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We are interested in the T cell repertoire in glioblastoma (GBM) patients who have been administered peripherally infused CART-EGFRvIII adoptive T cell therapy for recurrent EGFRvIII-positive GBM. On neuropathologic evaluation following GBM tumor resection, we noticed that several subjects had a robust lymphocytic infiltrate in their tumors following CART-EGFRvIII infusion, which was out of proportion to the level of CART-EGFRvIII genomic sequences observed. We sought to quantify the T cell infiltrate in five subjects whose tumors were resected at different time points following CART infusion (days 6-104). In these five subjects, we analyzed the T cell clonotypic repertoire in the infusion product and in pre- and post-infusion brain tumor specimens by deep sequencing the T cell receptor (TCR) Vβ chain of tumor-infiltrating T cells. Data analysis was performed using ImmunoSeq and exported data were further examined using Graphpad Prism. The infusion products in these patients were highly polyclonal with no clones represented above 1%. We found that in all five of these subjects, the number and frequency of unique TCRVβ clonotypes increased by several thousand (~20-fold) in post-infusion GBM specimens, and only a small portion of these unique TCRs were shared with pre-existing tumor-infiltrating specific T cells (TILs). A consistent finding in these patients was that the clonotype compositional diversity of T cells in the tumor bed post-infusion was considerably larger than the pre-CART vs. infusion product sharing frequency. These data indicate there was a significant increase in the number and clonotypic diversity of tumor-infiltrating T cells following intravenous CART-EGFRvIII infusion, but only a small proportion appears to be derived from the infusion product. Collectively, these results show that CART infusion can trigger accumulation of TILs. Whether or not these TILs respond to GBM neoantigens remains to be investigated. Citation Format: Donald M. O'Rourke, Fang Chen, Zev A. Binder, Irina Kulikovskaya, Minnal Gupta, Jeffrey Finklestein, David E. Ambrose, MacLean P. Nasrallah, Carl H. June, Simon F. Lacey, J. Joseph Melenhorst. Next-generation sequencing of T-cell receptor-beta gene rearrangements reveals dramatic expansion of T-cell clonotypes after CART-EGFRvIII therapy for glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-053. doi:10.1158/1538-7445.AM2017-LB-053

Background: Adoptive immunotherapy for cancer has been limited by lack of antigen specificity, low levels of target expression, and failure to break self-tolerance. We are conducting an early phase clinical trial (NCT01352286) attempting to overcome these barriers using T cells engineered with an HLA-A0201 restricted, affinity-enhanced TCR that recognizes an epitope expressed by the NY-ESO-1 and LAGE-1 cancer testis antigens; these cells are infused in the setting of profound lymphodepletion that accompanies high-dose chemotherapy with autologous stem cell transplant (aSCT) for patients with high risk or relapsed multiple myeloma (MM). Methods: Inclusion criteria include: 1) eligibility for aSCT, 2) PS of 0-2, 3) high risk MM or relapse after prior therapy, 4) HLA-A0201 positive, and 5) NY-ESO-1 and/or LAGE-1 positive tumor by PCR. CD25 depleted T cells are activated and expanded using anti-CD3/28 antibody conjugated microbeads, and genetically modified with a lentiviral vector. T cells are administered four days after high dose melphalan and two days following auto-SCT. Patients are evaluated for MM responses in accordance with the IMWG criteria at 6 weeks, and 3 and 6 months. At 3 months, patients with adequate marrow function start lenalidomide maintenance. Blood and marrow are monitored for persistence of engineered cells by qPCR and by surface expression of the NY-ESO-1 / LAGE-1 TCR using dextramerTM reagents. NY-ESO-1 and LAGE-1 antigen expression in marrow was assessed by qRT-PCR at baseline and post infusion. Results: As of November 2012, 21 patients have been enrolled, 15 have been infused; 4 were taken off study prior to infusion due to disease progression. An average of 2.7 x 109 engineered T cells were administered per patient (range 8.3 x 108-4.2 x 109), and the average transduction efficiency was 33% (range 30%-45%). More than 50% (8/15) of patients have high risk chromosomal abnormalities, and 3 (20%) have received prior aSCT. At 3 months post aSCT, 73% of patients were in a very good partial response (VGPR) or better. Gastrointestinal toxicity resulting from autologous GVHD (aGVHD) occurred in a subset of patients at a higher rate than reported following aSCT alone or aSCT and T cell infusion, and was resolved in all cases. Infused T cells typically showed peak expansion in blood at day 14, followed by durable persistence in blood and marrow at 6-12 months in all but one patient. Disease progression is typically accompanied by very low levels or loss of engineered T cell persistence or loss of target antigen on tumor. Conclusions: We report for the first time that possible correlates of clinical response in this study include persistence of engineered T cells and loss of antigen, suggesting specific activity of the infused cells. Infusions are well tolerated with a possible risk of manageable aGVHD. Citation Format: Aaron P. Rapoport, Edward A. Stadtmauer, Dan T. Vogl, Brendan Weiss, Gwendolyn K. Binder-Scholl, Dominic P. Smethurst, Jeffrey Finkelstein, Irina Kulikovskaya, Minnal Gupta, Erica Suppa, Tatiana Mikheeva, Joanna E. Brewer, Alan D. Bennett, Andrew B. Gerry, Nick J. Pumphrey, Helen K. Tayton-Martin, Lilliam Ribeiro, Elizabeth Veloso, Zhaohui Zheng, Ashraf Z. Bados, Saul Yanovich, Gorgun Akpek, Karen Dengel, Naseem Kerr, Sunita Philip, Kelly-Marie Betts, Sandra Westphal, Bruce L. Levine, Bent K. Jakobsen, Carl H. June, Michael Kalos. Correlates of clinical response following autologous stem cell transplant and adoptive immunotherapy with engineered T cells expressing an affinity-enhanced T cell receptor in patients with mutliple myeloma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2013-4575