As an alternative to TIL therapy, highly avid TCRs can be cloned from naturally occurring T cells and, by using gene transfer vectors, introduced into patient’s lymphocytes, creating the opportunity to generate large quantities of antigen specific T cells for treatment ()14, 15
. The first step in TCR gene therapy is to isolate a high affinity T cell clone for a defined target antigen. These TCRs can be isolated from patients with rare, highly reactive T cell clones that recognize and lyse target tumor cells16
. The isolation of these rare tumor reactive T cell clones is often the rate-limiting step in this procedure and these clones often have low affinity for the target antigen.
Clinical application of gene-modified T cells. Shown is a diagram of the use of both natural (top) and gene modified T cells (bottom) for treatment of cancer.
One of the most important applications of biotechnology to human immunology has been the development transgenic mice, which are engineered with human immune system genes. Transgenic mice containing the human leukocyte antigen system (HLA) can be used to generate TCRs against human antigens. This is done by immunizing HLA transgenic mice with human-specific antigenic peptides, and isolating the resultant mouse T cells, which will contain a TCR that recognizes a human peptide. Using this approach, investigators have been able to generate multiple murine TCRs against a variety of human tumor antigens from different histologies17, 18
. Another method that does not require patient material to obtain a tumor antigen reactive TCR, is the use of phage display technology for TCR isolation. Phage display technology has the advantage that it does not depend on the ability to generate T cell clones yet allows for the selection of high-affinity TCRs reactive against a variety of antigens19, 20
. One potential drawback to TCRs isolated by phage display is that caution must be exercised in the selection of very high-affinity TCRs, which have been shown to lose specificity21
. In theory, these nonhuman TCR isolation technologies create the possibility to provide the patient with a tailored therapy based on their unique antigen expression pattern, potentially ushering in a new era of personalized cancer immunotherapy.
With either method, after the high-avidity T-cell clone is obtained, the TCR alpha and beta chains are isolated and cloned into a gene expression vector (). To assure co-expression of both chains, the TCR alpha and beta genes are most commonly linked via a picornavirus 2A ribosomal skip peptide22
. For human applications, gene transfer platforms that can mediate stable gene transfer are the systems of choice (e.g., gamma-retroviral, lentiviral vectors, or transposons)23–25
. The two viral based systems are complex biologic reagents that require extensive safety testing for human applications but they mediate very high gene transfer efficiencies and have been used for over two decades in human studies. Transposons are a relative newcomer in the human gene therapy field and have the advantage that they are plasmid DNA-based, are much simpler to produce, and require less upfront safety testing. Ex vivo gene transfer is accomplished by first stimulating T cell growth and the activated cells are then transduced and expanded in culture to numbers sufficient for clinical applications (generally > 1X108
Figure 2 Producing anti-tumor T cells. Shown is the general schema for the construction of gene transfer reagents for the engineering of T cells with anti-tumor receptors. Step 1. Anti-tumor antigen receptor can be isolated as natural TCRs (left) or an antibody (more ...)
The genetic transfer of an antigen-specific TCR can generate antigen-specific T cells from any naturally occurring T cell. It has been shown that the transduced lymphocytes exhibit the specificity of the parental clone 26, 27
. These TCR gene engineered T cells can secrete cytokines upon encountering tumor antigen positive targets, exhibit tumor cell specific lysis, and expand upon antigenic stimulation.
Unlike antibodies, the affinity of many naturally occurring TCRs for their target peptide is low (in the μmolar range), and therefore, steps to improve the performance of TCRs through protein engineering have been made. These include strategies to improve TCR affinity, increase cell surface expression, and prevent mixed dimer formation between the introduced and endogenous TCR chains (such mixed dimmers would not target the tumor antigen)28
. Single or dual amino acid substitutions in the complementary determining region (CDR) of the alpha or beta chain has been shown to improve antigen specific reactivity in T cells29
. Development of hybrid TCRs where the human constant region is replaced by a murine constant region has been shown to improve specific chain pairing as well as facilitate stronger association with T cell signaling proteins of the CD3 complex. T cells engineered with these hybrid TCRs exhibit superior surface expression, cytokine release and cytolytic activity 30–32
. Introduction of an additional cysteine bridge in the constant region of the TCR alpha and beta chains was also shown to improve pairing 32, 33
. Inverse exchange of an amino acid pair at the interface the TCR alpha or beta constant regions that normally forms a “knob-into-hole” configuration into a “hole-into-knob”, has been shown to favor selective assembly of the introduced TCR with preserved function of the receptors34
. In addition, it is possible to produce a chimeric molecule by fusing the CD3zeta gene to the TCR alpha and beta chains, and in cell lines engineeered with these chimeric molecules, specific alpha-beta chain pairing was reported35
An alternative non-genetic approach is to use γδ T cells for adoptive therapy, in which αβ heterodimers can be intoduced without the concern for heterogenous pairing. However, whether γδ T cells will function and persist as well as in αβ T cells in the setting of adoptive T cell therapy is still under investigation36, 37
. All of these modifications have the potential to increase the anti-tumor activity of the engineered T cells. The main advantage of using TCRs to target tumors is that they function through well understood T cell signlaling pathways and are the natural means by which the body clears forgein elements. The main disadvantage of TCR-based anti-cancer therapies is that the biology of the TCR restircts it to one HLA type and alpha/beta TCRs cannot target non- protein tumor antignes (i.e., carbohydrate or lipid antigens).