were cross-reactive with two TCRm antibodies and two native TCRs and that were not easily predictable by other methods.

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 in vitro and in vivo, 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. ALK inhibition additionally decreased PD-L1 levels by 75%. Therefore, these oncogenes may enhance cancer formation by allowing tumors to evade the immune system by downregulating HLA expression. Altogether, RET and ALK inhibitors could enhance T-cell–based immunotherapies by upregulating HLA, decreasing checkpoint blockade ligands, and revealing new, immunogenic, cancer-associated antigens.

PDF file - 78KB, ESKM has a modified Fc glycosylation pattern, altering binding to FcgammaRs but not to the RMF/A2 target. Where applicable, data points are averages of each group, and error bars represent SEM. (A) Comparison of the oligosaccharide profile of ESK1 and ESKM. Peak assignment is based on the retention time and the monosaccharide composition analysis. G# indicates the number of terminal galactoses, F indicates presence of core fucose, Hex5GlcNAc2 denotes (GlcNAc)2 core with terminal Hexose 5 glycan structure (terminating in mannose and/or glucose). (B-C) Representative binding curves of ESK1 and ESKM against human FcgammaRI, FcgammaRIIa, FcgammaRIIb, FcgammaRIIIa - 158V, FcgammaRIIIa -158F, and FcRn (B), and mouse FcgammaRIIb and FcgammaRIV (C). (D) 125 I-labeled ESK1 and ESKM mAbs were titrated against JMN cells. All curves were fit with a non-linear single-site total binding saturation curve, and Kd was calculated using Prism software.

Includes peptide sequences for WT1 vaccine

Displays historical survival from multimodality cohorts that were used to set expected outcomes.

PDF file - 145KB, All human antibodies tested accumulated more in spleens of HLA-A2+ transgenic mice, but ESK1 did not bind specifically to isolated HLA-A2+ spleen, bone marrow or thymus cells. (A) Accumulation of 125 I-labeled antibodies in spleens of C57BL6/J or HLA-A2+ transgenic mice relative to antibody level in the blood. Mice were injected retroorbitally with 2microg indicated antibody, then sacrificed after 24 hours for blood and spleen collection. (B) Specific binding of 125I-labeled ESK1 to bone marrow, spleen, or thymus cells isolated from C57BL6/J or HLA-A2+ transgenic mice. Tissues were collected from 2 (C57) or 3 (HLA-A2+ transgenic) mice, then bound by 1microg/mL 125 I-labeled ESK1 either alone or after blocking with 50-fold excess unlabeled ESK1. Specific binding was determined, and #ESK1 bound per cell was calculated.

Data from an individual patient showing CD8 immune response to both native and heteroclitic peptides.

Purpose: Determine the 1-year progression-free survival (PFS) rate among patients with malignant pleural mesothelioma (MPM) receiving the WT1 peptide vaccine galinpepimut-S after multimodality therapy versus those receiving control adjuvants.Experimental Design: This double-blind, controlled, two center phase II trial randomized MPM patients after surgery and another treatment modality to galinpepimut-S with GM-CSF and Montanide or GM-CSF and Montanide alone. An improvement in 1-year PFS from 50% to 70% was the predefined efficacy threshold, and 78 patients total were planned. The study was not powered for comparison between the two arms.Results: Forty-one patients were randomized. Treatment-related adverse events were mild, self-limited, and not clinically significant. On the basis of a stringent prespecified futility analysis (futility = ≥10 of 20 patients on one arm experiencing progression < 1 year), the control arm closed early. The treatment arm was subsequently closed because of the resultant unblinding. The PFS rate at 1 year from beginning study treatment was 33% and 45% in the control and vaccine arms, respectively. Median PFS was 7.4 months versus 10.1 months and median OS was 18.3 months versus 22.8 months in the control and vaccine arms, respectively.Conclusions: The favorable safety profile was confirmed. PFS and OS were greater in those who received vaccine, but the trial was neither designed nor powered for comparison between the arms. On the basis of these promising results, the investigators are planning a larger randomized trial with greater statistical power to define the optimal use and benefit of galinpepimut-S in the treatment of MPM. Clin Cancer Res; 23(24); 7483–9. ©2017 AACR.

PDF file - 38KB, Histopathologic report from ESKM- or isotype control human IgG1-treated HLA-A*02:01+ transgenic mice.

