Tove Olafsen - Academia.edu (original) (raw)

Papers by Tove Olafsen

Antibody fragments with optimized pharmacokinetic profiles hold potential for detection and thera... more Antibody fragments with optimized pharmacokinetic profiles hold potential for detection and therapy of tumor malignan- cies. We studied the behavior of three anti-carcinoembryonic antigen (CEA)single-chain Fv-Fc (scFv-Fc)variants (I253A, H310A, and H310A/H435Q; Kabat numbering system)that exhibited differential serum persistence. Biodistribution studies done on CEA-positive tumor xenografted mice revealed that the 111In-labeled I253A fragment with the slowest clearance kinetics (T1/2B, 27.7 h)achieved

Journal of nuclear medicine : official publication, Society of Nuclear Medicine, Jan 7, 2015

The proliferation and trafficking of T lymphocytes in immune responses are crucial events in dete... more The proliferation and trafficking of T lymphocytes in immune responses are crucial events in determining inflammatory responses. To study whole body T lymphocyte dynamics non-invasively in vivo, we have generated anti-CD4 and -CD8 cys-diabodies (cDbs) derived from the parental antibody hybridomas GK1.5 and 2.43, respectively, for (89)Zr-immunoPET detection of helper and cytotoxic T cell populations. Anti-CD4 and -CD8 cys-diabodies were engineered, produced via mammalian expression, purified using immobilized metal affinity chromatography, and characterized for T cell binding. The cys-diabodies were site-specifically conjugated to maleimide-desferrioxamine for (89)Zr radiolabeling and subsequent microPET/CT acquisition and ex vivo biodistribution in both wild type mice and a model of hematopoietic stem cell (HSC) transplantation. ImmunoPET and biodistribution studies demonstrate targeting and visualization of CD4 and CD8 T cell populations in vivo in the spleen and lymph nodes of wil...

Nature protocols, 2006

Immunoglobulins (Igs) are large proteins of 150 kDa with prolonged residence time in blood. Their... more Immunoglobulins (Igs) are large proteins of 150 kDa with prolonged residence time in blood. Their half-life is controlled by their ability to interact with the protective neonatal Fc receptor (FcRn, Brambell receptor) present on endothelial cells. Here, we describe a protocol using site-specific mutagenesis of individual residues responsible for this interaction, resulting in engineered antibodies with distinct half-lives. The method is a powerful tool that enables manipulation of half-lives and is applicable to all antibodies and Fc fusion proteins for the development of agents with controlled pharmacokinetic properties. Moreover, the protocol is applicable to any situation where the structure and/or function of engineered proteins are to be studied. The protocol begins with the mutagenesis reaction at the DNA level and proceeds to describe mammalian expression and purification of recombinant proteins, radiolabeling and evaluation in vivo. The time frame for completing the procedur...

Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2007

In this study, we investigated the 18F-labeled anti-carcinoembryonic antigen (CEA) T84.66 diabody... more In this study, we investigated the 18F-labeled anti-carcinoembryonic antigen (CEA) T84.66 diabody, a genetically engineered noncovalent dimer of single-chain variable fragments, for small-animal PET imaging of CEA expression in xenograft-bearing mice. 18F labeling of the anti-CEA T84.66 diabody (molecular mass, 55 kDa) was achieved with N-succinimidyl-4-18F-fluorobenzoate (18F-SFB). The biodistribution of the 18F-fluorobenzyl-T84.66 diabody (18F-FB-T84.66 diabody) was evaluated in athymic nude mice bearing subcutaneous LS 174T human colon carcinoma and C6 rat glioma tumors. Serial small-animal PET imaging studies were performed to further evaluate in vivo targeting efficacy and pharmacokinetics. Radiolabeling required 35 +/- 5 (mean +/- SD) min starting from 18F-SFB, and the tracer 18F-FB-T84.66 diabody was synthesized with a specific activity of 1.83 +/- 1.71 TBq/mmol. The decay-corrected radiochemical yield was 1.40% +/- 0.16% (n = 4), and the radiochemical purity was greater than...

