AD822, AW199, AW200 and AW201 antibodies against the human Fas receptor (CD95) bind the surface of HEK293 cells as revealed by flow cytometry

Authors

  • Soyan Dawit
  • Meg-Mai-Ly Diep
  • Odyssee Ferrillo
  • Maxime Guertler
  • Catalina Gureu
  • Margaux Herren
  • Simon leyss
  • Karolina Liborova
  • Marie Maître
  • Livia Manghetti
  • Lucie Mencier
  • Lana Moudarres
  • Gaelle Najand
  • Laura Roch
  • Audrey Ruetsche
  • Valerie Schwitzguebel
  • Wissem Seddiki
  • Paola Soulie
  • Nneka Anagbogu
  • Kenza Bennani
  • Elise Brun
  • Hannah Butterworth
  • Jennifer Carry
  • Jean-Pierre Aubry-Lachainaye
  • Cécile Gameiro
  • Stéphane Durual
  • Tania Jauslin
  • Cyril Guilhen

DOI:

https://doi.org/10.24450/journals/abrep.2023.e1143

Abstract

The recombinant antibodies AD822, AW199, AW200 and AW201 bind HEK293 cells expressing the human Fas receptor protein as assessed by flow cytometry.

Introduction

The Fas receptor (or CD95, UniProt #P25445) is a transmembrane protein expressed at the cell surface. Binding of the Fas receptor to its ligand FasL leads to cell apoptosis (Itoh et al., 1991). Here, we tested by flow cytometry the ability of 6 recombinant antibodies (AD822, AW199, AW200 and AW201, AD825 and AJ757) to bind HEK293 cells that are known to express the Fas receptor (Uhlèn et al., 2015).

Materials & Methods

Antibodies: ABCD_AD822, ABCD_AD825, ABCD_AW199, ABCD_ AW200, ABCD_AW201, and ABCD_AJ757 antibodies (ABCD nomenclature, http://web.expasy.org/abcd/) were produced by the Geneva Antibody Facility (http:/unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding portion fused to a rabbit IgG Fc. (see Table 1 for clone names and references). HEK293 suspension cells (growing in HEK TF medium, Xell #861-0001, supplemented with 0.1% Pluronic F68, Sigma #P1300) were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants were collected after 4 days (see Table 1 for individual yields).

ABCD Clone Reference Yield (mg/L)
AD822 h-HFE7A Haruyama et al., 2002 10
AD825 huF919 Kamon et al., 2000 85
AW199 E09 Chodorge et al., 2012 90
AW200 E01 Chodorge et al., 2012 80
AW201 E03 Chodorge et al., 2012 80
AJ 757 APO-1 Nalivaiko et al., 2016 <5
Table 1: Clone number, reference and production yields for the antibodies used in this study.

Protocol: The whole procedure was carried out at 4°C. 1x106 HEK293 cells were pelleted and washed once in washing buffer (PBS + 0.2% BSA (w/v)). Cells were then incubated for 20 minutes with the recombinant antibodies (5 mg/L in 500 µL of PBS-BSA, excepted for the antibody AJ757 that was used undiluted). After two washes in 1 mL of washing buffer, cells were incubated for 20 minutes with 500 µL of secondary goat anti-rabbit IgG antibody conjugated to AlexaFluor-488 (1:400, Molecular Probes#A11034). After four washes in 1 mL of washing buffer, cells were resuspended in 500 µL of washing buffer and analyzed with a CytoFLEX S4 flow cytometer (Beckman Coulter). Dead cells were excluded after staining with Dapi (5 mg/L) and gating on Dapi negative cells.

Results

Antibodies AD822, AW199, AW200 and AW201 were able to bind to the HEK293 cells most probably via the Fas receptor protein present at the surface. No signal was detected when the primary antibody was omitted (Fig. 1). In our experimental conditions, AD825 and AJ757 did not label HEK293 cells and thus presumably did not recognize the Fas receptor protein. For AJ757, this might be due to the fact that this antibody is poorly produced. The same experiments performed using a lower antibody concentration (1 mg/L) yielded similar results (data not shown). Further experiments (ideally using specific KO cells) will be necessary to confirm the specific binding of AD822, AW199, AW200 and AW201 to the Fas receptor protein at the surface of the HEK293 cells suggested in this study.

Figure 1. Mono-parametric representation of flow cytometry analysis depicting the Alexa Fluor 488 signal. AD822, AW199, AW200 and AW201 antibodies labeled the Fas receptor protein in HEK293 cells. No signal was detected when primary antibody was omitted. AD825 and AJ757 did not recognize the Fas receptor by flow cytometry.

Conflict of interest

The authors declare no conflict of interest.

References

Chodorge M, Züger S, Stirnimann C, Briand C, Jermutus L, Grütter MG, et al. A series of Fas receptor agonist antibodies that demonstrate an inverse correlation between affinity and potency. Cell Death Differ. 2012 Jul;19(7): 1187‑95. PMID: 22261618.

Haruyama H, Ito S, Miyadai K, Takahashi T, Kawaida R, Takayama T, et al. Humanization of the Mouse Anti-Fas Antibody HFE7A and Crystal Structure of the Humanized HFE7A Fab Fragment. Biological and Pharmaceutical Bulletin. 2002;25(12): 1537‑45. PMID: 12499636.

Uhlén M, Fagerberg L, Hallström BM, et al. Proteomics. Tissue-based map of the human proteome. Science. 2015;347(6220):1260419. PMID: 25613900

Kamon S, Tomokazu M, Shigekazu N, Sung CM, Maximiliano V. Anti-Fas ligand antibody and assay method using the anti-Fas ligand antibody. United States, US6114507. 2000.

Nalivaiko K, Hofmann M, Kober K, Teichweyde N, Krammer PH, Rammensee HG, et al. A Recombinant Bispecific CD20×CD95 Antibody With Superior Activity Against Normal and Malignant B-cells. Mol Ther. 2016 Feb;24(2): 298‑305. PMID: 26581163.

Itoh N, Yonehara S, Ishii A, et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell. 1991;66(2):233-243. PMID: 1713127.

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Published

2023-03-29 — Updated on 2023-04-25

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1.
Dawit S, Diep M-M-L, Ferrillo O, Guertler M, Gureu C, Herren M, leyss S, Liborova K, Maître M, Manghetti L, Mencier L, Moudarres L, Najand G, Roch L, Ruetsche A, Schwitzguebel V, Seddiki W, Soulie P, Anagbogu N, Bennani K, Brun E, Butterworth H, Carry J, Aubry-Lachainaye J-P, Gameiro C, Durual S, Jauslin T, Guilhen C. AD822, AW199, AW200 and AW201 antibodies against the human Fas receptor (CD95) bind the surface of HEK293 cells as revealed by flow cytometry. Antib. Rep. [Internet]. 2023 Apr. 25 [cited 2024 Feb. 22];6(1):e1143. Available from: https://oap.unige.ch/journals/abrep/article/view/1143

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