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A multi-Fc-species system for recombinant antibody production.

Moutel S, El Marjou A, Vielemeyer O, Nizak C, Benaroch P, Dübel S, Perez F - BMC Biotechnol. (2009)

Bottom Line: We developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use.This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies.Altogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS UMR144, 26 rue d'Ulm, F75248 Paris Cedex 05, France. Sandrine.Moutel@curie.fr

ABSTRACT

Background: Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural antibodies.

Results: We developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use. This series enables the fusion of single chain Fv antibodies with human, mouse or rabbit Fc so that a given antibody is no longer restricted to a particular species. This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies. We also show that this multi-Fc species production system can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies and we converted the widely used 9E10 mouse anti-Myc-tag antibody into a human and a rabbit antibody.

Conclusion: Altogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use.

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Related in: MedlinePlus

Production and use of multi-species scFv-Fc antibodies. A: Schematic map of the generic pFuse-xIgG-Fc2 used to express scFvs (adapted from Invivogen). Three vectors are available bearing either a human (pFuse-hIgG-Fc2), mouse (pFuse-mIgG-Fc2) or rabbit IgG2 (pFuse-rIgG-Fc2). The scFv is inserted (NcoI/NotI) inframe between the IL2 secretion signal and the Fc portion. The vector can be selected using Zeocin both in E. coli and in mammalian cells. B: Western Blot analysis of the scFv F2C fused to Fc domains from 3 different species. hF2C, rF2C and mF2C secreted by CHO cells were purified on protein A and analyzed by western blot using either species-specific anti-Fc antibody, or using a polyclonal anti-scFv. Each F2C fusion antibody is detected in CHO supernatants and is respectively seen as a human, rabbit or mouse antibody.C: scFv-hFc are expressed as dimers. hF2C proteins were separated by SDS-PAGE either in reducing (left) or in non-reducing conditions (right) and analyzed by western blot. This confirmed that hF2C behaves as a dimer in non-reducing conditions. D: Using multi-species scFv-Fc antibodies for Western blot immuno-labeling. A HeLa cell protein lysate was separated by SDS-PAGE and transferred on a nitrocellulose membrane. The membrane was probed using anti-MyosinII SF9 (lane 1, 3 and 5) or anti-Tubulin F2C (2, 4, 6) antibodies fused to either human (h; 1,2), rabbit (r; 3,4) or mouse (m; 5,6) Fc. This experiment shows that each scFv kept its specificity when used as a human, mouse or rabbit antibody.
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Figure 1: Production and use of multi-species scFv-Fc antibodies. A: Schematic map of the generic pFuse-xIgG-Fc2 used to express scFvs (adapted from Invivogen). Three vectors are available bearing either a human (pFuse-hIgG-Fc2), mouse (pFuse-mIgG-Fc2) or rabbit IgG2 (pFuse-rIgG-Fc2). The scFv is inserted (NcoI/NotI) inframe between the IL2 secretion signal and the Fc portion. The vector can be selected using Zeocin both in E. coli and in mammalian cells. B: Western Blot analysis of the scFv F2C fused to Fc domains from 3 different species. hF2C, rF2C and mF2C secreted by CHO cells were purified on protein A and analyzed by western blot using either species-specific anti-Fc antibody, or using a polyclonal anti-scFv. Each F2C fusion antibody is detected in CHO supernatants and is respectively seen as a human, rabbit or mouse antibody.C: scFv-hFc are expressed as dimers. hF2C proteins were separated by SDS-PAGE either in reducing (left) or in non-reducing conditions (right) and analyzed by western blot. This confirmed that hF2C behaves as a dimer in non-reducing conditions. D: Using multi-species scFv-Fc antibodies for Western blot immuno-labeling. A HeLa cell protein lysate was separated by SDS-PAGE and transferred on a nitrocellulose membrane. The membrane was probed using anti-MyosinII SF9 (lane 1, 3 and 5) or anti-Tubulin F2C (2, 4, 6) antibodies fused to either human (h; 1,2), rabbit (r; 3,4) or mouse (m; 5,6) Fc. This experiment shows that each scFv kept its specificity when used as a human, mouse or rabbit antibody.

Mentions: Our plasmids are based on the pFUSE-Fc2(IL2ss)™ series from Invivogen (San Diego, USA) that contains the interleukin-2 (IL2) signal sequence and allows the secretion of Fc-Fusion proteins by mammalian cells. They are selectable using Zeocin™ (Zeo) both in prokaryotic and eukaryotic cells. These plasmids were modified by site directed mutagenesis and adaptor insertion (see Material and Methods, Figure 1A) to allow the easy one step cassette cloning of recombinant antibodies extracted from a large collection of common recombinant antibody selection and expression plasmids (e.g pHEN, pSEX, pHAL, pCANTAB, pHOG, pOPE, pSTE). Three plasmids were constructed enabling fusion of scFv at their C-terminus with either human IgG2 (h), mouse IgG2a (m) or the rabbit IgG (r) Fc domain (Fc regions comprise the CH2 and CH3 domains of the IgG heavy chain and the hinge region).


A multi-Fc-species system for recombinant antibody production.

