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Ribosome Display Selection of a Murine IgG(1) Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies
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
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.

Place, publisher, year, edition, pages
2011. Vol. 48, no 3, 263-276 p.
Keyword [en]
Mouse IgG1, Affibody, Selection, Fab fragment, Combinatorial protein engineering, Ribosome display
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-33435DOI: 10.1007/s12033-010-9367-1ISI: 000291656700008PubMedID: 21197589Scopus ID: 2-s2.0-80054079725OAI: oai:DiVA.org:kth-33435DiVA: diva2:415482
Funder
Swedish Research Council, 50548301
Note
QC 20110705Available from: 2011-05-06 Created: 2011-05-06 Last updated: 2017-12-11Bibliographically approved
In thesis
1. Ribosome display for selection and evolution of affibody molecules
Open this publication in new window or tab >>Ribosome display for selection and evolution of affibody molecules
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Affinity proteins are invaluable tools in biotechnological and medical applications. This thesis is about combinatorial protein engineering principles for the generation of novel affinity proteins to purify mouse immunoglobulin, detect a potential cancer marker protein or inhibit a cell proliferation pathway.

In a first study, ribosome display was for the first time applied to the selection of so-called affibody molecules, including the design of a ribosome display gene cassette, initial test enrichment experiments and the selection of binders against murine IgG1. One of the selected binders (ZMAB25) showed a highly selective binding profile to murine IgG1, which was exploited in the recovery of two different mouse monoclonal IgG1 antibodies from a bovine immunoglobulin-containing background. Ribosome display was further applied to the selection of affibody molecules binding to SATB1, a suggested marker protein for metastasizing adenocarcinoma. The study also included the selection of VHH antibody fragments from a naïve gene repertoire displayed on phage. Binders from both classes of protein scaffolds could be isolated that selectively recognized SATB1 but not its close homologue SATB2, and were used to detect endogenous SATB1 in Jurkat cells by immunofluorescence microscopy. The well-established phage display technology was used to select affibody molecules binding to H-Ras and Raf-1, both involved in the mitogen-activated protein kinase (MAPK) pathway and playing a central role in the control of cell proliferation, survival and differentiation. An isolated affibody molecule denoted ZRAF322 was found to selectively bind to Raf-1 and inhibit the interaction between H-Ras and Raf-1 in vitro. In a continued effort, ribosome display was applied to the affinity maturation of the ZRAF322 variant in a novel approach, based on repetitive cycles of diversification by error-prone PCR of the entire affibody gene and ribosome display selection, mimicking the principles of natural evolution. The method involved a monitoring of the progress of evolution and variants of ZRAF322 with 13- to 26-fold improved affinities were obtained, that contained different combinations of single or double amino acid substitutions in either previously randomized or framework positions. Implications of the substitutions for binder stability and selectivity were also investigated, showing that a higher affinity could be associated with a lower thermal melting point and that affinity-improved variants showed uncompromised binding selectivity to the hRaf-1 target.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2011. xii, 99 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2011:9
Keyword
affibody, ribosome display, phage display, combinatorial protein engineering, library, murine IgG1, SATB1, Ras, Raf
National Category
Other Industrial Biotechnology
Research subject
SRA - Molecular Bioscience
Identifiers
urn:nbn:se:kth:diva-33191 (URN)978-91-7415-952-3 (ISBN)
Public defence
2011-05-27, Fr4, AlbaNova University Center, Roslagstullsbacken 21, B3, Stockholm, 10:00 (English)
Opponent
Supervisors
Note
QC 20110506Available from: 2011-05-06 Created: 2011-04-29 Last updated: 2011-05-06Bibliographically approved
2. 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.
Series
TRITA-BIO-Report, ISSN 1654-2312 ; 2015:7
Keyword
Protein engineering
National Category
Natural Sciences
Research subject
Biotechnology
Identifiers
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)
Opponent
Supervisors
Note

QC 20150416

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

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