| 1. |
- Ding, Haozhong, 1990-
(författare)
-
Targeting HER2-expressing tumors with potent drug conjugates and fusion toxins based on scaffold proteins
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Targeted therapy is an emerging treatment for a variety of cancers. Small- sized scaffold proteins are an alternative to conventional antibody-based targeting molecules. Two small scaffold proteins—the 58-amino-acid protein class, the affibody molecules, and the 46-amino-acid protein class, the Albumin binding domain Derived Affinity Proteins (ADAPTs)—have previously been engineered to bind to a large variety of tumor-associated molecular targets with a high affinity. The human epidermal growth factor receptor 2 (HER2) is a membrane-bound receptor for growth signal transmission. Expression of a high level of HER2 can cause cells to proliferate and may ultimately lead to cancer. It has earlier been shown that HER2 is involved in several different types of cancers, e.g., breast, ovarian, bladder, and gastric cancers. HER2-targeted affibody and ADAPT molecules have previously been developed, such as ZHER2:2891 and ADAPT6 with strong affinity to HER2 with equilibrium dissociation constants of 76 pM and 2.5 nM, respectively. Their small size and high specificity have rendered these two scaffold proteins promising candidates for imaging of HER2-positive breast cancer tumors in clinical trials. Delivery of cytotoxic agents to cancer cells, using a cell-targeting domain, may potentially precisely kill the cancer cells while having very low cytotoxic effects on normal cells. Many cancer-targeted antibody drug conjugates (ADCs) and toxic proteins (immunotoxins) have advanced the field of cancer treatment. Small-sized scaffold proteins hold promise as alternative targeting domains to build novel drug conjugates or fusion toxins for cancer treatment. In this thesis, I first investigated an affibody-based drug conjugate (AffiDC) composed of an anti-HER2 affibody and an anti-mitotic maytansine-derived drug (DM1) for treatment of HER2-overexpressing cells. I studied a variety of targeting domain formats for efficacy optimization. All ZHER2:2891-based AffiDCs showed specific anti-tumor activity on HER2-overexpressing cancer cells in vitro as well as in mouse tumor xenografts. The hepatic uptake of the AffiDCs could be reduced by shielding the hydrophobic DM1 using a poly-glutamic-acid spacer, which might help to reduce potential liver toxicity allowing for administration of higher doses. In addition, tuning the valency of the affibody-targeting domain (ZHER2) from a divalent domain to a monovalent domain showed increased potency and reduced liver uptake. We also investigated the influence of the number of drug payloads on the pharmacokinetic profile of the AffiDCs. An AffiDC bearing three DM1s showed higher delivery of DM1 to the cancer cells in vivo, but fast blood clearance and an elevated liver retention was also observed. With regards to fusion toxin design, we constructed a variety of recombinant toxins. The targeting domains were ZHER2:2891 and/or ADAPT6, which were genetically fused with truncated versions of the highly cytotoxic Pseudomonas Exotoxin A (PE). All fusion toxins we studied showed potent HER2-specific anti-tumor activity. The results suggested that both ZHER2:2891 and ADAPT6 could direct the PE-based cytotoxins specifically to HER2- overexpressing cancer cells. In this work, we have demonstrated the potential of using ZHER2:2891 and ADAPT6 as targeting domains to carry the small molecule drug DM1, or cytotoxic PE-derived peptides to cancer cells. It can be concluded that careful molecular design of the targeting domain may considerably improve the potency and minimize the off-target uptake.
