Fast track to personalized TCR T cell therapies

In this issue of Cancer Cell, Hanada et al. leverage single-cell multi-omics of lung cancer resident lymphocytes to identify phenotypic and transcriptomic signatures differentially expressed by neoantigen-reactive clonotypes. These findings could substantially expedite the selection of neoantigen-specific T cell receptors (TCRs) for individualized T cell therapies. In this issue of Cancer Cell, Hanada et al. leverage single-cell multi-omics of lung cancer resident lymphocytes to identify phenotypic and transcriptomic signatures differentially expressed by neoantigen-reactive clonotypes. These findings could substantially expedite the selection of neoantigen-specific T cell receptors (TCRs) for individualized T cell therapies. Tumor-specific lymphocytes can specifically recognize and kill cancer cells through recognition of tumor antigens and adoptive transfer of ex vivo expanded tumor-infiltrating lymphocytes (TILs)—which are naturally-enriched in tumor-specific cells—and T cells genetically engineered to express cancer-specific T cell receptors (TCRs) can be harnessed to treat cancer. Cancer neoantigens, which are derived from somatically mutated gene products, are attractive targets for cancer immunotherapy. Mounting evidence suggests that neoantigen-specific T cells play an important role in the efficacy of cancer immunotherapies and neoantigen expression is restricted to tumor cells. Almost a decade ago now, the antitumor responses induced by ex vivo expanded neoantigen-specific lymphocytes in selected patients with epithelial cancers provided direct demonstration of the therapeutic potential of targeting neoantigens and laid the foundations for personalized T cell therapies (Tran et al., 2017Tran E. Robbins P.F. Rosenberg S.A. 'Final common pathway' of human cancer immunotherapy: targeting random somatic mutations.Nat. Immunol. 2017; 18: 255-262Crossref PubMed Scopus (295) Google Scholar). However, inconsistencies in the frequency and growth of neoantigen-reactive TILs have hindered the delivery of T cell products enriched for or enriched in neoantigen reactivity (Parkhurst et al., 2019Parkhurst M.R. Robbins P.F. Tran E. Prickett T.D. Gartner J.J. Jia L. Ivey G. Li Y.F. El-Gamil M. Lalani A. et al.Unique neoantigens Arise from somatic mutations in patients with gastrointestinal cancers.Cancer Discov. 2019; 9: 1022-1035Crossref PubMed Scopus (127) Google Scholar). This, along with concerns regarding the proliferative capacity and functionality of TILs, has urged the design of autologous T cell products that express neoantigen-specific TCRs; these are currently being tested in clinical trials (NCT03412877, NCT04102436, NCT03970382, NCT05194735). In this issue of Cancer Cell, Hanada et al. report a signature—leveraging the power of single-cell (sc) cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and TCR sequencing (TCR-seq)—to identify neoantigen-reactive T cells infiltrating non-small-cell lung cancer (NSCLC) samples (Hanada et al., 2022Hanada K. Zhao C. Gil-Hoyos R. Gartner J.J. Chow-Parmer C. Lowery F.J. Krishna S. Pricket T.D. Kivitz S. Parkhust M.R. et al.A phenotypic signature that Identifies neoantigen-reactive T cells in Fresh human lung cancers.Cancer Cell. 2022; 41Google Scholar). These findings could substantially expedite the selection of neoantigen-specific TCRs for individualized T cell therapies (Figure 1). One of the strategies commonly used to identify TCRs that target neoantigens entails culturing TILs from tumor biopsies in IL-2 and screening TILs for recognition of candidate neoantigens identified through tumor whole-exome sequencing (WES) using a personalized high-throughput screening approach (Tran et al., 2017Tran E. Robbins P.F. Rosenberg S.A. 'Final common pathway' of human cancer immunotherapy: targeting random somatic mutations.Nat. Immunol. 2017; 18: 255-262Crossref PubMed Scopus (295) Google Scholar). Subsequently, the nucleotide sequences that encode for the TCRs of neoantigen-reactive TILs are obtained, cloned, and used to genetically modify T cells to express candidate TCRs to (finally) validate neoantigen recognition. The results from Hanada et al. demonstrate that scTCR-seq coupled to transcriptomic and surface protein profiling of NSCLC-resident lymphocytes can lead to rapid identification of both CD8+ and CD4+ neoantigen-reactive TCRs (Figure 1). Selection of TCR clonotypes from cells that express CD39 and CXCL13 increased the number of identified neoantigen-reactive TCRs 3-fold compared to the previously mentioned TIL screening approach (from 11 to 34 TCRs). In addition, Hanada et al. describe further refined joint molecular and phenotypic profiles for the identification of CD8+ or CD4+ neoantigen-reactive lymphocytes from NSCLC lesions. In a recent related study, Lowery et al. reported valuable, prospectively validated, molecular