Purpose: RMFPNAPYL (RMF), a Wilms' tumor gene 1 (WT1)–derived CD8 T-cell epitope presented by HLA-A*02:01, is a validated target for T-cell–based immunotherapy. We previously reported ESK1, a high avidity (Kd < 0.2 nmol/L), fully-human monoclonal antibody (mAb) specific for the WT1 RMF peptide/HLA-A*02:01 complex, which selectively bound and killed WT1+ and HLA-A*02:01+ leukemia and solid tumor cell lines.Experimental Design: We engineered a second-generation mAb, ESKM, to have enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) function due to altered Fc glycosylation. ESKM was compared with native ESK1 in binding assays, in vitro ADCC assays, and mesothelioma and leukemia therapeutic models and pharmacokinetic studies in mice. ESKM toxicity was assessed in HLA-A*02:01+ transgenic mice.Results: ESK antibodies mediated ADCC against hematopoietic and solid tumor cells at concentrations below 1 μg/mL, but ESKM was about 5- to 10-fold more potent in vitro against multiple cancer cell lines. ESKM was more potent in vivo against JMN mesothelioma, and effective against SET2 AML and fresh ALL xenografts. ESKM had a shortened half-life (4.9 days vs. 6.5 days), but an identical biodistribution pattern in C57BL/6J mice. At therapeutic doses of ESKM, there was no difference in half-life or biodistribution in HLA-A*02:01+ transgenic mice compared with the parent strain. Importantly, therapeutic doses of ESKM in these mice caused no depletion of total WBCs or hematopoetic stem cells, or pathologic tissue damage.Conclusions: The data provide proof of concept that an Fc-enhanced mAb can improve efficacy against a low-density, tumor-specific, peptide/MHC target, and support further development of this mAb against an important intracellular oncogenic protein. Clin Cancer Res; 20(15); 4036–46. ©2014 AACR.

PDF file - 183KB, (A) ESKM reduced individual tumor burden during the treatment course in 3 of 5 mice. Alternate representation of data in Figure 2A, where signal for each mouse is normalized to starting luminescence. (B) ESKM had no effect against JMN in a NOG mouse model. (C) Comparison of BV173 (Ph+ ALL) and SET2 (AML) cell lines in NSG mice.

PDF file - 142KB, Human PBMCs were separated into effector cell populations and tested for in vitro ADCC activity against BV173 cells, mediated by ESKM or isotype control mAb. Only fractionated NK cells showed activity relative to isotype control antibody. % Lysis is calculated based on spontaneous release (target cells, no effectors or mAb) as 0% and maximal release (target cells + SDS) as 100%.

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.

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. We have developed a high-throughput genetic platform (termed “PresentER”) that encodes MHC-I peptide minigenes for functional immunologic assays and determines the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this article, we demonstrated that PresentER could be used to identify the on-and-off targets of T cells and TCR-mimic (TCRm) antibodies using in vitro coculture assays or binding assays. We found dozens of MHC-I ligands that were cross-reactive with two TCRm antibodies and two native TCRs and that were not easily predictable by other methods.

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 in vitro and in vivo, 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. ALK inhibition additionally decreased PD-L1 levels by 75%. Therefore, these oncogenes may enhance cancer formation by allowing tumors to evade the immune system by downregulating HLA expression. Altogether, RET and ALK inhibitors could enhance T-cell–based immunotherapies by upregulating HLA, decreasing checkpoint blockade ligands, and revealing new, immunogenic, cancer-associated antigens.

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. One of the mAbs, 3F8, was able to specifically mediate T cell- redirected cytotoxicity, in a bispecific T cell engager (BiTE) form. While further studies are required to assess the therapeutic potential of this approach, the study provided the proof of concept that TCRm mAb could be a therapeutic strategy for HPV-induced human cancers.

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 pointsCombination therapy of interferon gamma with epigenetic regulators leads to large increases in the immunopeptidome of DLBCL.HLA ligands from proteins RGS13 and E2F8 may provide DLBCL-specific targets for immunotherapy.

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. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01-restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.

