A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties.

Engagement of the T cell receptor (TCR) of mature T lymphocytes can lead either to activation/proliferation responses or programmed cell death. To understand the molecular regulation of these two fundamentally different outcomes of TCR signaling, we investigated the participation of various components of the TCR-CD3 complex. We found that the TCR-zeta chain, while not absolutely required, was especially effective at promoting mature T cell apoptosis compared with the CD3 epsilon, gamma, or delta chains. We also carried out mutagenesis to address the role of the immunoreceptor tyrosine-based activation motifs (ITAMs) that are the principal signaling components found three times in the TCR-zeta chain and once in each of the CD3 epsilon, gamma, or delta chains. We found that the ability of the TCR-zeta chain to promote apoptosis results both from a quantitative effect of the presence of multiple ITAMs as well as qualitatively different contributions made by individual ITAMs. Apoptosis induced by single chain chimeras revealed that the first zeta ITAM stimulated greater apoptosis than the third zeta ITAM, and the second zeta ITAM was unable to trigger apoptosis. Because microheterogeneity in the amino acid sequence of the various ITAM motifs found in the TCR-zeta and CD3 chains predicts interactions with distinct src-homology-2-domain signaling proteins, our results suggest the possibility that individual ITAM motifs might play unique roles in TCR responses by engaging specific signaling pathways.

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Safe and Effective Adoptive T-Cell Receptor Transfer with a High Affinity Single Chain p53(264–272)-Specific TCR

Blood ◽

10.1182/blood.v120.21.4226.4226 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4226-4226

Author(s):

Hakim Echchannaoui ◽

Jutta Petschenka ◽

Edite Antunes ◽

Matthias Theobald

Keyword(s):

T Cells ◽

T Cell ◽

Gene Transfer ◽

T Cell Receptor ◽

Cell Receptor ◽

Cell Transfer ◽

Single Chain ◽

Adoptive T Cell Transfer ◽

High Affinity ◽

T Cell Transfer

Abstract Abstract 4226 Several studies have demonstrated the clinical efficacy of adoptive T cell therapy for targeting cancer. Using HLA-A2.1 transgenic mice, we have demonstrated the feasibility of T-cell receptor (TCR) gene transfer into T cells to circumvent self-tolerance to the widely expressed human p53(264–272) tumor-associated antigen and developed approaches to generate high-affinity CD8-independent TCR. A safety concern of TCR gene transfer is the pairing of endogenous and introduced TCR chains resulting in the potential generation of self-reactive T cells (off-target autoimmunity). Several strategies to favor matched TCR chains pairing and thus enhancing TCR cell surface expression, including optimization of TCR encoding nucleotide sequences, introduction of an additional inter-chain disulfide bond between the TCR α and β chain constant domains, coexpression of both TCR α and β encoding-genes using self-cleaving 2A virus peptide-based retroviral vectors have been applied. However, adoptive transfer of mouse T cells transduced with modified p53-specific TCRs into p53-deficient humanized (A2Kb) mice was inducing lethal autoimmunity due to the formation of self-reactive TCRs infiltrating vital organs, such as spleen, liver and bone marrow. Therefore, an optimized single chain (sc) p53-specific TCR was engineered to avoid the formation of mismatched TCR heterodimers. The safety and therapeutic efficiency of this approach were evaluated in humanized mouse models of adoptive T cell transfer and successfully demonstrated that optimized p53-specific scTCR-redirected T cells (i) do not induce OFF-target autoimmunity and (ii) mediate antitumor reactivity. Importantly, because the expression of p53 antigen on normal tissues raises the concern of potential on-target toxicity, we performed adoptive T cell transfer experiments in humanized mice expressing the Human p53 protein (Hupki mice) and did not observe any sign of TCR gene transfer-mediated GvHD in this model. In conclusion, these mouse studies suggest that the optimized p53(264–272)-specific scTCR could represent a safe and efficient approach for TCR-based gene therapy. Disclosures: No relevant conflicts of interest to declare.

