T Cell Receptor Gene Transfer to Virus-Specific T Cells for Cellular Anti-Tumor Immunotherapy.
Authors: Griffioen Marieke , van Egmond H.M. Esther , van der Hoorn Menno A.W.G. , Hagedoorn Renate S. , Kester Michel , Willemze Roelof , Falkenburg J.H. Frederik , Heemskerk Mirjam 
Published: April 2020

Abstract
Patients with relapsed hematological malignancies after allogeneic stem cell transplantation (alloSCT) can be successfully treated by donor lymphocyte infusions (DLI). Since DLI consists of a variety of T cells with different specificities, the benificial anti-leukemic effect of DLI is often accompanied by Graft-versus-Host Disease (GvHD). Genetic modification of T cells to express T cell receptors (TCR) with defined anti-tumor specificity would be an attractive strategy to specifically eradicate the malignant cells without induction of GvHD. We previously demonstrated that transfer of the minor histocompatibility antigen HA-2 specific TCR to CMV specific T cells led to the generation of T cells with dual specificity for CMV as well as HA-2. CMV and EBV specific T cells are ideal target cells for TCR gene transfer, since the majority of human individuals have high frequencies of these T cells due to latent persistence of CMV and EBV. In addition, based on their virus specificity, these T cells do not induce GvHD in an alloSCT setting, and we hypothesize that due to frequent encounter with viral antigens, TCR transferred virus specific T cells will survive for a prolonged period of time in vivo. The aim of this study is to develop a clinical grade method for the generation of TCR transduced virus specific T cells for cellular immunotherapy. CMV and EBV specific T cells were isolated from healthy individuals using pentamers in combination with clinical grade available anti-biotin magnetic beads. Isolation by pentamer-coated beads induced stimulation, expansion and efficient transduction of virus specific T cells, leading to the generation of cell lines with high frequencies of virus specific (&gt;80%) and transduced (20–40%) T cells. T cells were transduced with multi-cistronic retroviral vectors encoding the α and β chains of the HA-2 TCR linked by an IRES or 2A-like sequence. No differences in transduction efficiency and TCR surface expression were observed between the IRES and 2A-like vectors. The transduced virus specific T cells were shown to exhibit dual specificity and tetramer staining of the introduced TCR correlated with specific lysis of target cells endogenously-expressing HA-2. Furthermore, variation in surface expression of the introduced TCR was observed between T cells with different virus specificities. T cells directed against the HLA-A1 epitope of CMV-pp50, for example, efficiently expressed the HA-2 TCR, whereas T cells specific for the HLA-B8 epitope of EBV-EBNA-3A did not express the introduced TCR. Functional analyses demonstrated that TCR-transduced pp50 specific T cells were dual specific, recognizing HA-2 as well as pp50 positive target cells, whereas TCR-engineered EBNA-3A specific T cells were primarily EBNA-3A specific. The efficiency of surface expression of the transferred TCR was shown to be determined by intrinsic properties of the TCRs, illustrating that for TCR gene transfer purposes TCRs need to be selected that exhibit high competition potential, whereas recipient T cells need to express endogenous TCRs with low competition potential. For clinical application, TCRs will be transferred to virus specific T cells selected for their capacity to efficiently express the introduced TCR without loss of virus specificity. The safety, clinical and immunological efficacy of TCR gene transfer to virus specific T cells as cellular anti-tumor immunotherapy after alloSCT will be investigated in a clinical phase I/II study.