Abstract
Monoclonal antibodies (mAbs) are biotherapeutics that have achieved outstanding success in treating many life-threatening and chronic diseases. The recognition of an antigen is mediated by the fragment antigen binding (Fab) regions composed by four different disulfide bridge-linked immunoglobulin domains. NMR is a powerful method to assess the integrity, the structure and interaction of Fabs, but site specific analysis has been so far hampered by the size of the Fabs and the lack of approaches to produce isotopically labeled samples. We proposed here an efficient in vitro method to produce [15N, 13C, 2H]-labeled Fabs enabling high resolution NMR investigations of these powerful therapeutics. As an open system, the cell-free expression mode enables fine-tuned control of the redox potential in presence of disulfide bond isomerase to enhance the formation of native disulfide bonds. Moreover, inhibition of transaminases in the S30 cell-free extract offers the opportunity to produce perdeuterated Fab samples directly in 1H2O medium, without the need for a time-consuming and inefficient refolding process. This specific protocol was applied to produce an optimally labeled sample of a therapeutic Fab, enabling the sequential assignment of 1HN, 15N, 13C′, 13Cα, 13Cβ resonances of a full-length Fab. 90% of the backbone resonances of a Fab domain directed against the human LAMP1 glycoprotein were assigned successfully, opening new opportunities to study, at atomic resolution, Fabs’ higher order structures, dynamics and interactions, using solution-state NMR.
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Data availability
The assignment of chemical shifts of backbone of the anti-LAMP1-Fab, together with all the NMR data sets used have been deposited into the BMRB (https://www.bmrb.wisc.edu/) with the accession number 52243.
References
Agarwal AK, Gude RP, Kalraiya RD (2014) Regulation of melanoma metastasis to lungs by cell surface lysosome associated membrane protein-1 (LAMP1) via galectin-3. Biochem Biophys Res Commun 449(3):332–337. https://doi.org/10.1016/j.bbrc.2014.05.028
Alessandrini F, Pezzè L, Ciribilli Y (2017) LAMPs: shedding light on cancer biology. Semin Oncol 44(4):239–253. https://doi.org/10.1053/j.seminoncol.2017.10.013
Arora A, Ha C, Park CB (2004) Inhibition of insulin amyloid formation by small stress molecules. FEBS Lett 564(1–2):121–125. https://doi.org/10.1016/S0014-5793(04)00326-6
Brinson RG, Marino JP, Delaglio F, Arbogast LW, Evans RM, Kearsley A, Gingras G, Ghasriani H, Aubin Y, Pierens GK, Jia X, Mobli M, Grant HG, Keizer DW, Schweimer K, Ståhle J, Widmalm G, Zartler ER, Lawrence CW et al (2018) Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics. mAbs 11(1):94–105. https://doi.org/10.1080/19420862.2018.1544454
Cameron B, Dabdoubi T, Berthou-Soulié L, Gagnaire M, Arnould I, Severac A, Soubrier F, Morales J, Leighton PA, Harriman W, Ching K, Abdiche Y, Radošević K, Bouquin T (2020) Complementary epitopes and favorable developability of monoclonal anti-LAMP1 antibodies generated using two transgenic animal platforms. PLoS ONE 15(7):e0235815. https://doi.org/10.1371/journal.pone.0235815
Cerofolini L, Ravera E, Fischer C, Trovato A, Sacco F, Palinsky W, Angiuoni G, Fragai M, Baroni F (2023) Integration of NMR spectroscopy in an analytical workflow to evaluate the effects of oxidative stress on abituzumab: beyond the fingerprint of mAbs. Anal Chem 95(24):9199–9206. https://doi.org/10.1021/acs.analchem.3c00317