|
|
| 2. |
- Güler, Rezan
(författare)
-
Affibody Molecules Targeting VEGFR2 - Two Turns Off and Four Turns On
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The notion of employing proteins as drugs traces back several decades. As recombinant DNA technology emerged, it became a powerful tool for the tailoring of protein traits via genetic approaches - so-called protein engineering. The application of such tools to develop proteins that bind specific molecular targets has seen remarkable clinical success, and today seven out of the ten top-selling drugs in the world belong to this class of proteins. A well-investigated protein scaffold for generating novel target-binding moieties is the small staphylococcal protein A-derived affibody molecule. This thesis revolves around the engineering of affibody-based binding proteins that aim to influence the signaling network in the biological process of blood vessel formation, so-called angiogenesis. The first study in this thesis describes the engineering of a heterodimeric affibody molecule that targets the principal regulating receptor of angiogenesis, vascular endothelial growth factor receptor-2 (VEGFR2). Two separate affibody molecules that bind adjacent VEGFR2 epitopes were previously fused into one biparatopic construct, leading to a remarkable increase in apparent affinity. Further, the biparatopic protein here demonstrated inhibition of vascular endothelial growth factor A (VEGF-A) binding to VEGFR2, and consequently inhibition of VEGFR2 phosphorylation, proliferation and in vitro sprouting of endothelial cells. In the second study, the aim was to evaluate the biparatopic protein as a molecular imaging probe for in vivo visualization of VEGFR2 expression in a glioblastoma multiforme (GBM) brain tumor-model. The results displayed significantly higher probe uptake in tumor compared to normal brain tissue, with a two-hour post injection tumor-to-brain ratio of 78. In the third study, the goal was to mimic the ability of the natural ligand to agonize VEGFR2 via receptor dimerization, and also simulate presentation as extracellular matrix (ECM) bound factors. To this end, the dimeric antagonist was reformatted into a tetrameric construct, hypothesized to bridge two receptor units, and fused to recombinant spider silk. Interestingly, whereas the tetramer displayed agonistic effects both in soluble and immobilized form, the activity of the dimer shifted from antagonistic to agonistic when immobilized. In the fourth study a combined in silico and directed evolution approach was used to increase the thermal stability and hydrophilicity of the biparatopic protein. The final construct demonstrated an increase in melting temperature of about 15°C, complete refolding after heat-induced denaturation and decreased uptake in normal tissues when evaluated in mouse biodistribution studies. In conclusion, this thesis covers the development, characterization and engineering of VEGFR2-binding affibody molecules, aimed for use in research, therapy and diagnosis.
|
|
| 3. |
- Hjelm, Linnea C., 1993-
(författare)
-
Development of new affinity proteins for neurodegenerative disorders
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Neurodegenerative disorders include a full spectrum of diagnoses, including dementias and other neuronal diseases, characterised by degradation of neurons in the brain occurring along with disease progression. Amongst the dementias, the most prevalent are Alzheimer’s (AD) and Parkinson’s disease (PD) that affect millions of people worldwide. During the last years, advancements in potential treatments have been made where the first two clinical antibodies have been approved by the US Food and Drug Administration (FDA) for a disease modifying effect on Alzheimer’s disease.As alternatives to antibodies, other types of affinity reagents that are based on non-immunoglobulin protein scaffolds are also investigated. Such alternative scaffolds often demonstrate distinct and complementary properties compared to antibodies. In this thesis, the development of a new type of affinity protein scaffold called sequestrin is described. Sequestrins are derived from the affibody molecule and comprise two heterogenic subunits with truncated N-terminals fused as a head-to-tail construct. Sequestrins undergo a structural rearrangement upon target binding and forms a stabile complex. The scaffold is designed for interactions with disease-related amyloidogenic peptides e.g. amyloid beta and alpha-synuclein involved in AD and PD, respectively. In the first paper, a sequestrin library was developed and its compatibility with phage display was investigated. Successful panning against the amyloid beta peptide resulted in binders with high affinity. Further on in paper II, the alpha-synuclein peptide was targeted and sequestrins with low nanomolar affinities were obtained. All sequestrins displayed structural rearrangement upon target engagement, which stabilized the interaction to the target peptides and further inhibited toxic aggregation, opening up for future studies of disease modifying effects in vivo.When targeting the brain, passage through the blood–brain barrier (BBB) is an obstacle that needs to be addressed to reach sufficiently high therapeutic concentrations. To overcome this barrier, brain shuttles have been developed with the capability to transport a cargo over the BBB. One such mechanism of transportation is by receptor-mediated transcytosis, which is utilized by e.g. the transferrin receptor (TfR). In paper III, a TfR-targeting shuttle was investigated for BBB passage when fused to a sequestrin targeting the amyloid beta peptide, resulting in a higher penetration through the BBB, and maintained functionality of the sequestrin.High-throughput in vitro methods would facilitate development of novel brain shuttles. Thus, in paper IV, a transwell system based on nanofibrillar silkmembranes with murine brain endothelial cells was developed. Evaluation of the method using a TfR-specific antibody demonstrated higher transfer over the barrier compared to an isotype control and the method has potential to facilitate screening of transcytosis capability of brain shuttles.In paper V, TfR-specific affibody-based brain shuttles were developed and investigated for transcytosis capability using the in vitro transcytosis assay. A panel of affibody molecules were evaluated, demonstrating both cross-species reactivity to murine and human TfR and active receptor-mediated transcytosis. These candidates could thus potentially be used in further development of CNS-targeting therapeutics.In conclusion, a new sequestrin scaffold was developed that can be utilised for targeting amyloidogenic peptides found in neurodegenerative disorders. An affibody-based brain shuttle was also developed, which showed transcytosis capability. In the future, the new brain shuttle might be combined with sequestrins to create multifunctional fusion proteins for facilitated delivery over the BBB, which hopefully can result in therapeutic concentrations in the brain even when administered with a lower dosage.
