| 1. |
- Thalén, Niklas, et al.
(författare)
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Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping
- 2024
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens.
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| 2. |
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| 3. |
- Aniander, Gustav
(författare)
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Improved candidate screening through tailored co-culture assays and precise tuning of protein expression
- 2024
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The field of biopharmaceuticals is a rapidly growing one. In the last ten years the number of approved biopharmaceuticals has more than doubled. A major hurdle to overcome for increased availability of all the new, effective biopharmaceuticals is the cost of treatment. Much of this can be attributed to the sheer time required for their development. Owing to this, interest in improvements to the biopharmaceuticals and their development process has also rapidly increased. As costs increase the further into development a drug candidate progresses, increasing the fidelity of screening at early stages could alleviate some of the exorbitant costs of development.In paper I, we showcase a novel way of targeting the tumor microenvironment (TME) to allow for TMElocalized CD40 activation. This is of interest as CD40 agonists have shown great potential for immune activation, but with systemic activation leading to severe adverse effects. The localized activation is achieved through the construction of an affinity fusion protein termed an AffiMab through fusion of a platelet derived growth factor receptor beta (PDGFRβ) targeting affibody to the heavy chain of a CD40 agonistic monoclonal antibody (mAb). We demonstrate PDGFRβ-dependent activation in a variety of assays, showing that the approach merits further investigation.Building on the activation assays set up in paper I, we aim to generate an in vitro screening platform for immune cell engagers in paper II. Screening candidates for on-target off-tumor activation is essential, as such activation would lead to adverse effects and be a doselimiting factor. To screen for this, we construct a series of plasmids which upon transfecting cells allow for different levels of a cell-surface target protein to be expressed, a so-called target density panel. This is achieved through the use of hairpin forming elements in the 5’ untranslated region of the mRNA dubbed regulatory elements (RgEs). Through use of different RgEs, we show that a target density panel can be generated and validate it in activation assays with the AffiMab developed in paper I. The platforms’ uniform cell surface background due to all different levels of target being expressed in the same host cell line and tunability through use of different RgEs are features that make it interesting for further research.Finally in paper III, we construct and test an improved translation initiation site (TIS) sequence. Using previous studies on the impact of the nucleotides in the sequence on the efficacy of the TIS, we constructed a novel sequence, TISNOV. This sequence enhanced titer and quality for recombinant production of IgG1 and IgG4 in both stable and transient settings. Further research into other TIS sequences and their uses in regulating protein expression, as well as usage of the TISNOV to improve expression of difficult to express proteins such as bispecifics remain interesting.In conclusion this thesis focuses on different manners to improve and hasten development of new biopharmaceuticals through usage of new workflows, platforms, and genetic engineering strategies.
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| 4. |
- Göstring, Lovisa, et al.
(författare)
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Cellular Effects of HER3-Specific Affibody Molecules
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:6, s. e40023-
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Tidskriftsartikel (refereegranskat)abstract
- Recent studies have led to the recognition of the epidermal growth factor receptor HER3 as a key player in cancer, and consequently this receptor has gained increased interest as a target for cancer therapy. We have previously generated several Affibody molecules with subnanomolar affinity for the HER3 receptor. Here, we investigate the effects of two of these HER3-specific Affibody molecules, Z05416 and Z05417, on different HER3-overexpressing cancer cell lines. Using flow cytometry and confocal microscopy, the Affibody molecules were shown to bind to HER3 on three different cell lines. Furthermore, the receptor binding of the natural ligand heregulin (HRG) was blocked by addition of Affibody molecules. In addition, both molecules suppressed HRG-induced HER3 and HER2 phosphorylation in MCF-7 cells, as well as HER3 phosphorylation in constantly HER2-activated SKBR-3 cells. Importantly, Western blot analysis also revealed that HRG-induced downstream signalling through the Ras-MAPK pathway as well as the PI3K-Akt pathway was blocked by the Affibody molecules. Finally, in an in vitro proliferation assay, the two Affibody molecules demonstrated complete inhibition of HRG-induced cancer cell growth. Taken together, our findings demonstrate that Z05416 and Z05417 exert an anti-proliferative effect on two breast cancer cell lines by inhibiting HRG-induced phosphorylation of HER3, suggesting that the Affibody molecules are promising candidates for future HER3-targeted cancer therapy.