Clark L, Dikiy I, Rosenbaum DM, Gardner KH (2018) On the use of Pichia pastoris for isotopic labeling of human GPCRs for NMR studies. J Biomol NMR 71(4):203–211. https://doi.org/10.1007/s10858-018-0204-3
de Marco A (2009) Strategies for successful recombinant expression of disulfide bond-dependent proteins in Escherichia coli. Microb Cell Fact 8(1):26. https://doi.org/10.1186/1475-2859-8-26
Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3):277–293. https://doi.org/10.1007/BF00197809
Dopp JL, Reuel NF (2020) Simple, functional, inexpensive cell extract for in vitro prototyping of proteins with disulfide bonds. Biochem Eng J 164:107790. https://doi.org/10.1016/j.bej.2020.107790
Etezady-Esfarjani T, Hiller S, Villalba C, Wüthrich K (2007) Cell-free protein synthesis of perdeuterated proteins for NMR studies. J Biomol NMR 39(3):229–238. https://doi.org/10.1007/s10858-007-9188-0
Favier A, Brutscher B (2019) NMRlib: user-friendly pulse sequence tools for Bruker NMR spectrometers. J Biomol NMR 73(5):199–211. https://doi.org/10.1007/s10858-019-00249-1
Gaciarz A, Veijola J, Uchida Y, Saaranen MJ, Wang C, Hörkkö S, Ruddock LW (2016) Systematic screening of soluble expression ofantibody fragments in the cytoplasm of E. Coli. Microb Cell Fact 15(1):22. https://doi.org/10.1186/s12934-016-0419-5
Gad W, Nair MG, Belle KV, Wahni K, Greve HD, Ginderachter JAV, Vandenbussche G, Endo Y, Artis D, Messens J (2013) The Quiescin Sulfhydryl Oxidase (hQSOX1b) Tunes the Expression of Resistin-Like Molecule Alpha (RELM-α or mFIZZ1) in a Wheat Germ Cell-Free Extract. PLOS ONE 8(1):e55621. https://doi.org/10.1371/journal.pone.0055621
Gagné D, Sarker M, Gingras G, Hodgson DJ, Frahm G, Creskey M, Lorbetskie B, Bigelow S, Wang J, Zhang X, Johnston MJW, Lu H, Aubin Y (2023) Strategies for the production of isotopically labelled Fab fragments of therapeutic antibodies in Komagataella phaffii (Pichia pastoris) and Escherichia coli for NMR studies. PLoS ONE 18:e0294406. https://doi.org/10.1371/journal.pone.0294406
Gardner KH, Kay LE (1998) The Use of 2 h, 13c, 15n multidimensional nmr gto study the structure and dynamics of proteins. Annu Rev BioPhys BioMol Struct 27(1):357–406. https://doi.org/10.1146/annurev.biophys.27.1.357
Garrett DS, Seok Y-J, Liao D-I, Peterkofsky A, Gronenborn AM, Clore GM (1997) Solution structure of the 30 kDa N-terminal domain of enzyme I of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system by multidimensional NMR. Biochemistry 36(9):2517–2530. https://doi.org/10.1021/bi962924y
Ghasriani H, Ahmadi S, Hodgson DJ, Aubin Y (2022) Backbone and side-chain resonance assignments of the NISTmAb-scFv and antigen-binding study. Biomol NMR Assignments 16(2):391–398. https://doi.org/10.1007/s12104-022-10109-z
Gupta SK, Shukla P (2017) Microbial platform technology for recombinant antibody fragment production: a review. Crit Rev Microbiol 43(1):31–42. https://doi.org/10.3109/1040841X.2016.1150959
Han B, Liu Y, Ginzinger SW, Wishart DS (2011) SHIFTX2: significantly improved protein chemical shift prediction. J Biomol NMR 50(1):43–57. https://doi.org/10.1007/s10858-011-9478-4
Hsu C-C, Thomas OR, Overton TW (2016) Periplasmic expression in and release of fab fragments from Escherichia coli using stress minimization. J Chem Technol Biotechnol 91(3):815–822. https://doi.org/10.1002/jctb.4672
Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of proton, carbon-13, and nitrogen-15 spectra of larger proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. application to calmodulin. Biochemistry 29(19):4659–4667. https://doi.org/10.1021/bi00471a022
Imbert L, Lenoir-Capello R, Crublet E, Vallet A, Awad R, Ayala I, Juillan-Binard C, Mayerhofer H (2021) In vitro production of perdeuterated proteins in H2O for biomolecular NMR studies. In: Chen Y. W., Yiu C.-P. B. (eds) Structural genomics: general applications. Springer, Berlin, pp 127–149. https://doi.org/10.1007/978-1-0716-0892-0_8
Kai L, Dötsch V, Kaldenhoff R, Bernhard F (2013) Artificial environments for the Co-translational stabilization of cell-free expressed proteins. PLoS ONE. https://doi.org/10.1371/journal.pone.0056637
Kaplon H, Crescioli S, Chenoweth A, Visweswaraiah J, Reichert JM (2023) Antibodies to watch in 2023. mAbs 15(1):2153410. https://doi.org/10.1080/19420862.2022.2153410
Katz JJ, Crespi HL (1966) Deuterated organisms: cultivation and uses. Science 151(3715):1187–1194. https://doi.org/10.1126/science.151.3715.1187
Kigawa T (2010) Cell-free protein preparation through prokaryotic transcription–translation methods. In: Endo Y, Takai K, Ueda T (eds) Cell-free protein production: methods and protocols. Humana Press, Totowa, pp 1–10. https://doi.org/10.1007/978-1-60327-331-2_1
Kigawa T, Yabuki T, Matsuda N, Matsuda T, Nakajima R, Tanaka A, Yokoyama S (2004) Preparation of Escherichia coli cell extract for highly productive cell-free protein expression. J Struct Funct Genomics 5(1):63–68. https://doi.org/10.1023/B:JSFG.0000029204.57846.7d
Kim DM, Choi CY (1996) A semicontinuous prokaryotic coupled transcription/translation system using a dialysis membrane. Biotechnol Prog 12(5):645–649. https://doi.org/10.1021/bp960052l
Kunert R, Reinhart D (2016) Advances in recombinant antibody manufacturing. Appl Microbiol Biotechnol 100(8):3451–3461. https://doi.org/10.1007/s00253-016-7388-9
Lescop E, Schanda P, Brutscher B (2007) A set of BEST triple-resonance experiments for time-optimized protein resonance assignment. J Magn Reson 187(1):163–169. https://doi.org/10.1016/j.jmr.2007.04.002
Lobstein J, Emrich CA, Jeans C, Faulkner M, Riggs P, Berkmen M (2012) SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm. Microb Cell Fact 11(1):753. https://doi.org/10.1186/1475-2859-11-56
Marino JP, Brinson RG, Hudgens JW, Ladner JE, Gallagher DT, Gallagher ES, Arbogast LW, Huang RY-C (2015) Emerging technologies to assess the higher order structure of monoclonal antibodies. ACS Symposium Series 1202(2):17–43. https://doi.org/10.1021/bk-2015-1202.ch002
Matsuda T, Watanabe S, Kigawa T (2013) Cell-free synthesis system suitable for disulfide-containing proteins. Biochem Biophys Res Commun 431(2):296–301. https://doi.org/10.1016/j.bbrc.2012.12.107
Matsuda T, Ito T, Takemoto C, Katsura K, Ikeda M, Wakiyama M, Kukimoto-Niino M, Yokoyama S, Kurosawa Y, Shirouzu M (2018) Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody–antigen interaction. PLoS ONE 13(2):e0193158. https://doi.org/10.1371/journal.pone.0193158
Michel E, Wüthrich K (2012) Cell-free expression of disulfide-containing eukaryotic proteins for structural biology: cell-free expression of disulfide-containing proteins. FEBS J 279(17):3176–3184. https://doi.org/10.1111/j.1742-4658.2012.08697.x