|
|
| 4. |
- Parks, Luke
(författare)
-
Development of directed-evolution methods utilizing combinatorial protein libraries in Escherichia coli
- 2024
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Directed evolution using combinatorial protein libraries is a tremendously powerful technique for the generation of proteins with new or improved properties. A key aspect in such techniques is the link between individual protein variants and their corresponding genetic information. To provide this link, the most successful combinatorial protein engineering methods employ microorganisms, such as bacteriophages, bacteria or yeast for the production and display of libraries. This thesis focuses on the development and application of new directed evolution methods utilizing the bacterium Escherichia coli (E. coli), for the engineering of affinity proteins and proteases.The first study aimed to engineer the substrate specificity of a protease from tobacco etch virus (TEV). For this purpose, a novel method was devised based on expression of intracellular protease libraries, and employed a reporter fusion protein consisting of amyloid β peptide fused to the N-terminus of enhanced green fluorescent protein (EGFP). Variants were screened for proteolytic activity on co-expressed target substrate by means of fluorescence-activated cell sorting (FACS). After three rounds of FACS, a set of TEV protease variants were enriched that exhibited improved proteolytic activity on the novel substrate.Studies two to four describe the development of an E. coli surface expression system that was explored for directed evolution applications. The method is based on display of recombinant proteins on the outer membrane via fusion to a bacterial autotransporter, adhesin involved in diffuse adherence I (AIDA-I). The second study focused on the optimization of the surface display system and its application to directed evolution. In this effort, several affinity protein classes were evaluated for surface display via AIDA-I in a panel of E. coli strains. Results showed that smaller and less complicated affibody molecules were displayed at high levels, while more complex proteins, such as antibody fragments, varied in their performance and functioned best in certain engineered strains. A mock affibody library was used to develop a high-throughput magnetic-assisted cell sorting (MACS) protocol for enrichment of binders from very large libraries.In the third and fourth study, the new E. coli display method combined with the MACS protocol was evaluated for generation of new affibody molecules.In the third study, a large naïve affibody library (>1.5×1011 members) was constructed, displayed on E. coli and characterized. The performance of the method and library was evaluated by selection of binders against two cancer-associated targets, tumor-associated calcium signal transducer 2 (TROP-2) and lymphocyte-activation gene 3 (LAG-3). MACS and FACS were performed, with flow cytometry assessment between rounds to monitor enrichment. Both selections produced high affinity binders to their respective targets.In the fourth study, a maturation library was constructed for improving the properties of an affibody molecule toward the renal cell carcinoma biomarker carbonic anhydrase IX (CAIX). Selections included stringent off-rate procedures and yielded variants with improved affinities and folding stability compared to previously reported binders.In summary, the work in this thesis demonstrate the potential of E. coli-based directed evolution methods for selection of new proteins with altered or improved properties.
