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PURPOSE Metastatic breast cancer (mBrCa) is most often an incurable disease with only modest responses to available immunotherapies. This study investigates the immunogenicity of somatic mutations in breast cancer and explores the therapeutic efficacy in a pilot trial of mutation-reactive tumor-infiltrating lymphocytes (TILs) in patients with metastatic disease. PATIENTS AND METHODS Forty-two patients with mBrCa refractory to previous lines of treatment underwent surgical resection of a metastatic lesion(s), isolation of TIL cultures, identification of exomic nonsynonymous tumor mutations, and immunologic screening for neoantigen reactivity. Clinically eligible patients with appropriate reactivity were enrolled into one cohort of an ongoing phase II pilot trial of adoptive cell transfer of selected neoantigen-reactive TIL, with a short course of pembrolizumab (ClinicalTrials.gov identifier: NCT01174121 ). RESULTS TILs were isolated and grown in culture from the resected lesions of all 42 patients with mBrCa, and a median number of 112 (range: 6-563) nonsynonymous mutations per patient were identified. Twenty-eight of 42 (67%) patients contained TIL that recognized at least one immunogenic somatic mutation (median: 3 neoantigens per patient, range: 1-11), and 13 patients demonstrated robust reactivity appropriate for adoptive transfer. Eight patients remained clinically eligible for treatment, and six patients were enrolled on a protocol of adoptive cell transfer of enriched neoantigen-specific TIL, in combination with pembrolizumab (≤ 4 doses). Objective tumor regression was noted in three patients, including one complete response (now ongoing over 5.5 years) and two partial responses (6 and 10 months). CONCLUSION Most patients with breast cancer generated a natural immune response targeting the expressed products of their cancer mutations. Adoptive transfer of TIL is a highly personalized experimental option for patients with mBrCa shown to be capable of mediating objective responses in this pilot trial and deserves further study.

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

Efforts to apply adoptive cell transfer (ACT) immunotherapy to patients with common epithelial cancers have been stimulated by the demonstration that the majority of these patients contain lymphocytes reactive against the expressed products of their cancer mutations. Early efforts to specifically target these antigens have been promising.

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

The number of each of the six classes of base substitutions resulting in non-synonymous changes in the whole exome screen is shown.

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.

A list of genes that were found to be somatically mutated in pt3713 and all of the major information needed for our genomic study

Supplementary Figures 1-9

Supplementary Materials and Methods

Immunotherapy treatment of patients with metastatic cancer has assumed a prominent role in the clinic. Durable complete response rates of 20% to 25% are achieved in patients with metastatic melanoma following adoptive cell transfer of T cells derived from metastatic lesions, responses that appear in some patients to be mediated by T cells that predominantly recognize mutated antigens. Here, we provide a detailed analysis of the reactivity of T cells administered to a patient with metastatic melanoma who exhibited a complete response for over 3 years after treatment. Over 4,000 nonsynonymous somatic mutations were identified by whole-exome sequence analysis of the patient's autologous normal and tumor cell DNA. Autologous B cells transfected with 720 mutated minigenes corresponding to the most highly expressed tumor cell transcripts were then analyzed for their ability to stimulate the administered T cells. Autologous tumor-infiltrating lymphocytes recognized 10 distinct mutated gene products, but not the corresponding wild-type products, each of which was recognized in the context of one of three different MHC class I restriction elements expressed by the patient. Detailed clonal analysis revealed that 9 of the top 20 most prevalent clones present in the infused T cells, comprising approximately 24% of the total cells, recognized mutated antigens. Thus, we have identified and enriched mutation-reactive T cells and suggest that such analyses may lead to the development of more effective therapies for the treatment of patients with metastatic cancer. Cancer Immunol Res; 4(8); 669–78. ©2016 AACR.

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.

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Purpose: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) can mediate regression of metastatic melanoma. However, many patients with cancer are ineligible for such treatment because their TIL do not expand sufficiently or because their tumors have lost expression of antigens and/or MHC molecules. Natural killer (NK) cells are large granular lymphocytes that lyse tumor cells in a non–MHC-restricted manner. Therefore, we initiated in a clinical trial to evaluate the efficacy of adoptively transferred autologous NK cells to treat patients with cancers who were ineligible for treatment with TIL.Experimental Design: Patients with metastatic melanoma or renal cell carcinoma were treated with adoptively transferred in vitro activated autologous NK cells after the patients received a lymphodepleting but nonmyeloablative chemotherapy regimen. Clinical responses and persistence of the adoptively transferred cells were evaluated.Results: Eight patients were treated with an average of 4.7 × 1010 (± 2.1 × 1010) NK cells. The infused cells exhibited high levels of lytic activity in vitro. Although no clinical responses were observed, the adoptively transferred NK cells seemed to persist in the peripheral circulation of patients for at least one week posttransfer and, in some patients, for several months. However, the persistent NK cells in the circulation expressed significantly lower levels of the key activating receptor NKG2D and could not lyse tumor cell targets in vitro unless reactivated with IL-2.Conclusions: The persistent NK cells could mediate antibody-dependent cell-mediated cytotoxicity without cytokine reactivation in vitro, which suggests that coupling adoptive NK cell transfer with monoclonal antibody administration deserves evaluation. Clin Cancer Res; 17(19); 6287–97. ©2011 AACR.