PurposeWilms’ tumor 1 (WT1) is overexpressed in several cancers, and WT1 expression levels are associated with poor prognosis. As a host protein that functions as an oncogene, it represents an important immunotherapeutic target. This study evaluated WT1 expression in Kaposi sarcoma (KS) tumors to assess whether immunotherapy targeting WT1 is a potential therapeutic approach for KS. We also investigated the role of the causal agent of KS, Kaposi sarcoma herpesvirus (KSHV/HHV-8) in regulating WT1 expression.Experimental designImmunohistochemistry for WT1, KSHV, and B and T cells subsets, followed by image analysis, was performed in 363 KS tumor biopsies. Expression of KSHV vFLIP was evaluated by immunofluorescence. Primary endothelial cell cultures and cell lines were infected with KSHV in vitro, or transduced with an inducible vFLIP vector and induced with doxycycline, and then assessed for WT1 expression. Binding of ESK-1, a T cell receptor mimic therapeutic antibody that recognizes WT1 peptides presented on MHC HLA-A0201, was assessed using flow cytometry.ResultsWe report overexpression of WT1 in KS tumors, which was associated with increased with increasing histopathologic stage and the proportion of KSHV-infected cells. Areas with high WT1 expression showed sparse T cell infiltrates. KSHV infection in vitro resulted in WT1 upregulation, mediated by the viral protein vFLIP, which resulted in stronger binding of ESK1.ConclusionsKS lesions express high levels of WT1, a process regulated by the KSHV-encoded vFLIP. These findings suggest that immunotherapy directed against WT1 may represent a therapeutic approach for this cancer.Translational RelevanceKaposi sarcoma (KS) is a vascular neoplasm caused by the Kaposi sarcoma herpesvirus (KSHV/HHV-8). People living with HIV are not only at a significantly higher risk of developing KS, but also often have a more aggressive clinical course. Although antiretroviral therapy may cause regression of HIV-associated KS lesions, advanced cases of KS also require chemotherapy, which is rarely curative. Wilms’ tumor 1 (WT1) has been reported to be overexpressed in various cancers, functioning as an oncogene and associated with a poor prognosis. WT1 is also an important immunotherapeutic target, with several WT1-directed therapies showing promising results in early clinical trials for leukemias and solid tumors. Here we report high expression of WT1 in KS, especially in higher histological stages. Our findings provide pre-clinical evidence that supports conducting anti-WT1 immunotherapy trials in KS, and evaluating WT1 expression as a potential biomarker to identify individuals most likely to benefit.

Context Target identification for CAR-T cell therapies, especially shared targets among different malignancies, 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 TCR-mimic antibodies. Objective and Design We hypothesized that mass spectrometry (MS)-based analysis of the presented HLA ligands of multiple cancer cell lines can be utilized to identify a shared, tumor-associated HLA ligand, which could then be targeted by TCR-mimic CAR-T cells specific for this particular HLA ligand. Results MS of HLA ligands from eight hematological and non-hematological cancer cell lines identified a shared, non-immunogenic, HLA-A*02 restricted ligand (ALNEQIARL) derived from the kinetochore-associated NDC80 gene, which was later identified in >90% (20/22) of all A*02-positive cell lines tested. A TCR-mimic scFv was prepared against this epitope, termed “NDC80-C.” CAR-T cells transduced with the NDC80-C, directed against the ALNEQIARL:HLA-A*02 complex on cancer cells, demonstrated high sensitivity and specificity for recognizing and killing multiple cancer types with a high preference for hematological malignancies (e.g., AML, ALL, DLBCL, and ALCL). In contrast, healthy leukocytes, activated B and T-cells, or hematopoietic stem cells from A*02-positive and -negative donors were not lysed. NDC80-C CAR-T cells suppressed colony formation of primary AML cells but not of healthy stem cells. Additionally, NDC80-C CAR-T cells were efficacious in mouse models against human mesothelioma and leukemia. Conclusions Our study demonstrates how MS can inform the design of tumor-agnostic TCR-mimic therapeutic platforms that target structures that are currently not druggable by small molecules, conventional CAR-T cells, T-cells, or antibodies. This strategy lays the groundwork for a potential antibody platform therapy or CAR-T cell with efficacy against highly proliferative A*02-positive cancer cells, independent of the respective cancer type.

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. In contrast, all six peptides failed to induce autologous peptide-specific response by T cells in peripheral blood or tumor-infiltrating lymphocytes from ascites of the patient. Surprisingly, T cell responses against a low-affinity p53-mutant Y220C epitope were consistently detected in the patient with either unprimed or in vitro peptide-stimulated T cells even though the patient's primary tumor did not bear this mutation. Our results demonstrated that tumor heterogeneity and distinct immune microenvironments within a patient should be taken into consideration for identification of immunogenic neoantigens. T cell responses to a driver gene-derived p53 Y220C mutation in ovarian cancer warrant further study.

