Team:Potsdam Bioware/Project/Part Antibody

Antibody Module

Antibody Module

Abstract

The function of the antibody module is to design and test antibody constructs which operate as sequence substrate for the AID, our mutating module. The antibody constructs were stably and transiently transfected into CHO cells. The antigen binding domains of the construct can directly interact with the specific antigen on the surface of the virus construct, the work object of our selection module. This interaction can lead to selective collection of a mutated and thus optimized antibody.

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-induced cytidine deaminase). Our two antibody constructs allow an easy handling and a straight forward integration into CHO cells. The use of Flp-In system realizes time saving incorporation of antibody construct 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 simple 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 and 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.

Fig. 1: Selected antibody forms relative to therapeutic applications (Peterson, Owens and Henry; 2006)

Nanobody

A specific form of immunoglobulin is the single domain antibody fragment, called by its inventor Ablynx - nanobody. Nanobodies are derived from camelid 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 built up from 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)

Fig. 2: Localization of the smaller antibody construct in transient transfected CHO cells

Advanced Antibody Construct

The advanced antibody construct was designed de novo via gene synthesis. It consists of two major building blocks represented by the actual antibody unit and the switchable membrane anchoring region. 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 combines 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 less 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 genetical 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. The cre recombinase is a trysosine recombinase enzyme which catalyzes reciprocal site-specific recombination between two specific 34-bp sites called loxP. The cre/loxP recombination system was derived from bacteriophage P1 and is frequently used in genetic manipulation. (Araki et al; 1997) Additionallly we integrated a switch on the protein level which is represented by the TEV protease cleavage site permitting the shift to a secretory antibody production. Expression of the antibody construct and its cellular localization can be monitored by the mCherry signal.

Fig. 3: Localization of the advanced antibody construct in transient transfected CHO cells

CHO Cells and Flp-In System

Chinese hamster ovary cells (CHO) are an immortal mammalian cell line which is frequently used for industrial recombinant protein expressions. Problems when working with mammalian cells are the varying transfection rate and the transitory nature of transient transfected plasmids. To overcome this problem we are working with a CHO expression cell line for the stable transfection of our construct of interest by using Flp recombinase-mediated integration.

Fig. 4: CHO cells in culture

Flp-In System

The FRT/Flp system (LifeTechnologies) is based on homologous recombination initiated by the enzyme flippase (Flp) at a specific FRT site in the genome. CHO cell containing a randomly but stably integrated FRT sequence can be selected by antibiotic resistance against Zeocin. Another FRT sequence is located on the shuttle plasmid that contains the gene of interest and a second antibiotic resistance against Hygromycin. The system is accomplished by the third plasmid carrying the recombinase flippase (Flp) which mediates the recombination of both FRT sequences leading to an exchange of Zeocin to Hygromycin resistance.

Fig. 5: Flp-In system of LifeTechnologies applied with our sequences in CHO cells

Both of our antibody constructs were also transfected transiently to get a first impression of the expression level, the cellular localization and the compatibility with the CHO cell metabolism.

Working with Other Cell Lines

In order to get a better insight into our system and its way of function we also transiently transfected HeLa and HEK293 cells with our constructs. HeLa cells are the oldest and most commonly used immortal human cell line and were derived in 1951 from cervical cancers cells of a patient called Henrietta Lacks. The other cell line we are using is HEK293 cells which are immortal human embryonic kidney cells.

Results

CHO cells

Cell Duplication Rate

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Sensitivity to Hygromycin for Flp-In System

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Smaller Antibody Construct

Cloning the Smaller Construct

The sequence from the human EGFR (ErbB-1) signal peptide was taken from the already existing BioBrick BBa_K157001 and was fused N-terminally to the scFv 425 against the epidermal growth factor receptor (EGFR) domain 3. The scFv425 has a shortened N-terminal FLAG-tag sequence of five amino acids (DYKDE) that allows us a handy purification and simplifies its detection. A TEV protease cleavage site was integrated between the scFv and the transmembrane domain by primer extension and permits the shift from surface presentation to secretion on protein level. The TEV protease recognition site ENLYFQG was used here and represents the most commonly used aa sequence for recognition by the 27kDA catalytic domain of Nuclear Inclusion a (NIa) protein encoded by the tobacco etch virus (TEV). For an unimpeded activity of the TEV protease a three amino acid linker at the N-terminal end and a two amino acid linker at the C-terminal end were added to the sequence. The helical single-span transmembrane domain of the B-cell receptor (BBa_K157010) was modified in terms of the shortening of the linker sequence to three amino acids at the C-terminal end. Expression of the scFv fusion protein and its cellular localization can be monitored by the enhanced YFP (BBa_E0030) signal with an excitation at 514 nm and an emission at 527 nm.

