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Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo–expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53–reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02–restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers. See related Spotlight by Klebanoff, p. 919

Supplementary Figure from Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor–Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors

Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo–expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53–reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02–restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers.See related Spotlight by Klebanoff, p. 919

Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen-reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T-cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic nonsynonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell-based immunotherapies for patients bearing these cancers.Significance:TILs cultured from 62 of 75 (83%) patients with gastrointestinal cancers recognized neoantigens encoded by 1.6% of somatic mutations expressed by autologous tumor cells, and 99% of the neoantigenic determinants appeared to be unique and not shared between patients.This article is highlighted in the In This Issue feature, p. 983

Gating strategy, Shannon entropy, PD-1 expression, additional co-culture data, primers sequence, Frequency of the top 10 CD8+PD-1+ TCRB in bulk unsorted TIL and sorted TIL subsets, Retrospective analysis of pairing the most frequent TCRB in the CD8+PD-1+ TIL subset with the most frequent TCRA from the same subset, Method of identification of TCR pairs, TCR expression on genetically engineered PBL.

Supplementary Figures 1-9

Supplementary Materials and Methods

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8+PD-1+ T-cell subset, matching of TCRA–TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734–43. ©2016 AACR.

Clinical details (S1) and neoepitope details (S2).

Purpose: This was a study prospectively evaluating intratumoral T-cell responses to autologous somatic mutated neoepitopes expressed by human metastatic ovarian cancers.Patients and Methods: Tumor-infiltrating lymphocytes (TIL) were expanded from resected ovarian cancer metastases, which were analyzed by whole-exome and transcriptome sequencing to identify autologous somatic mutations. All mutated neoepitopes, independent of prediction algorithms, were expressed in autologous antigen-presenting cells and then cocultured with TIL fragment cultures. Secretion of IFNγ or upregulation of 41BB indicated a T-cell response.Results: Seven women with metastatic ovarian cancer were evaluated, and 5 patients had clear, dominant T-cell responses to mutated neoantigens, which were corroborated by comparison with the wild-type sequence, identification of the minimal epitope, human leukocyte antigen (HLA) restriction element(s), and neoantigen-specific T-cell receptor(s). Mutated neoantigens were restricted by HLA-B, -C, -DP, -DQ, and/or -DR alleles and appeared to principally arise from random, somatic mutations unique to each patient. We established that TP53 “hotspot” mutations (c.659A>G; p.Y220C and c.733G>A; p.G245S) expressed by two different patients' tumors were both immunogenic in the context of HLA-DRB3*02:02.Conclusions: Mutation-reactive T cells infiltrated ovarian cancer metastases at sufficient frequencies to warrant their investigation as adoptive cell therapy. In addition, transfer of TP53 “hotspot” mutation-reactive T-cell receptors into peripheral blood T cells could be evaluated as a gene therapy for a diverse range of tumor histologies. Clin Cancer Res; 24(22); 5562–73. ©2018 AACR.See related commentary by McNeish, p. 5493

Additional materials and methods. Figure S1. CD4+ T cells responded to USP9X-Y2009C minimal epitope. Figure S2. CD4+ T cells recognized INPP5K-L272V neoantigen. Figure S3. T cells transduced with TCRs recognize Histone H1.5-A71D neoantigen. Figure S4. CD4+ T cells responded to RAPTOR-D654G minimal neoepitope. Figure S5. The p53-G245S minimal neoepitope was mapped to the N-terminus of the 25 amino acid neoantigen. Figure S6. Peptide parsing from Patient 7 demonstrated CTAGE5-E576V, p53-Y220C and HUWE1-F4353S responses.

Purpose: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved.Experimental Design: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing.Results: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1negVδ2neg subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1negVδ2neg>Vδ2) of survival of mice with ovarian cancer xenografts.Conclusions: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans. Clin Cancer Res; 20(22); 5708–19. ©2014 AACR.

PDF file - 1405KB, Supplementary Materials and Methods. Supplementary Figures - (1) Tumor cell line culture conditions and media formulations (2) Co-culture conditions for gamma/delta T cells and designer aAPC (3) Intracellular cytokine production, Luminex, and neutralizing antibody cytolysis assays (4) Lentivirus packaging and transduction of CAOV3 cells Contents of the Supplemental Data include figures: (1) An example of the gating strategy for gamma/delta T-cell analyses (2) Schematic of DNA plasmid pLVU3G-effLuc-T2A-mKateS158A used to co-express enhanced firefly luciferase (effLuc) and mKate (3) Expression of activation markers CD38 and CD95 on propagated gamma/delta T cells (4) aAPC developed for co-culture with gamma/delta T cells to determine the impact of introduced co-stimulatory molecules (5) Expansion of UCB-derived gamma/delta T cells on aAPC with IL-2 and IL-21 (6) Surface expression of TCRdelta1 and TCRdelta2 chains on gamma/delta T cells derived from PBMC prior to propagation (7) Abundance of Vdelta and Vgamma mRNA species in gamma/delta T cells prior to ex vivo numeric expansion (8) Surface expression of TCRdelta1 and TCRdelta2 chains on gamma/delta T cells derived from UCB and propagated on aAPC with IL-2 and IL-21 (9) mRNA expression of shared Valpha/Vdelta alleles in gamma/delta T cells separated and then propagated on aAPC, IL-2, and IL-21 (10) Abundance of mRNA species coding for Vgamma chains in gamma/delta T-cell subsets (11) In vitro lysis of tumor cell line panel by polyclonal gamma/delta T cells (12) Specific lysis of hematological and solid tumor cells by Vdelta T-cell subsets (13) Immunophenotypes associated with PBMC-derived Vdelta T-cell subsets expanded on aAPC/IL-2/IL-21. Supplemental Table 1 - details the antibodies used in this study, where they were purchased, and their hybridoma clone.