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

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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.

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Supplementary Data from Characterization of Genetically Modified T-Cell Receptors that Recognize the CEA:691-699 Peptide in the Context of HLA-A2.1 on Human Colorectal Cancer Cells

Purpose: Immunotherapies mediate the regression of human tumors through recognition of tumor antigens by immune cells that trigger an immune response. Mutations in the RAS oncogenes occur in about 30% of all patients with cancer. These mutations play an important role in both tumor establishment and survival and are commonly found in hotspots. Discovering T-cell receptors (TCR) that recognize shared mutated RAS antigens presented on MHC class I and class II molecules are thus promising reagents for “off-the-shelf” adoptive cell therapies (ACT) following insertion of the TCRs into lymphocytes. Experimental Design: In this ongoing work, we screened for RAS antigen recognition in tumor-infiltrating lymphocytes (TIL) or by in vitro stimulation of peripheral blood lymphocytes (PBL). TCRs recognizing mutated RAS were identified from the reactive T cells. The TCRs were then reconstructed and virally transduced into PBLs and tested. Results: Here, we detect and report multiple novel TCR sequences that recognize nonsynonymous mutant RAS hotspot mutations with high avidity and specificity and identify the specific class-I and -II MHC restriction elements involved in the recognition of mutant RAS. Conclusions: The TCR library directed against RAS hotspot mutations described here recognize RAS mutations found in about 45% of the Caucasian population and about 60% of the Asian population and represent promising reagents for “off-the-shelf” ACTs.

BACKGROUND: Therapeutic vaccinations against cancer have mainly targeted differentiation antigens, cancer-testis antigens, and overexpressed antigens and have thus far resulted in little clinical benefit. Studies conducted by multiple groups have demonstrated that T cells recognizing neoantigens are present in most cancers and offer a specific and highly immunogenic target for personalized vaccination. METHODS: We recently developed a process using tumor-infiltrating lymphocytes to identify the specific immunogenic mutations expressed in patients’ tumors. Here, validated, defined neoantigens, predicted neoepitopes, and mutations of driver genes were concatenated into a single mRNA construct to vaccinate patients with metastatic gastrointestinal cancer. RESULTS: The vaccine was safe and elicited mutation-specific T cell responses against predicted neoepitopes not detected before vaccination. Furthermore, we were able to isolate and verify T cell receptors targeting KRAS(G12D) mutation. We observed no objective clinical responses in the 4 patients treated in this trial. CONCLUSION: This vaccine was safe, and potential future combination of such vaccines with checkpoint inhibitors or adoptive T cell therapy should be evaluated for possible clinical benefit in patients with common epithelial cancers. TRIAL REGISTRATION: Phase I/II protocol (NCT03480152) was approved by the IRB committee of the NIH and the FDA. FUNDING: Center for Clinical Research, NCI, NIH.

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

A common theme across multiple successful immunotherapies for cancer is the recognition of tumor-specific mutations (neoantigens) by T cells. The rapid discovery of such antigen responses could lead to improved therapies through the adoptive transfer of T cells engineered to express neoantigen reactive T-cell receptors (TCR). Here, through CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) and TCR-seq of non-small cell lung cancer (NSCLC) tumor-infiltrating lymphocytes (TIL), we develop a neoantigen-reactive T-cell signature based on clonotype frequency and CD39 protein and CXCL13 mRNA expression. Screening of TCRs selected by the signature allows us to identify neoantigen-reactive TCRs with a success rate of 45% for CD8(+) and 66% for CD4(+) T cells. Because of the small number of samples analyzed (4 patients), generalizability remains to be tested. However, this approach can enable the quick identification of neoantigen-reactive TCRs and expedite the engineering of personalized neoantigen-reactive T cells for therapy.

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8 + and CD4 + neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature–derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.

