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• W3132168463 abstract "The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability–function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics. The fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold, which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability–function trade-off commonly associated with evolving a protein domain toward biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics. Developing a small, simple protein domain that includes a similar sized binding region to antibody complementarity determining regions (CDRs) is a successful strategy for overcoming the complexity of antibody structure. Non-antibody scaffolds are single domains, typically smaller than 20 kDa in molecular weight, and mostly free of glycosylation or disulfide bonds that require eukaryotic expression (1Crook Z.R. Nairn N.W. Olson J.M. Miniproteins as a powerful modality in drug development.Trends Biochem. Sci. 2020; 45: 332-346Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Critically, they exhibit comparable binding affinities to antibodies (2Vazquez-Lombardi R. Phan T.G. Zimmermann C. Lowe D. Jermutus L. Christ D. Challenges and opportunities for non-antibody scaffold drugs.Drug Discov. Today. 2015; 20: 1271-1283Crossref PubMed Scopus (132) Google Scholar). Monobodies based on the fibronectin type 3 domain (FN3) are a popular scaffold for developing non-antibody therapeutics (2Vazquez-Lombardi R. Phan T.G. Zimmermann C. Lowe D. Jermutus L. Christ D. Challenges and opportunities for non-antibody scaffold drugs.Drug Discov. Today. 2015; 20: 1271-1283Crossref PubMed Scopus (132) Google Scholar, 3Koide S. Koide A. Lipovsek D. Target-binding proteins based on the 10th human fibronectin type III domain (10Fn3).Methods Enzymol. 2012; 503: 135-156Crossref PubMed Scopus (62) Google Scholar, 4Hantschel O. Monobodies as possible next-generation protein therapeutics – a perspective.Swiss Med. Wkly. 2017; 147w14545PubMed Google Scholar, 5Chandler P.G. Buckle A.M. Development and differentiation in monobodies based on the fibronectin type 3 domain.Cells. 2020; 9: 610Crossref Scopus (15) Google Scholar). The FN3 domain has an Ig-like fold and thus retains three of the CDR-like loops of an antibody variable fragment, but is structurally simple enough to be engineered for advanced non-antibody functions as the monobody scaffold (4Hantschel O. Monobodies as possible next-generation protein therapeutics – a perspective.Swiss Med. Wkly. 2017; 147w14545PubMed Google Scholar, 5Chandler P.G. Buckle A.M. Development and differentiation in monobodies based on the fibronectin type 3 domain.Cells. 2020; 9: 610Crossref Scopus (15) Google Scholar, 6Lipovsek D. Adnectins: Engineered target-binding protein therapeutics.Protein Eng. Des. Sel. 2011; 24: 3-9Crossref PubMed Scopus (174) Google Scholar, 7Koide A. Bailey C.W. Huang X. Koide S. The fibronectin type III domain as a scaffold for novel binding proteins.J. Mol. Biol. 1998; 284: 1141-1151Crossref PubMed Scopus (389) Google Scholar). There are a number of unique monobody derivates in active development including clinical Adnectins by LIB therapeutics (8Stein E. Toth P. Butcher M. Kereiakes D. Magnu P. Bays H. Zhou R. Turner T.A. Safety, tolerability and Ldl-C reduction with A novel anti-Pcsk9 recombinant fusion protein (Lib003): Results of A randomized, double-blind, placebo-controlled, phase 2 study.Atherosclerosis. 2019; 287e7Abstract Full Text Full Text PDF Google Scholar) or ViiV Healthcare (9Wensel D. Sun Y. Davis J. Li Z. Zhang S. McDonagh T. Langley D. Mitchell T. Tabruyn S. Nef P. Cockett M. Krystal M. GSK3732394: A multi-specific inhibitor of HIV entry.J. Virol. 