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.

The N-propionylated group B meningococcal polysaccharide (NPrGBMP) mimics a unique protective epitope on the surface of group B meningococci (GBM) and Escherichia coli K1. Using a series of monoclonal antibodies (mAbs) induced by the NPrGBMP–monomeric tetanus toxoid (TT) conjugate vaccine it was demonstrated that mAbs having specificities for both extended and conventional short segments of the NPrGBMP were formed, but only the former were bactericidal, and/or gave passive protection against live challenge by GBM. The failure of mAbs specific for short epitopes to protect was further established when (NeuPr)4–TT was used as the vaccine. Of all the mAbs produced that were specific for short internal segments of the NPrGBMP, none were protective, despite the fact that most of them cross-react with the GBM capsular polysaccharide. In contrast, most of the protective mAbs produced by NPrGBMP– TT did not recognize the group B meningococcal polysaccharide (GBMP) unless it was present in its aggregated high molecular weight form. The bactericidal epitope mimicked by the NPrGBMP was shown to be ubiquitous in the capsule of both GBM and E. coli K1 using immunogold labeling techniques and, because of its unique properties, its identification could be significant in the development of a comprehensive conjugate vaccine against group B meningococcal meningitis. This is because most known human α(2–8)-polysialic acid self-antigens can be accommodated in 30–50 α(2–8)-linked sialic acid residues, which is roughly equivalent to an 11-kD length of the GBMP. It has been hypothesized that the formation of the protective epitope on the surface of GBM is due to the interaction of helical segments of the GBMP with another molecule and that the protective epitope is mimicked by the NPrGBMP. Support for the above hypothesis is provided by the fact that the protective NPrGBMP epitope has a similar unusual length dependency to that of the GBMP epitope.

The immunological properties of alpha(2-->8) polysialic acid have been rationalized in terms of the presence of an epitope situated on a unique extended helical segment (n approximately 9) of the polymer. The critical importance of the carboxylate group to the stability of the extended helical epitope can be ascertained from NMR spectrocopic studies and potential energy calculations on the carboxyl reduced alpha(2-->8) polysialic acid. These studies indicate that the extended helix (n approximately 9) is not stabilized in the reduced polymer and that the majority of conformers can only have helical parameters with n = 2 and 3. This result is also consistent with the fact that the reduced alpha(2-->8) polysialic acid, contrary to its acidic counterpart, exhibits conventional immunological properties. Only five to six reduced oligomers are required to inhibit the binding of the reduced polysialic acid to its homologous antiserum. NMR spectroscopic analysis and potential energy calculations on the N-propionyl, N-butanoyl, N-isobutanoyl, N-pentanoyl, N-hexanoyl, and N-glycolyl derivatives of alpha(2-->8) polysialic acid indicate that, despite the bulk of some of these substituents, they did not disrupt the extended helical conformer. The presence of the extended helical epitope in some of these N-acyl derivatives has also been confirmed from immunological data.

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