Isolation and characterization of human anti-CD20 single-chain variable fragment (scFv) from a Naive human scFv library
Tóm tắt
CD20 is a receptor expressed on B cells with anonymous functions. The receptor is the target of some food and drug administration (FDA) approved monoclonal antibodies (mAb), such as Rituximab and Obinutuzumab. Blocking CD20 using the aforementioned mAbs has improved Non-Hodgkin Lymphoma (NHL) therapy. All commercial mAbs on the market were raised in non-human animal models. Antibody humanization is inevitable to mitigate immune response. In order to keep the affinity of antibody intact, humanizations are only applied to frameworks which do not eliminate immune response to foreign CDRs sequences. To address this issue, human monoclonal antibody deemed imperative. Herein, we report the isolation and characterization of a fully human single-chain variable fragment (scFv) against the large loop of CD20 from naïve human antibody library. After three rounds of phage display, a library of enriched anti-CD20 scFv was obtained. The polyclonal phage ELISA demonstrated that after each round of phage display, the population of anti-CD20 scFv became dominant. The scFv, G7, with the most robust interaction with CD20 was selected for further characterization. The specificity of G7 scFv was evaluated by ELISA, western blot, and flow cytometry. Detecting CD20 in western blot showed that G7 binds to a linear epitope on CD20 large loop. Next, G7 scFv was also bound to Raji cell (CD20+) while no interaction was recorded with K562 cell line (CD20—). This data attested that the epitope recognized by G7 scFv is accessible on the cell membrane. The affinity of G7 scFv was estimated to be 63.41 ± 3.9 nM. Next, the sensitivity was evaluated to be 2 ng/ml. Finally, G7 scFv tertiary structure was modeled using Graylab software. The 3D structure illustrated two domains of variable heavy (VH) and variable light (VL) connected through a linker. Afterward, G7 scFv and CD20 were applied to in-silico docking using ClusPro to illustrate the interaction of G7 with the large loop of CD20. As the selected scFv from the human antibody library is devoid of interspecies immunogenic amino acids sequences, no humanization or any other modifications are required prior to clinical applications.
Tài liệu tham khảo
Bashford-Rogers R, et al. Analysis of the B cell receptor repertoire in six immune-mediated diseases. Nature. 2019;574(7776):122–6.
Greenfield AL, Hauser SL. B-cell therapy for multiple sclerosis: entering an era. Ann Neurol. 2018;83(1):13–26.
Payandeh Z, et al. The applications of anti-CD20 antibodies to treat various B cells disorders. Biomed Pharmacother. 2019;109:2415–26.
Casan J, et al. Anti-CD20 monoclonal antibodies: reviewing a revolution. Hum Vaccin Immunother. 2018;14(12):2820–41.
Petrie RJ, Deans JP. Colocalization of the B cell receptor and CD20 followed by activation-dependent dissociation in distinct lipid rafts. J Immunol. 2002;169(6):2886–91.
Polyak MJ, et al. CD20 homo-oligomers physically associate with the B cell antigen receptor: dissociation upon receptor engagement and recruitment of phosphoproteins and calmodulin-binding proteins. J Biol Chem. 2008;283(27):18545–52.
Luo C, et al. Efficacy and safety of new anti-CD20 monoclonal antibodies versus rituximab for induction therapy of CD20+ B-cell non-Hodgkin lymphomas: a systematic review and meta-analysis. Sci Rep. 2021;11(1):1–14.
Fanale, M.A., et al. (2018) Safety and efficacy of anti-CD20 immunotoxin MT-3724 in relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (NHL) in a phase I study. Am Soc Clin Oncol.
Leonard JP, et al. AUGMENT: a phase III study of lenalidomide plus rituximab versus placebo plus rituximab in relapsed or refractory indolent lymphoma. J Clin Oncol. 2019;37(14):1188.
Davids MS, et al. Efficacy and safety of duvelisib following disease progression on ofatumumab in patients with relapsed/refractory CLL or SLL in the DUO crossover extension study. Clin Cancer Res. 2020;26(9):2096–103.
