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Rational and combinatorial protein engineering for vaccine delivery and drug targeting
KTH, School of Biotechnology (BIO).
2005 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis describes recombinant proteins that have been generated by rational and combinatorial protein engineering strategies for use in subunit vaccine delivery and tumor targeting.

In a first series of studies, recombinant methods for incorporating immunogens into an adjuvant formulation, e.g. immunostimulating complexes (iscoms), were evaluated. Protein immunogens, which are not typically immunogenic in themselves, are normally administered with an adjuvant to improve their immunogenicity. To accomplish iscom incorporation of a Toxoplasma gondii surface antigen through hydrophobic interaction, lipids were added either in vivo via E. coli expression, or in vitro via interaction of an introduced hexahistidyl (His6) peptide and a chelating lipid. The possibility of exploiting the strong interaction between biotin and streptavidin was also explored, in order to couple a Neospora caninum surface antigen to iscom matrix, i.e. iscom particles without any antigen. Subsequent analyses confirmed that the immunogens were successfully incorporated into iscoms by the investigated strategies. In addition, immunization of mice with the recombinant Neospora antigen NcSRS2, associated with iscoms through the biotin-streptavidin interaction, induced specific antibodies to native NcSRS2 and reduced clinical symptoms following challenge infection. The systems described in this thesis might offer convenient and efficient methods for incorporating recombinant immunogens into adjuvant formulations that might be considered for the generation of future recombinant subunit vaccines.

In a second series of studies, Affibody® (affibody) ligands directed to the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu), which is known to be overexpressed in ∼ 20-30% of breast cancers, were isolated by phage display in vitro selection from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. Biosensor analyses demonstrated that one of the variants from the phage selection, denoted His6-ZHER2/neu:4, selectively bound with nanomolar affinity (KD ≈ 50 nM) to the extracellular domain of HER2/neu (HER2-ECD) at a different site than the monoclonal antibody trastuzumab. In order to exploit avidity effects, a bivalent affibody ligand was constructed by head-to-tail dimerization, resulting in a 15.6 kDa affibody ligand, termed His6-(ZHER2/neu:4)2, that was shown to have an improved apparent affinity to HER2-ECD (KD ≈ 3 nM) compared to the monovalent affibody. Moreover, radiolabeled monovalent and bivalent affibody ligands showed specific binding in vitro to native HER2/neu molecules expressed in human cancer cells. Biodistribution studies in mice carrying SKOV-3 xenografted tumors revealed that significant amounts of radioactivity were specifically targeted to the tumors in vivo, and the tumors could easily be visualized with a gamma camera. These results suggest that affibody ligands would be interesting candidates for specific tumor targeting in clinical applications, such as in vivo imaging and radiotherapy.

Place, publisher, year, edition, pages
2005.
Keyword [en]
Biomedicine, affibody, affinity chromatography, biotin, combinatorial, delivery, phage display
Keyword [sv]
Biomedicin
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:kth:diva-231ISBN: 91-7178-003-3 (print)OAI: oai:DiVA.org:kth-231DiVA: diva2:7988
Public defence
2005-06-03, Sal FD5, AlbaNova, Roslagstullsbacken 21, Stockholm, 13:00
Opponent
Supervisors
Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2012-03-22
List of papers
1. In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation
Open this publication in new window or tab >>In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation
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2001 (English)In: Journal of Immunological Methods, ISSN 0022-1759, Vol. 255, no 1-2, 135-148 p.Article in journal (Refereed) Published
Abstract [en]

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment ΔSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-ΔSAG1 and His6-ABP-ΔSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-ΔSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, ΔSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that ΔSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.

Keyword
Affinity purification, Iscom, Lipid tagging, Subunit vaccine, Toxoplasma gondii
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:kth:diva-5174 (URN)10.1016/S0022-1759(01)00430-6 (DOI)000170260300014 ()
Note
QC 20100917Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2010-09-17Bibliographically approved
2. Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens
Open this publication in new window or tab >>Applying biotin-streptavidin binding for iscom (immunostimulating complex) association of recombinant immunogens
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2005 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 41, 163-174 p.Article in journal (Refereed) Published
Abstract [en]

