Research Article: In Vitro and In Vivo Antitumor Effect of Anti-CD33 Chimeric Receptor-Expressing EBV-CTL against CD33+ Acute Myeloid Leukemia

Date Published: January 5, 2012

Publisher: Hindawi Publishing Corporation

Author(s): A. Dutour, V. Marin, I. Pizzitola, S. Valsesia-Wittmann, D. Lee, E. Yvon, H. Finney, A. Lawson, M. Brenner, A. Biondi, E. Biagi, R. Rousseau.


Genetic engineering of T cells with chimeric T-cell receptors (CARs) is an attractive strategy to treat malignancies. It extends the range of antigens for adoptive T-cell immunotherapy, and major mechanisms of tumor escape are bypassed. With this strategy we redirected immune responses towards the CD33 antigen to target acute myeloid leukemia. To improve in vivo T-cell persistence, we modified human Epstein Barr Virus-(EBV-) specific cytotoxic T cells with an anti-CD33.CAR. Genetically modified T cells displayed EBV and HLA-unrestricted CD33 bispecificity in vitro. In addition, though showing a myeloablative activity, they did not irreversibly impair the clonogenic potential of normal CD34+ hematopoietic progenitors. Moreover, after intravenous administration into CD33+ human acute myeloid leukemia-bearing NOD-SCID mice, anti-CD33-EBV-specific T cells reached the tumor sites exerting antitumor activity in vivo. In conclusion, targeting CD33 by CAR-modified EBV-specific T cells may provide additional therapeutic benefit to AML patients as compared to conventional chemotherapy or transplantation regimens alone.

Partial Text

Efforts to circumvent the limitations and improve the antitumor efficacy of current adoptive immunotherapy approaches have led to the development of novel strategies that combine the advantages of a T-cell-based therapy (tumor penetration, immune effector functions) and of antibody-based strategy (high specificity towards target antigen). The observation that engagement of a single T-cell receptor (TCRs) chain can induce cellular activation led to the design of chimeric antigen receptors (CAR), combining in a single molecule the antigen recognition (through motifs derived from single chain, highly variable antibody fragments, scFv) with signal transduction and activation motives (usually the TCR-ζ chain motif, CD3-ζ). CAR can be generated against every identified tumor-associated antigen (TAA) for which an antibody exists, including carbohydrates and glycolipids. The genetic modification of T cells to express CAR can redirect them toward tumor cells in a non-HLA-restricted manner. Nevertheless, the in vivo efficacy of CAR-expressing T cells is limited because the engagement of the CAR alone is usually not sufficient to mediate the critical costimulatory signals necessary for complete activation and persistence of genetically engineered T cells in vivo [1]. Expression of the CAR into antigen-specific cytotoxic T cells (CTLs) redirects the activated T cells (through their native TCR and costimulatory pathways) towards their new target [2–4]. The genetic modification of Epstein Barr Virus-specific-CTLs (EBV-CTLs) with tumor-specific CAR is particularly attractive because most individuals are persistently infected with EBV and express viral antigens in epithelial cells and B lymphocytes [5]. This approach has been validated in several recently published clinical trials [6–8].

Our results show that the expression of CD33 chimeric receptor in EBV-specific CTLs, while does not affect their phenotype and does not interfere with their ability to proliferate or to respond to autologous EBV-infected targets, renders them capable to specifically lyse CD33+ target cells and release Th1 and Tc cytokines upon encounter with target cells, even though the low average transduction efficiency. In order to further improve this percentage and possibly augmenting CAR+ T-cell response to its specific target, we might concentrate anti-CD33.CAR-specific retroviral supernatant administration by increasing the Multiplicity of Infection (MOI)/cells ratio. However, more efforts will be needed to find new regulatory elements such as insulators, tissue-specific promoters, or ribosome-binding sites, alone or in combination, to be included in the specific transgene plasmid in order to increase transcription efficiency in T cells. This will certainly be accompanied by a decreased of the in vitro E : T ratio, increasing the killing capacity of the transduced T cells, phenomenon that we can predict would also have an impact in the T-cell efficiency after clinical infusion. These results confirm that the engagement of the chimeric receptor by tumor antigen initiates signalling to the T-cell nucleus and induces effector functions, including cytokine secretion and specific lysis of antigen-expressing tumor cells in vitro. T-cell retargeting operates in an MHC unrestricted manner to attack the tumor, whereas it retains MHC-restricted specificity for the endogenous TCR. When we analyzed the in vivo activity of anti-CD33.CAR genetically modified EBV-CTLs in a mouse model of AML, we observed that intravenous administration of CD33-redirected EBV-CTLs in AML-bearing mice can exert a significant albeit partial antileukemic activity. The incomplete antitumoral effect exerted by anti-CD33.CAR-transduced EBV-CTLs might be related to their limited persistence in vivo in this animal model, an expected outcome given that CTLs were injected intravenously without concomitant administration of recombinant human IL-2 and the lack of chronic EBV stimulation in NOD/SCID mice. Indeed, studies reporting complete regression of tumor in mice were performed by intratumoral injection of T cells expressing chimeric receptors and/or addition of intravenous IL-2, or vaccination with autologous LCLs [3, 21, 22], which may have allowed for improved T-cell survival and in vivo expansion. Of note, despite the lack of cytokine addition or EBV stimulation, anti-CD33.CAR-transduced EBV-CTLs were able to home and infiltrate the tumor, where they exerted a significant anti-tumor activity. In addition, we observed that the repeated administrations of anti-CD33.CAR-transduced EBV-CTLs did not result in the selection of CD33-negative tumor clones, that would be resistant to our therapeutic approach.




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