Universal Artificial Antigen Presenting Cells to Selectively Propagate T Cells Expressing Chimeric Antigen Receptor Independent of Specificity

Abstract

T cells genetically modified to stably express immunoreceptors are being assessed for therapeutic potential in clinical trials. T cells expressing a chimeric antigen receptor (CAR) are endowed with a new specificity to target tumor-associated antigen (TAA) independent of major histocompatibility complex. Our approach to non-viral gene transfer in T cells uses ex vivo numeric expansion of CAR+ T cells on irradiated artificial antigen presenting cells (aAPC) bearing the targeted TAA. The requirement for aAPC to express a desired TAA limits the human application of CARs with multiple specificities when selective expansion through co-culture with feeder cells is sought. As an alternative to expressing individual TAAs on aAPC, we expressed one ligand that could activate CAR+ T cells for sustained proliferation independent of specificity. We expressed a CAR ligand (designated CARL) that binds the conserved IgG4 extracellular domain of CAR and demonstrated CARL+ aAPC propagate CAR+ T cells of multiple specificities. CARL avoids technical issues and costs associated with deploying clinical-grade aAPC for each TAA targeted by a given CAR. Employing CARL enables one aAPC to numerically expand all CAR+ T cells containing the IgG4 domain, and simplifies expansion, testing, and clinical translation of CAR+ T cells of any specificity.

Tuning Sensitivity of CAR to EGFR Density Limits Recognition of Normal Tissue While Maintaining Potent Antitumor Activity

“…Codon-optimized truncated human EGFR (amino acids 1–668, NP_005219.2; GeneArt, Regensburg, Germany) was cloned under expression of hEF1-α promoter followed by F2A cleavable peptide and neomycin phosphotransferase. CARL was derived from hybridoma clone 2D3 (17). …”

Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation

“…Propagation of PBMC with activating and propagating cells and IL-2 selectively expanded T cells and was repeated weekly. The T cells surviving drug treatment from days 2 to 14 were co-cultured without drug for another 3 weeks (9,20,21). On day 35 of propagation, T cells were stimulated with anti-CD3, anti-CD28, and IL-2 (9).…”

“…Flow Cytometry-T cells were prepared for flow cytometry by washing once in FACS buffer, and surface-stained for anti-human CD3-APC (BD Pharmingen, 340661, 1:33 dilution), as described previously (21). Intracellular staining for Myc tag Alexa Fluor 488 (MBL International, M047-A48, 1:33 dilution) and anti-human IL-12 p40/p70-PE (BD Pharmingen, 554575, 1:20 dilution) also proceeded as described previously (21).…”

“…Intracellular staining for Myc tag Alexa Fluor 488 (MBL International, M047-A48, 1:33 dilution) and anti-human IL-12 p40/p70-PE (BD Pharmingen, 554575, 1:20 dilution) also proceeded as described previously (21). …”

Cellular Immunotherapy in B-Cell Malignancy

“…T cells are activated and expanded using anti-CD3 and anti-CD28 antibodies, anti-CD3/CD28 beads, or artificial antigen-presenting cells [26][27][28]. Most studies have used viral vectors such as gammaretrovirus [29] or lentivirus [30] for transfer of the construct.…”