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic peptide or lipid tags to improve their capacity to be incorporated into an adjuvant formulation. In the present study, we have explored the strong interaction between biotin and SA (streptavidin) (K-D approximate to 10(-15) M) to couple recombinant immunogens to iscoms (immunostimulating complexes). Two different concepts were evaluated. In the first concept, a His(6)-tagged SA fusion protein (His(6)-SA) was bound to Ni2+-loaded iscom matrix (iscom without associated protein), and biotinylated immunogens were thereafter associated with the SA-coated iscoms. The immunogens were either biotinylated in vivo on E. coli expression or double biotinylated in vivo and in vitro. In the second concept, the recombinant immunogens were expressed as SA fusion proteins, which were directly bound to a biotinylated iscom matrix. A 53-amino-acid malaria peptide (M), derived from the central repeat region of the Plasmodium faiciparum blood-stage antigen Pf155/RESA, and a 232-amino-acid segment (SRS2') from the central region (from Pro-97 to Lys-328) of the major surface antigen NcSRS2 of the protozoan parasite Neospora caninum, served as model immunogens in the present study. All fusion proteins generated were found to be efficiently expressed and could be recovered to high purity using affinity chromatography. The association between the different immunogen-containing fusion proteins and the corresponding iscom matrix was demonstrated by analytical ultracentrifugation in a sucrose density gradient. However, some fusion proteins were, to a certain extent, also found to associate unspecifically with a regular iscom matrix. Furthermore, selected iscom fractions were demonstrated to induce high-titre antigen-specific antibody responses on immunization of mice. For the particular target immunogen SRS2', the induced antibodies demonstrated reactivity to the native antigen NcSRS2. We believe that the presented concepts offer convenient methods to achieve efficient adjuvant association of recombinant immunogens, and the advantages and disadvantages of the two concepts are discussed.

Keyword
biotin, iscom, Neospora caninum, Plasmodium falciparum, streptavidin, subunit vaccine
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-5175 (URN)10.1042/BA20040060 (DOI)000228313600009 ()2-s2.0-17244382312 (Scopus ID)
Note
QC 20111005Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2017-12-04Bibliographically approved
3. Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms
Open this publication in new window or tab >>Immunisation of mice against neosporosis with recombinant NcSRS2 iscoms
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2005 (English)In: Veterinary parasitology, ISSN 0304-4017, E-ISSN 1873-2550, Vol. 129, no 1-2, 25-34 p.Article in journal (Refereed) Published
Abstract [en]

The coccidian parasite Neospora caninum is an intracellular protozoan, causing abortion in cattle in many countries around the world. In this study, the protective potential of the major N. caninum surface antigen NcSRS2, expressed in Escherichia coli and formulated into immunostimulating complexes (iscoms), was investigated in an experimental mouse model. The recombinant protein was specially designed for binding to iscoms via biotin-streptavidin interaction. Two groups of 10 BALB/c mice were immunised twice, on days 0 and 28 with iscoms containing either the recombinant NcSRS2 (NcSRS2 iscoms) or similar iscoms with NcSRS2 substituted by an unrelated recombinant malaria peptide (M5) as a control (M5 iscoms). A third group of 10 age-matched BALB/c mice served as an uninfected control group. Immunisation with recombinant NcSRS2 iscoms resulted in production of substantial antibody titres against N. caninum antigen, while the mice immunised with M5 iscoms produced only very low levels of antibodies reacting with N. caninum antigen. After challenge infection with N. caninum tachyzoites on day 69, mice immunised with NcSRS2 iscoms showed only mild and transient symptoms, whereas the group immunised with M5 iscoms showed clinical symptoms until the end of the experiment at 31 days post inoculation. A competitive PCR assay detecting Nc5-repeats was applied to evaluate the level of parasite DNA in the brain. The amount of Nc5-repeats in the group vaccinated with NcSRS2 iscoms was significantly lower than in the control group given M5 iscoms. In conclusion, it was found that the recombinant NcSRS2 iscoms induced specific antibodies to native NcSRS2 and immunity sufficient to reduce the proliferation of N. caninum in the brains of immunised mice.

Keyword
Neospora caninum, subunit vaccine, recombinant immunogen, NcSRS2, iscom, antibody response, competetive PCR
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:kth:diva-5176 (URN)10.1016/j.vetpar.2004.12.004 (DOI)000228600600004 ()2-s2.0-16244380770 (Scopus ID)
Note
QC 20100917Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2017-12-04Bibliographically approved
4. Selection and characterization of HER2/neu-binding affibody ligands
Open this publication in new window or tab >>Selection and characterization of HER2/neu-binding affibody ligands
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2004 (English)In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 17, no 5, 455-462 p.Article in journal (Refereed) Published
Abstract [en]

Affibody® (affibody) ligands that are specific for the extracellular domain of human epidermal growth factor receptor 2 (HER2/neu) have been selected by phage display technology from a combinatorial protein library based on the 58 amino acid residue staphylococcal protein A-derived Z domain. The predominant variants from the phage selection were produced in Escherichia coli, purified by affinity chromatography, and characterized by biosensor analyses. Two affibody variants were shown to selectively bind to the extracellular domain of HER2/neu (HER2-ECD), but not to control proteins. One of the variants, denoted His6-ZHER2/neu:4, was demonstrated to bind with nanomolar affinity (∼50 nM) to the HER2-ECD molecule at a different site than the monoclonal antibody trastuzumab. Furthermore, radiolabeled His 6-ZHER2/neu:4 affibody showed specific binding to native HER2/neu, overexpressed on the SKBR-3 tumor cell line. Such affibody ligands might be considered in tumor targeting applications for radionuclide diagnostics and therapy of adenocarcinomas such as breast and ovarian cancers.