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.

Purpose: Determine the 1-year progression-free survival (PFS) rate among patients with malignant pleural mesothelioma (MPM) receiving the WT1 peptide vaccine galinpepimut-S after multimodality therapy versus those receiving control adjuvants. Experimental Design: This double-blind, controlled, two center phase II trial randomized MPM patients after surgery and another treatment modality to galinpepimut-S with GM-CSF and Montanide or GM-CSF and Montanide alone. An improvement in 1-year PFS from 50% to 70% was the predefined efficacy threshold, and 78 patients total were planned. The study was not powered for comparison between the two arms. Results: Forty-one patients were randomized. Treatment-related adverse events were mild, self-limited, and not clinically significant. On the basis of a stringent prespecified futility analysis (futility = ≥10 of 20 patients on one arm experiencing progression < 1 year), the control arm closed early. The treatment arm was subsequently closed because of the resultant unblinding. The PFS rate at 1 year from beginning study treatment was 33% and 45% in the control and vaccine arms, respectively. Median PFS was 7.4 months versus 10.1 months and median OS was 18.3 months versus 22.8 months in the control and vaccine arms, respectively. Conclusions: The favorable safety profile was confirmed. PFS and OS were greater in those who received vaccine, but the trial was neither designed nor powered for comparison between the arms. On the basis of these promising results, the investigators are planning a larger randomized trial with greater statistical power to define the optimal use and benefit of galinpepimut-S in the treatment of MPM. Clin Cancer Res; 23(24); 7483–9. ©2017 AACR.

Chimeric antigen receptor (CAR) T cells represent a novel class of FDA-approved drugs with high efficacy against refractory B cell derived malignancies and potentially other cancer types. However, target selection for CAR T cell therapy remains challenging as cell surface proteins are not cancer-specific and therefore often not adaptable for CAR T cell therapy. In contrast, many intracellular proteins can be highly tumor specific and are targetable after proteasomal degradation and presentation on human leukocyte antigen (HLA) complexes recognized by T cell receptor mimic antibodies. This class of antibodies recognizes peptide:HLA complexes with a similar mode of recognition as a TCR, but with the clinical versatility and applicability of an antibody. To identify a tumor specific target that is presented as a peptide in conjunction with the highly prevalent HLA allele A*02:01, we immunopurified peptide:HLA complexes from various cancer cell lines of different origins, separated HLA ligands from complexes and identified their peptide sequences via mass spectrometry. Network analysis of the resulting HLA ligand datasets identified shared biological processes among the tumor cell lines that were not present in network analyses of published datasets of healthy human tissue HLA ligandomes. Through this filtering process several potential targets were identified and an HLA ligand derived from kinetochore NDC80 protein homolog (NDC80) was selected as a target. The NDC80 derived peptide was detected in over 90% of the A*02 positive cell lines tested and never reported to be present in HLA ligand datasets of healthy human tissues. Furthermore, NDC80 has been shown to be differentially expressed in malignant compared to adjacent non-malignant tissues and is associated with poor prognosis in many cancer types. After utilizing E-ALPHA®phage library screening, one clone (NDC80-L1) was selected as the lead TCR mimic antibody. Overall, NDC80-L1 showed high specificity for the target HLA:peptide complex in both antibody and CAR T cell format in vitro and demonstrated binding primarily to the central region of the HLA ligand as determined by alanine screening assays. The exquisite specificity of NDC80-L1 was further illustrated by NDC80 knockdown experiments as well as successful immunopurification of the target peptide together with no relevant off-targets from BV173 ALL cells in mass spectrometry assays. Given the high specificity, sensitivity was assessed primarily in a potent CAR T cell format: Multiple tumor cell lines of different origin (e.g. ALL, AML, lymphoma, melanoma, mesothelioma, pancreatic and thyroid cancer) were successfully killed in vitro by NDC80-L1 CAR T cells, but no toxicity towards A*02:01 positive CAR T cells, healthy PBMCs or NDC80 target negative cell lines was observed. Interestingly, NDC80-L1 CAR T cells demonstrated highest efficacy in hematological malignancies most likely correlating with elevated expression of antigen presentation machinery and rapid cell division which leads to strong surface expression of NDC80 peptides. In summary, CAR T cells directed against peptide/HLA-A*02 derived from the NDC80 protein effectively kill multiple cancer cell lines in vitro without evidence of relevant off-target killing. However, the improved killing especially against ALL, AML and lymphomas highlights the potential of these CAR T cells to preferentially eliminate cancer cells with high proliferative capacity. Future in vivo studies with CAR T cell and antibody format will further investigate this TCR mimic antibody's potential as a tumor-agnostic therapeutic agent. Disclosures Klatt: MSKCC/EUREKA: Patents & Royalties: MSKCC AND EUREKA THERAPUETICS HAVE FILED A PATENT FOR THIS ANTIBODY/SCFV. Yang:Eureka Therapuetics: Current Employment, Current equity holder in private company, Patents & Royalties: MSKCC and Eureka have filed patent for this TCRm and ScvF. Liu:Eureka Therapue: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Dao:Eureka Therapeutics: Consultancy. Liu:Eureka Therapeutics: Current Employment, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm. Scheinberg:Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents & Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Arvenas: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Oncopep: Consultancy; Pfizer: Consultancy, Current equity holder in private company; Lantheus: Current equity holder in private company; Enscyse: Current equity holder in private company.

