Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

Sophie E. Roberts; Heather L. Martin; Danah Al-Qallaf; Anna A. Tang; Christian Tiede; Thembaninkosi G. Gaule; Albor Dobon-Alonso; Ross Overman; Sachin Shah; Hadrien Peyret; Keith Saunders; Robin Bon; Iain W. Manfield; Sandra M. Bell; George P. Lomonossoff; Valerie Speirs; Darren C. Tomlinson

doi:10.1016/j.isci.2024.110461

Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

Sophie E. Roberts, Heather L. Martin, Danah Al-Qallaf, Anna A. Tang, Christian Tiede, Thembaninkosi G. Gaule, Albor Dobon-Alonso, Ross Overman, Sachin Shah, Hadrien Peyret, Keith Saunders, Robin Bon, Iain W. Manfield, Sandra M. Bell, George P. Lomonossoff, Valerie Speirs, Darren C. Tomlinson^* (Corresponding Author)

^*Corresponding author for this work

Medical Sciences

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Abstract

Monoclonal antibodies have revolutionized therapies, but non-immunoglobulin scaffolds are becoming compelling alternatives owing to their adaptability. Their ability to be labeled with imaging or cytotoxic compounds and to create multimeric proteins is an attractive strategy for therapeutics. Focusing on HER2, a frequently overexpressed receptor in breast cancer, this study addresses some limitations of conventional targeting moieties by harnessing the potential of these scaffolds. HER2-binding Affimers were isolated and characterized, demonstrating potency as binding reagents and efficient internalization by HER2-overexpressing cells. Affimers conjugated with cytotoxic agent achieved dose-dependent reductions in cell viability within HER2-overexpressing cell lines. Bispecific Affimers, targeting HER2 and virus-like particles, facilitated efficient internalization of virus-like particles carrying enhanced green fluorescent protein (eGFP)-encoding RNA, leading to protein expression. Anti-HER2 affibody or designed ankyrin repeat protein (DARPin) fusion constructs with the anti-VLP Affimer further underscore the adaptability of this approach. This study demonstrates the versatility of scaffolds for precise delivery of cargos into cells, advancing biotechnology and therapeutic research.

Original language	English
Article number	110461
Number of pages	18
Journal	iScience
Volume	27
Issue number	8
Early online date	16 Jul 2024
DOIs	https://doi.org/10.1016/j.isci.2024.110461
Publication status	Published - 16 Aug 2024

Bibliographical note

We thank Dr Rachel George of the Mass Spectrometry Facility at the University of Leeds for her technical assistance and the Horticultural and Bioimaging at the John Innes Centre for their support.

HER2 Affimer screening was performed by D.C.T. D.A.Q. A.A.T. and C.T. Affimer characterization including Affimer-MMAE conjugates was carried out by S.E.R. S.P.R. was performed by S.E.R. and I.W.M. VLPs were produced by A.D.A. R.O. S.S. H.P. and K.S. Assessment of Affimer-VLP uptake was performed by H.L.M. and A.A.T. AlphaFold2 modeling was undertaken by T.G.G. S.E.R. H.L.M. R.O. S.M.B. G.P.L. V.S. and and D.C.T. designed the experiments in this manuscript. The manuscript was written by S.E.R. and H.L.M. All authors read and approved the final manuscript. The authors declare a potential conflict of interest. A patent application has been filed related to the work described in this manuscript. [Application number: LU505126, Inventors: George P. Lomonossoff, Sachin Shah, Keith Saunders, Darren C Tomlinson, Christian Tiede, Heather Martin, Ross Overman]. D.C.T. also is a named inventor on patent \u201Cscaffold protein derived from plant cystatins\u201D Europe 14705565.1, US 14/768,032, Australia 2014217628. G.P.L. also sits as a chair on the advisory board for Leaf Expression Systems.

Data Availability Statement

All data reported in this paper will be shared by the lead contact upon request
This paper does not report original code.
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Roberts, S. E., Martin, H. L., Al-Qallaf, D., Tang, A. A., Tiede, C., Gaule, T. G., Dobon-Alonso, A., Overman, R., Shah, S., Peyret, H., Saunders, K., Bon, R., Manfield, I. W., Bell, S. M., Lomonossoff, G. P., Speirs, V., & Tomlinson, D. C. (2024). Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles. iScience, 27(8), Article 110461. https://doi.org/10.1016/j.isci.2024.110461

Roberts, SE, Martin, HL, Al-Qallaf, D, Tang, AA, Tiede, C, Gaule, TG, Dobon-Alonso, A, Overman, R, Shah, S, Peyret, H, Saunders, K, Bon, R, Manfield, IW, Bell, SM, Lomonossoff, GP, Speirs, V & Tomlinson, DC 2024, 'Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles', iScience, vol. 27, no. 8, 110461. https://doi.org/10.1016/j.isci.2024.110461

