• Preparation of switchable universal chimeric antigen receptors for prostate cancer therapy
  • Sheila Seyed Motahari,1 Shiva Irani,2 Mohammad Ali Shokrgozar,3 Shahriyar Abdoli,4 Zahra Sharifzadeh,5,*
    1. aDepartment of Immunology, Pasteur Institute of Iran, Tehran, Iran and Department of Molecular Genetics, University of Tehran Science and research Branch, Islamic Azad University, Tehran, Iran
    2. Department of Molecular Genetics, University of Tehran Science and research Branch, Islamic Azad University, Tehran, Iran
    3. National Cell Bank of Iran, Pasteur Institute of Iran, Tehran, Iran
    4. School of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran
    5. Department of Immunology, Pasteur Institute of Iran, Tehran, Iran


  • Introduction: T cells expressing chimeric antigen receptors (CARs) are promising cancer therapeutic agents, with the prospect of becoming the ultimate smart cancer therapeutics. A traditional chimeric antigen receptor has a fixed design, and one type of CAR T cells can only target one antigen epitope. This rigid design limits clinical application and can lead to exceptionally high manufacturing cost. In universal CARs, the antigen recognition domain is split from the signaling domain of a conventional CAR, so the target antigen can be switched or re-directed without re-engineering the CAR T cells. The UniCAR platform has a modular design including a signaling module that binds to a specific epitope on the switching molecule and a switching module with an antigen-binding domain and a switching epitope specifically recognized by the signaling module. In this study the switchable modular CAR T cells will be produced using dimerizing leucine zippers.
  • Methods: UniCAR constructs were designed using CLC software and synthesized by Biomatik company. The switching module (ZipNb) sequence contains leucine zipper, anti-PSMA nanobody as the targeting moiety, as well as c-myc and his-tag sequences for detection and purification. The signaling module (ZipCAR) contains leucine zipper, CD28 extracellular and costimulatory domains and CD3ς intracellular signaling domain. Both constructs were validated by restriction enzymes and sequencing. The ZipNb gene was subcloned into pET28a vector and transformed into Rosetta DE3 cells. Then the ZipNb expression was induced by IPTG and the expressed protein was analyzed by SDS-PAGE and western blot with mouse anti-c-myc antibody. Protein purification was performed by Ni-NTA column, then the protein dialysis was performed. Two cell lines, LNCaP (PSMA +) and Du145 (PSMA-), were used for nanobody binding evaluation. The ZipCAR gene was subcloned into pLOX vector and transformed into Top10 cells. Extraction of ZipCAR plasmid was performed by Qiagen Maxi purification kit. By reverse transfection procedure, ZipCAR, psPAX and pMDG2 plasmids were tranfected to LentiX 293 T cell line to produce lentiviral particles. These transfected cells were incubated for ~3 days post-transfection. Then, the culture medium (virus-containing supernatants) was harvested and used to transduce HEK293 and target T cells. Finally, transduced Jurkat T cells were evaluated for the universal CAR T cell expression.
  • Results: In the present study, we prepared uiversal CAR T cells to targeted prostate cancer cells. The ZipNb containing anti-PSMA nanobody was expressed in different conditions. Our findings showed that the highest ZipNb expression was observed in Rosetta DE3 after 16 hours post-induction by 1mM IPTG, and in 37 ˚C. SDS-PAGE and western blot analysis proved the expression of the desired targeting module with the molecular weight of about 27 KDa. Flow cytometry results confirmed the ZipNb binding to PSMA antigen on target prostate cancer cells. The ZipCAR containing lentivirus were concentrated, titrated and used to transduce HEK293 and and Jurkat cell lines. Flow cytometry results showed the ZipCAR expression on the surface of transduced cell lines.
  • Conclusion: The modular UniCAR designs make it possible to re-engineer a variety of switchable and programmable UniCARs .The switch molecule makes a synapse between the CAR T cells and the target tumor cells. For CAR T immunotherapy to realize its potential target in solid tumors, addressing tumor heterogeneity and enhancing its safety profile are the main problems for its use in the clinic. An adaptable system such as modular CAR T cells conceivably could address these issues by tailoring CARs to a patient’s specific cancer and adapting treatment using a toolkit of adaptor targeting elements. The UniCAR T cells developed in this study could be promising tools to target different cancer antigens. However, functional assays are needed to prove the anti-tumor effect of these UniCAR T cells on prostate tumor cells.
  • Keywords: Cancer immunotherapy, Universal CAR T, PSMA, Prostate cancer, Nanobody.