BackgroundAutologous patient T cells engineered to express antitumor T cell receptors (TCRs) and chimeric antigen receptors (CARs) have been effective for the treatment of certain cancer types,1–4 and tumor neoantigens encoded by cancer-specific mutations have emerged as major targets of CD4+ and CD8+ T cells in immune checkpoint blockade (ICB) and in adoptive cell therapy (ACT).5–9 However, only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells.10–12 Conventional approaches to isolate tumor-reactive T cells and identify their TCRs from tumors rely on T cell function and can be impaired due to T cell exhaustion and dysfunction.13 14MethodsWe performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 46,000 T cells isolated from eleven archival metastatic tumor samples whose primary cancer types included colon, rectal, breast, anal, and melanoma. From these samples, 15 CD8+ and 17 CD4+ neoantigen-reactive TCR clonotypes (NeoTCRs) were known. We then performed transcriptomic clustering of these cells and mapped known NeoTCR clonotypes onto the transcriptomic map. Subsequently we predicted NeoTCRs from prospective metastatic colon cancer samples based on their presence within clusters sharing gene expression with NeoTCR+ clusters in the archival samples.ResultsProjecting known NeoTCRs onto the TIL transcriptomic map, we observed 325 total T cells bearing these NeoTCRs, and the majority (>80%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, that included by expression of CXCL13, ENTPD1 (CD39), TOX, TIGIT, LAG3, and PDCD1 (PD-1), indicating a dysfunctional state. Reasoning that T cells sharing phenotypes with those within the NeoTCR clusters could be novel NeoTCRs, we developed gene signatures (NeoTCR4 and NeoTCR8) of CD4 and CD8 NeoTCR+ cells, respectively, and four prospective patients' TIL were analyzed by scRNA/TCR-seq and scored according to NeoTCR signatures. We expressed predicted NeoTCRs in healthy donor PBL and screened them with antigen presenting cells (APCs) expressing candidate neoantigens. 33/73 predicted NeoTCRs (including both CD4 and CD8) were reactive against patients' tumors or candidate neoantigens.ConclusionsThis study enabled successful detection of tumor-specific NeoTCRs in the sequenced TIL of 14/14 patients for whom reactivity was studied. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapeutics, and prospective NeoTCR isolation based on TIL transcriptional phenotypes will allow for rapid development of personalized immunotherapy in the form of lymphocytes expressing these tumor-specific TCRs.AcknowledgementsWe thank the Surgery Branch TIL Laboratory and clinical team for generating TIL, and patients enrolled in our clinical protocols. Support from CCR Single Cell Analysis Facility was funded by FNLCR Contract HHSN261200800001E. This work utilized the computational resources of the NIH HPC Biowulf cluster (http://hpc.nih.gov). We also thank NIDAP for providing additional computational support and the CCR Genomics Core for next-generation sequencing supportReferencesRobbins PF, Morgan RA, Feldman SA, Yang JC, Sherry RM, Dudley ME, Wunderlich JR, Nahvi AV, Helman LJ, Mackall CL, Kammula US, Hughes MS, Restifo NP, Raffeld M, Lee CCR, Levy CL, Li YF, El-Gamil M, Schwarz SL, Laurencot C, Rosenberg SA.Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1. J Clin Oncol 2011;29:917–924.Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP, Zheng Z, Nahvi A, de Vries CR, Rogers-Freezer LJ, Mavroukakis SA, Rosenberg SA. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006;314:126–129.June CH, Sadelain M. Chimeric Antigen Receptor Therapy. N Engl J Med 2018;379:64–73.Kochenderfer JN, Yu Z, Frasheri D, Restifo NP, Rosenberg SA. Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. Blood 2010;116:3875–3886.Tran E, Robbins PF, Rosenberg SA, “Final common pathway” of human cancer immunotherapy: targeting random somatic mutations. Nat Immunol 2017;18:255–262.Robbins PF, Lu YC, El-Gamil M, Li YF, Gross C, Gartner J, Lin JC, Teer JK, Cliften P, Tycksen E, Samuels Y, Rosenberg SA, Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells. Nat Med 2013;19:747–752.Parkhurst MR, Robbins PF, Tran E, Prickett TD, Gartner JJ, Jia L, Ivey G, Li YF, El-Gamil M, Lalani A, Crystal JS, Sachs A, Groh E, Ray S, Ngo LT, Kivitz S, Pasetto A, Yossef R, Lowery FJ, Goff SL, Lo W, Cafri G, Deniger DC, Malekzadeh P, Ahmadzadeh M, Wunderlich JR, Somerville RPT, Rosenberg SA. Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers. Cancer Discov 2019;9:1022–1035.Gubin MM, Zhang X, Schuster H, Caron E, Ward JP, Noguchi T, Ivanova Y, Hundal J, Arthur CD, Krebber WJ, Mulder GE, Toebes M, Vesely MD, Lam SSK, Korman AJ, Allison JP, Freeman GJ, Sharpe AH, Pearce EL, Schumacher TN, Aebersold R, Rammensee HG, Melief CJM, Mardis ER, Gillanders WE, Artyomov MN, Schreiber RD. Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens. Nature 2014;515:577–581.van Rooij N, van Buuren MM, Philips D, Velds A, Toebes M, Heemskerk B, van Dijk LJA, Behjati S, Hilkmann H, el Atmioui D, Nieuwland M, Stratton MR, Kerkhoven RM, Keşmir C, Haanen JB, Kvistborg P, Schumacher TN. Tumor Exome Analysis Reveals Neoantigen-Specific T-Cell Reactivity in an Ipilimumab-Responsive Melanoma. Journal of Clinical Oncology 2013;31:e439–e442.Duhen T, Duhen R, Montler R, Moses J, Moudgil T, de Miranda NF, Goodall CP, Blair TC, Fox BA, McDermott JE, Chang SC, Grunkemeier G, Leidner R, Bell RB, Weinberg AD. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9:2724.Simoni Y, Becht E, Fehlings M, Loh CY, Koo SL, Teng KWW, Yeong JPS, Nahar R, Zhang T, Kared H, Duan K, Ang N, Poidinger M, Lee YY, Larbi A, Khng AJ, Tan E, Fu C, Mathew R, Teo M, Lim WT, Toh CK, Ong BH, Koh T, Hillmer AM, Takano A, Lim TKH, Tan EH, Zhai W, Tan DSW, Tan IB, Newell EW, Bystander CD8 T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature 2018;557:575–579.Scheper W, Kelderman S, Fanchi LF, Linnemann C, Bendle G, de Rooij MAJ, Hirt C, Mezzadra R, Slagter M, Dijkstra K, Kluin RJC, Snaebjornsson P, Milne K, Nelson BH, Zijlmans H, Kenter G, Voest EE, Haanen JBAG, Schumacher TN. Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers. 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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.