Protein engineering, design & selection : PEDS, 2004

An engineered antibody fragment (minibody; scFv-C H 3g 1 dimer, M r 80 000) specific for carcinoe... more An engineered antibody fragment (minibody; scFv-C H 3g 1 dimer, M r 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti-p185 HER-2 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V L -linker-V H and V H -linker-V L ) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C H 3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185 HER-2 overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K D $ 2-4 nM) were equivalent to that for the parental 10H8 mAb (K D $ 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (61.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (b-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.

Antibody Engineering, 2010

Intermediate-sized antibody fragments, such as scFv-CH3 (minibodies) and scFv-Fc, retain high avi... more Intermediate-sized antibody fragments, such as scFv-CH3 (minibodies) and scFv-Fc, retain high avidity for their antigen due to their nature of bivalency. In addition, the incorporation of the Fc region enables the generation of antibody fragments with a variety of biological functions and pharmacokinetic properties. This protocol describes the isolation of variable genes from hybridoma cells, gene assembly, cloning, and stable expression of these fragments in mammalian cells. These fragments are expressed as single peptide chains which bypass the challenges of coexpressing immunoglobulin heavy and light chains. Included is also a protocol to screen for high expressing clones, and suggestions for purification and biochemical characterization of the purified proteins.

Cancer research, Jan 15, 2005

Antibody fragments are recognized as promising vehicles for delivery of imaging and therapeutic a... more Antibody fragments are recognized as promising vehicles for delivery of imaging and therapeutic agents to tumor sites in vivo. The serum persistence of IgG1 and fragments with intact Fc region is controlled by the protective neonatal Fc receptor (FcRn) receptor. To modulate the half-life of engineered antibodies, we have mutated the Fc-FcRn binding site of chimeric anti-carcinoembryonic antigen (CEA) antibodies produced in a single-chain Fv-Fc format. The anti-CEA T84.66 single-chain Fv-Fc format wild-type and five mutants (I253A, H310A, H435Q, H435R, and H310A/H435Q, Kabat numbering system) expressed well in mammalian cell culture. After purification and characterization, effective in vitro antigen binding was shown by competition ELISA. Biodistribution studies in BALB/c mice using (125)I- and (131)I-labeled fragments revealed blood clearance rates from slowest to fastest as follows: wild-type > H435R > H435Q > I253A > H310A > H310A/H435Q. The terminal half-lives of ...

Tumor Biology, 2012

Combining the specificity of tumor-targeting antibodies with the sensitivity and quantification o... more Combining the specificity of tumor-targeting antibodies with the sensitivity and quantification offered by positron emission tomography (PET) provides tremendous opportunities for molecular characterization of tumors in vivo. Until recently, significant challenges have been faced when attempting to combine antibodies which show long biological half-lives and positron-emitting radionuclides with comparably short physical half-lives, in particular (18)F (half-life, 109 min). A fast and simple microwave-assisted method of generating N-succinimidyl-4-[(18)F]fluorobenzoate has been developed and employed for radiolabeling a small, rapidly targeting HER2-specific engineered antibody fragment, the cys-diabody. Using this tracer, HER2-positive tumor xenografts in mice were detected at 1-4 h post-injection by microPET. This confirms the rapid kinetics of [(18)F]fluorobenzoyl cys-diabody localization, and demonstrates the feasibility of same-day immunoPET imaging. This approach can be broadly applied to antibodies targeting cell surface biomarkers for molecular imaging of tumors and should be highly translatable for clinical use.

