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| 2. |
- 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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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
- 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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| 7. |
- Malm, Magdalena, 1983-
(författare)
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Generation and characterization of Affibody molecules targeting HER3
- 2013
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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.
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| 8. |
- Malm, Magdalena, 1983-, et al.
(författare)
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Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify bottlenecks limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant overexpression. Surprisingly, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. While most components of the secretory machinery showed comparable expression levels in both expression hosts, genes with significant expression variation were identified. Among these, ATF4, SRP9, JUN, PDIA3 and HSPA8 were validated as productivity boosters in CHO. Further, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components improving recombinant protein yield in HEK293 and CHO.
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| 9. |
- Malm, Magdalena, 1983-, et al.
(författare)
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Harnessing secretory pathway differences between HEK293 and CHO to rescue production of difficult to express proteins
- 2022
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Ingår i: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 72, s. 171-187
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Tidskriftsartikel (refereegranskat)abstract
- Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.
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