Affibodymolekyler är små affinitetsproteiner (6.5 kDa) som föreslås kunna ersätta monoklonala antikroppar i terapeutiska tillämpningsområden, exempelvis i antikropp-läkemedelskonjugat (ADCs) som kan navigera sig fram till biomarkörer som är uttryckta på cancerceller. Affibody-läkemedelskonjugat (AffiDC) kan användas för att målsöka just sådana överuttryckta proteiner, samtidigt som de erbjuder goda egenskaper, såsom snabb transportering och spridning i kroppen, och effektiv penetrering genom tumörer. Dessa AffiDC:er skulle kunna användas inom riktad cancerterapi for de cancersjukdomar som fortfarande är i behov av cancerhämmande behandlingar, såsom urotelial cancer.  Den här studien föreslog tillämpning av ABD-kopplade affibodymolekyler för att målsöka ett nytt målprotein som har visats vara överuttryckt i flera olika cancersjukdomar, exempelvis bröst-, pankreas- och urotelial cancer. Affibodykandidater mot målproteinet har valts ur ett rekombinant bibliotek med 1×1011 transformanter som uttrycks med hjälp av en så kallad metod med E. coli celldisplay där affibodymolekylen visas på cellens ytmembran. De slutliga kandidaterna var sedan identifierade och biokemiskt karaktäriserade i in vitro-studie på människoceller, som visade att två av kandidaterna verkade binda till cancercellinjerna BT-474 och MCF-7 med KD omkring 10 till 100 nM. / Affibody molecules are small affinity proteins (6.5 kDa) suggested to substitute monoclonal antibodies in therapeutic applications, e.g., antibody-drug conjugates (ADCs) targeting biomarker proteins expressed on cancer cells. An affibody-drug conjugate (AffiDC) could be used to target these types of overexpressed proteins on cancer cells while offering attractive properties, such as rapid transportation and distribution in the body, as well as efficient tumour penetration. These AffiDCs could be used as a targeted cancer therapy for cancers that are yet to be treatable and curable, like urothelial cancers.  This study suggested the use of ABD-fused affibodies to target a novel cancer protein that has been shown to be overexpressed on cancer cells, including breast, pancreatic and urothelial cancer. Affibody candidates toward this novel target were selected from a recombinant library, of 1×1011 transformants, that is expressed using E. coli cell display system. The final candidates were subsequently biochemically characterized and assessed for affinity for the target. Three affibodies were finally identified and assessed in in vitro studies on mammalian cells, revealing two affibodies that appear to bind to the cell lines BT-474 and MCF-7 with KD ranging 10 to 100 nM.

Affinity proteins constitute an important category of cancer therapeutics. Owing to properties such as high target affinity and selectivity, therapeutic proteins offer more targeted therapy than small molecule drugs. The target molecules are typically proteins that are overexpressed on the surface of tumour cells, such as membrane-bound receptors. However, these surface proteins are usually expressed in normal tissues as well, resulting in on-target off-tumour toxicity. Proteins with a higher tissue selectivity are thus needed. Here, this has been addressed by developing prodrug proteins dependent on cancer-specific proteases for activation. The prodrugs were composed of a target-binding affibody (active domain) connected to a masking affibody (masking domain) by a peptide linker including a protease substrate. The target of the prodrugs developed in this project was the HER2 receptor, which is overexpressed in several cancer types. Three prodrug candidates were developed, produced and characterised based on their ability to be activated by their respective protease. The hypothesis that the prodrugs could be activated and thus bind to HER2 in cancer cells was tested using biosensor assays, as well as preliminary cancer cell assays. One of the three candidates showed strong potential to be used as a targeted therapy for cancer treatment in the future. / Affinitetsproteiner utgör en viktig kategori av cancerläkemedel. Jämfört med småmolekylära läkemedel är affinitetsproteiner mer riktade, då de har högre affinitet och selektivitet än små molekyler. Oftast utgörs det molekylära målet av ett protein som överuttrycks på ytan av cancerceller, så som membranbundna receptorer. Dessvärre uttrycks de flesta cancerspecifika proteiner i mindre mängd även i normal vävnad. Detta leder till oönskade effekter som kan ge upphov till biverkningar. I syfte att utveckla mer vävnadsspecifika läkemedel har här affibody-baserade “prodrugs”, beroende av cancerspecifika proteaser för aktivering, tagits fram. Prodrug-proteinerna i detta projekt är riktade mot HER2-receptorn, som är överuttryckt i flera typer av cancer. Tre kandidater togs fram och utvärderades med avseende på deras förmåga att aktiveras av sina respektive proteaser. För att testa hypotesen att kandidaterna kunde binda till HER2 på cancerceller efter proteasaktivering användes biosensoranalys samt experiment med cancerceller. En av kandidaterna visade stark potential att kunna användas som ett riktat läkemedel mot cancer i framtiden.

