"
Team:Potsdam Bioware/Project/Part Antibody
From 2012.igem.org
(→Introduction) |
(→Introduction) |
||
| Line 20: | Line 20: | ||
==== Nanobody ==== | ==== Nanobody ==== | ||
A specific form of immunoglobulin is the single domain antibody fragment, so called nanobody by its inventor Ablynx. Nanobodies are derived from camellid antibodies that possess a single N-terminal domain, the VHH domain. The VHH domain is solely sufficient for a strong antigen binding and does not require a domain fusion. ( Harmsen, Haard; 2007) Thus, nanobodies have extremely small dimensions and show an elevated stability which both leads to the ability of recognizing epitopes that are not accessible for conventional antibody formats. ( de Marco; 2011) | A specific form of immunoglobulin is the single domain antibody fragment, so called nanobody by its inventor Ablynx. Nanobodies are derived from camellid antibodies that possess a single N-terminal domain, the VHH domain. The VHH domain is solely sufficient for a strong antigen binding and does not require a domain fusion. ( Harmsen, Haard; 2007) Thus, nanobodies have extremely small dimensions and show an elevated stability which both leads to the ability of recognizing epitopes that are not accessible for conventional antibody formats. ( de Marco; 2011) | ||
| + | === Generating the Antibody Constructs === | ||
| + | Two antibody constructs with different constitutions were generated by us to diversify our system and to guarantee a broadened scientific approach. They do also allow a directed troubleshoot of our concept. | ||
| + | ==== Smaller Antibody Construct ==== | ||
| + | We designed the smaller antibody construct out of already existing parts and BioBricks and added further functional units by assembly PCR. The smaller construct is build up by a single chain fragment (anti-EGRF scFv425) with a signal peptide for membrane translocation (BBa_K157001) on its N-terminal end, the transmembrane domain (BBa_K157010) with a flanking TEV recognition site and the eYFP reporter (BBa_E0030) at its C-terminal end. | ||
| + | The scFv425 is of murine origin and was derived against the human epidermal growth factor receptor domain 3. It was originally described in context of a publication dealing with immunotherapy of pancreatic carcinoma cells which consequently express the EGFR. The scFv was used as mediator of carcinoma cells and an endotoxin thus showing the high relevance of the single chain fragment for medical purposes. (Bruell et al; 2005) | ||
| + | <br> | ||
| + | <br> | ||
| + | picture | ||
| + | <br> | ||
| + | <br> | ||
| + | ==== Advanced Antibody Construct ==== | ||
| + | The advanced antibody construct consists of two major building blocks represented by the actual antibody unit and the switchable membrane anchoring region and were designed de novo via gene synthesis. Both elements guarantee the eligibility and easy handling of the construct in our CHO cell Flp-In expression system and are codon optimized. | ||
| + | The central part is the antibody cassette consisting of a replaceable anti-GFP nanobody and an IgG1 stem Fc domain (GenBank: J00228.1). A nanobody-Fc fusion protein unites the advantage of smaller dimensions with the ability to directly interact with the Fc receptor and complement proteins. The green fluorescent protein (GFP)-nanobody is a single-chain VHH antibody domain developed for specific binding activity to GFP and shows a Kd value of 1.4 nM. Its CDR3 loop is very short and has significantly fewer contacts with the GFP ligand compared to other nanobodies. Furthermore, the shortness of this CDR3 loop leads to the exposure of the framework 2 region, which has a major contribution to the binding with GFP. (Kubala et al; 2010) The Nanobody is framed by two specific restriction enzyme recognition sites on each terminal end allowing the easy exchange of this part with another suitable sequence. The membrane anchoring region ( modified BBa_K157010) is flanked by two LoxP sites that introduce a genetically cut to our system which will allow the modification from surface presentation to secretion of the antibody construct by activity of cre recombinase (Araki et al; 1997) in our CHO cells. An additional switch is represented by the TEV protease cleavage site permitting the shift to a secretory antibody production on protein level. Expression of the antibody construct and its cellular localization can be monitored by the mCherry signal. | ||
| + | <br> | ||
| + | <br> | ||
| + | picture | ||
| + | <br> | ||
| + | <br> | ||
</div> | </div> | ||
Revision as of 00:14, 26 September 2012
Antibody Module
Contents |
Introduction
Aim of the Antibody Module
The idea of the antibody module is to create antibody constructs which will specifically be mutated and optimized by the AID (activation induces deaminase). Our two antibody constructs allow an easy handling and a straightforwardly integration into CHO cells. The use of Flp-In system realizes time saving incorporation into the CHO genome. A switch from surface presentation to secretion of the antibodies can be enabled by TEV protease and cre recombinase and permits a comfortable harvest of selected antibodies.
