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Generation of fully human anti-GPC3 antibodies with high-affinity recognition of GPC3 positive tumors

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Summary

The acceleration of therapeutic antibody development has been motivated by the benefit to and their demand for human health. In particular, humanized transgenic antibody discovery platforms, combined with immunization, hybridoma fusion and/or single cell DNA sequencing are the most reliable and rapid methods for mining the human monoclonal antibodies. Human GPC3 protein is an oncofetal antigen, and it is highly expressed in most hepatocellular carcinomas and some types of squamous cell carcinomas. Currently, no fully human anti-GPC3 therapeutic antibodies have been reported and evaluated in extensive tumor tissues. Here, we utilized a new humanized transgenic mouse antibody discovery platform (CAMouse) that contains large V(D)J -regions and human gamma-constant regions of human immunoglobulin in authentic configurations to generate fully human anti-GPC3 antibodies. Our experiments resulted in four anti-GPC3 antibodies with high-specific binding and cytotoxicity to GPC3 positive cancer cells, and the antibody affinities are in the nanomolar range. Immunohistochemistry analysis demonstrated that these antibodies can recognize GPC3 protein on many types of solid tumors. In summary, the human anti-human GPC3 monoclonal antibodies described here are leading candidates for further preclinical studies of cancer therapy, further, the CAMouse platform is a robust tool for human therapeutic antibody discovery.

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Data availability

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Abbreviations

GPC3:

glypican-3

HCC:

hepatocellular carcinoma

ADCC:

antibody-dependent cellular cytotoxicity

CDC:

complement-dependent cytotoxicity

VH:

heavy chain variable

VL:

variable light chain

CDR:

complementarity-determining region

CAR-T:

chimeric antigen receptor T cells

BsAb:

bispecific antibody

ADC:

antibody drug conjugate

PBMCs:

peripheral blood mononuclear cells

ELISA:

enzyme-linked immunosorbent assay

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TDCC:

T cell–dependent cellular cytotoxicity

References

  1. Frenzel A, Schirrmann T, Hust M (2016) Phage display-derived human antibodies in clinical development and therapy. mAbs 8(7):1177–1194. https://doi.org/10.1080/19420862.2016.1212149

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Bruggemann M, Osborn MJ, Ma B, Hayre J, Avis S, Lundstrom B, Buelow R (2015) Human antibody production in transgenic animals. Arch Immunol Ther Exp 63(2):101–108. https://doi.org/10.1007/s00005-014-0322-x

    Article  CAS  Google Scholar 

  3. Bruggemann M, Osborn MJ, Ma B, Buelow R (2017) Strategies to obtain diverse and specific human monoclonal antibodies from transgenic animals. Transplantation 101(8):1770–1776. https://doi.org/10.1097/TP.0000000000001702

    Article  PubMed  Google Scholar 

  4. Chen WC, Murawsky CM (2018) Strategies for generating diverse antibody repertoires using transgenic animals expressing human antibodies. Front Immunol 9:460. https://doi.org/10.3389/fimmu.2018.00460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Filmus J, Capurro M, Rast J (2008) Glypicans. Genome Biol 9(5):224. https://doi.org/10.1186/gb-2008-9-5-224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhou F, Shang W, Yu X, Tian J (2017) Glypican-3: a promising biomarker for hepatocellular carcinoma diagnosis and treatment. Med Res Rev 38:741–767. https://doi.org/10.1002/med.21455

    Article  CAS  PubMed  Google Scholar 

  7. Hippo Y, Watanabe K, Watanabe A, Midorikawa Y, Yamamoto S, Ihara S, Tokita S, Iwanari H, Ito Y, Nakano K, Nezu J, Tsunoda H, Yoshino T, Ohizumi I, Tsuchiya M, Ohnishi S, Makuuchi M, Hamakubo T, Kodama T, Aburatani H (2004) Identification of soluble NH2-terminal fragment of glypican-3 as a serological marker for early-stage hepatocellular carcinoma. Cancer Res 64(7):2418–2423. https://doi.org/10.1158/0008-5472.Can-03-2191