<p>The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications.</p><p>This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of ¹²⁵I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR).</p><p>In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (Z_EGFR:955)₂ was selected and analysed both <i>in vitro</i> and <i>in vivo</i>. In papers II, III and V, the results obtained when using (Z_EGFR:955)₂ were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, <i>in vivo</i> studies were made, demonstrating that [¹¹¹In](Z_EGFR:955)₂ gave a tumour-specific ¹¹¹In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point. </p><p>The results of these studies indicated that the affibody molecule (Z_EGFR:955)₂ is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (Z_EGFR:955)₂ might be of interest for therapy applications.</p>

The human epidermal growth-factor like receptor (HER) family of receptor tyrosine kinases are important targets for cancer therapy. The family consists of four members - EGFR, HER2, HER3 and HER4 - that normally transfer stimulatory signals from extracellular growth factors to the intracellular signalling network. Over-activation of these receptors leads to uncontrolled cell proliferation and is seen in several types of tumours. The aim of the studies reported in this thesis was to study the uptake and effects of affibody molecules against EGFR, HER2 and HER3 in cultured cells. Affibody molecules are affinity proteins originally derived from one of the domains of protein A, and their small size and robust structure make them suitable agents for tumour targeting and therapy. Papers I and II of this thesis concern EGFR-specific affibody molecules, which were shown to be more similar to the antibody cetuximab than the natural ligand EGF in terms of cellular uptake, binding site and internalisation rate. In addition, fluorescence-based methods for the quantification of internalisation were evaluated. In the studies reported in papers III and IV, HER2-specific affibody molecules were utilised as carriers of radionuclides. Paper III reports that different cell lines exhibit different radiosensitivities to 211At-labelled affibody molecules; radiosensitivity was found to correlate with cell geometry and the rate of internalisation. Paper IV discusses the use of 17-AAG, an agent that induces HER2 internalisation and degradation, to force the internalisation of 211At- and 111In-labelled affibody molecules. Papers V and VI describe the selection and maturation of HER3-specific affibody molecules, which were found to compete with the receptor’s natural ligand, heregulin, for receptor binding. These affibody molecules were demonstrated to inhibit heregulin-induced HER3 activation and cell proliferation. The studies summarised in this paper will hopefully contribute to a better understanding of these affibody molecules and bring them one step closer to being helpful tools in the diagnosis and treatment of cancer.