A Short Review on Antibodies
Antibodies are highly specific targeting reagents and are the key defense system for recognizing pathogens and toxins. Antibodies are Y shaped multi domain proteins with two antigen binding sites displaying the Fab fragment and one effector domain represented by the Fc domain. (Holliger, Hudson; 2005) The Fab fragment is composed of two antigen recognition sites with one variable light (VL) and constant light (VC) chain and one variable heavy (VH) and constant heavy (CH) chain each which allow the specific an affine binding of antigens. By interacting with the complement and the Fc receptor, the Fc domain is able to mediate cytotoxic effector functions. (Holliger, Hudson; 2005)
Single Chain Fragment Variable
A single chain fragment variable is a small unit of immunoglobulin and consists of the variable heavy (VH) and the variable light (VL) domains which are joined together by a flexible peptide linker. (Alitheen and Hamid et al.; 2012) The scFv is a very popular format that shows a high antigen binding capacity and can be easily expressed.
picture
Nanobody
A specific form of immunoglobulin is the single domain antibody fragment, so called nanobody by its inventor Ablynx. Nanobodies are derived from camellid antibodies that possess a single N-terminal domain, the VHH domain. The VHH domain is solely sufficient for a strong antigen binding and does not require a domain fusion. ( Harmsen, Haard; 2007) Thus, nanobodies have extremely small dimensions and show an elevated stability which both leads to the ability of recognizing epitopes that are not accessible for conventional antibody formats. ( de Marco; 2011)
Generating the Antibody Constructs
Two antibody constructs with different constitutions were generated by us to diversify our system and to guarantee a broadened scientific approach. They do also allow a directed troubleshoot of our concept.
Smaller Antibody Construct
We designed the smaller antibody construct out of already existing parts and BioBricks and added further functional units by assembly PCR. The smaller construct is build up by a single chain fragment (anti-EGRF scFv425) with a signal peptide for membrane translocation (BBa_K157001) on its N-terminal end, the transmembrane domain (BBa_K157010) with a flanking TEV recognition site and the eYFP reporter (BBa_E0030) at its C-terminal end.
The scFv425 is of murine origin and was derived against the human epidermal growth factor receptor domain 3. It was originally described in context of a publication dealing with immunotherapy of pancreatic carcinoma cells which consequently express the EGFR. The scFv was used as mediator of carcinoma cells and an endotoxin thus showing the high relevance of the single chain fragment for medical purposes. (Bruell et al; 2005)
picture
Advanced Antibody Construct
The advanced antibody construct consists of two major building blocks represented by the actual antibody unit and the switchable membrane anchoring region and were designed de novo via gene synthesis. Both elements guarantee the eligibility and easy handling of the construct in our CHO cell Flp-In expression system and are codon optimized.
The central part is the antibody cassette consisting of a replaceable anti-GFP nanobody and an IgG1 stem Fc domain (GenBank: J00228.1). A nanobody-Fc fusion protein unites the advantage of smaller dimensions with the ability to directly interact with the Fc receptor and complement proteins. The green fluorescent protein (GFP)-nanobody is a single-chain VHH antibody domain developed for specific binding activity to GFP and shows a Kd value of 1.4 nM. Its CDR3 loop is very short and has significantly fewer contacts with the GFP ligand compared to other nanobodies. Furthermore, the shortness of this CDR3 loop leads to the exposure of the framework 2 region, which has a major contribution to the binding with GFP. (Kubala et al; 2010) The Nanobody is framed by two specific restriction enzyme recognition sites on each terminal end allowing the easy exchange of this part with another suitable sequence. The membrane anchoring region ( modified BBa_K157010) is flanked by two LoxP sites that introduce a genetically cut to our system which will allow the modification from surface presentation to secretion of the antibody construct by activity of cre recombinase (Araki et al; 1997) in our CHO cells. An additional switch is represented by the TEV protease cleavage site permitting the shift to a secretory antibody production on protein level. Expression of the antibody construct and its cellular localization can be monitored by the mCherry signal.
picture
