Thermostable designed ankyrin repeat proteins (DARPins) as building blocks for innovative drugs

Abstract

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins. Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins. Designed ankyrin repeat proteins (DARPins) are a class of antibody mimetics that have been conceived and developed about two decades ago at the University of Zurich (1Forrer P. Stumpp M.T. Binz H.K. Plückthun A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins.FEBS Lett. 2003; 539: 2-6Google Scholar, 2Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar, 3Binz H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. High-affinity binders selected from designed ankyrin repeat protein libraries.Nat. Biotechnol. 2004; 22: 575-582Google Scholar). Their application as research tool and protein therapeutic was recently reviewed (4Boersma Y.L. Protein scaffolds, design, synthesis, and applications.Methods Mol. Biol. 2018; 1798: 307-327Google Scholar). Originally devised as an alternative to immunoglobulins (“antibodies”), the potential of DARPins in protein engineering, directed evolution of binders, and drug development became obvious immediately at inception. Importantly, this potential extends beyond areas of applications that have classically been “occupied” by recombinant immunoglobulins. The DARPin scaffold was shown to serve as an alternative (5Foord E. Klynning C. Schoutrop E. Förster J.M. Krieg J. Mörtberg A. Müller M.R. Herzog C. Schiegg D. Villemagne D. Fiedler U. Snell D. Kebble B. Mattsson J. Levitsky V. Uhlin M. Profound functional suppression of tumor-infiltrating T-cells in ovarian cancer patients can be reversed using PD-1-blocking antibodies or DARPin proteins.J. Immunol. Res. 2020; 2020: 7375947Google Scholar), as a complementation, (6Akbari V. Chou C.P. Abedi D. New insights into affinity proteins for HER2-targeted therapy: Beyond trastuzumab.Biochim. Biophys. Acta Rev. Cancer. 2020; 1874: 188448Google Scholar) and as an expansion of what is possible with binders derived from immunoglobulins (7Münch R.C. Muth A. Muik A. Friedel T. Schmatz J. Dreier B. Trkola A. Plückthun A. Büning H. Buchholz C.J. Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors.Nat. Commun. 2015; 6: 6246Google Scholar, 8Plückthun A. Designed ankyrin repeat proteins (DARPins): Binding proteins for research, diagnostics, and therapy.Annu. Rev. Pharmacol. Toxicol. 2015; 55: 489-511Google Scholar). Translation of academic research in DARPin technology toward pharmaceutical benefits has been predominantly steered by Molecular Partners, who provided the fundamental clinical validation of the scaffold (9Stumpp M.T. Dawson K.M. Binz H.K. Beyond antibodies: The DARPin drug platform.BioDrugs. 2020; 34: 423-433Google Scholar). However, in light of the long generation cycles in drug development—especially in the case of biologics that typically require 10 years from concept to drug approval—the DARPin technology can still be regarded as young and emerging, and the full potential of DARPins as a class of biologics has yet to be realized. The recent development of ensovibep (10ensovibepum: WHO Drug Information. Vol. 34. WHO, Geneva, Switzerland2020: 968-970Google Scholar), a multi-specific anti-SARS-CoV-2 DARPin, which has entered clinical trials in November 2020 in less than 9 months after initial research and development activities had commenced, reinforces this high potential M. A. V. Villemagne D. M. T. A. A. P. M. M. M. anti-SARS-CoV-2 therapeutic 2020; Scholar, M. J. H. A. C. V. A. T. A. T. DARPin very high SARS-CoV-2 in Scholar). DARPins are based on ankyrin repeat proteins J. A. A Scholar) that have to various of in all of The ankyrin repeat as for protein 2004; Scholar). Their is than that of immunoglobulins. of and more than in recombinant C. C. U. M. P. therapeutic antibodies using Scholar), a single is to a DARPin H.K. A. C. T. Fiedler U. Stumpp M.T. and of a DARPin drug Scholar). ensovibep (10ensovibepum: WHO Drug Information. Vol. 34. WHO, Geneva, Switzerland2020: 968-970Google Scholar) five DARPin domains on a single in which two domains and three domains with the SARS-CoV-2 protein M. A. V. Villemagne D. M. T. A. A. P. M. M. M. anti-SARS-CoV-2 therapeutic 2020; Scholar). DARPins are from protein domains, which a modular that was derived by a consensus design H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar, of the ankyrin repeat U. A. Scholar, P. Binz H.K. Stumpp M.T. Plückthun A. design of repeat 2004; a of internal ankyrin of by and capping repeats and that to the of the protein domain these an ankyrin repeat present at at the of the internal repeats a the binding to proteins with high affinity and H.K. Amstutz P. Kohl A. Stumpp M.T. Briand C. Forrer P. Grütter M.G. Plückthun A. High-affinity binders selected from designed ankyrin repeat protein libraries.Nat. Biotechnol. 