Improved Anti-Leukemia Activities of Adoptively Transferred T Cells Expressing Bites
Authors: Liu Xiaojun , Barrett David M. , Jiang Shuguang , Fang Chongyun , Grupp Stephan A. , June Carl H. , Zhao Yangbing 
Published: October 2019

Abstract
Bispecific T cell engagers (BiTEs) that are specific for both a surface target antigen on cancer cells and CD3 on T cells have been used to engage T cells for cancer therapies. However, the development of BiTEs to treat cancer patients still faces enormous challenges. BiTEs need to penetrate well into tumor tissue, where the reactivate tumor resident T cells might have already been tolerized or energized by tumor microenvironments. In addition, the therapeutic potential of exogenously administered BiTEs could be limited by their short half-life and their dissociation from the triggering receptor within a relatively short period of time. RNA electroporation or lentiviral transduction of BiTEs into T cell could potentially enhance cancer treatment efficiency of both BiTEs and T cell adoptive immunotherapy of leukemia.
In this study, we examined whether BiTEs could be produced by aCD3/aCD28 Dynal bead stimulated and expanded T cells that were electroporated with mRNA encoding BiTEs (Bis-RNA) and engage T cells specifically targeting tumors, both in vitro and in an aggressive leukemia animal model. We found that T cells with Bis-RNA encoding a CD19-CD3 BiTEs (Blinatumomab) could as efficiently recognize tumor as those T cells with CD19 CAR. We also discovered that the BiTEs secreted by Blinatumomab Bis-RNA transfected T cells could enable non-transfected T cells to specifically recognize CD19 positive tumors in vitro. Moreover, T cells with Bis-RNA showed increased sensitivity of tumor lysis and prolonged functional persistence, comparing to the CD19 CAR T cells. Upon specific antigen stimulation, T cells transfected with Bis-RNA secreted higher cytokines, showed more potent proliferation and less co-stimulation dependency, as well as more resistance to Treg and PD1 induced T cell suppression, than the T cells expressing CD19 CAR. In a Nalm6 leukemia mouse model, the anti-tumor activity of Blinatumomab Bis-RNA T cells showed more sustained anti-tumor activity comparing with CD19 RNA CAR T cells. A fully human CD19 RNA BiTE construct was successfully generated utilizing scFvs of both CD19 and CD3 fully human antibodies and the T cells transferred with this fully human CD19 BiTEs showed similar anti-tumor activities both in vitro and in Nalm6 leukemia model as Blinatumomab BiTEs.
To maximize the dose of Bis-RNA electroporated T cells, we used the rapid T cell expansion protocol (REP) that uses OKT3 and IL-2 to stimulate T cells with irradiated allogeneic PBMCs as feeders. We found that this approach expands T cells up to 1000-fold in 10 days, which is 10 times more than aCD3/aCD28 Dynal bead stimulation. The T cells expanded by REP showed enhanced anti-tumor activities in vitro for both CD19 RNA CAR T or Bis-RNA T cells, as evidenced by increased CD137 up-regulation when the T cells were stimulated by CD19+ cell lines, and increased lytic ability, especially when the RNA input dose was limited. Moreover, CAR expression was higher for REP T cells than aCD3/aCD28 Dynal Beads T cells at different times after CAR RNA electroporation, indicating stronger and more persistent anti-tumor activities of REP T cells. To test if the improved anti-tumor activity of REP T cells evidenced in vitro correlates with their in vivo activity, T cells from the same normal donor were stimulated and expanded by either aCD3/aCD28 Dynal beads or REP and electroporated with CD19 CAR RNA or Blinatumomab Bis-RNA. The therapeutic results of those T cells in the NSG/Nalm6 model showed that better control of tumor growth was achieved by using REP CD19 CAR RNA T cells compared to CD19 CAR RNA T cells prepared by bead stimulation (P<0.01). Furthermore, using REP Bis-RNA T cells further improved the treatment results (P<0.05). It was found that treating NALM6 leukemia using REP Bis-RNA T cells led to significantly prolonged survival of the treated mice, which was comparable to what we have seen in the same mouse model treated with CD19 CAR lentivirally transduced T cells.
In sumary, BiTEs could be efficiently produced and delivered by T cells to overcome the limitation of short half-life when using exogenously administered BiTEs. Introducing Bis-RNA into T cells for adoptive cell therapy, which not only takes the advantages of CAR based tumor antigen recognition that avoids most common tumor immune evasion mechanism, but also avoids the potential CAR based T cell activation defects, could be developed into a next generation adoptive immunotherapy.

Disclosures
Liu: University of Pennsylevania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Grupp:Novartis: Consultancy, Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Zhao:Novartis: Patents & Royalties, Research Funding.