Purpose:The purpose of this study was to evaluate antigen experienced T cells in peripheral blood lymphocytes (PBL) for responses to p53 neoantigens.Experimental Design:PBLs from patients with a mutated TP53 tumor were sorted for antigen-experienced T cells and in vitro stimulation (IVS) was performed with p53 neoantigens. The IVS cultures were stimulated with antigen-presenting cells expressing p53 neoantigens, enriched for 41BB/OX40 and grown with rapid expansion protocol.Results:T-cell responses were not observed in the PBLs of 4 patients who did not have tumor-infiltrating lymphocyte (TIL) responses to mutated TP53. In contrast, 5 patients with TIL responses to mutated TP53 also had similar T-cell responses in their PBLs, indicating that the PBLs and TILs were congruent in p53 neoantigen reactivity. CD4+ and CD8+ T cells were specific for p53R175H, p53Y220C, or p53R248W neoantigens, including a 78% reactive T-cell culture against p53R175H and HLA-A*02:01. Tracking TCRB clonotypes (clonality, top ranked, and TP53 mutation-specific) supported the enrichment of p53 neoantigen–reactive T cells from PBLs. The same T-cell receptor (TCR) from the TIL was found in the IVS cultures in three cases and multiple unique TCRs were found in another patient. TP53 mutation–specific T cells also recognized tumor cell lines bearing the appropriate human leukocyte antigen restriction element and TP53 mutation, indicating these T cells could recognize processed and presented p53 neoantigens.Conclusions:PBL was a noninvasive source of T cells targeting TP53 mutations for cell therapy and can provide a window into intratumoral p53 neoantigen immune responses.See related commentary by Olivera et al., p. 1203

Table 1: Antibodies used in the study. Table 2: Normalized initial screening data from PBL with a T cell response to mutated TP53. Table 3: TCRB tracking of p53 neoantigen-reactive cells before and after IVS, co-culture, 41BB/OX40 sorting and REP.

Purpose: The purpose of this study was to evaluate antigen experienced T cells in peripheral blood lymphocytes (PBL) for responses to p53 neoantigens. Experimental Design: PBLs from patients with a mutated TP53 tumor were sorted for antigen-experienced T cells and in vitro stimulation (IVS) was performed with p53 neoantigens. The IVS cultures were stimulated with antigen-presenting cells expressing p53 neoantigens, enriched for 41BB/OX40 and grown with rapid expansion protocol. Results: T-cell responses were not observed in the PBLs of 4 patients who did not have tumor-infiltrating lymphocyte (TIL) responses to mutated TP53. In contrast, 5 patients with TIL responses to mutated TP53 also had similar T-cell responses in their PBLs, indicating that the PBLs and TILs were congruent in p53 neoantigen reactivity. CD4+ and CD8+ T cells were specific for p53R175H, p53Y220C, or p53R248W neoantigens, including a 78% reactive T-cell culture against p53R175H and HLA-A*02:01. Tracking TCRB clonotypes (clonality, top ranked, and TP53 mutation-specific) supported the enrichment of p53 neoantigen–reactive T cells from PBLs. The same T-cell receptor (TCR) from the TIL was found in the IVS cultures in three cases and multiple unique TCRs were found in another patient. TP53 mutation–specific T cells also recognized tumor cell lines bearing the appropriate human leukocyte antigen restriction element and TP53 mutation, indicating these T cells could recognize processed and presented p53 neoantigens. Conclusions: PBL was a noninvasive source of T cells targeting TP53 mutations for cell therapy and can provide a window into intratumoral p53 neoantigen immune responses. See related commentary by Olivera et al., p. 1203

Adoptive T cell therapies (ACT) have been curative for a limited number of cancer patients. The sensitization of cancer cells to T cell killing may expand the benefit of these therapies for more patients. To this end, we use a three-step approach to identify cancer genes that disfavor T cell immunity. First, we profile gene transcripts upregulated by cancer under selection pressure from T cell killing. Second, we identify potential tumor gene targets and pathways that disfavor T cell killing using signaling pathway activation libraries and genome-wide loss-of-function CRISPR-Cas9 screens. Finally, we implement pharmacological perturbation screens to validate these targets and identify BIRC2, ITGAV, DNPEP, BCL2, and ERRα as potential ACT-drug combination candidates. Here, we establish that BIRC2 limits antigen presentation and T cell recognition of tumor cells by suppressing IRF1 activity and provide evidence that BIRC2 inhibition in combination with ACT is an effective strategy to increase efficacy.

BackgroundEGFR, KRAS and TP53 have frequent somatic hotspot mutations giving rise to biologically relevant amino acid substitutions in EGFR, KRAS and p53 proteins, respectively, that can be processed and presented on the cell surface by human leukocyte antigen (HLA) molecules as neoantigens to T cells through their T-cell receptor (TCR). These mutations are critical for the cancer cell and are absent in normal tissue; thus, these shared neoantigens are attractive and likely safe targets. Given the complexity of different neoantigen/HLA combinations needed to effectively target a large patient population, a TCR library approach is warranted and can be used ”off-the-shelf” for any patient with matching somatic hotspot mutation and HLA restriction. Sleeping Beauty transposition is the most advanced non-viral gene transfer technology for TCR-T cells and is appealing for TCR libraries given its low cost, speed, and flexibility.MethodsIn this study, Sleeping Beauty transposons were constructed with TCRs targeting EGFR, KRAS and p53 neoantigens restricted by either or both HLA Class-I and HLA Class-II molecules. Donor T cells from peripheral blood were co-electroporated with TCR transposon and Sleeping Beauty transposase and grown in vitro to clinical scale quantities (>109 TCR-T cells) with high expression (>60%) of the introduced neoantigen-specific TCRs.ResultsThe specificity of TCRs to neoantigens was confirmed in TCR-T cell co-cultures with antigen-presenting cells pulsed with peptides, which demonstrated interferon-γ secretion and/or up-regulation of 41BB on the TCR-T cell surface in response to the neoantigen with high avidity (Sleeping Beauty transposition, and TCR-T cells will be adoptively transferred for the treatment of bile duct, colon, lung, pancreas and gynecological cancers.ConclusionsZiopharm’s library TCR-T cell program has the potential to result in safe, durable, objective clinical regressions of cancer at a commercial scale.

