A Novel Protein-Based Approach to Generate Allogeneic CAR-T Cells with Simultaneous TCR and MHC Class 1 Downregulation

Introduction Autologous chimeric antigen receptor (CAR)-T cells are highly effective against B cell malignancies. However, autologous T cells have significant drawbacks, including time delays, manufacturing failures, poor T cell quality and cost/logistic issues. A solution could be allogeneic CAR-T cells from healthy donors, if these could be prevented from causing graft versus host disease (GvHD) and being rejected by the host immune system. GvHD is mediated by T cell receptors (TCR) of donor T cells while rejection is largely caused by host T cell recognition of foreign MHC class 1 molecules on allo-CAR-T. Previous approaches have used genome-editing to delete TCR +/- MHC class 1, introducing complexity, expense and unpredictable genotoxicity. Here we introduce a novel approach to express CAR and simultaneously downregulate TCR and MHC class 1, in a single vector without genome editing.

Results We designed a synthetic protein where the anti-CD3 scFv UCHT1 was fused to the N-terminus of US11, a protein from human cytomegalovirus (HCMV) which binds to nascent MHC class 1 chains and directs them for ubiquitination and degradation. In cell lines and primary T cells, expression of UCHT1-US11 led to complete downregulation of both TCR and MHC class 1. This approach was not specific to TCR: binder-US11 fusion proteins could also downregulate other protein targets (eg PD-1) along with MHC class 1.

Next, we cloned UCHT1-US11 into a vector also encoding RQR8 (a sort-suicide gene with CD34 and rituximab-binding epitopes) and anti-CD19 CAR. After CD34 magnetic enrichment, T cells >95% TCR-negative, MHC-class 1 negative, CAR+, RQR8+ (UCHT1-US11 or VHH-US11 CAR-T cells) were isolated. In co-cultures with CD19+ and CD19- cell lines, UCHT1-US11 CAR-T cells demonstrated efficient antigen-specific cytotoxicity equivalent to conventional anti-CD19 CAR-T cells. Although robust cytokine secretion (IFN-G, IL-2) was also seen with UCHT1-US11, this was partially reduced compared to CAR19, likely reflecting lower CAR expression due to larger vector size. However, in 7-day proliferation assays, anti-CD19 and UCHT1-US11 demonstrated similar expansion and expression of markers of exhaustion and activation.

We tested UCHT1-US11 CAR-T cells in 10-day mixed lymphocyte reactions (MLRs) with peripheral blood mononuclear cells. While CAR19 control cells expanded robustly in response to allogeneic but not autologous donor cells, UCHT1-US11 CAR-T did not, indicating lack of TCR function. However, T cells incubated with UCHT1-US11 CAR-T cells showed increased TCR internalisation, proliferation and activation compared to those incubated with CAR19 control cells. This was true for both autologous and allogeneic T cells, confirming this was not an allo-response against UCHT1-US11 CAR-T, but instead was likely due to bystander T cell stimulation by low levels of secreted UCHT1-US11.

To address this isue, we designed a new fusion protein where UCHT1 was replaced with a single-domain VHH anti-CD3 binder to make VHH-US11,reasoning that a compact VHH binder was unlikely to be mitogenic. This was cloned into an identical vector as previously. Robust TCR and MHC class 1 downregulation was again seen, and performance was similar to UCHT1-US11 in co-cultures and MLR. However, bystander T cell stimulation was not seen. Indeed, allogeneic donor T cell proliferation was markedly lower with VHH-US11 CAR-T than CAR19 cells, suggesting reduced allogeneic T cell recognition of these cells. Instead, expansion of allogeneic NK cells was seen, suggesting NK recognition of MHC class 1 null cells.

Next, we tested UCHT-US11 and VHH-US11 CAR-T cells in the Nalm6 NSG xenograft stress test model (0.2M CAR-T cells). Compared to control anti-GFP, CAR19, UCHT1-US11 or VHH-US11 CAR-T cells substantially delayed leukemia progression and prolonged overall survival, with no differences demonstrated in terms of tumour burden or T cell infiltration.

Conclusions and further work We have demonstrated that anti-CD3 binders fused to HCMV-US11 can simultaneously downregulate both TCR and MHC class 1 without genome editing, and that CAR-T cells co-expressing these modules are robustly functional both in vitro and in vivo. We are now testing the ability of our lead candidate, VHH-US11 CAR-T, to resist rejection and prevent GvHD in novel humanised murine models, along with further modifications to prevent MHC class 2 expression and NK-based rejection.

Kassimatis:Autolus Ltd: Current Employment. Maciocia:Autolus Ltd: Current equity holder in publicly-traded company. Pule:Autolus Ltd: Current Employment, Current equity holder in publicly-traded company. Maciocia:Autolus Ltd: Current equity holder in publicly-traded company.