| 1. |
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| 2. |
- Binz, Hans, et al.
(författare)
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Method for enhancing the immunogenicity of an immunogenic compound or hapten, and use thereof for preparing vaccines
- 1994
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Patent (populärvet., debatt m.m.)abstract
- The present invention relates to a process for improving the immunogenicity of an immunogen, an antigen or a hapten, when it is administered to a host, independently of the mode of administration, characterized in that the said antigen or hapten is coupled covalently to a support molecule in order to form a complex, and in that this support molecule is a polypeptide fragment which is able to bind specifically to mammalian serum albumin. The invention also relates to the use, as a medicament, of the product which can be obtained in this way.
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| 3. |
- Binz, Hans, et al.
(författare)
-
Respiratory syncytial virus protein g expressed on bacterial membrane
- 1994
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Patent (populärvet., debatt m.m.)abstract
- A method for preparing a peptide or protein, wherein (a) a DNA sequence coding for a heterologous polypeptide on a peptide sequence between amino acid residues 130 and 230 of respiratory syncytial virus protein G, sub-groups A and B, or a peptide sequence at least 80 % homologous thereto, and (b) means enabling the expression of the polypeptide on the bacterial membrane surface, are inserted into a bacterium which is not pathogenic for mammals. The resulting conjugate polypeptide and a live bacterium expressing same, pharmaceutical compositions containing them and their use for preparing a vaccine, as well as a DNA sequence coding for said polypeptide, are also disclosed.
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| 4. |
- Cena-Diez, Rafael, et al.
(författare)
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Naturally occurring dipeptide from elite controllers with dual anti-HIV-1 mechanism
- 2023
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Ingår i: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579 .- 1872-7913. ; 61:5, s. 106792-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Enhanced levels of a dipeptide, WC-am, have been reported among elite controllers - patients who spontaneously control their HIV-1 infection. This study aimed to evaluate anti-HIV-1 activity and mechanism of action of WC-am.Methods: Drug sensitivity assays in TZM.bl cells, PBMCs and ACH-2 cells using WT and mutated HIV-1 strains were performed to evaluate the antiviral mechanism of WC-am. Mass spectrometry-based proteomics and Real-time PCR analysis of reverse transcription steps were performed to unravel the second anti-HIV-1 mechanism of WC-am.Results: The data suggest that WC-am binds to the CD4 binding pocket of HIV-1 gp120 and blocks its binding to the host cell receptors. Additionally, the time course assay showed that WC-am also inhibited HIV-1 at 4-6 hours post-infection, suggesting a second antiviral mechanism. Drug sensitivity assays under acidic wash conditions confirmed the ability of WC-am to internalise into the host cell in an HIV independent manner. Proteomic studies showed a clustering of all samples treated with WC-am independent of the number of doses or presence or absence of HIV-1. Differentially expressed proteins due to the WC-am treatment indicated an effect on HIV-1 reverse transcription, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR).Conclusion: Naturally occurring in HIV-1 elite controllers, WC-am stands out as a new kind of antiviral compound with two independent inhibitory mechanisms of action on HIV-1 replication. WC-am halts HIV-1 entry to the host cell by binding to HIV-1 gp120, thereby blocking the binding of HIV-1 to the host cell. WC-am also exerts a post-entry but pre-integration antiviral effect related to RT-activity.
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| 5. |
- Duru, Adil Doganay, et al.
(författare)
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Tuning antiviral CD8 T-cell response via proline-altered peptide ligand vaccination
- 2020
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Ingår i: PLoS Pathogens. - : PUBLIC LIBRARY SCIENCE. - 1553-7366 .- 1553-7374. ; 16:5
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Tidskriftsartikel (refereegranskat)abstract
- Viral escape from CD8(+) cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8(+) T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape. Author summary Viral escape mutagenesis correlates often with disease progression and represents a major hurdle for vaccination-based therapies. Here, we have designed and developed a novel generation of altered epitopes that re-establish and enhance significantly CD8(+) T cell recognition of a naturally occurring viral immune escape variant. Biophysical and structural analyses provide a clear understanding of the molecular mechanisms underlying this reestablished recognition. We believe that this approach can be implemented to currently available or novel vaccination approaches to efficiently restore T cell recognition of virus escape variants to control disease progression.
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| 6. |
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| 7. |
- Giang, Kim Anh, et al.
(författare)
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Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells
- 2023
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Ingår i: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 77, s. 139-148
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Tidskriftsartikel (refereegranskat)abstract
- We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (KD) for CD16a in the range of 100 nM–3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15–23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles.
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| 8. |
- Giang, Kim Anh, et al.
