Neoantigen-reactive Tumor-infiltrating Lymphocytes Research Articles

Combination of Expanded Allogeneic NK Cells and T Cell-Based Immunotherapy Exert Enhanced Antitumor Effects.

Immunotherapies based on immune checkpoint blockade, neoantigen-reactive tumor-infiltrating lymphocytes and T cell receptor-engineered T cells (TCR-T) have achieved favorable clinical outcomes in tumor treatment. However, sustained immune response and tumor regression have been observed only in a few patients due to immune escape. Natural killer (NK) cells can mediate direct tumor lysis and target cancer cells with low or no expression of human leukocyte antigen class I (HLA-I) that are no longer recognized by T cells during immune escape. Therefore, the combination of T cell-based immunotherapy and NK cell therapy is a promising strategy for improving antitumor response and response rate. However, allogeneic NK cells for adoptive cell therapy have been limited by both the required cell number and quality. Here, we developed an efficient manufacturing system that relies on genetically modified K562 cells for the expansion of high-quality NK cells derived from peripheral blood mononuclear cells. NK cells with the optimal expansion and activity were identified by comparing the different culture systems. Furthermore, we demonstrated that the cooperation of NK cells with tumor-reactive T cells or with NY-ESO-1-specific TCR-T cells further enhanced tumors lysis, especially against tumors with downregulated HLA-I expression. The advantages of HLA-mismatch and non-rejection by other allogeneic immune cells demonstrated the potential of "off-the-shelf" NK cells with the capacity to target tumors for immunotherapy. Our results indicate that the combination strategy based on T cell and allogeneic NK cell immunotherapy might have potential for overcoming the barrier of immune incompetence caused by HLA-I downregulation.

Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers.

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.

64 A cloning and expression system of the neoantigen-specific TCRs from tumor-infiltrating lymphocytes by single-cell sequencing of paired TCRα and TCRβ chains

BackgroundT-cells that target tumor neoantigens arising from cancer mutations are the primary mediators of cancer immunotherapies. Identifying neoantigens and T-cells that recognize them is essential for T-cell-based immunotherapy. However, neoantigen-reactive Tumor-infiltrating lymphocytes (TILs) are highly differentiated or exhausted with a limited proliferative capacity; it is challenging to expand them for a sufficient number to probe their specificity. Therefore, we developed a novel cloning and expression system to examine TCRs discovered by single-cell sequencing of TILs for their neoantigen-specificity.MethodsTILs of lung cancer and sarcoma were analyzed. Surgically removed tumors were divided into several pieces. They were enzymatically digested to prepare fresh tumor digest (FTD) and cryopreserved. They were used to generate TIL cultures and perform WES and RNA-Seq to identify tumor-specific mutations. MHCflurry was used to predict the binding affinity of potential epitopes arising from these mutations to HLA class I. Peptides that were predicted to bind to patients‘ own MHC class I molecules strongly were then synthesized. Single TILs isolated with the ICELL8® cx system (Takara Bio) were dispensed into a nanowell TCR chip containing preprinted barcodes. Barcoded cDNAs were PCR-amplified in-chip, pooled off-chip, and used as a template in the TCR-specific PCR or for the whole transcriptome library generation of 5’ ends of all transcripts. Based on single-cell transcriptome data and TCR profiles of TILs, we predict and prioritize neoantigen-specific TCRs and cloned them into siTCR® retrovirus vectors. These TCRs were transduced into SUP-T1-based reporter cells in which ZsGreen fluorescent protein expression is controlled by AP-1 and NFAT binding sites. TCR-expressing reporter cells were cocultured with patient autologous APCs pulsed with a pool of candidate neoantigen peptides. ZsGreen expression indicates that TCRs match their cognate neoantigens.ResultsIn a lung cancer patient, we set up 18 TIL cultures and obtained 12 TILs. TILs were cocultured with FTD; IFN-γ production was measured by ELISA to evaluate their reactivity to the autologous tumor. NGS identified 197 somatic mutations, 4 fusion genes, and 8 highly expressed cancer-testis antigens. Among them, 339 candidate peptides were synthesized and screened. In addition, we cloned 3 pairs of TCRαβ chains from most expanded TIL cultures and 4 TCRs from ex vivo TILs with exhausted phenotype. Two reporter cells that express TCRs from exhausted TILs responded to the same neoantigen peptide.ConclusionsGenerating TCR expressing cell lines facilitated the identifying neoantigens and their cognate TCR sequences from patients.Ethics ApprovalG3545

