Investigational New Drugs

, Volume 36, Issue 2, pp 171–186 | Cite as

Hersintuzumab: A novel humanized anti-HER2 monoclonal antibody induces potent tumor growth inhibition

  • Mohammad Mehdi Amiri
  • Forough Golsaz-Shirazi
  • Tahereh Soltantoyeh
  • Reza Hosseini-Ghatar
  • Tannaz Bahadori
  • Jalal Khoshnoodi
  • Shadi Sadat Navabi
  • Samira Farid
  • Mohammad Hossein Karimi-Jafari
  • Mahmood Jeddi-Tehrani
  • Fazel Shokri


Humanized monoclonal antibodies (mAbs) against HER2 including trastuzumab and pertuzumab are widely used to treat HER2 overexpressing metastatic breast cancers. These two mAbs recognize distinct epitopes on HER2 and their combination induces a more potent blockade of HER2 signaling than trastuzumab alone. Recently, we have reported characterization of a new chimeric mAb (c-1T0) which binds to an epitope different from that recognized by trastuzumab and significantly inhibits proliferation of HER2 overexpressing tumor cells. Here, we describe humanization of this mAb by grafting all six complementarity determining regions (CDRs) onto human variable germline genes. Humanized VH and VL sequences were synthesized and ligated to human γ1 and κ constant region genes using splice overlap extension (SOE) PCR. Subsequently, the humanized antibody designated hersintuzumab was expressed and characterized by ELISA, Western blot and flow cytometry. The purified humanized mAb binds to recombinant HER2 and HER2-overexpressing tumor cells with an affinity comparable with the chimeric and parental mouse mAbs. It recognizes an epitope distinct from those recognized by trastuzumab and pertuzumab. Binding of hersintuzumab to HER2 overexpressing tumor cells induces G1 cell cycle arrest, inhibition of ERK and AKT signaling pathways and growth inhibition. Moreover, hersintuzumab could induce antibody-dependent cell cytotoxicity (ADCC) on BT-474 cells. This new humanized mAb is a potentially valuable tool for single or combination breast cancer therapy.


