Transduktion primärer humaner T-Zellen mit einem CD44v7/8-spezifischen T-Zellrezeptor

Zusammenfassung

Fragestellung

Das variante CD44 v7/8-Epitop wird in ca. 70 % der invasiven Zervixkarzinome an der Zelloberfläche exprimiert und bietet sich somit als tumorspezifisches Antigen zur gentechnischen In-vitro-Manipulation der Erkennungsspezifität von T-Lymphozyten an. Dies wird durch die Klonierung und Transduktion des Gens für einen chimären T-Zell-Rezeptor (cTCR) erreicht, in welchem das Einzelketten-Fv-Fragment des CD44v7/8-spezifischen monoklonalen Antikörpers VFF 17 mit dem Gen der signaltransduzierenden Zeta-Kette des cTCR fusioniert wird. Die Schwierigkeit liegt darin, dass viele Gentransfermethoden sich nur begrenzt für primäre humane T-Zellen eignen. Ziel unserer Arbeit war somit primär die Etablierung der Methodik der Gentransduktion von primären humanen T-Zellen, um die Voraussetzung für die Expression des chimären T-Zell-Rezeptors in primäre humane T-Zellen zu schaffen und CD44v7/8-positive Tumorzellen zu erkennen.

Methodik

Das scFv(VFF 17)y:α:ζ-Fusions-Gen wurde zunächst mittels retroviraler Transduktion in die Galv-pseudotypische amphotrope Verpackungs-Zelllinie PG13 eingebracht. Primäre humane T-Zellen wurden durch Inkubation mit anti-CD3- und anti-CD28-Antikörpern aktiviert. Der retrovirale Gentransfer erfolgte auf mit rekombinantem Fibronektin beschichteten Platten.

Ergebnisse

Der retrovirale Gentransfer des scFv(VFF 17)y:α:ζ-Fusions-Gens in die Verpackungs-Zelllinie PG13 war erfolgreich. Die Expression des chimären Rezeptors konnte auf Transkriptionsebene in den transduzierten naiven T-Zellen nachgewiesen werden. Eine Oberflächenexpression des cTCR war nicht detektierbar.

Schlussfolgerung

Die Transduktion der primären humanen T-Zellen war erfolgreich. Zukünftige Untersuchungen werden zeigen, ob die nicht detektierbare Oberflächenexpression des chimären Rezeptors auf Proteinebene an einer Pseudotransduktion, nicht induzierten Translation oder an einer fehlerhaften Proteinfaltung lag.

Abstract

Purpose

The variant epitope CD44v7/8 is frequently expressed on cervical carcinoma. Therefore CD44v7/8 is a tumour-specific antigen and may play a role as a promising target for tumour-specific immunotherapy. Since many gene transfer methods in human T-cells are limited and problematical, the primary aim of our study was to develop a method for the transduction of T-lymphocytes with a CD44v7/8 target specifity.

Material and Methods

The genes coding for the single chain fragment scFv of the mononuclear antibody VFF 17 and of the ζ-chain of the TCR (T-cell receptor) complex were fused and inserted into a retroviral vector. We retrovirally transduced primary human T-cells. The gene transfer was tested by PCR (polymerase chain reaction). A FACS (fluorescent activated cell sorter) and a functionality test were performed to investigate the expression of the chimeric receptor of primary human T-cells.

Results

The mRNA expression of the chimeric receptor on the transduced human T-cells could be demonstrated. But on the protein level no expression of the scFv(VFF 17)y:α:ζ-fusion protein could be detected.

Discussion

Further studies will show whether the undetected expression of the surface protein on the transduced T-cells is due to a pseudotransduction, a not performed translation or an incorrect folding of the protein and consequently a flawed transportation of the protein to the surface.

CD44v7/8 - T-Zellen - Gentherapie - Zervixkarzinom

CD44v7/8 - T-cells - gene therapy - cervical carcinoma

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