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High-Throughput Generation of In Silico Derived Synthetic Antibodies via One-step Enzymatic DNA Assembly of Fragments

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Abstract

Phage-display technology offers robust methods for isolating antibody (Ab) molecules with specificity for different target antigens. Recent advancements couple Ab selections with in silico strategies, such as predictive computational models or next-generation sequencing metadata analysis of Ab selections. These advancements result in enhanced Ab clonal diversities with potential for enlarged epitope coverage of the target antigen. A current limitation however, is that de novo Ab sequences must undergo DNA gene synthesis, and subsequent expression as Ab proteins for downstream validations. Due to the high costs and time for commercially generating large sets of DNA genes, we report a high-throughput platform for the synthesis of in silico derived Ab clones. As a proof of concept we demonstrate the simultaneous synthesis of 96 unique Abs with varied lengths and complementary determining region compositions. Each of the 96 Ab clones undergoes a one-step enzymatic assembly of distinct DNA fragments that combine into a circularized Fab expression plasmid. This strategy allows for the rapid and efficient synthesis of 96 DNA constructs in a 3 day window, and exhibits high percentage fidelity—greater than 93%. Accordingly, the synthesis of Ab DNA constructs as Fab expression plasmids allow for rapid execution of downstream Ab protein validations, with potential for implementation into high-throughput Ab protein characterization pipelines. Altogether, the platform presented here proves rapid and also cost-effective, which is important for labs with limited resources, since it utilizes standard laboratory equipment and molecular reagents.

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Abbreviations

Ab:

Antibody

CDRs:

Complementary determining regions

NGS:

Next-generation sequencing

AmpR:

β-Lactamase gene

KanR:

Neomycin phosphotransferase II gene

Mid:

Middle Fab sequence region

References

  1. Winter, G., & Milstein, C. (1991). Man-made antibodies. Nature,349(6307), 293–299. https://doi.org/10.1038/349293a0.

    Article  CAS  PubMed  Google Scholar 

  2. Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D., & Winter, G. (1991). By-passing immunization. Journal of Molecular Biology,222(3), 581–597. https://doi.org/10.1016/0022-2836(91)90498-U.

    Article  CAS  PubMed  Google Scholar 

  3. Goding, J. W. (1980). Antibody production by hybridomas. Journal of Immunological Methods,39(4), 285–308. https://doi.org/10.1016/0022-1759(80)90230-6.

    Article  CAS  PubMed  Google Scholar 

  4. Tang, D. C., DeVit, M., & Johnston, S. A. (1992). Genetic immunization is a simple method for eliciting an immune response. Nature,356(6365), 152–154. https://doi.org/10.1038/356152a0.

    Article  CAS  PubMed  Google Scholar 

  5. Köhler, G., & Milstein, C. (1975). Continuous cultures of fused cells secreting antibody of predefined specificity. Nature,256, 495–497. https://doi.org/10.1038/256495a0.

    Article  PubMed  Google Scholar 

  6. Winter, G., Griffiths, A. D., Hawkins, R. E., & Hoogenboom, H. R. (1994). Making antibodies by phage display technology. Annual Review of Immunology,12(1), 433–455. https://doi.org/10.1146/annurev.iy.12.040194.002245.

    Article  CAS  PubMed  Google Scholar 

  7. Sidhu, S. S., & C. R. Geyer (Eds.). (2005). Phage display in biotechnology and drug discovery (Vol. 55). Boca Raton, FL: CRC Press. https://doi.org/10.1201/9780849359125.

  8. Bradbury, A. R. M., & Marks, J. D. (2004). Antibodies from phage antibody libraries. Journal of Immunological Methods,290(1–2), 29–49. https://doi.org/10.1016/j.jim.2004.04.007.

    Article  CAS  PubMed  Google Scholar 

  9. Yang, G.-H., Yoon, S. O., Jang, M. H., & Hong, H. J. (2007). Affinity maturation of an anti-hepatitis B virus PreS1 humanized antibody by phage display. Journal of Microbiology (Seoul, Korea),45(6), 528–533.

    CAS  Google Scholar 

  10. Bradbury, A. R. M., Sidhu, S. S., Dübel, S., McCafferty, J., & Dubel, S. (2011). Beyond natural antibodies: The power of in vitro display technologies. Nature Biotechnology,29(3), 245–254. https://doi.org/10.1038/nbt.1791.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Benhar, I. (2007). Design of synthetic antibody libraries. Expert Opinion on Biological Therapy,7(5), 763–779. https://doi.org/10.1517/14712598.7.5.763.

