Background We constructed RediTac, novel biologics designed to selectively activate and expand virus-specific CD8 T cells (CTL) and redirect them to eliminate cancer cells (figure 1A). RediTac utilize an Fc-fusion protein scaffold to dimerize an HLA-A2 pMHC linked to cancer-targeting scFvs (figure 1B). The pMHC presents a tethered virus peptide to target its cognate TCR and selectively stimulate virus-specific CTL. The scFv redirects virus-specific CTLs to eliminate cancer cells. As a proof-of-concept, we developed RediTac with a pMHC presenting a CMV-derived peptide, NLV, capable of expanding CMV-specific CTLs with potent cytotoxic activity.1 This pMHC was linked to a CD19-specific scFv capable of targeting malignant B cells.
Methods We first confirmed the functionality of the NLV-pMHC domain and CD19-specific scFv domain by demonstrating NLV-CD19 RediTac-mediated expansion of NLV-specific CTLs and selective binding to a CD19 on cells, respectively. The NLV-CD19 RediTac was then evaluated for its capacity to expand and redirect CMV-specific CTLs to eliminate NALM6, a CD19-expressing leukemia cell-line, in vitro and in vivo. To do this, donor PBMC with expanded NLV-specific CTLs were cocultured with NALM6 cells or coinjected with NALM6 cells into spleens of NSG mice. NLV-CD19 RediTac or control treatments were administered and NALM6 cell elimination was measured as a readout for functional activity.
Results NLV-CD19 was detected binding to the surface of CD19+ NALM6 cells (figure 2A,B). Twelve days after NLV-CD19 RediTac treatment, NLV-specific CTLs expanded by >40-fold as compared to untreated controls (figure 3A). After expansion, treatment with fresh NLV-CD19 RediTac induced IFNγ secretion by these expanded CTLs (figure 3B). Expanded CTLs eliminated ~60% of NALM6 cells when treated with NLV-CD19 RediTac during a two-day coculture, but no significant NALM6 cell elimination was observed when treated with control RediTac, either without a targeting scFv or with a pMHC linked to an irrelevant peptide (figure 4A). Five days after expanded CTLs and NALM6 cells were coinjected into NSG mice and NLV-CD19 was administered intravenously, the NALM6 population in the spleens was reduced by >99% when compared to a vehicle control treatment or a no-effector-cell control (figure 4B).
Conclusions NLV-CD19 RediTac expands CMV-specific CTLs and redirects their cytotoxic activity to eliminate NALM6 leukemia cells. We can use the RediTac modular design to expand and redirect other virus-specific T cells and target other cancers with scFvs. These results support RediTac-mediated virus-specific CTL expansion and redirection as a novel immunotherapy strategy to eliminate cancer cells and an effective platform for future treatments.
Acknowledgements We would like to acknowledge Dr. Steve Almo and Dr. Scott Garforth for their work in conceiving and developing the RediTac technology.
Li M, Garforth SJ, O’Connor KE, Su H, Lee DM, Celikgil A, Chaparro RJ, Seidel RD, Jones RB, Arav-Boger R, Almo SC, Goldstein H. T cell receptor-targeted immunotherapeutics drive selective in vivo HIV- and CMV-specific T cell expansion in humanized mice. J Clin Invest. 2021 Dec 1;131(23):e141051
Ethics Approval All human PBMC samples and animal work was done with ethics approval by the Albert Einstein College of Medicine. All participants gave informed consent. This study was approved by the Albert Einstein College of Medicine institution’s Ethics Board; approval number 2017–8116. Animal use was approved by the AECOM Institute of Animal Studies animal use protocol number 00001105.
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