(författare)
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Selection of Affibody Affinity Proteins from Phagemid Libraries
- 2023
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Ingår i: Methods in Molecular Biology. - : Springer Nature. - 1064-3745 .- 1940-6029. ; 2702, s. 373-392
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Tidskriftsartikel (refereegranskat)abstract
- Herein, we describe a general protocol for the selection of target-binding affinity protein molecules from a phagemid-encoded library. The protocol is based on our experience with phage display selections of non-immunoglobulin affibody affinity proteins but can in principle be applied to perform biopanning experiments from any phage-displayed affinity protein library available in a similar phagemid vector. The procedure begins with an amplification of the library from frozen bacterial glycerol stocks via cultivation and helper phage superinfection, followed by a step-by-step instruction of target protein preparation, selection cycles, and post-selection analyses. The described procedures in this standard protocol are relatively conservative and rely on ordinary reagents and equipment available in most molecular biology laboratories.
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| 9. |
- Hafstrand, Ida, et al.
(författare)
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Successive crystal structure snapshots suggest the basis for MHC class I peptide loading and editing by tapasin
- 2019
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 116:11, s. 5055-5060
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Tidskriftsartikel (refereegranskat)abstract
- MHC-I epitope presentation to CD8(+) T cells is directly dependent on peptide loading and selection during antigen processing. However, the exact molecular bases underlying peptide selection and binding by MHC-I remain largely unknown. Within the peptide-loading complex, the peptide editor tapasin is key to the selection of MHC-I-bound peptides. Here, we have determined an ensemble of crystal structures of MHC-I in complex with the peptide exchange-associated dipeptide GL, as well as the tapasin-associated scoop loop, alone or in combination with candidate epitopes. These results combined with mutation analyses allow us to propose a molecular model underlying MHC-I peptide selection by tapasin. The N termini of bound peptides most probably bind first in the N-terminal and middle region of the MHC-I peptide binding cleft, upon which the peptide C termini are tested for their capacity to dislodge the tapasin scoop loop from the F pocket of the MHC-I cleft. Our results also indicate important differences in peptide selection between different MHC-I alleles.
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| 10. |
- Hartmann, R. W., et al.
(författare)
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The Wittig bioconjugation of maleimide derived, water soluble phosphonium ylides to aldehyde-tagged proteins
- 2021
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 19:47, s. 10417-10423
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Tidskriftsartikel (refereegranskat)abstract
- Herein we disclose the transformation of maleimides into water-soluble tris(2-carboxyethyl)phosphonium ylides and their subsequent application in the bioconjugation of protein- and peptide-linked aldehydes. The new entry into Wittig bioconjugate chemistry proceeds under mild conditions and relies on highly water soluble reagents, which are likely already part of most biochemists' inventory.
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| 11. |
- Hjelm, Linnea C., et al.
(författare)
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Lysis of Staphylococcal Cells by Modular Lysin Domains Linked via a n-covalent Barnase-Barstar Interaction Bridge
- 2019
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Ingår i: Frontiers in Microbiology. - : Frontiers Media SA. - 1664-302X. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- Bacteriophage endolysins and bacterial exolysins are capable of enzymatic degradation of the cell wall peptidoglycan layer and thus show promise as a new class of antimicrobials. Both exolysins and endolysins often consist of different modules, which are responsible for enzymatic functions and cell wall binding, respectively. Individual modules from different endo- or exolysins with different binding and enzymatic activities, can via gene fusion technology be re-combined into novel variants for investigations of arrangements of potential clinical interest. The aim of this study was to investigate if separately produced cell wall binding and enzyme modules could be assembled into a functional lysin via a non-covalent affinity interaction bridge composed of the barnase ribonuclease from Bacillus amyloliquefaciens and its cognate inhibitor barstar, known to form a stable heterodimeric complex. In a proof-of-principle study, using surface plasmon resonance, flow cytometry and turbidity reduction assays, we show that separately produced modules of a lysin cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) from Staphylococcus aureus bacteriophage K endolysin (LysK) fused to barnase and a cell wall binding Src homology 3 domain (SH3b) from the S. simulans exolysin lysostaphin fused to barstar can be non-covalently assembled into a functional lysin showing both cell wall binding and staphylolytic activity. We hypothesize that the described principle for assembly of functional lysins from separate modules through appended hetero-dimerization domains has a potential for investigations of also other combinations of enzymatically active and cell wall binding domains for desired applications.
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| 12. |
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| 13. |
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| 14. |
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| 15. |
- Lindberg, Jakob, et al.