    Article  CAS  PubMed  Google Scholar 

  8. Capurro M, Wanless IR, Sherman M, Deboer G, Shi W, Miyoshi E, Filmus J (2003) Glypican-3: a novel serum and histochemical marker for hepatocellular carcinoma. Gastroenterology 125(1):89–97. https://doi.org/10.1016/s0016-5085(03)00689-9

    Article  CAS  PubMed  Google Scholar 

  9. Sung YK, Hwang SY, Park MK, Farooq M, Han IS, Bae HI, Kim JC, Kim M (2003) Glypican-3 is overexpressed in human hepatocellular carcinoma. Cancer Sci 94(3):259–262

    Article  CAS  Google Scholar 

  10. Baumhoer D, Tornillo L, Stadlmann S, Roncalli M, Diamantis EK, Terracciano LM (2008) Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples. Am J Clin Pathol 129(6):899–906. https://doi.org/10.1309/hcqwpwd50xhd2dw6

    Article  PubMed  Google Scholar 

  11. Liu X, Wang SK, Zhang K, Zhang H, Pan Q, Liu Z, Pan H, Xue L, Yen Y, Chu PG (2015) Expression of glypican 3 enriches hepatocellular carcinoma development-related genes and associates with carcinogenesis in cirrhotic livers. Carcinogenesis 36(2):232–242. https://doi.org/10.1093/carcin/bgu245

    Article  CAS  PubMed  Google Scholar 

  12. Kinoshita Y, Tanaka S, Souzaki R, Miyoshi K, Kohashi K, Oda Y, Nakatsura T, Taguchi T (2015) Glypican 3 expression in pediatric malignant solid tumors. Eur J Pediatr Surg 25(1):138–144. https://doi.org/10.1055/s-0034-1393961

  13. Iglesias BV, Centeno G, Pascuccelli H, Ward F, Peters MG, Filmus J, Puricelli L, de Kier Joffe EB (2008) Expression pattern of glypican-3 (GPC3) during human embryonic and fetal development. Histol Histopathol 23(11):1333–1340

    CAS  PubMed  Google Scholar 

  14. Capurro MI, Xiang Y-Y, Lobe C, Filmus J (2005) Glypican-3 promotes the growth of hepatocellular carcinoma by stimulating canonical Wnt signaling. Cancer Res 65:6245–6254

    Article  CAS  Google Scholar 

  15. Song HH, Shi W, Xiang YY, Filmus J (2005) The loss of glypican-3 induces alterations in Wnt signaling. J Biol Chem 280(3):2116–2125. https://doi.org/10.1074/jbc.M410090200

    Article  CAS  PubMed  Google Scholar 

  16. Capurro M, Martin T, Shi W, Filmus J (2014) Glypican-3 binds to frizzled and plays a direct role in the stimulation of canonical Wnt signaling. J Cell Sci 127(Pt 7):1565–1575. https://doi.org/10.1242/jcs.140871

    Article  CAS  PubMed  Google Scholar 

  17. Li N, Wei L, Liu X, Bai H, Ye Y, Li D, Li N, Baxa U, Wang Q, Lv L, Chen Y, Feng M, Lee B, Gao W, Ho M (2019) A frizzled-like cysteine rich domain in glypican-3 mediates Wnt binding and regulates hepatocellular carcinoma tumor growth in mice. Hepatology. 70:1231–1245. https://doi.org/10.1002/hep.30646

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Midorikawa Y, Ishikawa S, Iwanari H, Imamura T, Sakamoto H, Miyazono K, Kodama T, Makuuchi M, Aburatani H (2003) Glypican-3, overexpressed in hepatocellular carcinoma, modulates FGF2 and BMP-7 signaling. Int J Cancer 103(4):455–465. https://doi.org/10.1002/ijc.10856

