Systematic Activity Maturation of a Single-Domain Antibody with Non-canonical Amino Acids through Chemical Mutagenesis

•Chemical mutagenesis was pursued along the CDR3 loop of a single-domain antibody•Sites deemed accessible had diverse side chains screened for activity enhancement•Final mutant greatly enhanced and maintained other desired properties Great advances have been made over last four decades in therapeutic diagnostic applications antibodies. The maturation antibody candidates, however, remains significant challenge. To address this problem, we present method that enables systematic enhancement through post-translational installation non-canonical by chemical mutagenesis. We illustrate approach performing structure-activity relationship study beyond 20 naturally occurring amino acids on designed silico to inhibit aggregation amyloid-? peptide, process closely linked Alzheimer's disease. found can improve, five orders magnitude, anti-aggregation starting antibody, without affecting its stability. These results show expansion space available antibodies be exploited these molecules. Antibodies become cornerstone modern medicine biotechnology are increasingly used as agents wide range diseases (Carter Lazar, 2018Carter P.J. Lazar G.A. Next generation drugs: pursuit ‘high-hanging fruit’.Nat. Rev. Drug Disc. 2018; 17: 197Crossref PubMed Scopus (354) Google Scholar; Grilo Mantalaris, 2019Grilo A.L. Mantalaris A. human profitable monoclonal market.Trends Biotechnol. 2019; 37: 9-16Abstract Full Text PDF (215) Kaplon et al., 2020Kaplon, H., Muralidharan, M., Schneider, Z., Reichert, J.M. (2020). watch 2020. Paper presented at: MAbs (Taylor & Francis).Google Scholar). A arsenal technologies is currently utilized discovery, including immunization display methods (Boder 2000Boder E.T. Midelfort K.S. Wittrup K.D. Directed evolution fragments with monovalent femtomolar antigen-binding affinity.Proc. Natl. Acad. Sci. U S 2000; 97: 10701-10705Crossref (530) Bradbury 2011Bradbury A.R. Sidhu S. Dübel McCafferty J. Beyond natural antibodies: power vitro technologies.Nat. 2011; 29: 245Crossref (381) Hoogenboom, 2005Hoogenboom H.R. Selecting screening recombinant libraries.Nat. 2005; 23: 1105-1116Crossref (772) Miersch Sidhu, 2012Miersch Synthetic concepts, potential practical considerations.Methods. 2012; 57: 486-498Crossref (79) 2000Sidhu S.S. Phage pharmaceutical biotechnology.Curr. Op Biotechmol. 11: 610-616Crossref (204) Winter 1994Winter G. Griffiths A.D. Hawkins R.E. Hoogenboom Making phage technology.Annu. Immunol. 1994; 12: 433-455Crossref (1338) However, many procedures require amount time resources development fully functional One particular area has proven difficult their biological while preserving important properties, such epitope selectivity, conformational stability, solubility (Bradbury Plückthun, 2015Bradbury Plückthun Reproducibility: standardize research.Nature. 2015; 518: 27-29Crossref (401) Lerner, 2016Lerner R.A. Combinatorial libraries: new advances, immunological insights.Nat. 2016; 16: 498Crossref (59) Liu, 2014Liu J.K. history development—progress, remaining challenges future innovations.Ann. Med. Surg. 2014; 3: 113-116Crossref (223) high-throughput nature (up 1010 variants case display) exploration at various positions antibody-target interface (Sidhu, While leads, variety cases, discovery effective antibodies, further expand scope it would desirable able perform large molecules traditional (SAR) studies typically carried out small (Cherkasov 2014Cherkasov Muratov E.N. Fourches D. Varnek Baskin I.I. Cronin M. Dearden Gramatica P. Martin Y.C. Todeschini R. QSAR modeling: where you been? Where going to?.J. Chem. 4977-5010Crossref (957) Dobson, 2004Dobson C.M. Chemical Space Biology. Nature Publishing Group, 2004Crossref (703) Kolb 2001Kolb H.C. Finn Sharpless K.B. Click chemistry: function from few good reactions.Angew. Int. Ed. 2001; 40: 2004-2021Crossref (10908) Tropsha, 2010Tropsha Best practices model development, validation, exploitation.Mol. Inform. 