Mishra A, Mody RS, Pandey A, Somani S (2018) An improved refolding process for antibody’s fragments (Brevet AU2016307976A1). https://patents.google.com/patent/AU2016307976A1/en
Missiakas D, Georgopoulos C, Raina S (1994) The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation. EMBO J 13(8):2013–2020
Morgan WD, Kragt A, Feeney J (2000) Expression of deuterium-isotope-labelled protein in the yeast Pichia pastoris for NMR studies. J Biomol NMR 17(4):337–347. https://doi.org/10.1023/A:1008313530207
Nakamoto H, Bardwell JCA (2004) Catalysis of disulfide bond formation and isomerization in the Escherichia coli periplasm. Biochim Biophys Acta (BBA) - Mol Cell Res 1694(1):111–119. https://doi.org/10.1016/j.bbamcr.2004.02.012
Pratt C (1980) Kinetics and regulation of cell-free alkaline phosphatase synthesis. J Bacteriol 143(3):1265–1274. https://doi.org/10.1128/jb.143.3.1265-1274.1980
Pruvost T, Mathieu M, Dubois S, Maillère B, Vigne E, Nozach H (2023) Deciphering cross-species reactivity of LAMP-1 antibodies using deep mutational epitope mapping and alphafold. mAbs 15(1):2175311. https://doi.org/10.1080/19420862.2023.2175311
Rietsch A, Belin D, Martin N, Beckwith J (1996) An in vivo pathway for disulfide bond isomerization in Escherichia coli. Proc Natl Acad Sci USA 93(23):13048–13053. https://doi.org/10.1073/pnas.93.23.13048
Sawasaki T, Hasegawa Y, Tsuchimochi M, Kamura N, Ogasawara T, Kuroita T, Endo Y (2002) A bilayer cell-free protein synthesis system for high-throughput screening of gene products. FEBS Lett 514(1):102–105. https://doi.org/10.1016/S0014-5793(02)02329-3
Schmidt E, Güntert P (2012) A new algorithm for reliable and general NMR resonance assignment. J Am Chem Soc 134(30):12817–12829. https://doi.org/10.1021/ja305091n
Schwarz D, Klammt C, Koglin A, Löhr F, Schneider B, Dötsch V, Bernhard F (2007) Preparative scale cell-free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies. Methods 41(4):355–369. https://doi.org/10.1016/j.ymeth.2006.07.001
Segelke BW, Schafer J, Coleman MA, Lekin TP, Toppani D, Skowronek KJ, Kantardjieff KA, Rupp B (2004) Laboratory scale structural genomics. J Struct Funct Genomics 5(1):147–157. https://doi.org/10.1023/B:JSFG.0000029193.82120.d1
Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44(4):213–223. https://doi.org/10.1007/s10858-009-9333-z
Skinner SP, Fogh RH, Boucher W, Ragan TJ, Mureddu LG, Vuister GW (2016) CcpNmr analysisassign: a flexible platform for integrated NMR analysis. J Biomol NMR 66(2):111–124. https://doi.org/10.1007/s10858-016-0060-y
Solomon TL, Chao K, Gingras G, Aubin Y, O’Dell WB, Marino JP, Brinson RG (2023) Backbone NMR assignment of the yeast expressed fab fragment of the NISTmAb reference antibody. Biomol NMR Assignments 17(1):75–81. https://doi.org/10.1007/s12104-023-10123-9
Spirin AS, Baranov VI, Ryabova LA, Ovodov S, Alakhov YB (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242(4882):1162–1164. https://doi.org/10.1126/science.3055301
Su X-C, Loh C-T, Qi R, Otting G (2011) Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15 N-labelling and production of perdeuterated proteins in H2O. J Biomol NMR 50(1):35–42. https://doi.org/10.1007/s10858-011-9477-5
Suzuki R, Sakakura M, Mori M, Fujii M, Akashi S, Takahashi H (2018) Methyl-selective isotope labeling using α-ketoisovalerate for the yeast Pichia pastoris recombinant protein expression system. J Biomol NMR 71(4):213–223. https://doi.org/10.1007/s10858-018-0192-3
