A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells

Abstract

Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53. An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide. In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex. To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38). The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide. Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner. These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex. Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy.

Quantifying and Imaging NY-ESO-1/LAGE-1-Derived Epitopes on Tumor Cells Using High Affinity T Cell Receptors

“…HLA class I-associated Ags have also been detected on cells using Abs (15)(16)(17) or Fab monomers/tetramers (18 -20) specific for given peptide-HLA complexes, allowing semiquantitative determination of surface Ag levels by FACS. A similar approach has used soluble monomers (21,22), dimers (23), or tetramers (24,25) of soluble TCRs to detect class I-restricted Ags, although given the naturally low affinity of TCR for Ag, it remains uncertain how quantitative and Ag sensitive such measurements are.…”

Engineered T Cell Receptors and their Potential in Molecular Medicine

“…In these studies soluble, natural TcRs were fused with an immunomodulatory molecule that redirected the immune system to engage the malignancy. In the first study, exotoxin A was attached to a single-chain TcR and this fusion was shown to selectively kill cells presenting the p53 peptide in vitro [44]. In the second study, interleukin-2 was fused with a single-chain, p53-specific TcR and this fusion was shown to reduce lung metastases in mice [45].…”

“…These receptors could be used as competitive T cell blockers in the treatment of autoimmunity, or for receptor-guided therapies through the artificial attachment of molecular fusion partners for treating malignancies or viral infection. Such fusions could include radionuclides [147], toxins [44], cytokines [45,148,149], mAb fragments [46], gold nanoparticles/nanorods [150,151], viral particules [134], drug-enclosing liposomes [152], and magnetic nanoparticles [153,154]. The attachment of magnetic nanoparticles is of particular interest for immunodiagnostics and immunotherapy [155] since target cells coated in magnetic nanoparticles can be clearly visualized in vivo through magnetic resonance imaging [156,157], and a recent study has claimed that single cell resolution can be achieved [158].…”

Stable, soluble, high-affinity, engineered T cell receptors: novel antibody-like proteins for specific targeting of peptide antigens

“…However, there are reports of wild‐type single‐chain TCRs fused to, e.g. exotoxin A protein, specifically killing cells presenting p53 peptide antigens [56], and high‐affinity engineering may improve the efficacy of such approaches.…”

“…However, there are reports of wildtype single-chain TCRs fused to, e.g. exotoxin A protein, specifically killing cells presenting p53 peptide antigens [56], and high-affinity engineering may improve the efficacy of such approaches. Fusion of wild-type single-chain TCRs to IL-2 have been reported to reduce lung metastases in an HLA-A*0201 transgenic mouse tumour model [57], and immune effector function may also be provided by fusion to IgG1 H [58].…”