2010; 476-488Crossref (1078) SAR allow accurate assessment effects brought about physicochemical changes structure, optimizing molecule atom-by-atom thanks synthetic medicinal chemistry Guha, 2013Guha On exploring relationships.in: In Silico Models Discovery. Humana Press, Totowa, NJ2013: 81-94Crossref (62) If same kind exquisite control chemists could achieved acid similarly rational key residues paratope enable direct path maturing initial candidates. At time, possible predict biophysical traits, thus offering more manageable system. genetic codon technology previously incorporate unnatural (UAAs) into unique ultimately relies methods, each UAA expression system only increase lexicon one residue (Chin 2003Chin J.W. Cropp T.A. Anderson J.C. Mukherji Zhang Z. Schultz P.G. An expanded eukaryotic code.Science. 2003; 301: 964-967Crossref (596) Lang Chin, 2014Lang K. Chin Cellular incorporation bioorthogonal labeling proteins.Chem. 114: 4764-4806Crossref (632) For efficiently explored, there should ideally rapid efficient site interest (Krall 2016Krall N. Da Cruz F.P. Boutureira O. Bernardes G.J. Site-selective protein-modification basic biology drug development.Nat. 8: 103Crossref (313) Prescher Bertozzi, 2005Prescher J.A. Bertozzi C.R. Chemistry living systems.Nat. Biol. 1: 13-21Crossref (1052) Wright 2016bWright T.H. Vallée M.R.J. Davis B.G. From post-expression mutagenesis: 50 year odyssey.Angew. 55: 5896-5903Crossref (25) Here, report use post-translationally installed synthetically versatile dehydroalanine (Dha) create platform precise augmentation inhibiting 42-residue form peptide (A?42), protein fragment associated disease (AD) (Hardy Selkoe, 2002Hardy Selkoe D.J. amyloid hypothesis disease: progress problems road therapeutics.Science. 2002; 297: 353-356Crossref (10472) Knowles 2014Knowles T.P. Vendruscolo Dobson state association misfolding diseases.Nat. Mol. Cell 15: 384-396Crossref (1388) Dha suitable intermediate side-chain due ease conversion cysteine precursors, well ability react bioorthogonally vast number reagents (Bernardes 2008Bernardes Chalker Errey Facile alkyl cysteines surfaces: switchable access functionalized proteins.J. Am. Soc. 2008; 130: 5052-5053Crossref (250) 2011Chalker Gunnoo S.B. Gerstberger S.C. Fernández-González Griffin L. Hailu H. Schofield C.J. Methods converting peptides 2: 1666-1676Crossref (222) Freedy 2017Freedy A.M. Matos M.J. Corzana F. Guerreiro Akkapeddi Somovilla V.J. Rodrigues T. Nicholls Xie B. Chemoselective amine bonds proteins aza-Michael ligation.J. 2017; 139: 18365-18375Crossref (54) Tamura Hamachi, 2018Tamura Hamachi I. covalent modification endogenous/native proteins: test tubes complex systems.J. 141: 2782-2799Crossref (110) 2016aWright Bower B.J. Wiewiora Ng W.-L. Raj Faulkner Phanumartwiwath Posttranslational strategy diversity.Science. 354: aag1465Crossref (139) Yang 2019Yang Wang Schnier P.D. Zheng Zhu Polizzi N.F. Ittuveetil Saikam V. DeGrado W.F. Q. Genetically introducing biochemically reactive dehydrobutyrine 7698-7703Crossref (34) indeed similar manner enhance enzyme (Windle 2017Windle C.L. Simmons K.J. Ault J.R. Trinh C.H. Nelson Pearson Berry Extending molecular recognition an alphabet.Proc. 2610-2615Crossref (21) Scholar) complementarity-determining region (CDR) loops nanobody Boolean logic gates response (Gunnoo 2014Gunnoo Finney H.M. Baker T.S. Lawson Anthony D.C. Creation gated conditionally protein.Nat. Commun. 5: 1-8Crossref (13) proof-of-concept study, (Jov?evska Muyldermans, 2020Jov?evska Muyldermans nanobodies.BioDrugs. 2020; 34: 11-26Crossref (184) Peyvandi 2016Peyvandi Scully Kremer Hovinga Cataland Knöbl Wu Artoni Westwood J.-P. Mansouri Taleghani Jilma Caplacizumab acquired thrombotic thrombocytopenic purpura.New Engl. 374: 511-522Crossref (296) Scholar), called DesAb-A?(3–9), carrying in-silico-designed grafted third CDR (CDR3) targeting N terminus (residues 3–9) A?42 (Aprile 2017Aprile F.A. Sormanni Perni Arosio Linse Selective primary secondary nucleation pathways using scanning method.Sci. Adv. e1700488Crossref (74) Scholar, Aprile 2020Aprile Podpolny Chhangur Needham L.-M. Ruggeri F.S. Limbocker Heller G.T. Sneideris Rational design conformation-specific quantification A? oligomers.Proc. 117: 13509-13518Crossref (23) 2015Sormanni specific epitopes within intrinsically disordered proteins.Proc. 112: 9902-9907Crossref (69) 2018Sormanni Third methods: design.Chem. 47: 9137-9157Crossref As fibrils fundamental processes underlying AD, inhibition emerged major AD (Knowles since consisting tightly coupled microscopic mechanisms, perturbation haphazard potentially exacerbate toxicity formation (Arosio 2014Arosio kinetics combat diseases.Trends Pharmacol. 