This thesis describes the development and use ofnon-immunoglobulin affinity proteins denoted affibodies asalternatives to antibodies in different immunotechnologyapplications. A 58 aa IgG Fc binding three-helix bundle domainZ, derived from staphylococcal protein A has been used asframework for library constructions, in which the face of themolecule involved in the native binding activity has beenengineered by combinatorial protein engineering. Recruting 13surface-located positions for simultanenous substitutionmutagenesis, using degenerated oligonucleotides for libraryassembly at the genetic level, two libraries differing in thechoice of codons were constructed to serve as general sourcesof novel affinity proteins. The libraries were adapted fordisplay onE. colifilamentous phage particles allowingin vitroselection of desired variants capable ofbinding a given target molecule. In selections using human IgAas target, several new IgA specific affibodies could beidentified. One variant ZIgA1, was further investigated and showed binding toboth IgA1 and IgA2 human subclasses as well as to secretoryIgA. This variant was further demonstrated uesful as ligand inaffinity chromatography purification for recovery of IgA fromdifferent samples including unconditioned human plasma.Affibodies of different specificities were also fused to otherprotein domains to construct fusion proteins of relevance forimmunotechnology applications. Using Fc of human IgG as genefusion partner, "artificial antbodies" could be produced inE. colias homodimeic proteins, where the antigenbinding was confered by N-terminally positioned affibodymoieties of different valencies. One area of application forthis type of constructs was demonstrated through specificdetection of the target protein by Western blotting. Exploitingthe uncomplicated structure of affibody affinity proteins, genefusions between affibodies and the homotetrameric reporterenzyme β-galactosidase were constructed, which could beproduced as soluble proteins intracellularly inE. coli. The potential use of such recombinantimmunoconjugates in immunotechnology was demonstrated in ELISAdot-blot and immunohistochemistry, where in the latter case IgAdepositions in the glomeruli of a human kidney biopsy could bespecfically detected with low background staining ofsurrounding tissues. In a novel format for sandwich ELISA, thepossible advantage of the bacterial origin of the affibodyclass of affinity proteins was investigated. As a means tocircumvent problems associated with the presence of humanheterophilic antibodies in serum, causing bakground signals dueto analyte-independent crosslinking of standard capture anddetection antibody reagents, assay formats based oncombinations of antibody and affibody reagents for capture anddetection were investigated and found to be of potentialuse. <b>Keywords:</b>phage display, combinatorial, affinity, IgAligand, immunohistochemistry, affibody-fusions

Proteins are essential components in most processes in living organisms. The detection and quantification of specific proteins can be used e.g. as measures of certain physiological conditions, and are therefore of great importance. This thesis focuses on development of affinity-based bioassays for specific protein detection. The use of Affibody molecules for specific molecular recognition has been central in all studies in this thesis. Affibody molecules are affinity proteins developed by combinatorial protein engineering of the 58-residue protein A-derived Z domain scaffold. In the first paper, solid phase peptide synthesis is investigated as a method to generate functional Affibody molecules. Based on the results from this paper, chemical synthesis has been used throughout the following papers to produce Affibody molecules tailored with functional groups for protein detection applications in vitro and in vivo.   In paper I, an orthogonal protection scheme was developed to enable site-specific chemical introduction of three different functional probes into synthetic Affibody molecules. Two of the probes were fluorophores that were used in a FRET-based binding assay to detect unlabeled target proteins. The third probe was biotin, which was used as an affinity handle for immobilization onto a solid support. In paper II, a panel of Affibody molecules carrying different affinity handles were synthesized and evaluated as capture ligands on microarrays. Paper III describes the synthesis of an Affibody molecule that binds to the human epidermal growth factor receptor type 2, (HER2), and the site-specific incorporation of a mercaptoacetyl-glycylglycylglycine (MAG3) chelating site in the peptide sequence to allow for radiolabeling with 99mTc. The derivatized Affibody molecule was found to retain its binding capacity, and the 99mTc-labeling was efficient and resulted in a stable chelate formation. 99mTc-labeled Affibody molecules were evaluated as in vivo HER2-targeting imaging agents in mice. In the following studies, reported in papers IV-VI, the 99mTc-chelating sequence was engineered in order to optimize the pharmacokinetic properties of the radiolabeled Affibody molecules and allow for high-contrast imaging of HER2-expressing tumors and metastatic lesions. The main conclusion from these investigations is that the biodistribution of Affibody molecules can be dramatically modified by amino acid substitutions directed to residues in the MAG3-chelator. Finally, paper VII is a report on the chemical synthesis and chemoselective ligation to generate a cross-linked HER2-binding Affibody molecule with improved thermal stability and tumor targeting capacity.   Taken together, the studies presented in this thesis illustrate how peptide synthesis can be used for production and modification of small affinity proteins, such as Affibody molecules for protein detection applications. / QC 20100719

