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Supplementary Figure 5

Supplementary Tables 1 and 2

Supplementary Table 3

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.

Supplementary Figures 1, 2, 3, 4, 6 and 7

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

Supplementary Figures 1-9

Supplementary Materials and Methods

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.

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.

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

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.

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.

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.

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