|
|
| 5. |
- Dahlsson Leitao, Charles
(författare)
-
Affibody-mediated targeting of HER-family receptors for cancer imaging and therapy
- 2022
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are remarkable molecules with diverse and specialized functions playing essential roles in most biological processes. One such function is protecting us from diseases by the action of antibodies in our immune system that can recognize and mediate the destruction of invading pathogens by binding to foreign epitopes found on non-self proteins. The concept of utilizing specific protein-protein interactions to achieve a therapeutic effect has for several decades been a cornerstone for the development of cancer-directed treatments. While antibodies have formed a basis for the development of such drugs, other protein alternatives may be engineered to complement current antibody-based treatments, and may even prove to possess superior features. This thesis focuses on the engineering of affibody molecules, a small alternative scaffold protein, for design and development of novel cancer-targeting therapeutic and diagnostic drugs. There are many different strategies that have been investigated for inhibiting cancer progression and tumour growth with perhaps one of the most straightforward involving disruption of dysregulated growth-promoting signalling pathways. Members of the human epidermal growth factor receptor (HER) family is prominently expressed in various cancer types and have been shown to be intricately involved in tumorigenesis. One of the members (HER3) often becomes upregulated in cancer and have been shown to mediate acquired resistance to targeted therapies by the mechanism of ligand-induced activation. We have designed five novel affibody-based HER3-targeting molecules able to prevent ligand-binding and consequently activation of HER3. We investigated the targeting properties and biodistribution profiles of these molecules in vivo and subsequently evaluated the anti-tumour efficacy for the most promising variants in direct comparison to a HER3-targeting antibody with a similar inhibitory mechanism. We observed a large influence of design on both the biodistribution properties and the in vivo efficacy of different affibody molecules. Moreover, we demonstrated that two of the affibody-formats were equally effective as the antibody in inhibiting tumour growth and prolonging survival of mice bearing HER3-positive xenografts. The effectiveness of cancer treatments depends on efficient diagnostic approaches that can reliably stratify patients based on these targetable biomarkers, which is possible using radionuclide molecular imaging. We have performed a direct comparison of the diagnostic potential for visualizing HER3-expressing tumours of affibody- and antibody-based imaging probes. We concluded that affibody molecules provide superior imaging quality with higher diagnostic potential and enable early visualization of HER3-expression in tumours. Another member of the HER family that is of interest for cancer therapy is HER1 (or EGFR) but due to substantial expression in healthy tissues, targeted therapies may lead to severe side-effects. One possible solution to this is taking advantage of the distinct milieu of the tumour microenvironment to design EGFR-targeting drugs that become conditionally activated at the tumour site, but not in normal tissues, with the aim of drastically reducing systemic toxicity. We have generated an affibody molecule with anti-idiotypic binding specificity for a previously generated EGFR-binding affibody molecule, which we used to construct an affibody-based prodrug. We were able to show that, in a proof-of-concept format, this anti-idiotypic masking domain was able to block the binding to EGFR until removed by protease-mediated cleavage. We subsequently developed and characterized a more refined version of this prodrug, which we call a pro-affibody, and could show that activation by cancer-associated proteases confers binding to EGFR-expressing cancer cells and enables conditional cytotoxic payload delivery in vitro. The pro-affibody was further evaluated in vivo using tumour-bearing mice to investigate the feasibility for masked uptake in healthy tissues while retaining binding-activity in tumours. We observed a substantial reduction in EGFR-specific liver uptake compared to a control construct without a masking domain, and a strong indication of protease-mediated EGFR-binding in tumours. In conclusion, the experimental work presented in this thesis provides a rationale for designing effective affibody-based cancer therapeutics and diagnostics with different targeting strategies and demonstrates the potential of such drugs from preclinical in vivo data.
