Abstract 1066: PD-1 expressing ex-vivo cultured tumor infiltrating lymphocytes confers tumor reactivity in cervical cancer: Possible implication for adoptive cell therapy
Authors: Sayem Mohammad A. , Sunthamala Nuchsupha , Yaguchi Tomonori , Iwata Takashi , Fujita Tomonobu , Kawakami Yutaka 
Published: September 2020

Adoptive cell transfer (ACT) of ex-vivo tumor-infiltrating lymphocytes (TILs) has been shown to achieve durable clinical response in cervical cancer (CC). However, objective response is still limited. One of the ways to improve the efficacy of ACT is to enrich tumor reactive TILs for adoptive transfer. In this study, we aim to identify tumor-reactive CD8+ TILs by their ability to recognize antigen presenting cells loaded with pools of human papillomavirus (HPV) E6/E7 derived proteins, HPV E6/E7 overexpressing cells, or autologous tumors.

TILs were generated from CC tumor specimens. Tissues were minced into 1-2 mm fragments and cultured in 6000 IU per mL of rhIL-2. Autologous dendritic cells (DCs) were generated from CD14+ cells isolated from patients' peripheral blood mononuclear cells (PBMCs). Autologous fibroblast cells were transfected with the E6/E7 genes. In some cases, autologous tumor cells were generated using monolayer or matrigel based 3D culture system. DCs loaded with pools of peptides derived from HPV16 or HPV18 E6/E7 oncoproteins, E6/E7 transfected autologous fibroblast cells, and autologous tumor cells were cocultured with TILs, and IFN-γ production was measured by enzyme-linked immunosorbent assay (ELISA). MHC class I or II restriction was evaluated using blocking antibodies. Knockdown of HPV gene was performed by using small interfering RNA (siRNA). Tumor-reactive and non-reactive T cells were then analyzed for the surface expression of PD-1, TIM-3, LAG-3, TIGIT, CD39, CD103, and CD69 by flow cytometry. In certain cases, T-cells were sorted using flow cytometry and cocultured with autologous tumor cells.

We were able to generate TILs from 76% of the CC patients (n=101). The cultured TILs were reactive to DCs loaded with pools of peptides derived from HPV16 or HPV18 E6/E7 oncoproteins, autologous fibroblast cells transfected with HPV16 E6/E7 gene, and autologous tumor cells. IFN-γ was produced by TILs in either MHC class I or class II restricted manner. Production of IFN-γ was inhibited when HPV16 E6/E7 genes in the stimulator cells were silenced by siRNA. We found that only expression of PD-1 among tested molecules was significantly higher on tumor-reactive TILs than non-reactive TILs. As expected sorted PD-1 positive T cells produced significantly higher IFN-γ compared to PD-1 negative T cells.

These data indicate that tumor-reactive CD8+ T-cells were enriched in the PD-1 positive T cells. Use of PD-1 positive tumor-reactive TILs may improve the efficacy of ACT for patients with CC compared with bulk culture TILs.

Citation Format: Mohammad A. Sayem, Nuchsupha Sunthamala, Tomonori Yaguchi, Takashi Iwata, Tomonobu Fujita, Yutaka Kawakami. PD-1 expressing ex-vivo cultured tumor infiltrating lymphocytes confers tumor reactivity in cervical cancer: Possible implication for adoptive cell therapy [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 1066.