Fox E, et al. A phase 2 multicenter study of ublituximab, a novel glycoengineered anti-CD20 monoclonal antibody, in patients with relapsing forms of multiple sclerosis. Mult Scler J. 2021;27(3):420–9.
Marinov AD, et al. The type II anti-CD20 antibody obinutuzumab (GA101) is more effective than rituximab at depleting B cells and treating disease in a Murine Lupus model. Arthritis Rheumatol. 2021;73(5):826–36.
Golay J, et al. CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: further regulation by CD55 and CD59. Blood, J Am Soc Hematol. 2001;98(12):3383–9.
Golay J, et al. Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis. Blood, The Journal of the American Society of Hematology. 2000;95(12):3900–8.
Clynes RA, et al. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med. 2000;6(4):443–6.
Reff ME, et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood. 1994;83(2):435–45.
Tang X, et al. Rituximab (anti-CD20)-modified AZD-2014-encapsulated nanoparticles killing of B lymphoma cells. Artificial Cells Nanomed Biotechnol. 2018;46(sup2):1063–73.
Shadman M, et al. CD20 targeted CAR-T for high-risk B-cell non-Hodgkin lymphomas. Blood. 2019;134:3235.
Ye S, et al. Early clinical results of a novel anti-CD20 chimeric antigen receptor (CAR)-T cell therapy for B-cell NHL patients who are relapsed/resistant following CD19 CAR-T therapy. Blood. 2020;136:8–9.
Hosseini I, et al. Mitigating the risk of cytokine release syndrome in a Phase I trial of CD20/CD3 bispecific antibody mosunetuzumab in NHL: impact of translational system modeling. NPJ Syst Biol App. 2020;6(1):1–11.
Hutchings M, et al. Epcoritamab (GEN3013; DuoBody-CD3× CD20) to induce complete response in patients with relapsed/refractory B-cell non-Hodgkin lymphoma (B-NHL): complete dose escalation data and efficacy results from a phase I/II trial. J Clin Oncol. 2020;38:8009.
Yuan AQ, et al. Isolation of and characterization of neutralizing antibodies to Covid-19 from a large human naïve scFv phage display library. BioRxiv. 2020;25:527.
Khajeh S, et al. Phage display selection of fully human antibody fragments to inhibit growth-promoting effects of glycine-extended gastrin 17 on human colorectal cancer cells. Artif Cells, Nanomed Biotechnol. 2018;46(sup2):1082–90.
Glumac PM, et al. The identification of a novel antibody for CD133 using human antibody phage display. Prostate. 2018;78(13):981–91.
Xu M, et al. Development of a novel, fully human, anti-PCSK9 antibody with potent hypolipidemic activity by utilizing phage display-based strategy. EBioMedicine. 2021;65:103250.
Pansri P, et al. A compact phage display human scFv library for selection of antibodies to a wide variety of antigens. BMC Biotechnol. 2009;9(1):1–16.
Harper S, Speicher DW. Purification of proteins fused to glutathione S-transferase. In: Protein chromatography. Springer; 2011. p. 259–80.
Brochet X, Lefranc M-P, Giudicelli V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized VJ and VDJ sequence analysis. Nucleic Acids Res. 2008;36:W503–8.
Ko J, et al. GalaxyWEB server for protein structure prediction and refinement. Nucleic Acids Res. 2012;40(W1):W294–7.
Comeau SR, et al. ClusPro: a fully automated algorithm for protein–protein docking. Nucleic Acids Res. 2004;32:W96–9.
DeLano WL. Pymol: an open-source molecular graphics tool. CCP4 Newslett Protein Crystallogr. 2002;40(1):82–92.
Klein C, et al. Response to: monoclonal antibodies targeting CD20. MAbs. 2013;5:337–8.
Klein C, et al. Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties. MAbs. 2013;5:22–33.
Rougé L, et al. Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab. Science. 2020;367(6483):1224–30.
