Clinical-Scale Selection of Anti-CD3/CD28–Activated T Cells After Transduction with a Retroviral Vector Expressing Herpes Simplex Virus Thymidine Kinase and Truncated Nerve Growth Factor Receptor

Abstract

Activation of T cells is necessary for efficient retroviral-mediated gene transfer. In addition, if the population of infused cells is to be limited to transduced cells, a means of positive selection is required. We describe a clinical scale procedure for activation of donor T cells with anti-CD3/CD28 beads followed by transduction with a retroviral construct expressing the herpes simplex virus thymidine kinase (HSV-tk) and human nerve growth factor receptor (NGFR). Optimization of transduction parameters was performed, testing the timing of transduction, centrifugation, and the use of serum. In large-scale experiments, 3-5 x 10(8) peripheral blood mononuclear cells (PBMC) were activated with anti-CD3/CD28 beads and expanded to day 13. Transduction was accomplished using MFG-TKiNG supernatant produced from the PG13 packaging line 48 hr after T-cell activation. The mean transduction frequency was 37.5% based on NGFR expression, and the mean expansion observed was 42.6-fold (mean final cell number 1.85 x 10(10)). A comparison of the ability of the Baxter Isolex 300i and the Miltenyi CliniMACS to perform purification of NGFR+ cells suggests that greater purity can be achieved with the CliniMACS device (67.4% vs. 97.7%), while the yield of transduced cells appears higher with the Isolex 300i (41.3% vs. 23.5%). We conclude that a strategy based on activation of human T cells with anti-CD3/CD28 beads can result in sufficient transduction, expansion, and purification based on NGFR expression for clinical trials.

Human T Cells Engineered To Express a Programmed Death 1/28 Costimulatory Retargeting Molecule Display Enhanced Antitumor Activity

“…The a-and b-chains from the previously characterized TCRs specific for MART-1 [26][27][28][29][30][31][32][33][34][35] termed F4 (or DMF4) (18,27) and for p53 [264][265][266][267][268][269][270][271][272] (25) were subcloned into the pGEM-4Z/64A vector, as described previously (28). The chimeras PD1/28 and PD1/28BB were created by overlapping PCR, and their amino acid composition is indicated in Fig.…”

Incorporation of Transmembrane Hydrophobic Mutations in the TCR Enhance Its Surface Expression and T Cell Functional Avidity

“…The sequences of the peptides used in this study are as follows: MART-1 [26][27][28][29][30][31][32][33][34][35] (ELAGIGILTV), gp100 209M (IMDQVPFSV), and p53 264-272 (LLGRNSFEV).…”

“…The a-and b-chains from the previously characterized TCRs specific for MART-1 [26][27][28][29][30][31][32][33][34][35] termed F4 (or DMF4) (7,27), for p53 (4), and for gp100 (28) were subcloned into the pGEM-4Z/64A vector as described previously (11). In addition, we created mutant TCRs in which we mutated residues in the TM region (20,29) of the TCRa-and TCRb-chains.…”

“…To study the impact of hydrophobic mutations in the TCR TM domain, we used the F4 TCR (27), specific for HLA-A2/MART-1 [26][27][28][29][30][31][32][33][34][35] , which was initially demonstrated to mediate tumor regression in patients (7). We generated the poly-La and poly-Lb…”

Adoptive cellular immunotherapy for childhood malignancies

“…120 Gene transfer can also be undertaken to render T cells resistant to the anti-inflammatory tumor micro-environment, such as by expressing a dominant-negative TGF-b receptor 121,122 or decreasing the sensitivity of the adoptively-transferred T cells to Fas-induced apoptosis. 123 Finally, given the potential for gene transfer to cause unwanted genotoxicity, investigators have included suicide genes [124][125][126][127][128][129][130][131][132] which conditionally render the cells sensitive to ablation in the event of an adverse event such as GVHD. [133][134][135][136] Adoptive cellular immunotherapy in the future There are a number of challenges to broadening the application of adoptive cellular immunotherapy for childhood cancers.…”