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Generating Affinity Proteins for Biotechnological, Diagnostic and Therapeutic Applications
KTH, School of Biotechnology (BIO), Protein Technology.
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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:7
Keyword [en]
Protein engineering
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-164320ISBN: 978-91-7595-492-9 (print)OAI: oai:DiVA.org:kth-164320DiVA: diva2:805688
Public defence
2015-05-08, FD5 (The Swedbergssalen), Roslagstullsbacken 21, KTH, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

QC 20150416

Available from: 2015-04-16 Created: 2015-04-16 Last updated: 2015-04-16Bibliographically approved
List of papers
1. Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG(1)
Open this publication in new window or tab >>Tailor-Making a Protein A-Derived Domain for Efficient Site-Specific Photocoupling to Fc of Mouse IgG(1)
2013 (English)In: PLoS ONE, ISSN 1932-6203, E-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.

Keyword
Antibody-Binding, Biotinylation, Substrate, Fragment
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-121127 (URN)10.1371/journal.pone.0056597 (DOI)000315965100057 ()2-s2.0-84873724699 (Scopus ID)
Note

QC 20130419

Available from: 2013-04-19 Created: 2013-04-19 Last updated: 2017-12-06Bibliographically approved
2. Site-specific photoconjugation of betalactamase split-protein fragments to monoclonal antibodies for homogeneous assay development
Open this publication in new window or tab >>Site-specific photoconjugation of betalactamase split-protein fragments to monoclonal antibodies for homogeneous assay development
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-164404 (URN)
Note

QS 2015

Available from: 2015-04-16 Created: 2015-04-16 Last updated: 2015-04-16Bibliographically approved
3. Ribosome Display Selection of a Murine IgG(1) Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
Open this publication in new window or tab >>Ribosome Display Selection of a Murine IgG(1) Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
2011 (English)In: Molecular Biotechnology, ISSN 1073-6085, E-ISSN 1559-0305, Vol. 48, no 3, 263-276 p.Article in journal (Refereed) Published
Abstract [en]

Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG(1), the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 10(11) affibody molecules. Four rounds of selection using three different mouse IgG(1) mAbs as alternating targets resulted in the identification of binders with broad mIgG(1) recognition and dissociation constants (K (D)) in the low nanomolar to low micromolar range. For one of the binders, denoted Z(mab25), competition in binding to full length mIgG(1) by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG(1) Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG(1) had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG(1) nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Z(mab25). This selective binding profile towards murine IgG(1) was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.

Keyword
Mouse IgG1, Affibody, Selection, Fab fragment, Combinatorial protein engineering, Ribosome display
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-33435 (URN)10.1007/s12033-010-9367-1 (DOI)000291656700008 ()21197589 (PubMedID)2-s2.0-80054079725 (Scopus ID)
Funder
Swedish Research Council, 50548301
Note
QC 20110705Available from: 2011-05-06 Created: 2011-05-06 Last updated: 2017-12-11Bibliographically approved
4. An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo
Open this publication in new window or tab >>An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo
Show others...
2014 (English)In: mAbs, ISSN 1942-0862, Vol. 6, no 6, 1598-1607 p.Article in journal (Refereed) Published
Abstract [en]

In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor a (sIL-6R alpha) and IL6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, Z(IL-6_13) with an affinity (K-D) for IL-6 of similar to 500 pM, showed the best performance. To construct fusion proteins ("AffiMabs") with dual cytokine specificities, Z(IL-6_13) was fused to either the N-or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira (R)). One AffiMab construct with Z(IL-6_13) positioned at the N-terminus of the heavy chain, denoted Z(IL-6_13)-HCAda, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the Z(IL-6_13)-HCAda AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.

Keyword
AffiMab, Antibody, IL-6, TNF, adalimumab, affibody, affinity, fusion, inflammation
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-159385 (URN)10.4161/mabs.36089 (DOI)000346878600024 ()2-s2.0-84920827599 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150203

Available from: 2015-02-03 Created: 2015-01-29 Last updated: 2015-04-16Bibliographically approved

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