Antibody humanization Monoclonal antibody Breast cancer Immunotherapy HER2 


  1. 1.
    Hynes NE, MacDonald G (2009) ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol 21(2):177–184. CrossRefPubMedGoogle Scholar
  2. 2.
    Ladjemi MZ, Jacot W, Chardes T, Pelegrin A, Navarro-Teulon I (2010) Anti-HER2 vaccines: new prospects for breast cancer therapy. Cancer Immunol Immunother 59(9):1295–1312. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kruser TJ, Wheeler DL (2010) Mechanisms of resistance to HER family targeting antibodies. Exp Cell Res 316(7):1083–1100. CrossRefPubMedGoogle Scholar
  4. 4.
    Amiri MM, Jeddi-Tehrani M, Kazemi T, Bahadori M, Maddah M, Hojjat-Farsangi M, Khoshnoodi J, Rabbani H, Shokri F (2013) Construction and characterization of a new chimeric antibody against HER2. Immunotherapy 5(7):703–715. CrossRefPubMedGoogle Scholar
  5. 5.
    Ceran C, Cokol M, Cingoz S, Tasan I, Ozturk M, Yagci T (2012) Novel anti-HER2 monoclonal antibodies: synergy and antagonism with tumor necrosis factor-alpha. BMC Cancer 12:450. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Baselga J, Cortes J, Kim SB, Im SA, Hegg R, Im YH, Roman L, Pedrini JL, Pienkowski T, Knott A, Clark E, Benyunes MC, Ross G, Swain SM (2012) Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer. N Engl J Med 366(2):109–119. CrossRefPubMedGoogle Scholar
  7. 7.
    Traynor K (2012) FDA approves pertuzumab for breast cancer. Am J Health Syst Pharm 69(14):1178. Google Scholar
  8. 8.
    Kazemi T, Tahmasebi F, Bayat AA, Mohajer N, Khoshnoodi J, Jeddi-Tehrani M, Rabbani H, Shokri F (2011) Characterization of novel murine monoclonal antibodies directed against the extracellular domain of human HER2 tyrosine kinase receptor. Hybridoma (Larchmt) 30(4):347–353. CrossRefGoogle Scholar
  9. 9.
    Tahmasebi F, Kazemi T, Amiri MM, Khoshnoodi J, Bayat AA, Jeddi-Tehrani M, Rabbani H, Shokri F (2014) In vitro assessment of the effects of anti-HER2 monoclonal antibodies on proliferation of HER2-overexpressing breast cancer cells. Immunotherapy 6(1):1–7CrossRefGoogle Scholar
  10. 10.
    Schroff RW, Foon KA, Beatty SM, Oldham RK, Morgan AC Jr (1985) Human anti-murine immunoglobulin responses in patients receiving monoclonal antibody therapy. Cancer Res 45(2):879–885PubMedGoogle Scholar
  11. 11.
    Khazaeli MB, Conry RM, LoBuglio AF (1994) Human immune response to monoclonal antibodies. J Immunother Emphasis Tumor Immunol 15(1):42–52CrossRefPubMedGoogle Scholar
  12. 12.
    Marcatili P, Rosi A, Tramontano A (2008) PIGS: automatic prediction of antibody structures. Bioinformatics 24(17):1953–1954. CrossRefPubMedGoogle Scholar
  13. 13.
    Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(1):33–38 27-38CrossRefPubMedGoogle Scholar
  14. 14.
    Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (2008) Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modelling. J Biochem 144(1):115–120. CrossRefPubMedGoogle Scholar
  15. 15.
    Almagro JC, Fransson J (2008) Humanization of antibodies. Front Biosci 13:1619–1633PubMedGoogle Scholar
  16. 16.
    Hu WG, Chau D, Wu J, Jager S, Nagata LP (2007) Humanization and mammalian expression of a murine monoclonal antibody against Venezuelan equine encephalitis virus. Vaccine 25(16):3210–3214. CrossRefPubMedGoogle Scholar
  17. 17.
    Queen C, Schneider WP, Selick HE, Payne PW, Landolfi NF, Duncan JF, Avdalovic NM, Levitt M, Junghans RP, Waldmann TA (1989) A humanized antibody that binds to the interleukin 2 receptor. Proc Natl Acad Sci 86(24):10029–10033CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Jones ML, Barnard RT (2005) Chimerization of multiple antibody classes using splice overlap extension PCR. BioTechniques 38(2):181–182CrossRefPubMedGoogle Scholar
  19. 19.
    Saboor-Yaraghi AA, Ghods R, Gharagozlou S, Roohi A, Khoshnoodi J, Towfighi F, Jeddi-Tehrani M, Shokri F (2004) Identification of cross-reactive and restricted epitopes localized on human chorionic gonadotropin beta-subunit by monoclonal antibodies. Hybrid Hybridomics 23(2):101–107. CrossRefPubMedGoogle Scholar
  20. 20.
    García-Morales P, Hernando E, Carrasco-García E, Menéndez-Gutierrez MP, Saceda M, Martínez-Lacaci I (2006) Cyclin D3 is down-regulated by rapamycin in HER-2-overexpressing breast cancer cells. Mol Cancer Ther 5(9):2172–2181CrossRefPubMedGoogle Scholar
  21. 21.
    Caromile LA, Dortche K, Rahman MM, Grant CL, Stoddard C, Ferrer FA, Shapiro LH (2017) PSMA redirects cell survival signaling from the MAPK to the PI3K-AKT pathways to promote the progression of prostate cancer. Sci Signal 10(470).