(författare)
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Progress and Future Directions with Peptide-Drug Conjugates for Targeted Cancer Therapy
- 2021
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Ingår i: Molecules. - : MDPI AG. - 1431-5157 .- 1420-3049. ; 26:19
-
Forskningsöversikt (refereegranskat)abstract
- We review drug conjugates combining a tumor-selective moiety with a cytotoxic agent as cancer treatments. Currently, antibody-drug conjugates (ADCs) are the most common drug conjugates used clinically as cancer treatments. While providing both efficacy and favorable tolerability, ADCs have limitations due to their size and complexity. Peptides as tumor-targeting carriers in peptide-drug conjugates (PDCs) offer a number of benefits. Melphalan flufenamide (melflufen) is a highly lipophilic PDC that takes a novel approach by utilizing increased aminopeptidase activity to selectively increase the release and concentration of cytotoxic alkylating agents inside tumor cells. The only other PDC approved currently for clinical use is Lu-177-dotatate, a targeted form of radiotherapy combining a somatostatin analog with a radionuclide. It is approved as a treatment for gastroenteropancreatic neuroendocrine tumors. Results with other PDCs combining synthetic analogs of natural peptide ligands with cytotoxic agents have been mixed. The field of drug conjugates as drug delivery systems for the treatment of cancer continues to advance with the application of new technologies. Melflufen provides a paradigm for rational PDC design, with a targeted mechanism of action and the potential for deepening responses to treatment, maintaining remissions, and eradicating therapy-resistant stem cells.
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| 16. |
- Lundqvist, Magnus, et al.
(författare)
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Chromophore pre-maturation for improved speed and sensitivity of split-GFP monitoring of protein secretion
- 2019
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Ingår i: Scientific Reports. - : NATURE PUBLISHING GROUP. - 2045-2322. ; 9
-
Tidskriftsartikel (refereegranskat)abstract
- Complementation-dependent fluorescence is a powerful way to study co-localization or interactions between biomolecules. A split-GFP variant, involving the self-associating GFP 1-10 and GFP 11, has previously provided a convenient approach to measure recombinant protein titers in cell supernatants. A limitation of this approach is the slow chromophore formation after complementation. Here, we alleviate this lag in signal generation by allowing the GFP 1-10 chromophore to mature on a solid support containing GFP 11 before applying GFP 1-10 in analyses. The pre-maturated GFP 1-10 provided up to 150-fold faster signal generation compared to the non-maturated version. Moreover, pre-maturated GFP 1-10 significantly improved the ability of discriminating between Chinese hamster ovary (CHO) cell lines secreting GFP 11-tagged erythropoietin protein at varying rates. Its improved kinetics make the pre-maturated GFP 1-10 a suitable reporter molecule for cell biology research in general, especially for ranking individual cell lines based on secretion rates of recombinant proteins.
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| 17. |
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| 18. |
- Mortensen, Anja, et al.
(författare)
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Selection, characterization and in vivo evaluation of novel CD44v6-targeting antibodies for targeted molecular radiotherapy
- 2023
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Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.
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| 19. |
- Myrhammar, Anders, 1987-
(författare)
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Investigations of chemical and enzymatic functionalization of affinity proteins
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- AbstractAffinity proteins are important reagents in research, diagnostics and therapeutic settings. The focus of this thesis has been on investigating different chemical and enzymatic strategies for engineering of affinity proteins to generate affinity reagents with improved or changed functionality. The modifications introduced in affibodies, representing a class of small, three-helix engineered scaffold proteins, and antibodies were selected and implemented through rational design, using a combination of solid phase peptide synthesis, genetic engineering and enzymatic conjugation, depending on the case.In a first study, thioether crosslinks were introduced between internally positioned lysines and cysteines of the human epidermal growth factor receptor (hEGFR)-targeting affibody ZEGFR:1907, to test the possibility to increase the proteolytic stability of the affibody scaffold. Three different variants of crosslinked affibodies were produced, containing one or two crosslinks. All three variants showed similar affinities to EFGR, and secondary structure contents, as the unmodified control protein. The crosslinked affibodies were challenged with the endopeptidases pepsin, found in the stomach, and trypsin and chymotrypsin, found in the gut. All affibodies showed improved stability towards at least one of the proteases, but the largest improvement was seen for the affibody harboring two crosslinks, which displayed the greatest stability in both assays.Improvement in proteolytic stability of affibodies was further explored. In another study a sortase A-catalyzed intramolecular head-to-tail conjugation of the dimeric human epidermal growth factor 2 (HER2)-targeting affibody (ZHER2:342)2 was performed. Analysis showed no change in α-helicity for the cyclic dimer compared to the linear control, and a slight increase in melting temperature. Interestingly, in contrast to the linear variant, the cyclic dimer showed no signs of proteolytic degradation after 60 min exposure to the exopeptidase carboxypeptidase A.The ability to change protein functionality by chemical modification was explored in two studies. The immunoglobulin-binding Z domain, from which the affibody scaffold is derived, was used as a model protein in one study, where light-induced affinity modulation was investigated. An azobenzene switch that isomerizes from a trans to a cis state was introduced end-to-end to one of the helices in three different designs of the Z domain. The conformational change induced by