No abstract

Chimeric antigen receptor (CAR) development involves extensive empirical characterization of antigen-binding domain (ABD)/CAR constructs for clinical suitability. Here, we present a cost-efficient and rapid method for evaluating CARs in human Jurkat T cells. Using a modular CAR plasmid, a highly efficient ABD cloning strategy, plasmid electroporation, shortterm co-culture, and flow-cytometric detection of CD69, this assay (referred to as CAR-J) evaluates sensitivity and specificity for ABDs. Assessing 16 novel anti-CD22 single-chain variable fragments derived from mouse monoclonal antibodies, CAR-J stratified constructs by response magnitude to CD22-expressing target cells. We also characterized 5 novel anti-EGFRvIII CARs for preclinical development, identifying candidates with varying tonic and target-specific activation characteristics. When evaluated in primary human T cells, tonic/auto-activating (without target cells) EGFRvIII-CARs induced targetindependent proliferation, differentiation toward an effector phenotype, elevated activity against EGFRvIII-negative cells, and progressive loss of target-specific response upon in vitro re-challenge. These EGFRvIII CAR-T cells also showed anti-tumor activity in xenografted mice. In summary, CAR-J represents a straightforward method for high-throughput assessment of CAR constructs as genuine cell-associated antigen receptors that is particularly useful for generating large specificity datasets as well as potential downstream CAR optimization.

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-associated MMP that has been recently reported to have a central role in tumour cell invasion. Here we report that both the native and overexpressed recombinant forms of MT1-MMP are highly enriched in low-density Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin 1. Moreover, the MT1-MMP-dependent activation of proMMP-2 induced by concanavalin A and cytochalasin D was correlated with the processing of MT1-MMP to its proteolytically inactive 43kDa fragment in U-87 glioblastoma and HT-1080 fibrosarcoma tumour cell lines; this processing was also preferentially observed within the caveolar fraction. Interestingly, whereas the expression of caveolin 1had no effect on the MT1-MMP-dependent activation of proMMP-2, its co-expression with MT1-MMP antagonized the MT1-MMP-increased migratory potential of COS-7 cells. Taken together, our results provide evidence that MT1-MMP is preferentially compartmentalized and proteolytically processed in caveolae of cancer cells. The inhibition of MT1-MMP-dependent cell migration by caveolin 1 also suggests that the localization of MT1-MMP to caveolin-enriched domains might have an important function in the control of its enzymic activity.

We have recently shown that green tea polyphenols, and especially (-)-epigallocatechin 3-gallate (EGCg), acted as potent inhibitors of matrix metalloproteinase activities as well as of proMMP-2 activation (M. Demeule, M. Brossard, M. Page, D. Gingras, R. Beliveau, Biochim. Biophys. Acta 1478 (2000)). In the present work, we sought to examine the involvement of MT1-MMP in the EGCg-induced inhibition of proMMP-2 activation. The incubation of U-87 glioblastoma cells in the presence of concanavalin A or cytochalasin D, two potent activators of MT1-MMP, resulted in proMMP-2 activation that was correlated with the cell surface proteolytic processing of MT1-MMP to its inactive 43 kDa form. Addition of EGCg strongly inhibited the MT1-MMP-dependent proMMP-2 activation. The inhibitory effect of EGCg on MT1-MMP was also demonstrated by the down-regulation of MT1-MMP transcript levels and by the inhibition of MT1-MMP-driven cell migration of transfected COS-7 cells. These observations suggest that this catechin may act at both the MT1-MMP gene and protein expression levels. In addition, treatment of cells with non-cytotoxic doses of EGCg significantly reduced the amount of secreted proMMP-2, and led to a concomitant increase in intracellular levels of that protein. This effect was similar to that observed using well-characterized secretion inhibitors such as brefeldin A and manumycin, suggesting that EGCg could also potentially act on intracellular secretory pathways. Taken together, these results indicate that EGCg targets multiple MMP-mediated cellular events in cancer cells and provides a new mechanism for the anticancer properties of that molecule.