Adoptive cell therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from rare mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. Here, we describe a library of T cell receptors (TCRs) that can target TP53 mutations shared among 7.3% of patients with solid cancers. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner both in vitro and in vivo. Patients with chemorefractory epithelial cancers treated with ex vivo-expanded autologous tumor infiltrating lymphocytes (TILs) naturally reactive with mutant p53 experienced limited clinical responses (2 PRs/12 patients), and we detected low frequencies, exhausted phenotypes, and poor persistence of the infused mutant p53-reactive TILs. Alternatively, we treated one patient with a chemorefractory breast cancer with ACT by transducing autologous peripheral blood lymphocytes with an HLA-A*02-restricted anti-p53R175H TCR. The infused cells exhibited an improved immunophenotype and prolonged persistence compared to the TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these data demonstrate the feasibility of off-the-shelf TCR-engineered cell therapies targeting shared p53 neoantigens to treat human cancers.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) fails to consistently elicit tumor rejection. Manipulation of intrinsic factors that inhibit T cell effector function and neoantigen recognition may therefore improve TIL therapy outcomes. We previously identified the cytokine-induced SH2 protein (CISH) as a key regulator of T cell functional avidity in mice. Here, we investigate the mechanistic role of CISH in regulating human T cell effector function in solid tumors and demonstrate that CRISPR/Cas9 disruption of CISH enhances TIL neoantigen recognition and response to checkpoint blockade.Single-cell gene expression profiling was used to identify a negative correlation between high CISH expression and TIL activation in patient-derived TIL. A GMP-compliant CRISPR/Cas9 gene editing process was developed to assess the impact of CISH disruption on the molecular and functional phenotype of human peripheral blood T cells and TIL. Tumor-specific T cells with disrupted Cish function were adoptively transferred into tumor-bearing mice and evaluated for efficacy with or without checkpoint blockade.CISH expression was associated with T cell dysfunction. CISH deletion using CRISPR/Cas9 resulted in hyper-activation and improved functional avidity against tumor-derived neoantigens without perturbing T cell maturation. Cish knockout resulted in increased susceptibility to checkpoint blockade in vivo.CISH negatively regulates human T cell effector function, and its genetic disruption offers a novel avenue to improve the therapeutic efficacy of adoptive TIL therapy.This study was funded by Intima Bioscience, U.S. and in part through the Intramural program CCR at the National Cancer Institute.

Background Neoantigen-specific T cells isolated from tumors have shown promise clinically but fail to consistently elicit durable tumor regression. Expression of the intracellular checkpoint CISH is elevated in human tumor infiltrating lymphocytes (TIL) and has been shown to inhibit neoantigen reactivity in murine TIL. Methods To explore CISH function in human T cells we developed a CRISPR/Cas9-based strategy to knockout (KO) CISH in human T cells with high-efficiency (>90%) and without detectable off-target editing. Results CISH KO in peripheral blood T cells enhanced proliferation, cytokine polyfunctionality, and cytotoxicity in vitro. To determine if CISH KO similarly enhances TIL function, we developed a clinical-scale, GMP-compliant manufacturing process for CISH disruption in primary human TIL. In process validation runs we achieved CISH KO efficiencies >90% without detectable off-target editing while maintaining high viability and expansion. Compared to WT controls, CISH KO in patient-derived TIL demonstrated increased proliferation, T cell receptor (TCR) avidity, neoantigen recognition, and unmasked reactivity to common p53 mutations. Hyperactivation in CISH KO TIL did not increase differentiation, suggesting that CISH KO may uncouple activation and differentiation pathways. Single cell profiling identifies a pattern of CISH expression inverse to key regulators of activation, and CISH KO in human TIL increases PD1 expression. Adoptive transfer of Cish KO T cells synergistically combines with PD1 inhibition resulting in durable tumor regression in mice, highlighting orthogonal dual cell surface and intracellular checkpoint inhibition as a novel combinatorial approach for T cell immunotherapy. Conclusions These pre-clinical data offer new insight into neoantigen recognition and serve as the basis for a recently initiated human clinical trial at the University of Minnesota (NCT04426669) evaluating inhibition of the novel intracellular immune checkpoint CISH in a CRISPR-engineered, neoantigen-specific T cell therapy for solid tumors. Updates from the clinical trial will be highlighted. Trial Registration NCT04426669

Engineered T cells expressing tumor-specific T cell receptors (TCRs) are emerging as a mode of personalized cancer immunotherapy that requires identification of TCRs against the products of known driver mutations and novel mutations in a timely fashion. We present a non-viral and non-next generation sequencing (NGS) platform for rapid, and efficient neoantigen-specific TCR identification and evaluation that doesn’t require the use of recombinant cloning techniques. The platform includes an innovative method of TCR alpha (TCRα) detection using Sanger sequencing, TCR pairings and the use of TCRα/β gene fragments for putative TCR evaluation. Using patients’ samples, we validated and compared our new methods head-to-head with conventional approaches used for TCR discovery. Development of a unique de-multiplexing method for identification of TCRα, adaptation of synthetic TCRs for gene transfer, and a reliable reporter system significantly shortens TCR discovery time over conventional methods and increases throughput to facilitate testing prospective personalized TCRs for adoptive cell therapy (ACT).

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

T cells targeting shared oncogenic mutations can induce durable tumor regression in epithelial cancer patients. Such T cells can be detected in tumor infiltrating lymphocytes, but whether such cells can be detected in the peripheral blood of patients with the common metastatic epithelial cancer patients is unknown. Using a highly sensitive in vitro stimulation and cell enrichment of peripheral memory T cells from six metastatic cancer patients, we identified and isolated CD4+, and CD8+ memory T cells targeting the mutated KRASG12D and KRASG12V variants, respectively, in three patients. In an additional two metastatic colon cancer patients, we detected CD8+ neoantigen-specific cells targeting the mutated SMAD5 and MUC4 proteins. Therefore, memory T cells targeting unique as well as shared somatic mutations can be detected in the peripheral blood of epithelial cancer patients and can potentially be used for the development of effective personalized T cell-based cancer immunotherapy across multiple patients., Adoptive cell therapy (ACT) using neoantigen-specific T cells can lead to tumor regression. Here the authors use an in vitro stimulation approach to isolate tumor specific memory T cells from peripheral blood of metastatic epithelial cancer patients targeting unique as well as shared mutations in the KRAS oncogene.

