Investigational New Drugs

, Volume 37, Issue 5, pp 876–889 | Cite as

A novel tetravalent bispecific antibody targeting programmed death 1 and tyrosine-protein kinase Met for treatment of gastric cancer

  • Weihua Hou
  • Qingyun Yuan
  • Xingxing Yuan
  • Yuxiong Wang
  • Wei Mo
  • Huijie WangEmail author
  • Min YuEmail author


Background Redirecting T cells to tumor cells using bispecific antibodies (BsAbs) is emerging as a potent cancer therapy. The main concept of this strategy is to cross-link tumor cells and T cells by simultaneously binding to cell surface tumor-associated antigen (TAA) and the CD3ƹ chain. However, immune checkpoint programmed cell death ligand-1 (PD-L1) on tumor cells or other myeloid cells upreglulated remarkablely after the treatment of CD3-binding BsAbs, leads to the generation of suppressed microenvironment for immune evasion and tumor progression. Although this resistance could be partially reversed by anti-PD-L1 treatment, targeting two pathways through one antibody-based molecule may provide a strategic advantage over the combination of BsAbs and immune checkpoint inhibitors. Methods We developed two novel BsAbs PD-1/c-Met DVD-Ig and IgG-scFv both targeting PD-1 to restore the immune effector function of T cells and engaging them to tumor cells via binding to cellular-mesenchymal to epithelial transition factor (c-Met). Binding activities, T cell activation and proliferation were analyzed by flow cytometry. Cell Cytotoxicity and cytokine release were measured using LDH release assay and ELISA, respectively. Anti-tumor response in vivo was evaluated by generate xenograft models in NOD-SCID mice. Results These bispecific antibodies exhibited effective antitumor activity against high- and low- c-Met-expressing gastric cancer cell lines in vitro and mediated strong tumor growth inhibition in human gastric cancer xenograft models. Conclusion The engagement of the PD-1/PD-L1 blockade to c-Met-overexpressing cancer cells is a promising strategy for the treatment of gastric cancer and potentially other malignancies.


PD-1 C-MET Bispecific antibody Cancer immunotherapy 



This work was supported financially by the National Key Research Project Bio-safety Key Technology Development Program 2016YFC1201501.

Compliance with ethical standards

Conflict of interest


Ethical approval

All procedures involving human blood products were in accordance with the 1964 Helsinki declaration and its later amendments. Study involving mice use was carried out according to the protocols approved by Institutional Animal Care and Use Committee of Fudan University.

Informed consent

All individual participants who donated blood samples for analysis signed an informed consent form.