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Characterization of a single-chain T-cell receptor expressed in Escherichia coli.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.10.4759 ◽

1992 ◽

Vol 89 (10) ◽

pp. 4759-4763 ◽

Cited By ~ 41

Author(s):

W. F. Hoo ◽

M. J. Lacy ◽

L. K. Denzin ◽

E. W. Voss ◽

K. D. Hardman ◽

...

Keyword(s):

Escherichia Coli ◽

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Single Chain

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Display of functional αβ single-chain T-cell receptor molecules on the surface of bacteriophage

Journal of Immunological Methods ◽

10.1016/s0022-1759(98)00153-7 ◽

1998 ◽

Vol 221 (1-2) ◽

pp. 59-76 ◽

Cited By ~ 23

Author(s):

Jon A Weidanz ◽

Kimberlyn F Card ◽

Ana Edwards ◽

Ethan Perlstein ◽

Hing C Wong

Keyword(s):

T Cell ◽

T Cell Receptor ◽

Cell Receptor ◽

Single Chain

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Anti-graft-versus-host disease effect of DT390-anti-CD3sFv, a single- chain Fv fusion immunotoxin specifically targeting the CD3 epsilon moiety of the T-cell receptor

Blood ◽

10.1182/blood.v88.6.2342.bloodjournal8862342 ◽

1996 ◽

Vol 88 (6) ◽

pp. 2342-2353 ◽

Cited By ~ 24

Author(s):

DA Vallera ◽

A Panoskaltsis-Mortari ◽

C Jost ◽

S Ramakrishnan ◽

CR Eide ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Fusion Protein ◽

Enzymatic Activity ◽

T Cell Receptor ◽

Graft Versus Host Disease ◽

Cell Receptor ◽

Single Chain ◽

Graft Versus Host ◽

Single Chain Fv

In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3 epsilon moiety of the T-cell receptor (TCR) had a potent, but partial, graft-versus-host disease (GVHD) effect (Vallera et al, Blood 86:4367, 1995). Therefore, in this current study, we determined whether a fusion immunotoxin made with anti-CD3 single-chain Fv (sFv), the smallest unit of antibody recognizing antigen, would have anti-GVHD activity. A fusion protein was synthesized from a construct made by splicing sFv cDNA from the hybridoma 145–2C11 to a truncated form of the diphtheria toxin (DT390) gene. DT390 encodes a molecule that retains full enzymatic activity, but excludes the native DT binding domain. The DT390-anti-CD3sFv hybrid gene was cloned into a vector under the control of an inducible promoter. The protein was expressed in Escherichia coli and then purified from inclusion bodies. The DT390 moiety of the protein had full enzymatic activity compared with native DT and DT390-anti-CD3sFv, with an IC50 of 1 to 2 nmol/L against phytohemagglutinin-stimulated and alloantigen-stimulated T cells. Specificity was shown (1) by blocking the IT with parental anti-CD3 antibody, but not with a control antibody; (2) by failure of DT390-anti-CD3sFv to inhibit lipopolysaccharide-stimulated murine B cells; (3) by failure of an Ig control fusion protein, DT390-Fc, to inhibit T-cell responses; and (4) with in vivo immunohistochemisty studies. GVHD was studied in a model in which C57BL/6 (H-2b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B.-17 scid (H-2d) mice to assess GVHD effects in the absence of irradiation toxicity. Flow cytometry studies showed that donor T cells were expanded 57-fold and histopathologic analysis showed the hallmarks of a lethal model of GVHD. Control mice receiving phosphate-buffered saline showed 17% survival on day 80 after bone marrow transplantation, and mice receiving 2 micrograms DT390-Fc fusion toxin control administered in 2 daily doses for 6 days (days 0 through 5) had a 43% survival rate. In contrast, 86% of mice receiving the same dose of DT390-anti-CD3sFv were survivors on day 80, a significant improvement, although survivors still showed histopathologic signs of GVHD. These findings suggest that new anti-GVHD agents can be genetically engineered and warrant further investigation of fusion proteins for GVHD treatment.

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