Durable Complete Response from Metastatic Melanoma after Transfer of Autologous T Cells Recognizing 10 Mutated Tumor Antigens

Abstract

Immunotherapy treatment of patients with metastatic cancer has assumed a prominent role in the clinic. Durable complete response rates of 20% to 25% are achieved in patients with metastatic melanoma following adoptive cell transfer of T cells derived from metastatic lesions, responses that appear in some patients to be mediated by T cells that predominantly recognize mutated antigens. Here, we provide a detailed analysis of the reactivity of T cells administered to a patient with metastatic melanoma who exhibited a complete response for over 3 years after treatment. Over 4,000 nonsynonymous somatic mutations were identified by whole-exome sequence analysis of the patient's autologous normal and tumor cell DNA. Autologous B cells transfected with 720 mutated minigenes corresponding to the most highly expressed tumor cell transcripts were then analyzed for their ability to stimulate the administered T cells. Autologous tumor-infiltrating lymphocytes recognized 10 distinct mutated gene products, but not the corresponding wild-type products, each of which was recognized in the context of one of three different MHC class I restriction elements expressed by the patient. Detailed clonal analysis revealed that 9 of the top 20 most prevalent clones present in the infused T cells, comprising approximately 24% of the total cells, recognized mutated antigens. Thus, we have identified and enriched mutation-reactive T cells and suggest that such analyses may lead to the development of more effective therapies for the treatment of patients with metastatic cancer. Cancer Immunol Res; 4(8); 669-78. ©2016 AACR.

Figure 1

Clinical response of patient 3713 (pt.3713) after treatment with adoptive cell therapy. Computed tomography (CT) scans showing the disease present in both lobes of the lung (A-B) prior to surgical resection and TIL therapy (C-D). The 3cm lesion in the left lobe (A) was resected and used to derived patient specific TIL. Panel (D) shows patients lungs clear of disease. White arrows show areas of metastatic disease.

Figure 2

Screening of pt.3713 autologous infusion bag TIL for reactivity to tandem minigenes (TMGs). (A) Autologous B cells transformed with EBV were transfected with IVT RNA from TMG-1 to TMG-62 individually and then co-cultured with autologous infusion bag TIL. IVT RNA expressing GFP was a negative control and autologous 3713 mel cell line (*) was used as positive control. (B) HEK-293T cells were transiently co-transfected with positive pt.3713 TMGs in the presence of patient specific HLAs (A*020101, A*290201, B*440301, B*510101, C*150101, or C*160101). GFP expression construct was used a negative control. (C) COS7 cells were transiently co-transfected with positive pt.3713 TMGs in the presence of patient specific HLAs (A*020101, A*290201, B*440301, B*510101, C*150101, or C*160101). GFP expression construct was used a negative control.

Figure 3

Minimal neo-epitope dose curve analysis with pulsed EBV co-cultured with autologous TIL. EBV cells were pulsed with between 1,000 and 0.1 ng/ml of mutant or wild type HPLC purified peptides with the exception of TPX2 peptides, which were pulsed at concentrations ranging between 10,000 and 1 ng/ml, and the release of IFNγ following an overnight culture with TIL 3713 determined. Each neo-epitope dose response curve was run three independent times. The gene names, HLA restriction elements, and TMG constructs encoding the mutated epitopes are shown above each graph.

Figure 4

Reactivity of clones or TCRs corresponding to predominant clonotypes in TIL 3713. Responses of allogeneic whole PBMC (A-C) or CD8-enriched T cells (D) transduced with TCRs reactive with the mutated SRPX (A), AFMID (B) HELZ2 (C) or CENPL epitopes were evaluated by measuring the release of IFNγ in response to T2 cells (A) or autologous EBV B cells (B-D) pulsed with the appropriate peptides. Transduced T cells were also evaluated for their ability to release IFNγ in response to either, the autologous TC line or an allogeneic melanoma TC line. In addition, T-cell clones isolated from TIL that were reactive with the mutated SRPX epitope (E) or the mutated SEC22C epitope (F) evaluated for their ability to up-regulate 4-1BB in response to autologous normal B cells (E) or EBV B cell (F) targets pulsed with the appropriate peptides or the autologous TC line (graphs represent 2 independent experimental repeats).