Fig. 6: Smaller antibody construct cloned into pSB1C3 (BBa_K929101)

Fluorescence Microscopy (expression)

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Immune fluorescence

Immune blotting

Confocal microscopy

FACS

Transfection in HeLa and HEK293 cells

Advanced Antibody Construct

Gene synthesis

The advanced antibody construct was designed by us de novo on the basis of sequence information delivered by Genebank and UniProt files and already existing Biobrick parts. The construct consists of two major building blocks represented by the actual antibody unit and the switchable membrane anchoring region. The antibody unit is represented by the signal peptide, the anti-GFP Nanobody and the Fc region. TEV protease recognition site, 2 LoxP sites, the B-cell receptor transmembrane domain and the mCherry reporter display the switchable membrane anchoring region. The signal peptide was taken from a human immunoglobulin G kappa variable chain I (UniProt: P01601) and functions as translocation signal to the cell membrane. As antibody component for this construct we used a single-chain VHH antibody domain, also called nanobody. The anti- green fluorescent protein (GFP)-nanobody was developed for specific binding activity to GFP. (see Introduction) (Kubala et al; 2010) Our advanced construct possesses a special feature that allows the replacement of the nanobody with another antigen binding domain. The nanobody is framed by two specific restriction enzyme recognition sites, SacI on the N-terminal end and KasI at the C-terminal end, which will allow the easy exchange with another favored and suitable sequence. The nanobody is fused to a human immunoglobulin gamma 1 heavy chain constant region (Fc) (UniProt: P01857) which enables the direct interaction of the antibody unit with the Fc receptor and complement proteins. Similar to the smaller construct a TEV protease cleavage site was integrated between the Fc region and the transmembrane domain permitting as well the shift from surface presentation to secretion on protein level (see smaller construct). For an unimpeded activity of the TEV protease two amino acids at the N-terminal end were added as spacer. Additionally to this switch on protein level, the whole membrane anchoring region is flanked by two LoxP sites introducing a non-reversible genetically switch to our system. It allows the modification from surface presentation to secretion on the DNA level. Both LoxP sites are oriented in the same direction leading to the elimination of the enclosed region by cre recombinase activity. We choose two LoxP site sequences of 34 base pairs (ATAACTTCGTATA GCATACAT TATACGAAGTTAT) for recognition by cre recombinase. The helical single-span transmembrane domain sequence of the B-cell receptor was taken from the registry (BBa_K157010) and modified by shortening of the linker sequence to three amino acids at the C-terminal end. Expression of the advanced antibody construct and its cellular localization can be monitored by the mCherry signal with an excitation at 587 nm and an emission at 610 nm.

Fig. 7: Advanced antibody construct cloned into pSB1C3 (BBa_K929107)

Fluorescence microscopy

Immune fluorescence

Immune blotting

FACS

Transfection in Hela and HEK cells

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Discussion

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References

• de Marco A. Biotechnological applications of recombinant single-domain antibody fragments. Microb Cell Fact. 2011 Jun 9;10:44. Review. [http://www.ncbi.nlm.nih.gov/pubmed?term=21658216 PubMed PMID: 21658216.]
• Harmsen MM, De Haard HJ. Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol. 2007 Nov;77(1):13-22. Epub 2007 Aug 18. Review. [http://www.ncbi.nlm.nih.gov/pubmed?term=17704915 PubMed PMID: 17704915.]
• Peterson E, Owens SM, Henry RL. Monoclonal antibody form and function: manufacturing the right antibodies for treating drug abuse. AAPS J. 2006 May 26;8(2):E383-90. Review. [http://www.ncbi.nlm.nih.gov/pubmed?term=16796389 PubMed PMID: 16796389.]
• Holliger P, Hudson PJ. Engineered antibody fragments and the rise of single domains. Nat Biotechnol. 2005 Sep;23(9):1126-36. Review. [http://www.ncbi.nlm.nih.gov/pubmed?term=16151406 PubMed PMID: 16151406.]
• Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. Clin Dev Immunol. 2012;2012:980250. Epub 2012 Mar 15. [http://www.ncbi.nlm.nih.gov/pubmed?term=22474489 PubMed PMID: 22474489.]
• Bruell D, Bruns CJ, Yezhelyev M, Huhn M, Müller J, Ischenko I, Fischer R, Finnern R, Jauch KW, Barth S. Recombinant anti-EGFR immunotoxin 425(scFv)-ETA' demonstrates anti-tumor activity against disseminated human pancreatic cancer in nude mice. Int J Mol Med. 2005 Feb;15(2):305-13. [http://www.ncbi.nlm.nih.gov/pubmed?term=15647848 PubMed PMID: 15647848.]
• Invitrogen (2011) Flp-In System Manual. Publication Part Number 25-0366