Purpose: This was a study prospectively evaluating intratumoral T-cell responses to autologous somatic mutated neoepitopes expressed by human metastatic ovarian cancers. Patients and Methods: Tumor-infiltrating lymphocytes (TIL) were expanded from resected ovarian cancer metastases, which were analyzed by whole-exome and transcriptome sequencing to identify autologous somatic mutations. All mutated neoepitopes, independent of prediction algorithms, were expressed in autologous antigen-presenting cells and then cocultured with TIL fragment cultures. Secretion of IFNγ or upregulation of 41BB indicated a T-cell response. Results: Seven women with metastatic ovarian cancer were evaluated, and 5 patients had clear, dominant T-cell responses to mutated neoantigens, which were corroborated by comparison with the wild-type sequence, identification of the minimal epitope, human leukocyte antigen (HLA) restriction element(s), and neoantigen-specific T-cell receptor(s). Mutated neoantigens were restricted by HLA-B, -C, -DP, -DQ, and/or -DR alleles and appeared to principally arise from random, somatic mutations unique to each patient. We established that TP53 “hotspot” mutations (c.659A>G; p.Y220C and c.733G>A; p.G245S) expressed by two different patients' tumors were both immunogenic in the context of HLA-DRB3*02:02. Conclusions: Mutation-reactive T cells infiltrated ovarian cancer metastases at sufficient frequencies to warrant their investigation as adoptive cell therapy. In addition, transfer of TP53 “hotspot” mutation-reactive T-cell receptors into peripheral blood T cells could be evaluated as a gene therapy for a diverse range of tumor histologies. Clin Cancer Res; 24(22); 5562–73. ©2018 AACR. See related commentary by McNeish, p. 5493

While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

SummaryAll current highly effective anti-tumor immunotherapeutics depend on the activity of T cells, but tumor cells can escape immune recognition by several mechanisms including loss of function in antigen presentation and inflammatory response genes, expression of immunomodulatory proteins and an immunosuppressive tumor microenvironment. In contrast, the comprehensive identification of strategies that sensitize tumor cells to immunotherapy in vivo has remained challenging. Here, we combine a two-cell type (2CT) whole-genome CRISPR-Cas9 screen with dynamic transcriptional analysis (DTA) of tumor upon T cell encounter to identify a set of genes that tumor cells express as an active defense against T cell-mediated killing. We then employed small molecule and biologic screens designed to antagonize gene products employed by tumor cells to actively defend against T cell-mediated tumor destruction and found that the inhibition of BIRC2, ITGAV or DNPEP enhanced tumor cell destruction by T cells. Mechanistically, we found that BIRC2 promoted immunotherapy resistance through inhibiting non-canonical NF-κB signaling and limiting inflammatory chemokine production. These findings show the path forward to improving T cell-mediated tumor destruction in the clinic.

Mutations in critical genes for cell survival and proliferation, e.g., KRAS and TP53, are found as clonal events in multiple tumor types of unrelated people likely due to their importance to the malignant phenotype. T cells recognize products of mutated genes, termed neoantigens, because they are expressed in the tumor but not in the normal tissues; thus, neoantigens are foreign entities from an immunological perspective. T-cell receptors (TCRs) with specificity to the neoantigen in the context of human leukocyte antigen (HLA) on the tumor cell surface can be isolated from the neoantigen-reactive T cell and potentially used for genetically-modified adoptive immunotherapy for any patient with matching mutation and HLA. The purpose of this study was to evaluate the ability of the non-viral Sleeping Beauty transposon/transposase gene transfer system to re-direct the specificity of T cells towards p53 and KRAS neoantigens and to characterize the resultant engineered TCR-T cell populations for specificity and function. Genes encoding the alpha and beta chains of p53 or KRAS neoantigen-specific TCRs were linked by a 2A ribosomal slip site linker and cloned into the clinical Sleeping Beauty transposon. These transposons were co-electroporated into donor peripheral blood leukocytes (PBL) with a DNA plasmid encoding the SB11 transposase and expanded in vitro. Logarithmic proliferation was observed and resulted in large numbers of highly pure TCR-T cells (>80% by introduced TCR expression). The TCR-T cells upregulated 41BB on the T-cell surface and secreted interferon-γ in response to antigen presenting cells with the appropriate HLA molecule pulsed with KRAS or p53 neoantigen peptides but not the cognate wild type peptide, confirming re-directed specificity to mutated KRAS or TP53 genes, respectively. Similarly, TCR-engineered T cells demonstrated cytolysis of tumor cells expressing the neoantigen and appropriate HLA restriction, suggesting that adoptive transfer of these TCR-T cells could mediate anti-tumor responses. In all, we demonstrated that multiple TCRs with unique specificities targeting recurrent p53 and KRAS substitutions in frequent HLA haplotypes could be stably expressed using Sleeping Beauty transposition to re-direct peripheral blood T cells towards tumor cells. Translation of these TCR-T cells into adoptive immunotherapy could result in safe and effective treatments for any cancer patient with matching HLA and KRAS or TP53 hotspot mutations. Citation Format: Ana B. Korngold, Lin-Kin Yong, Ming Zhang, Ugochi Ibekwe, Lenka V. Hurton, Yaoyao Shi, Julissa Simmons, Matthew R. Collinson-Pautz, Eleanor De Groot, Laurence J. Cooper, Drew C. Deniger. Hotspot mutations in KRAS and TP53 targeted by TCR-T cells genetically modified with the Sleeping Beauty transposon/transposase system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1515.

Purpose: This pilot feasibility clinical trial evaluated the coadministration of vemurafenib, a small-molecule antagonist of BRAFV600 mutations, and tumor-infiltrating lymphocytes (TIL) for the treatment of metastatic melanoma. Experimental Design: A metastatic tumor was resected for growth of TILs, and patients were treated with vemurafenib for 2 weeks, followed by resection of a second lesion. Patients then received a nonmyeloablative preconditioning regimen, infusion of autologous TILs, and high-dose interleukin-2 administration. Vemurafenib was restarted at the time of TIL infusion and was continued for 2 years or until disease progression. Clinical responses were evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) 1.0. Metastases resected prior to and after 2 weeks of vemurafenib were compared using TCRB deep sequencing, immunohistochemistry, proliferation, and recognition of autologous tumor. Results: The treatment was well tolerated and had a safety profile similar to that of TIL or vemurafenib alone. Seven of 11 patients (64%) experienced an objective clinical response, and 2 patients (18%) had a complete response for 3 years (one response is ongoing at 46 months). Proliferation and viability of infusion bag TILs and peripheral blood T cells were inhibited in vitro by research-grade vemurafenib (PLX4032) when approaching the maximum serum concentration of vemurafenib. TCRB repertoire (clonotypes numbers, clonality, and frequency) did not significantly change between pre- and post-vemurafenib lesions. Recognition of autologous tumor by T cells was similar between TILs grown from pre- and post-vemurafenib metastases. Conclusions: Coadministration of vemurafenib and TILs was safe and feasible and generated objective clinical responses in this small pilot clinical trial. Clin Cancer Res; 23(2); 351–62. ©2016 AACR. See related commentary by Cogdill et al., p. 327

