Dual Engineering of Human CD8+ T-Cells with WT1-Specific TCR Gene and Chemokine Receptor Gene Transfer Confers Functionally Strengthened Anti-Cancer Reactivity
Authors: Miyazaki Yukihiro , Fujiwara Hiroshi , Ochi Toshiki , Jun An , Shirakata Toshiaki , Nagai Kozo , Okamoto Sachiko , Mineno Junichi , Kuzushima Kiyotaka , Shiku Hiroshi , Yasukawa Masaki 
Published: October 2019

Abstract
Abstract 4295

Background & Purpose:
Currently a novel adoptive therapy with engineered T-cells using cancer antigen-specific T-cell receptor (TCR) gene transfer has attracted the attention as a challenging option for cancer treatment. However, reported efficacy from clinical trials using TCR-gene transfer was generally less than expected. In concurrent with the major issue of generation of mispaired TCRs between introduced and endogenous TCR α/β chains, the less accumulation of adoptively infused engineered T-cells at local tumor site in number could also impede the clinical efficacy. Therefore, for the purpose of sufficient accumulation of adoptively transferred tumor-specific T cells into local tumor sites, we set out to examine the feasibility of dual transduction of cancer antigen-specific TCR gene and chemokine receptor gene into human T-cells.

Methods:
HLA-A*2402-restricted and WT1235-243-specific TCR α/β genes were cloned into a novel GaLV-pseudotyped retroviral vector carrying silencers for endogenous TCR-a/b chains. With retronectin (TakaraBio, Inc.)-coated plate, WT1-specific TCR-α/β genes were introduced into normal CD8+ T-cells with upto more than 50% of WT1-tetramer positivity. On the other hand, using QRT-PCR, we comprehensively screened the chemokine expression profiles of pre-determined HLA-A*2402+ human leukemia cell lines (n=13) and lung cancer cell lines (n=4) which were all WT1-expressing and sensitive to WT1235-243-specific TCR-transduced CD8+ T-cells. Then, the receptor gene for selected chemokine was cloned and inserted into retroviral vector. Expression and function of induced chemokine receptor into Jurkat cells was examined by flowcytometer and transwell experiment. Next, the chemokine receptor gene was introduced into WT1-specific TCR introduced CD8+ T-cells. Migration ability toward target chemokine, and cytotoxicity against the target tumor cells of double-gene transfectans were examined by transwell experiment and 51Cr-releasing assay, respectively. Finally, the combined anti-cancer effect of double-gene transfectants was examined in a novel assay consisted of transwell part and cytotoxicity assay determined by concentration of LDH released from target tumor cells in the bottom well, which were killed by migrated double-gene transfectants from the upper well towards the chemokine produced by target tumor cells.

Results:
CCL2 chemokine was abundantly produced by some of human lung cancer cell lines and leukemia cell lines. Additionally, receptor for CCL2 (CCR2) was not expressed in activated CD8+ T-cells. Thus, we selected CCL2/CCR2 interaction for this system. CCR2 gene was successfully transduced into Jurkat cells and conferred migration activity towards CCL2 and CCL2 producing lung cnacer cell line LK79 and leukemia cell line KH88. CCR2 gene was also successfully introduced into WT1-specific TCR gene trnasduced CD8+ T-cells. The double-gene transfectants successfully migrated towards CCL2-producing LK79 and KH88 cells. Eventually, in our novel assay system, only double genes-transduced CD8+ T cells in the upper-well, but not single WT1-specific TCR gene-transduced CD8+ T-cells, could migrate towards LK79 cells in the bottom-well and efficiently killed LK79 cells.

Background:
Although further investigations are warranted, the dual T-cell engineering of human T-cells with chemokine receptor gene and our novel avidity-enhanced tumor-specific TCR gene may be able to more efficiently accumulate engineered T-cells at local tumor sites, which may enhance the immediate anti-tumor effect of adoptively transferred such engineered T-cells.

Disclosures:
No relevant conflicts of interest to declare.