Abstract 6539: Bioinformatics algorithm of mRNA-4157 identifies neoantigens with pre-existing TIL reactivities in colorectal tumors

Abstract Background: Personalized cancer vaccines (PCVs) encode mutation-derived epitopes (neoantigens) predicted from each tumor's unique set of somatic mutations, and can trigger the activation of neoantigen-specific CD4+ and/or CD8+ T cells to target cancer cells. The accurate prediction of immunogenic neoantigens from a tumor's mutanome, which may contain hundreds of somatic mutations, is crucial for designing an effective PCV. In this study, we evaluated the performance of the bioinformatics algorithm for mRNA-4157, an mRNA-based therapeutic PCV, in identifying neoantigens that are recognized by pre-existing tumor infiltrating lymphocytes (TILs) in a cohort of colorectal cancers shared by the National Cancer Institute (NCI) [1]. Methods: Whole exome and transcriptome sequencing data from 29 patients with microsatellite stable, mismatch repair (MMR)-proficient metastatic colorectal cancer were shared by NCI. All patients had received at least one previous systemic therapy, and neoantigen-reactive TIL populations had been identified from these patients by high-throughput immunologic screening [1]. The same bioinformatics algorithm as used for mRNA-4157 was run on the sequencing data, to generate a vaccine design for each patient that included up to 34 neoantigens. Results: Overall, 41% (26/64) of the neoantigens recognized by CD8+ or CD4+ TILs according to the NCI assay were included in the vaccines designed by the mRNA-4157 bioinformatics algorithm. For 61% (18/29) of the patients, at least one neoantigen with demonstrated CD8+ or CD4+ TIL reactivity was included in the vaccines designed. For 28% (8/29) of the patients, all neoantigens associated with demonstrated CD8+ or CD4+ TIL reactivities were included in the vaccines designed. Conclusion: The bioinformatics algorithm for mRNA-4157 is able to predict and select neoantigens with pre-existing TIL reactivities based on human ex vivo assays for vaccine designs with high accuracy. [1] Parkhurst MR, et al. Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers. Cancer Discov. 2019 Aug;9(8):1022-1035. Citation Format: Shan Zhong, Ben Breton, Wei Zheng, Iain McFadyen, Kristen Hopson, Joshua Frederick, Robert S. Meehan, Tal Zaks. Bioinformatics algorithm of mRNA-4157 identifies neoantigens with pre-existing TIL reactivities in colorectal tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6539.

Identification of Neoantigen-Reactive Tumor-Infiltrating Lymphocytes in Primary Bladder Cancer.

Immune checkpoint inhibitors are effective in treating a variety of malignancies, including metastatic bladder cancer. A generally accepted hypothesis suggests that immune checkpoint inhibitors induce tumor regressions by reactivating a population of endogenous tumor-infiltrating lymphocytes (TILs) that recognize cancer neoantigens. Although previous studies have identified neoantigen-reactive TILs from several types of cancer, no study to date has shown whether neoantigen-reactive TILs can be found in bladder tumors. To address this, we generated TIL cultures from patients with primary bladder cancer and tested their ability to recognize tumor-specific mutations. We found that CD4+ TILs from one patient recognized mutated C-terminal binding protein 1 in an MHC class II-restricted manner. This finding suggests that neoantigen-reactive TILs reside in bladder cancer, which may help explain the effectiveness of immune checkpoint blockade in this disease and also provides a rationale for the future use of adoptive T cell therapy targeting neoantigens in bladder cancer.