  1. 1.
    Siegel RL, Miller KD, Jemal A (2016) Cancer statistics, 2016. CA Cancer J Clin 66(1):7–30. CrossRefGoogle Scholar
  2. 2.
    Arrington AK, Nelson R, Patel SS, Luu C, Ko M, Garcia-Aguilar J, Kim J (2013) Timing of chemotherapy and survival in patients with resectable gastric adenocarcinoma. World J Gastrointest Surg 5(12):321–328. CrossRefGoogle Scholar
  3. 3.
    Qiu MZ, Xu RH (2013) The progress of targeted therapy in advanced gastric cancer. Biomark Res 1(1):32. CrossRefGoogle Scholar
  4. 4.
    Organ SL, Tsao MS (2011) An overview of the c-MET signaling pathway. Ther Adv Med Oncol 3(1 Suppl):S7–S19. CrossRefGoogle Scholar
  5. 5.
    Lennerz JK, Kwak EL, Ackerman A, Michael M, Fox SB, Bergethon K, Lauwers GY, Christensen JG, Wilner KD, Haber DA, Salgia R, Bang YJ, Clark JW, Solomon BJ, Iafrate AJ (2011) MET amplification identifies a small and aggressive subgroup of esophagogastric adenocarcinoma with evidence of responsiveness to crizotinib. J Clin Oncol Off J Am Soc Clin Oncol 29(36):4803–4810. CrossRefGoogle Scholar
  6. 6.
    Erichsen R, Kelsh MA, Oliner KS, Nielsen KB, Froslev T, Laenkholm AV, Vyberg M, Acquavella J, Sorensen HT (2016) Prognostic impact of tumor MET expression among patients with stage IV gastric cancer: a Danish cohort study. Ann Epidemiol 26(7):500–503. CrossRefGoogle Scholar
  7. 7.
    Lee HE, Kim MA, Lee HS, Jung EJ, Yang HK, Lee BL, Bang YJ, Kim WH (2012) MET in gastric carcinomas: comparison between protein expression and gene copy number and impact on clinical outcome. Br J Cancer 107(2):325–333. CrossRefGoogle Scholar
  8. 8.
    Zhu M, Tang R, Doshi S, Oliner KS, Dubey S, Jiang Y, Donehower RC, Iveson T, Loh EY, Zhang Y (2015) Exposure-response analysis of rilotumumab in gastric cancer: the role of tumour MET expression. Br J Cancer 112(3):429–437. CrossRefGoogle Scholar
  9. 9.
    Dreier T, Lorenczewski G, Brandl C, Hoffmann P, Syring U, Hanakam F, Kufer P, Riethmuller G, Bargou R, Baeuerle PA (2002) Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody. Int J Cancer 100(6):690–697. CrossRefGoogle Scholar
  10. 10.
    Garber K (2014) Bispecific antibodies rise again. Nat Rev Drug Discov 13(11):799–801. CrossRefGoogle Scholar
  11. 11.
    Brinkmann U, Kontermann RE (2017) The making of bispecific antibodies. mAbs 9(2):182–212. CrossRefGoogle Scholar
  12. 12.
    Frankel SR, Baeuerle PA (2013) Targeting T cells to tumor cells using bispecific antibodies. Curr Opin Chem Biol 17(3):385–392. CrossRefGoogle Scholar
  13. 13.
    Cartellieri M, Arndt C, Feldmann A, von Bonin M, Ewen EM, Koristka S, Michalk I, Stamova S, Berndt N, Gocht A, Bornhauser M, Ehninger G, Schmitz M, Bachmann M (2013) TCR/CD3 activation and co-stimulation combined in one T cell retargeting system improve anti-tumor immunity. Oncoimmunology 2(12):e26770. CrossRefGoogle Scholar
  14. 14.
    Bargou R, Leo E, Zugmaier G, Klinger M, Goebeler M, Knop S, Noppeney R, Viardot A, Hess G, Schuler M, Einsele H, Brandl C, Wolf A, Kirchinger P, Klappers P, Schmidt M, Riethmuller G, Reinhardt C, Baeuerle PA, Kufer P (2008) Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science 321(5891):974–977. CrossRefGoogle Scholar
  15. 15.
    Osada T, Patel SP, Hammond SA, Osada K, Morse MA, Lyerly HK (2015) CEA/CD3-bispecific T cell-engaging (BiTE) antibody-mediated T lymphocyte cytotoxicity maximized by inhibition of both PD1 and PD-L1. Cancer Immunol Immunother: CII 64(6):677–688. CrossRefGoogle Scholar
  16. 16.
