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1.  Magnetic-Activated Cell Sorting of TCR-Engineered T Cells, Using tCD34 as a Gene Marker, but Not Peptide–MHC Multimers, Results in Significant Numbers of Functional CD4+ and CD8+ T Cells 
Human Gene Therapy Methods  2012;23(3):213-224.
Abstract
T cell-sorting technologies with peptide–MHC multimers or antibodies against gene markers enable enrichment of antigen-specific T cells and are expected to enhance the therapeutic efficacy of clinical T cell therapy. However, a direct comparison between sorting reagents for their ability to enrich T cells is lacking. Here, we compared the in vitro properties of primary human T cells gene-engineered with gp100280–288/HLA-A2-specific T cell receptor-αβ (TCRαβ) on magnetic-activated cell sorting (MACS) with various peptide–MHC multimers or an antibody against truncated CD34 (tCD34). With respect to peptide–MHC multimers, we observed that Streptamer®, when compared with pentamers and tetramers, improved T cell yield as well as level and stability of enrichment, of TCR-engineered T cells (>65% of peptide–MHC-binding T cells, stable for at least 6 weeks). In agreement with these findings, Streptamer, the only detachable reagent, revealed significant T cell expansion in the first week after MACS. Sorting TCR and tCD34 gene-engineered T cells with CD34 monoclonal antibody (mAb) resulted in the most significant T cell yield and enrichment of T cells (>95% of tCD34 T cells, stable for at least 6 weeks). Notably, T cells sorted with CD34 mAb, when compared with Streptamer, bound about 2- to 3-fold less peptide–MHC but showed superior antigen-specific upregulated expression of CD107a and production of interferon (IFN)-γ. Multiparametric flow cytometry revealed that CD4+ T cells, uniquely present in CD34 mAb-sorted T cells, contributed to enhanced IFN-γ production. Taken together, we postulate that CD34 mAb-based sorting of gene-marked T cells has benefits toward applications of T cell therapy, especially those that require CD4+ T cells.
Govers and colleagues perform direct comparisons between T cell sorting reagents for their ability to enrich T cells. They compare in vitro properties of primary human T cells gene-engineered with gp100280-288 Human Leukocyte Antigen A2-specific T-cell receptorαβ undergoing Magnetic-Activated Cell Sorting either with different peptide-MHC multimers or with an antibody against truncated CD34 (tCD34). Among the multimers, streptamers resulted in the best T cell yield, stability, and expansion ability after sorting, while tCD34-sorted cells resulted in the most significant T cell yield and enrichment.
doi:10.1089/hgtb.2012.074
PMCID: PMC4015082  PMID: 22871260
2.  TCR Gene Transfer: MAGE-C2/HLA-A2 and MAGE-A3/HLA-DP4 Epitopes as Melanoma-Specific Immune Targets 
Adoptive therapy with TCR gene-engineered T cells provides an attractive and feasible treatment option for cancer patients. Further development of TCR gene therapy requires the implementation of T-cell target epitopes that prevent “on-target” reactivity towards healthy tissues and at the same time direct a clinically effective response towards tumor tissues. Candidate epitopes that meet these criteria are MAGE-C2336-344/HLA-A2 (MC2/A2) and MAGE-A3243-258/HLA-DP4 (MA3/DP4). We molecularly characterized TCRαβ genes of an MC2/A2-specific CD8 and MA3/DP4-specific CD4 T-cell clone derived from melanoma patients who responded clinically to MAGE vaccination. We identified MC2/A2 and MA3/DP4-specific TCR-Vα3/Vβ28 and TCR-Vα38/Vβ2 chains and validated these TCRs in vitro upon gene transfer into primary human T cells. The MC2 and MA3 TCR were surface-expressed and mediated CD8 T-cell functions towards melanoma cell lines and CD4 T-cell functions towards dendritic cells, respectively. We intend to start testing these MAGE-specific TCRs in phase I clinical trial.
doi:10.1155/2012/586314
PMCID: PMC3287115  PMID: 22400038

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