Transduction with Allorestricted TCR as a Mean to Overcome Central Tolerance

Normally negative selection ablates high-avidity T cell clones that can react with self antigens of individual 1 in the context of self MHC molecules (H-2x-P1). But the clones specific to H-2x-P1 can be presented in allogeneic individual 2 because negative selection in this organism deletes the clones specific to tumor-associated antigens in the context of "another self' (i.e., allogeneic to P1) MHC (H-2y-P2). Therefore, allorestricted recognition can supposedly provide the basis for obtaining clones of individual 2 specific to the combination of MHC molecule with H-2x-P1 peptide of allogeneic recipient for adoptive immunotherapy (Figure 1).

One example of successful use of allorestricted recognition of tumor-associated antigens is the work of Elena Sadovnikova and Hans Stauss who obtained allorestricted CTL clones of H-2d mice. These clones were specific to the complex of H-2Kb plus mdm-2-derived peptide; it is noteworthy that mdm-2 is frequently overexpressed in tumor cells. In culture, these clones selectively reacted with tumor versus normal cells and killed melanoma and lymphoma cells but not normal H-2Kb- expressing dendritic cells. In vivo, the allorestricted clones caused retardation of growth of melanoma and lymphoma in syngeneic (H-2b) recipients (Sadovnikova and Stauss 1996). The same authors attempted to obtain allorestricted clones specific to a cyclin D1 peptide in the context of human HLA-A2. The clones lysed cyclin D1 overexpressing breast carcinoma cells but not Epstein-Barr-transformed lymphoblastoid cells (Sadovnikova et al. 1998). Allorestricted recognition became an efficient means of breaking tolerance to tumor-associated antigens and to get responses to leukemia-associated markers such as WT1, CD68, and CD45 (Gao et al. 2000; Sadovnikova et al. 2002; Amrolia et al. 2003).

Figure 1. Negative selection ablates high-avidity clones specific to tumor-associated antigens of individual 1. This process makes antitumor immune response and vaccination of individual 1 inefficient. Clones with required specificity may be isolated from allogeneic individual 2 for subsequent cloning of T cell receptors(TCRs) and retroviral transduction of T lymphocytes of individual 1.

Figure 1. Negative selection ablates high-avidity clones specific to tumor-associated antigens of individual 1. This process makes antitumor immune response and vaccination of individual 1 inefficient. Clones with required specificity may be isolated from allogeneic individual 2 for subsequent cloning of T cell receptors(TCRs) and retroviral transduction of T lymphocytes of individual 1.

Then, the TCR genes isolated from antigen-specific T cells can be exploited as generic therapeutic molecules for antigen-specific immunotherapy. Retroviral TCR gene transfer into patient T cells can readily produce populations of antigen-specific lymphocytes after a single round of polyclonal T cell stimulation. The TCR gene-modified lymphocytes are functionally competent in vitro and can have therapeutic efficacy in murine models in vivo. The TCR gene expression is stable, and the modified lymphocytes can develop into the memory T cells. Introduction of TCR genes into CD8+ and CD4+ lymphocytes provides an opportunity to use the same TCR specificity to produce antigen-specific killer and helper T lymphocytes. Thus, TCR gene therapy provides an attractive strategy to develop antigen-specific immunother-apy with autologous lymphocytes as a generic treatment option (Morris et al. 2005; Xue et al. 2005).

The ultimate goal of cancer immunotherapy is to utilize the immune system to eliminate malignant cells. Recently published research has mainly focused on the generation of effective antigen-specific T cell responses because of the general belief that T cell immunity is essential in controlling tumor growth and protection against viral infections. However, isolation of antigen-specific T cells for therapeutic application is a laborious task. Therefore, strategies were developed to genetically transfer tumor-specific immune-receptors into patients' T cells. To this end, the chimeric receptors were constructed that comprise the antibody fragments specific for tumor associated antigens linked to genes encoding signaling domains of TCR or Fc receptor. T cells with such chimeric antibody receptors recapitulate the immune-specific responses mediated by the receptor (Willemsen et al. 2003).

It is noteworthy that murine TCRs are highly functional when expressed in human lymphocytes. Recently published work compared human and mouse TCR function and expression to delineate the molecular basis for the apparent superior biological activity of murine receptors in human T lymphocytes. To this end, authors created hybrid TCRs where they swapped the original constant regions with either human or mouse ones. Murine or "murinized" receptors were overexpressed on the surface of human lymphocytes compared with their human/humanized counterparts and were able to mediate higher levels of cytokine secretion when cocultured with peptide-pulsed antigen-presenting cells. Preferential pairing of murine constant regions and improved CD3 stability seemed to be responsible for these observations. This approach allowed circumventing the natural low avidity of class I MHC TCR in CD4+ cells by introducing the murinized TCR into CD4+ lymphocytes, giving them the ability to recognize melanoma. These findings have implications for human TCR gene therapy (Sommermeyer et al. 2006).

In another work, the TCR gene transfer was investigated as a convenient method to produce antigen-specific T cells for adoptive therapy. The authors focused on the expression of two TCRs in T cells, which could impair their function or cause unwanted effects of mixed TCR heterodimers. With five different TCRs and four different T cells, either mouse or human, they have shown that some TCRs were strong—in terms of cell surface expression—and replaced weak TCRs on the cell surface, resulting in exchange of antigen specificity. Two strong TCRs were coex-pressed. The mouse TCR replaced human TCR on human T cells. Even though it is still poorly understood why some TCRaP combinations are preferentially expressed on T cells, the data suggest that T cells with exclusive tumor reactivity can potentially be generated by T cell engineering (Cohen et al. 2006).

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