    Article  CAS  PubMed  Google Scholar 

  19. Wong C-M, Gao W, Kim H, Ho M (2015) Human monoclonal antibody targeting the heparan sulfate chains of glypican-3 inhibits HGF-mediated migration and motility of hepatocellular carcinoma cells. PLoS One 10(9):e0137664. https://doi.org/10.1371/journal.pone.0137664

    Article  CAS  Google Scholar 

  20. Takai H, Ashihara M, Ishiguro T, Terashima H, Watanabe T, Kato A, Suzuki M (2009) Involvement of glypican-3 in the recruitment of M2-polarized tumor-associated macrophages in hepatocellular carcinoma. Cancer Biol Ther 8(24):2329–2338

    Article  CAS  Google Scholar 

  21. Wu Y, Liu H, Weng H, Zhang X, Li P, Fan CL, Li B, Dong PL, Li L, Dooley S, Ding HG (2015) Glypican-3 promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells through ERK signaling pathway. Int J Oncol 46(3):1275–1285. https://doi.org/10.3892/ijo.2015.2827

    Article  CAS  PubMed  Google Scholar 

  22. Ishiguro T, Sugimoto M, Kinoshita Y, Miyazaki Y, Nakano K, Tsunoda H, Sugo I, Ohizumi I, Aburatani H, Hamakubo T, Kodama T, Tsuchiya M, Yamada-Okabe H (2008) Anti-glypican 3 antibody as a potential antitumor agent for human liver cancer. Cancer Res 68(23):9832–9838. https://doi.org/10.1158/0008-5472.CAN-08-1973

    Article  CAS  PubMed  Google Scholar 

  23. Phung Y, Gao W, Man YG, Nagata S, Ho M (2012) High-affinity monoclonal antibodies to cell surface tumor antigen glypican-3 generated through a combination of peptide immunization and flow cytometry screening. mAbs 4(5):592–599. https://doi.org/10.4161/mabs.20933

    Article  PubMed  PubMed Central  Google Scholar 

  24. Nakano K, Ishiguro T, Konishi H, Tanaka M, Sugimoto M, Sugo I, Igawa T, Tsunoda H, Kinoshita Y, Habu K, Orita T, Tsuchiya M, Hattori K, Yamada-Okabe H (2010) Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization. Anti-Cancer Drugs 21(10):907–916. https://doi.org/10.1097/CAD.0b013e32833f5d68

    Article  CAS  PubMed  Google Scholar 

  25. Zhang YF, Ho M (2016) Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma. Sci Rep 6:33878. https://doi.org/10.1038/srep33878

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Feng M, Gao W, Wang R, Chen W, Man YG, Figg WD, Wang XW, Dimitrov DS, Ho M (2013) Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma. Proc Natl Acad Sci 110(12):E1083–E1091. https://doi.org/10.1073/pnas.1217868110

    Article  CAS  PubMed  Google Scholar 

  27. Hanaoka H, Nagaya T, Sato K, Nakamura Y, Watanabe R, Harada T, Gao W, Feng M, Phung Y, Kim I, Paik CH, Choyke PL, Ho M, Kobayashi H (2015) Glypican-3 targeted human heavy chain antibody as a drug carrier for hepatocellular carcinoma therapy. Mol Pharm 12(6):2151–2157. https://doi.org/10.1021/acs.molpharmaceut.5b00132

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Abou-Alfa GK, Puig O, Daniele B, Kudo M, Merle P, Park JW, Ross P, Peron JM, Ebert O, Chan S, Poon TP, Colombo M, Okusaka T, Ryoo BY, Minguez B, Tanaka T, Ohtomo T, Ukrainskyj S, Boisserie F, Rutman O, Chen YC, Xu C, Shochat E, Jukofsky L, Reis B, Chen G, Di Laurenzio L, Lee R, Yen CJ (2016) Randomized phase II placebo controlled study of codrituzumab in previously treated patients with advanced hepatocellular carcinoma. J Hepatol 65(2):289–295. https://doi.org/10.1016/j.jhep.2016.04.004