2019; 93: 1-21Crossref Scopus (7) Google Scholar), the stability-enhanced Centyrins under ARO therapeutics (10Addis, R. C., Kolakowski, R., Kulkarni, S., Gorsky, J., Meyer, R., Xin, Y., Mortezavi, E., O’Neil, K. T., and Nadler, S. G. Tumor-targeted knockdown of KRAS mutants with novel Centyrin:siRNA conjugates [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 April 27-28 and June 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1825.Google Scholar, 11Addis, R., Kolakowski, R., Kulkarni, S., Gorsky, J., Meyer, R., and ONeil, K. Abstract 4830: ABX9xx: A bispecific centyrin that synergizes to attenuate intracellular signaling in Met/EGFR positive tumors. IN: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 March 29-April 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract 4830.Google Scholar) or in a CAR-T format by Poseida Therapeutics (12Gregory, T. K., Berdeja, J. G., Patel, K. K., Ali, S. A., Cohen, A. D., Costello, C., Ostertag, E. M., Silva, N. de, Shedlock, D. J., Resler, M., Spear, M. A., and Orlowski, R. Z. Clinical trial of P-BCMA-101 T stem cell memory (Tscm) CAR-T cells in relapsed/refractory (r/r) multiple myeloma (MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 April 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT130.Google Scholar), and also the related TN3 monobodies under Viela Bio (13Karnell J.L. Albulescu M. Drabic S. Wang L. Moate R. Baca M. Oganesyan V. Gunsior M. Thisted T. Yan L. Li J. Xiong X. Eck S.C. De Los Reyes M. Yusuf I. et al.A CD40L-targeting protein reduces autoantibodies and improves disease activity in patients with autoimmunity.Sci. Transl. Med. 2019; 11eaar6584Crossref PubMed Scopus (32) Google Scholar). An important consideration in the ability to evolve a non-antibody scaffold for binding is the combination of a high initial stability and a mutationally robust framework. The small size and lack of redundant framework regions in non-antibody scaffolds result in protein domains that will accumulate only a few mutations to their variable regions before stability becomes compromised (2Vazquez-Lombardi R. Phan T.G. Zimmermann C. Lowe D. Jermutus L. Christ D. Challenges and opportunities for non-antibody scaffold drugs.Drug Discov. Today. 2015; 20: 1271-1283Crossref PubMed Scopus (132) Google Scholar, 14Löfblom J. Frejd F.Y. Ståhl S. Non-immunoglobulin based protein scaffolds.Curr. Opin. Biotechnol. 2011; 22: 843-848Crossref PubMed Scopus (109) Google Scholar). Most monobody derivatives typically lose ∼40 °C of thermostability upon evolution for binding (2Vazquez-Lombardi R. Phan T.G. Zimmermann C. Lowe D. Jermutus L. Christ D. Challenges and opportunities for non-antibody scaffold drugs.Drug Discov. Today. 2015; 20: 1271-1283Crossref PubMed Scopus (132) Google Scholar), which often results in insoluble expression in bacteria that must be resolved through later rounds of evolution (15Olson C.A. Liao H.I. Sun R. Roberts R.W. mRNA display selection of a high-affinity, modification-specific phospho-iκbα-binding fibronectin.ACS Chem. Biol. 2008; 3: 480-485Crossref PubMed Scopus (38) Google Scholar). Critical to the design of next-generation therapeutics, poor biophysical properties such as thermostability and poor or insoluble expression hinder scaffold “developability” and correlate to higher risk of failure in during clinical development (16Jain T. Sun T. Durand S. Hall A. Houston N.R. Nett J.H. Sharkey B. Bobrowicz B. Caffry I. Yu Y. Cao Y. Lynaugh H. Brown M. Baruah H. Gray L.T. et al.Biophysical properties of the clinical-stage antibody landscape.