isomerization was hypothesized to be large enough to cause a loss in binding affinity in the conjugated affibody, which was tested in an affinity chromatography assay in which one of the affibodies captured to an IgG-sepharose column showed loss of affinity during illumination.Peptide nucleic acid (PNA) probes have previously successfully been used for selective hybridization between the primary, tumor-targeting agent and the secondary agent in a pretargeting set-up for in vivo tumor imaging or directed therapy. In a last study, a Z domain-PNA conjugate produced via sortase A-mediated conjugation was photoconjugated to a lactosaminated antibody for possible use as an in vivo clearing agent for clearance of excess of primary probes via an hepatic route. The clearing agent showed partial success in a mouse model but the concept needs further work.The work in this thesis shows the diverse possibilities available for changing the functionality of affinity proteins through chemical and enzymatic methods for different applications, and provides a framework for potential further improvement of both affibody and antibody functionality.
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| 20. |
- Nilsson, Björn, et al.
(författare)
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Bacterial receptor structures
- 1994
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Patent (populärvet., debatt m.m.)abstract
- Novel proteins obtainable by mutagenesis of surface-exposed amino acids of domains of natural bacterial receptors, said proteins being obtained without substantial loss of basic structure and stability of said natural bacterial receptors; proteins which have been selected from a protein library embodying a repertoire of said novel proteins; and methods for the manufacture of artificial bacterial receptor structures.
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| 21. |
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| 22. |
- Nygren, Per-Åke, 1961-
(författare)
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SANDWICH ASSAY
- 2002
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Patent (populärvet., debatt m.m.)abstract
- A sandwich assay method for detecting the presence of a target molecule in a sample comprising a complex biological fluid is provided. The assay comprises providing a first affinity ligand with affinity for the target molecule, which affinity ligand is capable of being immobilized to a solid support; applying the sample in such a way that binding of a target molecule, if present in the sample, to the first affinity ligand is enabled; applying a second affinity ligand with affinity for the target molecule, the application enabling binding of the second affinity ligand to the target molecule; removing second affinity ligand not bound to target molecule; and detecting the presence of the second affinity ligand, such presence being an indicator of the presence of a target molecule in the sample. The first affinity ligand is immobilized to the solid support at any stage before said detection, and at least one of the first and second affinity ligands is an affinity ligand other than an antibody. The invention also provides a kit for use in the method. The kit comprises: a first affinity ligand with affinity for the target molecule and capable of being immobilized to a slid support; a second affinity ligand with affinity for the target molecule, the presence of which ligand is detectable; and a solid support to which the first affinity ligand is capable of being immobilized. In he kit, at least one of the first and second affinity ligands is an affinity ligand other than an antibody.
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| 23. |
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| 24. |
- Ozawa, Takayuki, et al.
(författare)
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Systemic administration of monovalent follistatin-like 3-Fc-fusion protein increases muscle mass in mice
- 2021
-
Ingår i: iScience. - : Elsevier BV. - 2589-0042. ; 24:5
-
Tidskriftsartikel (refereegranskat)abstract
- Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor beta (TGF-beta) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-beta family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration ofmono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo. Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies.
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| 25. |
- Persson, Jonas, et al.
(författare)
-
Discovery, optimization and biodistribution of an Affibody molecule for imaging of CD69
- 2021
-
Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 11:1
-
Tidskriftsartikel (refereegranskat)abstract
- Due to the wide scale of inflammatory processes in different types of disease, more sensitive and specific biomarkers are required to improve prevention and treatment. Cluster of differentiation 69 (CD69) is one of the earliest cell surface proteins expressed by activated leukocytes. Here we characterize and optimize potential new imaging probes, Affibody molecules targeting CD69 for imaging of activated immune cells. Analysis of candidates isolated in a previously performed selection from a Z variant E. coli library to the recombinant extracellular domain of human CD69, identified one cross-reactive Z variant with affinity to murine and human CD69. Affinity maturation was performed by randomization of the primary Z variant, followed by selections from the library. The resulting Z variants were evaluated for affinity towards human and murine CD69 and thermal stability. The in vivo biodistribution was assessed by SPECT/CT in rats following conjugation of the Z variants by a DOTA chelator and radiolabeling with Indium-111. A primary Z variant with a K-d of approximately 50 nM affinity to human and murine CD69 was identified. Affinity maturation generated 5 additional Z variants with improved or similar affinity. All clones exhibited suitable stability. Radiolabeling and in vivo biodistribution in rat demonstrated rapid renal clearance for all variants, while the background uptake and washout varied. The variant Z(CD69:4) had the highest affinity for human and murine CD69 (34 nM) as well as the lowest in vivo background binding. In summary, we describe the discovery, optimization and evaluation of novel Affibody molecules with affinity for CD69. Affibody molecule Z(CD69:4) is suitable for further development for imaging of activated immune cells.