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abstract = "Monoclonal antibodies have revolutionized therapies, but non-immunoglobulin scaffolds are becoming compelling alternatives owing to their adaptability. Their ability to be labeled with imaging or cytotoxic compounds and to create multimeric proteins is an attractive strategy for therapeutics. Focusing on HER2, a frequently overexpressed receptor in breast cancer, this study addresses some limitations of conventional targeting moieties by harnessing the potential of these scaffolds. HER2-binding Affimers were isolated and characterized, demonstrating potency as binding reagents and efficient internalization by HER2-overexpressing cells. Affimers conjugated with cytotoxic agent achieved dose-dependent reductions in cell viability within HER2-overexpressing cell lines. Bispecific Affimers, targeting HER2 and virus-like particles, facilitated efficient internalization of virus-like particles carrying enhanced green fluorescent protein (eGFP)-encoding RNA, leading to protein expression. Anti-HER2 affibody or designed ankyrin repeat protein (DARPin) fusion constructs with the anti-VLP Affimer further underscore the adaptability of this approach. This study demonstrates the versatility of scaffolds for precise delivery of cargos into cells, advancing biotechnology and therapeutic research.",

author = "Roberts, {Sophie E.} and Martin, {Heather L.} and Danah Al-Qallaf and Tang, {Anna A.} and Christian Tiede and Gaule, {Thembaninkosi G.} and Albor Dobon-Alonso and Ross Overman and Sachin Shah and Hadrien Peyret and Keith Saunders and Robin Bon and Manfield, {Iain W.} and Bell, {Sandra M.} and Lomonossoff, {George P.} and Valerie Speirs and Tomlinson, {Darren C.}",

note = "We thank Dr Rachel George of the Mass Spectrometry Facility at the University of Leeds for her technical assistance and the Horticultural and Bioimaging at the John Innes Centre for their support. HER2 Affimer screening was performed by D.C.T. D.A.Q. A.A.T. and C.T. Affimer characterization including Affimer-MMAE conjugates was carried out by S.E.R. S.P.R. was performed by S.E.R. and I.W.M. VLPs were produced by A.D.A. R.O. S.S. H.P. and K.S. Assessment of Affimer-VLP uptake was performed by H.L.M. and A.A.T. AlphaFold2 modeling was undertaken by T.G.G. S.E.R. H.L.M. R.O. S.M.B. G.P.L. V.S. and and D.C.T. designed the experiments in this manuscript. The manuscript was written by S.E.R. and H.L.M. All authors read and approved the final manuscript. The authors declare a potential conflict of interest. A patent application has been filed related to the work described in this manuscript. [Application number: LU505126, Inventors: George P. Lomonossoff, Sachin Shah, Keith Saunders, Darren C Tomlinson, Christian Tiede, Heather Martin, Ross Overman]. D.C.T. also is a named inventor on patent \u201Cscaffold protein derived from plant cystatins\u201D Europe 14705565.1, US 14/768,032, Australia 2014217628. G.P.L. also sits as a chair on the advisory board for Leaf Expression Systems.",

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AU - Roberts, Sophie E.

AU - Martin, Heather L.

AU - Al-Qallaf, Danah

AU - Tang, Anna A.

AU - Tiede, Christian

AU - Gaule, Thembaninkosi G.

AU - Dobon-Alonso, Albor

AU - Overman, Ross

AU - Shah, Sachin

AU - Peyret, Hadrien

AU - Saunders, Keith

AU - Bon, Robin

AU - Manfield, Iain W.

AU - Bell, Sandra M.

AU - Lomonossoff, George P.

AU - Speirs, Valerie

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N2 - Monoclonal antibodies have revolutionized therapies, but non-immunoglobulin scaffolds are becoming compelling alternatives owing to their adaptability. Their ability to be labeled with imaging or cytotoxic compounds and to create multimeric proteins is an attractive strategy for therapeutics. Focusing on HER2, a frequently overexpressed receptor in breast cancer, this study addresses some limitations of conventional targeting moieties by harnessing the potential of these scaffolds. HER2-binding Affimers were isolated and characterized, demonstrating potency as binding reagents and efficient internalization by HER2-overexpressing cells. Affimers conjugated with cytotoxic agent achieved dose-dependent reductions in cell viability within HER2-overexpressing cell lines. Bispecific Affimers, targeting HER2 and virus-like particles, facilitated efficient internalization of virus-like particles carrying enhanced green fluorescent protein (eGFP)-encoding RNA, leading to protein expression. Anti-HER2 affibody or designed ankyrin repeat protein (DARPin) fusion constructs with the anti-VLP Affimer further underscore the adaptability of this approach. This study demonstrates the versatility of scaffolds for precise delivery of cargos into cells, advancing biotechnology and therapeutic research.

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Affimer reagents enable targeted delivery of therapeutic agents and RNA via virus-like particles

Abstract

Bibliographical note

Data Availability Statement

UN SDGs

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