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| 5. |
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| 6. |
- Karlander, Maximilian
(författare)
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Methods for engineering and characterization of advanced therapeutics
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein therapeutics are used worldwide to treat a multitude of diseases. Most therapeutic proteins on the market today are antibodies, and one of the characteristics that has made antibodies so useful for therapeutic use is their propensity to specifically and selectively bind other proteins with high affinity. While antibodies themselves can be very effective for treatment of different diseases, the field has also started to progress towards more advanced formats and entities. Monoclonal antibodies are generally monospecific. In many cases, however, binding to two or more therapeutically relevant proteins simultaneously can be beneficial. To overcome this limitation, different bispecific antibody formats have been engineered. The AffiMab, for example, is a bispecific antibody format, where a monoclonal antibody is fused to a much smaller affinity protein called an affibody molecule, allowing two independent affinity proteins to be combined in a modular fashion. To achieve this, the individual affinity proteins first needs to be developed, and this can be achieved using protein engineering methods, such as phage display. While phage display enables selection of affinity proteins binding new target proteins, simply binding may not be enough, and methods for discovering more diverse affinity proteins and characterizing their binding are therefore essential.Bispecific antibodies constitute only one example where advanced therapeutic entities have emerged. Another example of advanced therapeutics are gene therapies, where adeno-associated viruses (AAV) have transpired as one of the most successful platforms. AAV-based gene therapy has, in several recent approvals, proved its ability to be used to cure inherited genetic disorders, such as spinal muscle atrophy and haemophilia, with a single treatment. The lack of precise targeting of AAV vectors, however, has prompted researchers to develop engineered AAVs with improved targeting. While these engineered variants are yet to reach the clinic, they hold promise of gene therapies with less side-effects and higher effective doses in the desired tissues. Compared to protein therapeutics, AAVs add new dimensions to characterization of quality of the produced therapeutic. As AAVs are a combination of proteins and DNA, both these components need to be verified, and as such, methods used for characterization of proteins only cover a part of it. Analysis and characterization of the DNA inside the AAV capsids is crucial, both for drug discovery, to ensure the correct gene is being delivered, and for understanding the observed effects of treatment. Standardized methods for sequencing and analysis of recombinant AAV genomes are yet to be established but this, combined with novel AAV variants, could help leverage a new generation of AAV-based therapies. All of these topics are explored in this thesis. In Study I, AffiMabs were developed for treatment of HER2 resistant gastric carcinoma, with improved cytotoxic effect observed in vitro compared to the parental proteins trastuzumab and ZEGFR. In Study II, a method for rapid, residue level, eptiope mapping was developed, and the eptiopes of three new antibodies targeting the SARS-CoV-2 spike protein were determined. In Study III, phage display was combined with deep sequencing to enable discovery of affibody molecules with different binding kinetics and epitopes. In Study IV, affibody molecules were fused to AAV capsids to achieve receptor specific targeting. The capsids were shown to be both functional, superior to the parental serotype for receptor-specific transduction, and modular, as different affibody molecules could be incorporated. Finally, in Study V, a method for deep sequencing and analysis of AAV genomes was established, allowing identification of a wide array of different DNA encapsidated in AAVs.In summary, the studies presented in this thesis explores a broad selection of new and improved methods for both engineering and characterization of advanced therapeutics.
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| 7. |
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| 8. |
- Kronqvist, Nina, et al.
