Construction of a Natural Human Fab Phage Antibody Library and Screening of Phage Antibody against PD-L1

Construction of a Natural Human Fab Phage Antibody Library and Screening of Phage Antibody against PD-L1

Loading document ...
Loading page ...


Author(s): Jiangtao Gu, Raoqing Guo, Ligang Zhang, Ning Deng

Download Full PDF Read Complete Article

DOI: 10.18483/ijSci.2561 23 84 29-38 Volume 11 - Feb 2022


Programmed Death Ligand-1(PD-L1) is an important target to drive T cell dysfunction when it connects with Programmed Death-1(PD-1), leading to immune escape of tumor cells, thus anti-PD-L1 antibody shows a promising prospect in the treatment of tumor. In order to construct a large natural antibody library, we collected a large number of lymphocytes of adults and children. The light chain and Fd genes of antibody were amplified by PCR, and the Fab phage antibody library with a library capacity of 4.27×109 was constructed. The insertion rates of the light chain library and the Fab library were 90% and 70%, respectively. The cloned sequences identified by PCR showed that all the sequences analyzed were unique, and the amino acid sequences of the CDR regions were diverse, which proved that there was good diversity in the antibody library. The positive clones that bind to PD-L1 were screened by phage ELISA, PCR identification and sequence analysis. In the end, two high-affinity positive clones were determined. The successful construction of this natural phage antibody library provided an effective method for screening human PD-L1 antibodies, which was expected to screen humanized antibodies against various antigens.


