Development and Optimisation of a CLEC12A-Targeting Inhibitory CAR to Improve the Safety Profile of SEMA4A CAR-T Cells in Multiple Myeloma

Immunotherapeutics have revolutionised the treatment of multiple myeloma (MM), but BCMA remains the only approved CAR T-cell target. Furthermore, despite impressive responses to these agents, relapses are still inevitable. There is thus an unmet need to expand our repertoire of targets. We have previously used proteomic approaches to demonstrate that classical CAR T-cell targets are inevitably expressed on healthy tissue, resulting in on-target/off-tumour toxicity. Further engineering is therefore required to enable better discrimination between healthy and malignant cells.

We have previously identified SEMA4A as an attractive MM immunotherapeutic target, owing to its ubiquitous, obligate expression in primary MM. We also demonstrated that a SEMA4A-targeting CAR T-cell was highly effective in eliminating myeloma cells. However, SEMA4A has modest expression on monocytes, granulocytes, and CD34+ hematopoietic stem and progenitor cells (HSPCs), so on-target/off-tumour pancytopenia was a major risk of our original CAR T-cell. We therefore describe here our use of NOT-gating to enable much safer CAR T-cell targeting of SEMA4A in MM.

To identify a suitable inhibitory CAR (iCAR) protein to pair with our primary SEMA4A CAR, we used a high-resolution mass-spectrometry-based proteomics dataset of primary human immune cells to identify cell-surface proteins that were absent on myeloma cells, but which were expressed at levels three-fold greater than SEMA4A, or more, on monocytes, granulocytes, and CD34+ cells. To prevent constitutive cis-inhibition of NOT-gated T-cells, we excluded all potential iCAR proteins also expressed on T-cells. We identified multiple potential iCAR partners, the most promising of which was the protein CLEC12A, based on its expression profile across the different lineages. We profiled multiple healthy and MM-associated bone marrow samples using flow cytometry to confirm the expression profile of this protein. As expected, we saw ubiquitous expression of CLEC12A on SEMA4A-positive monocytes, granulocytes and HSPCs, but a complete absence of expression on T-cells and on MM-associated plasma cells.

Having identified CLEC12A as a high-quality iCAR protein partner, we proceeded to engineer the inhibitory machinery. We identified two single domain VHHs against CLEC12A. To confirm that they could bind the extracellular protein in its natural conformation and trigger downstream T-cell signalling, we overexpressed CLEC12A in K562 cells and co-cultured these with a second-generation CAR T-cell engineered with the VHHs to target CLEC12A. This led to robust T-cell activation, confirming the utility of the VHHs. We then cloned the VHHs upstream of the dual intracellular inhibitory regions of LIR-1 and PD-1 and co-expressed these iCARs in both Jurkat reporter cells and primary human T-cells expressing the SEMA4A targeting CAR. Parallel cell-surface expression of both constructs was validated through flow cytometry. Importantly, when co-cultured with CLEC12A-/SEMA4A+ target cells, there was robust T-cell activation and cell killing by the SEM4A-CAR/CLEC12A-iCAR demonstrating that the CLEC12A iCAR does not prevent cytotoxicity in the absence of its target. When co-cultured with CLEC12A+/SEMA4A+ target cells, T-cell activation was reduced, though not completely, indicating partial function of the CLEC12A iCAR.

We reasoned that our failure to achieve complete inhibitory function was due to the relatively smaller size of the extracellular domain of CLEC12A relative to that of SEMA4A, leading to steric hindrance. We therefore experimented with different hinge lengths on the iCAR and targeting CAR to enhance performance. By increasing the iCAR/CAR hinge ratio, we were able to demonstrate significantly more inhibition of T-cell activation in the presence of CLEC12A on the target cell, whilst maintaining robust killing when CLEC12A was absent. Ongoing work is seeking to test our SEMA4A-CAR/CLEC12A-iCAR in vivo.

In conclusion, we confirm that logic-gating can increase the targeting repertoire of CAR T-cells, and we demonstrate a NOT-gated CAR T-cell that can safely kill myeloma cells via their SEMA4A expression, while limiting on-target off-tumour toxicity of other immune cells. However, this study also illustrates that the increased targeting repertoire comes at the cost of increased engineering complexity.