Redirection of human T cells to tumor cells via nanobody-based target modules using the universal chimeric antigen receptor system


Redirection of human T cells to tumor cells via nanobody-based target modules using the universal chimeric antigen receptor system

Albert, S.; Bergmann, R.; Koristka, S.; Feldmann, A.; Arndt, C.; Aliperta, R.; Ehninger, A.; Cartellieri, M.; Ehninger, G.; Steinbach, J.; Bachmann, M.

In general, adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells has an impressive immunotherapeutic potential. However, due to the time-consuming establishment of new CAR constructs, the risk of life-threatening side effects, and the lack of control mechanisms once infused into patients, we developed a switchable modular CAR platform technology termed UniCAR.
The UniCAR system is composed of two individual components, the universal signal-transducing UniCAR and an exchangeable target module (TM). In contrast to conventional CARs, the single-chain fragment variable (scFv) of the UniCAR binds to a small peptide epitope, which is physiologically not accessible on intact cells. The cross-linkage to tumor cells is mediated via TMs comprising the antigen-specifity and the epitope recognized by the UniCAR. Consequently, UniCAR-engrafted T cells are inert in the absence of redirecting TMs and only switched on in their presence. In addition to the increased safety, the modular structure enables a flexible targeting of different tissue antigens. New TMs can be easily constructed just by fusing the UniCAR epitope to a targeting entity. So far we produced a series of functional scFv-based TMs against different tumor-associated antigens like PSCA, PSMA, GD2 and CD33.
Here we demonstrate that TMs can alternatively contain a nanobody (nb) domain instead of an scFv. Nbs are derived from camelid heavy-chain antibodies, consist of a single variable domain and form the smallest known antigen binding fragments. For redirection of UniCAR T cells to epithelial tumors the frequently overexpressed EGFR is a suitable target antigen. Thus, we generated a nb-based -EGFR TM. As shown by in vitro assays with EGFR+ tumor cell lines, the novel TM efficiently activates UniCAR T cells in a strictly target-specific manner and induces the release of pro-inflammatory cytokines. Furthermore, the data reveal that the -EGFR TM triggers a highly potent tumor lysis at low pM concentrations and redirects UniCAR-engrafted T cells to tumor cells in immunodeficient mice. Using dynamic PET analysis we observed a short half-life of the TM and could confirm its release from UniCAR-TM-complexes. Thereby, it is possible to precisely dose the TM concentration and to rapidly switch the system off in case of adverse side effects. Finally, the -EGFR TM also offers the possibility to image the tumor during therapy. For an increased anti-tumor response we additionally generated a bivalent -EGFR-EGFR TM that shows improved in vitro and in vivo functionality compared to the monovalent construct.
In summary, we established a novel mono- and bivalent nb-based TM for EGFR-specific recruitment of UniCAR T cells which results in an efficient, target-specific and -dependent killing of EGFR+ tumor cells. Thus, we could prove that instead of scFvs also other binding moieties can be used and confirmed the high flexibility of the modular UniCAR platform.

Keywords: CAR T cell therapy; nanobodies; EGFR-targeting

  • Poster
    Third CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference, 06.09.2017, Mainz, Deutschland

Permalink: https://www.hzdr.de/publications/Publ-26020
Publ.-Id: 26020