Protein Engineering Design and Selection, 2004

An engineered anti-carcinoembryonic antigen (CEA) diabody (scFv dimer, 55 kDa) was previously con... more An engineered anti-carcinoembryonic antigen (CEA) diabody (scFv dimer, 55 kDa) was previously constructed from the murine anti-CEA T84.66 antibody. Tumor targeting, imaging and biodistribution studies in nude mice bearing LS174T xenografts with radiolabeled anti-CEA diabody demonstrated rapid tumor uptake and fast blood clearance, which are favorable properties for an imaging agent. Current radiolabeling approaches result in random modi®cation of the protein surface, which may impair immunoreactivity especially for smaller antibody fragments. Site-speci®c conjugation approaches can direct modi®cations to reactive groups located away from the binding site. Here, cysteine residues were introduced into the anti-CEA diabody at three different locations, to provide speci®c thiol groups for chemical modi®cation. One version (with a C-terminal Gly-Gly-Cys) existed exclusively as a disul®de-bonded dimer. This cysteine-modi®ed diabody (Cys-diabody) retained high binding to CEA and demonstrated tumor targeting and biodistribution properties identical to the non-covalent diabody. Furthermore, following reduction of the disul®de bond, the Cys-diabody could be chemically modi®ed using a thiol-speci®c bifunctional chelating agent, for radiometal labeling. Thus, the Cys-diabody provides a covalently linked alternative to conventional diabodies, which can be reduced and modi-®ed site-speci®cally. This format will provide a versatile platform for targeting a variety of agents to CEA-positive tumors.

Protein Engineering Design and Selection, 2004

An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for ca... more An engineered antibody fragment (minibody; scFv-C(H)3gamma(1) dimer, M(r) 80 000) specific for carcinoembryonic antigen (CEA) has previously demonstrated excellent tumor targeting coupled with rapid clearance in vivo. In this study, variable (V) genes from the anti- p185(HER-2) 10H8 antibody were similarly assembled and expressed. Four constructs were made: first, the V genes were assembled in both orientations (V(L)-linker-V(H) and V(H)-linker-V(L)) as single chain Fvs (scFvs). Then each scFv was fused to the human IgG1 C(H)3 domain, either by a two amino acid linker (ValGlu) that resulted in a non-covalent, hingeless minibody, or by IgG1 hinge and a GlySer linker peptide to produce a covalent, hinge-minibody. The constructs, expressed in NS0 mouse myeloma cells at levels of 20-60 mg/l, demonstrated binding to the human p185(HER-2) overexpressing breast cancer cell line, MCF7/HER2. Binding affinities (K(D) approximately 2-4 nM) were equivalent to that for the parental 10H8 mAb (K(D) approximately 1.6 nM). Radioiodinated 10H8 hinge-minibody was evaluated in athymic mice, bearing MCF7/HER2 xenografts. Maximum tumor uptake was 5.6 (+/-1.65)% injected dose/g (ID/g) at 12 h, which was lower than that of the anti-CEA minibody, whereas the blood clearance (beta-phase, 5.62 h) was similar. Thus, minibodies with different specificities display similar pharmacokinetics, while tumor uptake may vary depending on the antigen-antibody system.

Protein Engineering Design and Selection, 2010

Rapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time... more Rapid clearing engineered antibody fragments for immunoPET promise high sensitivity at early time points. Here, tumor targeting of anti-CD20 diabodies (scFv dimers) for detection of low-grade B-cell lymphomas were evaluated. In addition, the effect of linker length on oligomerization of the diabody was investigated. Four rituximab scFv variants in the V L -V H orientation with different linker lengths between the V domains (scFv-1, scFv-3, scFv-5, scFv-8), plus the scFv-5 with a C-terminal cysteine (Cys-Db) for site-specific modification were generated. The scFv-8 and Cys-Db were radioiodinated with 124 I for PET imaging, and biodistribution of 131 I-Cys-Db was carried out at 2, 4 10 and 20 h. The five anti-CD20 scFv variants were expressed as fully functional dimers. Shortening the linker to three or one residue did not produce higher order of multimers. Both 124 I-labeled scFv-8 and Cys-Db exhibited similar tumor targeting at 8 h post injection, with significantly higher uptakes than in control tumors (P < 0.05). At 20 h, less than 1% ID/g of 131 I-labeled Cys-Db was present in tumors and tissues. Specific tumor targeting and high contrast images were achieved with the anti-CD20 diabodies. These agents extend the repertoire of reagents that can potentially be used to improve detection of low-grade lymphomas.