(författare)
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Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules
- 2011
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press (OUP). - 1741-0126 .- 1741-0134. ; 24:4, s. 385-396
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Tidskriftsartikel (refereegranskat)abstract
- Emerging evidence suggests that the catalytically inactive ErbB3 (HER3) protein plays a fundamental role in normal tyrosine kinase receptor signaling as well as in aberrant functioning of these signaling pathways, resulting in several forms of human cancers. ErbB3 has recently also been implicated in resistance to ErbB2-targeting therapies. Here we report the generation of high-affinity ErbB3-specific Affibody molecules intended for future molecular imaging and biotherapeutic applications. Using a high-complexity phage-displayed Affibody library, a number of ErbB3 binders were isolated and specific cell-binding activity was demonstrated in immunofluorescence microscopic studies. Subsequently, a second-generation library was constructed based on sequences of the candidates from the phage display selection. By exploiting the sensitive affinity discrimination capacity of a novel bacterial surface display technology, the affinity of candidate Affibody molecules was further increased down to subnanomolar affinity. In summary, the demonstrated specific targeting of native ErbB3 receptor on human cancer cell lines as well as competition with the heregulin/ErbB3 interaction indicates that these novel biological agents may become useful tools for diagnostic and therapeutic targeting of ErbB3-expressing cancers. Our studies also highlight the powerful approach of combining the advantages of different display technologies for generation of functional high-affinity protein-based binders. Potential future applications, such as radionuclide-based diagnosis and treatment of human cancers are discussed.
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| 9. |
- Leitao, Charles Dahlsson, 1992-, et al.
(författare)
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Display of a naïve affibody library on staphylococci for selection of binders by means of flow cytometry sorting
- 2023
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 655, s. 75-81
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Tidskriftsartikel (refereegranskat)abstract
- Within the field of combinatorial protein engineering there is a great demand for robust high-throughput selection platforms that allow for unbiased protein library display, affinity-based screening, and amplification of selected clones. We have previously described the development of a staphylococcal display system used for displaying both alternative-scaffolds and antibody-derived pro-teins. In this study, the objective was to generate an improved expression vector for displaying and screening a high-complexity naive affibody library, and to facilitate downstream validation of isolated clones. A high-affinity normalization tag, consisting of two ABD-moieties, was introduced to simplify off-rate screening procedures. In addition, the vector was furnished with a TEV protease substrate recog-nition sequence upstream of the protein library which enables proteolytic processing of the displayed construct for improved binding signal. In the library design, 13 of the 58 surface-exposed amino acid positions were selected for full randomization (except proline and cysteine) using trinucleotide tech-nology. The genetic library was successfully transformed to Staphylococcus carnosus cells, generating a protein library exceeding 109 members. De novo selections against three target proteins (CD14, MAPK9 and the affibody ZEGFR:2377) were successfully performed using magnetic bead-based capture followed by flow-cytometric sorting, yielding affibody molecules binding their respective target with nanomolar affinity. Taken together, the results demonstrate the feasibility of the staphylococcal display system and the proposed selection procedure to generate new affibody molecules with high affinity.
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| 10. |
- Malm, Magdalena, 1983-, et al.
(författare)
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Evolution from adherent to suspension: systems biology of HEK293 cell line development
- 2020
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 10:1
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Tidskriftsartikel (refereegranskat)abstract
- The need for new safe and efficacious therapies has led to an increased focus on biologics produced in mammalian cells. The human cell line HEK293 has bio-synthetic potential for human-like production attributes and is currently used for manufacturing of several therapeutic proteins and viral vectors. Despite the increased popularity of this strain we still have limited knowledge on the genetic composition of its derivatives. Here we present a genomic, transcriptomic and metabolic gene analysis of six of the most widely used HEK293 cell lines. Changes in gene copy and expression between industrial progeny cell lines and the original HEK293 were associated with cellular component organization, cell motility and cell adhesion. Changes in gene expression between adherent and suspension derivatives highlighted switching in cholesterol biosynthesis and expression of five key genes (RARG, ID1, ZIC1, LOX and DHRS3), a pattern validated in 63 human adherent or suspension cell lines of other origin.
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