    Article  CAS  PubMed  Google Scholar 

  12. Birtalan, S., Zhang, Y., Fellouse, F. A., Shao, L., Schaefer, G., & Sidhu, S. S. (2008). The intrinsic contributions of tyrosine, serine, glycine and arginine to the affinity and specificity of antibodies. Journal of Molecular Biology,377(5), 1518–1528. https://doi.org/10.1016/j.jmb.2008.01.093.

    Article  CAS  PubMed  Google Scholar 

  13. Glanville, J., Zhai, W., Berka, J., Telman, D., Huerta, G., Mehta, G. R., ..., Pons, J. (2009). Precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire. Proceedings of the National Academy of Sciences of the United States of America,106(48), 20216–20221. https://doi.org/10.1073/pnas.0909775106.

    Article  CAS  Google Scholar 

  14. Miersch, S., & Sidhu, S. S. (2012). Synthetic antibodies: Concepts, potential and practical considerations. Methods,57(4), 486–498. https://doi.org/10.1016/j.ymeth.2012.06.012.

    Article  CAS  PubMed  Google Scholar 

  15. Das, R., Baker, D., Nussinov, R., Wolfson, H. J., Corn, J. E., Strauch, E.-M., ..., Baker, D. (2008). Macromolecular modeling with rosetta. Annual Review of Biochemistry,77(4), 363–382. https://doi.org/10.1146/annurev.biochem.77.062906.171838.

    Article  CAS  PubMed  Google Scholar 

  16. Fleishman, S. J., Whitehead, T. A., Ekiert, D. C., Dreyfus, C., Corn, J. E., Strauch, E.-M., ..., Baker, D. (2011). Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science,332(6031), 816–821. https://doi.org/10.1126/science.1202617.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Huang, P.-S., Love, J. J., & Mayo, S. L. (2007). A de novo designed protein–protein interface. Protein Science,16(12), 2770–2774. https://doi.org/10.1110/ps.073125207.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kries, H., Blomberg, R., & Hilvert, D. (2013). De novo enzymes by computational design. Current Opinion in Chemical Biology,17(2), 221–228. https://doi.org/10.1016/j.cbpa.2013.02.012.

    Article  CAS  PubMed  Google Scholar 

  19. Der, B. S., & Kuhlman, B. (2011). From computational design to a protein that binds. Science,332(6031), 801–802. https://doi.org/10.1126/science.1207082.

    Article  CAS  PubMed  Google Scholar 

  20. Kortemme, T., Joachimiak, L. A., Bullock, A. N., Schuler, A. D., Stoddard, B. L., & Baker, D. (2004). Computational redesign of protein–protein interaction specificity. Nature Structural and Molecular Biology,11(4), 371–379. https://doi.org/10.1038/nsmb749.

    Article  CAS  PubMed  Google Scholar 

  21. Lazar, G. A., Dang, W., Karki, S., Vafa, O., Peng, J. S., Hyun, L., ..., Dahiyat, B. I. (2006). Engineered antibody Fc variants with enhanced effector function. Proceedings of the National Academy of Sciences of the United States of America,103(11), 4005–4010. https://doi.org/10.1073/pnas.0508123103.

    Article  CAS  Google Scholar 

  22. Hwang, I., & Park, S. (2008). Computational design of protein therapeutics. Drug Discovery Today: Technologies,5(2–3), e43–e48. https://doi.org/10.1016/j.ddtec.2008.11.004.

    Article  PubMed  Google Scholar 

  23. Caravella, J., & Lugovskoy, A. (2010). Design of next-generation protein therapeutics. Current Opinion in Chemical Biology,14(4), 520–528. https://doi.org/10.1016/j.cbpa.2010.06.175.

    Article  CAS  PubMed  Google Scholar 

  24. Marshall, S. A., Lazar, G. A., Chirino, A. J., & Desjarlais, J. R. (2003). Rational design and engineering of therapeutic proteins. Drug Discovery Today,8(5), 212–221. https://doi.org/10.1016/S1359-6446(03)02610-2.

    Article  CAS  PubMed  Google Scholar 

  25. Ravn, U., Didelot, G., Venet, S., Ng, K. T., Gueneau, F., Rousseau, F., ..., Fischer, N. (2013). Deep sequencing of phage display libraries to support antibody discovery. Methods,60(1), 99–110. https://doi.org/10.1016/j.ymeth.2013.03.001.

    Article  CAS  PubMed  Google Scholar 

  26. ’t Hoen, P. A. C., Jirka, S. M. G., Ten Broeke, B. R., Schultes, E. A., Aguilera, B., Pang, K. H., …, den Dunnen, J. T., (2012). Phage display screening without repetitious selection rounds. Analytical Biochemistry,421(2), 622–631. https://doi.org/10.1016/j.ab.2011.11.005.

