Optimization of the HA-1-Specific T Cell Receptor for Gene Therapy of Hematological Malignancies.

Abstract 4093

Poster Board III-1028

Relapsed hematological malignancies after HLA-matched allogeneic stem cell transplantation (allo-SCT) are treated by donor lymphocyte infusion (DLI), inducing long-lasting complete remissions. However, treatment of relapsed hematological malignancies after allo-SCT with DLI is associated with induction of graft versus host disease (GvHD). It has been demonstrated that T cells recognizing minor histocompatibility antigens (mHags) selectively expressed on hematopoietic cells mediate anti-leukemic reactivity after allo-SCT without causing GvHD. mHags are derived from genetically polymorphic proteins that can be differentially expressed between donor and recipient. The mHag HA-1 is presented in the context of HLA-A2. The HA-1 tissue distribution is restricted to hematopoietic cells and carcinomas, making it an attractive target antigen to treat hematological malignancies relapsing after allo-SCT when the patient is HA-1⁺ and the donor is HA-1^-. Therefore, adoptive transfer of HA-1-TCR gene modified T cells might be an attractive strategy to separate GvHD from graft versus leukemia effect (GvL). For optimal anti-leukemic reactivity, high expression of introduced TCRs and persistence of the gene modified T cells is important. Based on the previously reported low HA-1-TCR expression on HA-1-TCR modified T cells, optimization of the strategy is required. Several strategies to improve expression of the introduced TCR have been described. Protein expression of the TCR chains can be enhanced by codon optimization. In addition, preferential pairing facilitated by introduction of an extra disulfide bond in the constant regions of the TCR chains can increase the cell surface expression of the transferred TCR. Another strategy based on the fact that TCRs differ in their capacity to compete for cell surface expression, is to select recipient T cells with weak competitor phenotypes. In this study, we investigated the cause of low HA-1-TCR expression after gene transfer, and used the different strategies to increase HA-1-TCR expresssion. To study whether low HA-1-TCR expression was due to inefficiency of the TCRα and β chains to pair, TCR-deficient jurkat cells were transduced with the individual TCRα and TCRβ chains in combination with 17 different TCRα chains and TCRβ chains. Results indicated that low HA-1-TCR expression was not due to inefficient pairing of the HA-1-TCR chains, but caused by low HA-1-TCRβ chain expression on the cell surface. To investigate whether low cell surface expression of the HA-1-BV6S4 chain was due to intrinsic properties, the CDR1 and CDR3 region of the HA-1-TCR BV6S4 chain were exchanged with the CDR1 and CDR3 region of the HA-2-TCRβ (BV6S2) chain. We demonstrated that exchange of the HA-1-TCRβ CDR1 region with the HA-2-TCRβ CDR1 region resulted in improved TCR-expression, however, the HA-1-specificity was completely abolished, indicating that the HA-1-TCRβ CDR1 region is crucial for HA-1-specificity. Furthermore, since there is exclusive TCRBV6S4 chain usage of HA-1-specific T cells, we were unable to select for another HA-1-TCR for clinical use, and were pressed to optimize the HA-1-TCRβ chain. Both codon optimization of the HA-1-TCR chains aiming at improving protein expression and inclusion of cysteine residues in the HA-1-TCR chains aiming at inducing preferential pairing resulted in a significant increase in HA-1-TCR expression. Combining the two strategies increased the HA-1-TCR expression even more, resulting in 70% and 35% of tetramer positive HA-1-TCR transferred weak competitor and strong competitor T cells, respectively. In addition, the HA-1-TCR engineered T cells were able to efficiently recognize target cells that endogenously process and present HA-1, independent of whether the recipient T cells were strong or weak competitor T cells. These results illustrate that engineering of the HA-1-TCR by codon optimization and introduction of an extra cysteine bond resulted in high numbers of high-avidity HA-1-TCR in any T cell of choice irrespective of the properties of the endogenous TCR.