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| 26. |
- Schulte, T., et al.
(författare)
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Assigned NMR backbone resonances of the ligand-binding region domain of the pneumococcal serine-rich repeat protein (PsrP-BR) reveal a rigid monomer in solution
- 2020
-
Ingår i: Biomolecular NMR Assignments. - : Springer. - 1874-2718 .- 1874-270X.
-
Tidskriftsartikel (refereegranskat)abstract
- The pneumococcal serine rich repeat protein (PsrP) is displayed on the surface of Streptococcus pneumoniae with a suggested role in colonization in the human upper respiratory tract. Full-length PsrP is a 4000 residue-long multi-domain protein comprising a positively charged functional binding region (BR) domain for interaction with keratin and extracellular DNA during pneumococcal adhesion and biofilm formation, respectively. The previously determined crystal structure of the BR domain revealed a flat compressed barrel comprising two sides with an extended β-sheet on one side, and another β-sheet that is distorted by loops and β-turns on the other side. Crystallographic B-factors indicated a relatively high mobility of loop regions that were hypothesized to be important for binding. Furthermore, the crystal structure revealed an inter-molecular β-sheet formed between edge strands of two symmetry-related molecules, which could promote bacterial aggregation during biofilm formation. Here we report the near complete 15N/13C/1H backbone resonance assignment of the BR domain of PsrP, revealing a secondary structure profile that is almost identical to the X-ray structure. Dynamic 15N-T1, T2 and NOE data suggest a monomeric and rigid structure of BR with disordered residues only at the N- and C-termini. The presented peak assignment will allow us to identify BR residues that are crucial for ligand binding.
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| 27. |
- Schulte, Tim, et al.
(författare)
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Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH
- 2023
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Ingår i: Open Biology. - : The Royal Society. - 2046-2441. ; 13:5
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Tidskriftsartikel (refereegranskat)abstract
- G3BP is the central node within stress-induced protein-RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.
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| 28. |
- Shibasaki, Seiji, et al.
(författare)
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Inhibitory effects of H-Ras/Raf-1–binding affibody molecules on synovial cell function
- 2014
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Ingår i: AMB Express. - : Springer Berlin/Heidelberg. - 2191-0855. ; 4:82
-
Tidskriftsartikel (refereegranskat)abstract
- Affibody molecules specific for H-Ras and Raf-1 were evaluated for their ability to inhibit synovial cell function. Affibody molecules targeting H-Ras (Zras122, Zras220, and Zras521) or Raf-1 (Zraf322) were introduced into the MH7A synovial cell line using two delivery methods: transfection with plasmids encoding the affibody molecules or direct introduction of affibody protein using a cell-penetrating peptide reagent. Interleukin-6 (IL-6) and prostaglandin E2 (PGE2) production by MH7A cells were analyzed by enzyme-linked immunosorbent assay after stimulation with tumor necrosis factor-alpha (TNF-α). Cell proliferation was also analyzed. Phosphorylation of extracellular signal-regulated kinase (ERK) was analyzed by western blot. All affibody molecules could inhibit IL-6 and PGE2 production in TNF-α-stimulated MH7A cells. The inhibitory effect was stronger when affibody molecules were delivered as proteins via a cell-penetrating peptide reagent than when plasmid-DNA encoding the affibody moelcules was transfected into the cells. Plasmid-expressed Zras220 inhibited phosphorylation of ERK in TNF-α-stimulated MH7A cells. Protein-introduced Zraf322 inhibited the production of IL-6 and PGE2 and inhibited cell proliferation in MH7A cells. These findings suggest that affibody molecules specific for H-Ras and Raf-1 can affect intracellular signal transduction through the MAP kinase pathway to inhibit cell proliferation and production of inflammatory mediators by synovial cells.
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| 29. |
- Ståhl, Stefan, et al.