    Article  PubMed  Google Scholar 

  27. Matochko, W. L., Chu, K., Jin, B., Lee, S. W., Whitesides, G. M., & Derda, R. (2012). Deep sequencing analysis of phage libraries using Illumina platform. Methods,58(1), 47–55. https://doi.org/10.1016/j.ymeth.2012.07.006.

    Article  CAS  PubMed  Google Scholar 

  28. Barreto, K., Maruthachalam, B. V., Hill, W., Hogan, D., Sutherland, A. R., Kusalik, A., ..., Geyer, C. R. (2019). Next-generation sequencing-guided identification and reconstruction of antibody CDR combinations from phage selection outputs. Nucleic Acids Research,47(9), e50. https://doi.org/10.1093/nar/gkz131.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Spiliotopoulos, A., Owen, J. P., Maddison, B. C., Dreveny, I., Rees, H. C., & Gough, K. C. (2015). Sensitive recovery of recombinant antibody clones after their in silico identification within NGS datasets. Journal of Immunological Methods,420, 50–55. https://doi.org/10.1016/J.JIM.2015.03.005.

    Article  CAS  PubMed  Google Scholar 

  30. D’Angelo, S., Kumar, S., Naranjo, L., Ferrara, F., Kiss, C., & Bradbury, A. R. M. (2014). From deep sequencing to actual clones. Protein Engineering Design and Selection,27(10), 301–307. https://doi.org/10.1093/protein/gzu032.

    Article  CAS  Google Scholar 

  31. Ravn, U., Gueneau, F., Baerlocher, L., Osteras, M., Desmurs, M., Malinge, P., ..., Fischer, N. (2010). By-passing in vitro screening—Next generation sequencing technologies applied to antibody display and in silico candidate selection. Nucleic Acids Research,38(21), e193–e193. https://doi.org/10.1093/nar/gkq789.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Yang, W., Yoon, A., Lee, S., Kim, S., Han, J., & Chung, J. (2017). Next-generation sequencing enables the discovery of more diverse positive clones from a phage-displayed antibody library. Experimental and Molecular Medicine,49(3), e308. https://doi.org/10.1038/emm.2017.22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang, H., Torkamani, A., Jones, T. M., Ruiz, D. I., Pons, J., & Lerner, R. A. (2011). Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems. Proceedings of the National Academy of Sciences of the United States of America,108(33), 13456–13461. https://doi.org/10.1073/pnas.1111218108.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Kunkel, T. A. (1985). Rapid and efficient site-specific mutagenesis without phenotypic selection. Proceedings of the National Academy of Sciences of the United States of America,82(2), 488–492. https://doi.org/10.1073/pnas.82.2.488.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Scholle, M. D., Kehoe, J. W., & Kay, B. K. (2005). Efficient construction of a large collection of phage-displayed combinatorial peptide libraries. Combinatorial Chemistry and High Throughput Screening,8(6), 545–551. https://doi.org/10.2174/1386207054867337.

    Article  Google Scholar 

  36. Gallo, E. (2019). A high-throughput platform for the generation of synthetic Ab clones by single-strand site-directed mutagenesis. Molecular Biotechnology,61(6), 410–420. https://doi.org/10.1007/s12033-019-00171-9.

    Article  CAS  PubMed  Google Scholar 

  37. Sanmark, H., Huovinen, T., Matikka, T., Pettersson, T., Lahti, M., & Lamminmäki, U. (2015). Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes. Journal of Immunological Methods,426, 134–139. https://doi.org/10.1016/j.jim.2015.08.005.

    Article  CAS  PubMed  Google Scholar 

  38. Nelson, R. S., & Valadon, P. (2017). A universal phage display system for the seamless construction of Fab libraries. Journal of Immunological Methods,450, 41–49. https://doi.org/10.1016/j.jim.2017.07.011.

    Article  CAS  PubMed  Google Scholar 

  39. Kato, M., & Hanyu, Y. (2013). Construction of an scFv library by enzymatic assembly of VL and VH genes. Journal of Immunological Methods,396(1–2), 15–22. https://doi.org/10.1016/j.jim.2013.07.003.

    Article  CAS  PubMed  Google Scholar 

  40. Wiedmann, M., Wilson, W. J., Czajka, J., Luo, J., Barany, F., & Batt, C. A. (1994). Ligase chain reaction (LCR)—Overview and applications. PCR Methods and Applications,3(4), S51–S64. https://doi.org/10.1101/gr.3.4.s51.