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 944-949Crossref PubMed Scopus (193) Google Scholar, 17Jarasch A. Koll H. Regula J.T. Bader M. Papadimitriou A. Kettenberger H. Developability assessment during the selection of novel therapeutic antibodies.J. Pharm. Sci. 2015; 104: 1885-1898Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). There are multiple approaches of improving the stability of protein folds (18Chandler P.G. Broendum S.S. Riley B.T. Spence M.A. Jackson C.J. McGowan S. Buckle A.M. Strategies for increasing protein stability.Methods Mol. Biol. 2020; 2073: 163-181Crossref PubMed Scopus (7) Google Scholar). Protein stability exists between two critical thresholds, where a protein is able to fold into a stable, native three-dimensional shape but is also still dynamic enough to perform its functions (19Stimple S.D. Smith M.D. Tessier P.M. Directed evolution methods for overcoming trade-offs between protein activity and stability.AIChE J. 2020; 66e16814Crossref PubMed Scopus (11) Google Scholar, 20Petrovic D. Risso V.A. Kamerlin S.C.L. Sanchez-Ruiz J.M. Conformational dynamics and enzyme evolution.J. R. Soc. Interface. 2018; 15: 20180330Crossref PubMed Scopus (68) Google Scholar). As proteins evolve over time, the resulting mutations are more likely to be detrimental to stability of the native fold than neutral or positive and may reduce protein stability below the folding threshold (21Tokuriki N. Stricher F. Schymkowitz J. Serrano L. Tawfik D.S. The stability effects of protein mutations appear to be universally distributed.J. Mol. Biol. 2007; 369: 1318-1332Crossref PubMed Scopus (270) Google Scholar). This is often considered as a natural trade-off between stability and activity and can be a severe limitation to directed evolution experiments (22Tokuriki N. Tawfik D.S. Stability effects of mutations and protein evolvability.Curr. Opin. Struct. Biol. 2009; 19: 596-604Crossref PubMed Scopus (444) Google Scholar, 23Bloom J.D. Labthavikul S.T. Otey C.R. Arnold F.H. Protein stability promotes evolvability.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5869-5874Crossref PubMed Scopus (748) Google Scholar). Therefore, as a protein scaffold takes on mutations to a variable region to improve binding, it is at risk of deleterious losses in thermostability. Accordingly, proteins with improved initial thermostability are able to sample a larger proportion of this destabilizing sequence space as neutral mutations—they are more mutationally robust—which in turn increases the chance of reaching novel functions (24Besenmatter W. Kast P. Hilvert D. Relative tolerance of mesostable and thermostable protein homologs to extensive mutation.Proteins. 2006; 66: 500-506Crossref Scopus (51) Google Scholar, 25Romero P.A. Arnold F.H. Exploring protein fitness landscapes by directed evolution.Nat. Rev. Mol. Cell Biol. 2009; 10: 866-876Crossref PubMed Scopus (587) Google Scholar, 26Ota N. Kurahashi R. Sano S. Takano K. The direction of protein evolution is destined by the stability.Biochimie. 2018; 150: 100-109Crossref PubMed Scopus (9) Google Scholar, 27Zheng J. Guo N. Wagner A. Selection enhances protein evolvability by increasing mutational robustness and foldability.Science. 2020; 370eabb5962Crossref PubMed Scopus (12) Google Scholar). In this way, prestabilization can enhance both evolvability—the ability of a protein to evolve new functions (23Bloom J.D. Labthavikul S.T. Otey C.R. Arnold F.H. Protein stability promotes evolvability.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5869-5874Crossref PubMed Scopus (748) Google Scholar), and developability—the biophysical likelihood of successful development from a lead protein into a therapeutic drug (17Jarasch A. Koll H. Regula J.T. Bader M. Papadimitriou A. Kettenberger H. Developability assessment during the selection of novel therapeutic antibodies.J. Pharm. Sci. 2015; 104: 1885-1898Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). From a drug development perspective, the native thermostability of a protein correlates with expression titre and improves critical quality attributes such as shelf-life (16Jain T. Sun T. Durand S. Hall A. Houston N.R. Nett J.H. Sharkey B. Bobrowicz B. Caffry I. Yu Y. Cao Y. Lynaugh H. Brown M. Baruah H. Gray L.T. et al.Biophysical properties of the clinical-stage antibody landscape.