Keyword
Affibody, HER2, Ligand, Phage display, Selection
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:kth:diva-5177 (URN)10.1093/protein/gzh053 (DOI)000223949100006 ()2-s2.0-4644247278 (Scopus ID)
Note
QC 20100929Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2017-12-04Bibliographically approved
5. In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics
Open this publication in new window or tab >>In vitro characterization of a bivalent anti-HER-2 affibody with potential for radionuclide-based diagnostics
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2005 (English)In: Cancer Biotherapy and Radiopharmaceuticals, ISSN 1084-9785, E-ISSN 1557-8852, Vol. 20, no 3, 239-248 p.Article in journal (Refereed) Published
Abstract [en]

The 185 kDa transmembrane glycoprotein human epidermal growth factor receptor 2 (HER-2) (p185/neu, c-ErbB-2) is overexpressed in breast and ovarian cancers. Overexpression in breast cancer correlates with poor patient prognosis, and visualization of HER-2 expression might provide valuable diagnostic information influencing patient management. We have previously described the generation of a new type of affinity ligand, a 58-amino-acid affibody (Z HER2:4) with specific binding to HER-2. In order to benefit from avidity effects, we have created a bivalent form of the affibody ligand, (Z HER2:4)2. The monovalent and bivalent ligands were compared in various assays. The new bivalent affibody has a molecular weight of 15.6 kDa and an apparent affinity (KD) against HER-2 of 3 nM. After radioiodination, using the linker molecule N-succinimidyl p-(trimethylstannyl) benzoate (SPMB), in vitro binding assays showed specific binding to HER-2 overexpressing cells. Internalization of 125I was shown after delivery with both the monovalent and the bivalent affibody. The cellular retention of 125I was longer after delivery with the bivalent affibody when compared to delivery with the monovalent affibody. With approximately the same affinity as the monoclonal antibody trastuzumab (Herceptin™) but only one tenth of the size, this new bivalent molecule is a promising candidate for radionuclide-based detection of HER-2 expression in tumors. 125I was used in this study as a surrogate marker for the diagnostically relevant radioisotopes 123I for single photon emission computed tomography (SPECT)/gamma-camera imaging and 124I for positron emission tomography (PET).

Keyword
Affibody, c-ErbB-2, HER-2, Iodine, Radionuclide, SKBR-3, Tumor
National Category
Microbiology
Identifiers
urn:nbn:se:kth:diva-5178 (URN)10.1089/cbr.2005.20.239 (DOI)000230696400001 ()2-s2.0-22344445345 (Scopus ID)
Note
QC 20100917. Uppdaterad från In press till Published (20100917)Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2017-12-04Bibliographically approved
6. Affibody mediated tumor targeting of HER-2 expressing xenografts in mice
Open this publication in new window or tab >>Affibody mediated tumor targeting of HER-2 expressing xenografts in mice
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2006 (English)In: European Journal of Nuclear Medicine and Molecular Imaging, ISSN 1619-7070, E-ISSN 1619-7089, Vol. 33, no 6, 631-638 p.Article in journal (Refereed) Published
Abstract [en]

PURPOSE:

Targeted delivery of radionuclides for diagnostic and therapeutic applications has until recently largely been limited to receptor ligands, antibodies and antibody-derived molecules. Here, we present a new type of molecule, a 15-kDa bivalent affibody called (Z(HER2:4))(2), with potential for such applications. The (Z(HER2:4))(2) affibody showed high apparent affinity (K (D)=3 nM) towards the oncogene product HER-2 (also called p185/neu or c-erbB-2), which is often overexpressed in breast and ovarian cancers. The purpose of this study was to investigate the in vivo properties of the new targeting agent.

METHODS:

The biodistribution and tumour uptake of the radioiodinated (Z(HER2:4))(2) affibody was studied in nude mice carrying tumours from xenografted HER-2 overexpressing SKOV-3 cells.

RESULTS:

The radioiodinated (Z(HER2:4))(2) affibody was primarily excreted through the kidneys, and significant amounts of radioactivity were specifically targeted to the tumours. The blood-borne radioactivity was, at all times, mainly in the macromolecular fraction. A tumour-to-blood ratio of about 10:1 was obtained 8 h post injection, and the tumours could be easily visualised with a gamma camera at this time point.

CONCLUSION:

The results indicate that the (Z(HER2:4))(2) affibody is an interesting candidate for applications in nuclear medicine, such as radionuclide-based tumour imaging and therapy.

Keyword
affibody, HER-2, imaging, SKOV-3, tumour, breast-cancer, in-vitro, cellular retention, human tissues, antibody, proteins, binding, overexpression, localization, penetration
National Category
Medical Engineering
Identifiers
urn:nbn:se:kth:diva-5179 (URN)10.1007/s00259-005-0012-3 (DOI)000238023000002 ()2-s2.0-33646259546 (Scopus ID)
Note
Updated from submitted to published. QC 20111007Available from: 2005-05-30 Created: 2005-05-30 Last updated: 2017-12-04Bibliographically approved

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