    Article  CAS  PubMed  Google Scholar 

  29. Jain T, Sun T, Durand S, Hall A, Houston NR, Nett JH, Sharkey B, Bobrowicz B, Caffry I, Yu Y, Cao Y, Lynaugh H, Brown M, Baruah H, Gray LT, Krauland EM, Xu Y, Vasquez M, Wittrup KD (2017) Biophysical properties of the clinical-stage antibody landscape. Proc Natl Acad Sci U S A 114(5):944–949. https://doi.org/10.1073/pnas.1616408114

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ramos-Vara JA (2011) Principles and methods of immunohistochemistry. Methods Mol Biol (Clifton, NJ) 691:83–96. https://doi.org/10.1007/978-1-60761-849-2_5

    Article  CAS  Google Scholar 

  31. Barnes DM, Harris WH, Smith P, Millis RR, Rubens RD (1996) Immunohistochemical determination of oestrogen receptor: comparison of different methods of assessment of staining and correlation with clinical outcome of breast cancer patients. Br J Cancer 74(9):1445–1451. https://doi.org/10.1038/bjc.1996.563

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ishiguro T, Sano Y, S-i K, Kamata-Sakurai M, Kaneko A, Kinoshita Y, Shiraiwa H, Azuma Y, Tsunenari T, Kayukawa Y, Sonobe Y, Ono N, Sakata K, Fujii T, Miyazaki Y, Noguchi M, Endo M, Harada A, Frings W, Fujii E, Nanba E, Narita A, Sakamoto A, Wakabayashi T, Konishi H, Segawa H, Igawa T, Tsushima T, Mutoh H, Nishito Y, Takahashi M, Stewart L, ElGabry E, Kawabe Y, Ishigai M, Chiba S, Aoki M, Hattori K, Nezu J (2017) An anti–glypican 3/CD3 bispecific T cell–redirecting antibody for treatment of solid tumors. Sci Transl Med 9(410):eaal4291. https://doi.org/10.1126/scitranslmed.aal4291

    Article  CAS  PubMed  Google Scholar 

  33. Oberst MD, Fuhrmann S, Mulgrew K, Amann M, Cheng L, Lutterbuese P, Richman L, Coats S, Baeuerle PA, Hammond SA (2014) CEA/CD3 bispecific antibody MEDI-565/AMG 211 activation of T cells and subsequent killing of human tumors is independent of mutations commonly found in colorectal adenocarcinomas. mAbs 6(6):1571–1584. https://doi.org/10.4161/19420862.2014.975660

    Article  PubMed  PubMed Central  Google Scholar 

  34. Ho DT, Park JF, Ho DE, Nunez B, Bang J, Ni JHT (2016) Fucosylation of a therapeutic antibody: effects on antibody-dependent, cell-mediated cytotoxicity (ADCC) potency and efficacy. BioProcess International 14(4):30–38

    Article  CAS  Google Scholar 

  35. Wu J, Han D, Shi S, Zhang Q, Zheng G, Wei M, Han Y, Li G, Yang F, Jiao D, Xie P, Zhang L, Yang AG, Zhao A, Qin W, Wen W (2019) A novel fully human antibody targeting extracellular domain of PSMA inhibits tumor growth in prostate cancer. Mol Cancer Ther 18:1289–1301. https://doi.org/10.1158/1535-7163.MCT-18-1078

    Article  CAS  PubMed  Google Scholar 

  36. Jiang Z, Jiang X, Chen S, Lai Y, Wei X, Li B, Lin S, Wang S, Wu Q, Liang Q, Liu Q, Peng M, Yu F, Weng J, Du X, Pei D, Liu P, Yao Y, Xue P, Li P (2016) Anti-GPC3-CAR T cells suppress the growth of tumor cells in patient-derived xenografts of hepatocellular carcinoma. Front Immunol 7:690. https://doi.org/10.3389/fimmu.2016.00690

    Article  CAS  PubMed  Google Scholar 

  37. Fu Y, Urban DJ, Nani RR, Zhang Y-F, Li N, Fu H, Shah H, Gorka AP, Guha R, Chen L, Hall MD, Schnermann MJ, Ho M (2018) Glypican-3 specific antibody drug conjugates targeting hepatocellular carcinoma. Hepatology. 70:563–576. https://doi.org/10.1002/hep.30326

