Adoptive immunotherapy using human peripheral blood lymphocytes transferred with RNA encoding Her-2/neu-specific chimeric immune receptor in ovarian cancer xenograft model

Abstract

The current gene transfer technology for single chain (scFv)-based chimeric immune receptor (CIR) has relied on retrovirus and lentivirus vectors which require a long time to obtain sufficient number of transduced cells and stably incorporate into genome. To ameliorate these limitations, we applied RNA electroporation to human peripheral blood lymphocytes (PBLs) activated with anti-CD3 antibody and interleukin-2 (IL-2) for 3 days and assessed that PBL transiently expressing anti-Her-2/neu CIR (CIR-PBL) containing signaling portion of CD28 and CD3z could elicit strong cytotoxicity in vitro and antitumor responses in vivo. The CIR-PBL expressed high level of CIR in CD4 þ , CD8 þ and CD56 þ cells. Her-2/neu-specific stimulation induced secretion of type-I cytokines including interferon-g (IFN-g), IL-8 and granulocyte-macrophage colony-stimulating factor, and IFN-g secretion was mainly mediated by CD8 þ T cells. CIR-PBL specifically killed SKOV3 cell line expressing Her-2/neu. Adoptive transfer of CIR-PBL in SKOV3 xenograft model led to significant inhibition of tumor growth compared with transfer of mock-transduced PBL and showed higher inhibition than those with Herceptin, humanized monoclonal antibody specific for Her-2/neu. These results provided evidence that electroporation of CIR RNA to human PBLs could be used for rapid generation and high number of therapeutic antigen-specific T cells for adoptive immunotherapy.

Direct and indirect antitumor effects by human peripheral blood lymphocytes expressing both chimeric immune receptor and interleukin-2 in ovarian cancer xenograft model

“…Because electroporated PBLs mainly included T and NK cells and allowed high efficiency of gene expression in CD4 þ , CD8 þ , CD56 þ T cells (495%), 9 IL-2 may further activate NK cells in response to SKOV3 to secrete IFN-g. 45 In a nude mouse xenograft model, CIR/IL-2-PBL showed enhanced infiltration of mouse NK cells by secreting IL-2 in addition to tumorspecific trapping by CIR. Because the number of NK cells in spleen was not significantly changed by IL-2-PBL or by CIR/IL-2-PBL, IL-2 secreted from infused PBLs may induce paracrine effects localized in the tumor site rather than systemic effects.…”

“…46 Although high levels of CIR expression were observed in CD4 þ , CD8 þ and CD56 þ populations, the percentage of IFN-g-secreting cells in CIR-PBL on antigen stimulation with SKOV3 was 17% for CD8 þ , 3% for CD4 þ and 0.7% for CD56 þ cells, indicating that, even in the mixture of subsets, the response is mainly mediated by CD8 þ T cells. 9 Furthermore, the construct of CIR contains the signaling portions of CD28 and CD3z, which provide co-stimulatory signals and promotes secretion of additional type I cytokines, including IFN-g and TNF-a, in T cells. The absence of signaling domain from DAP12 may explain why NK cells expressing CIR were not as effective as CD8 þ T cells.…”

“…22 Peripheral blood lymphocytes or cytokine-induced killer cells electroporated with RNA encoding anti-Her-2/ neu-specific CIR have been shown to elicit potent immune responses against Her-2/neu-overexpressing SKOV3 tumors. 9,23 The main problem with this approach has been that, due to lack of survival and gradual decrease in cell number, the antitumor effects of PBLs were not maintained long enough to target the tumors. In this study, we co-transfected dual RNAs encoding CIR and IL-2 to induce antigen-specific PBLs as well as maintain their activity by means of secretion of the survival factor in the physical location of the cells.…”

“…8 CIR genes that contain the coding sequence of an antibody-derived single-chain Fv directed against a tumor-associated antigen, connected to the transmembrane and intracytoplasmic sequences of T-cell signaling molecules, have been generated. 9 Subsequent transfection of these genes into cytotoxic T cells provides them the ability to specifically recognize and kill tumor cells. 10,11 To improve the effectiveness of adoptive immunotherapy, we can use the infusion of interleukin-2 (IL-2) to activate host antigen-presenting cells with toll-like receptor agonists and stimulate the transferred cells in vivo, thus increasing their function and persistence.…”

Chimeric antigen receptor modified T cell therapy for B cell malignancies

“…CARs generally comprise a tumor-targeting domain, a spacer, a transmembrane domain, and one or more intracellular T cell signaling domains. The most commonly used approaches to introduce CARs into T cells involve gamma retroviral or lentiviral transduction; however, other strategies such as the Sleeping Beauty system and electroporation of naked DNA plasmid or mRNA have been employed [12][13][14][15][16]. CAR-modified T cells can be activated by any surfaceexpressed antigen to which a scFv can be generated, rather than being restricted to TCR-mediated recognition of short peptide antigens that are processed and presented by HLA molecules.…”