T Cell Receptor Gene Transfer into Naive and Central Memory Lymphocytes by Lentiviral Vectors for a Safe and Effective Adoptive Immune Therapy of Leukemia

The potency of cellular adoptive immunotherapy against cancer has been revealed by persistent and complete clinical responses obtained with allogeneic hemopoietic cell transplantation (allo-HSCT) followed by the adoptive transfer of donor T lymphocytes and by initial clinical responses observed with the adoptive transfer of tumor specific cytotoxic T lymphocytes (CTLs) in cancer patients. Major hurdles limiting adoptive T cell therapy relate to toxicity (i.e. graft-versus-host disease-GvHD in allo-HSCT) and efficacy (i.e. difficulty in expanding rare, high-avidity tumor-specific CTLs in conditions that can preserve their function and prevent exhaustion). The transfer of the T cell receptor (TCR) from high-avidity tumor-specific CTLs to polyclonal lymphocytes may overcome these difficulties, but is still limited by

1. low and transient transgene expression,

2. unpredictable pairing of the exogenous and endogenous TCR chains and

3. poor survival and expansion potential of gene-modified effector lymphocytes.

To overcome these limitations, we cloned genes encoding a high-avidity TCR specific for an HLA-A2-restricted peptide from the oncogenic Wilms tumor antigen 1 (WT1126-135), in a third generation lentiviral vector under the control of a bi-directional PGK or a bi-directional EF1α promoter. To increase TCR expression and facilitate appropriate TCR pairing, we used a codon-optimized TCR, modified with point mutations to introduce cysteines into the constant regions of the α and 7β chains. Human T lymphocytes were efficiently transduced by both vectors, following activation with anti-CD3 and anti-CD28 antibody-conjugated beads (bCD3/CD28) and culture with low doses of IL-7/IL-15. However, the PGK promoter was superior to EF1α in sustaining stochiometric expression of WT1-specific TCR chains, at levels appropriate for efficient HLA-A2/WT1 pentamer binding (16%), for up to 50 days, in the absence of further T cell stimulation. Additionally, we observed that a phenotype consistent with early (naïve and central memory) T cell differentiation (CD45RA−/+CD62L+, CD28+CD27+, IL7Ra+, IL-2+ γIFN±) was preserved in TCR-modified lymphocytes generated in these culture conditions. Sorted naïve (CD45RA+/CD62L+) and central memory (CD45RA−/CD62L+) lymphocytes were efficiently transduced by TCR-LV and maintained the original T cell phenotype. Accordingly, TCR-modified lymphocytes showed excellent survival and expansion capacity, and, upon antigenic stimulation mediated high WT1-specific γIFN production and cytotoxic activity. To improve the safety of the strategy, we attempted sitespecific integration of transgenes using of the ZFN technology. Adenoviral transfer of a set of ZFN specific for the putative safe-harbor locus CCR5 coupled with integrase defective lentiviral vectors carrying the donor DNA flanked transgene, enables efficient site-specific integration in human lymphocytes resulting in stable transgene expression. Analyses of sorted gene-modified cells is currently ongoing. Site-specific integration of an optimized, leukemia-specific TCR into both naïve and central memory lymphocytes may represent an effective method for the generation of robust engineered tumor-specific CTLs.