Adoptive transfer of T cells with engineered T-cell receptor (TCR) genes that target tumor-specific antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. To test this hypothesis, we developed a multistep strategy that involved TCRB deep sequencing of the CD8+PD-1+ T-cell subset, matching of TCRA–TCRB pairs by pairSEQ and single-cell RT-PCR, followed by testing of the TCRs for tumor-antigen specificity. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes, which included reactivity against neoantigens. These data show the feasibility of developing a rapid, personalized TCR-gene therapy approach that targets the unique set of antigens presented by the autologous tumor without the need to identify their immunologic reactivity. Cancer Immunol Res; 4(9); 734–43. ©2016 AACR.

Adoptive cell therapy (ACT) with T cells targeting neoantigens can mediate durable responses in patients with metastatic cancer. Cell therapies targeting common shared antigens for epithelial cancers are not yet broadly available. Here, we report the identification and characterization in one patient of T-cell receptors (TCRs) recognizing mutated p53 p.R175H, which is shared among a subset of patients with cancer. Tumor-infiltrating lymphocytes were screened for recognition of mutated neoantigens in a patient with metastatic colorectal cancer. HLA-A*0201–restricted recognition of mutated p53 p.R175H was identified, and the minimal peptide epitope was HMTEVVRHC. Reactive T cells were isolated by tetramer sorting, and three TCRs were identified. These TCRs mediated recognition of commercially available ovarian cancer, uterine carcinoma, and myeloma cell lines, as well as an NIH patient–derived esophageal adenocarcinoma line that endogenously expressed p53 p.R175H and HLA-A*0201. They also mediated recognition of p53 p.R175H+ colon, breast, and leukemia cell lines after transduction with a retrovirus encoding HLA-A*0201. This work demonstrates that common shared mutated epitopes such as those found in p53 can elicit immunogenic responses and that the application of ACT may be extended to patients with any cancer histology that expresses both HLA-A*0201 and the p53 p.R175H mutation.

CD4(+) regulatory T (T(reg)) cells have an essential function in maintaining self-tolerance; however, they may also play a detrimental role in antitumor immune responses. The presence of elevated frequencies of T(reg) cells in tumors correlates with disease progression and poor survival in patients with cancer. The antigen specificity of T(reg) cells that have expanded in the tumor microenvironment is poorly understood; answering this question may provide important insights for immunotherapeutic approaches. To address this, we used a novel combinatorial approach to characterizing the T cell receptor (TCR) profiles of intratumoral T(reg) cells from patients with metastatic melanoma, gastrointestinal, and ovarian cancers and elucidated their antigen specificities. The TCR repertoires of tumor-resident T(reg) cells were diverse yet displayed significant overlap with circulating T(reg) cells but not with conventional T cells in tumor or blood. TCRs isolated from T(reg) cells displayed specific reactivity against autologous tumors and mutated neoantigens, suggesting that intratumoral T(reg) cells act in a tumor antigen–selective manner leading to their activation and clonal expansion in the tumor microenvironment. Tumor antigen–specific T(reg)-derived TCRs resided in the tumor and in the circulation, suggesting that both T(reg) cell compartments may serve as a source for tumor-specific TCRs. These findings provide insights into the TCR specificity of tumor-infiltrating human T(reg) cells that may have potential implications for cancer immunotherapy.

Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen-reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T-cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic nonsynonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell-based immunotherapies for patients bearing these cancers. Significance: TILs cultured from 62 of 75 (83%) patients with gastrointestinal cancers recognized neoantigens encoded by 1.6% of somatic mutations expressed by autologous tumor cells, and 99% of the neoantigenic determinants appeared to be unique and not shared between patients. This article is highlighted in the In This Issue feature, p. 983

Adoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of neoantigen-reactive TILs could be enhanced by enriching T cells that express PD-1 and/or T cell activation markers followed by microwell culturing to avoid overgrowth of nonreactive T cells. In 6 patients with metastatic epithelial cancer, this method led to the detection of CD4+ and CD8+ T cells targeting 18 and 1 neoantigens, respectively, compared with 6 and 2 neoantigens recognized by CD4+ and CD8+ T cells, respectively, when using our standard TIL fragment screening approach. In 2 patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of 5 neoantigen-reactive T cell receptors (TCRs) against 5 different mutations from one patient and a highly potent MHC class II-restricted KRASG12V-reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated 3 MHC class II-restricted TCRs targeting the TP53G245S hot-spot mutation. In conclusion, this approach provides a highly sensitive platform to isolate clinically relevant neoantigen-reactive T cells or their TCRs for cancer treatment.

Purpose: To activate and propagate populations of γδ T cells expressing polyclonal repertoire of γ and δ T-cell receptor (TCR) chains for adoptive immunotherapy of cancer, which has yet to be achieved. Experimental Design: Clinical-grade artificial antigen-presenting cells (aAPC) derived from K562 tumor cells were used as irradiated feeders to activate and expand human γδ T cells to clinical scale. These cells were tested for proliferation, TCR expression, memory phenotype, cytokine secretion, and tumor killing. Results: γδ T-cell proliferation was dependent upon CD137L expression on aAPC and addition of exogenous IL2 and IL21. Propagated γδ T cells were polyclonal as they expressed TRDV1, TRDV2-2, TRDV3, TRDV5, TRDV7, and TRDV8 with TRGV2, TRGV3F, TRGV7, TRGV8, TRGV9*A1, TRGV10*A1, and TRGV11 TCR chains. IFNγ production by Vδ1, Vδ2, and Vδ1negVδ2neg subsets was inhibited by pan-TCRγδ antibody when added to cocultures of polyclonal γδ T cells and tumor cell lines. Polyclonal γδ T cells killed acute and chronic leukemia, colon, pancreatic, and ovarian cancer cell lines, but not healthy autologous or allogeneic normal B cells. Blocking antibodies demonstrated that polyclonal γδ T cells mediated tumor cell lysis through combination of DNAM1, NKG2D, and TCRγδ. The adoptive transfer of activated and propagated γδ T cells expressing polyclonal versus defined Vδ TCR chains imparted a hierarchy (polyclonal>Vδ1>Vδ1negVδ2neg>Vδ2) of survival of mice with ovarian cancer xenografts. Conclusions: Polyclonal γδ T cells can be activated and propagated with clinical-grade aAPCs and demonstrate broad antitumor activities, which will facilitate the implementation of γδ T-cell cancer immunotherapies in humans. Clin Cancer Res; 20(22); 5708–19. ©2014 AACR.

