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Conversion of murine antibodies to human antibodies and their optimization for ovarian cancer therapy targeted to the folate receptor

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Abstract

We previously developed murine and chimeric antibodies against a specific epithelial ovarian carcinoma (EOC) marker, named folate receptor (FR), and promising results were obtained in phase II trials. More recently, we successfully generated a completely human Fab fragment, C4, by conversion of one of the murine anti-FR antibodies to human antibody using phage display and guided selection. However, subsequent efforts to obtain C4 in a dimer format, which seems especially desirable for EOC locoregional treatment, resulted in a highly heterogeneous product upon natural dimerization and in a very poor production yield upon chemical dimerization by a non-hydrolyzable linker to a di-Fab-maleimide (DFM). We therefore designed, constructed and characterized a large Fab dual combinatorial human antibody phage display library obtained from EOC patients and potentially biased toward an anti-tumor response in an effort to obtain new anti-FR human antibodies suitable for therapy. Using this library and guiding the selection on FR-expressing cells with murine/human antibody chains, we generated four new human anti-FR antibody (AFRA) Fab fragments, one of which was genetically and chemically manipulated to obtain a chemical dimer, designated AFRA-DFM5.3, with high yield production and the capability for purification scaled-up to clinical grade. Overall affinity of AFRA-DFM5.3 was in the 2-digit nanomolar range, and immunohistochemistry indicated that the reagent recognized the FR expressed on EOC samples. 131I-AFRA-DFM5.3 showed high immunoreactivity, in vitro stability and integrity, and specifically accumulated only in FR-expressing tumors in subcutaneous preclinical in vivo models. Overall, our studies demonstrate the successful conversion of murine to completely human anti-FR antibodies through the combined use of antibody phage display libraries biased toward an anti-tumor response, guided selection and chain shuffling, and point to the suitability of AFRA5.3 for future clinical application in ovarian cancer.

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Abbreviations

EOC:

Epithelial ovarian carcinoma

FR:

Folate receptor

DFM:

Di-Fab-maleimide

AFRA:

Anti-FR antibody

mAb(s):

Monoclonal antibody/ies

PBMC:

Peripheral blood mononuclear cells

chi:

Chimeric

TCEP:

Tris (2-carboxyethyl) phosphine hydrochloride

BMOE:

Bismaleimidoethane

RP-HPLC:

Reverse phase-high performance liquid chromatography

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

IC50 :

Concentration inhibiting 50% of binding

s.c.:

Subcutaneous

i.v.:

Intravenous

%ID/g:

Percentage of the injected dose per unit mass of organ

References

  1. Committee on cancer research (UKCCCR), United Kingdom (1998) Guidelines for the welfare of animals in experimental neoplasia, 2nd edn. Br J Cancer 77: 1–10

  2. Adamczyk M, Gebler JC, Wu J, Yu Z (2002) Complete sequencing of anti-vancomycin Fab fragment by liquid chromatography-electrospray ion trap mass spectrometry with a combination of database searching and manual interpretation of the MS/MS spectra. J Immunol Methods 260:235–249

    Article  PubMed  CAS  Google Scholar 

  3. Alberti S, Miotti S, Fornaro M, Mantovani L, Walter S, Canevari S, Ménard S, Colnaghi MI (1990) The Ca-MOv18 molecule, a cell-surface marker of human ovarian carcinomas, is anchored to the cell membrane by phosphatidylinositol. Biochem Biophys Res Commun 171:1051–1055

    Article  PubMed  CAS  Google Scholar 

  4. Brack SS, Silacci M, Birchler M, Neri D (2006) Tumor-targeting properties of novel antibodies specific to the large isoform of tenascin-C. Clin Cancer Res 12:3200–3208

    Article  PubMed  CAS  Google Scholar 

  5. Canevari S, Pupa SM, Menard S (1996) 1975–1995 revised anti-cancer serological response: biological significance and clinical implications. Ann Oncol 7:227–232