(författare)
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Affibody Molecules in Biotechnological and Medical Applications
- 2017
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Ingår i: Trends in Biotechnology. - : Elsevier. - 0167-7799 .- 1879-3096. ; 35:8, s. 691-712
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Forskningsöversikt (refereegranskat)abstract
- Affibody molecules are small (6.5-kDa) affinity proteins based on a three-helix bundle domain framework. Since their introduction 20 years ago as an alternative to antibodies for biotechnological applications, the first therapeutic affibody molecules have now entered clinical development and more than 400 studies have been published in which affibody molecules have been developed and used in a variety of contexts. In this review, we focus primarily on efforts over the past 5 years to explore the potential of affibody molecules for medical applications in oncology, neurodegenerative, and inflammation disorders, including molecular imaging, receptor signal blocking, and delivery of toxic payloads. In addition, we describe recent examples of biotechnological applications, in which affibody molecules have been exploited as modular affinity fusion partners.
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| 30. |
- Ståhl, Stefan, et al.
(författare)
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Recombinant DNA coding for signal peptide, selective interacting polypeptide and membrane anchoring sequence
- 1991
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Patent (populärvet., debatt m.m.)abstract
- Tripartite recombinant DNA encoding fusion proteins which comprise three sequences, i.e., a signal peptide which is operable in a Gram positive bacterium, an immunogenic polypeptide linked thereto which is not normally expressed in a Gram positive bacterium, and a cell wall spanning and a membrane anchoring sequence, as well as their use in Gram positive bacteria to express the resultant fusion protein on their surface are described. The preferred cell wall spanning and anchoring polypeptides include Staphylococcus protein A and Streptococcus protein G.
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| 31. |
- Subramanian, Karthik, et al.
(författare)
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Pneumolysin binds to the mannose receptor C type 1 (MRC-1) leading to anti-inflammatory responses and enhanced pneumococcal survival
- 2019
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Ingår i: Nature Microbiology. - : Nature Publishing Group. - 2058-5276. ; 4:1, s. 62-70
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pneumoniae (the pneumococcus) is a major cause of mortality and morbidity globally, and the leading cause of death in children under 5 years old. The pneumococcal cytolysin pneumolysin (PLY) is a major virulence determinant known to induce pore-dependent pro-inflammatory responses. These inflammatory responses are driven by PLY-host cell membrane cholesterol interactions, but binding to a host cell receptor has not been previously demonstrated. Here, we discovered a receptor for PLY, whereby pro-inflammatory cytokine responses and Toll-like receptor signalling are inhibited following PLY binding to the mannose receptor C type 1 (MRC-1) in human dendritic cells and mouse alveolar macrophages. The cytokine suppressor SOCS1 is also upregulated. Moreover, PLY-MRC-1 interactions mediate pneumococcal internalization into non-lysosomal compartments and polarize naive T cells into an interferon-gamma(low), interleukin-4(high) and FoxP3(+) immunoregulatory phenotype. In mice, PLY-expressing pneumococci colocalize with MRC-1 in alveolar macrophages, induce lower pro-inflammatory cytokine responses and reduce neutrophil infiltration compared with a PLY mutant. In vivo, reduced bacterial loads occur in the airways of MRC-1-deficient mice and in mice in which MRC-1 is inhibited using blocking antibodies. In conclusion, we show that pneumococci use PLY-MRC-1 interactions to downregulate inflammation and enhance bacterial survival in the airways. These findings have important implications for future vaccine design.