2004; 22: 575-582Google Scholar, M. M. A the a designed protein and J. Scholar, C. E. J.M. D. Plückthun A. A designed ankyrin repeat protein to affinity to Mol. Biol. Scholar). are in DARPin which are as for in by of J. Plückthun A. In and evolution of functional proteins by using U. A. Scholar), to the and of DARPins from the protein as could be to to the internal repeats H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar). and are present in the DARPin that became clinically validated abicipar pegol. the clinical of the of these caps was indicating that be for in for that DARPin thermostability. In we three that the for increased thermostability of of and of of and the and of drug and of of for protein to for design, for M. A. V. Villemagne D. M. T. A. A. P. M. M. M. anti-SARS-CoV-2 therapeutic 2020; Scholar), K.M. M. J. D. of in Scholar), and J. A. A. V. M. A. J. A. D. Designed protein to with of 2020; Scholar). the thermostability of the of the capping the shown by Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar). five of which are at the to the internal repeat and two at the very to the and shown to the of a DARPin, of an a single full-consensus repeat and a by about 17 that from °C to °C was to as the Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar). this was with the H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar), a of the toward the internal repeat was as by Plückthun A. Grütter M.G. for of designed ankyrin repeat proteins with a Mol. Biol. Scholar). in an increased and a of the the internal repeat and the which the thermostability. the of DARPins was we are of a analysis or thermostability of the as in the which is still predominantly by the research thermostability improvements of the have been in the application and are clinically validated in the domains of ensovibep (10ensovibepum: WHO Drug Information. Vol. 34. WHO, Geneva, Switzerland2020: 968-970Google Scholar). The of is as in the with the in a of is that this from the present in which the the by the from the DARPin and this for The analysis of was to the of we to the thermostability of DARPins the in silico and at we Asp17 as an of DARPin Molecular dynamics simulations and analysis an for the increased Asp17 replacement in DARPin We a three repeat DARPin domain as in the of full-consensus internal repeat by an and as a DARPin to for thermostability for an of the DARPin domains as as for The of this DARPin has a has the DARPin of three repeats with two repeat the and the and the and the using a consensus that an of all the ankyrin repeats from at or possible that be present in P. Binz H.K. Stumpp M.T. Plückthun A. design of repeat 2004; Scholar). DARPins more stable with of we to have internal repeat and a thermostability M. Binz H.K. Plückthun A. and of stable full-consensus ankyrin repeat proteins.J. Mol. Biol. Scholar) such that improvements are of DARPin domains in the present with and as in the domain with full consensus to of Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar) M. Binz H.K. Plückthun A. and of stable full-consensus ankyrin repeat proteins.J. Mol. Biol. domain based on an DARPin domain to C. E. J.M. D. Plückthun A. A designed ankyrin repeat protein to affinity to Mol. Biol. domain based on an DARPin domain of the protein of abicipar WHO Drug Information. Vol. WHO, Geneva, domain based on the DARPin domain of ensovibep (10ensovibepum: WHO Drug Information. Vol. 34. WHO, Geneva, Switzerland2020: 968-970Google Scholar). an to the by Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. a of of all domain in a a of of all domain By analysis of the DARPin we identified the to be of potential for the repeat and for the overall domain are at the and of the or at the and the that a mutation improves the thermostability of we analysis on and Asp17 on an the to is possible to the stable to of and five showed in with