Protein Engineering Design and Selection, 2006

An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the ... more An anti-carcinoembryonic antigen (CEA) antibody fragment, the anti-CEA diabody, was fused to the bioluminescence enzyme Renilla luciferase (RLuc) to generate a novel optical imaging probe. Native RLuc or one of two stabilized variants (RLucC124A, RLuc8) was used as the bioluminescent moiety. A bioluminescence ELISA showed that diabody-luciferase could simultaneously bind to CEA and emit light. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-RLuc8 to CEA-positive xenografts, with a tumor:background ratio of 6.0 +/- 0.8 at 6 h after intravenous injection, compared with antigen-negative tumors at 1.0 +/- 0.1 (P = 0.05). Targeting and distribution was also evaluated by microPET imaging using (124)I-diabody-RLuc8 and confirmed that the optical signal was due to antibody-mediated localization of luciferase. Renilla luciferase, fused to biospecific sequences such as engineered antibodies, can be administered systemically to provide a novel, sensitive method for optical imaging based on expression of cell surface receptors in living organisms.

Protein Engineering Design and Selection, 2008

We have previously demonstrated preclinical in vivo targeting of prostate stem cell antigen (PSCA... more We have previously demonstrated preclinical in vivo targeting of prostate stem cell antigen (PSCA) using a humanized anti-PSCA 2B3 monoclonal antibody (mAb). However, humanization resulted in 5-fold loss of apparent affinity relative to the parental mAb (1 nM). In this study, diabodies (scFv dimers of 55 kDa) were generated from 2B3 including variants with different linker lengths as well as back-mutations to original murine residues to improve affinity. Parental 2B3 ( p2B3) and back-mutated 2B3 (bm2B3) diabodies (Dbs) with five-or eight-amino acid linkers ( p2B3-Db5, p2B3-Db8, bm2B3-Db5 and bm2B3-Db8) were evaluated for binding to PSCA by flow cytometry and affinities were determined by surface plasmon resonance. Back-mutation restored the affinity from 5.4 to 1.9 nM. Stability, evaluated by size exclusion, revealed that diabodies with eight-residue linkers existed as a mixture of dimeric and monomeric species at low concentrations ( 1 mg/ml). Shortening the linker from eight to five residues improved dimer stability, notably in the bm2B3-Db8 compared with bm2B3-Db5. Both p2B3-Db8 and bm2B3-Db8 were radioiodinated with 124 I and evaluated by serial micro-positron emission tomography imaging in mice bearing LAPC-9 human prostate cancer xenografts. Localization in LAPC-9 xenografts was seen at 4 h, whereas at 20 h most of the activity had cleared from the tumor. Highest tumor-to-background contrast ratios and best images were obtained at 12 h. Although the higher affinity bm2B3-Db8 demonstrated improved tumor retention at later time points (20 h), it did not improve tumor targeting or imaging compared with p2B3-Db8 at 12 h.

Protein Engineering Design and Selection, 2001

A series of single-chain anti-CD20 antibodies was produced by fusing single-chain Fv (scFv) with ... more A series of single-chain anti-CD20 antibodies was produced by fusing single-chain Fv (scFv) with human IgG1 hinge and Fc regions, designated scFv-Fc. The initial scFv-Fc construct was assembled using an 18 amino acid (aa) linker between the antibody light- and heavy-chain variable regions, with the Cys residue in the upper hinge region (Kabat 233) mutagenized to Ser. Anti-CD20 scFv-Fc retained specific binding to CD20-positive cells and was active in mediating complement-dependent cytolysis. Size-exclusion HPLC analysis revealed that the purified scFv-Fc included multimeric as well as monomeric components. Variant scFv-Fcs were constructed incorporating four different hinges between the scFv and Fc regions, or three different linkers in the scFv domain. All formed multimers, with the highest level of multimerization found in the scFv-Fc with the shortest linker (8 aa). Elimination of an unusual salt bridge between residues L38 and H89 in the V(L)-V(H) domain interface failed to reduce the formation of higher order forms. Structural analysis of the scFv-Fc constructed with 18 or 8 aa linkers by pepsin or papain cleavage suggested the proteins contained a form in which scFv units had cross-paired to form a &amp;#39;diabody&amp;#39;. Thus, domain exchange or cross-pairing appears to be the basis of the observed multimerization.

