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Adapter CARs: Estimation of the affinity between adapter and CAR domain required for function

Bartsch, T.; Arndt, C.; González Soto, K. E.; Wodtke, R.; Brandt, F.; Loureiro, L. R.; Mitwasi, N.; Kegler, A.; Feldmann, A.; Bachmann, M.


As next generation for CAR T cells, adaptor CAR platforms have been developed, which are designed to improve safety, but at the same time maintain the high efficiency of the CAR T cell approach. In our lab, we developed the UniCAR system, which consists of universal (Uni)CAR T cells and tumor-specific target modules (TMs), which work as bridging molecules between the UniCAR T cells and the target cells. Until now, type 1 and type 2 UniCARs were developed. Both UniCAR types consist of an extracellular binding domain derived from a monoclonal antibody (mAb) directed to the nuclear La/SS-B protein. Type 1 UniCARs are derived from the anti-La mAb 5B9. Type 2 UniCARs from the anti-La mAb 7B6. As both anti-La mAbs are not able to precipitate native La protein, both anti-La mAbs are directed to a specific cryptic epitope which is not accessible on the cell surface. Thus, the UniCAR T cell is per se inert. To activate the UniCAR T cell for tumor cell killing a TM is needed as a second component. Typically, a TM is composed of a tumor-specific binding domain and the respective La epitope.
Consequently, the affinity of the TM towards the target antigen but also towards the UniCAR T cell via the E5B9-tag plays an important role for functionality of the respective UniCAR system.
In this study, we representatively aimed to elucidate if and how the affinity of the type 1 UniCAR domain to the E5B9 epitope impacts the functionality of the UniCAR system. To alter the interaction of UniCAR and TM, we designed different mutated E5B9 peptides (M1-M3) carrying one or two amino acid (aa) changes. In detail, aspartic acid and/or glutamic acid were mutated to glycine residues as they most probably are involved in epitope/paratope interactions. We subsequently fused these mutated peptides to an scFv domain, resulting in three different mutated TM versions.
By conducting ELISA and flow-cytometry based binding studies, we showed that a single aa exchange (D3>G3) in M1 did not alter the affinity towards the mAb 5B9. However, replacing two aa resulted in a 4-fold (M2: E2>G2, D3>G3) or even 50-fold reduced affinity (M3: E2>G2, E6>G6) of the mAb 5B9 towards the mutated E5B9 epitopes. By chromium release assay, we could show that only the TM with M1 was able to induce efficient lysis with EC50 values comparable to the original TM containing the non-mutated E5B9-tag. The TMs with the mutated peptides M2 or M3, showing a lower affinity, were not able to redirect UniCAR T cells for tumor cell killing.
In summary, our data revealed that lowering the affinity between E5B9 peptide and mAb 5B9/ UniCAR T cell by a factor of four already impedes the functionality of the UniCAR system and that affinity of around 0.1 nM is required for proper functionality.

  • Open Access Logo Lecture (Conference)
    TUMOR IMMUNOLOGY MEETS ONCOLOGY (TIMO) XVI 2022 HALLE, 07.-09.07.2022, Halle, Deutschland


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