Combined Genetic Ablation of CD54 and CD58 in CAR Engineered Cytotoxic Lymphocytes Effectively Averts Allogeneic Immune Cell Rejection

Allogeneic cell therapies hold promise to be cost effective with scaled manufacturing for multi-dosing and on-demand off-the-shelf availability. A critical consideration for allogeneic cell products is their ability to persist, maintain function and avoid rejection by the patient's immune system. Genetic knockout (KO) of beta-2-microglobulin (B2M) leads to complete loss of cell-surface human leukocyte antigen (HLA) class I expression and efficiently abrogates CD8⁺ T-cell reactivity. However, loss of HLA class I triggers NK cell-mediated missing-self recognition and manipulation of B2M must therefore be combined with other immune-modulating strategies to limit recipient NK cell reactivity.

We hypothesized that rejection by the patient's immune system can be diminished in primary CAR T cells, iPSC-derived T (iT) and NK (iNK) cells by reverse-engineering common tumor escape mechanisms. The adhesion molecules CD54 and CD58 are both present at the target cell side of the immune synapse, and loss of either of these molecules have previously been reported to elicit immune escape. Here, we show that the combined deletion of CD54 and CD58 in allogeneic immune effector cells makes them resistant to rejection by recipient immune cells through unidirectional reduced synapse formation (Figure 1A).

HLA class I down-regulation by B2M silencing in primary T and NK cells triggered potent cytotoxicity by resting allogeneic NK cells. This response was mostly driven by educated NK cells expressing either NKG2A or killer cell immunoglobulin-like receptors (KIR) binding to HLA-E and HLA-C, respectively. However, over-expression of HLA-E or single HLA-C ligands in a K562 screening model only shut down the specific response of the NK cell subset carrying the cognate inhibitory receptor, resulting in only partial resistance to NK cells at the bulk level. Notably, the introduction of HLA-E was particularly detrimental in donors with expanded NKG2C⁺ NK cell subsets, due to its stimulatory effect through the activating NKG2C receptor. In contrast, combined deletion of CD54 and CD58 in target cells uniquely decreased the response of all tested NK cell subsets and showed universal reduction across NK cell populations from 18 healthy donors (Figure 1B). To delineate the mechanisms behind the increased resistance of target cells carrying these edits, we studied NK cell-target cell interactions at the single cell level by confocal microscopy in microchips. Allogeneic NK cells formed fewer conjugates and failed to form productive immune synapses with CD54^-/-CD58^-/- target cells, supporting the notion that they are more resistant to NK-cell mediated killing by unidirectional altered adhesion.

We next introduced these edits in primary B2M^-/- T cells engineered to express a second generation CAR19 from the TRAC locus. Corroborating the K562 screen, CD54^-/-CD58^-/-B2M^-/- CAR-T cells had a selective survival advantage over B2M^-/- CAR T cells and HLA-E-over-expressing B2M^-/- CAR T cells in conventional mixed lymphocyte reaction (MLR) assays in vitro. Furthermore, we established an in vivo model to probe the effect of different genetic edits on the persistence of allogeneic cell therapy products. To this end, a mixed population of B2M^-/- CAR T cells additionally bearing either CD54 and/or CD58 KO, HLA-E over-expression, or no further edits were infused into mice harboring allogeneic healthy donor PBMC. We found that CD54^-/-CD58^-/-B2M^-/- CAR T cells had significantly better in vivo persistence compared to both B2M^-/- CAR T cells and HLA-E⁺B2M^-/- CAR T cells in the presence of PBMC from healthy donors (Figure 1B).

Although multiplexed editing is feasible in primary CAR T cells, the iPSC platform has an unmatched capacity for homogenously introducing multiple immune-evasion strategies for off-the-shelf cell therapy. Similar to primary CAR T cells, multiplexed edited CD54^-/-CD58^-/-B2M^-/-CIITA^-/- iNK cells showed normal growth kinetics and were resistant to rejection by activated allogeneic NK cells in MLR assays.

Together, these data demonstrate that reverse-engineering of common tumor escape mechanisms, which render target cells less susceptible to immune synapse formation, is an effective strategy to avert immune rejection of allogeneic CAR T and iPSC-derived CAR NK cells.

Perica:NextImmune: Patents & Royalties. Mbofung:Fate Therapeutics Inc: Current Employment. Varady:Fate Therapeutics: Current Employment. Jelcic:Fate Therapeutics: Current Employment. Pan:Fate Therapeutics: Current Employment. Groff:Fate Therapeutics: Current Employment. Abujarour:Fate Therapeutics: Current Employment. Lee:Fate Therapeutics: Current Employment. Williams:Fate Therapeutics: Current Employment. Goodridge:Fate Therapeutics: Current Employment. Valamehr:Fate Therapeutics: Current Employment. Onfelt:Vycellix: Consultancy; Affimed: Research Funding. Sadelain:Atara Biotherapeutics: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics: Research Funding; Mnemo Therapeutics: Research Funding; Takeda Pharmaceuticals: Research Funding. Malmberg:Vycellix: Consultancy; Fate Therapeutics: Consultancy, Patents & Royalties, Research Funding; Merck: Research Funding; Oncopeptides: Research Funding.