CAR-redirected T cells have demonstrated clinical effectiveness in early phase clinical trials, with persistence of adoptively transferred CD19-specific T cells correlated with positive outcomes. Notwithstanding the successes for some hematological malignancies, CAR-T targets a limited number of cell-surface antigens that curtails their appeal for solid tumors. This can be overcome by TCR gene transfer with specificity for intracellular tumor-associated antigens such as NY-ESO-1 expressed by hematologic malignancies and solid tumors. The predominant technologies for both CAR- and TCR-redirection of T cells utilize viral genetic modification as well as require a lengthy period of in vitro propagation with resultant deleterious differentiation to achieve clinically relevant cell numbers. A major impediment with current TCR-T is the high-cost and lengthy time associated with a viral-based manufacture of a library of TCRs that can address a multitude of desired targets and match HLA restriction to meet the need to infuse personalized TCR-T products with multiple specificities in each recipient. The Sleeping Beauty (SB) platform is the most clinically advanced non-viral gene transfer technology and overcomes the issues of scalability with viral based manufacture of TCR-T. We initially showed in pre-clinical models that co-expression of membrane-bound interleukin-15 (mbIL15) enhanced in vivo persistence of CAR-T (PMID: 27849617). This technology has been recently advanced to produce CD19-specific T cells in ≤ 2 days after electro-transfer of DNA plasmids using so-called "rapid personalized manufacture" (RPM). This was based on the SB system to stably co-express CAR and mbIL15 with a kill switch (HER1t). We have now adapted these technologies to address current limitations for T-cell therapy by using the RPM process to very rapidly generate TCR-modified T cells. The rationale for RPM of TCR-T is based on: (i) SB to genetically modify resting T cells thus eliminating the need to propagate cells prior to, or after, genetic modification, (ii) introduction of TCR to redirect T-cell specificity to tumor-associated antigens, (iii) mbIL15-HER1t to support T-cell persistence and enable selective elimination to increase safety, and (iv) manufacture within two days of gene transfer which limits T-cell differentiation and decreases time to manufacture. Mononuclear cells were electroporated with SB-derived DNA plasmids expressing (a) HLA A2-restricted NY-ESO-1-specific TCR or (b) the TCR and mbIL15-HER1t in separate plasmids. Following electroporation, cells were directly (unpropagated) injected into NSG (immunocompromised) mice bearing established HLA A2+ NY-ESO-1+ tumor. Administration of RPM TCR-mbIL15 T cells exhibited superior anti-tumor activity compared with RPM TCR T cells (Figure). Though engraftment of TCR+ T cells was not significantly different between the two groups, the RPM TCR-mbIL15 T cell-treated mice exhibited increased frequency of CD27+TCR+ T cells (p = 0.035, n = 6-7, Mann Whitney test), a phenotype that is correlated with improved therapeutic responses in human subjects. The RPM technology can thus be adapted to co-express TCR with mbIL15 (and HER1t), which can now be scaled to provide a cost-effective approach to manufacturing a multitude of TCR-T products from a library of TCRs with the necessary complexity to manage the range of specificities and HLA restrictions to treat multiple patients. Disclosures Hurton: • M.D. Anderson Cancer Center: Patents & Royalties; Intrexon: Patents & Royalties: US 9,629,877 B2 ; Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties: US 9,629,877 B2 . Zhang:Intrexon: Patents & Royalties: US 9,629,877 B2; Ziopharm Oncology: Patents & Royalties: US 9,629,877 B2. Deniger:Ziopharm Oncology: Employment, Equity Ownership. Olivares:Ziopharm Oncology: Patents & Royalties: US9629877B2, US20160158285A1, WO2009091826A2, US20190085079A1, US20170158749A1, US20170333480A1, US20190055299A1; Intrexon: Patents & Royalties: US9629877B2, US20160158285A1, WO2009091826A2, US20190085079A1, US20170158749A1, US20170333480A1, US20190055299A1. Cooper:CytoSen: Equity Ownership; Targazyme: Equity Ownership; MD Anderson Cancer Center: Patents & Royalties; Sangamo BioSciences: Patents & Royalties; Immatics: Equity Ownership, Patents & Royalties; City of Hope: Patents & Royalties; Ziopharm Oncology: Employment, Equity Ownership, Other: Contracted research, Patents & Royalties; Secure Transfusion Services: Equity Ownership; CellChorus: Equity Ownership. Singh:Ziopharm Oncology: Patents & Royalties: US9629877B2, US20160096902A1, US20170333480A1, US10125193B2; Intrexon: Patents & Royalties: US9629877B2, US20160096902A1, US20170333480A1, US10125193B2.

Even though other γδ T-cell subsets exhibit antitumor activity, adoptive transfer of γδ Tcells is currently limited to one subset (expressing Vγ9Vδ2 T-cell receptor (TCR)) due to dependence on aminobisphosphonates as the only clinically appealing reagent for propagating γδ T cells. Therefore, we developed an approach to propagate polyclonal γδ T cells and rendered them bispecific through expression of a CD19-specific chimeric antigen receptor (CAR). Peripheral blood mononuclear cells (PBMC) were electroporated with Sleeping Beauty (SB) transposon and transposase to enforce expression of CAR in multiple γδ T-cell subsets. CAR(+)γδ T cells were expanded on CD19(+) artificial antigen-presenting cells (aAPC), which resulted in10(9) CAR(+)γδ T cells from10(6) total cells. Digital multiplex assay detected TCR mRNA coding for Vδ1, Vδ2, and Vδ3 with Vγ2, Vγ7, Vγ8, Vγ9, and Vγ10 alleles. Polyclonal CAR(+)γδ T cells were functional when TCRγδ and CAR were stimulated and displayed enhanced killing of CD19(+) tumor cell lines compared with CAR(neg)γδ T cells. CD19(+) leukemia xenografts in mice were reduced with CAR(+)γδ T cells compared with control mice. Since CAR, SB, and aAPC have been adapted for human application, clinical trials can now focus on the therapeutic potential of polyclonal γδ T cells.