the from °C to °C and the melting temperature at However, the mutation showed a of the from °C to °C we alternative at the position 17 of and and that from the of protein in a with the for the and Asp17Leu from °C to °C, °C, and °C, Overall, Asp17 in to Val, Leu, Ile, Met, Ala, or to an of the 8 °C to 10 that is an at position and that are alternative in a thermostability these alternative Asp17Leu provided of the which we of DARPin domains by °C and all at a of 10 in to The °C for the °C for the °C for the and °C for the the Asp17Leu has a of °C and is with DARPin, designed ankyrin repeat of various at position in a the improvements derived from the Asp17 are and of the we transferred the Asp17Leu mutation the and the that in from The Asp17Leu mutation the thermostability of in the and by more than °C we in based on the and the based on the Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar) be We that the in with a in a of about °C or 9 °C in an or the benefits of the Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar). In the of the with the in that the improvements of are and the to °C in that by about the of Asp17Leu in we to °C when we the of this in we the for and in a and of °C and °C, the very and the could be by the Asp17Leu mutation to in a of about °C in Overall, the Asp17Leu mutation about 9 °C to °C to the of of indicating that this is a for DARPin of or at position 17 of the in or in a of or at position 17 of the in and in in in a We simulations to the for the or at position 17 of from the of ankyrin repeat proteins of and A. Binz H.K. Forrer P. Stumpp M.T. Plückthun A. Grütter M.G. Designed to be of a consensus ankyrin repeat U. A. 2003; Scholar), Plückthun A. Grütter M.G. for of designed ankyrin repeat proteins with a Mol. Biol. Scholar), and Plückthun A. Grütter M.G. for of designed ankyrin repeat proteins with a Mol. Biol. Scholar), we Asp17 with in the and the and and the with the and the in be for which is of the that for N-terminal the for In the to the position 17 are for and for and from the three simulations for two at and at for a of can be from the simulations and the of at position 17 with in all to with the the analysis on the protein at high and on two and that the at position 17 than Asp17 The of the Asp17Leu mutation are more on the than on the The the full protein the in the and the at the The of the of the is for The in the of the are less as to The of the as with Asp17 is in with the increased thermostability of Asp17Leu DARPins in on the the Asp17Leu mutation reduces in the the and in of the flexible of a DARPin domain from the of the to the at the of the internal In the and the the Asp17Leu mutation are more in the of position The be to the mutation of the the protein are in the and the and which be by the by the that position 17 is an with to the and the overall DARPin domain 17 and the of the in in analysis was on the of at in in a The analysis was on the of at the thermostability derived from the Asp17Leu is on the of in the DARPin present in the and to DARPin, we this mutation on binders selected growth C. E. J.M. D. Plückthun A. A designed ankyrin repeat protein to affinity to Mol. Biol. Scholar), vascular endothelial growth A WHO Drug Information. Vol. WHO, Geneva, Scholar), and T. Stumpp M.T. Binz H.K. D. Forrer P. D. M. J. Designed ankyrin repeat protein domains with binding for Scholar) The selected DARPin domains are a mutation in as in and and are clinically validated DARPin domains, as are present in abicipar and ensovibep, In all the three the Asp17Leu mutation increased the thermostability of the DARPin domains and to about °C in Overall, the in thermostability of the Asp17Leu mutation to be and is to and and selected for high including clinically validated DARPin of DARPin binding to and at in a The in this position 17 of the DARPin as a key position thermostability of DARPins. The of the capping repeats as a for the high and of the has been the of this antibody M. Binz H.K. Plückthun A. and of stable full-consensus ankyrin repeat proteins.J. Mol. Biol. Scholar, A. Binz H.K. Forrer P. Stumpp M.T. Plückthun A. Grütter M.G. Designed to be of a consensus ankyrin repeat U. A. 2003; Scholar). the has been for for Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar), an of the in the with of has been The of the capping repeats to the overall of the DARPin