Moutel S, El Marjou A, Vielemeyer O, Nizak C, Benaroch P, Dübel S, Perez F - BMC Biotechnol. (2009)

Production and use of multi-species scFv-Fc antibodies. A: Schematic map of the generic pFuse-xIgG-Fc2 used to express scFvs (adapted from Invivogen). Three vectors are available bearing either a human (pFuse-hIgG-Fc2), mouse (pFuse-mIgG-Fc2) or rabbit IgG2 (pFuse-rIgG-Fc2). The scFv is inserted (NcoI/NotI) inframe between the IL2 secretion signal and the Fc portion. The vector can be selected using Zeocin both in E. coli and in mammalian cells. B: Western Blot analysis of the scFv F2C fused to Fc domains from 3 different species. hF2C, rF2C and mF2C secreted by CHO cells were purified on protein A and analyzed by western blot using either species-specific anti-Fc antibody, or using a polyclonal anti-scFv. Each F2C fusion antibody is detected in CHO supernatants and is respectively seen as a human, rabbit or mouse antibody.C: scFv-hFc are expressed as dimers. hF2C proteins were separated by SDS-PAGE either in reducing (left) or in non-reducing conditions (right) and analyzed by western blot. This confirmed that hF2C behaves as a dimer in non-reducing conditions. D: Using multi-species scFv-Fc antibodies for Western blot immuno-labeling. A HeLa cell protein lysate was separated by SDS-PAGE and transferred on a nitrocellulose membrane. The membrane was probed using anti-MyosinII SF9 (lane 1, 3 and 5) or anti-Tubulin F2C (2, 4, 6) antibodies fused to either human (h; 1,2), rabbit (r; 3,4) or mouse (m; 5,6) Fc. This experiment shows that each scFv kept its specificity when used as a human, mouse or rabbit antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654441&req=5

Figure 1: Production and use of multi-species scFv-Fc antibodies. A: Schematic map of the generic pFuse-xIgG-Fc2 used to express scFvs (adapted from Invivogen). Three vectors are available bearing either a human (pFuse-hIgG-Fc2), mouse (pFuse-mIgG-Fc2) or rabbit IgG2 (pFuse-rIgG-Fc2). The scFv is inserted (NcoI/NotI) inframe between the IL2 secretion signal and the Fc portion. The vector can be selected using Zeocin both in E. coli and in mammalian cells. B: Western Blot analysis of the scFv F2C fused to Fc domains from 3 different species. hF2C, rF2C and mF2C secreted by CHO cells were purified on protein A and analyzed by western blot using either species-specific anti-Fc antibody, or using a polyclonal anti-scFv. Each F2C fusion antibody is detected in CHO supernatants and is respectively seen as a human, rabbit or mouse antibody.C: scFv-hFc are expressed as dimers. hF2C proteins were separated by SDS-PAGE either in reducing (left) or in non-reducing conditions (right) and analyzed by western blot. This confirmed that hF2C behaves as a dimer in non-reducing conditions. D: Using multi-species scFv-Fc antibodies for Western blot immuno-labeling. A HeLa cell protein lysate was separated by SDS-PAGE and transferred on a nitrocellulose membrane. The membrane was probed using anti-MyosinII SF9 (lane 1, 3 and 5) or anti-Tubulin F2C (2, 4, 6) antibodies fused to either human (h; 1,2), rabbit (r; 3,4) or mouse (m; 5,6) Fc. This experiment shows that each scFv kept its specificity when used as a human, mouse or rabbit antibody.
Mentions: Our plasmids are based on the pFUSE-Fc2(IL2ss)™ series from Invivogen (San Diego, USA) that contains the interleukin-2 (IL2) signal sequence and allows the secretion of Fc-Fusion proteins by mammalian cells. They are selectable using Zeocin™ (Zeo) both in prokaryotic and eukaryotic cells. These plasmids were modified by site directed mutagenesis and adaptor insertion (see Material and Methods, Figure 1A) to allow the easy one step cassette cloning of recombinant antibodies extracted from a large collection of common recombinant antibody selection and expression plasmids (e.g pHEN, pSEX, pHAL, pCANTAB, pHOG, pOPE, pSTE). Three plasmids were constructed enabling fusion of scFv at their C-terminus with either human IgG2 (h), mouse IgG2a (m) or the rabbit IgG (r) Fc domain (Fc regions comprise the CH2 and CH3 domains of the IgG heavy chain and the hinge region).

Bottom Line: We developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use.This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies.Altogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS UMR144, 26 rue d'Ulm, F75248 Paris Cedex 05, France. Sandrine.Moutel@curie.fr

ABSTRACT

Background: Genomic, transcriptomic and proteomic projects often suffer from a lack of functional validation creating a strong demand for specific and versatile antibodies. Antibody phage display represents an attractive approach to select rapidly in vitro the equivalent of monoclonal antibodies, like single chain Fv antibodies, in an inexpensive and animal free way. However, so far, recombinant antibodies have not managed to impose themselves as efficient alternatives to natural antibodies.

Results: We developed a series of vectors that allow one to easily fuse single chain Fv antibodies to Fc domains of immunoglobulins, improving their sensitivity and facilitating their use. This series enables the fusion of single chain Fv antibodies with human, mouse or rabbit Fc so that a given antibody is no longer restricted to a particular species. This opens up unlimited multiplexing possibilities and gives additional value to recombinant antibodies. We also show that this multi-Fc species production system can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies and we converted the widely used 9E10 mouse anti-Myc-tag antibody into a human and a rabbit antibody.

Conclusion: Altogether, this new expression system, that brings constant quality, sensitivity and unique versatility, will be important to broaden the use of recombinant and natural monoclonal antibodies both for laboratory and diagnosis use.

Show MeSH
Related in: MedlinePlus