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 944-949Crossref PubMed Scopus (193) Google Scholar), while also expanding the range of storage formulations which can be used in a drug product (28Falconer R.J. Advances in liquid formulations of parenteral therapeutic proteins.Biotechnol. Adv. 2019; 37: 107412Crossref PubMed Scopus (19) Google Scholar). Biological formulations need to remain stable for at least 2 years at 5 °C, and storage buffers are usually applied to reach this target. This has resulted in a standard set of buffer formulations across industry that are well-validated and focused on reducing aggregation or other loss of active protein (29Falconer R.J. Chan C. Hughes K. Munro T.P. Stabilization of a monoclonal antibody during purification and formulation by addition of basic amino acid excipients.J. Chem. Technol. Biotechnol. 2011; 86: 942-948Crossref Scopus (40) Google Scholar). However, this sole focus on protein stability limits the exploration of nonstandard formulations, which benefit other critical quality attributes (28Falconer R.J. Advances in liquid formulations of parenteral therapeutic proteins.Biotechnol. Adv. 2019; 37: 107412Crossref PubMed Scopus (19) Google Scholar), such as controlling viscosity or osmolarity to lower the pain associated with injection (30Berteau C. Filipe-Santos O. Wang T. Roja H.E. Granger C. Schwarzenbach F. Evaluation of the impact of viscosity, injection volume, and injection flow rate on subcutaneous injection tolerance.Med. Devices (Auckl.). 2015; 8: 473-484PubMed Google Scholar, 31Wang W. Tolerability of hypertonic injectables.Int. J. Pharm. 2015; 490: 308-315Crossref PubMed Scopus (44) Google Scholar, 32Zbacnik T.J. Holcomb R.E. Katayama D.S. Murphy B.M. Payne R.W. Coccaro R.C. Evans G.J. Matsuura J.E. Henry C.S. Manning M.C. Role of buffers in protein formulations.J. Pharm. Sci. 2017; 106: 713-733Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Multiple, well-established techniques use phylogenetic information to derive thermostable homologs for a given protein family (18Chandler P.G. Broendum S.S. Riley B.T. Spence M.A. Jackson C.J. McGowan S. Buckle A.M. Strategies for increasing protein stability.Methods Mol. Biol. 2020; 2073: 163-181Crossref PubMed Scopus (7) Google Scholar, 33Wilding M. Hong N. Spence M. Buckle A.M. Jackson C.J. Protein engineering: The potential of remote mutations.Biochem. Soc. Trans. 2019; 47: 701-711Crossref PubMed Scopus (23) Google Scholar, 34Porebski B.T. Buckle A.M. Consensus protein design.Protein Eng. Des. Sel. 2016; 29: 245-251Crossref PubMed Scopus (77) Google Scholar, 35Kumar S. Nussinov R. How do thermophilic proteins deal with heat?.Cell Mol. Life Sci. 2001; 58: 1216-1233Crossref PubMed Scopus (352) Google Scholar, 36Goldenzweig A. Fleishman S. Principles of protein stability and their application in computational design.Annu. Rev. Biochem. 2018; 87: 105-129Crossref PubMed Scopus (84) Google Scholar, 37Sternke M. Tripp K.W. Barrick D. Consensus sequence design as a general strategy to create hyperstable, biologically active proteins.Proc. Natl. Acad. Sci. U. S. A. 2019; 166: 11275-11284Crossref Scopus (30) Google Scholar). Here we have specifically applied consensus design, which identifies conserved residues within a protein family that are likely to be stabilizing (34Porebski B.T. Buckle A.M. Consensus protein design.Protein Eng. Des. Sel. 2016; 29: 245-251Crossref PubMed Scopus (77) Google Scholar, 37Sternke M. Tripp K.W. Barrick D. Consensus sequence design as a