*8.2. Turning Lymphocytes into HLA-Independent Killers with Virus-Deployed Bispecific T-Cell Engagers*

Bispecific T-cell engagers (BiTEs) crosslink endogenous CD3 on T-cells to surface targets on cancer cells, creating an activating pseudosynapse through clustering and

leading to rapid and efficient target-specific cytotoxicity. The T-cells then detach from the target cell and can bind to a new target cell, earning them the epithet serial killer T-cells. This powerful approach is reminiscent of converting endogenous T-cells into antigenspecific chimaeric antigen receptor (CAR) T cell-like cells in situ, recognising any chosen surface antigen.

BiTEs are difficult to deploy through conventional intravenous delivery because of their short plasma half-life, and fine tuning of affinity of the CD3 binding arm is crucial to prevent sequestration to T-cells outside the tumor. When it comes to the choice of the antigen-binding arm for a systemic therapy, there is a trade-off between using broadly expressed antigens that could mediate on-target off-disease side effects, or highly cancerselective antigens that may not be universally expressed throughout the tumor. Expressing BiTES locally and exclusively in the tumor microenvironment from an oncolytic virus may avoid these complications, allowing for a far broader range of target and effector arms to be considered. A constantly renewed supply of BiTEs might be expected to synergize with newly arriving T-cells encouraged to the tumor through chemokines or oncolysis.

Several groups have now demonstrated the successful delivery of BiTEs using a range of oncolytic viruses. The earliest approaches used oncolytic vaccinia and adenovirus to express BiTEs targeting receptors on the cancer cell surface, allowing cancer cells to be targeted simultaneously by two distinct cytotoxic entities—the oncolytic virus and the BiTEtargeted T-cell [77–79]. It would be difficult to use any other strategy to target these surface markers with the anatomical selectivity provided by oncolytic viruses. However, despite the potency and selectivity of the approach, it was soon considered suboptimal because the rapid cytotoxicity can decrease the pool of therapeutic viruses by eradicating the tumor cells that produce them. Accordingly, more recent innovations have explored combining the direct cytotoxicity of oncolytic viruses against cancer cells with BiTEs that retarget endogenous T-cells to attack other cellular components of the tumor microenvironment. To date, these have included tumor-associated macrophages via targeting to folate receptor b or CD206 [80], or tumor-associated fibroblasts via fibroblast activation protein (FAP) [81,82]. This latter approach forms the basis of an ongoing clinical trial which uses an oncolytic adenovirus to express a FAP BiTE alongside alpha interferon (which should increase immune stimulation and may restore HLA function), together with two chemokines intended to recruit T-cells into the tumor (Clinical Trials identifier NCT04053283). Finally, harnessing endogenous T-cells in this way should provide greater therapeutic potential for heterogeneous solid tumors than exogenously applied CAR T-cells, since every T-cell within the tumor may be redeployed against the chosen target antigen, whereas CAR T-cells will always be a subset of those engrafting into solid tumors.