PD-L1, Phage Antibody Library, Fab Antibody, Solid-Phase Screening


  1. Aksentijevich, I., Flinn, I.W., 2002. Monoclonal antibody therapy with autologous peripheral blood stem cell transplantation for non-Hodgkin’s lymphoma. Cancer Control 9, 99–105.
  2. Armand, P., Nagler, A., Weller, E.A., Devine, S.M., Avigan, D.E., Chen, Y.-B., Kaminski, M.S., Holland, H.K., Winter, J.N., Mason, J.R., Fay, J.W., Rizzieri, D.A., Hosing, C.M., Ball, E.D., Uberti, J.P., Lazarus, H.M., Mapara, M.Y., Gregory, S.A., Timmerman, J.M., Andorsky, D., Or, R., Waller, E.K., Rotem-Yehudar, R., Gordon, L.I., 2013. Disabling immune tolerance by programmed death-1 blockade with pidilizumab after autologous hematopoietic stem-cell transplantation for diffuse large B-cell lymphoma: results of an international phase II trial. J Clin Oncol 31, 4199–4206.
  3. Bishop, K.D., Harris, J.E., Mordes, J.P., Greiner, D.L., Rossini, A.A., Czech, M.P., Phillips, N.E., 2009. Depletion of the programmed death-1 receptor completely reverses established clonal anergy in CD4(+) T lymphocytes via an interleukin-2-dependent mechanism. Cell Immunol 256, 86–91.
  4. Boland, J.M., Kwon, E.D., Harrington, S.M., Wampfler, J.A., Tang, H., Yang, P., Aubry, M.C., 2013. Tumor B7-H1 and B7-H3 expression in squamous cell carcinoma of the lung. Clin Lung Cancer 14, 157–163.
  5. Bostrom, J., Yu, S.-F., Kan, D., Appleton, B.A., Lee, C.V., Billeci, K., Man, W., Peale, F., Ross, S., Wiesmann, C., Fuh, G., 2009. Variants of the antibody herceptin that interact with HER2 and VEGF at the antigen binding site. Science 323, 1610–1614.
  6. Brahmer, J.R., Drake, C.G., Wollner, I., Powderly, J.D., Picus, J., Sharfman, W.H., Stankevich, E., Pons, A., Salay, T.M., McMiller, T.L., Gilson, M.M., Wang, C., Selby, M., Taube, J.M., Anders, R., Chen, L., Korman, A.J., Pardoll, D.M., Lowy, I., Topalian, S.L., 2010. Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates. J Clin Oncol 28, 3167–3175.
  7. Bylicki, O., Paleiron, N., Rousseau-Bussac, G., Chouaïd, C., 2018. New PDL1 inhibitors for non-small cell lung cancer: focus on pembrolizumab. Onco Targets Ther 11, 4051–4064.
  8. Cai, Y., Yao, S., Zhong, J., Zhang, J., Jiang, H., Deng, Y., Deng, N., 2017. Inhibition activity of a disulfide-stabilized diabody against basic fibroblast growth factor in lung cancer. Oncotarget 8, 20187–20197.
  9. Chikuma, S., Terawaki, S., Hayashi, T., Nabeshima, R., Yoshida, T., Shibayama, S., Okazaki, T., Honjo, T., 2009. PD-1-mediated suppression of IL-2 production induces CD8+ T cell anergy in vivo. J Immunol 182, 6682–6689.
  10. Dantas-Barbosa, C., Brígido, M.M., Maranhão, A.Q., 2005. Construction of a human Fab phage display library from antibody repertoires of osteosarcoma patients. Genet Mol Res 4, 126–140.
  11. Deantonio, C., Cotella, D., Macor, P., Santoro, C., Sblattero, D., 2014. Phage display technology for human monoclonal antibodies. Methods Mol Biol 1060, 277–295.
  12. Ebrahimizadeh, W., Rajabibazl, M., 2014. Bacteriophage vehicles for phage display: biology, mechanism, and application. Curr Microbiol 69, 109–120.
  13. Fox, K.M., Dias, J.A., Van Roey, P., 2001. Three-dimensional structure of human follicle-stimulating hormone. Mol Endocrinol 15, 378–389.
  14. Gurung, S., Khan, F., Gunassekaran, G.R., Yoo, J.D., Poongkavithai Vadevoo, S.M., Permpoon, U., Kim, S.-H., Kim, H.-J., Kim, I.-S., Han, H., Park, J.-H., Kim, S., Lee, B., 2020. Phage display-identified PD-L1-binding peptides reinvigorate T-cell activity and inhibit tumor progression. Biomaterials 247, 119984.
  15. Jiao, Y., Zhao, P., Zhu, J., Grabinski, T., Feng, Z., Guan, X., Skinner, R.S., Gross, M.D., Hay, R.V., Tachibana, H., Cao, B., 2005. Construction of human naïve Fab library and characterization of anti-met Fab fragment generated from the library. Mol Biotechnol 31, 41–54.
  16. Kambayashi, Y., Fujimura, T., Hidaka, T., Aiba, S., 2019. Biomarkers for Predicting Efficacies of Anti-PD1 Antibodies. Front Med (Lausanne) 6, 174.
  17. Kügler, J., Wilke, S., Meier, D., Tomszak, F., Frenzel, A., Schirrmann, T., Dübel, S., Garritsen, H., Hock, B., Toleikis, L., Schütte, M., Hust, M., 2015. Generation and analysis of the improved human HAL9/10 antibody phage display libraries. BMC Biotechnol 15, 10.
  18. Kwok, G., Yau, T.C.C., Chiu, J.W., Tse, E., Kwong, Y.-L., 2016. Pembrolizumab (Keytruda). Hum Vaccin Immunother 12, 2777–2789.
  19. Li, S., Jiang, K., Wang, T., Zhang, W., Shi, M., Chen, B., Hua, Z., 2020. Nanobody against PDL1. Biotechnol Lett 42, 727–736.
  20. Liu, mei jun, Gao, X., Xu, C., 2014. Research advances on Fabs antibodies. J Int Pharm Res 41, 318–347.
  21. Mellman, I., Coukos, G., Dranoff, G., 2011. Cancer immunotherapy comes of age. Nature 480, 480–489.