general strategy to create hyperstable, biologically active proteins.Proc. Natl. Acad. Sci. U. S. A. 2019; 166: 11275-11284Crossref Scopus (30) Google Scholar). Previously, we used consensus design to produce the prestabilized FN3Con monobody, which has substantially increased thermostability compared with similar fibronectin domains (38Porebski B.T. Nickson A.A. Hoke D.E. Hunter M.R. Zhu L. McGowan S. Webb G.I. Buckle A.M. Structural and dynamic properties that govern the stability of an engineered fibronectin type III domain.Protein Eng. Des. Sel. 2015; 28: 67-78Crossref PubMed Scopus (26) Google Scholar). The mutational robustness of FN3Con was then demonstrated by grafting binding loops from an FN3 scaffold that had been selected by laboratory evolution to bind lysozyme with high affinity (39Porebski B.T. Conroy P.J. Drinkwater N. Schofield P. Vazquez-Lombardi R. Hunter M.R. Hoke D.E. Christ D. McGowan S. Buckle A.M. Circumventing the stability-function trade-off in an engineered FN3 domain.Protein Eng. Des. Sel. 2016; 29: 1-9Crossref PubMed Scopus (10) Google Scholar). Whereas binding function was completely transferred, the trade-off in thermostability was negligible compared with that which occurred upon directed evolution of the original scaffold. In the current study, we aimed to establish whether FN3Con can harbor valuable loop sequences that confer clinical inhibition of a target but were detrimental to stability in established scaffolds. For this study we chose an Adnectin domain that was previously subjected to directed evolution for high-affinity binding to the therapeutic target VEGFR2, with function gained at the expense of large losses in thermostability and increased oligomerization propensity (40Parker M.H. Chen Y. Danehy F. Dufu K. Ekstrom J. Getmanova E.V. Gokemeijer J. Xu L. Lipovsek D. Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two.Protein Eng. Des. Sel. 2005; 18: 435-444Crossref PubMed Scopus (69) Google Scholar, 41Mamluk R. Carvajal I.M. Morse B.A. Wong H.K. Abramowitz J. Aslanian S. Lim A.-C. Gokemeijer J. Storek M.J. Lee J. Gosselin M. Wright M.C. Camphausen R.T. Wang J. Chen Y. et al.Anti-tumor effect of CT-322 as an Adnectin inhibitor of vascular endothelial growth factor receptor-2.mAbs. 2010; 2: 199-208Crossref PubMed Scopus (57) Google Scholar). We grafted binding loops from Adnectin-anti-VEGFR2 onto the FN3Con scaffold to produce a recombined domain that retained binding affinity. The FN3Con-anti-VEGFR2 graft was expressed in E. coli with little aggregation and maintained characteristically high thermostability, including 24-month stability at 37 °C. An early exploration of buffer excipients produced further stability improvements. We discuss the implications of generating clinical leads by salvaging loop sequences from scaffolds with challenging biophysical features and the importance of designing highly “evolvable” constructs on downstream factors of scaffold “developability.” We chose the Adnectin-anti-VEGFR2 monobody “CT-322” as a candidate for loop grafting to the hyperstable FN3Con in order to test our hypothesis that a stabilized scaffold can rescue stability losses accrued after evolutionary selection for high-affinity binding. This Adnectin was generated from mRNA display against a construct of seven extracellular domains of VEGFR2 fused with a human antibody Fc region, generating high-affinity binding (KD = 0.31 nM) but with a 34 °C loss in thermostability (Tm) and also at cost to oligomerization resistance (40Parker M.H. Chen Y. Danehy F. Dufu K. Ekstrom J. Getmanova E.V. Gokemeijer J. Xu L. Lipovsek D. Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two.Protein Eng. Des. Sel. 2005; 18: 435-444Crossref PubMed Scopus (69) Google Scholar, 41Mamluk R. Carvajal I.M. Morse B.A. Wong H.K. Abramowitz J. Aslanian S. Lim A.