35: 127-135Abstract (153) fluorescence-based assays framework elucidate inhibitory mechanism different variants. Thus, performed two successive rounds gain improvement magnitude rate aggregation, compromising stability antibody. describe work based three steps: (1) scan sequence select most modifications (Figure 1), (2) screen panel mutants potent 2), (3) 3).Figure 2Screen Panels Mutants Three Most Accessible Positions Loop Non-canonical Side ChainsShow full caption(A) List tested (E137, T138, L139) selected accessibility 1). confirmed LC-MS, validated structural integrity after circular dichroism (CD), assessed potency ThT fluorescence stoichiometries (1:2, 1:4, 1:8). cysteamine position T138 studies.(B C) Comparison profiles DesAb-A?(3–9) (T138cysteamine); continuous lines represent fit data integrated law aggregation. scatterplot represents apparent decoupled constants dilution antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Activity Maturation (T138cysteamine) Using Panel derivatives T138.(B) LC-MS confirming addition captamine (T138Dha).(C) CD (T138captamine).(D) Inhibitory profile (T138captamine); aggregation.(E) Scatterplot representing ratio rates varying equivalents (T138captamine).(F) knk+ 1:2 dilutions (black) (T138captamine) (turquoise).(G) thermal denaturation (turquoise), monitored signal 207 nm, indicate did not decrease (PPT) (A) studies. (B T138. (B) (T138Dha). (C) (T138captamine). (D) (E) (F) (turquoise). (G) Our inspiration came considering how More specifically, aimed extend potentiation 1A). end, excellent candidate, easily post-translationally, electrophile readily selectively modified array nucleophiles, importantly purpose minimal linking group just single carbon 1B). Many explored several precursors our efforts here, chose rely bis-alkylation/elimination reagent 2,5-dibromohexadiamide (DBHDA) shown camelid nanobodies added organic solvents (Chalker With approach, started characterized VH scaffold whose generated target linear peptides, 1C) incorporated newly identified thermally stabilizing mutations promote reaction sequences (Julian 2017Julian M.C. Li Garde Wilen Tessier P.M. Efficient affinity variable domains requires co-selection compensatory maintain thermodynamic stability.Sci. Rep. 7: 45259Crossref (51) install Dha, were created seven engineered loop, all expressed purified yields comparable construct. Conversion free first attempted treating 100-?M solutions (20 mM NaPi pH 8) 500 powdered DBHDA 37°C 5 h 1C). reactions end liquid chromatography-mass spectrometry (LC-MS) formation. By h, observed undergo complete (>95%) 1C), allowing us explore subsequently modify site. assess benchmark nucleophile, ?-mercaptoethanol, completion (50 ?M protein, 100 ?L total volume, [pH 8], 37°C). This investigation revealed that, rather uniform regardless position, subsequent Michael highly dependent local environment 1D). varied widely <15 min certain (E137Dha L139Dha) 6 others (R142Dha) feature explain some proximity negatively charged groups (D E), which unfavorable electrostatic interactions between carboxylic attacking thiolate anion. guide us, decided move forward (E137Dha, T138Dha, evaluation. Each ?-mercaptoethanol classes chains, charged, polar, hydrophobic, respectively. completely verify screen, conjugation sites LC-tandem MS (LC-MS/MS) S1). LC-MS/MS modification, disulfide bridge subjected treatment, no detected. creation mutants, chemistries thiol-Michael addition. example, initially piperidine-based (Freedy efficient, also led denaturation, likely high excess N-nucleophile needed (data shown). Side-chain radical C-C bond then 2-iodopropane coupling (Wright Scholar); trying conditions both NaBH4 Zn(0) powder initiators, never conversions thiol-based availability reliable conjugate Dha. chosen positions, set disparate activity. start, largely overarching physiochemical property they substituting (i.e., hydrophobic L139 position) 2A). Reaction kept 37°C) but optimized position. polar thiol completion, detected 1 h. thiols took considerably longer (12 h). Upon reactions, buffer exchanged standard phosphate-buffered saline examined (CD). All displayed compatible unmodified next step, testing aggregates. thioflavin T (ThT), dye increases upon binding (Cohen 2013Cohen S.I. Luheshi L.M. Hellstrand E. White D.A. Rajah Otzen D.E. Proliferation amyloid-?42 aggregates occurs mechanism.Proc. USA. 2013; 110: 9758-9763Crossref (833) protocol reproducible curves A?42, and, together solution differential equations growth 2009Knowles