Tumor neoepitopes presented by major histocompatibility complex (MHC) class I are recognized by tumor-infiltrating lymphocytes (TIL) and are targeted by adoptive T-cell therapies. Identifying which mutant neoepitopes from tumor cells are capable of recognition by T cells can assist in the development of tumor-specific, cell-based therapies and can shed light on antitumor responses. Here, we generate a ranking algorithm for class I candidate neoepitopes by using next-generation sequencing data and a dataset of 185 neoepitopes that are recognized by HLA class I–restricted TIL from individuals with metastatic cancer. Random forest model analysis showed that the inclusion of multiple factors impacting epitope presentation and recognition increased output sensitivity and specificity compared to the use of predicted HLA binding alone. The ranking score output provides a set of class I candidate neoantigens that may serve as therapeutic targets and provides a tool to facilitate in vitro and in vivo studies aimed at the development of more effective immunotherapies. Robbins and colleagues develop and test a machine learning neoantigen ranking model using experimentally validated neoantigens from human tumors, providing a resource of targetable neoantigens for future immunotherapies.

BACKGROUND: Immunotherapies mediate the regression of human tumors through recognition of tumor antigens by immune cells that trigger an immune response. Mutations in the RAS oncogenes occur in about 30% of all cancer patients. These mutations play an important role in both tumor establishment and survival and are commonly found in hotspots. PURPOSE: Discovering T cell receptors (TCR) that recognize shared mutated RAS antigens presented on major histocompatibility complex (MHC) class I and class II molecules are thus promising reagents for “off-the-shelf” adoptive cell therapies (ACT) following insertion of the TCR into lymphocytes. EXPERIMENTAL DESIGN: In this ongoing work, we screened for RAS antigen recognition in tumor-infiltrating lymphocytes (TIL) or by in-vitro stimulation (IVS) of peripheral blood lymphocytes (PBLs). TCRs recognized mutated RAS were identified from the reactive T cells. The TCRs were then reconstructed and virally transduced into PBLs and tested. RESULTS: Here, we detect and report multiple novel TCRs sequences that recognize non-synonymous mutant RAS hotspot mutations with high avidity and specificity and identify the specific class-I and II MHC restriction elements involved in the recognition of mutant RAS. CONCLUSIONS: The TCR library directed against RAS hotspot mutations described here recognize RAS mutations found in about 45% of the Caucasian population and about 60% of the Asian population and represent promising reagents for “off-the-shelf” adoptive cell therapies (ACT).

