Human CC chemokines macrophage inflammatory protein (MIP)-1␣, MIP-1␤, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1␣ variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1␣ residues Asp 26 and Glu 66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1␤ and RAN-TES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1␣ has enabled comparison of these residues in hMIP-1␤ and RANTES. Aggregated and disaggregated forms of hMIP-1␣, hMIP-1␤, and RANTES generally have equivalent G-protein-coupled receptormediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1␣, hMIP-1␤, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M-and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SF-PacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications.

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.