Agez M, et al. Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab. Sci Rep. 2019;9(1):1–13.
Pandey A, et al. Current strategies for protein production and purification enabling membrane protein structural biology. Biochem Cell Biol. 2016;94(6):507–27.
Lacapère J-J, Robert J-C, Thomas-soumarmon A. Efficient solubilization and purification of the gastric H+, K+-ATPase for functional and structural studies. Biochem J. 2000;345(2):239–45.
Asano T, Kaneko MK, Kato Y. RIEDL tag: a novel pentapeptide tagging system for transmembrane protein purification. Biochem Biophys Rep. 2020;23:100780.
Vogler I, et al. An improved bicistronic CD20/tCD34 vector for efficient purification and in vivo depletion of gene-modified T cells for adoptive immunotherapy. Mol Ther. 2010;18(7):1330–8.
Niederfellner G, et al. Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies. Blood J Am Soc Hematol. 2011;118(2):358–67.
Clackson T, et al. Making antibody fragments using phage display libraries. Nature. 1991;352(6336):624–8.
Noppe W, et al. Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries. BMC Biotechnol. 2009;9(1):1–9.
Bratkovič T. Progress in phage display: evolution of the technique and its applications. Cell Mol Life Sci. 2010;67(5):749–67.
Kang TH, Seong BL. Solubility, stability, and avidity of recombinant antibody fragments expressed in microorganisms. Front Microbiol. 2020. https://doi.org/10.3389/fmicb.2020.01927.
de Aguiar RB, et al. Generation and functional characterization of a single-chain variable fragment (scFv) of the anti-FGF2 3F12E7 monoclonal antibody. Sci Rep. 2021;11(1):1–11.
Xiong S, et al. Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia coli and its “oxidizing” mutant. World J Gastroenterol: WJG. 2005;11(7):1077.
Nikkhoi SK, Rahbarizadeh F, Ahmadvand D. Oligo-clonal nanobodies as an innovative targeting agent for cancer therapy: new biology and novel targeting systems. Protein Expr Purif. 2017;129:115–21.
Berkmen M. Production of disulfide-bonded proteins in Escherichia coli. Protein Expr Purif. 2012;82(1):240–51.
Manta B, Boyd D, Berkmen M. Disulfide bond formation in the periplasm of Escherichia coli. EcoSal Plus. 2019. https://doi.org/10.1128/ecosalplus.ESP-0012-2018.
Javadian FS, et al. Solubility assessment of single-chain antibody fragment against epithelial cell adhesion molecule extracellular domain in four Escherichia coli strains. J Genet Eng Biotechnol. 2021;19(1):1–8.
Sandomenico A, Sivaccumar JP, Ruvo M. Evolution of Escherichia coli expression system in producing antibody recombinant fragments. Int J Mol Sci. 2020;21(17):6324.
Beatty JD, Beatty BG, Vlahos WG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods. 1987;100(1–2):173–9.
Beatty JD, et al. Method of analysis of non-competitive enzyme immunoassays for antibody quantification. J Immunol Methods. 1987;100(1–2):161–72.
Kazemi-Lomedasht F, et al. Production and characterization of novel camel single domain antibody targeting mouse vascular endothelial growth factor. Monoclonal Antibodies in Immunodiagnosis and Immunotherapy. 2016;35(3):167–71.
Raghava G, Agrewala JN. Method for determining the affinity of monoclonal antibody using non-competitive ELISA: a computer program. J Immunoassay Immunochem. 1994;15(2):115–28.
Fadlalla MH, et al. Development of ELISA and lateral flow immunoassays for ochratoxins (OTA and OTB) detection based on monoclonal antibody. Front Cell Infect Microbiol. 2020;10:80.
de Ridder GG, Ray R, Pizzo SV. A murine monoclonal antibody directed against the carboxyl-terminal domain of GRP78 suppresses melanoma growth in mice. Melanoma Res. 2012;22(3):225–35.
Adams GP, et al. High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules. Can Res. 2001;61(12):4750–5.