signatures that are capable of identifying CD8+ or CD4+ neoantigen-specific TILs (NeoTCR8 and NeoTCR4, respectively) across cancer types. Although the molecular profiles of the latter were solely generated using scRNA-seq data, and these studies differ in additional experimental aspects, both articles define parallel signatures to identify neoantigen-specific TCRs, and both will be discussed hereafter. Overall, approximately 50% of the TCRs predicted through the use of the respective signatures were neoantigen-reactive (Hanada et al., 2022Hanada K. Zhao C. Gil-Hoyos R. Gartner J.J. Chow-Parmer C. Lowery F.J. Krishna S. Pricket T.D. Kivitz S. Parkhust M.R. et al.A phenotypic signature that Identifies neoantigen-reactive T cells in Fresh human lung cancers.Cancer Cell. 2022; 41Google Scholar; Lowery et al., 2022Lowery F.J. Krishna S. Yossef R. Parikh N.B. Chatani P.D. Zacharakis N. Parkhurst M.R. Levin N. Sindiri S. Sachs A. et al.Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers.Science. 2022; 375: 877-884Crossref PubMed Scopus (51) Google Scholar). More importantly, this strategy also led to the identification of neoantigens that were missed when screening cultured TILs, and it enabled the identification of reactive TCRs from both CD8+ and CD4+ TILs in patients for whom reactivities were initially detected uniquely in one subset. In addition, isolating TCRs predicted with specific molecular and phenotypic signatures using sc multi-omics considerably reduces the turnover time from tumor obtention to neoantigen-specific TCR identification because this omits TIL growth and screening. Overall, these data further provide evidence that the proliferative capacity, functionality, and/or stochastic expansion of TILs following culture in high dose IL-2 hinders the identification of reactive TILs. This underscores the promise of the use of the proposed gene and surface protein signatures to rapidly and more consistently isolate both CD8+ and CD4+ TCRs that target one or more neoantigens. Moreover, given that Hanada et al. focused on identifying TCRs that target only neoantigens, this study likely underestimates the number of tumor-reactive TCRs and additional tumor antigens recognized. Indeed, results reported by Lowery et al. demonstrate that some predicted TCRs are capable of recognizing tumors, but do not recognize any of the neoantigens screened, and this suggests that evaluating reactivity to additional tumor-specific antigens, such as cancer germline antigens and cryptic neoantigens, could further increase the number of tumor-reactive TCR candidates identified. Including Hanada et al., six recent studies on patients with different types of solid tumors elegantly leveraged the power of sc multi-omics to delineate the functional state of tumor-reactive TILs (Caushi et al., 2021Caushi J.X. Zhang J. Ji Z. Vaghasia A. Zhang B. Hsiue E.H. Mog B.J. Hou W. Justesen S. Blosser R. et al.Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers.Nature. 2021; 596: 126-132Crossref PubMed Scopus (113) Google Scholar; Hanada et al., 2022Hanada K. Zhao C. Gil-Hoyos R. Gartner J.J. Chow-Parmer C. Lowery F.J. Krishna S. Pricket T.D. Kivitz S. Parkhust M.R. et al.A phenotypic signature that Identifies neoantigen-reactive T cells in Fresh human lung cancers.Cancer Cell. 2022; 41Google Scholar; Lowery et al., 2022Lowery F.J. Krishna S. Yossef R. Parikh N.B. Chatani P.D. Zacharakis N. Parkhurst M.R. Levin N. Sindiri S. Sachs A. et al.Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers.Science. 2022; 375: 877-884Crossref PubMed Scopus (51) Google Scholar; Oliveira et al., 2021Oliveira G. Stromhaug K. Klaeger S. Kula T. Frederick D.T. Le P.M. Forman J. Huang T. Li S. Zhang W. et al.Phenotype, specificity and avidity of antitumour CD8(+) T cells in melanoma.Nature. 2021; 596: 119-125Crossref PubMed Scopus (122) Google Scholar; Veatch et al., 2022Veatch J.R. Lee S.M. Shasha C. Singhi N. Szeto J.L. Moshiri A.S. Kim T.S. Smythe K. Kong P. Fitzgibbon M. et al.Neoantigen-specific CD4+ T cells in human melanoma have diverse differentiation states and correlate with CD8+ T cell, macrophage, and B cell function.Cancer Cell. 2022; 40: 393-409.e399Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar; Zheng et al., 2022Zheng C. Fass J.N. Shih Y.-P. Gunderson A.J. Sanjuan Silva N. Huang H. Bernard B.M. Rajamanickam V. Slagel J. Bifulco C.B. et al.Transcriptomic profiles of neoantigen-reactive T cells in human gastrointestinal cancers.Cancer Cell. 2022; 40: 410-423.e417Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Indeed, as expected based on prior publications (Thommen et al., 2018Thommen D.S. Koelzer V.H. Herzig P. Roller A. Trefny M. Dimeloe S. Kiialainen A. Hanhart J. Schill C. Hess C. et al.A transcriptionally and functionally distinct PD-1(+) CD8(+) T cell pool with predictive potential in non-small-cell lung cancer treated with PD-1 blockade.Nat. Med. 2018; 24: 994-1004Crossref PubMed Scopus (559) Google Scholar, Van der Leun et al., 2020Van der Leun A.M. Thommen D.S. Schumacher T.N. CD8+ T cell states in human cancer: insights from single-cell analysis.Nat Rev Cancer. 