Genetic modification of human T lymphocytes for the treatment of hematologic malignancies

Abstract

Modern chemotherapy regimens and supportive care have produced remarkable improvements in the overall survival of patients with hematologic malignancies. However, the development of targeted small molecules, monoclonal antibodies, and biological therapies that demonstrate greater efficacy and lower toxicity remains highly desirable in hematology, and oncology in general. In the context of biological therapies, T-lymphocyte based treatments have enormous potential. Donor lymphocyte infusion in patients relapsed after allogeneic hematopoietic stem cell transplant pioneered the concept that T lymphocytes can effectively control tumor growth, and this was then followed by the development of cell culture strategies to generate T lymphocytes with selective activity against tumor cells. Over the past decade, it has become clear that the adoptive transfer of ex vivo expanded antigen-specific cytotoxic T lymphocytes promotes sustained antitumor effects in patients with virus-associated lymphomas, such as Epstein-Barr virus related post-transplant lymphomas and Hodgkin's lymphomas. Because of this compelling clinical evidence and the concomitant development of methodologies for robust gene transfer to human T lymphocytes, the field has rapidly evolved, offering new opportunities to extend T-cell based therapies. This review summarizes the most recent biological and clinical developments using genetically manipulated T cells for the treatment of hematologic malignancies.

Figure 1.

Expression of suicide genes in T lymphocytes. The figure illustrates the two suicide systems that have been tested in clinical trials. (A) HSV-tk is an enzyme that phosphorylates specific nucleoside analogs (gancyclovir) to nucleoside monophosphate. A second cellular kinase phosphorylates this product into nucleoside triphosphate, a molecule that inhibits DNA synthesis and therefore leads to death of dividing cells. (B) Caspase-9 is a key player in the mitochondrial apoptosis pathway. This molecule dimerizes upon binding with apaf1 and leads to cleavage of the effector caspases 3, 6 and 7. (C) The inducible caspase-9 (iC9) is a fusion protein in which the native CARD domain has been replaced by an FKBP12 domain that dimerizes in the presence of a specific chemical inducer of dimerization (CID).

Figure 2.

Inducible caspase 9 suicide gene. (A) Schematic representation of the bicistronic vector that encodes the inducible caspase 9 (iC9) and the truncated form of human CD19. The two genes are linked through the inclusion of a 2A-like peptide sequence. (B) Expression of CD19 in activated T lymphocytes transduced with the bicistronic vector and enrichment of CD19⁺ cells obtained after positive selection using a CD19-specific antibody. (C) Induction of apoptosis of T cells 24 h after exposure to the AP1903 (CID) that activates the inducible caspase 9. (D) Elimination of transduced T cells in 4 patients after one single dose of AP1903 as measured by phenotypic analysis and copy numbers of the iC9 transgene in peripheral blood mononuclear cells.

Figure 3.

Redirecting T-cell specificity. T-cell specificity can be redirected by the expression of chimeric antigen receptors (CARs) or transgenic α-and β-TCR chains. (A) CARs are composed of two distinct fragments. The first is obtained by linking the variable heavy and light regions of a monoclonal antibody of known specificity into a single chain (scFv) and the second is the signaling region of the native TCR ζ-chain. (B) Second generation CARs incorporate co-stimulatory molecules such as CD28 and 4-1BB. (C) Third generation CARs include 2 or more co-stimulatory molecules. (D) CARs are activated after the scFv portion binds to an extracellular antigen thus the antigen recognition is MHC-independent. (E) Transgenic TCRs recognize specific peptides presented in the context of MHC class I molecules.

Figure 4.

Enhancing T-cell persistence and trafficking. The figure illustrates some of the genetic modifications that have been implemented with the purpose of improving the anti-tumor effects of adoptively transferred T cells. (A) Expression of constitutively activated Akt (caAkt) supports cytokine production and upregulation of anti-apoptotic molecules in T cells. (B) IL-7Rα expression that is lost in effector-memory T cells can be restored rendering effector-memory T cells responsive to the homeostatic cytokine IL-7. (C) T cells can be modified to produce cytokine such as IL-2, IL-15 and IL-12 to support their own proliferation and function. (D) CD95(Fas) that leads to apoptosis of activated T lymphocytes by engaging its ligand Fas-L can be down-regulated by siRNA in T cells in order to provide them with resistance to Fas-L-mediated apoptosis. (E) T cells genetically modified to express a dominant negative TGF-β receptor (dnTGF-βII) are resistant to TGF-β inhibition. (F) T cells forced to express specific chemokine receptors show enhanced migration towards tumor chemokine gradients.