    Zou W, Wolchok JD, Chen L (2016) PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: mechanisms, response biomarkers, and combinations. Sci Transl Med 8(328):328rv324. CrossRefGoogle Scholar
  17. 17.
    Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, Chen S, Klein AP, Pardoll DM, Topalian SL, Chen L (2012) Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med 4(127):127ra137. CrossRefGoogle Scholar
  18. 18.
    Chang CH, Wang Y, Li R, Rossi DL, Liu D, Rossi EA, Cardillo TM, Goldenberg DM (2017) Combination therapy with bispecific antibodies and PD-1 blockade enhances the antitumor potency of T cells. Cancer Res 77(19):5384–5394. CrossRefGoogle Scholar
  19. 19.
    Junttila TT, Li J, Johnston J, Hristopoulos M, Clark R, Ellerman D, Wang BE, Li Y, Mathieu M, Li G, Young J, Luis E, Lewis Phillips G, Stefanich E, Spiess C, Polson A, Irving B, Scheer JM, Junttila MR, Dennis MS, Kelley R, Totpal K, Ebens A (2014) Antitumor efficacy of a bispecific antibody that targets HER2 and activates T cells. Cancer Res 74(19):5561–5571. CrossRefGoogle Scholar
  20. 20.
    Sun LL, Wang P, Clark R, Hristopoulos M, Ellerman D, Mathieu M, Chu Y-W, Wang H, Totpal K, Ebens AJ, Polson AG, Gould S (2016) Preclinical characterization of combinability and potential synergy of anti-CD20/CD3 T-cell dependent bispecific antibody with chemotherapy and PD-1/PD-L1 blockade. Blood 128(22):4168–4168Google Scholar
  21. 21.
    Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N (2006) Immunohistochemical localization of programmed death-1 ligand-1 (PD-L1) in gastric carcinoma and its clinical significance. Acta Histochem 108(1):19–24. CrossRefGoogle Scholar
  22. 22.
    Jiang J, Zhu Y, Wu C, Shen Y, Wei W, Chen L, Zheng X, Sun J, Lu B, Zhang X (2010) Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer. Cancer Immunol Immunother: CII 59(11):1707–1714. CrossRefGoogle Scholar
  23. 23.
    Eto S, Yoshikawa K, Nishi M, Higashijima J, Tokunaga T, Nakao T, Kashihara H, Takasu C, Iwata T, Shimada M (2016) Programmed cell death protein 1 expression is an independent prognostic factor in gastric cancer after curative resection. Gastric Cancer 19(2):466–471. CrossRefGoogle Scholar
  24. 24.
    Bilgin B, Sendur MA, Bulent Akinci M, Sener Dede D, Yalcin B (2017) Targeting the PD-1 pathway: a new hope for gastrointestinal cancers. Curr Med Res Opin 33(4):749–759. CrossRefGoogle Scholar
  25. 25.
    Wu C, Ying H, Grinnell C, Bryant S, Miller R, Clabbers A, Bose S, McCarthy D, Zhu RR, Santora L, Davis-Taber R, Kunes Y, Fung E, Schwartz A, Sakorafas P, Gu J, Tarcsa E, Murtaza A, Ghayur T (2007) Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol 25(11):1290–1297. CrossRefGoogle Scholar
  26. 26.
    Orcutt KD, Ackerman ME, Cieslewicz M, Quiroz E, Slusarczyk AL, Frangioni JV, Wittrup KD (2010) A modular IgG-scFv bispecific antibody topology. Protein Eng Des Sel: PEDS 23(4):221–228. CrossRefGoogle Scholar
  27. 27.
    Gan Y, Shi C, Inge L, Hibner M, Balducci J, Huang Y (2010) Differential roles of ERK and Akt pathways in regulation of EGFR-mediated signaling and motility in prostate cancer cells. Oncogene 29(35):4947–4958. CrossRefGoogle Scholar
  28. 28.
    Nagai T, Arao T, Furuta K, Sakai K, Kudo K, Kaneda H, Tamura D, Aomatsu K, Kimura H, Fujita Y, Matsumoto K, Saijo N, Kudo M, Nishio K (2011) Sorafenib inhibits the hepatocyte growth factor-mediated epithelial mesenchymal transition in hepatocellular carcinoma. Mol Cancer Ther 10(1):169–177. CrossRefGoogle Scholar
  29. 29.
    Wu Z, Cheung NV (2018) T cell engaging bispecific antibody (T-BsAb): from technology to therapeutics. Pharmacol Ther 182:161–175. CrossRefGoogle Scholar