2020; 20: 218-232Crossref PubMed Scopus (443) Google Scholar), emerging sc multi-omics data on tumor-reactive lymphocytes confirm that CD8+ neoantigen and tumor-reactive lymphocytes in human tumor lesions predominantly exist in an exhausted state—regardless of the specific tumor type—which is characterized by co-expression of CD39, PD-1, and CXCL13, among other markers associated with T cell dysfunction. Importantly, both Hanada et al. and Lowery et al. revealed that CD8+ and CD4+ neoantigen-reactive TILs transcriptomic profiles bear resemblance, exhibiting upregulation of genes related to exhaustion, but also of new genes with largely unknown function—potentially making them novel targets to reinvigorate exhausted reactive cells. Genes related with T follicular helper cell maintenance and activation were also coordinately upregulated, and CXCL13 was captured as a convergent functional trait of tumor-reactive cells in the tumor microenvironment. Both Hanada et al. and Lowery et al. initially identified a limited number of TCR clonotypes through screening of ex vivo cultured TILs. This represents a limitation in both studies, especially given that the initial projections of these clonotypes onto the sc data were exploited to predict additional neoantigen-reactive T cells, which were subsequently pooled together to generate the protein and gene neoantigen-reactivity signatures. Although this strategy demonstrably delineated signatures to detect neoantigen-reactive TCRs, it could underestimate the transcriptomic and phenotypic complexity of tumor-reactive TILs. The use of more high-throughput strategies to screen a larger number of TCRs from each patient, including TCRs from different memory and effector clusters, could reveal greater functional complexity. An additional limitation is the well-recognized bias in transcript capture efficiency and sequencing coverage that can be introduced by scRNA-seq, which could miss relevant genes. Hanada et al. used CITE-seq, including a panel of 12 barcoded antibodies to detect surface protein expression, in addition to transcriptome. Interestingly, unsupervised clustering of TILs through the use of either cell-surface expression or transcriptomic sc data and projection of the neoantigen-specific TCRs revealed that protein expression more accurately grouped the neoantigen-reactive cells together compared to gene expression. This study and an additional report analyzing melanoma TILs (Oliveira et al., 2021Oliveira G. Stromhaug K. Klaeger S. Kula T. Frederick D.T. Le P.M. Forman J. Huang T. Li S. Zhang W. et al.Phenotype, specificity and avidity of antitumour CD8(+) T cells in melanoma.Nature. 2021; 596: 119-125Crossref PubMed Scopus (122) Google Scholar) give evidence that cell surface protein profiling provides an additional dimension that complements, but could also potentially substitute for, the transcriptomic data. If further validated, this could greatly decrease the cost of obtaining the sc data needed for candidate TCR identification. In summary, the signatures reported by Hanada et al. and Lowery et al. hold promise to accelerate the development of individualized TCR gene-engineered T cell therapies, and they may also facilitate the dissection and better understanding of functional state of tumor-reactive TILs in human cancer patients. P.L.L. received funding from the Spanish Ministry of Science and Innovation (PID2020-118529RB-100). A.G. was funded by the Comprehensive Program of Cancer Immunotherapy & Immunology II (CAIMI-II) supported by the BBVA Foundation (53/2021), La Fundació La Marató de TV3 (201919-30), the Spanish Ministry of Science and Innovation (PID2020-118529RB-100), and Instituto de Salud Carlos III (CP15/00058). We thank the CERCA Program/Generalitat de Catalunya for institutional support. A.G. is member of the scientific advisory boards (SABs) of Achilles Therapeutics plc, SingulaBIO, RootPath, Inc., and BioNTech SE, and consults for PACT Pharma, Inc. and Instil BIo. A.G. is co-inventor of patents licensed and with royalties related to this work: E−059-2013/0, E−085-2013/0, and E−149-2015/0. P.L. declares no competing interests. A phenotypic signature that identifies neoantigen-reactive T cells in fresh human lung cancersHanada et al.Cancer CellApril 21, 2022In BriefThe discovery of T cell receptors (TCRs) that recognize tumor-specific neoantigens can lead to improved therapies through the adoptive transfer of T cells engineered to express these TCRs. Here, Hanada et al. design a signature of neoantigen-reactive T cells that enables the rapid identification of both CD4 and CD8 neoantigen-reactive TCRs. Full-Text PDF Open Archive