|
|
| 6. |
|
|
| 7. |
- Liu, Hao
(författare)
-
Tumor targeted delivery of cytotoxic payloads using affibody molecules and ABD-derived affinity proteins
- 2018
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer treatment cost billions of dollars every year, but the mortality rate is still high. An ideal treatment is the so-called “magic bullets” that recognize and kill tumor cells while leaving normal cells untouched. In recent years, some nonimmunoglobulin alternative scaffold affinity proteins, such as affibody molecules and ADAPTs, have emerged and been used to specifically recognize different tumor antigens. In this thesis, I studied the properties and anti-tumor activities of affibody and ADAPT fusion toxins and affibody drug conjugates. In the first two papers, I studied a panel of recombinant affitoxins (affibody toxin fusion proteins) consisting of an anti-HER2 affibody molecule (ZHER2), an albumin binding domain (ABD) and a truncated version of Pseudomonas Exotoxin A(PE38X8). The affitoxins demonstrated specific anti-tumor activity on HER2-overexpressing tumor cells in vitro. A biodistribution experiment showed that addition of an ABD increased the blood retention by 28-fold and a (HE)3 N-terminal purification tag decreased hepatic uptake of the affitoxin compared with a His6 tag. In paper III, I studied immunotoxins consisting of an anti-HER2 ABD-derived affinity protein (ADAPT), an ABD and a minimized and deimmunized version of Pseudomonas exotoxin A (PE25). These immunotoxins demonstrated potent and specific cytotoxicity toward HER2 overexpressing tumor cells in vitro similar to affitoxins. In paper IV, I produced a panel of affibody drug conjugates consisting of ZHER2, ABD and malemidocaproylmertansine (mc-DM1). The conjugates had selective toxic activity on HER2-overexpressing tumor cells in vitro comparable with the approved drug trastuzumab emtansine. The conjugate, ZHER2-ZHER2-ABD-mc-DM1 was found to prolong the life span of tumor bearing mice and delayed the growth ofxenografted SKOV-3 tumors. In conclusion, affibody molecules and ADAPTs are promising alternatives to antibodies for targeted tumor therapy.
|
|
| 8. |
- Malm, Magdalena, 1983-
(författare)
-
Generation and characterization of Affibody molecules targeting HER3
- 2013
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the field of oncology, the ability to target specific tumor cells using highly selective targeting molecules is an attractive and emerging concept. In this context, the epidermal growth factor receptor HER3 has proven central to the biology behind many different human cancers and inhibition of the signaling mediated by this receptor could provide antitumoral effects. Consequently, this receptor has emerged as a suitable target for imaging, functional blocking or delivery of toxic payloads. A promising targeting-molecule for such applications is the small non-immunoglobulin derived Affibody molecule. The work upon which this thesis is based, revolves around HER3 with the aim to generate and characterize Affibody molecules targeting this receptor. In the first study, HER3-specific Affibody molecules were generated by combinatorial protein engineering using a combined approach where first generation binders were isolated from a phage-displayed naive library, followed by affinity maturation of these binders using a focused staphylococcal surface-displayed library and flow-cytometric cell sorting. This engineering strategy enabled the successful isolation of HER3-specific Affibody molecules with subnanomolar affinities for the receptor and the ability to compete with the natural ligand heregulin (HRG) for binding to HER3. In the second study, the cellular effects of these Affibody molecules were characterization in vitro. The results demonstrated that the ability to inhibit HRG-binding to the receptor translated into inhibition of ligand-induced phosphorylation of HER3, HER2 as well as the downstream signaling molecules Akt and Erk. As a result, the HER3-specific Affibody molecules also inhibited HRG-induced cell growth of two different breast cancer cell lines in vitro. These promising results, suggested that the HER3-targeting Affibody molecules could have a therapeutic effect in tumors that are dependent on ligand-induced signaling of HER3. However, due to the relatively low expression level of HER3 on tumor cells, we explored two different engineering approaches of the HER3-specific Affibody molecules in order to potentially improve its tumor targeting ability. One approach was to construct bispecific Affibody molecules where a HER3- and a HER2-specific Affibody molecule were fused on each side of an albumin-binding domain (ABD). In the third study, one such bispecific construct was shown to have increased ability to inhibit ligand-induced phosphorylation of HER2 and retained ability to inhibit HRG-induced activation of HER3, as compared to the monomeric anti-HER3 Affibody. Another strategy was to further increase the affinity of the HER3-specific Affibody molecules towards the receptor through a semi-rational affinity maturation approach. In the fourth study, a staphylococcal displayed affinity maturation library was screened by FACS using an off-rate selection procedure. This approach resulted in the successful isolation of picomolar HER3-binders with improved potency of inhibiting HRG-induced cell growth as compared to a first generation binder. Moreover, in the fifth study, in vivo characterization of these HER3-specific Affibody molecules was performed in both normal and xenograft mice. The results suggested specific targeting of HER3 in vivo and provided the first evidence of successful tumor imaging using a HER3-specific Affibody. Taken together, the work included in this thesis describes (to our knowledge) the first non-immunoglobulin derived affinity protein targeting HER3, with promising features for both therapeutic and imaging applications.
