A deletion variant of epidermal growth factor receptor (EGFRvIII) is a known driver mutation in a subset of primary and secondary glioblastoma multiforme. Adoptive transfer of genetically modified chimeric-antigen receptor (CAR) lymphocytes has demonstrated efficacy in hematologic malignancies but is still early in development for solid cancers. The surface expression of the truncated extracellular ligand domain created by EGFRvIII makes it an attractive target for a CAR-based cancer treatment. Patients with recurrent glioblastoma expressing EGFRvIII were enrolled in a dose escalation phase I trial, using a third-generation chimeric antigen receptor construct derived from a human antibody. Transduced cells were administered after lymphodepleting chemotherapy and supported post-transfer with intravenous interleukin-2. The dose escalation proceeded at half-log increments from 10 7 to >10 10 cells. Primary endpoints were safety and progression-free survival. Eighteen patients were treated with final infusion products ranging from 6.3×10 6 to 2.6×10 10 anti-EGFRvIII-CAR+ T cells. Median progression free survival was 1.3 months (interquartile range 1.1-1.9), with a single outlier of 12.5 months. Two patients experienced severe hypoxia, including one treatment related mortality after cell administration at the highest dose level. All patients developed expected transient hematologic toxicities from preparative chemotherapy. Median overall survival was 6.9 months (interquartile range 2.8-10). Two patients survived over one year, and a third patient was alive at 59 months. Persistence of CAR+ cells correlated with cell dose, but there were no objective responses. Administration of anti-EGFRvIII CAR-transduced T cells did not demonstrate clinically meaningful impact in patients with glioblastoma multiforme in this phase I pilot trial.

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 which 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 non-synonymous 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.

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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 HLAA*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.

COUNTS Statement of Translational RelevanceThis study demonstrated that T cells with specificity to mutated neoantigens were present in tumor infiltrating lymphocytes in patients with metastatic ovarian cancer, sometimes at high frequencies and with multivalent reactivity, suggesting that these T cells could be used for adoptive cell therapy. Furthermore, T cells from two of the patients were specific for TP53 "hotspot" mutations (c.659A>G; p.Y220C and c.733G>A; p.G245S), which are also expressed in a broad range of tumor types in unrelated individuals. Genetic transfer of the TP53 "hotspot" mutation-specific T-cell receptors into autologous lymphocytes could be used to generate cells for use in the adoptive cell transfer immunotherapy of cancer.Research. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on May 31, 2018; DOI: 10.1158/1078-0432.CCR-18-0573 3 AbstractPurpose: This was a study prospectively evaluating intratumoral T-cell responses to autologous somatic mutated neoepitopes expressed by human metastatic ovarian cancers.Experimental Design: Tumor infiltrating lymphocytes (TILs) were expanded from resected ovarian cancer metastases, which were analyzed by whole exome and transcriptome sequencing to identify autologous somatic mutations. All mutated neoepitopes, independent of prediction algorithms, were expressed in autologous antigen presenting cells then co-cultured with TIL fragment cultures. Secretion of interferon-gamma or up-regulation of 41BB indicated a T-cell response.Results: Seven women with metastatic ovarian cancer were evaluated and 5 patients had clear, dominant T-cell responses to mutated neoantigens, which were corroborated by comparison to the wild type sequence, identification of the minimal epitope, human leukocyte antigen (HLA) restriction element(s) and neoantigen-specific T-cell receptor(s). Mutated neoantigens were restricted by HLA-B, -C, -DP, -DQ and/or -DR alleles and appeared to principally arise from random, somatic mutations unique to each patient. We established that TP53 "hotspot" mutations (c.659A>G; p.Y220C and c.733G>A; p.G245S) expressed by two different patient's tumors were immunogenic both in the context of HLA-DRB3*02:02.Conclusions: Mutation-reactive T cells infiltrated ovarian cancer metastases at sufficient frequencies to warrant their investigation as adoptive cell therapy. Additionally, transfer of TP53 "hotspot" mutation-reactive T-cell receptors into peripheral blood T cells could be evaluated as a gene therapy for a diverse range of tumor histologies.Research.on April 28,