T Cell Activation by Antibody-Like Immunoreceptors: Increase in Affinity of the Single-Chain Fragment Domain above Threshold Does Not Increase T Cell Activation against Antigen-Positive Target Cells but Decreases Selectivity

Abstract

Chimeric TCRs with an Ab-derived binding domain confer predefined specificity and MHC-independent target binding to T cells for use in adoptive immunotherapy. We investigated the impact of receptor binding affinity on the activation of grafted T cells. A series of anti-ErbB2 single-chain fragment binding domains with a Kd ranging from 3.2 × 10−7 to 1.5 × 10−11 M was linked to CD3ζ-derived immunoreceptors and expressed in human PBL. Solid phase bound ErbB2 protein triggered activation of receptor-grafted T cells in a dose-dependent manner. The activation threshold inversely correlated with the affinity of the receptor binding domain. The maximum level of cellular activation, however, was the same and independent of the binding affinity. Upon binding to ErbB2+ cells, T cells grafted with immunoreceptors carrying a single-chain fragment of Kd < 10−8 M were activated in a similar fashion against cells with different amounts of ErbB2 on the surface. T cells with a low affinity receptor (Kd > 10−8 M), however, were activated exclusively by cells with high amounts of ErbB2. In conclusion, recombinant immunoreceptors of higher affinity do not necessarily induce a more potent activation of T cells than low affinity immunoreceptors, but the higher affinity immunoreceptors exhibit less discrimination between target cells with high or low Ag expression levels.

Chimeric antigen receptor-modified T cells for the immunotherapy of patients with EGFR-expressing advanced relapsed/refractory non-small cell lung cancer

“…EGFR expression has been observed in both epithelium-derived malignancies and most epithelial cells, raising great concerns in the target-mediated toxicity on EGFR-expressing normal tissues. However, the lower affinity of CAR molecule with EGFR antigen may discriminate the damage activities between normal cells with relatively lower level expression of EGFR and tumors with higher expression (Caruso et al, 2015;Chmielewski et al, 2014;Liu et al, 2015). In this study, tolerable and controllable EGFR targeting-related toxicities were just observed in 11 NSCLC cases as shown in Table 2, implying an appropriate affinity of EGFR-CAR epitope we adopted.…”

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

“…21 To generate the retroviral expression cassettes for the anti-ErbB2-scFv-Fc-CD28-CD3z CARs with combined CD28-CD3z signalling domain, the C6.5 scFv DNA and its derivatives (kindly provided by Dr J Marks, UCSF) ( Table 1) were amplified by PCR using the following set of primer oligonucleotides (NcoI and BamHI restriction sites are underlined): 5¢-CGTACCATGGATTTTGANTIGENGT GCANTIGENATTTTCANTIGENCTTCCTGCTAATCANTIGENTGCCTCAGT CATAATGTCTANTIGENACCGGCGATGGCCCANTIGENGTG-3¢ (sense) and 5¢-TTCTGGATCCGCACCTANTIGENGACGGTGACCTT-3¢ (antisense). The amplified anti-ErbB2 scFv DNA replaced the scFv DNA of the BW431/ 26scFv-Fc-CD28-CD3z CAR, which was previously described.…”

“…The amplified PCR product CD8-CD3z was excised with BamHI and XhoI and cloned between the BamHI and XhoI sites of the anti-ErbB2 CAR encoding vectors. 21 T cells were transduced with recombinant retroviruses as described in detail elsewhere. 35 Cells were analyzed by flow cytometry.…”

“…20 CD28-CD3z CARredirected T-cell activation, however, differs from physiological TCR-CD28 activation in so far that the CAR delivers the costimulatory signal independently of B7 recruitment to the triggering synapse. Although CD3z CAR-mediated T-cell activation strongly depends on the affinity of the antibody-derived binding domain, 21 it remains unclear whether CD28 cosignalling in second-generation CARs without B7 recruitment alters the affinity-dependent threshold in redirected T-cell activation. The issue is of major relevance with respect to the fact that most target antigens for redirected adoptive immunotherapy are not exclusively expressed on tumor cells but broadly on a variety of healthy tissues, although at lower levels.…”

Chimeric Antigen Receptor T Cells for the Treatment of Cancer and the Future of Preclinical Models for Predicting their Toxicities

“…When the antigen was immobilised on a solid surface CAR-mediated T-cell activation was reported to be dependent on the strength of the interaction, because of the correlation between the affinity of different CAR T-cellconstructs and IFN-release by the CAR T-cells [20] .…”