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November 8, 2016

Domain Swaps Lessen Autoimmunity in TCR Gene Therapy

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    A domain-swapped T-cell receptor (TCR) engaging a peptide presented by a tumor cell on a major histocompatibility complex (MHC) molecule. In TCR gene therapy, genes encoding a tumor-specific TCR are introduced to a patient's T cells, retargeting them to attack cancer cells. Swapping the constant domains between the alpha and beta chains of the tumor-specific TCR prevents these chains from mispairing with the TCR chains resident to each T cell, which can result in deleterious autoimmunity. [Caltech]

    On the surface of the T cell, one T-cell receptor (TCR) type has the makings of an immune party, but two TCRs could be a crowd. A second TCR type can be disruptive, a cause of autoimmunity. Or it can be the life of the party, the instigator of an amplified immune response. Rancor or good times—it all depends on the second TCR’s engineering and its potential for mispairing between therapeutic αβ chains and endogenous αβ chains. Mispairing reduces therapeutic TCR surface expression and generates self-reactive TCRs—limiting the effectiveness of TCR gene therapy, a cancer immunotherapy approach.

    To prevent TCR mispairing and improve the safety of TCR gene therapy, researchers based at the California Institute of Technology have developed a new kind of TCR-engineered T cell. It is the result of a new technique—domain swapping.

    Details of the new technique appeared November 8 in the journal eLife, in an article entitled, “Domain-Swapped T Cell Receptors Improve the Safety of TCR Gene Therapy.” The new technique involves the generation of hybrid genes encoding TCR chains with their α and β constant domains swapped in a compensatory fashion.

    “When paired, domain-swapped (ds)TCRs assemble with CD3, express on the cell surface, and mediate antigen-specific T cell responses,” wrote the authors of the eLife article. “By contrast, dsTCR chains mispaired with endogenous chains cannot properly assemble with CD3 or signal, preventing autoimmunity.”

    The specificity of the TCR in each T cell results from the pairing of two protein chains—the α chain and the β chain—each of which has constant domains (shared between all TCRs) and variable domains (unique to each T cell). Normally, each T cell encodes only one α chain and one β chain, which pair to form a single TCR. In TCR gene therapy, the introduction of genes encoding a tumor-reactive TCR results in T cells that express two α chains and two β chains, with four possible pairings. This nonphysiological situation poses a risk of autoimmunity.

    "As T cells are produced, the immune system 'auditions' them, eliminating those that react to healthy cells and selecting those with potential to recognize diseased cells," explained Michael Bethune, a postdoctoral scholar at CalTech, and lead author of the current study. "However, in T cells engineered to express a second TCR, the introduced chains can mispair with the resident chains, resulting in TCRs with unintended and unpredictable specificity. These mispaired TCRs are not auditioned by the immune system, and some will target healthy cells causing autoimmunity."

    Up to 90% of mice administered TCR-engineered T cells develop autoimmune disease, and cultured human T cells that are engineered to express two TCRs also react with healthy cells.

    Autoimmunity may be avoided by means of domain swapping because “mispairing between domain-swapped chains and resident chains results in TCRs that lack domains needed for functional assembly of the TCR complex," Bethune asserted. "This ensures that only correctly paired domain-swapped TCRs function at the surface of the cell."

    “We validate [the domain-swapping] approach in cell-based assays and in a mouse model of TCR gene transfer-induced graft-versus-host disease,” reported the eLife article’s authors. “We also validate a related approach whereby replacement of αβ TCR domains with corresponding γδ TCR domains yields a functional TCR that does not mispair.”

    Indeed, the researchers found that domain-swapped TCRs and unmodified TCRs both function in human T cells, and they prevented tumor growth in mice to a similar extent. However, whereas unmodified TCRs mispaired with resident TCR chains in both mouse and human T cells, and caused autoimmunity in mice, domain-swapped TCRs did not.

    In addition to preventing mispairing, domain-swapped TCRs highlight a surprising robustness to the function of the TCR complex. The Caltech group, which was led by David Baltimore, the senior author of the eLife article, teamed with Mike Kuhns at the University of Arizona to determine that domain-swapped TCRs assemble in a similar manner to unmodified TCRs, despite significant structural rearrangement of the constituent protein chains. Domain-swapped TCRs may be useful tools for further study of the structure and function of the TCR complex.

    Finally, in collaboration with Wolfgang Uckert at the Max Delbrück Center for Molecular Medicine, the researchers showed that domain-swapped TCRs were expressed at higher levels on the T-cell surface when the resident TCR genes were silenced.

    "Our paper focuses on the increased safety afforded by domain-swapping,” noted Bethune. “But combining these two solutions [domain swapping and gene silencing] may result in a therapy with improved safety and efficacy compared to current practice."

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