  22. 22.
    Yeung YG, Stanley ER (2009) A solution for stripping antibodies from polyvinylidene fluoride immunoblots for multiple reprobing. Anal Biochem 389(1):89–91. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Abhinandan KR, Martin AC (2007) Analyzing the "degree of humanness" of antibody sequences. J Mol Biol 369(3):852–862. CrossRefPubMedGoogle Scholar
  24. 24.
    Harris LJ, Larson SB, Hasel KW, McPherson A (1997) Refined structure of an intact IgG2a monoclonal antibody. Biochemistry 36(7):1581–1597. CrossRefPubMedGoogle Scholar
  25. 25.
    Burmester J, Spinelli S, Pugliese L, Krebber A, Honegger A, Jung S, Schimmele B, Cambillau C, Pluckthun A (2001) Selection, characterization and x-ray structure of anti-ampicillin single-chain Fv fragments from phage-displayed murine antibody libraries. J Mol Biol 309(3):671–685. CrossRefPubMedGoogle Scholar
  26. 26.
    Fiszman GL, Jasnis MA (2011) Molecular Mechanisms of Trastuzumab Resistance in HER2 Overexpressing Breast Cancer. Int J Breast Cancer 2011:352182. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Cortes J, Fumoleau P, Bianchi GV, Petrella TM, Gelmon K, Pivot X, Verma S, Albanell J, Conte P, Lluch A, Salvagni S, Servent V, Gianni L, Scaltriti M, Ross GA, Dixon J, Szado T, Baselga J (2012) Pertuzumab monotherapy after trastuzumab-based treatment and subsequent reintroduction of trastuzumab: activity and tolerability in patients with advanced human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol 30(14):1594–1600. CrossRefPubMedGoogle Scholar
  28. 28.
    Pohlmann PR, Mayer IA, Mernaugh R (2009) Resistance to Trastuzumab in Breast Cancer. Clin Cancer Res 15(24):7479–7491. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Harwerth IM, Wels W, Schlegel J, Muller M, Hynes NE (1993) Monoclonal antibodies directed to the erbB-2 receptor inhibit in vivo tumour cell growth. Br J Cancer 68(6):1140–1145CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Drebin JA, Link VC, Greene MI (1988) Monoclonal antibodies reactive with distinct domains of the neu oncogene-encoded p185 molecule exert synergistic anti-tumor effects in vivo. Oncogene 2(3):273–277PubMedGoogle Scholar
  31. 31.
    Kasprzyk PG, Song SU, Di Fiore PP, King CR (1992) Therapy of an animal model of human gastric cancer using a combination of anti-erbB-2 monoclonal antibodies. Cancer Res 52(10):2771–2776PubMedGoogle Scholar
  32. 32.
    Chames P, Van Regenmortel M, Weiss E, Baty D (2009) Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol 157(2):220–233. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    De Groot AS, Martin W (2009) Reducing risk, improving outcomes: bioengineering less immunogenic protein therapeutics. Clin Immunol 131(2):189–201. CrossRefPubMedGoogle Scholar
  34. 34.
    Ahmadzadeh V, Farajnia S, Feizi MA, Nejad RA (2014) Antibody humanization methods for development of therapeutic applications. Monoclon Antib Immunodiagn Immunother 33(2):67–73. CrossRefPubMedGoogle Scholar
  35. 35.
    Dennis MS (2010) CDR repair: A novel approach to antibody humanization. In: Current trends in monoclonal antibody development and manufacturing. Springer, New York, pp 9–28Google Scholar
  36. 36.
    Gonzales NR, Padlan EA, De Pascalis R, Schuck P, Schlom J, Kashmiri SV (2004) SDR grafting of a murine antibody using multiple human germline templates to minimize its immunogenicity. Mol Immunol 41(9):863–872. CrossRefPubMedGoogle Scholar
  37. 37.
    Verhoeyen M, Milstein C, Winter G (1988) Reshaping human antibodies: grafting an antilysozyme activity. Science 239(4847):1534–1536CrossRefPubMedGoogle Scholar
  38. 38.
    Yoon SO, Lee TS, Kim SJ, Jang MH, Kang YJ, Park JH, Kim KS, Lee HS, Ryu CJ, Gonzales NR, Kashmiri SV, Lim SM, Choi CW, Hong HJ (2006) Construction, affinity maturation, and biological characterization of an anti-tumor-associated glycoprotein-72 humanized antibody. J Biol Chem 281(11):6985–6992. CrossRefPubMedGoogle Scholar
  39. 39.
    Jones PT, Dear PH, Foote J, Neuberger MS, Winter G (1986) Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature 321(6069):522–525Google Scholar
  40. 40.
    Tiwari A, Khanna N, Acharya SK, Sinha S (2009) Humanization of high affinity anti-HBs antibody by using human consensus sequence and modification of selected minimal positional template and packing residues. Vaccine 27(17):2356–2366. CrossRefPubMedGoogle Scholar
  41. 41.