<p>This thesis describes the development and use ofnon-immunoglobulin affinity proteins denoted affibodies asalternatives to antibodies in different immunotechnologyapplications. A 58 aa IgG Fc binding three-helix bundle domainZ, derived from staphylococcal protein A has been used asframework for library constructions, in which the face of themolecule involved in the native binding activity has beenengineered by combinatorial protein engineering. Recruting 13surface-located positions for simultanenous substitutionmutagenesis, using degenerated oligonucleotides for libraryassembly at the genetic level, two libraries differing in thechoice of codons were constructed to serve as general sourcesof novel affinity proteins. The libraries were adapted fordisplay on<i>E. coli</i>filamentous phage particles allowing<i>in vitro</i>selection of desired variants capable ofbinding a given target molecule. In selections using human IgAas target, several new IgA specific affibodies could beidentified. One variant Z_IgA1, was further investigated and showed binding toboth IgA1 and IgA2 human subclasses as well as to secretoryIgA. This variant was further demonstrated uesful as ligand inaffinity chromatography purification for recovery of IgA fromdifferent samples including unconditioned human plasma.Affibodies of different specificities were also fused to otherprotein domains to construct fusion proteins of relevance forimmunotechnology applications. Using Fc of human IgG as genefusion partner, "artificial antbodies" could be produced in<i>E. coli</i>as homodimeic proteins, where the antigenbinding was confered by N-terminally positioned affibodymoieties of different valencies. One area of application forthis type of constructs was demonstrated through specificdetection of the target protein by Western blotting. Exploitingthe uncomplicated structure of affibody affinity proteins, genefusions between affibodies and the homotetrameric reporterenzyme β-galactosidase were constructed, which could beproduced as soluble proteins intracellularly in<i>E. coli</i>. The potential use of such recombinantimmunoconjugates in immunotechnology was demonstrated in ELISAdot-blot and immunohistochemistry, where in the latter case IgAdepositions in the glomeruli of a human kidney biopsy could bespecfically detected with low background staining ofsurrounding tissues. In a novel format for sandwich ELISA, thepossible advantage of the bacterial origin of the affibodyclass of affinity proteins was investigated. As a means tocircumvent problems associated with the presence of humanheterophilic antibodies in serum, causing bakground signals dueto analyte-independent crosslinking of standard capture anddetection antibody reagents, assay formats based oncombinations of antibody and affibody reagents for capture anddetection were investigated and found to be of potentialuse.</p><p><b>Keywords:</b>phage display, combinatorial, affinity, IgAligand, immunohistochemistry, affibody-fusions</p>

Optimization of Affibody molecules would allow for high contrast imaging of cancer associated surface receptors using molecular imaging. The primary aim of the thesis was to develop Affibody-based PET imaging agents to provide the highest possible sensitivity of RTK detection in vivo. The thesis evaluates the effect of radiolabelling chemistry on biodistribution and targeting properties of Affibody molecules directed against HER2 and PDGFRβ. The thesis is based on five published papers (I-V). Paper I. The targeting properties of maleimido derivatives of DOTA and NODAGA for site-specific labelling of a recombinant HER2-binding Affibody molecule radiolabelled with 68Ga were compared in vivo. Favourable in vivo properties were seen for the Affibody molecule with the combination of 68Ga with NODAGA. Paper II. The aim was to compare the biodistribution of 68Ga- and 111In-labelled HER2-targeting Affibody molecules containing DOTA, NOTA and NODAGA at the N-terminus. This paper also demonstrated favourable in vivo properties for Affibody molecules in combination with 68Ga and NODAGA placed on the N-terminus. Paper III.  The influence of chelator positioning on the synthetic anti-HER2 affibody molecule labelled with 68Ga was investigated. The chelator DOTA was conjugated either at the N-terminus, the middle of helix-3 or at the C-terminus of the Affibody molecules. The N-terminus placement provided the highest tumour uptake and tumour-to-organ ratios. Paper IV. The aim of this study was to evaluate if the 68Ga labelled PDGFRβ-targeting Affibody would provide an imaging agent suitable for PDGFRβ visualization using PET. The 68Ga labelled conjugate provided high-contrast imaging of PDGFRβ-expressing tumours in vivo using microPET as early as 2h after injection. Paper V. This paper investigated if the replacement of IHPEM with IPEM as a linker molecule for radioiodination of Affibody molecules would reduce renal retention of radioactivity. Results showed that the use of the more lipophilic linker IPEM reduced the renal radioactivity retention for radioiodinated Affibody molecules. In conclusion, this thesis clearly demonstrates that the labelling strategy is of great importance with a substantial influence on the targeting properties of Affibody molecules and should be taken under serious considerations when developing new imaging agents.

