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Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG(1)
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.
KTH, School of Biotechnology (BIO), Molecular Biotechnology.ORCID iD: 0000-0003-4214-6991
2013 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 8, no 2, e56597- p.Article in journal (Refereed) Published
Abstract [en]

Affinity proteins binding to antibody constant regions have proved to be invaluable tools in biotechnology. Here, protein engineering was used to expand the repertoire of available immunoglobulin binding proteins via improvement of the binding strength between the widely used staphylococcal protein A-derived Z domain and the important immunoglobulin isotype mouse IgG(1) (mIgG(1)). Addressing seven positions in the 58-residue three-helix bundle Z domain by single or double amino acid substitutions, a total of 170 variants were individually constructed, produced in E. coli and tested for binding to a set of mouse IgG(1) monoclonal antibodies (mAbs). The best variant, denoted Z(F5I) corresponding to a Phe to Ile substitution at position 5, showed a typical ten-fold higher affinity than the wild-type as determined by biosensor technology. Eight amino acid positions in the Z(F5I) variant were separately mutated to cysteine for incorporation of a photoactivable maleimide-benzophenone (MBP) group as a probe for site-specific photoconjugation to Fc of mIgG(1), The best photocoupling efficiency to mIgG(1) Fc was seen when the MBP group was coupled to Cys at position 32, resulting in adduct formation to more than 60% of all heavy chains, with no observable non-selective conjugation to the light chains. A similar coupling yield was obtained for a panel of 19 different mIgG(1) mAbs, indicating a general characteristic. To exemplify functionalization of a mIgG(1) antibody via site-specific biotinylation, the Z(F5I-Q32C-MBP) protein was first biotinylated using an amine reactive reagent and subsequently photoconjugated to an anti-human interferon-gamma mIgG(1) mAb. When comparing the specific antigen binding ability of the probe-biotinylated mAb to that of the directly biotinylated mAb, a significantly h`igher bioactivity was observed for the sample biotinylated using the Z(F5I-Q32C-MBP) probe. This result indicates that the use of a site-specific and affinity probe-mediated conjugation strategy can result in antibody reagents with increased assay sensitivity.

Place, publisher, year, edition, pages
2013. Vol. 8, no 2, e56597- p.
Keyword [en]
Antibody-Binding, Biotinylation, Substrate, Fragment
National Category
Biological Sciences
URN: urn:nbn:se:kth:diva-121127DOI: 10.1371/journal.pone.0056597ISI: 000315965100057ScopusID: 2-s2.0-84873724699OAI: diva2:616906

QC 20130419

Available from: 2013-04-19 Created: 2013-04-19 Last updated: 2015-04-16Bibliographically approved
In thesis
1. Generating Affinity Proteins for Biotechnological, Diagnostic and Therapeutic Applications
Open this publication in new window or tab >>Generating Affinity Proteins for Biotechnological, Diagnostic and Therapeutic Applications
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein engineering is a powerful tool to modify proteins to generate novel and desired properties that could be applied in biotechnological, diagnostics and therapeutic areas. In this thesis, both rational design and library based engineering principles have been exploited to develop affinity proteins with desired traits.

One study was focused on the use of site-directed mutagenesis to obtain variants of the staphylococcal protein A-derived 58-residue immunoglobulin binding Z domain with improved affinity for mouse IgG1 Fc. Screening of ca. 170 constructed variants revealed one variant with a single F5I amino acid substitution, denoted ZF5I, with a ten-fold higher affinity. The Fc binding ZF5I variant was further investigated for use in affinity-driven site-specific covalent photoconjugation to mIgG1 monoclonal antibodies. Here, nine candidate positions in the domain were investigated for introduction of a UV-activatable maleimide benzophenone (MBP) group via conjugation to an introduced cysteine residue. The best photo-conjugation results were obtained for a variant in which the MBP was introduced at position 32, denoted ZF5I-Q32C-MBP, which could be conjugated at high yields to all nineteen mouse IgG1s tested. The use of a biotinylated Z-based probe for biotinylation via photoconjugation of a monoclonal anti-interferon gamma antibody resulted in a higher antigen binding activity than if a conventional amine directed biotinylation strategy was used.

In a second study, the goal was to develop a new homogeneous immunoassay for quick antigen detection, based on split-protein complementation and pairs of antigen recognizing proteins. In one of the formats investigated, separate fragments of a split-beta-lactamase enzyme reporter were genetically linked to ZF5I-Q32C-MBP units which were individually photo-conjugated to two different mAbs recognizing different epitopes on a human interferon gamma model target analyte. Simultaneous binding of the two mAb-enzyme half probes to the analyte resulted in an analyte concentration-dependent enzyme fragment complementation which could be spectrophotometrically detected using a nitrocefin substrate.

Using ribosome display technology, Z-domain based binders to mouse IgG1 were selected from an affibody library. One binder denoted Zmab25 was shown capable of selective binding to mouse IgG in a background of bovine IgG, and could be used for species-selective recovery of monoclonal antibodies from complex samples, resembling hybridoma culture supernatants. Epitope mapping experiments showed that that the binding site on mouse IgG was located in the Fab fragment and was overlapping with that of streptococcal protein G.

In a final study, phage display technology was used to select affibodies binding to human interleukin 6 (IL-6), for potential use in rheumatoid arthritis (RA) therapy via blocking of the signaling involving the ternary complex between IL-6, the IL-6 receptor α (IL-6R α) and the gp130 co-receptor. Several affibodies were shown to be capable of blocking the interaction between gp130 and preformed complexes of IL-6 and soluble IL-6R α (IL-6/sIL-6R α) in vitro, corresponding to the so-called trans-signaling interaction. One of these affibody variants denoted ZIL-6_13 showed a KD of approx. 500 pM for IL-6 and was genetically fused to different chain ends of the monoclonal anti-TNF antibody adalimumab to build bi-specific “AffiMab” constructs. One construct, ZIL-6_13-HCAda,in which the affibody was fused to the N-terminus of the adalimumab heavy chain had the most optimal properties in different cell assays and was also evaluated in vivo in an acute serum amyloid A (SAA) mouse RA model, involving a dual challenge of animals with both IL-6 and TNF. Compared to adalimumab that could only reduce SAA levels to 50% at the highest dose, the bi-functional AffiMab reduced SAA levels to below the detection level. 

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2015. 103 p.
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:7
Protein engineering
National Category
Natural Sciences
Research subject
urn:nbn:se:kth:diva-164320 (URN)978-91-7595-492-9 (ISBN)
Public defence
2015-05-08, FD5 (The Swedbergssalen), Roslagstullsbacken 21, KTH, Stockholm, 10:00 (English)

QC 20150416

Available from: 2015-04-16 Created: 2015-04-16 Last updated: 2015-04-16Bibliographically approved

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