Enhanced antilymphoma efficacy of CD19-redirected influenza MP1–specific CTLs by cotransfer of T cells modified to present influenza MP1

Abstract

IntroductionAdoptive transfer of ex vivo-expanded T cells specific for immunodominant viral epitopes into immunocompromised hosts can reconstitute protective antiviral immunity and can result in the long-term persistence of transferred cells. [1][2][3][4][5] In contrast, the application of adoptive T-cell transfer to the successful cellular immunotherapy of malignancy has proved to be significantly more challenging, in part because of the difficulty of isolating high-affinity, tumor-specific T cells that can mediate effective antitumor in vivo effector functions and the potential for tumors to evade immunologic clearance through a variety of escape mechanisms, including the down-regulation of restricting HLA molecules. [6][7][8] Several groups, including ours, are developing alternative strategies for targeting tumors using genetically modified T cells that are endowed with redirected antigen specificity through the expression of chimeric antigen receptors (CARs), such as a CD19-specific chimeric immunoreceptor. These chimeras typically use HLA-independent, highaffinity antigen recognition domains consisting of extracellular singlechain immunoglobulin variable fragments (scFvs) linked to cytoplasmic T-cell activation domain(s), such as CD3-. [9][10][11][12][13][14][15][16][17][18][19] Strategies to enhance the antitumor activity of adoptively transferred CAR ϩ cytotoxic T lymphocytes (CTLs) and to overcome the potentially deleterious impact of in vivo recycling of these cells solely through CAR-redirected engagement of tumor cells will likely be critical for achieving therapeutic efficacy. Because CAR-redirected T cells retain the specificity and function of their endogenous T-cell antigen receptor (TcR), expressing CARs on virus-specific T cells, such as commonly acquired latent viruses (Epstein-Barr virus [EBV] and cytomegalovirus [CMV]), is a potential approach to maintain persistence in vivo through re-encounter of these bispecific T cells with viral antigen presented by professional antigen-presenting cells (APCs). 9,20,21 Although the timing and magnitude of latent virus reactivation makes the in vivo restimulation of bispecific T cells difficult to control, we hypothesize that the grafting of antitumor CARs to T cells specific for common nonlatent viruses and the delivery of a viral antigen vaccine boost(s) after adoptive transfer (transfer-boost strategy) is an approach amenable to iatrogenic regulation.Here we describe the usefulness of ex vivo-expanded CD8 ϩ and CD4 ϩ T cells to function as APCs by their genetic modification to express a model viral antigen (influenza A MP1) for eliciting the in vitro expansion of MP1-specific CTLs and for augmenting the clearance of CD19 ϩ Daudi lymphoma in vivo, by CD19ϫMP1-bispecific CTLs by post-transfer boosting. Our finding that human Materials and methods Plasmid expression vectorsThe pMG expression vector (InvivoGen, San Diego, CA) was modified by site-directed mutagenesis to remove a PacI restriction enzyme (RE) site at position 307 to generate pMGP ac ( Fig...

Antitumor activity of dual-specific T cells and influenza virus

“…Immunogens used to date are alloantigen 8 in mouse cells, and Epstein-Barr (virus) EBV 27 and cytomegalovirus (CMV) 28 and influenza virus 29 in human cells. Importantly, the potential utility of dual-specific T cells was highlighted by the demonstration of tumor inhibition in mice using mouse dual-specific T cells recognizing alloantigen and the ovarian cancerassociated antigen FBP.…”

“…The feasibility of generating and transducing influenza-specific human T cells has been previously demonstrated. 29 Alternatively, an attenuated virus may be suitable, or a widespread persistent virus such as EBV or CMV may be an effective second specificity to use. The possibility of generating human dual-specific T cells with reactivity against tumor and EBV or CMV has been previously demonstrated in vitro.…”

Adoptive cellular immunotherapy for childhood malignancies

“…For example, to increase the proliferative potential (i) the CAR endodomain can be systematically altered to provide a fully competent activation signal, [105][106][107][108][109][110][111] or (ii) T cells can be modified to enforce expression of stimulatory cytokine transgenes [112][113][114][115][116] or (iii) the CAR can be introduced into T cells which can respond/ proliferate via endogenous TCR to defined (often viral) antigens. [117][118][119] Recognizing that tumor-specific T cells may not traffic to sites of disease, cells can be genetically modified to express desired homing receptors. 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.…”

“…[28][29][30][31][32][33] As an alternative source of APC, investigators have also used T cells, which can be nonspecifically propagated ex vivo to vast numbers by cross-linking CD3 with OKT3, as a source of antigen present cells (T-APC). [34][35][36][37] (iv) The safe and efficacious infusions of EBV-specific T cells for LPD has facilitated the targeting of other EBV-derived antigens associated with Hodgkin's disease (HD), and nasopharyngeal carcinoma (type II EBV malignancies) with investigators developing technologies to generate T cells with specificity for latency membrane protein (LMP) 1, LMP2 and EBNA-1. 38-46 (v) Since the generation of EBV-specific T cells on LCL requires weeks of cell-culture time, investigators have recently developed a paradigm for the a priori manufacture of HLA-restricted EBV-specific T cells.…”

Chimeric T-cell receptors: new challenges for targeted immunotherapy in hematologic malignancies

“…[25][26][27][28][29] The initial construct was composed of a CD19-specific single-chain immunoglobulin extracellular targeting domain, fused to a CD3-zeta intracellular signaling domain. It was shown that CD19-redirected CTL were capable of potently killing primary B-ALL blasts and also of producing Th1 cytokines and were consistently proliferating after recognition of the targeted molecule.…”