The optimal way to detect and treat cancer is to target cancer cells exclusively without affecting the surrounding tissue. One promising approach is to use radiolabelled molecules to target receptors that are overexpressed in cancer cells. Since the epidermal growth factor receptor (EGFR) family is overexpressed in many types of cancer, it is an attractive target for both diagnostic and therapeutic applications. This thesis can be divided into two parts. In part one (paper I), studies were conducted to modulate radionuclide uptake in tumour cells. The results showed that it was possible to modulate the cellular uptake of 125I delivered by trastuzumab (targeting HER2) by adding EGF (targeting EGFR). In part two (papers II-V) a high affinity EGFR-targeting affibody molecule (ZEGFR:955)2 was selected and analysed both in vitro and in vivo. In papers II, III and V, the results obtained when using (ZEGFR:955)2 were compared with those obtained with the two EGFR-binding molecules, EGF and cetuximab. These studies demonstrated that the affibody molecule bound specifically to EGFR (probably to subdomain III) with high affinity (~50 nM in biosensor analysis and ~1 nM in cellular studies) and produced intracellular signalling changes similar to those with cetuximab. In paper IV, in vivo studies were made, demonstrating that [111In](ZEGFR:955)2 gave a tumour-specific 111In uptake of 3.8±1.4% of injected dose per gram tumour tissue, 4 h post-injection. The tumours could be easily visualized with a gamma camera at this time-point. The results of these studies indicated that the affibody molecule (ZEGFR:955)2 is a possible candidate for radionuclide-based imaging of EGFR-expressing tumours. The biological effects of (ZEGFR:955)2 might be of interest for therapy applications.

Abnormal expression and signaling of the ErbB receptors is associated with the development and progression of several forms of cancer. In this thesis, new ErbB-targeting affibody molecules are evaluated regarding their cellular effects in vitro. Since ligand binding to an ErbB receptor might have an impact on the cell it is important to be aware of these effects as they may have consequences for the continued growth of the tumor when used in vivo. The affibody molecules are intended for tumor targeting with the prospect of clinical use in imaging or therapy. Three types of affibody molecules were studied, HER2-binding, EGFR-binding and bispecific binders that target both EGFR and HER2. The HER2-targeting (ZHER2:342)2 showed promising characteristics. It sensitized SKBR-3 cells to irradiation and decreased cell growth to the same extent as the clinically approved antibody Herceptin. The monomeric version, ZHER2:342, did not induce any large effects on intracellular signaling or biological outcome. This makes (ZHER2:342)2 interesting for therapy purposes, while ZHER2:342 may be better suited for imaging. The bispecific affibody molecules were all able to simultaneously bind to both EGFR and HER2, but none of the six constructs resulted in any large effects on cellular outcome. Interestingly, all three monovalent binders are more functional when positioned at the N-terminal part of the construct and the (S4G)3 linker renders higher affinity of the bispecific binders compared to (G4S)3. Tumors that co-express several ErbB receptors are often more aggressive and associated with a worse prognosis, suggesting that the total ErbB expression pattern might be more informative than the expression level of one receptor regarding cancer prognosis and prediction of response to targeted therapies. Bispecific ligands could thus be used as imaging agents with prognostic value. Another aspect of dual targeting is the possibility of increased tumor specificity since tumors are more likely than healthy tissue to express high amounts of two receptors.