Use Of Chimeric Antigen Receptor Research Articles

GUCY2C-targeted cancer immunotherapy: past, present and future.

For the last decade, we have focused on guanylyl cyclase C (GUCY2C) as a potentially ideal target antigen for colorectal cancer immunotherapy. GUCY2C is expressed only in intestinal epithelial cells and by nearly 100% of colorectal cancers. We have developed and tested a recombinant adenoviral vector possessing GUCY2C (Ad5-GUCY2C) as a candidate vaccine for colorectal cancer patients. Murine studies have revealed that this vaccine is safe and effective against GUCY2C-expressing targets, and Ad5-GUCY2C is poised for phase I clinical testing in colorectal cancer patients with minimal residual disease. Moreover, we are developing second-generation GUCY2C-targeted therapeutics, including the use of chimeric antigen receptor (CAR)-expressing T cells, for treatment of patients with advanced colorectal cancer for whom Ad5-GUCY2C immunization is not appropriate. Thus, a family of GUCY2C-targeted immunotherapeutics may bridge the gap in effective treatments for the 500,000 patients worldwide who die annually from colorectal cancer.

Adoptive T-cell therapy of B-cell malignancies: Conventional and physiological chimeric antigen receptors

Hematological malignancies remain incurable diseases because of the high risk of relapse, even after complete remission. Adoptive T-cell therapy (ACT) using modified T cells with chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens expressed by B-cell malignancies represents an attractive approach for cancer immunotherapy. Investigators optimized the design of CARs to enhance receptor mediated T cell signaling and demonstrated that second and third generation CARs, including various costimulatory molecules, resulted in enhanced T-cell persistence and sustained antitumor activity in both in vitro and in vivo mouse models as well as clinical trials. We highlight advances in the use of CARs in the treatment of B-cell malignancies and future challenges in the use of adoptive therapy with CAR-engineered T cells.

Carving the CAR

Chimeric antigen receptors (CARs) are major histocompatibility complex (MHC)-independent one-polypeptide chain receptor molecules that, when expressed in T cells, can redirect specificity towards tumor or viral antigens. The antigen-binding domain of the CAR is derived from a single-chain antibody fragment and the transmembrane and signalling domains from the T-cell receptor CD3z chain, and optionally includes a costimulatory molecule such as CD28. CARs therefore combine the antigen specificity of an antibody and the property to activate a cytotoxic T cell in a single fusion molecule. A spacer is required between the extracellular antigen-binding domain and the transmembrane moiety. Most commonly, the constant immunoglobulin G (IgG)1 hingeCH2-CH3 Fc domain is used as a spacer domain, resulting in dimerization and thereby increasing CAR expression on the cell surface. In this issue, Hombach et al. reveal that the IgG1 spacer used in most CARs may also bind to surface IgG Fc gamma receptors expressed on macrophages and natural killer cells. This binding activates both the engineered T cells and the innate immune cells independent of the specificity of the CAR binding domain. Activation of CAR-positive T cells and Fc receptor-bearing cells through FcgR binding led to an unwanted, ‘off-target’ pro-inflammatory immune response. Hombach et al. provide a solution to this problem by modifying the sequences required for Fc binding in the constant immunoreceptor Fc domain, which eliminated interaction with Fc-positive cells but preserved stable expression of CARs on the surface of T cells and specific antigen recognition. In recent years, a variety of CARs with different scFv-binding domains have been designed. These CARs possess binding specificities for tumor-associated antigens, such as carcinoembryonic antigen and melanoma-associated antigens, CD30 and CD19, or for virus-infected cells, such as HIV or hepatitis B virus. Clinical trials that use CAR-modified T cells for adoptive immunotherapy of malignant diseases have been implemented and additional trials are planned. Owing to its one-polypeptide chain design and modular composition, the use of CARs substantially simplifies redirecting T cells with defined specificity. Upon antigen engagement, the intracellular signalling domain initiates T-cell activation, resulting in T-cell proliferation, secretion of pro-inflammatory cytokines and cytolysis of the antigen-positive target cell. There are major differences between the natural T cell receptor and a CAR, which confer potential advantages and disadvantages. CAR recognition is independent from antigen presentation on MHC, and allows broad clinical application for patients of any HLA haplotype. The lack of MHC restriction may also allow effective therapy of tumors that escape MHC-restricted T cells by downregulating MHC expression or through defects in antigen processing. However, early versions of CARs often provided weak activation of engineered T cells, mainly because CAR expression was unstable and costimulatory molecules were not, or only inadequately, engaged. The modular nature of CARs has enabled researchers to modify the CAR structure to achieve better activity. In the early research on CARs, it became obvious that inserting a ‘spacer’ domain, the IgG1 Fc being most commonly used, between the antigen-binding and transmembrane moieties improved expression and binding. Later on, research focussed on improving the signalling capacity by fusing costimulatory domains, such as CD28, OX40, 4-1BB or the src family kinase lck, to the CD3z signalling domain. T cells carrying CARs with CD3z and CD28 signalling domains proved to be superior in vivo and were able to reject established tumors in mice. Using T cells with natural specificity for Epstein–Barr virus is another strategy to provide appropriate costimulation through intermittent T-cell receptor ligation, and improved the persistence and probably also the activity of CARgrafted T cells in tumor patients. Severe side effects reported from recent clinical trials of adoptive immunotherapy with CAR-redirected T cells are a poignant reminder that much remains to be learned to realize the therapeutic promise of this approach. In one case, reactivity of the CAR antibody-binding domain with normal antigen-expressing cells was implicated as the cause of a cytokine storm and organ toxicity, and an unexplained sepsis-like syndrome occurred in a second case. The work of Hombach et al. illustrates the potential for ‘offtarget’ activation of engineered T cells via FcR binding to induce unwanted cytokine release and tissue damage—and, importantly, provides an improvement in CAR design that may help to avoid these side effects. It is anticipated that further improvements in CAR design will focus on increasing the specificity of the CARredirected T-cell response and minimizing the inflammatory response that is elicited. This should improve the safety and efficacy of adoptive immunotherapy with CAR-engineered T cells.

Sleeping Beauty Transposon‐mediated Engineering of Human Primary T Cells for Therapy of CD19+ Lymphoid Malignancies

We have reported earlier that the non-viral Sleeping Beauty (SB) transposon system can mediate genomic integration and long-term reporter gene expression in human primary peripheral blood (PB) T cells. In order to test whether this system can be used for genetically modifying both PB T cells and umbilical cord blood (UCB) T cells as graft-versus-leukemia effector cells, an SB transposon was constructed to coexpress a single-chain chimeric antigen receptor (CAR) for human CD19 and CD20. PB and UCB were nucleofected with the transposon and a transposase plasmid, activated and then expanded in culture using anti-CD3/CD28 beads. Stable dual-gene expression was confirmed in both T-cell types, permitting enrichment by positive selection with Rituxan. The engineered CD4(+) T cells and CD8(+) T cells both exhibited specific cytotoxicity against CD19(+) leukemia and lymphoma cell lines, as well as against CD19 transfectants, and produced high-levels of antigen-dependent Th1 (but not Th2) cytokines. The in vivo adoptive transfer of genetically engineered T cells significantly reduced tumor growth and prolonged the survival of the animal. Taken together, these data indicate that T cells from PB and UCB can be stably modified using a non-viral DNA transfer system, and that such modified T cells may be useful in the treatment of refractory leukemia and lymphoma.