CD19-directed chimeric antigen receptor (CAR) T cells are clinically effective in a limited set of leukemia patients. However, CAR T-cell therapy thus far has been largely restricted to targeting extracellular tumor-associated antigens (TAA). Herein, we report a T-cell receptor-mimic (TCRm) CAR, termed WT1-28z, that is reactive to a peptide portion of the intracellular onco-protein Wilms Tumor 1(WT1), as it is expressed on the surface of the tumor cell in the context of HLA-A*02:01. T cells modified to express WT1-28z specifically targeted and lysed HLA-A*02:01+ WT1+ tumors and enhanced survival of mice engrafted with HLA-A*02:01+, WT1+ leukemia or ovarian tumors. This in vivo functional validation of TCRm CAR T cells provides the proof-of-concept necessary to expand the range of TAA that can be effectively targeted for immunotherapy to include attractive intracellular targets, and may hold great potential to expand on the success of CAR T-cell therapy.

Objectives Adoptive transfer of autologous Tcells specifically targeting neoepitopes derived from tumor mutations has achieved responses in melanoma and other cancers. We sought to identify ovarian cancer neoepitopes. Methods We collected tumor, blood and ascites specimens from ovarian cancer patients. We combined mass spectrometry analysis of MHC class1 peptidomes with parallel sequencing of whole exome and RNA of cancer cells, to identify neoepitopes. Missense mutations were identified by whole exome sequencing and epitopes confirmed by mass spectrometry from ascites. We selected peptides predicted to have high binding affinity to HLA-A*02:01, for in vitro Tcell priming. We first validated the immunogenicity of these peptides for priming Tcells with synthesized peptides in healthy HLA-A*02:01+ donors. Results 32 missense mutations were identified in patient’s ascites. 7 epitopes were confirmed by mass spectroscopy; 5 were predicted to have high binding affinity to HLA-A*02:01. 3/5 epitopes induced peptide-specific Tcell responses not cross-reactive with native sequences. However, these 3 peptides failed to induce autologous peptide-specific Tcell response in TILs to patient‘s ascites, nor to autologous tumor cells, suggesting immunosuppression. Interestingly, when autologous tumor cells were pre-treated with IFN-gamma, Tcell responses against tumor cells were observed. Conclusions We demonstrated effective T cell responses against tumor neoantigens, is not only dependent on stimulation of Tcells, but also on efficient epitope processing and presentation on cell surface. While efforts have been focused on activation of Tcell responses, such as immune checkpoint blockade, our study suggests that strategies to modulate tumor cells to efficiently present neoepitopes should be considered for successful immunotherapy against neoantigens.

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. We have generated a T cell receptor mimic antibody, “Foxp3-#32,” recognizing a Foxp3-derived epitope in the context of HLA-A*02:01. The mAb Foxp3-#32 selectively recognizes CD4 + CD25 + CD127low and Foxp3 + Tregs also expressing HLA-A*02:01 and depletes these cells via antibody-mediated cellular cytotoxicity. Foxp3-#32 mAb depleted Tregs in xenografts of PBMCs from a healthy donor and ascites fluid from a cancer patient. A TCRm mAb targeting intracellular Foxp3 epitope represents an approach to deplete Tregs.