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Giang, K. A., Boxaspen, T., Diao, Y., Nilvebrant, J., Kosugi-Kanaya, M., Kanaya, M., . . . Nygren, P.-Å. (2023). Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells. New Biotechnology, 77, 139-148
Open this publication in new window or tab >>Affibody-based hBCMA x CD16 dual engagers for NK cell-mediated killing of multiple myeloma cells
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2023 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 77, p. 139-148Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier BV, 2023
Keywords
Affibody, BCMA, CD16, Dual engager, Multiple myeloma, NK cells
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:kth:diva-337461 (URN)10.1016/j.nbt.2023.09.002 (DOI)001075124700001 ()37673373 (PubMedID)2-s2.0-85170430125 (Scopus ID)
Note

QC 20231006

Available from: 2023-10-06 Created: 2023-10-06 Last updated: 2023-10-24Bibliographically approved
Schulte, T., Panas, M. D., Han, X., Williams, L., Kedersha, N., Fleck, J. S., . . . McInerney, G. M. (2023). Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH. Open Biology, 13(5), Article ID 220369.
Open this publication in new window or tab >>Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH
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2023 (English)In: Open Biology, E-ISSN 2046-2441, Vol. 13, no 5, article id 220369Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
The Royal Society, 2023
Keywords
stress granule, G3BP, caprin-1, crystal structure, condensate, pH
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-328289 (URN)10.1098/rsob.220369 (DOI)000984747000003 ()37161291 (PubMedID)2-s2.0-85159669448 (Scopus ID)
Note

QC 20230607

Available from: 2023-06-07 Created: 2023-06-07 Last updated: 2023-08-17Bibliographically approved
Giang, K. A., Sidhu, S. S. & Nilvebrant, J. (2023). Construction of Synthetic Antibody Phage Display Libraries. Methods in Molecular Biology, 2702, 59-75
Open this publication in new window or tab >>Construction of Synthetic Antibody Phage Display Libraries
2023 (English)In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 2702, p. 59-75Article in journal (Refereed) Published
Abstract [en]

Synthetic antibody libraries provide a vast resource of renewable antibody reagents that can rival natural antibodies and be rapidly isolated through controlled in vitro selections. Use of highly optimized human frameworks enables the incorporation of defined diversity at positions that are most likely to contribute to antigen recognition. This protocol describes the construction of synthetic antibody libraries based on a single engineered human autonomous variable heavy domain scaffold with diversity in all three complementarity-determining regions. The resulting libraries can be used to generate recombinant domain antibodies targeting a wide range of protein antigens using phage display. Furthermore, analogous methods can be used to construct antibody libraries based on larger antibody fragments or second-generation libraries aimed to fine-tune antibody characteristics including affinity, specificity, and manufacturability. The procedures rely on standard reagents and equipment available in most molecular biology laboratories.

Place, publisher, year, edition, pages
Springer Nature, 2023
Keywords
Antibody fragment, Degenerate oligonucleotide, Domain antibody, Human antibody, Phage display, Protein engineering
National Category
Biochemistry and Molecular Biology Immunology in the medical area
Identifiers
urn:nbn:se:kth:diva-337791 (URN)10.1007/978-1-0716-3381-6_4 (DOI)37679615 (PubMedID)2-s2.0-85170160841 (Scopus ID)
Note

QC 20231009

Available from: 2023-10-09 Created: 2023-10-09 Last updated: 2023-10-09Bibliographically approved
Nilvebrant, J., Hober, S. & Kanje, S. (2023). Ligand and use thereof. us 11820802.
Open this publication in new window or tab >>Ligand and use thereof
2023 (English)Patent (Other (popular science, discussion, etc.))
Abstract [en]

The present invention is within the field of protein engineering and purification. The invention relates to a target-binding polypeptide mutant of an IgG binding polypeptide, such as Protein A, Protein G, Protein L or Protein M, comprising a metal binding motif. More closely the invention relates to an Fc binding ligand comprising an engineered protein based on the Protein A derived Z domain, to which a calcium binding EF-loop has been introduced.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-342007 (URN)
Patent
US 11820802 (2023-11-21)
Note

QC 20240109

Available from: 2024-01-09 Created: 2024-01-09 Last updated: 2024-02-13Bibliographically approved
Cena-Diez, R., Narayanan, A., Ray, S., van de Klundert, M., Rodriguez, J. E., Nilvebrant, J., . . . Sonnerborg, A. (2023). Naturally occurring dipeptide from elite controllers with dual anti-HIV-1 mechanism. International Journal of Antimicrobial Agents, 61(5), 106792, Article ID 106792.
Open this publication in new window or tab >>Naturally occurring dipeptide from elite controllers with dual anti-HIV-1 mechanism
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2023 (English)In: International Journal of Antimicrobial Agents, ISSN 0924-8579, E-ISSN 1872-7913, Vol. 61, no 5, p. 106792-, article id 106792Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Elsevier BV, 2023
Keywords
Antiviral, HIV, Dual mechanism, Entry, Retrotranscription, Therapy
National Category
Infectious Medicine
Identifiers
urn:nbn:se:kth:diva-327401 (URN)10.1016/j.ijantimicag.2023.106792 (DOI)000983254500001 ()36931610 (PubMedID)2-s2.0-85151745660 (Scopus ID)
Note

