Structure-function relationships of chimeric antigen receptors in acute T cell responses to antigen

A multivariate, quantitative assay that disentangles key kinetic parameters of primary human T cell function in vitro

“…It can be used to generate dose-response curves that reveal tonic signaling, general cellular off-target signaling, sensitivity of response (EC50), and maximal response (Emax). All of these parameters have relevance to the receptor under study; for instance, EC50 relates to the probability that the receptor will engage and be activated by a pMHC ligand on the T2 surface; and Emax relates in part to the level of functional receptor protein [20,21].…”

Design of TCR Structural Variants That Retain or Invert the Normal Activation Signal

“…Notably, our assays were performed in Jurkat cells, which express many, but not all, of the proteins used to create the fusions. Although Jurkat cells do not contain all the components identified through study of normal T cell biology [e.g., PTEN, SYK, SHP-1, CTLA-4 ( 42)], we have shown that acute receptor sensitivity measured in Jurkat cells correlates with primary T cells (32). Thus, the lack of acute effect in Jurkat cells may be explained by the parent molecules availability for recruitment into the signaling complex.…”

“…Second-and third-generation CARs have been used in the clinic as components of cell therapeutics and achieved noteworthy success (28)(29)(30)(31). Most engineering for therapeutic purposes has been restricted to CARs, as opposed to TCRs, given the CARs tolerance for alterations (32). TCRs have been typically used in their native form or with minor sequence changes that involve introduction of stabilizing disulfide bonds or swapping of mouse constant domains to facilitate desirable H-L-chain pairing in human cells (33)(34)(35).…”

“…We should point out that this statement may be controversial, but we believe that many studies focus on binding affinity, rather than functional sensitivity. We and others have shown that these parameters are only weakly correlated in CARs, as in TCRs [ (12,32); see Ref. 43 for review].…”

Potent, Selective CARs as Potential T-Cell Therapeutics for HPV-positive Cancers

“…We also noted here a disconnect between binding and function that we have observed generally. 16 Though binding is necessary, and further optimization rounds yielded improved function, the relationship between binding and function is apparently not simple. Finally, in the case of HPV E6 29–38 , we completed 2 optimization cycles on top of the initial primary binder selection.…”