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| 32. |
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| 33. |
- Tano, Hanna
(författare)
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PNA and affinity protein tools for selective tumor targeting of radiopharmaceuticals
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Targeted radiotherapy of cancer intends to selectively deliver cytotoxic radionuclides to tumor cells. Affinity proteins of various kinds are explored for this task, and depending on the affinity protein used, different challenges arise. Full-length antibodies are typically associated with long serum half-life, leading to high systemic toxicity, while smaller affinity ligands such as engineered scaffold proteins, antibody fragments or peptides, usually demonstrate high radioactive uptake in kidneys. The smallest affinity ligands furthermore suffer from low therapeutic efficacy due to their fast wash-out, thus demanding frequent administrations of the radio-conjugate to reach a therapeutic effect. These issues were addressed in this thesis, where small affinity ligands (an Affibody molecule, a single domain antibody fragment and a peptide) have been explored as targeting agents for the cancer targets HER2, CD38 and GRPR, respectively. The Affibody molecule and the single domain antibody fragment were used in a pretargeting setting where high selective hybridization are used as recognition tags between peptide nucleic acid (PNA) strands on the tumor targeting primary agent and the radiolabelled secondary agent. In papers I and II, different sets of PNA hybridization probes were evaluated, in vitro and in vivo. In paper I, we demonstrate that the shortest tested secondary PNA probe (the 9-mer HP16) had the most favourable biodistribution profile with high tumor uptake along with the lowest kidney uptake. In paper II, we produced a set of shorter primary PNA probes, aiming for simplified production, and new sets of even shorter secondary PNA probes. A secondary 8-mer was identified as suitable for testing in cell assays and in vivo together with HER2-binding Affibody-PNA conjugates with varying length of the primary PNA probe, in order to determine if the smaller hydrodynamic range would further improve the biodistribution properties. In paper III, the Affibody-mediated PNA-based pretargeting strategy was evaluated as a monotherapy and as a co-treatment strategy with trastuzumab, to treat mice bearing HER2-positive tumors. Mice treated with the co-treatment strategy had significantly longer survival compared to other groups. In paper IV, the feasibility of using the PNA pretargeting strategy in combination with another affinity protein (a single domain antibody fragment) was evaluated in a CD38-expressing cell line. In paper V, the GRPR-binding peptide RM26 was conjugated to an albumin-binding domain, with the aim to achieve a high tumor uptake over time. The RM26-ABD conjugate did demonstrate good tumor uptake over time. However, the conjugate also demonstrated high kidney uptake, limiting its use as a therapeutic construct. In conclusion, the work presented in this thesis shows strategies for selective tumor targeting of radiopharmaceuticals using affinity proteins and PNA-mediated pretargeting.
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| 34. |
- Yu, Feifan, et al.
(författare)
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Site-Specific Photoconjugation of Beta-Lactamase Fragments to Monoclonal Antibodies Enables Sensitive Analyte Detection via Split-Enzyme Complementation
- 2018
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Ingår i: Biotechnology Journal. - : WILEY-V C H VERLAG GMBH. - 1860-6768 .- 1860-7314. ; 13:7
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Tidskriftsartikel (refereegranskat)abstract
- Protein fragment complementation assays (PCA) rely on a proximity-driven reconstitution of a split reporter protein activity, typically via interaction between bait and prey units separately fused to the reporter protein halves. The PCA principle can also be formatted for use in immunossays for analyte detection, e.g., via the use of small immunoglobulin binding proteins (IgBp) as fusion partners to split-reporter protein fragments for conversion of pairs of antibodies into split-protein half-probes. However, the non-covalent binding between IgBp and antibodies is not ideal for development of robust assays. Here, the authors describe how split-enzyme reporter halves can be both site-specifically and covalently photoconjugated at antibody Fc-parts for use in homogeneous dual-antibody in vitro immunoassays based on analyte-dependent split-enzyme fragment complementation. The half-probes consist of parts of a beta-lactamase split-protein reporter fused to an immunoglobulin Fc binding domain equipped with a unique cysteine residue at which a photoactivable maleimide benzophenone group (MBP) is attached. Using such antibody conjugates the authors obtain an analyte-driven complementation of the reporter enzyme fragments monitored via conversion of a chromogenic substrate. Results from detection of human interferon-gamma and the extracellular domain of HER2 is shown. The described principles for site-specific conjugation of proteins to antibodies should be broadly applicable.
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| 35. |
- Yu, Wendy, et al.
(författare)
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Efficient Labeling of Native Human IgG by Proximity-Based Sortase-Mediated Isopeptide Ligation
- 2021
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Ingår i: Bioconjugate chemistry. - : American Chemical Society (ACS). - 1043-1802 .- 1520-4812. ; 32:6, s. 1058-1066
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Tidskriftsartikel (refereegranskat)abstract
- Antibody-drug conjugates (ADCs) have demonstrated great therapeutic potential due to their ability to target the delivery of potent cytotoxins. However, the heterogeneous nature of conventional drug conjugation strategies can affect the safety, efficacy, and stability of ADCs. Site-specific conjugations can resolve these issues, but often require genetic modification of Immunoglobulin G (IgG), which can impact yield or cost of production, or require undesirable chemical linkages. Here, we describe a near-traceless conjugation method that enables the efficient modification of native IgG, without the need for genetic engineering or glycan modification. This method utilizes engineered variants of sortase A to catalyze noncanonical isopeptide ligation. Sortase A was fused to an antibody-binding domain to improve ligation efficiency. Antibody labeling is limited to five lysine residues on the heavy chain and one on the light chain of human IgG1. The ADCs exhibit conserved antigen and Fc-receptor interactions, as well as potent cytolytic activity.
