A conformation-specific ON-switch for controlling CAR T cells with an orally available drug

Abstract

<jats:p>Molecular ON-switches in which a chemical compound induces protein–protein interactions can allow cellular function to be controlled with small molecules. ON-switches based on clinically applicable compounds and human proteins would greatly facilitate their therapeutic use. Here, we developed an ON-switch system in which the human retinol binding protein 4 (hRBP4) of the lipocalin family interacts with engineered hRBP4 binders in a small molecule-dependent manner. Two different protein scaffolds were engineered to bind to hRBP4 when loaded with the orally available small molecule A1120. The crystal structure of an assembled ON-switch shows that the engineered binder specifically recognizes the conformational changes induced by A1120 in two loop regions of hRBP4. We demonstrate that this conformation-specific ON-switch is highly dependent on the presence of A1120, as demonstrated by an ∼500-fold increase in affinity upon addition of the small molecule drug. Furthermore, the ON-switch successfully regulated the activity of primary human CAR T cells in vitro. We anticipate that lipocalin-based ON-switches have the potential to be broadly applied for the safe pharmacological control of cellular therapeutics.</jats:p>

Engineering AvidCARs for combinatorial antigen recognition and reversible control of CAR function

“…6 ). Of note, recently we introduced a small molecule-regulated heterodimerization system, which is based on human retinol binding protein 4, an engineered protein scaffold and the orally available small-molecule drug A1120 29 . Thus, this molecular ON-switch represents an alternative to the AP21967-based system, potentially enabling the future design of AND-gate AvidCARs that can additionally be controlled with a small-molecule drug shown to be nontoxic in mice, even when administered at high doses up to 30 mg/kg for several months 56 .…”

“…To address the lack of control and tumor specificity associated with CAR-T cells, several promising strategies have been developed including CARs for combinatorial antigen recognition [8][9][10][11][12][13][14] , affinity-tuned CARs to minimize killing of healthy cells expressing low levels of target antigen [15][16][17][18][19][20] and CARs whose function can be regulated by administration of small molecules or soluble proteins 10,[21][22][23][24][25][26][27][28][29] . Despite these important advances, there is still a need for improvement.…”

Biological Functions of RBP4 and Its Relevance for Human Diseases

“…By lowering retinol levels in serum and its non-retinoid nature, which may confer a more favorable safety profile compared to fenretinide, also A1120 is an investigational therapy for Stargardt macular dystrophy ( Dobri et al, 2013 ; Hussain et al, 2018 ). Moreover, human RBP4 was shown to interact with engineered RBP4 protein scaffolds in an A1120-dependent manner, allowing for a synthetic and conformation-specific ON-switch system with a broad potential for pharmacological applications ( Zajc et al, 2020 ).…”

CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?

“…Several dimerizer systems have been developed; 178 of these, the inducible caspase-9 (iC9) system has been evaluated in humans with encouraging results. 179,180 Other promising strategies to increase the safety profile of CAR T cells include splitting CARs into two domains, which need to be linked with a small molecule to be active, 181,182 or taking advantage of the small molecule-assisted shut-off (SMAsh) technology. 183 The third approach relies on expressing a molecule on the cell surface of T cells that can be targeted with a clinically approved mAb (e.g., CD20 and truncated EGFR).…”