|
|
| 9. |
- Meister, Sebastian W., 1988-
(författare)
-
Amyloid Beta – Biotherapy Target and Biotechnological Tool
- 2020
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Alzheimer’s disease is a progressive neurodegenerative disease characterized primarily by memory impairment and cognitive decline. As of 2020, an estimated 50 million people suffer from Alzheimer’s disease or related dementia and no disease-modifying treatment options are yet approved for clinical practice. A major pathological feature of Alzheimer’s disease is the presence of cerebral senile plaques with aggregated amyloid beta (Aβ) peptides as the main constituent. In this thesis, Aβ is used in five separate studies either as a target for the development of potential biotherapeutical interventions or as a tool in biotechnological research.In the first study, a high-throughput screening method was developed that enables functiondriven selection of protein-based aggregation inhibitors from combinatorial libraries. The method employs a reporter protein consisting of Aβ42 fused to the N-terminus of green fluorescent protein (GFP). The reporter protein misfolds due to the aggregating nature of Aβ42. Following protein expression in Escherichia coli a low whole-cell GFP fluorescence signal was detected using flow cytometry. However, when co-expressed with an affibodybased aggregation inhibitor, the reporter protein was rescued from aggregation and an increased whole-cell GFP fluorescence signal was detected in flow cytometry. By combining the screening method with flow cytometric cell sorting, the aggregation-inhibiting affibody molecule could successfully be enriched from a large background of non-inhibiting affibody molecules. The results thus demonstrated that the developed method enables highthroughput screening and sorting of combinatorial protein libraries based on the Aβ aggregation inhibiting ability.The second study explored a strategy to increase the uptake of a biotherapeutical candidate protein into the central nervous system (CNS) via receptor-mediated transcytosis across the blood-brain barrier (BBB). The affibody-based candidate ZSYM73 binds monomeric Aβ and inhibits Aβ aggregation. Here, ZSYM73 was fused to the C-terminus of a single-chain variable fragment (scFv8D3) binding the transferrin receptor (TfR); a receptor expressed on the BBB. An engineered albumin-binding domain (ABD) was fused to ZSYM73 to extend the circulatory half-life of the fusion protein. In a mouse study, the tri-specific fusion protein scFv8D3-ZSYM73-ABD exhibited increased cerebrospinal fluid (CSF) bioavailability compared to the control protein ZSYM73-ABD, indicating an active transport mechanism into the CNS.In the third study, a novel method for combinatorial protease engineering was developed and applied to generate highly proteolytic active variants of the coxsackievirus 3C protease. The method is based on the findings form the first study and employs a reporter protein consisting of Aβ42 fused to the N-terminus of GFP via a peptide linker containing a protease substrate sequence. The reporter protein misfolds upon expression in E. coli, which resulted in a low whole-cell GFP fluorescence signal detected in flow cytometry. Co-expression of a protease with activity on the substrate sequence led to proteolytic separation of the aggregation-prone Aβ42 peptide from GFP and restored whole-cell fluorescence. This method was used in combination with flow cytometric cell sorting to isolate highly proteolytic active variants from a randomly mutated 3C protease library. The aim of the fourth study was to evaluate the potential of the newly developed method from the third study to engineer the substrate specificity of proteases. A semi-rational tobacco etch virus (TEV) protease library was screened for variants with proteolytic activity on a novel target substrate. The target substrate differed substantially from the wild-type TEV consensus substrate and exhibited high sequence similarity to the aggregation-inducing hydrophobic core region of Aβ. After three rounds of flow cytometric cell sorting, a set of TEV protease variants was enriched that exhibited proteolytic activity on the novel substrate.In the fifth study, a methodology employing flow cytometric sorting of multiprotein aggregates was developed to investigate the protein interactome related to Aβ plaques. It was demonstrated that in human serum or human CSF, Aβ aggregates bound to a fluorescent probe can be detected and isolated using flow cytometry. Quantitative mass spectrometry analysis was performed on Aβ aggregates isolated from human CSF. The abundances and functional features of proteins found in the isolated aggregates were investigated, and a hypothetical model of the layered architecture of Aβ aggregates was proposed. In conclusion, this thesis describes the development of new concepts and methods that will hopefully contribute to increasing the understanding and improving the therapy of Alzheimer’s disease and other diseases associated with protein aggregation.
