Forced Expression of CC Chemokine Receptor 2 Enhances Anti-Cancer Reactivity Mediated by T Lymphocytes Beforehand Redirected Toward WT1 Inside the Tumor Microenvironment
Authors: Asai Hiroaki , Fujiwara Hiroshi , Ochi Toshiki , An Jun , Shirakata Toshiaki , Nagai Kozo , Miyazaki Yukihiro , Mineno Junichi , Kuzushima Kiyotaka , Shiku Hiroshi , Yasukawa Masaki 
Published: October 2019

Abstract
Abstract 2059

Background & Purpose:
Redirected T-cell based adoptive therapy using cancer antigen-specific T-cell receptor (TCR) gene transfer has proven promise, however its clinical efficacy still remains unsatisfactory. The less accumulation in number of infused redirected T cells at the local tumor site is one of the causes. In order to accumulate those tumor-responsive T cells inside tumor microenvironment, chemokine produced by tumor cells and/or tumor associated cells is an attractive target. In this study, we examined the feasibility of CC chemokine receptor 2 (CCR2) gene transfer into T cells beforehand redirected toward WT1 in order to enhance the anti-cancer reactivity, both in vitro and in vivo.

Methods:
HLA-A*24:02-restricted and WT1235–243-specific TCR-a/b genes were introduced into normal CD8+ T cells using our novel retroviral TCR-gene expression vector encompassing silencers for endogenous TCRs (WT1-si-TCR vector). mRNA expression of total 11 chemokines expressed by 10 human lung cancer cell lines was examined using QRT-PCR, then CCL2 (variably produced in 7 out of 10 examined cell lines) and the small lung cancer cell line, LK79 which abundantly produced CCL2 was chosen for the proof of concept. Cloned CCR2 gene was retrovirally introduced into Jurkat cells, Jurkat/MA cells and normal CD8+ T cells similarly redirected beforehand using WT1-siTCR vector. Introduced CCR2 was validated using flow cytometer and transwell experiments. Cytotoxicity was examined using standard 51chromium release assay. Cooperative functionality composed of CCL2-directinality and WT1-specific antitumor cytotoxicity mediated by those double gene transfectants was examined in vitro; values of LDH released from destroyed LK79 cells in the bottom well by migrated double gene transduced CD8+ T cells from the upper well were measured. Antitumor reactivity in vivo was assessed using xenograft mouse model using luciferase-transduced LK79 cells (LK79-luc). Direct effect of CCL2 ligation on WT1-TCR signaling in double gene transfectant was assessed using luciferase assay with double gene transduced TCR− Jurkat/MA cell line, which stably expresses hCD8a and NFAT-luciferase reporter genes (Jurkat/MA/CD8a/luc; kindly provided by Prof. Erik Hooijberg, Netherlands).

Results:
CCL2 sensitivity was successfully introduced by CCR2 gene transfer; CCR2 transduced Jurkat successfully directed toward CCL2 producing cell line, LK79 cells. CCR2 gene transduction did not impede WT1-specific cytotoxicity mediated by CD8+ T cells beforehand redirected using WT1-siTCR gene transfer, rather double gene transduction cooperatively endowed those transfectants with CCL2 sensitivity and WT1-specific cytotoxicity against LK79 cells. Furthermore, in vivo assay using xenograft mouse model, growth of subcutaneously inoculated LK79-luc cells was more efficiently suppressed by those double gene transduced CD8+ T cells than WT1-siTCR single gene trnasfectants, in particular, immediately after adoptive transfer. Finally, CCL2 synergistically enhanced the magnitude of cognate peptide evoked WT1-specific TCR signaling in a dose dependent manner. Even without WT1 peptide ligation, such TCR signaling was similarly evoked by CCL2 to some extent.

Conclusion:
In this study, our results demonstrate that forced expression of CCR2 on CTL beforehand redirected toward WT1 enhanced its anti-cancer reactivity both in vitro and in vivo. This in vivo enhancement of antitumor reactivity may be caused by increased number of effector cells and enhanced WT1-TCR signaling generated both by CCL2 in the local tumor microenvironment. Although further studies are warranted, CCR2 gene transfer into redirected WT1-specific tumor-reactive CTL may be feasible for the treatment of human cancers.

Disclosures:
No relevant conflicts of interest to declare.