QC 20230526

Available from: 2023-05-26 Created: 2023-05-26 Last updated: 2023-05-26Bibliographically approved
Mortensen, A. C., Berglund, H., Segerström, L., Walle, M., Hofström, C., Persson, H., . . . Nestor, M. (2023). Selection, characterization and in vivo evaluation of novel CD44v6-targeting antibodies for targeted molecular radiotherapy. Scientific Reports, 13(1), Article ID 20648.
Open this publication in new window or tab >>Selection, characterization and in vivo evaluation of novel CD44v6-targeting antibodies for targeted molecular radiotherapy
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2023 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 13, no 1, article id 20648Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Springer Nature, 2023
National Category
Cancer and Oncology Radiology, Nuclear Medicine and Medical Imaging
Identifiers
urn:nbn:se:kth:diva-340841 (URN)10.1038/s41598-023-47891-2 (DOI)001136085000078 ()38001360 (PubMedID)2-s2.0-85177659141 (Scopus ID)
Note

QC 20231218

Available from: 2023-12-18 Created: 2023-12-18 Last updated: 2024-02-06Bibliographically approved
Giang, K. A., Nygren, P.-Å. & Nilvebrant, J. (2023). Selection of Affibody Affinity Proteins from Phagemid Libraries. Methods in Molecular Biology, 2702, 373-392
Open this publication in new window or tab >>Selection of Affibody Affinity Proteins from Phagemid Libraries
2023 (English)In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 2702, p. 373-392Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Springer Nature, 2023
Keywords
Affibody, Antigen preparation, Phage display, Phage-ELISA, Signal normalization
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-337793 (URN)10.1007/978-1-0716-3381-6_19 (DOI)37679630 (PubMedID)2-s2.0-85170169986 (Scopus ID)
Note

QC 20231009

Available from: 2023-10-09 Created: 2023-10-09 Last updated: 2023-10-09Bibliographically approved
Giang, K. A., Kanaya, M., Boxaspen, T., Kanaya, M., Krokeide, S. Z., Diao, Y., . . . Nygren, P.-Å. (2022). Affibody-Based BCMA x CD16 Dual Engagers for Activation of NK Cells Towards Multiple Myeloma. Blood, 140, 10699-10700
Open this publication in new window or tab >>Affibody-Based BCMA x CD16 Dual Engagers for Activation of NK Cells Towards Multiple Myeloma
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2022 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 140, p. 10699-10700Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
American Society of Hematology, 2022
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-324899 (URN)10.1182/blood-2022-164753 (DOI)000893230303312 ()
Note

QC 20230321

Available from: 2023-03-21 Created: 2023-03-21 Last updated: 2023-03-21Bibliographically approved
Sherpa, D., Mueller, J., Karayel, O., Xu, P., Yao, Y., Chrustowicz, J., . . . Alpi, A. F. (2022). Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation. eLIFE, 11, Article ID e77937.
Open this publication in new window or tab >>Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation
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2022 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 11, article id e77937Article in journal (Refereed) Published
Abstract [en]

The development of haematopoietic stem cells into mature erythrocytes - erythropoiesis - is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed maturation stage-dependent assemblies of topologically homologous RANBP9- and RANBP10-CTLH complexes. Moreover, protein abundance of CTLH's cognate E2 ubiquitin conjugating enzyme UBE2H increased during terminal differentiation, and UBE2H expression depended on catalytically active CTLH E3 complexes. CRISPR-Cas9-mediated inactivation of CTLH E3 assemblies or UBE2H in erythroid progenitors revealed defects, including spontaneous and accelerated erythroid maturation as well as inefficient enucleation. Thus, we propose that dynamic maturation stage-specific changes of UBE2H-CTLH E2-E3 modules control the orderly progression of human erythropoiesis.

Place, publisher, year, edition, pages
eLife Sciences Publications, Ltd, 2022
Keywords
Ubiquitylation, erythropoiesis, E3 ubiquitin ligase, CTLH E3 complex, UBE2H, proteomics, Human
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-322617 (URN)10.7554/eLife.77937 (DOI)000893592500001 ()36459484 (PubMedID)2-s2.0-85143379786 (Scopus ID)
Note

QC 20221223

Available from: 2022-12-23 Created: 2022-12-23 Last updated: 2023-09-25Bibliographically approved
Nilvebrant, J., Åstrand, M. & Hober, S. (2021). An orthogonal fusion tag for efficient protein purification (Volume 2178ed.). In: Methods in Molecular Biology: (pp. 159-166). Springer Nature
Open this publication in new window or tab >>An orthogonal fusion tag for efficient protein purification
2021 (English)In: Methods in Molecular Biology, Springer Nature , 2021, Volume 2178, p. 159-166Chapter in book (Refereed)
Abstract [en]

In this chapter, we present an efficient method for stringent protein purification facilitated by a dual affinity tag referred to as ABDz1, which is based on a 5 kDa albumin-binding domain from Streptococcal Protein G. The small fusion tag enables an orthogonal affinity purification approach based on two successive and highly specific affinity purification steps. This approach is enabled by native binding of ABDz1 to human serum albumin and engineered binding to Staphylococcal Protein A, respectively. The ABDz1-tag can be fused to either terminus of a protein of interest and the purification steps can be carried out using standard laboratory equipment.

Place, publisher, year, edition, pages
Springer Nature, 2021 Edition: Volume 2178
Keywords
ABDz1, Albumin binding, Fusion tag, Human serum albumin, Orthogonal affinity purification, Protein A, Protein G, abdz1 fusion protein, bacterial protein, bevifimod, fusion protein, protein tag, signal peptide, unclassified drug, affinity labeling, amino acid sequence, amino terminal sequence, binding site, cell lysate, combinatorial mutagenesis, nonhuman, polyacrylamide gel electrophoresis, protein binding, protein domain, protein engineering, protein expression, protein purification, Streptococcus
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-290280 (URN)10.1007/978-1-0716-0775-6_13 (DOI)33128750 (PubMedID)2-s2.0-85095127442 (Scopus ID)
Note

QC 20210217

Available from: 2021-02-17 Created: 2021-02-17 Last updated: 2022-06-25Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-6104-6446

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