|
|
| 10. |
- Sandersjöö, Lisa, 1984-
(författare)
-
Bacteria-based methods for engineering and characterization of proteases and affinity proteins
- 2015
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is focused on the development of methods for characterization and engineering of both proteases and affinity proteins. In addition, a prodrug concept for small affinity proteins is developed.Two of the developed methods are for engineering and/or characterization of proteases. First, a method for substrate profiling and engineering of proteases was investigated (paper I). In this method, a protease and a reporter are co-expressed in E. coli. The reporter is comprised of an enzyme, which confers resistance to an antibiotic, fused to a substrate, and a degradation tag. In absence of site-specific proteolysis within the reporter, the degradation tag renders the entire reporter a substrate for the intracellular degradation machinery. Thus, by applying competitive growth in presence of the antibiotic, a substrate that is preferred by a model protease could be enriched relative to less efficiently hydrolyzed substrates. Then, an alternative method for substrate profiling was developed (paper III). Here, the substrate is instead displayed on the surface of bacteria, and located between two anti-idiotypic domains, where one blocks the other from interacting with a reporter. Site-specific proteolysis releases the blocking domain and is therefore reflected in reporter binding. After incubation with fluorescently labeled reporter, the proteolysis can be analyzed by flow cytometry. When large libraries of potential substrates for matrix metalloprotease 1 (MMP-1) were screened, a panel of substrates with the previously reported motif PXXXHy was enriched, thereby demonstrating the potential of the method. This method offers the possibility for high-throughput substrate profiling of proteases as well as engineering of substrates for use in for example protease-activated prodrugs.In another study, a new prodrug concept for small affinity proteins was developed to improve the tissue selectivity in future in vivo studies (paper II). This concept takes advantage of the local upregulation of proteases in the diseased tissue in various disorders. By fusing a targeting domain to an anti-idiotypic binding partner via a protease-sensitive linker, the targeting domain is masked from interacting with its target until activation by site-specific proteolysis within the linker. The concept was demonstrated for a small affinity protein (Affibody molecule). Bacterial display was employed to engineer the so-called pro-Affibody. When displayed on the bacterial surface, the pro-Affibody showed over 1.000-fold increase in apparent binding affinity upon activation by a disease-associated protease. Additionally, the activated pro-Affibody could bind to its target expressed on cancer cells, as opposed to the non-activated pro-Affibody. This concept is likely to be extendable to other small affinity proteins and opens up for the possibility to develop new such prodrugs to previously non-druggable targets.In the last study, a screening method for protein-based aggregation inhibitors was developed (paper IV). In this method, a reporter and an inhibitor are co-expressed in E. coli. The reporter is comprised of green fluorescent protein (GFP) fused to an aggregation prone peptide. Upon aggregation, the fluorescence is decreased, but it is then restored when the reporter is co-expressed with an inhibitor. In a model screening experiment, an Affibody molecule that targets the Aβ peptide (involved in Alzheimer’s disease) could be enriched from a background of non-inhibiting Affibody molecules. Also this method is likely to be extendable to other types of affinity proteins, and also to different aggregation prone peptides/proteins involved in other diseases.In conclusion, the methods and concepts presented in this thesis could in the future yield new means for the engineering and characterization of proteins with desired properties to be used in both biotechnological and medical applications.
|
|