  30. 30.
    Nagorsen D, Baeuerle PA (2011) Immunomodulatory therapy of cancer with T cell-engaging BiTE antibody blinatumomab. Exp Cell Res 317(9):1255–1260. CrossRefGoogle Scholar
  31. 31.
    Motz GT, Coukos G (2011) The parallel lives of angiogenesis and immunosuppression: cancer and other tales. Nat Rev Immunol 11(10):702–711. CrossRefGoogle Scholar
  32. 32.
    Brezski RJ, Georgiou G (2016) Immunoglobulin isotype knowledge and application to fc engineering. Curr Opin Immunol 40:62–69. CrossRefGoogle Scholar
  33. 33.
    Dahan R, Sega E, Engelhardt J, Selby M, Korman AJ, Ravetch JV (2015) FcgammaRs modulate the anti-tumor activity of antibodies targeting the PD-1/PD-L1 Axis. Cancer Cell 28(3):285–295. CrossRefGoogle Scholar
  34. 34.
    Linke R, Klein A, Seimetz D (2010) Catumaxomab: clinical development and future directions. mAbs 2(2):129–136CrossRefGoogle Scholar
  35. 35.
    Correia I, Sung J, Burton R, Jakob CG, Carragher B, Ghayur T, Radziejewski C (2013) The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen. mAbs 5(3):364–372. CrossRefGoogle Scholar
  36. 36.
    Klein C, Sustmann C, Thomas M, Stubenrauch K, Croasdale R, Schanzer J, Brinkmann U, Kettenberger H, Regula JT, Schaefer W (2012) Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies. mAbs 4(6):653–663. CrossRefGoogle Scholar
  37. 37.
    Jakob CG, Edalji R, Judge RA, DiGiammarino E, Li Y, Gu J, Ghayur T (2013) Structure reveals function of the dual variable domain immunoglobulin (DVD-Ig) molecule. mAbs 5(3):358–363. CrossRefGoogle Scholar
  38. 38.
    Smyth EC, Sclafani F, Cunningham D (2014) Emerging molecular targets in oncology: clinical potential of MET/hepatocyte growth-factor inhibitors. Oncotargets Ther 7:1001–1014. CrossRefGoogle Scholar
  39. 39.
    Cecchi F, Rabe DC, Bottaro DP (2012) Targeting the HGF/Met signaling pathway in cancer therapy. Expert Opin Ther Targets 16(6):553–572. CrossRefGoogle Scholar
  40. 40.
    Gherardi E, Birchmeier W, Birchmeier C, Vande Woude G (2012) Targeting MET in cancer: rationale and progress. Nat Rev Cancer 12(2):89–103. CrossRefGoogle Scholar
  41. 41.
    Michaud NR, Jani JP, Hillerman S, Tsaparikos KE, Barbacci-Tobin EG, Knauth E, Putz H Jr, Campbell M, Karam GA, Chrunyk B, Gebhard DF, Green LL, Xu JJ, Dunn MC, Coskran TM, Lapointe JM, Cohen BD, Coleman KG, Bedian V, Vincent P, Kajiji S, Steyn SJ, Borzillo GV, Los G (2012) Biochemical and pharmacological characterization of human c-Met neutralizing monoclonal antibody CE-355621. mAbs 4(6):710–723. CrossRefGoogle Scholar
  42. 42.
    Pacchiana G, Chiriaco C, Stella MC, Petronzelli F, De Santis R, Galluzzo M, Carminati P, Comoglio PM, Michieli P, Vigna E (2010) Monovalency unleashes the full therapeutic potential of the DN-30 anti-Met antibody. J Biol Chem 285(46):36149–36157. CrossRefGoogle Scholar
  43. 43.
    Tolbert WD, Daugherty-Holtrop J, Gherardi E, Vande Woude G, Xu HE (2010) Structural basis for agonism and antagonism of hepatocyte growth factor. Proc Natl Acad Sci U S A 107(30):13264–13269. CrossRefGoogle Scholar
  44. 44.
    Sanchez-Martin D, Sorensen MD, Lykkemark S, Sanz L, Kristensen P, Ruoslahti E, Alvarez-Vallina L (2015) Selection strategies for anticancer antibody discovery: searching off the beaten path. Trends Biotechnol 33(5):292–301. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular Biology, School of Basic MedicineFudan UniversityShanghaiChina
  2. 2.Department of Medical Oncology, Shanghai Cancer Center, School of Basic MedicineFudan UniversityShanghaiChina

Personalised recommendations