-C. Gokemeijer J. Storek M.J. Lee J. Gosselin M. Wright M.C. Camphausen R.T. Wang J. Chen Y. et al.Anti-tumor effect of CT-322 as an Adnectin inhibitor of vascular endothelial growth factor receptor-2.mAbs. 2010; 2: 199-208Crossref PubMed Scopus (57) Google Scholar). Given the absence of structural information for the binding mechanism of Adnectin-anti-VEGFR2 to its large multidomain target, we used previously established loop sequence boundaries (39Porebski B.T. Conroy P.J. Drinkwater N. Schofield P. Vazquez-Lombardi R. Hunter M.R. Hoke D.E. Christ D. McGowan S. Buckle A.M. Circumventing the stability-function trade-off in an engineered FN3 domain.Protein Eng. Des. Sel. 2016; 29: 1-9Crossref PubMed Scopus (10) Google Scholar) to guide the transfer of evolved binding loops to FN3Con, designing FN3Con-anti-VEGFR2 [Fig. 1A and B]. Additionally, the entire Adnectin C-terminal tail was reported to be critical to high-affinity binding and was also transferred (40Parker M.H. Chen Y. Danehy F. Dufu K. Ekstrom J. Getmanova E.V. Gokemeijer J. Xu L. Lipovsek D. Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two.Protein Eng. Des. Sel. 2005; 18: 435-444Crossref PubMed Scopus (69) Google Scholar). FN3Con-anti-VEGFR2 displayed high-affinity binding to VEGFR2 (KD = 0.72 nM) [Table 1], very similar to the published affinity of Adnectin-anti-VEGFR2 (KD = 0.31 nM) (41Mamluk R. Carvajal I.M. Morse B.A. Wong H.K. Abramowitz J. Aslanian S. Lim A.-C. Gokemeijer J. Storek M.J. Lee J. Gosselin M. Wright M.C. Camphausen R.T. Wang J. Chen Y. et al.Anti-tumor effect of CT-322 as an Adnectin inhibitor of vascular endothelial growth factor receptor-2.mAbs. 2010; 2: 199-208Crossref PubMed Scopus (57) Google Scholar). We carried out affinity measurement through an orthogonal approach, where two independent methods provided a KD range of 0.72 to 48.79 nM [Fig. 1, C and D and Table 1], with the KD of 0.72 nM derived from Biacore data presenting the most robust fits to derive underlying equilibrium constants while controlling for confounding nonspecific binding and mass transport effects. The ELISA data validated this 2- to 3-fold difference in affinity between binders, although nonspecific binding likely increased the measured KD for both monobodies.Table 1Methodology and results for VEGFR2 binding experiments in Figure 1ProteinSurface plasmon resonance (SPR) KDELISA KDAdnectin-anti-VEGFR20.31 nMaSPR KD from (41), ELISA values are KD ± SE.16.87 nM ± 4.26FN3Con-anti-VEGFR20.72 nM ±0.2148.79 nM ± 6.78Analysis Fold Difference2.322.89 FN3Con-anti-VEGFR2 Model FitR2 = 0.95R2 = 0.98 Immobilized proteinVEGFR2VEGFR2 MeasureChange in refractive indexConcentration of Biotinylated proteina SPR KD from (41Mamluk R. Carvajal I.M. Morse B.A. Wong H.K. Abramowitz J. Aslanian S. Lim A.-C. Gokemeijer J. Storek M.J. Lee J. Gosselin M. Wright M.C. Camphausen R.T. Wang J. Chen Y. et al.Anti-tumor effect of CT-322 as an Adnectin inhibitor of vascular endothelial growth factor receptor-2.mAbs. 2010; 2: 199-208Crossref PubMed Scopus (57) Google Scholar), ELISA values are KD ± SE. Open table in a new tab The Adnectin-anti-VEGFR2 undergoes irreversible thermal denaturation with a Tm of 50 °C ± 0.4 °C, as measured by circular dichroism (CD), with visible precipitate upon cooling [Fig. 2A]. In striking contrast, FN3Con-anti-VEGFR2 unfolds reversibly with a Tm of 89 °C ± 0.2 °C [Fig. 2B]. The Tm of the Adnectin-anti-VEGFR2 was not previously published (41Mamluk R. Carvajal I.M. Morse B.A. Wong H.K. Abramowitz J. Aslanian S. Lim A.