  22. Powles, T., Eder, J.P., Fine, G.D., Braiteh, F.S., Loriot, Y., Cruz, C., Bellmunt, J., Burris, H.A., Petrylak, D.P., Teng, S., Shen, X., Boyd, Z., Hegde, P.S., Chen, D.S., Vogelzang, N.J., 2014. MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer. Nature 515, 558–562.
  23. R, H., K, K., Y, K., H, Y., M, N., T, H., T, O., Y, T., 2010. Tumor cell expression of programmed cell death-1 ligand 1 is a prognostic factor for malignant melanoma. Cancer 116.
  24. Robert, C., Long, G.V., Brady, B., Dutriaux, C., Maio, M., Mortier, L., Hassel, J.C., Rutkowski, P., McNeil, C., Kalinka-Warzocha, E., Savage, K.J., Hernberg, M.M., Lebbé, C., Charles, J., Mihalcioiu, C., Chiarion-Sileni, V., Mauch, C., Cognetti, F., Arance, A., Schmidt, H., Schadendorf, D., Gogas, H., Lundgren-Eriksson, L., Horak, C., Sharkey, B., Waxman, I.M., Atkinson, V., Ascierto, P.A., 2015. Nivolumab in previously untreated melanoma without BRAF mutation. N Engl J Med 372, 320–330.
  25. Schwimmer, L.J., Huang, B., Giang, H., Cotter, R.L., Chemla-Vogel, D.S., Dy, F.V., Tam, E.M., Zhang, F., Toy, P., Bohmann, D.J., Watson, S.R., Beaber, J.W., Reddy, N., Kuan, H.-F., Bedinger, D.H., Rondon, I.J., 2013. Discovery of diverse and functional antibodies from large human repertoire antibody libraries. J Immunol Methods 391, 60–71.
  26. Shi, L., Chen, S., Yang, L., Li, Y., 2013. The role of PD-1 and PD-L1 in T-cell immune suppression in patients with hematological malignancies. J Hematol Oncol 6, 74.
  27. Smith, G.P., 1985. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315–1317.
  28. Tajiri, K., Kishi, H., Tokimitsu, Y., Kondo, S., Ozawa, T., Kinoshita, K., Jin, A., Kadowaki, S., Sugiyama, T., Muraguchi, A., 2007. Cell-microarray analysis of antigen-specific B-cells: single cell analysis of antigen receptor expression and specificity. Cytometry A 71, 961–967.
  29. Tian, X., Shou, C., Dong, Z., 2000. Construction of human phage antibody library and preliminary screening of anti VEGF antibody. Chinese Journal of Biochemistry and molecular biology 16, 200–205.
  30. Tiller, T., Schuster, I., Deppe, D., Siegers, K., Strohner, R., Herrmann, T., Berenguer, M., Poujol, D., Stehle, J., Stark, Y., Heßling, M., Daubert, D., Felderer, K., Kaden, S., Kölln, J., Enzelberger, M., Urlinger, S., 2013. A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties. MAbs 5, 445–470.
  31. Topalian, S.L., Hodi, F.S., Brahmer, J.R., Gettinger, S.N., Smith, D.C., McDermott, D.F., Powderly, J.D., Carvajal, R.D., Sosman, J.A., Atkins, M.B., Leming, P.D., Spigel, D.R., Antonia, S.J., Horn, L., Drake, C.G., Pardoll, D.M., Chen, L., Sharfman, W.H., Anders, R.A., Taube, J.M., McMiller, T.L., Xu, H., Korman, A.J., Jure-Kunkel, M., Agrawal, S., McDonald, D., Kollia, G.D., Gupta, A., Wigginton, J.M., Sznol, M., 2012. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366, 2443–2454.
  32. Topalian, S.L., Sznol, M., McDermott, D.F., Kluger, H.M., Carvajal, R.D., Sharfman, W.H., Brahmer, J.R., Lawrence, D.P., Atkins, M.B., Powderly, J.D., Leming, P.D., Lipson, E.J., Puzanov, I., Smith, D.C., Taube, J.M., Wigginton, J.M., Kollia, G.D., Gupta, A., Pardoll, D.M., Sosman, J.A., Hodi, F.S., 2014. Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab. J Clin Oncol 32, 1020–1030.
  33. Wang, L., Qian, J., Lu, Y., Li, H., Bao, H., He, D., Liu, Z., Zheng, Y., He, J., Li, Y., Neelapu, S., Yang, J., Kwak, L.W., Yi, Q., Cai, Z., 2013. Immune evasion of mantle cell lymphoma: expression of B7-H1 leads to inhibited T-cell response to and killing of tumor cells. Haematologica 98, 1458–1466.
  34. Weiner, L.M., Surana, R., Wang, S., 2010. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat Rev Immunol 10, 317–327.
  35. Zarei, B., Javidan, Z., Fatemi, E., Rahimi Jamnani, F., Khatami, S., Khalaj, V., 2020. Targeting c-Met on gastric cancer cells through a fully human fab antibody isolated from a large naive phage antibody library. Daru 28, 221–235.
  36. Zhang, S., Huang, J., Zhang, L., Gu, J., Song, Q., Cai, Y., Zhong, J., Zhong, H., Deng, Y., Zhu, W., Zhao, J., Deng, N., 2021. Fermentation, Purification, and Tumor Inhibition of a Disulfide-Stabilized Diabody Against Fibroblast Growth Factor-2. Front Oncol 11, 585457.

Cite this Article:

International Journal of Sciences is Open Access Journal.
This article is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) License.
Author(s) retain the copyrights of this article, though, publication rights are with Alkhaer Publications.

Search Articles

Issue June 2023

Volume 12, June 2023

Table of Contents

World-wide Delivery is FREE

Share this Issue with Friends:

Submit your Paper