Abstract A095: New genes for enhancing T cell function in adoptive cell therapy of cancer
Authors: Gross Gideon , Pato Aviad , Weinstein-Marom Hadas , Levin Noam , Margalit Alon , Itzhaki Orit , Besser Michal , Peretz Tamar , Machlenkin Arthur , Lotem Michal 
Published: May 2016

Abstract
A growing number of clinical trials point to the exquisite ability of adoptively transferred T cells to eradicate vast masses of tumor cells, including aggressive metastatic tumors. In one therapeutic approach T cells are derived from tumor biopsies (tumor-infiltrating lymphocytes, or TILs) and express endogenous TCRs. In others T cells are expanded from the peripheral blood lymphocyte pool and redirected to recognize tumor antigens by virtue of exogenous TCR or chimeric antigen receptor (CAR) genes. Although reports on enduring clinical responses are encouraging, a significant portion of patients who do not respond, or respond only partially and transiently, underscore the critical challenge of improving T cell function and survival at the tumor site.
In attempt to improve the clinical efficacy of adoptive cell therapy we created three classes of genetic adjuvants designed to operate autonomously in gene-modified T cells. One class encodes constitutively-active (ca) toll-like receptors devoid of their ligand-binding domain, the other produces membrane-anchored immunostimulatory cytokines and the third encodes a novel homo-oligomerizing configuration of tumor necrosis factor receptors, inducing constitutive ligand-free activation. For gene delivery into human TILs or peripheral blood T cells we have been using electroporation of in-vitro-transcribed mRNA.
Here we present data obtained in preliminary ex-vivo experiments designed to assess the function of caTLR4, membrane IL-2, IL-12 (single chain) and IL-15 and trimeric caCD40 and their level of cooperation. The mere expression of caTLR4 mRNA in polyclonal human CD8 and CD4 T cells induced the production of IFN-γ, triggered the surface expression of CD25, CD69 and 4-1BB and upregulated a panel of cytokines and chemokines. In all samples of anti-melanoma TILs tested, caTLR4 induced a burst of IFN-γ secretion, which usually ceased within 24 hours post-transfection. Furthermore, caTLR4 enhanced the cytolytic activity of TILs and augmented the secretion of IFN-γ, TNF-α and GM-CSF in the presence of autologous, but not mismatched, melanoma for at least 4 days post-transfection. The membrane cytokines were found to act mainly in-cis and each could support the proliferation of human CD8 and CD4 T cells for at least 6 days post-transfection, at a magnitude comparable to a saturating dose of soluble IL-2. Following mRNA electroporation of anti-melanoma TILs, membrane cytokines cooperated with caTLR4 in inducing IFN-γ secretion and upregulating the activation and costimulatory molecules CD25, CD69, 4-1BB and OX40. Different combinations of caTLR4, membrane cytokines and caCD40 mRNA induced spontaneous cytokine secretion in mRNA-transfected TILs, typically acting in a synergistic manner. Even when the spontaneous, initial effect waned, a considerably higher fraction of mRNA-transfected, compared to control-transfected TILs could still respond robustly to autologous, but not allogeneic, melanoma. In the presence of the three classes of adjuvants, transfected short-term cultured ‘young’ TILs revealed exceptional upregulation of 4-1BB, OX40, CD25 and CD28 and an increase in IFN-γ production and no, or only minimal change in the inhibitory receptors PD-1 and CTLA-4. Here, too, the different adjuvants displayed marked synergy, with caCD40 often exerting a prominent effect even when delivered as a single gene. Enhanced killing of autologous melanoma cells by electroporated young TILs was observed up to ten days post-electroporation.
Our findings suggest that this new panel of genetic adjuvants can substantially improve the functional properties of antitumor T cells for extended periods, offering a new tool in all approaches for cancer immunotherapy employing adoptive T cell transfer.
Citation Format: Gideon Gross, Aviad Pato, Hadas Weinstein-Marom, Noam Levin, Alon Margalit, Orit Itzhaki, Michal Besser, Tamar Peretz, Arthur Machlenkin, Michal Lotem. New genes for enhancing T cell function in adoptive cell therapy of cancer. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A095.