Developing rcSso7d-based inhibitory receptors for improving the specificity and safety of NKG2D-CAR-T cells

Background.

CAR-T cell therapy has recently shown groundbreaking success in the therapy of previously incurable forms of B-cell leukemia. The principle of this type of immunotherapy is based on redirecting T cells against tumor cells by expression of chimeric antigen receptors (CARs), which can be directed against any desired tumor associated antigen (TAA) on the surface of tumor cells. These CARs are fusion proteins of TAA binding domains, which are mostly derived from antibodies, and signaling domains, derived from the TCR-complex and costimulatory receptors, which together strongly activate the T cells upon TAA binding on the tumor cell.

The exploitation of the potential of CAR-T cell therapy for the treatment of e.g. high-risk childhood cancers, which are characterized by essentially unchanged low cure rates since more than two decades, is highly desirable. One promising target on most of these pediatric tumors could be the cell stress-induced ligands of NKG2D (NKG2D-Ls), against which we have developed a CAR (Lehner et al., 2012). However, as is the case with all TAAs, their expression is not absolutely confined to tumors but also occurs on some healthy tissue cells of epithelial type, which would then be targeted by CAR-T cells, too. Such so-called on-target/off-tumor toxicity is the biggest hurdle regarding the broad application of CAR-T cell therapy and has already caused deadly incidences in several clinical pilot trials with some CARs.

One promising approach for improving the tumor-targeting specificity of CAR-T cells is to coexpress an inhibitory chimeric receptor, in which a binder domain is fused to the cytoplasmic domain of the inhibitory receptor PD1 (Fedorov et al, 2013). This strategy is particularly attractive for the NKG2D-CAR-T cell therapy of Ewing sarcoma, Osteosarcoma and Rhabdomyosarcoma, which - contrary to the vast majority of tumors - are not derived from epithelial cells but, as sarcomas, are derived from mesenchymal cells. Thus, a pan-epithelial-specific inhibitory receptor could protect healthy NKG2D-L expressing epithelial cells in NKG2D-CAR-T cell therapy.
The aim of this project is to generate binders directed against antigens enabling specific targeting of sarcoma cells by NKG2D-CAR-T cells. These binders will be based on a charge-reduced recombinant protein variant of Sso7d, which is characterized by very small size (7 kDa) and high thermal stability (3).

Aims and methods.

The aim of the project is the development of an rcSso7d-based inhibitory receptor. This receptor will be coexpressed with an NKG2D-based CAR in T cells, which will be tested for target cell specificity in vitro and in vivo.
After identifying a promising pan-epithelial candidate antigen, mutants of rcSso7d will be selected for binding to this antigen by using yeast surface display technology. In this process, mutants with specificity for different epitopes of the antigen will be selected by competition assays. The affinity of the binders will be determined by titration on yeast and biolayer interferometry. Furthermore, the binders will be evaluated for their stability by differential scanning calorimetry (DSC) as well as for their aggregation behavior by using size exclusion chromatography (SEC).

In the next step, chimeric inhibitory receptors will be generated by substitution of the ectodomain of PD1 with the most promising binder candidates. The design of these fusion proteins will be optimized for maximum expression in T cells. The function of the receptors in the T cells will be tested by determining their ability to inhibit CAR-mediated activation of the T cells by a second antigen. Activation of the T cells will be assayed by measuring degranulation using FACS analysis, release of interferon-gamma and cytolysis of target cells. Potentially different functionality of the receptors recognizing different epitopes of the antigen will be identified by titration of the antigen density. Finally, the target cell specificity of the CAR-T cells will be analyzed in vivo in an NSG-mouse model for sarcoma.

Lehner M, Gotz G, Proff J, et al. Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor expressed by lentiviral transduction or mRNA transfection. PLoS One. 2012;7(2):e31210.
Fedorov VD, Themeli M, Sadelain M. PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) divert off-target immunotherapy responses. Sci Transl Med. 2013;5(215):215ra172.
Traxlmayr, M. W. et al. Strong enrichment of aromatic residues in binding sites from a charge-neutralized hyperthermostable Sso7d scaffold library. submitted