    Article  CAS  PubMed  Google Scholar 

  41. Au, L. C., Yang, F. Y., Yang, W. J., Lo, S. H., & Kao, C. F. (1998). Gene synthesis by a LCR-based approach: High-level production of leptin-L54 using synthetic gene in Escherichia coli. Biochemical and Biophysical Research Communications,248(1), 200–203. https://doi.org/10.1006/bbrc.1998.8929.

    Article  CAS  PubMed  Google Scholar 

  42. Dillon, P. J., & Rosen, C. A. (1990). A rapid method for the construction of synthetic genes using the polymerase chain reaction. BioTechniques,9(3), 298–300. https://www.ncbi.nlm.nih.gov/pubmed/2223068.

    CAS  PubMed  Google Scholar 

  43. Sandhu, G. S., Aleff, R. A., & Kline, B. C. (1992). Dual asymmetric PCR: One-step construction of synthetic genes. BioTechniques,12(1), 14–16. https://www.ncbi.nlm.nih.gov/pubmed/1734918.

    CAS  PubMed  Google Scholar 

  44. Wu, G., Wolf, J. B., Ibrahim, A. F., Vadasz, S., Gunasinghe, M., & Freeland, S. J. (2006). Simplified gene synthesis: A one-step approach to PCR-based gene construction. Journal of Biotechnology,124(3), 496–503. https://doi.org/10.1016/j.jbiotec.2006.01.015.

    Article  CAS  PubMed  Google Scholar 

  45. Gao, X., Gulari, E., & Zhou, X. (2004). In situ synthesis of oligonucleotide microarrays. Biopolymers,73(5), 579–596. https://doi.org/10.1002/bip.20005.

    Article  CAS  PubMed  Google Scholar 

  46. Richmond, K. E., Li, M.-H., Rodesch, M. J., Patel, M., Lowe, A. M., Kim, C., ..., Cerrina, F. (2004). Amplification and assembly of chip-eluted DNA (AACED): A method for high-throughput gene synthesis. Nucleic Acids Research,32(17), 5011–5018. https://doi.org/10.1093/nar/gkh793.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Moorcroft, M. J., Meuleman, W. R. A., Latham, S. G., Nicholls, T. J., Egeland, R. D., & Southern, E. M. (2005). In situ oligonucleotide synthesis on poly(dimethylsiloxane): A flexible substrate for microarray fabrication. Nucleic Acids Research,33(8), e75. https://doi.org/10.1093/nar/gni075.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Cox, J. C., Lape, J., Sayed, M. A., & Hellinga, H. W. (2007). Protein fabrication automation. Protein Science,16(3), 379–390. https://doi.org/10.1110/ps.062591607.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. de Wildt, R. M. T., Tomlinson, I. M., Mundy, C. R., & Gorick, B. D. (2000). Antibody arrays for high-throughput screening of antibody–antigen interactions. Nature Biotechnology,18(9), 989–994. https://doi.org/10.1038/79494.

    Article  CAS  PubMed  Google Scholar 

  50. Krebs, B., Rauchenberger, R., Reiffert, S., Rothe, C., Tesar, M., Thomassen, E., ..., Kretzschmar, T. (2001). High-throughput generation and engineering of recombinant human antibodies. Journal of Immunological Methods,254(1–2), 67–84. https://doi.org/10.1016/S0022-1759(01)00398-2.

    Article  CAS  PubMed  Google Scholar 

  51. Gupta, P. B., Onder, T. T., Jiang, G., Tao, K., Kuperwasser, C., Weinberg, R. A., et al. (2009). Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell,138(4), 645–659. https://doi.org/10.1016/j.cell.2009.06.034.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Bleicher, K. H., Böhm, H.-J., Müller, K., & Alanine, A. I. (2003). A guide to drug discovery: Hit and lead generation: Beyond high-throughput screening. Nature Reviews Drug Discovery,2(5), 369–378. https://doi.org/10.1038/nrd1086.

    Article  CAS  PubMed  Google Scholar 

  53. Dietrich, J. A., McKee, A. E., & Keasling, J. D. (2010). High-throughput metabolic engineering: Advances in small-molecule screening and selection. Annual Review of Biochemistry,79(1), 563–590. https://doi.org/10.1146/annurev-biochem-062608-095938.

    Article  CAS  PubMed  Google Scholar 

  54. Fellouse, F. A., Esaki, K., Birtalan, S., Raptis, D., Cancasci, V. J., Koide, A., ..., Sidhu, S. S. (2007). High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. Journal of Molecular Biology,373(4), 924–940. https://doi.org/10.1016/j.jmb.2007.08.005.