Immunotherapies mediate the regression of human tumors through the recognition of tumor antigens by immune cells, which triggers an immune response. Mutations in the RAS oncogene occur across various cancer histologies and are found in about 30% of all cancer patients. These mutations play an essential role in tumor establishment and survival and are most commonly found in hotspots - specifically at amino acid positions 12, 13, and 61. We have recently treated a patient who underwent a durable objective response after receiving tumor-infiltrating lymphocytes (TILs) that recognized a KRAS G12D mutation. T cell receptors (TCR) that recognize shared mutated RAS antigens presented on major histocompatibility complex (MHC) class I and class II molecules are thus promising reagents for “off-the-shelf” adoptive cell therapies (ACT) following insertion of the TCR into lymphocytes. We recently published a paper describing the discovery of anti-mutated RAS reactivities by using screening TIL and by performing in-vitro stimulation (IVS) of peripheral blood lymphocytes (PBLs). Interestingly by those two methods, we sometimes found reactivity or TCR in a patient in one method but not with the other method. In this work, we described the discovery of anti-mutated RAS TCRs by TIL IVS, which combines the best of the two described methods: using TILs coming from the tumor and therefore assumed to have a higher proportion of anti-tumor-reactive cells and stimulate them, which could enrich smaller populations. The specific anti-mutated RAS CD4 and CD8 TCRs were isolated by coculture of the reactive T cells with dendritic cells (DC) loaded with peptides or transfected with mRNA encoding RAS mutated antigens. The reactive T cells were subjected to single-cell sequencing to identify TCRs. The TCRs were reconstructed as a viral plasmid and retrovirally transduced into PBLs. The transduced TCRs were tested for avidity, MHC restriction, and specific recognition of the mutation. In the same patients tested by both methods, by TIL IVS, identified 12 anti-mutated RAS TCRs in addition the 9 TCRs that we could find by TIL screening. Overall, we present here the discovery of multiple mutation-specific anti-RAS TCRs and its MHC class I and class II restriction elements involved in tumor recognition. Each of the newly discovered TCRs recognized mutated RAS with high avidity and specificity and low to no reactivity against wild-type RAS. Testing of the TCR transduced cells to recognize tumor cell lines in-vitro and in-vivo demonstrated tumor cell line recognition. The anti-RAS mutated TCRs we discovered by all of our methods can potentially be used as an “off the shelf” treatment for the evaluation of TCR-based ACT in 45-60% of patients were tumor bears RAS mutations. Citation Format: Noam Levin, Frank J. Lowery, Maria R. Parkhurst, Zhiya Yu, Nolan R. Vale, Sanghyun P. Kim, Steven A. Rosenberg. Identification of T-cell receptors targeting RAS hotspot mutations using TIL IVS in human cancers for use in cell-based immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3576.

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.

BackgroundAdoptive cellular transfer (ACT) of autologous tumor-infiltrating lymphocytes (TIL) is capable of inducing durable clinical responses in patients with advanced solid malignancies,1 however response rates are low. Limitations in personalized cancer modeling have been obstacles to understanding tumor-specific mechanisms of immune evasion. Patient-derived tumor organoids (PDTO) can be efficiently grown from common solid tumors and show genetic fidelity to whole exomic sequencing (WES) of the source tumor. We investigated their use in evaluating patient-specific immune responses in vitro and selecting T-cells for adoptive cellular immunotherapy.MethodsPDTO were established from metastatic deposits from patients with colorectal, breast, and pancreatic cancers and with their tumors of origin, were subjected to WES and RNAseq. They were then included in immunologic recognition assays with autologous TIL and cloned T-cell receptors (TCR) against shared mutations. TIL neoantigen reactivity was defined by screening against mutations using minigenes or synthetic peptides expressed by autologous antigen presenting cells.ResultsPDTO were successfully grown from 18/22 tumors from 15 patients. These cultures demonstrated a high degree of genetic fidelity to their parental tumors with near-complete retention of clonally expressed mutations. Organoid lines from 11 of these patients were utilized in recognition screening with autologous TIL or PBL transduced with relevant TCRs. TIL recognized 5/7 organoids tested, and this allowed the isolation and cloning of many of the TCRs responsible. Nineteen available TCRs predicted to be reactive with patient-specific neoantigens were also tested against relevant organoids, and only nine were found to be reactive. In most instances where such TCRs could not recognize organoid, tumor-specific defects in neoantigen processing or presentation or HLA-LOH events were either functionally or genetically identified.ConclusionsTo improve response rates to neoantigen-directed adoptive T-cell therapy, a better understanding of tumor immune evasion mechanisms is needed. The inability to grow autologous tumor lines from common epithelial cancers has been a major obstacle. We demonstrate that PDTO can be efficiently established and are genetically representative of their parental tumors. Importantly, these organoids can reveal defects in neoantigen processing or surface presentation as well as HLA loss-of-heterozygosity that would preclude immune recognition. PDTO may be a valuable tool for screening for tumor reactivity and selecting T-cells and TCRs for clinical use.Trial RegistrationThe study protocol was registered under https://clinicaltrials.gov under NCT00068003.ReferenceZacharakis N, Chinnasamy H, Black M, et al. Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. Nat Med. 2018;24:724–730.Ethics ApprovalAll samples were derived from study participants who granted written, informed consent to be enrolled on a clinical protocol approved by the Institutional Review Board at the NCI (Bethesda, MD).

