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Neoepitope-reactive T cells achieve complete tumor regression in metastatic breast cancer

June 20, 2018

In a paper recently published in Nature Medicine, Zacharakis et al. report the case of a 49-year-old woman with estrogen receptor (ER)-positive, HER2-negative metastatic breast cancer, refractory to multiple lines of chemotherapy, who was enrolled in a phase II clinical trial that evaluated the efficacy of autologous tumor-infiltrating lymphocytes (TILs) in reducing tumor burden in metastatic epithelial cancers. Selection of TIL subpopulations reactive against tumor-specific neoepitopes may have dramatically improved efficacy in this low-mutation-rate tumor type.

Zacharakis et al. performed whole-exome sequencing and RNA-seq on one of the lesions and found 62 nonsynonymous somatic mutations. To determine which of these mutations yielded antigenic peptides, the team cultured TILs with high-dose IL-2 and screened them for reactivity using pulsed peptide pools or transfected mRNA of tandem minigenes expressed in autologous antigen-presenting cells (APCs). The screening revealed three neoantigen-reactive TIL fragments, with CD4+ TILs recognizing mutant SLC3A2 protein and CD8+ TILs reactive against mutant KIAA0368 protein. In order to isolate neoantigen-reactive T cell clonotypes, the team sorted reactive TILs by TCR-β variable region (TRBV) allotypes or high expression of the 4-1BB activation marker and performed TCR-focused deep sequencing. This led to the identification of seven TCR clonotypes that recognized SLC3A2 and one TCR that recognized KIAA0368.

Following the identification of neoepitope-specific TCRs, the patient was treated with a total of 8.2x1010 TILs from the three neoantigen-positive TIL fragments administered in 7 doses every 8 hours. Prior to TIL infusion, the patient underwent nonmyeloablative lymphodepletion via chemotherapy (cyclophosphamide and fludarabine) and a single infusion of pembrolizumab (anti-PD-1). Pembrolizumab was also administered 3, 6, and 9 weeks after TIL infusion. TILs consisted of 62.5% CD4+ T cells with an effector memory phenotype and 33% CD8+ T cells. About 21% of TILs expressed PD-1 at the time of infusion. Six weeks after TIL infusion, target tumor lesions shrunk by 51%. Even more remarkably, at the most recent evaluation, 22 months after cell transfer, the patient achieved a complete radiographic resolution of all target and nontarget lesions.

The researchers had originally added pembrolizumab to TIL treatment in order to prevent T cell exhaustion in the tumor microenvironment. Having achieved tumor regression, they retrospectively assessed the role that anti-PD-1 may have had on the success of the therapy. They analyzed two tumor samples obtained prior to cell transfer and found no PD-L1 expression in tumor cells of either lesion, while PD-L1 was expressed in the stromal cells of one tumor sample. PD-1 expression on peripheral blood T cells remained relatively constant over the course of 2 years. Based on this data the authors suspect that pembrolizumab did not play a significant role in antitumor response.

The team analyzed the clonotypes of infused TILs and peripheral blood T cells and found that the eight originally identified reactive TCRs, constituting 23% of TRBV sequences at the time of transfer, were present at about 2% in peripheral blood 6 weeks after transfer. Additionally, the researchers identified two other TRBV families that expanded significantly in the blood after treatment and contained at least one dominant TCR clonotype. Further analysis revealed a CD8+ T cell clone recognizing mutant CADPS2 protein and two CD4+ T cell clones reactive against mutant CTSB protein. Of the 11 total neoantigen-reactive TCRs identified, eight persisted in peripheral blood for at least 17 months after treatment.

Overall, Zacharakis et al. show in a single patient that autologous TIL transfer can be highly effective in a tumor with a relatively low mutational burden when the TILs are enriched for neoantigen-reactive T cells that target multiple tumor antigens to prevent immune escape, further opening the door to personalized TIL or TCR therapies.

by Anna Scherer


Zacharakis N., Chinnasamy H., Black M., Xu H., Lu Y.C., Zheng Z., Pasetto A., Langhan M., Shelton T., Prickett T., Gartner J., Jia L., Trebska-McGowan K., Somerville R.P., Robbins P.F., Rosenberg S.A., Goff S.L., Feldman S.A. Immune recognition of somatic mutations leading to complete durable regression in metastatic breast cancer. NatMed. 2018 Jun.

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