Genetic engineering of T cells for adoptive immunotherapy

Abstract

To be effective for the treatment of cancer and infectious diseases, T cell adoptive immunotherapy requires large numbers of cells with abundant proliferative reserves and intact effector functions. We are achieving these goals using a gene therapy strategy wherein the desired characteristics are introduced into a starting cell population, primarily by high efficiency lentiviral vector-mediated transduction. Modified cells are then expanded using ex vivo expansion protocols designed to minimally alter the desired cellular phenotype. In this article, we focus on strategies to (1) dissect the signals controlling T cell proliferation; (2) render CD4 T cells resistant to HIV-1 infection; and (3) redirect CD8 T cell antigen specificity.

Fig. 1

Overview of our laboratory strategy. Human mononuclear cell subsets are obtained from the apheresis product from a patient. We use magnetic bead or high-speed MOFLO sorting to obtain the required T cell subsets. After stimulation and transduction with viral vectors, the engineered cells are utilized for in vitro and in vivo studies. Finally, the technologies developed are “scaled-up” in a GMP-compliant manner to reinfuse in patients participating in clinical trials

Fig. 2

Transduction and characterization of mCD28/h28 chimera. (a) Shown is a cartoon depicting the CD28 chimera with murine CD28 extracellular domain and human CD28 cytoplasmic tail. (b) High-titer lentiviral vector was generated and used to transduce human CD4 T cells, and these cells were stained for expression of mouse CD28 six days post-transduction. Once cells returned to a near resting state, they were stimulated with beads coated with antibodies against CD3/MHC-I, CD3/mCD28, or CD3/hCD28 for 24 h. (c, d) RNA was harvested and cDNA was synthesized and probed with primers to detect IL-2, CCR5, and 28 s rRNA. Real-time PCR amplification and product detection were performed on an ABI Prism 7700 (PE Biosystems) as recommended by the manufacturer. Results for IL-2 and CCR5 were normalized to 28 s rRNA and relative expression was determined by arbitrarily assigning the value obtained for unstimulated cells to 1

Fig. 3

Gene disruption by Zinc-finger nucleases. (a) ZFNs are composed of three or four zinc-finger domains that recognize and bind to a three-base pair sequence, such that a protein including more zinc fingers targets a longer sequence and therefore has greater specificity for its target gene. The non-specific endonuclease Fok I is ligated to the zinc-finger domains and comprises the nuclease domain. (b) While one ZFN molecule binds its target sequence on one DNA strand, another ZFN molecule binds its target sequence on the opposite strand, as shown. The nuclease domains dimerize and each cleaves its own strand, producing a double-stranded break. Once the DSB is generated, it is repaired by error-prone non-homologous end-joining mechanisms, resulting in a defective gene

Fig. 4

Chimeric antigen receptors. Schematic representation of chimeric antigen receptors compared to a T cell receptor. We are studying the contribution of the CD28 and 4-1BB signaling domains, alone or in combination. From the left, tripartite CAR (CD28, 4–1BB and CD3ζ), 28ζ CAR, BBζ CAR, CD3ζ CAR, and truncated-ζ CAR. T cells redirected by the T-body approach are bi-specific, since they maintain expression of their endogenous TCR