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- von Witting, Emma
(författare)
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The ADAPT scaffold as a tool for diagnostic imaging and targeted therapy
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Molecular recognition, or the specific interactions between a protein and its ligand, is central to biology and a key factor for many different clinical and technical applications. Despite antibodies being only one of many different affinity proteins, it has by far been the most successful. However, their large size and complex structure can be limiting in terms of cost and stability. Furthermore, their effector functions can sometimes be undesired or even detrimental. Over the past decades, many alternative affinity proteins have emerged to overcome some of these limitations.The Albumin Binding Domain (ABD), originally present on the surface of certain bacterial cells, has previously been subjected to combinatorial protein engineering for the generation of ADAPTs (ABD Derived Affinity ProTeins) that bind to different targets. One of these, the ADAPT6, targets HER2 and has shown great promise as a tracer for radionuclide molecular imaging for diagnosis and stratification of HER2 positive patients. The work in this thesis has aimed to optimise the ADAPT6 tracer further and also describes the first-inhuman clinical trial for imaging of HER2-overexpressing breast cancer. The results establish that ADAPT6 is safe and well-tolerated by patients and able to detect primary tumours as well as metastases with very high contrast already 2 hours after injection. However, the high kidney uptake associated with its fast blood clearance prevents further use of ADAPT6 also in a therapeutic setting. By engineering the ADAPT6 to prolong its circulatory half-life and reduce the kidney uptake, this thesis has also aimed to explore the therapeutic potential of this molecule. As a first step towards this goal, the ADAPT6 was genetically fused to an ABD to allow for binding to a patient’s own serum albumin and hence avoid the same extent of renal filtration. Indeed, when evaluated in mice, fusion to ABD increased the retention in circulation by more than 200-fold and exhibited a dramatically decreased renal activity. Treatment of tumour-bearing mice with the ABD-fused ADAPT6 conjugated to a cytotoxic radionuclide significantly prolonged survival by more than two-fold and was not associated with any observable toxicity. Finally, this thesis also describes a novel combinatorial library from which several bispecific ADAPTs have been identified, binding to both albumin and other clinically relevant targets simultaneously. This miniature bispecific scaffold offers an opportunity to combine the benefits associated with small size such as good tissue extravasation and alternative administration routes while still maintaining a sufficient in vivo half-life.
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| 2. |
- Kanje, Sara, 1986-, et al.
(författare)
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Engineering of Protein A for improved purification of antibodies and Fc-fused proteins
- 2020
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Ingår i: Approaches to the Purification, Analysis and Characterization of Antibody-Based Therapeutics. - : Elsevier. ; , s. 35-54
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- In industrial scale downstream processing of antibodies and Fc-fusion proteins, Protein A chromatography is the most commonly used purification method. As the treatments for many severe diseases shift toward biological drugs, in particular antibodies, the need for robust purification methods has increased. Since the 1970s, when Protein A debuted as an affinity ligand, the full protein and its domains have been extensively studied. Even if matrices based on unmodified Protein A historically met the process demands of industrial antibody manufacturing, there is room for improvement. The cleaning in place (CIP) methods required for safe reuse of the matrix in therapeutic protein purification pose a problem since the harsh conditions required can be harmful to the proteinaceous purification ligand. Further, the low pH used for elution can sometimes be detrimental to the target protein. Here, efforts to improve these features as well as capacity improvements for Protein A resins will be discussed.
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