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, CARJ 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 proapoptotic protein Bad is a key player in cell survival decisions, and is regulated post-translationally by several signaling networks. We expressed Bad in mouse embryonic fibroblasts to sensitize them to apoptosis, and tested cell lines derived from knock-out mice to establish the significance of the interaction between the adaptor protein Grb10 and the Raf-1 protein kinase in anti-apoptotic signaling pathways targeting Bad. When compared with wild-type cells, both Grb10 and Raf-1-deficient cells exhibit greatly enhanced sensitivity to apoptosis in response to Bad expression. Structure-function analysis demonstrates that, in this cellular model, the SH2, proline-rich, and pleckstrin homology domains of Grb10, as well as its Akt phosphorylation site and consequent binding by 14-3-3, are all necessary for its anti-apoptotic functions. As for Raf-1, its kinase activity, its ability to be phosphorylated by Src on Tyr-340/341 and the binding of its Ras-associated domain to the Grb10 SH2 domain are all necessary to promote cell survival. Silencing the expression of either Grb10 or Raf-1 by small interfering RNAs as well as mutagenesis of specific serine residues on Bad, coupled with signaling inhibitor studies, all indicate that Raf-1 and Grb10 are required for the ability of both the phosphatidylinositol 3-kinase/Akt and MAP kinase pathways to modulate the phosphorylation and inactivation of Bad. Because total Raf-1, ERK, and Akt kinase activities are not impaired in the absence of Grb10, we propose that this adapter protein creates a subpopulation of Raf-1 with specific anti-apoptotic activity.

Uveal melanoma is the most common primary intraocular tumor in adults and exclusively disseminates haematogenously in order to form metastases. The aim of this study was to measure the transcriptional profiles of human uveal melanoma cells isolated from a primary intraocular tumor, circulating malignant cells (CMCs), and metastases in order to elucidate the changes in gene expression associated with this progression. Human EST microarrays and universal reference RNA were used to measure the differences between tissue samples isolated from an immunosuppressed xenograft rabbit model of uveal melanoma. Cells were isolated from a single rabbit at the time of sacrifice from an intraocular tumor, peripheral blood, and metastasis. RNA was extracted from each sample and subjected to transcriptional profiling analysis. Results were compared to the transcriptional profiles previously obtained from the original cell line used for intraocular injections. Changes were verified using real-time PCR analysis. A total of 314 significant changes in gene expression were seen from the intraocular tumor to metastasis, as determined by transcript abundance. Principle Components Analysis was used to cluster these changes into four distinct groups. An additional 61 statistically significant changes were observed between the recultured and CMCs, with the latter believed to represent an intermediate step in the progression from intraocular tumor to metastasis. In conclusion, we have produced a detailed analysis of the transcriptional changes that take place as human uveal melanoma cells evolve from a primary tumor to metastasis in a xenograft animal model, including the decrease in expression of specific melanoma markers.

Proc Amer Assoc Cancer Res, Volume 47, 2006 4169 Purpose: Uveal melanoma is the most common intraocular tumor in adults and is exclusively disseminated via a haematogenous route in order to form metastasis. The aim of this study is to measure the transcriptional profiles of human uveal melanoma cells isolated and cultured from the intraocular primary tumor, circulating malignant cells and metastasic locations. Materials and Methods: Human single-spotted 19k microarrays and universal human reference RNA were used to measure the differences between cultured cells isolated from various locations in an immunosuppressed rabbit model of uveal melanoma. Cells were isolated at the time of sacrifice from intraocular, peripheral blood and metastasis. RNA was then extracted from each sample and subjected to transcriptional profiling analysis. Results were compared to the transcriptional profiles previously obtained from the original 92.1 cell line injected into the eye of the rabbits. Results: Statistical analysis using an ANOVA cut off of 0.05 revealed 3123 transcripts that were modulated between the re-cultured cells from each location and the original 92.1 cell line. 207 of those transcripts had at least a two fold increase or decrease. Included in the transcripts of interest that were up regulated in the circulating malignant cells were Septin 7, Alpha Kinase 3, and Insulin receptor substrate 2. Melanoma specific markers such as Melan A were included in the group of transcripts that were down regulated. Conclusion: For the first time we describe proteins that are significantly up or down regulated between primary, circulating malignant cells and metastasis in uveal melanoma. These changes included transcription factors, ribosomal proteins, and melanoma specific markers. Moreover, cells that were re-cultured after isolation from blood maintained these significant changes. These changes allow us to investigate the expression patterns that uveal melanoma cells may require to survive through circulation until the time of implantation at a distant organ. Future work will investigate the role of these factors in the survival of cells in circulation and their role in metastasis.