    Article  CAS  Google Scholar 

  38. Shimizu Y, Suzuki T, Yoshikawa T, Endo I, Nakatsura T (2019) Next-generation cancer immunotherapy targeting glypican-3. Front Oncol 9:248. https://doi.org/10.3389/fonc.2019.00248

    Article  PubMed  PubMed Central  Google Scholar 

  39. Yu X, Li Y, Chen SW, Shi Y, Xu F (2015) Differential expression of glypican-3 (GPC3) in lung squamous cell carcinoma and lung adenocarcinoma and its clinical significance. Genet Mol Res 14(3):10185–10192. https://doi.org/10.4238/2015.August.28.2

  40. Wang D, Gao Y, Zhang Y, Wang L, Chen G (2019) Glypican-3 promotes cell proliferation and tumorigenesis through up-regulation of beta-catenin expression in lung squamous cell carcinoma. Biosci Rep 39(6):BSR20181147. https://doi.org/10.1042/BSR20181147

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ortiz MV, Roberts SS, Glade Bender J, Shukla N, Wexler LH (2019) Immunotherapeutic targeting of GPC3 in pediatric solid embryonal tumors. Front Oncol 9:108. https://doi.org/10.3389/fonc.2019.00108

    Article  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by National Major Scientific Research Instrument Development Project of NSFC (21827812), The Key R & D Project in Agriculture and Animal Husbandrys of Rongchang (19255), and Chongqing Agriculture Development Grant (17406).

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Authors and Affiliations

  1. Key Laboratory of Biorheological Science and Technology (Ministry of Education), College of Bioengineering, Chongqing University, No. 174 Shazheng Street, Shapingba District, Chongqing, 400044, China

    Lin Yu, Heng Sun & Jianhua Wang

  2. Chongqing Academy of Animal Sciences, No.51 Changlong Street, Rongchang District, Chongqing, 402460, China

    Xi Yang, Nan Huang, Meng Wu, Qilin He, Qiaoli Lang, Zuohua Liu & Liangpeng Ge

  3. Chongqing Engineering Technology Research Center for Medical Animal Resources Development and Application, Chongqing, 402460, China

    Xi Yang, Nan Huang, Meng Wu, Qilin He, Qiaoli Lang, Zuohua Liu & Liangpeng Ge

  4. Chongqing CAMAB Biotech Ltd., Chongqing, 402460, China

    Xiangang Zou & Liangpeng Ge

Authors
  1. Lin Yu
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  2. Xi Yang
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  3. Nan Huang
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  4. Meng Wu
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  5. Heng Sun
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  6. Qilin He
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  7. Qiaoli Lang
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  8. Xiangang Zou
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  9. Zuohua Liu
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  10. Jianhua Wang
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  11. Liangpeng Ge
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Contributions

Lin Yu, Liangpeng Ge and Xiangang Zou designed the experiments and Zuohua Liu helped in design; Lin Yu, Xi Yang, Nan Huang, Meng Wu, Heng Sun, Qilin He, Qiaoli Lang performed the experiments. Lin Yu wrote the manuscript. Xiangang Zou and Jianhua Wang helped in the writing.

Corresponding authors

Correspondence to Jianhua Wang or Liangpeng Ge.

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Ethical approval

All protocols performed in studies involving animals were approved by the Ethics Committee of Chongqing Academy of Animal Sciences.

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Conflict of interest

A provisional patent application that covers the fully human anti-GPC3 antibodies and its usage has been filed jointly by Chongqing Academy of Animal Sciences and Chongqing CAMAB Biotech Ltd.

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Yu, L., Yang, X., Huang, N. et al. Generation of fully human anti-GPC3 antibodies with high-affinity recognition of GPC3 positive tumors. Invest New Drugs 39, 615–626 (2021). https://doi.org/10.1007/s10637-020-01033-x

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