    PubMed  CAS  Google Scholar 

  6. Canevari S, Stoter G, Arienti F, Bolis G, Colnaghi MI, Di Re E, Eggermont AMM, Goey SH, Gratama JW, Lamers CHJ, Nooy MA, Parmiani G, Raspagliesi F, Ravagnani F, Scarfone G, Trimbos JB, Warnaar SO, Bolhuis RLH (1995) Regression of advanced ovarian carcinoma by intraperitoneal treatment with autologous T-lymphocytes retargeted by a bispecific monoclonal antibody. J Natl Cancer Inst 87:1463–1469

    Article  PubMed  CAS  Google Scholar 

  7. Carter PJ (2006) Potent antibody therapeutics by design. Nat Rev Immunol 6:343–357

    Article  PubMed  CAS  Google Scholar 

  8. Casalini P, Luison E, nard S, Colnaghi MI, Paganelli G, Canevari S (1997) Tumor pretargeting: role of avidin/streptavidin on monoclonal antibody internalization. J Nucl Med 38:1378–1381

    PubMed  CAS  Google Scholar 

  9. Casalini P, Mezzanzanica D, Canevari S, Della Torre G, Miotti S, Colnaghi MI, Matzku S (1991) Use of combination of monoclonal antibodies directed against three distinct epitopes of a tumor-associated antigen: analysis of cell binding and internalization. Int J Cancer 48:284–290

    Article  PubMed  CAS  Google Scholar 

  10. Casey JL, King DJ, Chaplin LC, Haines AM, Pedley RB, Mountain A, Yarranton GT, Begent RH (1996) Preparation, characterisation and tumour targeting of cross-linked divalent and trivalent anti-tumour Fab′ fragments. Br J Cancer 74:1397–1405

    PubMed  CAS  Google Scholar 

  11. Coliva A, Zacchetti A, Luison E, Tomassetti A, Bongarzone I, Seregni E, Bombardieri E, Martin F, Giussani A, Figini M, Canevari S (2005) 90Y labeling of monoclonal antibody MOv18 and preclinical validation for radioimmunotherapy of human ovarian carcinomas. Cancer Immunol Immunother 54:1200–1213

    Article  PubMed  CAS  Google Scholar 

  12. Coney LR, Mezzanzanica D, Sanborn D, Casalini P, Colnaghi MI, Zurawski VR Jr (1994) Chimeric murine-human antibodies directed against folate binding receptor are efficient mediators of ovarian carcinoma cell killing. Cancer Res 54:2448–2455

    PubMed  CAS  Google Scholar 

  13. Coney LR, Tomassetti A, Carayannopoulos L, Frasca V, Kamen BA, Colnaghi MI, Zurawski VR Jr (1991) Cloning of a tumor-associated antigen: MOv18 and MOv19 antibodies recognize a folate-binding protein. Cancer Res 51:6125–6132

    PubMed  CAS  Google Scholar 

  14. Coronella-Wood JA, Hersh EM (2003) Naturally occurring B-cell responses to breast cancer. Cancer Immunol Immunother 52:715–738

    Article  PubMed  Google Scholar 

  15. Crippa F, Bolis G, Seregni E, Gavoni N, Bombardieri E, Scarfone G, Ferrari C, Buraggi GL (1995) Single dose intraperitoneal radioimmunotherapy with the murine monoclonal antibody 131I-MOv18: clinical results in patients with minimal residual disease of ovarian cancer. Eur J Cancer 31A:686–690

    Article  PubMed  CAS  Google Scholar 

  16. de Kruif J, van der Vuurst de Vries AR, Cilenti L, Boel E, van Ewijk W, Logtenberg T (1996) New perspectives on recombinant human antibodies. Immunol Today 17:453–455

    Article  PubMed  Google Scholar 

  17. Figini M, Ferri R, Mezzanzanica D, Bagnoli M, Luison E, Miotti S, Canevari S (2003) Reversion of transformed phenotype in ovarian cancer cells by intracellular expression of anti-folate receptor antibodies. Gene Ther 10:1018–1025

    Article  PubMed  CAS  Google Scholar 

  18. Figini M, Marks JD, Winter G, Griffiths AD (1994) In vitro assembly of repertoires of antibody chains on the surface of phage by renaturation. J Mol Biol 239:68–78