Purpose: The adoptive transfer of lymphocytes genetically modified to express tumor reactive T-cell receptors (TCR) can mediate tumor regression. Some tumor-infiltrating lymphocytes (TIL) recognize somatic mutations expressed only in the patient's tumors, and evidence suggests that clinically effective TILs target tumor-specific neoantigens. Here we attempted to isolate neoantigen-reactive TCRs as a prelude to the treatment of patients with autologous T cells genetically modified to express such TCRs. Experimental Design: Mutations expressed by tumors were identified using whole-exome and RNA sequencing. Tandem minigene (TMG) constructs encoding 12–24 mutated gene products were synthesized, each encoding the mutated amino acid flanked by 12 amino acids of the normal protein sequence. TILs were cultured with autologous dendritic cells (DC) transfected with in vitro transcribed (IVT) mRNAs encoding TMGs and were evaluated for IFNγ secretion and CD137 expression. Neoantigen-reactive T cells were enriched from TILs by sorting for CD137+ CD8+ T cells and expanded in vitro. Dominant TCR α and β chains were identified in the enriched populations using a combination of 5′ rapid amplification of cDNA ends, deep sequencing of genomic DNA, PairSeq analysis, and single-cell RT-PCR analysis. Human PBL retrovirally transduced to express the TCRs were evaluated for recognition of relevant neoantigens. Results: We identified 27 TCRs from 6 patients that recognized 14 neoantigens expressed by autologous tumor cells. Conclusions: This strategy provides the means to generate T cells expressing neoantigen-reactive TCRs for use in future adoptive cell transfer immunotherapy trials for patients with cancer. Clin Cancer Res; 23(10); 2491–505. ©2016 AACR.

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.

Tumor-resident lymphocytes can mount a response against neoantigens expressed in microsatellite-stable gastrointestinal (GI) cancers, and adoptive transfer of neoantigen-specific lymphocytes has demonstrated antitumor activity in selected patients. However, whether peripheral blood could be used as an alternative minimally invasive source to identify lymphocytes targeting neoantigens in patients with GI cancer with relatively low mutation burden is unclear. We used a personalized high-throughput screening strategy to investigate whether PD-1 expression in peripheral blood could be used to identify CD8(+) or CD4(+) lymphocytes recognizing neoantigens identified by whole-exome sequencing in 7 patients with GI cancer. We found that neoantigen-specific lymphocytes were preferentially enriched in the CD8(+)PD-1(+/hi) or CD4(+)PD-1(+/hi) subsets, but not in the corresponding bulk or PD-1(–) fractions. In 6 of 7 individuals analyzed we identified circulating CD8(+) and CD4(+) lymphocytes targeting 6 and 4 neoantigens, respectively. Moreover, neoantigen-reactive T cells and a T cell receptor (TCR) isolated from the CD8(+)PD-1(+) subsets recognized autologous tumor, albeit at reduced levels, in 2 patients with available cell lines. These data demonstrate the existence of circulating T cells targeting neoantigens in GI cancer patients and provide an approach to generate enriched populations of personalized neoantigen-specific lymphocytes and isolate TCRs that could be exploited therapeutically to treat cancer.

The correlations between tumor mutational load and higher response rates to immune-checkpoint inhibitor (ICI) therapies revealed the importance of targeting tumor neoantigens in cancer immunotherapy. Although ICI therapy has been revolutionary, many patients still do not respond to these treatments. One reason for this may be the absence of an adequate anti-tumor T-cell repertoire. A potential solution is the adoptive transfer of T-cells targeting neoantigens. Despite having some common shared neoantigens such as KRAS and p53, the majority of patients have highly individualized neoantigens and personalized T-cell responses. One method for identifying neoantigen-reactive T-cells from a patient's tumor-infiltrating lymphocytes (TIL) involves: (1) identifying non-synonymous mutations via whole-exome sequencing (WES), (2) using autologous dendritic cells (DC) to display these mutations in the form of electroporated minigenes (as RNA) or pulsed synthetic peptides (to create a tumor cell surrogate) and (3) co-culturing the TIL with these DCs to detect neoantigen-reactive T-cells through the upregulation of T-cell activation markers (e.g., 4-1BB). Each of these steps is labor-intensive, costly, and time-consuming. To address this, we sought to identify neoantigen-reactive TCRs directly from fresh tumors by developing a signature based on the cell surface protein and transcriptomic phenotype of cells proven to be mutation reactive. Knowing the V(D)J sequences for T-cells known to be neoantigen reactive (by the above screening), we performed 10x Genomics single cell RNA-Seq with CITE-Seq analysis on TIL isolated from frozen samples of those fresh tumors. Using the known CDR3 sequences, we compared neoantigen-reactive T-cells (six CD8 reactivities and two CD4 reactivities) versus other T-cells. This analysis led to identifying a set of cell surface proteins and genes that are specifically over- or under-expressed (including but not limited to CD62L-, CD45RA-, IL7Rlow, CD39+, CD27+, CD74+, TIGIT+, CXCL13+, LAYN+, HMOX1+, BATF+) on neoantigen-reactive T-cells. We tested whether CD39+ and CXCL13+ signature in combination with the TCR clonotype frequency could prospectively identify other neoantigen-reactive TCRs. This proved to be the case and applied not only to CD8 TIL but strongly to CD4 reactivities. In all cases, we could identify neoantigen-reactive TCRs, and in one case, 8 out of 10 candidate receptors identified by phenotype signature proved to be neoantigen-reactive. Not only were they neoantigen specific, but new, previously unidentified neoantigens were found when their specificities were analyzed. This method can open the path to treating patients with custom-made TCRs in a timely manner and expand the repertoire of tumor-specific neoantigens and the diversity of the T-cell response discoverable in human tumors. Citation Format: Kenichi Hanada, Chihao Zhao, Raul Gil-Hoyos, Jared Gartner, Christopher Chow-Parmer, Frank Lowery, Sri Krishna, Samuel Chatmon, Prickett D. Todd, Scott Kivitz, Maria Parkhurst, Michelle Langhan, Thomas Shelton, Zulmarie Franco, Robert Somerville, John Wunderlich, David Danforth, Zachary Rae, Kelly Michael, Nathan Wong, Paul Robbins, Steven Rosenberg, James Yang. A signature for tumor neoantigen-reactive T-cells in fresh human lung cancers allows rapid cloning of their receptors [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 1509.