    Article  CAS  PubMed  Google Scholar 

  55. Miersch, S., Maruthachalam, B. V., Geyer, C. R., & Sidhu, S. S. (2017). Structure-directed and tailored diversity synthetic antibody libraries yield novel anti-EGFR antagonists. ACS Chemical Biology,12(5), 1381–1389. https://doi.org/10.1021/acschembio.6b00990.

    Article  CAS  PubMed  Google Scholar 

  56. Chung, C. T., & Miller, R. H. (1988). A rapid and convenient method for the preparation and storage of competent bacterial cells. Nucleic Acids Research,16(8), 3580. https://doi.org/10.1093/nar/16.8.3580.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods,6(5), 343–345. https://doi.org/10.1038/nmeth.1318.

    Article  CAS  PubMed  Google Scholar 

  58. Gibson, D. G. (2011). Enzymatic assembly of overlapping DNA fragments. Methods in Enzymology,498, 349–361. https://doi.org/10.1016/B978-0-12-385120-8.00015-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Sayers, J. R., & Eckstein, F. (1991). A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease. Nucleic Acids Research,19(15), 4127–4132. https://doi.org/10.1093/nar/19.15.4127.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Takahashi, M., Yamaguchi, E., & Uchida, T. (1984). Thermophilic DNA ligase. Purification and properties of the enzyme from Thermus thermophilus HB8. The Journal of Biological Chemistry,259(16), 10041–10047. https://www.ncbi.nlm.nih.gov/pubmed/6469954.

    CAS  PubMed  Google Scholar 

  61. Wang, Y., Prosen, D. E., Mei, L., Sullivan, J. C., Finney, M., & Vander Horn, P. B. (2004). A novel strategy to engineer DNA polymerases for enhanced processivity and improved performance in vitro. Nucleic Acids Research,32(3), 1197–1207. https://doi.org/10.1093/nar/gkh271.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Nixon, A. M. L., Duque, A., Yelle, N., McLaughlin, M., Davoudi, S., Pedley, N. M., ..., Moffat, J. (2019). A rapid in vitro methodology for simultaneous target discovery and antibody generation against functional cell subpopulations. Scientific Reports,9(1), 842. https://doi.org/10.1038/s41598-018-37462-1.

  63. Persson, H., Ye, W., Wernimont, A., Adams, J. J., Koide, A., Koide, S., ..., Sidhu, S. S. (2013). CDR-H3 diversity is not required for antigen recognition by synthetic antibodies. Journal of Molecular Biology,425(4), 803–811. https://doi.org/10.1016/j.jmb.2012.11.037.

    Article  CAS  PubMed  Google Scholar 

  64. Verma, R., Boleti, E., & George, A. J. (1998). Antibody engineering: Comparison of bacterial, yeast, insect and mammalian expression systems. Journal of Immunological Methods,216(1–2), 165–181. https://doi.org/10.1016/S0022-1759(98)00077-5.

    Article  CAS  PubMed  Google Scholar 

  65. Braun, P., & LaBaer, J. (2003). High throughput protein production for functional proteomics. Trends in Biotechnology,21(9), 383–388. https://doi.org/10.1016/S0167-7799(03)00189-6.

    Article  CAS  PubMed  Google Scholar 

  66. Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., ..., Rout, M. P. (2014). A robust pipeline for rapid production of versatile nanobody repertoires. Nature Methods,11(12), 1253–1260. https://doi.org/10.1038/nmeth.3170.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Na, H., Laver, J. D., Jeon, J., Singh, F., Ancevicius, K., Fan, Y., ..., Sidhu, S. S. (2016). A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes. RNA (New York, NY),22(4), 636–655. https://doi.org/10.1261/rna.055186.115.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Miethe, S., Meyer, T., Wöhl-Bruhn, S., Frenzel, A., Schirrmann, T., Dübel, S., et al. (2013). Production of single chain fragment variable (scFv) antibodies in Escherichia coli using the LEXTM bioreactor. Journal of Biotechnology,163(2), 105–111. https://doi.org/10.1016/j.jbiotec.2012.07.011.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

The author would like to thank A. Kelil for the generation of Ab clone sequences utilized in this report. This work was supported by the Charles H. Best foundation.

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  1. Department of Molecular Genetics, Charles Best Institute, University of Toronto, 112 College Street, 112 College Street, Room 70, Toronto, ON, M5G 1L6, Canada

    Eugenio Gallo

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Gallo, E. High-Throughput Generation of In Silico Derived Synthetic Antibodies via One-step Enzymatic DNA Assembly of Fragments. Mol Biotechnol 62, 142–150 (2020). https://doi.org/10.1007/s12033-019-00232-z

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