T-cells targeting shared oncogenic mutations can induce durable tumor regression in epithelial cancer patients. These T-cells can be detected in tumor-infiltrating lymphocytes, but whether such cells can be detected in the peripheral blood of patients with common metastatic epithelial cancer is unknown. Using a highly sensitive in vitro stimulation and enrichment of peripheral memory T-cells from six metastatic cancer patients, we identified and isolated memory T-cells targeting the mutated KRASG12D and KRASG12V variants, in three patients. In an additional two metastatic colon cancer patients, we detected CD8+ neoantigen-specific cells targeting the unique mutated SMAD5 and MUC4 proteins. Therefore, memory T-cells targeting KRAS and neoantigens can be detected in the peripheral blood of epithelial cancer patients and can potentially be used for the development of effective personalized T-cell-based cancer immunotherapy across multiple patients. Citation Format: Gal Cafri, Rami Yossef, Anna Pasetto, Drew Deniger, Jared J. Gartner, Todd Prickett, Paul F. Robbins, Steven A. Rosenberg. Memory T-cells targeting unique and shared oncogenic mutations detected in peripheral blood of epithelial cancer patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A175.

Adoptive cell transfer (ACT) of selected tumor-infiltrating lymphocytes (TILs) targeting neoantigens can mediate tumor regression in selected patients with metastatic epithelial cancer. However, effectively identifying and harnessing neoantigen-reactive T-cells for patient treatment remains a challenge and it is unknown whether current methods to detect neoantigen-reactive T-cells are missing potentially clinically relevant neoantigen reactivities. We thus investigated whether the detection of tumor-neoantigen reactive TILs in epithelial cancers could be enhanced by enriching T-cells that express PD-1 and/or T-cell activation markers (CD134, CD137) followed by microwell culturing at limiting dilution cell concentrations to avoid overgrowth of non-reactive T-cells. Using this approach, in six patients with metastatic epithelial cancer including stomach, colon pancreatic and ovarian cancers, this method led to the detection of CD4 and CD8 T-cells targeting 19 neoantigens compared to only eight neoantigens recognized using our standard TIL fragment screening approach. In two patients, no recognition of mutated peptides was observed using our conventional screen, while our high-throughput approach led to the identification of five neoantigen reactive-TCRs against five different mutations from one patient and a highly potent MHC-II-restricted KRASG12V reactive TCR from a second patient. In addition, in a metastatic tumor sample from a patient with serous ovarian cancer, we isolated three MHC class-II-restricted TCRs targeting the TP53G245S “hot-spot” mutation. In conclusion, this approach provides a highly sensitive and specific platform to isolate clinically relevant neoantigen-reactive T-cells or their TCRs for cancer treatment. Citation Format: Rami Yossef, Eric Tran, Alena Gros, Drew Deniger, Gal Cafri, Steven A. Rosenberg. Enhanced detection of T-cells targeting unique neoantigens and shared mutated oncogenes for personalized cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B055.

Respiratory syncytial virus (RSV) is a common cause of respiratory tract infections in infants and the elderly. Like many other pH-independent enveloped viruses, RSV is thought to enter at the cell surface, independently of common endocytic pathways. We have used a targeted small interfering RNA (siRNA) library to identify key cellular genes involved in cytoskeletal dynamics and endosome trafficking that are important for RSV infection. Surprisingly, RSV infection was potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis, including clathrin light chain. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. We conclude that, while RSV may be competent to enter at the cell surface, clathrin function and endocytosis are a necessary and important part of a productive RSV infection, even though infection is strictly independent of pH. These findings raise the possibility that other pH-independent viruses may share a similar dependence on endocytosis for infection and provide a new potential avenue for treatment of infection.

Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens.

Adoptive immunotherapy infusing T cells with engineered specificity for CD19 expressed on B- cell malignancies is generating enthusiasm to extend this approach to other hematological malignancies, such as acute myelogenous leukemia (AML). CD123, or interleukin 3 receptor alpha, is overexpressed on most AML and some lymphoid malignancies, such as acute lymphocytic leukemia (ALL), and has been an effective target for T cells expressing chimeric antigen receptors (CARs). The prototypical CAR encodes a VH and VL from one monoclonal antibody (mAb), coupled to a transmembrane domain and one or more cytoplasmic signaling domains. Previous studies showed that treatment of an experimental AML model with CD123-specific CAR T cells was therapeutic, but at the cost of impaired myelopoiesis, highlighting the need for systems to define the antigen threshold for CAR recognition. Here, we show that CARs can be engineered using VH and VL chains derived from different CD123-specific mAbs to generate a panel of CAR+ T cells. While all CARs exhibited specificity to CD123, one VH and VL combination had reduced lysis of normal hematopoietic stem cells. This CAR's in vivo anti-tumor activity was similar whether signaling occurred via chimeric CD28 or CD137, prolonging survival in both AML and ALL models. Co-expression of inducible caspase 9 eliminated CAR+ T cells. These data help support the use of CD123-specific CARs for treatment of CD123+ hematologic malignancies.