-C. Gokemeijer J. Storek M.J. Lee J. Gosselin M. Wright M.C. Camphausen R.T. Wang J. Chen Y. et al.Anti-tumor effect of CT-322 as an Adnectin inhibitor of vascular endothelial growth factor receptor-2.mAbs. 2010; 2: 199-208Crossref PubMed Scopus (57) Google Scholar), but our results closely match a precursor Adnectin-anti-VEGFR2 variant of similar affinity, thermostability, and loop sequences (40Parker M.H. Chen Y. Danehy F. Dufu K. Ekstrom J. Getmanova E.V. Gokemeijer J. Xu L. Lipovsek D. Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two.Protein Eng. Des. Sel. 2005; 18: 435-444Crossref PubMed Scopus (69) Google Scholar). This trajectory of loss in Adnectin thermostability presents the trade-offs that take place as affinity is further matured. In contrast, while the FN3Con scaffold loses ∼10 °C of thermostability after loop grafting, scaffold stability remains substantially higher than the parent Adnectin molecules, while also retaining reversible refolding. We next investigated the effect of FN3Con-anti-VEGFR2 hyperstability on long-term stability (LTS) and binding activity, as a proxy for extended shelf-life. The Adnectin-anti-VEGFR2 sample completely aggregates within 1 month’s storage at 36 °C in PBS [Fig. 2C]. In contrast, the FN3Con-anti-VEGFR2 remained in solution for at least 24 months, with ∼30% of the total sample remaining as a monomer after 2 years storage at 36 °C [Fig. 2D]. Up to 50% of the FN3Con-anti-VEGFR2 high-order species observed formed between 0 and 6 months at 36 °C, after which oligomer formation stabilized. Accordingly, FN3Con-anti-VEGFR2 presented extended LTS at 4 °C, remaining as a monomeric protein in extended trials up to 24 months of storage in PBS buffer [Fig. 2E]. Strikingly, high-affinity binding (KD ∼157 nM) to VEGFR2 was maintained after 24 months at 36 °C [Fig. 2F], although the observed affinity for the total sample is threefold lower than “fresh” FN3Con-anti-VEGFR2 (KD ∼49 nM). This suggests that only the ∼30% monomeric fraction retains binding affinity to the target. Given the results from accelerated stability testing, our final investigation explored the effect of stabilizing excipients on further improving the shelf-life properties of the FN3Con-anti-VEGFR2 construct. After incubation at 40 °C for 30 days with five different buffer excipients (16Jain T. Sun T. Durand S. Hall A. Houston N.R. Nett J.H. Sharkey B. Bobrowicz B. Caffry I. Yu Y. Cao Y. Lynaugh H. Brown M. Baruah H. Gray L.T. et al.Biophysical properties of the clinical-stage antibody landscape.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 944-949Crossref PubMed Scopus (193) Google Scholar), size-exclusion chromatography revealed that amino-acid excipients arginine, histidine, glycine, and aspartic acid produced a doubling of monomeric FN3Con-anti-VEGFR2 sample [Fig. 3]. Excipients such as Tween80 provide resistance to factors such as hydrophobic unfolding from shaking during storage (28Falconer R.J. Advances in liquid formulations of parenteral therapeutic proteins.Biotechnol. Adv. 2019; 37: 107412Crossref PubMed Scopus (19) Google Scholar, 42Agarkhed M. O’Dell C. Hsieh M.C. Zhang J. Goldstein J. Srivastava A. Effect of polysorbate 80 concentration on thermal and photostability of a monoclonal antibody.AAPS PharmSciTech. 2013; 14: 1-9Crossref PubMed Scopus (46) Google Scholar), and was added with no significantly greater detrimental effect on accelerated thermal stability than with PBS buffer alone. Previously, we have shown that FN3Con is robust to the stability–function trade-off (39Porebski B.T. Conroy P.J. Drinkwater N. Schofield P. Vazquez-Lombardi R. Hunter M.R. Hoke D.E. Christ D. McGowan" @default.
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• W3132168463 title "Mutational and biophysical robustness in a prestabilized monobody" @default.
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• W3132168463 doi "https://doi.org/10.1016/j.jbc.2021.100447" @default.
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