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| 36. |
- Zhang, Jie
(författare)
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Targeting Human Epidermal Growth Factor Receptors with Drug Conjugates Based on Affibody Molecules
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer is a major public health challenge and the second leading cause of death in the world, with millions of new cases being diagnosed each year. Traditional cancer treatments such as surgery, radiation therapy, and chemotherapy are many times effective, but may also cause damage to healthy cells, leading to side effects. Targeted therapy is a more precise and focused approach to cancer treatment, where the aim is to target the cancer cells while leaving the normal cells unaffected. It is particularly effective in cancers where specific molecular targets are known, such as the subset of breast cancer patients with HER2 over-expression or in the subset of patients with pancreatic cancer with HER3 over-expression. Antibody-drug conjugates (ADCs) are an important addition to tumor-targeted therapy, with twelve drugs approved for clinical use by the FDA. They utilize the high specificity of monoclonal antibodies conjugated with highly cytotoxic small molecules to enhance the accumulation of the drugs in the tumor, for highly specific and efficient killing. However, traditional ADCs may not be the optimal delivery format for the directed delivery of cytotoxic drugs. They are limited by their relatively large molecular weights, resulting in relatively low penetration of solid tumors. Recently, a novel type of drug conjugates, affibody-drug conjugates, has been described. These combined an engineered scaffold affinity protein, an affibody molecule, with an albumin binding domain (ABD) for half-life extension, to which a cytotoxic payload has been conjugated. Previous studies show that these novel drug conjugates have a potent and tumor-cell-specific cytotoxic effect. In the future, they may therefore become complementary or alternatives to current targeted cancer therapies. This thesis focuses on the optimization of affibody-drug conjugates targeting HER2 and HER3, members of the human epidermal growth factor receptor family. The thesis presents in vitro and in vivo preclinical data, showing the potential for further clinical development. In paper I, we investigated the influence of the drug-to-affibody ratio (DAR) on the pharmacokinetic profile of affibody-drug conjugates targeting HER2. Increasing the drug load resulted in an elevated delivery of the DM1 drug to the tumors; however, it also led to increased uptake by the liver. Further optimization of the molecular design is necessary to enable highly efficient delivery to tumors while minimizing the uptake in normal organs and tissues. In paper II, the effect of the length and composition of the linker between the HER2 targeting affibody molecule and the ABD was investigated. The use of a 12 amino acids linker reduced hepatic uptake compared with the use of a 5 amino acids linker. This finding offers an important insight into the influence of the linker on the properties of the affibody drug conjugates. In paper III, we investigated the influence of different cytotoxic payloads, as part of an affibody-drug conjugate targeting HER2, on binding properties, cytotoxicity, biodistribution, and anti-tumor effect. The combination of a potent cytotoxic effect in vitro, and a high tumor uptake in vivo, resulted in a superior anti-tumor effect for ZHER2- ABD-mcMMAF at lower doses compared to the previously investigated ZHER2-ABD- mcDM1. Importantly, it maintained a favorable toxicity profile with lower liver uptake compared to ZHER2-ABD-mcDM1. The affibody-drug conjugate ZHER2-ABD- mcMMAF holds great promise as a valuable agent for HER2-targeted cancer therapy. In paper IV, we generated a series of HER2-targeted affibody-drug conjugates fused with different PAS or XTEN polypeptides. We evaluated the ability of the XTEN and PAS polypeptides to extend the plasma half-life, and their influence on tumor uptake, and tissue biodistribution. We compared our new constructs with the previously developed construct, ZHER2-ABD-mcDM1, where an albumin binding domain was used for half-life extension. It was found that the ABD-fused affibody-drug conjugate demonstrated superior tumor uptake and tumor-to-normal-organ ratios compared to the PASylated and XTENylated affibody-drug conjugates. It is possible that ABD is better also for other cancer-targeting strategies where a high tumor uptake while maintaining comparable accumulation in normal tissues is desired. In paper V, we compared the binding properties and cytotoxic potential of a monovalent and a bivalent HER3-targeting affibody-drug conjugate. The biodistribution and therapeutic potential of the bivalent drug construct were evaluated. We found that the bivalent ZHER3-ABD-ZHER3- mcDM1 is a highly potent drug conjugate with favorable biodistribution and anti-tumor efficacy. These results suggest that ZHER3-ABD-ZHER3-mcDM1 holds promise for future clinical development as a potential therapeutic option for patients with HER3 over-expressing cancer. In summary, the potential for modification and optimization through the design of diverse components within HER2 and HER3-targeting affibody-drug conjugates significantly enhances therapeutic effectiveness, thereby encouraging prospective advancements in the development of targeted drug conjugates.
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