Molecular Imaging and Biology, 2007

The bioluminescent protein Gaussia luciferase (GLuc) was fused to an anti-carcinoembryonic antige... more The bioluminescent protein Gaussia luciferase (GLuc) was fused to an anti-carcinoembryonic antigen (CEA) antibody fragment, the diabody, for in vivo optical tumor imaging. A 15-amino acid N-terminal truncation (GLDelta15) resulted in a brighter protein. Fusions of the anti-CEA diabody to full-length GLuc and GLDelta15 retained high affinity for the antigen, emitted light, and exhibited excellent enzymatic stability. In vivo optical imaging of tumor-bearing mice demonstrated specific targeting of diabody-GLDelta15 to CEA-positive xenografts, with a tumor/background ratio of 3.8 +/- 0.4 at four hours after tail-vein injection, compared to antigen-negative tumors at 1.3 +/- 0.1 (p = 0.001). MicroPET imaging using (124)I-diabody-GLDelta15 demonstrated specific uptake in the CEA-positive tumor (2.6% ID [injected dose]/g) compared to the CEA-negative tumor (0.4% ID/g) at 21 hours. Although further optimization of this fusion protein may be needed to improve in vivo performance, the diabody-GLDelta15 is a promising optical imaging probe for tumor detection in vivo.

Journal of Surgical Research, 2011

Reporter gene imaging has great potential for many clinical applica- tions including the tracking... more Reporter gene imaging has great potential for many clinical applica- tions including the tracking of transplanted cells and monitoring of gene therapy. However, currently available reporter gene- reporter probe combinations have significant limitations with the biodistribution of the reporter probe and the specificity and immu- nogenicity of the reporter gene. The objective of the present study was to evaluate a

Journal of Nuclear Medicine, 2009

The CD20 cell surface antigen is expressed at high levels by over 90% of B cell non-Hodgkin lymph... more The CD20 cell surface antigen is expressed at high levels by over 90% of B cell non-Hodgkin lymphomas (NHL), and is the target of the anti-CD20 monoclonal antibody rituximab. To provide more sensitive, tumor-specific positron emission tomography (PET) imaging of NHL, we sought to develop PET imaging agents targeting CD20.

Journal of Immunotherapy, 2001

Regrowth of drug-resistant tumor cells is responsible for approximately half of an unselected ost... more Regrowth of drug-resistant tumor cells is responsible for approximately half of an unselected osteosarcoma population still dying of the disease despite aggressive combination therapy. Two monoclonal antibodies, TP-1 (immunoglobulin 2a) and TP-3 (immunoglobulin 2b) are available, which specifically recognize an antigen on osteosarcoma cells. In this work, we have fused the variable (V) genes of TP-3 to a truncated fragment of Pseudomonas exotoxin A, referred to as PE38. Two immunotoxins were made that differed in the Fv portion: TP-3(scFv)-PE38, which contains a peptide linker, and TP-3(dsFv)-PE38, which contains a disulfide bond for stabilization of the association between the V domains. Recombinant TP-3 immunotoxins were expressed in Escherichia coli and purified from inclusion bodies. We describe the design and expression of these immunotoxins, and their properties with regard to antigen binding, stability, and cytotoxicity. Toxicity studies were done in mice. We found that the immunotoxins exhibited very similar in vitro properties, whereas in vivo TP-3(dsFv)-PE38 was much better tolerated than TP-3(scFv)-PE38.

Journal of nuclear medicine : official publication, Society of Nuclear Medicine, Jan 7, 2015

Nature protocols, 2006

Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 2007

Protein engineering, design & selection : PEDS, 2004

Antibody Engineering, 2010

Cancer research, Jan 15, 2005

Tumor Biology, 2012

Protein Engineering Design and Selection, 2004

Protein Engineering Design and Selection, 2010

Protein Engineering Design and Selection, 2006

Protein Engineering Design and Selection, 2008

Protein Engineering Design and Selection, 2001

Molecular Imaging and Biology, 2007

Journal of Surgical Research, 2011

Journal of Nuclear Medicine, 2009

Journal of Immunotherapy, 2001