Bivalent nanobodies anchoring to tumor extracellular matrix extend localized IL-12 produced by thermogenetic CAR T cells

Introduction: : Chimeric antigen receptors (CAR) T cell therapy has yet to achieve as robust outcomes in solid tumors as in hematological cancers. One of the major hurdles for CAR T cell therapy is the immunosuppressive tumor microenvironment (TME). Potent proinflammatory cytokines, such as interleukin-12 (IL-12), have the potential to overturn such barrier and to reinvigorate antitumoral activities of CAR T cells. However, due to the potent pleiotropic effects of IL-12 on the immune system, a series of studies have demonstrated that systemic exposure of IL-12 elicits severe immunotoxicity. Therefore, to fully leverage the benefits of IL-12's immune-modulating activities, we aim to impose tight control: produce and anchor IL-12 within tumors to improve the therapeutic outcome. To locally produce IL-12, we engineered CAR T cells with temperature sensitive genetic switch (TS) and photothermally induce the production at the tumor site. To anchor IL-12 within tumor, tumor extracellular matrix (ECM) component, fibronectin extra domain B (FN-EDB), has gained popularity as a tumor target due to its high abundance in tumor. We fused IL-12 with nanobodies (VHH) with high binding affinity toward tumor ECM component, fibronectin extra domain B (FN-EDB). The spatial thermal control of CAR T cells producing tumor anchored IL-12 will thereby prolong the retention in tumor, and we foresee that this approach will augment the efficacy of CAR T cell therapy against solid tumor while maintaining the safety profile.

Materials and

Methods: : We engineered primary mouse T cells with retrovirus encoding for TS with various constructs with IL-12, NanoLuc luciferase (NLuc) and VHH against FN-EDB (EDB VHH) or non-binding VHH (nb VHH) with HER2 CAR (Fig 1a). To validate the binding of different constructs against FN-EDB in vitro, we used thermal cycler to heat the mouse T cells to 42°C and collected the conditioned media 1 day post heating. The luminescence signal from different constructs was normalized and then incubated on high binding plate coated with recombinant FN-EDB. The plate was washed with PBS and In Vivo Imaging System (IVIS) was used to measure the NLuc signal in each well (Fig. 1b). To validate the functionality of IL-12, we stimulated freshly activated mouse CD3+ T cells with conditioned media collected 1 day post heating of engineered mouse T cells (collected as described above). We then intracellularly stained for phosphorylated signal transducer and activator of transcription 4 (pSTAT4) and assessed its expression by flow cytometry (Fig. 1c-d). To evaluate the anchoring of IL-12 in tumor in vivo, C57BL/6 mice were inoculated with 5x105 B16 HER2 tumor on the left flank. 9 days post inoculation, mice received an adoptive cell transfer (ACT) of 5x106 TS. EDB VHHx2 NLuc IL-12 HER2 CAR T cells (Fig. 1e). Photothermal laser was used to heat the tumor site to 42°C for 20 minutes. NLuc activity was measured using IVIS at different timepoints post heating (Fig. 1f-g).

Results, Conclusions, and Discussions:: We hypothesized that IL-12 with tumor ECM binders, EDB VHH, secreted by thermally sensitive CAR T cells will prolong the retention at tumor site and therefore enhance tumor immunotherapy (Fig. 1a). We first evaluated the binding efficiency of the protein constructs with different numbers of EDB VHH (Fig. 1b). Comparing with monovalent EDB VHH, the bivalent construct shows a 14-fold increase in NLuc retention, resulting in >800-fold over background anchoring against plate-bound recombinant FN-EDB (Fig. 1b). We next validated the functionality of IL-12 in this compound protein construct by stimulating freshly activated mouse T cells with the conditioned media containing EDB VHHx2 or nb VHHx2 NLuc IL-12. Stimulated mouse T cells were fixed and permed, and intracellular pSTAT4 were evaluated post stimulation (Fig. 1c-d). Elevated pSTAT4 level demonstrated that IL-12 functionality was maintained in this design. We next evaluated in vivo retention efficiency of this approach in an immunocompetent murine model with B16 HER2 tumor. Following ACT of TS. EDB VHHx2 NLuc IL-12 HER2 CAR T cells and photothermal heating (Fig. 1e), we observed a long retention in tumor, with signal observed ~1 week post heating (Fig. 1f-g).
Taken together, our studies demonstrated thermally controlled local production of IL-12 with bivalent EDB VHHs by mouse CAR T cells can effectively prolong the IL-12 retention in B16 tumor model in vivo. This approach combines the benefits of precise control of drug payload production site and the anchoring ability of the payload towards tumor, showing potential of enhancing cancer cell therapy in solid tumors.

Acknowledgements and/or References (Optional)::