Engineering a small molecule regulated heterodimerization system for the control of CAR T cells

Background.

Adoptive transfer of CAR T cells resulted in groundbreaking success in the treatment of B cell malignancies (June and Sadelain 2018). This highly promising immunotherapy is based on the engineering of T cells to express chimeric antigen receptors (CARs) able to target any desired tumor associated antigen (TAA) on the surface of tumor cells. CARs are fusion proteins of antigen binding domains, mostly derived from antibodies, linked to the signaling domains of costimulatory receptors and CD3ζ to enable efficient activation of T cells. Hence, upon binding of TAAs, CARs trigger T cell effector functions like cytotoxic activity, proliferation and cytokine release.

However, the success of this therapy has been mitigated by severe side effects such as cytokine release syndrome (CRS) and neurotoxicity (Labanieh, Majzner et al. 2018). In order to avoid such potentially fatal toxicity, different strategies for regulating CAR T cell activation have emerged.

A new approach recently developed in our group is based on a small molecule binding carrier protein and an engineered conformation-specific binder scaffold. This system uses, instead of a single-chain variable fragment (scFv), alternative binder scaffolds based on either the tenth type III domain of human fibronectin (FN3) or a charge-reduced version (rcSso7d) of the Sso7d protein derived from the hyperthermophilic archaeon Sulfolobus solfataricus (Traxlmayr, Kiefer et al. 2016). Our novel approach for small molecule regulated protein heterodimerization could enable for the first time the regulation of CAR signaling by drugs with appropriate tolerability and pharmacokinetics for long term in vivo application. This would be a major step forward compared to other heterodimerization systems such as the one from Wu et al., where the small molecule used for regulation is not suited for broad clinical application (Wu, Roybal et al. 2015).

Aims and methods.

The aim of this project is to improve the function of our small molecule regulated heterodimerization system for the control of CAR T cell function. To this end, we want to (i) increase the affinities of the previously generated conformation-specific binders and (ii) improve the expression of the regulatable CAR constructs in primary human T cells.

Yeast surface display will be used for improving the affinities of our previously generated rcSso7d- and FN3-based binders. In this strategy, mutants of these binder proteins will be generated by error prone PCR and expressed on the surface of yeast. After the yeast display selection, several candidates with different mutations will be sub-cloned individually. The corresponding protein variants will be expressed solubly and subsequently analyzed for their thermal stability and tendency to aggregate by differential scanning calorimetry (DSC) and size exclusion chromatography (SEC), respectively. To analyze the interaction of the binders with the small molecule binding carrier protein, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) will be used for a detailed characterization of binding affinity, stoichiometry and kinetic parameters. In addition, the affinities of the binders will be verified by flow cytometry by titration on yeast and, after incorporation into the CAR, also in primary human T cells.

The most promising protein mutants will be incorporated into CARs and functionally tested in vitro. In order to maximize CAR expression, we will test CAR constructs with different designs with respect to length and type of linkers and arrangement of protein domains. Expression levels will be quantified in molecules per cell by using flow cytometry. CAR function will be evaluated by measuring T cell effector functions such as cytolytic activity and cytokine release. In addition, CAR signaling capacity will be investigated in a Jurkat cell line with fluorescent reporters for the transcription factors NFAT and NFκB.

Finally, to better understand our heterodimerization system, we will investigate the molecular switching mechanism of the interaction between one of our highly conformation-selective binders with the small molecule binding protein. Based on an available crystal structure recently resolved by us, we will analyze in collaboration with Prof. Oostenbrink the molecular dynamics of the protein complex, in particular in the regions involved in the small molecule induced conformational changes.

June, C. H. and M. Sadelain (2018). "Chimeric Antigen Receptor Therapy." New England Journal of Medicine 379(1): 64-73.
Labanieh, L., R. G. Majzner and C. L. Mackall (2018). "Programming CAR-T cells to kill cancer." Nature Biomedical Engineering 2(6): 377-391.
Traxlmayr, M. W., J. D. Kiefer, R. R. Srinivas, E. Lobner, A. W. Tisdale, N. K. Mehta, N. J. Yang, B. Tidor and K. D. Wittrup (2016). "Strong Enrichment of Aromatic Residues in Binding Sites from a Charge-neutralized Hyperthermostable Sso7d Scaffold Library." Journal of Biological Chemistry 291(43): 22496-22508.
Wu, C.-Y., K. T. Roybal, E. M. Puchner, J. Onuffer and W. A. Lim (2015). "Remote control of therapeutic T cells through a small molecule–gated chimeric receptor." Science 350(6258): aab4077.