domain is M. Binz H.K. Plückthun A. and of stable full-consensus ankyrin repeat proteins.J. Mol. Biol. Scholar). The of repeat protein domains on in which is a fundamental to In repeat and are a repeat and In to the capping repeats have repeat of ankyrin repeats in 2003; Scholar) and designed ankyrin repeat proteins M. Binz H.K. Plückthun A. and of stable full-consensus ankyrin repeat proteins.J. Mol. Biol. Scholar) have been shown to in a with the capping to an internal repeat a in of the repeat the and of DARPins to the caps of H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google and be for the capping repeats can the overall of a DARPin as was shown for the and the Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar). The of a mutation when with the thermostability of DARPin a than Leu, into the at the the and the internal mutation has the that an from the DARPin of and on the of biologics is H. of therapeutic Res. 2004; Scholar). We a analysis of the for that the overall DARPin domain in silico we of DARPins that position 17 of the is an of a DARPin domain and that the at position 17 of the is to overall DARPin thermostability. We showed that the on thermostability of Asp17 can be by to Leu, Val, Ile, Ala, Met, or to a in by about 8 °C to 16 °C on the Importantly, the of an the of was increased by about 9 °C when the Asp17Leu the beneficial of this mutation with the of about °C for Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar) that the is a for the thermostability of the DARPin that this is the case for the The position 17 is at the of two at the of the the and the the are at the N-terminal of as protein for at the of Scholar), which the of at position The DARPin that the of Asp17 can and be in the and or into the Asp17 is is in van-der-Waals with to the and of and is with a which Asp17 the from the to the The in are with the of of Asp17 with the of the protein The of at and the 17 and at that in Asp17 the of the Asp17 and is as the electrostatic to the Importantly, the simulations that at position 17 and that the Asp17Leu mutation improves the and the van-der-Waals by about and and The of are with the in thermostability by Importantly, the of about 8 °C to 16 °C on the that was by the Asp17Leu mutation to be generically applicable to various as by and could be on a of on the DARPin The the of the two domains of ensovibep, of the are in to and present in the binding domain of abicipar the Asp17Leu mutation was beneficial in the of the Asp17Leu a when transferred to three DARPin binders directed C. E. J.M. D. Plückthun A. A designed ankyrin repeat protein to affinity to Mol. Biol. Scholar), WHO Drug Information. Vol. WHO, Geneva, Scholar), or T. Stumpp M.T. Binz H.K. D. Forrer P. D. M. J. Designed ankyrin repeat protein domains with binding for Scholar), indicating that the is of the the DARPin of the Asp17Leu mutation be of the that all DARPin domains are based on a the A. Binz H.K. Forrer P. Stumpp M.T. Plückthun A. Grütter M.G. Designed to be of a consensus ankyrin repeat U. A. 2003; Scholar). The for this of the is the consensus design of in the of (1Forrer P. Stumpp M.T. Binz H.K. Plückthun A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins.FEBS Lett. 2003; 539: 2-6Google Scholar, 2Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar, P. Binz H.K. Stumpp M.T. Plückthun A. design of repeat 2004; Scholar). However, the DARPin domains and that which are key for binding still from the Asp17Leu that this single be beneficial for DARPin domains in when these have a on the domain The of of DARPins in with have shown that and to the DARPin in of the A. H. Grütter M.G. and of a DARPin with Scholar). 17 is of these and based that the overall of the domain the Asp17Leu mutation is we that the Asp17Leu has a on the binding of DARPin of the Asp17Leu mutation into DARPin binders could thermostability The by this novel a of 8 °C to 16 °C for a DARPin is in to that for the by Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar). the and in an a of °C when the of the novel mutation Asp17Leu is to the the mutation with the DARPins (1Forrer P. Stumpp M.T. Binz H.K. Plückthun A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins.FEBS Lett. 2003; 539: 2-6Google Scholar, 2Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar), in the two improvements at the the by 10 °C) and the at the can a of °C for a DARPin Asp17Leu to capping repeat improvements We the of DARPins as the for the modular of thermostability improvements DARPins are the of repeat that are yet are by with and repeat to the overall domain M. Binz H.K. Plückthun A. and of stable full-consensus ankyrin repeat proteins.J. Mol. Biol. Scholar). DARPins a of key beneficial for drug development from to and clinical development and high high and the high drug to (9Stumpp M.T. Dawson K.M. Binz H.K. Beyond antibodies: The DARPin drug platform.BioDrugs. 