Abstract
Adoptive cellular therapy (ACT), as a kind of biological treatment, has played an important role in immunotherapy for malignancies, by which an individual passively acquires the specific immunological rejection to tumor molecular target through cellular infusion, resulting in the suppression or destruction of malignancies. In the early clinical studies, immunotherapies based on tumor-infiltrating lymphocytes (TILs) have demonstrated definitively therapeutic effect on a variety of tumors, especially on melanoma, which underlined feasibility and necessity of ACT in anti-tumor therapies. However, the difficulties on cellular material acquisition, relatively complex cellular constitute, undefined anti-tumor target and unstable efficacy, all of which affected and limited the clinical practice with TILs, in addition to the immune tolerance-inducing behaviors of the tumor itself, such as the immune escape, suppression of the immune effects via peri-tumor microenvironment, down-regulation of MHC to minimize antigen exposure, et al.
Chimeric antigen receptor (CAR) has effectively integrated the specific recognition of Ab-Ag and signal transfer function of TCR to form a transmembrane molecule with different functional domains. The expression fragment of CAR could be formed by aligning single chain antibody fragment (scFv) cDNA and costimulatory domain in tandem, which is subsequently transferred via lentiviral vectors to modify autologous T lymphocytes. After gene expression and translation, the CAR has an MHC-independent specific recognition to tumor-associated antigen (TAA), resulting in T cell activation, anti-tumor cytotoxic effort and memory cell differentiation. CAR-modified lymphocytes (CART) are effective ACT tools cells with anti-tumor specificity through gene transfection. Currently, CD19-targeted CART therapy has yielded promising therapeutic effect.
Our study selected a lineage-specific CD33 and another tumor-specific CD30 as CAR-associated targets, with which we designed and constructed a series of third generation CAR containing CD28 activation domain and 4-1BB costimulatory domain. The CAR-containing expression vectors were transfected into the Jurkat cell line via lentiviral system and nucleofector electroporation system, respectively.The target gene fragments, anti-CD30-scFv, anti-CD33-scFv, and CD28-4-1BB-CD3ζ sequence encoding T cell activation plus costimulatory signals were amplified from pUC57-CD30-scFv, pUC57-CD33-scFv and pUC57-CD2841BBZ via PCR. The separated scFv and signal sequence were linked into a continuous fragment, CAR-CD30 and CAR-CD33 cDNA, by overlapping PCR, which were subsequently subcloned into a lentiviral expression vector, pCDH-CMV-MCS-EF1-Puro, to construct recombinant lentiviral vector pCDH-CAR30 and pCDH-CAR33; the electric transfection vector pmax-CAR30 and pmax-CAR33 were constructed as well.With virus packaging, the corresponding CARs/GFP/Blank-containing virus were added into Jurkat cell medium, which were immediately centrifuged to facilitate infection; puromycin was used to screen Jurkat strains highly expressing CAR, CAR-modified Jurkat strains were thus harvested one week from antibiotic screening, named as: Jurkat-pCDH-Blank, Jurkat-pCDH-GFP, Jurkat-pCDH-CAR30 and Jurkat-pCDH-CAR33, whose protein samples were then extracted for CAR detection.
The Jurkat cells could also be effectively transfected by applying an electrical pulse to momentarily create small pores in cell membranes, with the aid of pmax-CAR30 or pmax-CAR33, moreover, different discharge mode was designed to yield higher cell viability, and otherwise higher transfection efficiency. The fluorescent signal could be observed 24h from electric pulse and protein samples could be extracted at the same time.
After one-week screening with puromycin, fluorescence from Jurkat cells infected with GFP-containing virus were detected. Electroporation was used as an alternative procedure to produce CAR-modified Jurkat cell line, and the transfection efficiency was higher than lentiviral system did, approximately 90%, by GFP observation. The expression of CAR-CD30 and CAR-CD33 were also successfully detected by western blot in Jurkat cells, indicating a preliminary and technical basis ahead of in-depth study on ACT and CART agent preparation.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