    Wedemayer GJ, Patten PA, Wang LH, Schultz PG, Stevens RC (1997) Structural insights into the evolution of an antibody combining site. Science 276(5319):1665–1669CrossRefPubMedGoogle Scholar
  42. 42.
    Foote J, Winter G (1992) Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol 224(2):487–499CrossRefPubMedGoogle Scholar
  43. 43.
    An Z (2011) Therapeutic monoclonal antibodies: from bench to clinic. Wiley, New YorkGoogle Scholar
  44. 44.
    Kipriyanov SM, Le Gall F (2004) Generation and production of engineered antibodies. Mol Biotechnol 26(1):39–60. CrossRefPubMedGoogle Scholar
  45. 45.
    Graves SS, Goshorn SC, Stone DM, Axworthy DB, Reno JM, Bottino B, Searle S, Henry A, Pedersen J, Rees AR (1999) Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody. Clin Cancer Res 5(4):899–908PubMedGoogle Scholar
  46. 46.
    Li B, Wang H, Zhang D, Qian W, Hou S, Shi S, Zhao L, Kou G, Cao Z, Dai J (2007) Construction and characterization of a high-affinity humanized SM5-1 monoclonal antibody. Biochem Biophys Res Commun 357(4):951–956CrossRefPubMedGoogle Scholar
  47. 47.
    S-o L, Goldenberg DM, Dion AS, Pellegrini MC, Shevitz J, Shih LB, Hansen HJ (1995) Construction and characterization of a humanized, internalizing, B-cell (CD22)-specific, leukemia/lymphoma antibody, LL2. Mol Immunol 32(17):1413–1427Google Scholar
  48. 48.
    Hu WG, Yin J, Chau D, Negrych LM, Cherwonogrodzky JW (2012) Humanization and characterization of an anti-ricin neutralization monoclonal antibody. PLoS One 7(9):e45595. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Kuroda D, Shirai H, Jacobson MP, Nakamura H (2012) Computer-aided antibody design. Protein Eng Des Sel 25(10):507–521. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, Rowland AM, Kotts C, Carver ME, Shepard HM (1992) Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci U S A 89(10):4285–4289CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Adams CW, Allison DE, Flagella K, Presta L, Clarke J, Dybdal N, McKeever K, Sliwkowski MX (2006) Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab. Cancer Immunol Immunother 55(6):717–727CrossRefPubMedGoogle Scholar
  52. 52.
    Hu S, Zhu Z, Li L, Chang L, Li W, Cheng L, Teng M, Liu J (2008) Epitope mapping and structural analysis of an anti-ErbB2 antibody A21: Molecular basis for tumor inhibitory mechanism. Proteins 70(3):938–949. CrossRefPubMedGoogle Scholar
  53. 53.
    Ko BK, Lee SY, Lee YH, Hwang IS, Persson H, Rockberg J, Borrebaeck C, Park D, Kim KT, Uhlen M, Lee JS (2015) Combination of novel HER2-targeting antibody 1E11 with trastuzumab shows synergistic antitumor activity in HER2-positive gastric cancer. Mol Oncol 9(2):398–408. CrossRefPubMedGoogle Scholar
  54. 54.
    Sherr CJ, Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 13(12):1501–1512CrossRefPubMedGoogle Scholar
  55. 55.
    Le XF, Claret FX, Lammayot A, Tian L, Deshpande D, LaPushin R, Tari AM, Bast RC Jr (2003) The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. J Biol Chem 278(26):23441–23450. CrossRefPubMedGoogle Scholar
  56. 56.
    Valabrega G, Montemurro F, Aglietta M (2007) Trastuzumab: mechanism of action, resistance and future perspectives in HER2-overexpressing breast cancer. Ann Oncol 18(6):977–984. CrossRefPubMedGoogle Scholar
  57. 57.
    Li R, Hu S, Chang Y, Zhang Z, Zha Z, Huang H, Shen G, Liu J, Song L, Wei W (2016) Development and Characterization of a Humanized Anti-HER2 Antibody HuA21 with Potent Anti-Tumor Properties in Breast Cancer Cells. Int J Mol Sci 17(4):563. CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Nahta R, Hung MC, Esteva FJ (2004) The HER-2-targeting antibodies trastuzumab and pertuzumab synergistically inhibit the survival of breast cancer cells. Cancer Res 64(7):2343–2346CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Mohammad Mehdi Amiri
    • 1
  • Forough Golsaz-Shirazi
    • 1
  • Tahereh Soltantoyeh
    • 1
  • Reza Hosseini-Ghatar
    • 1
  • Tannaz Bahadori
    • 2
  • Jalal Khoshnoodi
    • 1
  • Shadi Sadat Navabi
    • 1
  • Samira Farid
    • 2
  • Mohammad Hossein Karimi-Jafari
    • 3
  • Mahmood Jeddi-Tehrani
    • 2
  • Fazel Shokri
    • 1
    • 2
  1. 1.Department of Immunology, School of Public HealthTehran University of Medical SciencesTehranIran
  2. 2.Monoclonal Antibody Research CenterAvicenna Research Institute, ACECRTehranIran
  3. 3.Department of Bioinformatics, Institute of Biochemistry and BiophysicsUniversity of TehranTehranIran

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