The adoptive transfer of tumor-infiltrating lymphocytes (TILs) expanded in vitro can mediate regressions of established tumors, yet only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells. This complexity underpins the need to phenotypically characterize T cells infiltrating the tumor microenvironment. In this study, we performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 45,000 T cells isolated from ten archival metastatic tumor samples whose primary histologies included colon, rectal, breast, and melanoma. By transcriptomic clustering of the T cells and mapping TCR identity data onto these blueprints, we observed a diversity of T cell phenotypes spanning from memory-like to dysfunctional effector-like, with clonal expansion occurring more widely in the effector-like cells. Through sequencing of peripheral blood lymphocytes (PBL) obtained at the time of tumor resection, we found T cell clusters that were either tumor enriched or distinctly PBL enriched, potentially representing recent tumor immigrants. Prior knowledge of 30 CD4 and CD8 tumor neoantigen-reactive TCRs (NeoTCRs) from eight patients allowed us to project these NeoTCRs onto the TIL transcriptomic map where we observed 380 total T cells bearing these NeoTCRs. The majority (>85%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, marked by expression of CXCL13, ENTPD1 (CD39), TOX, and PDCD1 (PD-1), indicating a dysfunctional state. We reasoned that other dominant T cell clonotypes within the NeoTCR clusters could contain additional novel NeoTCR clonotypes. We expressed these predicted NeoTCRs in donor PBL and screened them in vitro with antigen presenting cells (APCs) pulsed with peptides or tandem minigenes encoded by patient-specific tumor mutations, yielding 24 new NeoTCR clones (251 cells), which included neoantigen reactivities undetected in the same patients' fragment TIL screens. Using a NeoTCR signature score derived from the archival samples, three prospective patients' TIL were analyzed by scRNA/TCRseq and clusters scored according to their NeoTCR signature. TCRs reconstructed from these clusters and tested against patients' tumor-specific reagents yielded novel CD4 and CD8 NeoTCRs. Altogether, this study enabled successful enrichment and detection of tumor-specific NeoTCRs in the sequenced TIL of 12/12 patients for whom reactivity was either previously assessed or prospectively identified. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapy, as prospective NeoTCR isolation based on transcriptional phenotype of TIL will allow for rapid therapeutic development in the form of T cells expressing these tumor-specific TCRs. Citation Format: Frank J. Lowery, Sri Krishna, Rami Yossef, Praveen D. Chatani, Neilesh B. Parikh, Yong-Chen Lu, Nikolaos Zacharakis, Noam Levin, Ken-ichi Hanada, James C. Yang, Paul F. Robbins, Maria R. Parkhurst, Steven A. Rosenberg. Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer [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 127.

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.

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.

Adoptively transferred tumor-infiltrating T lymphocytes (TILs) that mediate complete regression of metastatic melanoma have been shown to recognize mutated epitopes expressed by autologous tumors. Here, in an attempt to develop a strategy for facilitating the isolation, expansion, and study of mutated antigen–specific T cells, we performed whole-exome sequencing on matched tumor and normal DNA isolated from 8 patients with metastatic melanoma. Candidate mutated epitopes were identified using a peptide-MHC–binding algorithm, and these epitopes were synthesized and used to generate panels of MHC tetramers that were evaluated for binding to tumor digests and cultured TILs used for the treatment of patients. This strategy resulted in the identification of 9 mutated epitopes from 5 of the 8 patients tested. Cells reactive with 8 of the 9 epitopes could be isolated from autologous peripheral blood, where they were detected at frequencies that were estimated to range between 0.4% and 0.002%. To the best of our knowledge, this represents the first demonstration of the successful isolation of mutation-reactive T cells from patients’ peripheral blood prior to immune therapy, potentially providing the basis for designing personalized immunotherapies to treat patients with advanced cancer.

