Synthesis of chimeric T cell receptor with OX40 co-stimulatory receptor targeted against breast cancer cells

Chimeric antigen T cell receptors provide a good approach for adoptive immunotherapy of cancer. In this new kind of chimeric T cell receptor, nanobodies are replaced as variable fragment of T cell receptor. Nanobodies (VHH) are the smallest fragments of antibodies that have great homology to human VH and low immunogenic potential. VHH-hing-CD28-CD3و construct was made in our laboratory. Tumor cells rarely provide costimulatory signals and hence CAR receptors that transmit just a CD3و signal can only initiate target cell killing and interferon γ release and fail to induce full activation. Although incorporation of a CD28 component results in IL-2 (Interlukin 2) release and limited proliferation, T cell activation remains incomplete. To optimize CAR signaling, tripartite endodomains were constructed which consist of CD28-OX40-CD3ζ. During T cell activation, OX40 transmits a potent and prolonged T cell activation signal and it is crucial for maintaining an immunological response. We designed overlapping primers that contain 60% of OX40 sequence, then, CD28-Ox40-CD3ζ region was synthesized by SOE-PCR, consequently, it was replaced with CD28-CD3ζ region in VHH-Hing-CD28-CD3ζ construct. Then, this construct was transfected in Jurkat T cells, so IL-2 secretion and T cells proliferation were increased. CD28-OX40-CD3ζ tripartite cytoplasmic domain provided a full complement of activation, proliferation, and survival signals for enhanced anti-tumor activity. With IL-2 and semi-quantitative RT PCR, it is concluded that mRNA expression of pCDNA-1-hinge-CAR-OX40 and pCDNA-2-hinge-CAR-OX40 constructs are the same but the function of pCDNA-2-hinge-CAR-OX40 construct is more than the others.