Abstract
Membrane FasL is the natural trigger of Fas-mediated apoptosis. A soluble homotrimeric counterpart (sFasL) also exists which is very weakly active, and needs oligomerization beyond its trimeric state to induce apoptosis. We recently generated a soluble FasL chimera by fusing the immunoglobulin-like domain of the leukemia inhibitory factor receptor gp190 to the extracellular region of human FasL, which enabled spontaneous dodecameric homotypic polymerization of FasL. This polymeric soluble human FasL (pFasL) displayed anti-tumoral activity in vitro and in vivo without systemic cytotoxicity in mouse. In the present work, we focused on the improvement of pFasL, with two complementary objectives. First, we developed more complex pFasL-based chimeras that contained a cell-targeting module. Secondly, we attempted to improve the production and/or the specific activity of pFasL and of the cell-targeting chimeras. We designed two chimeras by fusing to pFasL the extracellular portions of the HLA-A2 molecule or of a human gamma-delta TCR, and analyzed the consequences of co-expressing these molecules or pFasL together with sFasL on their heterotopic cell production. This strategy significantly enhanced the production of pFasL and of the two chimeras, as well as the cytotoxic activity of the two chimeras but not of pFasL. These results provide the proof of concept for an optimization of FasL-based chimeric proteins for a therapeutic use.
Highlights
FasL (CD95L) is a type II homotrimeric transmembrane protein of the TNF family [1]
We observed that the larger chimeric proteins HLA-pfFasL and TCR-pfFasL were produced at much lower levels (36±18 ng/ml and 133±46 ng/ml respectively) than sfFasL (17±8.5 μg/ml), whereas pfFasL was secreted at an intermediate level (3.3±2.9 μg/ml)
We described the design of polymeric FasLbased chimeric proteins with better heterotopic cellular production and enhanced biological activity
Summary
FasL (CD95L) is a type II homotrimeric transmembrane protein of the TNF family [1]. FasL is expressed on activated T lymphocytes and natural killer cells, as a mean to eliminate transformed or infected cells expressing the transmembrane receptor Fas (CD95/APO-1) [2]. A humanized anti-Fas antibody did not display such toxicity but frequently triggered anti-idiotype sensitization in monkeys [11] Besides this approach, numerous research teams have designed sFasL-derived agonists devoid of the toxicity of the anti-Fas antibodies and with at least comparable apoptotic activity [reviewed in references 12,13, using either spontaneously polymeric (i.e. at least hexameric) or oligomeric (i.e. smaller than the hexamer) sFasL-based fusion proteins. Numerous research teams have designed sFasL-derived agonists devoid of the toxicity of the anti-Fas antibodies and with at least comparable apoptotic activity [reviewed in references 12,13, using either spontaneously polymeric (i.e. at least hexameric) or oligomeric (i.e. smaller than the hexamer) sFasL-based fusion proteins These chimeras contained or not, N-terminally to sFasL, a targeting module able to recognize the target cell to be destroyed or to be recognized by a carrier cell, which will present FasL to the target cell, thereby mimicking transmembrane FasL. Representative examples were the hexameric MegaFasL [9] which associates the collagen domain of adiponectin (ACRP30) to sFasL, the streptavidin-FasL (SAFasL) [14], and the CTLA-4-FasL [15,16] chimeras, which respectively bind to biotin-labeled or to CD80-expressing cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