Immunotherapy treatment of patients with metastatic cancer has assumed a prominent role in the clinic. Durable complete response rates of 20% to 25% are achieved in patients with metastatic melanoma following adoptive cell transfer of T cells derived from metastatic lesions, responses that appear in some patients to be mediated by T cells that predominantly recognize mutated antigens. Here, we provide a detailed analysis of the reactivity of T cells administered to a patient with metastatic melanoma who exhibited a complete response for over 3 years after treatment. Over 4,000 nonsynonymous somatic mutations were identified by whole-exome sequence analysis of the patient's autologous normal and tumor cell DNA. Autologous B cells transfected with 720 mutated minigenes corresponding to the most highly expressed tumor cell transcripts were then analyzed for their ability to stimulate the administered T cells. Autologous tumor-infiltrating lymphocytes recognized 10 distinct mutated gene products, but not the corresponding wild-type products, each of which was recognized in the context of one of three different MHC class I restriction elements expressed by the patient. Detailed clonal analysis revealed that 9 of the top 20 most prevalent clones present in the infused T cells, comprising approximately 24% of the total cells, recognized mutated antigens. Thus, we have identified and enriched mutation-reactive T cells and suggest that such analyses may lead to the development of more effective therapies for the treatment of patients with metastatic cancer. Cancer Immunol Res; 4(8); 669–78. ©2016 AACR.

Wilms tumor gene 1 (WT1) is an attractive target antigen for cancer immunotherapy because it is overexpressed in many hematologic malignancies and solid tumors but has limited, low-level expression in normal adult tissues. Multiple HLA class I and class II restricted epitopes have been identified in WT1, and multiple investigators are pursuing the treatment of cancer patients with WT1-based vaccines and adoptively transferred WT1-reactive T cells. Here we isolated an HLA-A*0201-restricted WT1-reactive T-cell receptor (TCR) by stimulating peripheral blood lymphocytes of healthy donors with the peptide WT1:126-134 in vitro. This TCR mediated peptide recognition down to a concentration of ∼0.1 ng/mL when pulsed onto T2 cells as well as recognition of HLA-A*0201 target cells transfected with full-length WT1 cDNA. However, it did not mediate consistent recognition of many HLA-A*0201 tumor cell lines or freshly isolated leukemia cells that endogeneously expressed WT1. We dissected this pattern of recognition further and observed that WT1:126-134 was more efficiently processed by immunoproteasomes compared with standard proteasomes. However, pretreatment of WT1 tumor cell lines with interferon gamma did not appreciably enhance recognition by our TCR. In addition, we highly overexpressed WT1 in several leukemia cell lines by electroporation with full-length WT1 cDNA. Some of these lines were still not recognized by our TCR suggesting possible antigen processing defects in some leukemias. These results suggest WT1:126-134 may not be a suitable target for T-cell based tumor immunotherapies.

Purpose: Adoptive transfer of tumor-infiltrating lymphocytes (TIL) can mediate regression of metastatic melanoma. However, many patients with cancer are ineligible for such treatment because their TIL do not expand sufficiently or because their tumors have lost expression of antigens and/or MHC molecules. Natural killer (NK) cells are large granular lymphocytes that lyse tumor cells in a non–MHC-restricted manner. Therefore, we initiated in a clinical trial to evaluate the efficacy of adoptively transferred autologous NK cells to treat patients with cancers who were ineligible for treatment with TIL. Experimental Design: Patients with metastatic melanoma or renal cell carcinoma were treated with adoptively transferred in vitro activated autologous NK cells after the patients received a lymphodepleting but nonmyeloablative chemotherapy regimen. Clinical responses and persistence of the adoptively transferred cells were evaluated. Results: Eight patients were treated with an average of 4.7 × 1010 (± 2.1 × 1010) NK cells. The infused cells exhibited high levels of lytic activity in vitro. Although no clinical responses were observed, the adoptively transferred NK cells seemed to persist in the peripheral circulation of patients for at least one week posttransfer and, in some patients, for several months. However, the persistent NK cells in the circulation expressed significantly lower levels of the key activating receptor NKG2D and could not lyse tumor cell targets in vitro unless reactivated with IL-2. Conclusions: The persistent NK cells could mediate antibody-dependent cell-mediated cytotoxicity without cytokine reactivation in vitro, which suggests that coupling adoptive NK cell transfer with monoclonal antibody administration deserves evaluation. Clin Cancer Res; 17(19); 6287–97. ©2011 AACR.