HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species
Tóm tắt
The production of recombinant monoclonal antibodies in mammalian cell culture is of high priority in research and medical fields. A critical step in this process is the isolation of the antigen-binding domain sequences of antibodies possessing the desired properties. Many different techniques have been described to achieve this goal, but all have shortcomings; most techniques have problems with robustness, are time-consuming and costly, or have complications in the transfer from isolation to production phase. Here, we report a novel HybriFree technology for the development of monoclonal antibodies from different species that is robust, rapid, inexpensive and flexible and can be used for the subsequent production of antibodies in mammalian cell factories. HybriFree technology is illustrated herein via detailed examples of isolating mouse, rabbit and chicken monoclonal antibody sequences from immunized animals. Starting from crude spleen samples, antigen capturing of specific B-cells is performed initially. cDNA of antibody variable domains is amplified from the captured cells and used a source material for simple and rapid restriction/ligation free cloning of expression vector library in order to produce scFv-Fc or intact IgG antibodies. The vectors can be directly used for screening purposes as well as for the subsequent production of the developed monoclonal antibodies in mammalian cell culture. The antibodies isolated by the method have been shown to be functional in different immunoassays, including ELISA, immunofluorescence and Western blot. In addition, we demonstrate that by using a modified method including a negative selection step, we can isolate specific antibodies targeting the desired epitope and eliminate antibodies directed to undesired off-targets. HybriFree can be used for the reliable development of monoclonal antibodies and their subsequent production in mammalian cells. This simple protocol requires neither the culturing of B-cells nor single-cell manipulations, and only standard molecular biology laboratory equipment is needed. In principle, the method is applicable to any species for which antibody cDNA sequence information is available.
Tài liệu tham khảo
Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495–7.
Chen G, Sidhu SS. Design and generation of synthetic antibody libraries for phage display. Methods Mol Biol. 2014;1131:113–31.
Koenig P, Fuh G. Selection and screening using antibody phage display libraries. Methods Mol Biol. 2014;1131:133–49.
He M, Edwards BM, Kastelic D, Taussig MJ. Eukaryotic ribosome display with in situ DNA recovery. Methods Mol Biol. 2012;805:75–85.
Doerner A, Rhiel L, Zielonka S, Kolmar H. Therapeutic antibody engineering by high efficiency cell screening. FEBS Lett. 2014;588(2):278–87.
Bowers PM, Horlick RA, Kehry MR, Neben TY, Tomlinson GL, Altobell L, et al. Mammalian cell display for the discovery and optimization of antibody therapeutics. Methods. 2014;65(1):44–56.
Beerli RR, Rader C. Mining human antibody repertoires. MAbs. 2010;2(4):365–78.
Tiller T. Single B cell antibody technologies. N Biotechnol. 2011;28(5):453–7.
Wen L, Hanvanich M, Werner-Favre C, Brouwers N, Perrin LH, Zubler RH. Limiting dilution assay for human B cells based on their activation by mutant EL4 thymoma cells: total and antimalaria responder B cell frequencies. Eur J Immunol. 1987;17(6):887–92.
Zubler RH, Erard F, Lees RK, Van Laer M, Mingari C, Moretta L, et al. Mutant EL-4 thymoma cells polyclonally activate murine and human B cells via direct cell interaction. J Immunol. 1985;134(6):3662–8.
Banchereau J, de Paoli P, Valle A, Garcia E, Rousset F. Long-term human B cell lines dependent on interleukin-4 and antibody to CD40. Science. 1991;251(4989):70–2.
Kasturirangan S, Reasoner T, Schulz P, Boddapati S, Emadi S, Valla J, et al. Isolation and characterization of antibody fragments selective for specific protein morphologies from nanogram antigen samples. Biotechnol Prog. 2013;29(2):463–71.
Tian H, Davidowitz E, Lopez P, He P, Schulz P, Moe J, et al. Isolation and characterization of antibody fragments selective for toxic oligomeric tau. Neurobiol Aging. 2015;36(3):1342–55.
Chinestra P, Olichon A, Medale-Giamarchi C, Lajoie-Mazenc I, Gence R, Inard C, et al. Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation. PLoS One. 2014;9(11):e111034.
Axis-Shield. Isolation of mononuclear cells (lymphocytes) from tissues. Application Sheet C40; 6th edition, March 2013. In: Axis-Shield Application Sheet Index for Prokaryotic and Eukaryotic cells. http://www.axis-shield-density-gradient-media.com/C40.pdf. Accessed Sept 2012.
Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas 3rd CF. Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods. 2000;242(1–2):159–81.
Schaefer JV, Honegger A, Plückthun A. Construction of scFv Fragments from Hybridoma or Spleen Cells by PCR Assembly. In: Kontermann R, Dübel S, editors. Antibody Engineering. vol. 1, 2nd ed. Heidelberg: Springer; 2010. p. 21–44.
IMGT® Web resources. [http://www.imgt.org/IMGTrepertoire/ Accessed May 2012]
Rader C, Ritter G, Nathan S, Elia M, Gout I, Jungbluth AA, et al. The rabbit antibody repertoire as a novel source for the generation of therapeutic human antibodies. J Biol Chem. 2000;275(18):13668–76.
IMGT® Web resources. [http://www.imgt.org/IMGTrepertoire/ Accessed Aug 2015]
Quan J, Tian J. Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries. Nat Protoc. 2011;6(2):242–51.
Karro K, Männik T, Männik A, Ustav M. DNA Transfer into animal cells using stearylated CPP based transfection reagent. Methods Mol Biol. 2015;1324:435–45.
Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser. 1999;41:95–8.
Kodituwakku AP, Jessup C, Zola H, Roberton DM. Isolation of antigen-specific B cells. Immunol Cell Biol. 2003;81(3):163–70.
Reth M. Antigen receptors on B lymphocytes. Annu Rev Immunol. 1992;10:97–121.
Lightwood DJ, Carrington B, Henry AJ, McKnight AJ, Crook K, Cromie K, et al. Antibody generation through B cell panning on antigen followed by in situ culture and direct RT-PCR on cells harvested en masse from antigen-positive wells. J Immunol Methods. 2006;316(1–2):133–43.
Seeber S, Ros F, Thorey I, Tiefenthaler G, Kaluza K, Lifke V, et al. A robust high throughput platform to generate functional recombinant monoclonal antibodies using rabbit B cells from peripheral blood. PLoS One. 2014;9(2):e86184.
Clargo AM, Hudson AR, Ndlovu W, Wootton RJ, Cremin LA, O'Dowd VL, et al. The rapid generation of recombinant functional monoclonal antibodies from individual, antigen-specific bone marrow-derived plasma cells isolated using a novel fluorescence-based method. MAbs. 2014;6(1):143–59.
Loh L, Hudson JB. Interaction of murine cytomegalovirus with separated populations of spleen cells. Infect Immun. 1979;26(3):853–60.
Kushnir N, Streatfield SJ, Yusibov V. Virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development. Vaccine. 2012;31(1):58–83.