Adoptive transfer of T-cell receptor (TCR) gene engineered T-cells targeting tumor-associated antigens can mediate cancer regression. Accumulating evidence suggests that the clinical success of many immunotherapies is mediated by T-cells targeting mutated neoantigens unique to the patient. We hypothesized that the most frequent TCR clonotypes infiltrating the tumor were reactive against tumor antigens. We thus developed a multi-step strategy that involved CDR3-TCRB deep sequencing of tumor infiltrating lymphocytes and matching of TCRA-TCRB pairs by pairSEQ and single cell RT-PCR. CDR3 TCRA-TCRB pairs identified with this approach, were then reconstructed to full length based on IMGT database [http://www.imgt.org], synthetized and cloned into non-viral and retroviral expression vectors. T-cells were gene-engineered with these TCRs and tested against tumor cell lines and antigen presenting cells expressing tandem minigenes encoding mutated tumor neoantigens and/or pulsed with corresponding mutated peptides. Analysis of 12 fresh metastatic melanomas revealed that in 11 samples, up to 5 tumor-reactive TCRs were present in the 5 most frequently occurring clonotypes including reactivity against neoantigens. These data demonstrate the feasibility of developing a rapid approach for the identification of tumor-reactive TCRs that can be used for personalized TCR-gene therapy.

Somatic mutations expressed by the tumor can serve as neoantigens for autologous T cells. Tumor infiltrating lymphocytes (TIL) with varying degrees of neoantigen-reactivity infused for the treatment of melanoma resulted in 50% overall objective response and 20% complete response rates. A largely oligoclonal population of ERBB2IPE805G mutated neoantigen-specific T cells resulted in a long-term, ongoing partial regression of metastatic cholangiocarcinoma suggesting that infusion of selected mutation-reactive TIL could be efficacious in the treatment of common epithelial cancers. We now studied whether TIL obtained from metastatic ovarian cancer recognized tumor mutations. Exome and transcriptome sequencing was performed from resected metastatic ovarian cancer deposits in parallel with growth of TIL fragment cultures in interleukin-2. Long peptides and tandem minigenes encompassing all mutations were synthesized, introduced into autologous antigen presenting cells, co-cultured with individual TIL fragments and T-cell reactivity was determined by interferon-y ELISPOT and surface expression of 41BB. In the 8 ovarian tumors evaluated, there was a median of 227 mutations (range: 63-332) and an average of 94% of 24 fragments initiated (range: 58% - 100%) available for testing. Six of eight (75%) patients had T cell responses to mutated neoantigens at >5% of the fragment culture. Antigens identified to date were unique to each patient, i.e. no overlapping mutations between patients, and both CD4 and CD8 responses have been detected. One patient had a CD4 T cell response to p53G245S hotspot mutation, which opens opportunities for treatment of other cancer patients with TCR-transduced T cells because this mutation is present in 2.8% of all cancers. The average time from resection to identification of mutation-reactive T cell fragment culture was 8 weeks indicating that this strategy could be used for prospective therapy. In summary, mutation-specific T-cell responses were found in 6 of 8 patients with metastatic ovarian cancer, which opens the opportunity to use these cells for adoptive T cell treatment of advanced ovarian cancer.

Functionally pleiotropic human γδ T cells are a unique set of immune cells that exhibit qualities of both innate and adaptive immune responses. In contrast to αβ T cells, γδ T cells recognize their ligands independent of MHC and are infrequent (1%–5% of T cells) in peripheral blood. Studies have shown that γδ T cells exhibit endogenous cytotoxicity towards tumor cells by directly recognizing a diverse array of Tumor Associated Antigen (TAA) and have the ability to present TAA to elicit an antitumor response. This broad recognition of antitumor activity is achieved because these cells express a diverse TCR γδ repertoire (combination of Vδ1, Vδ2, or Vδ3 with one of fourteen Vγ chains). Because of their characteristics γδ T cells are attractive targets for clinical manipulation and tumor immunotherapy. To obtain clinically significant numbers of cells for immunotherapy current ex vivo γδ T cells expansion protocol uses Zoledronic acid, an aminobisphosphonate that results only in selective propagation of Vγ9Vδ2. These cells however, showed clinical response against both solid and hematologic tumors. We have previously shown a novel ex vivo expansion protocol using artificial antigen presenting cells (aAPC), IL2 and IL21 assist aAPC in activating and expanding polyclonal γδ T-cells (Vδ1, Vδ2 and Vδ1neg Vδ2neg T cell subtypes) to clinically significant numbers. These expanded cell subsets secrete proinflammatory cytokines, lysed a broad range of malignancies and improved survival in ovarian cancer xenograft model. To better define the role of the subsets and their application in immunotherapy here we present high-resolution transcriptome, microRNA and secreted cytokine-chemokine signatures of ex vivo expanded subsets. Our data provide an atlas of transcriptional, epigenetic and secretome of three defined subsets, and subset-specific signatures. The data presented here will have a broader impact in understanding the immunobiology of γδ T cell subsets and facilitate improving the human application of these cells in cancer immunotherapy. Citation Format: Sourindra Maiti, Drew Deniger, Jianliang Dai, Laurence Cooper. Transcriptional and epigenetic signatures of ex vivo propagated three distinct TCR Vδ1, TCR Vδ2 and TCR Vδ3 cell subtypes with broad specificity for malignancies. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B028.

One of the most important steps in a productive viral infection is when the virus fuses to a cell membrane and delivers its genome into the cell cytosol. This dynamic event is mediated by interactions between specific virus envelope proteins with their cell-bound receptors. This process is exemplified by Moloney murine leukemia virus (Mo-MLV) where envelope protein interaction with its receptor, mCAT-1, leads to virus-cell membrane fusion and infection of cells. Here, fluorescent nanoparticles (NPs) were coated with Mo-MLV derived membranes (Mo-NPs) by extrusion. Electron microscopy and biochemical analysis showed tight association of the virus membranes and NPs. The coated NPs mimic native virus by binding and entering only cells expressing the virus receptor. Confocal microscopy revealed that the coated NPs were taken up into endocytic compartments containing receptor and were also seen associated with caveolin, a marker of caveolae. To demonstrate that the Mo-NPs could escape endosomes and deliver a protein cargo into the cell cytosol, beta-lactamase (betalac) was covalently coupled to the Mo-NP cores and incubated with cells. betalac activity was only detected in the cytosol of mCAT-1-expressing cells. This is the first time that virus proteins have been used to specifically target NPs to receptor-bearing cells as well as penetration into the cell cytosol. Extrusion provides a rapid, detergent-free method to couple virus membranes to NPs and should be readily applicable for many other virus and NP types.

Meeting abstracts Adoptive cell therapy with T cells bearing mutation specific T cell receptors (TCR) can be an effective method for treating metastatic cancers. The objective of this study was to identify mutation reactive T cells in the circulation of patients with different types of metastatic