Novel Cellular Immunotherapy Using Allogeneic Vγ9/δ2-T Cells Gene-Modified to Express HTLV-1 P40Tax-Specific TCR for the Treatment of Adult T Cell Leukemia
Authors: Fujiwara Hiroshi , Okumura Satoshi , Miyahara Yoshihiro , Wan Linan , Tawara Isao , Jo Tatsuro , Tanaka Yuetsu , Tanaka Yoshimasa , Ikeda Hiroaki , Shiku Hiroshi 
Published: November 2019

Background: Adult T-cell leukemia/lymphoma (ATL) is a refractory peripheral T-cell malignancy caused by human T-lymphotropic virus type 1 (HTLV-1) infection. Although only allogeneic hematopoietic stem cell transplantation (allo-HSCT) displaying the graft-vs. ATL (GvATL) can bring durable remission, allo-HSCT is largely ineligible for newly diagnosed ATL patients due to disease aggressiveness and advanced age-related conditions. Thus, a novel treatment with safety and efficacy instead of allo-HSCT still remains an unmet need, and a cellular immunotherapy using TCR or CAR gene-modified immune cells exerting GvATL could be such an option. However, to generate those effector cells from autologous T cells of heavily pre-treated ATL patients faces many obstacles. To circumvent those hurdles, an employment of unconventional allogeneic Vγ9/δ2-T cells which are potentially free from the risk of GVHD could provide greater treatment opportunity for ATL patients due to highly extended donor availability. Taking above, here, we have newly devised an adoptive immunotherapy using a novel HTLV-1 p40Tax-specific TCR gene-modified allogeneic Vγ9/δ2 T cells against ATL.
Methods: After written informed conscent, we firstly established novel HLA-A24 restricted TCR-α/β genes from HTLV-1 P40Tax301-309 (SFHSLHLLF)/HLA-A24 tetramer-positive peripheral CD8+T-lymphocytes of ATL patinets in durable remission using a single cell cloning method. Then, we confirmed that T cells gene-modified with these TCR-α/β genes exerted the epitope-specific and HLA-A24-restricted responses. Next, in order to achieve highly stable expression of this TCR-α/β heterodimer on gene-modified Vγ9/δ2-T cells, we newly developed a retroviral vector co-expressing TCR-α/β and CD8 α/β genes using self-cleaving P2A and E2A peptides. Using this vector, allogeneic Vγ9/δ2-T cells from healthy donors numerously expanded with high purity in our novel culture system were subjected to gene-transfer to express relevant TCR α/β complex. Thereafter, we asssessed target-reactive cytokine production and cytocidal activity mediated by those gene-modified allogeneic Vγ9/δ2-T cells both in vitro and in vivo. Finally, we additionally assessed a potential risk of GVHD using intravenous administration of another TCR gene-modified Vγ9/δ2 T-cells in vivo.
Results: To start with PBMCs from healthy donors, allogeneic Vγ9/δ2-T cells were stably multiplied greater than thousandfold with a quite high purity (≥95%) using our novel bisphosphonate derivative PTA (tetrakis-pivaloyloxymethyl2-(thiazole-2-ylamino) ethylidene-1,1-bisphosphonate) combined with both 25 ng/ml of IL-7 and IL-15 in culture for 8 to 10 days. The stable expression of introduced TCR α/β heterodimer on Vγ9/δ2-T cells were successfully achieved by co-expression of CD8 α/β molecule. Those gene-modified Vγ9/δ2-T cells successfully recognized target peptide (SFHSLHLLF) in an HLA-A24 restricted fashion, and similarly demonstrated a cytocidal activity both in vitro and in vivo against HLA-A24 positive HTLV-1 infected cell lines (TL-Su and ILT#Hod), but not HLA-A24-negtive/Tax-positive cell line ILT#37 or HLA-A24-positve/Tax-negative cell line ATN-1. Furthermore, intravenously administered those TCR gene-modified Vγ9/δ2-T cells quickly and durably eradicated luciferase-gene modified TL-Su cells, but not ATN-1 cells in xenografted immunodeficient (NOG) mice, examined by in vivo imaging system. Finally, infused HLA-A2 restricted and NY-ESO-1 specific TCR (G50) gene-modified Vγ9/δ2-T cells exerted durable antitumor activity without causing GVHD using NOG mice xenografted with HLA-A2 positive melanoma cell line cells (NW-MEL-38).
Conclusions: Our preclinical observations here obviously demonstrated the potential utility of TCR-α/β gene-modified allogeneic Vγ9/δ2-T cells for the treatment of ATL without causing GVHD. Further studies regarding biological behaviors of HTLV-1 Tax specific TCR-α/β gene-modified allogeneic Vγ9/δ2-T cells following target recognition in vivo are warranted, however, based on these lines of evidence and currently conducting assessments using clinical samples, we are planning to launch a novel clinical trial, particularly focusing on the applicability of HLA partially matched relative donors, as the source of gene-modified allogeneic Vγ9/δ2-T cells, which could highly extend the donor availability.

Disclosures
Fujiwara: BrightPath Biotherapeutics, Co.,Ltd.: Other: member of the department endowed by BrightPath Bio. Okumura:BrightPath Biotherapeutics, Co.,Ltd.: Other: member of the department endowed by BrightPth Bio.. Miyahara:BirghtPath Biotherapeutics, Co., Ltd.: Other: member of the department endowed by BrightPath Bio.. Wan:BrightPath Biotherapeutics, Co., Ltd.: Other: member of the department endowed by BrightPath Bio.. Tawara:Astellas Pharma: Research Funding; Ono Pharmaceutical: Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding. Shiku:BrightPath Biotherapeutics, Co., Ltd.: Other: Chair of the department endowed by BrightPath Bio..