2020; 34: 423-433Google Scholar). modular DARPins H.K. A. C. T. Fiedler U. Stumpp M.T. and of a DARPin drug Scholar) can be with a single and as the binding domains are Their high and high with and are the for Their high in and for and very to protein to molecules with novel of such as K.M. M. J. D. of in Scholar). these DARPins therapeutic antibodies and the of innovative drugs the of and applications that can be conceived and with this on the of this an increased thermostability as in this have a on A recent of drug development at is the development of the anti-SARS-CoV-2 DARPin ensovibep that from of binders to entry into the in less than 9 months M. J. H. A. C. V. A. T. A. T. DARPin very high SARS-CoV-2 in Scholar). Interestingly, ensovibep the Asp17Leu mutation in three of five DARPin domains (10ensovibepum: WHO Drug Information. Vol. 34. WHO, Geneva, Switzerland2020: 968-970Google Scholar). clinical validation to the Asp17Leu the and of the The high thermostability of °C and of for to °C) for ensovibep M. A. V. Villemagne D. M. T. A. A. P. M. M. M. anti-SARS-CoV-2 therapeutic 2020; Scholar) be partly by the of the Asp17Leu beneficial are as this drug is of five DARPin domains with on a single In an drug with require to be at that Molecular is that ensovibep has the potential to and that a alternative to antibody for M. J. H. A. C. V. A. T. A. T. DARPin very high SARS-CoV-2 in Scholar). the three DARPin domains binding to three of the of that all Asp17Leu in ensovibep is in of two DARPins for two domains are to of by the overall of ensovibep be by replacement of Asp17 with Leu, Val, Ile, Ala, Met, or in the of the two DARPins. of to the ankyrin repeat domain of abicipar that is a generation DARPin drug to patients with clinical trials and that abicipar is to showed a of or in than D. A. H. E. M. J. and of abicipar in of 2020; Scholar). The of these that the abicipar was to proteins with the to the of by the thermostability of the ankyrin repeat domain of abicipar could be increased by about °C when with the A abicipar be to more that in with We have shown a of various DARPin domains to 16 °C) by Asp17 in the with Leu, Val, Ile, Ala, Met, or Thr. for the of the capping repeats for the and of this with the by Plückthun A. A. and of designed ankyrin repeat proteins by dynamics simulations and Mol. Biol. Scholar) and the melting temperature of DARPin domains by about the initial DARPin design (1Forrer P. Stumpp M.T. Binz H.K. Plückthun A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins.FEBS Lett. 2003; 539: 2-6Google Scholar, 2Binz H.K. Stumpp M.T. Forrer P. Amstutz P. Plückthun A. Designing repeat proteins: Well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins.J. Mol. Biol. 2003; 332: 489-503Google Scholar) is by the research and is clinically validated this of the that the capping repeats of the which based on have high that can be by a in the capping antibody mimetics could the for the development of innovative increased thermostability of biologics is to with a H. M. B. P. T. protein thermostability using a the Commun. Scholar). we an of DARPins the the thermostability DARPins more to protein We that this is of high when the of a drug to be for to the of K.M. M. J. D. of in Scholar). the increased thermostability into more development and and and to the of The recent development of the DARPin ensovibep the Asp17Leu in less than 9 months from to entry into the this potential M. A. V. Villemagne D. M. T. A. A. P. M. M. M. anti-SARS-CoV-2 therapeutic 2020; Scholar, M. J. H. A. C. V. A. T. A. T. DARPin very high SARS-CoV-2 in Scholar). the we that a high thermostability the development of or of DARPin that be of for the delivery of ensovibep for the of In drug development for more and very and we that DARPins be the scaffold for

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