Terasawa K, Tomabechi Y, Ikeda M, Ehara H, Kukimoto-Niino M, Wakiyama M, Podyma-Inoue KA, Rajapakshe AR, Watabe T, Shirouzu M, Hara-Yokoyama M (2016) Lysosome-associated membrane proteins-1 and – 2 (LAMP-1 and LAMP-2) assemble via distinct modes. Biochem Biophys Res Commun 479(3):489–495. https://doi.org/10.1016/j.bbrc.2016.09.093
Torizawa T, Shimizu M, Taoka M, Miyano H, Kainosho M (2004) Efficient production of isotopically labeled proteins by cell-free synthesis: a practical protocol. J Biomol NMR 30(3):311–325. https://doi.org/10.1007/s10858-004-3534-2
Tugarinov V, Kanelis V, Kay LE (2006) Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy. Nat Protoc. https://doi.org/10.1038/nprot.2006.101
Venters RA, Farmer II, Fierke BT, C. A., Spicer LD (1996) Characterizing the use of perdeuteration in NMR studies of large proteins:13 C,15 N and1H assignments of human carbonic anhydrase II. J Mol Biol 264(5):1101–1116. https://doi.org/10.1006/jmbi.1996.0699
Venturi M, Seifert C, Hunte C (2002) High level production of functional antibody fab fragments in an oxidizing bacterial cytoplasm11 edited by J. Karn. J Mol Biol 315(1):1–8. https://doi.org/10.1006/jmbi.2001.5221
Welsh DT, Herbert RA (1999) Osmotically induced intracellular trehalose, but not glycine betaine accumulation promotes desiccation tolerance in Escherichia coli. FEMS Microbiol Lett 174(1):57–63. https://doi.org/10.1111/j.1574-6968.1999.tb13549.x
Yagi H, Zhang Y, Yagi-Utsumi M, Yamaguchi T, Iida S, Yamaguchi Y, Kato K (2015) Backbone 1H, 13 C, and 15 N resonance assignments of the fc fragment of human immunoglobulin G glycoprotein. Biomol Nmr Assignments 9(2):257–260. https://doi.org/10.1007/s12104-014-9586-7
Yin G, Swartz JR (2004) Enhancing multiple disulfide bonded protein folding in a cell-free system. Biotechnol Bioeng 86(2):188–195. https://doi.org/10.1002/bit.10827
Zubay G (1973) In vitro synthesis of protein in microbial systems. Annu Rev Genet 7(1):267–287. https://doi.org/10.1146/annurev.ge.07.120173.001411
Acknowledgements
The authors thank Isabel Ayala and Rida Awad for advice and stimulating discussions. This work is supported by the French National Research Agency in the framework of the “Investissements d’avenir” program (ANR-15-IDEX-02), NMR4mAbs project n° ANR-22-CE29-0024-01 and by the CEA/SANOFI collaborative research program C36478/191073. This work used the high field NMR and Cell-Free facilities at the Grenoble Instruct-ERIC Center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB). Platform access was supported by FRISBI (ANR-10-INBS-05-02) and GRAL, a project of the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003) and IR INFRANALYTICS FR2054. IBS acknowledges integration into the Interdisciplinary Research Institute of Grenoble (IRIG, CEA).
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AG, LI, FD. and CS and EC prepared the samples. AG, AF, FH, OF and JB. analyzed NMR data. AG, LI, OF, EC and JB wrote the main manuscript text and AG, LI, FH prepared the figures. All authors reviewed the manuscript.
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Giraud, A., Imbert, L., Favier, A. et al. Enabling site-specific NMR investigations of therapeutic Fab using a cell-free based isotopic labeling approach: application to anti-LAMP1 Fab. J Biomol NMR (2024). https://doi.org/10.1007/s10858-023-00433-4
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DOI: https://doi.org/10.1007/s10858-023-00433-4