    Article  PubMed  CAS  Google Scholar 

  19. Figini M, Obici L, Mezzanzanica D, Griffiths AD, Colnaghi MI, Winter G, Canevari S (1998) Panning phage antibody libraries on cells: isolation of human Fab fragments against ovarian carcinoma using guided selection. Cancer Res 58:991–996

    PubMed  CAS  Google Scholar 

  20. Groulet A, Dorvillius M, Pelegrin A, Barbet J, Baty D (2002) Pharmacokinetic and tumor-seeking properties of recombinant and nonrecombinant anti-carcinoembryonic antigen antibody fragments. Int J Cancer 100:367–374

    Article  PubMed  CAS  Google Scholar 

  21. Jain M, Venkatraman G, Batra SK (2007) Optimization of radioimmunotherapy of solid tumors: biological impediments and their modulation. Clin Cancer Res 13:1374–1382

    Article  PubMed  CAS  Google Scholar 

  22. Knutson KL, Krco CJ, Erskine CL, Goodman K, Kelemen LE, Wettstein PJ, Low PS, Hartmann LC, Kalli KR (2006) T-cell immunity to the folate receptor alpha is prevalent in women with breast or ovarian cancer. J Clin Oncol 24:4254–4261

    Article  PubMed  CAS  Google Scholar 

  23. Liu B, Conrad F, Roth A, Drummond DC, Simko JP, Marks JD (2007) Recombinant full-length human IgG1s targeting hormone-refractory prostate cancer. J Mol Med 85:1113–1123

    Article  PubMed  CAS  Google Scholar 

  24. Loo L, Robinson MK, Adams GP (2008) Antibody engineering principles and applications. Cancer J 14(3):149–153

    Article  PubMed  CAS  Google Scholar 

  25. Macor P, Mezzanzanica D, Cossetti C, Alberti P, Figini M, Canevari S, Tedesco F (2006) Complement activated by chimeric anti-folate receptor antibodies is an efficient effector system to control ovarian carcinoma. Cancer Res 66:3876–3883

    Article  PubMed  CAS  Google Scholar 

  26. Marks JD, Ouwehand WH, Bye JM, Finnern R, Gorick BD, Voak D, Thorpe S, Hughes-Jones NC, Winter G (1993) Human antibody fragments specific for human blood group antigens from a phage display library. Biotechnology 11:1145–1149

    PubMed  CAS  Google Scholar 

  27. Mayor S, Rothberg KG, Maxfield FR (1994) Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science 264:1948–1951

    Article  PubMed  CAS  Google Scholar 

  28. Meredith RF, Buchsbaum DJ, Alvarez RD, LoBuglio AF (2007) Brief overview of preclinical and clinical studies in the development of intraperitoneal radioimmunotherapy for ovarian cancer. Clin Cancer Res 13:5643s–5645s

    Article  PubMed  CAS  Google Scholar 

  29. Miotti S, Canevari S, Ménard S, Mezzanzanica D, Porro G, Pupa SM, Regazzoni M, Tagliabue E, Colnaghi MI (1987) Characterization of human ovarian carcinoma-associated antigens defined by novel monoclonal antibodies with tumor-restricted specificity. Int J Cancer 39:297–303

    Article  PubMed  CAS  Google Scholar 

  30. Miotti S, Negri DR, Valota O, Calabrese M, Bolhuis RL, Gratama JW, Colnaghi MI, Canevari S (1999) Level of anti-mouse antibody response induced by bispecific monoclonal antibody OC/TR in ovarian carcinoma patients is associated with longer survival. Int J Cancer 84:62–68

    Article  PubMed  CAS  Google Scholar 

  31. Mirick GR, Bradt BM, Denardo SJ, Denardo GL (2004) A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words. Q J Nucl Med Mol Imaging 48:251–257

    PubMed  CAS  Google Scholar 

  32. Ottone F, Miotti S, Bottini C, Bagnoli M, Perego P, Colnaghi MI, Ménard S (1997) Relationship between folate binding protein expression and cisplatin sensitivity in ovarian carcinoma cell lines. Br J Cancer 76:77–82

    PubMed  CAS  Google Scholar 

  33. Presta LG (2006) Engineering of therapeutic antibodies to minimize immunogenicity and optimize function. Adv Drug Deliv Rev 58:640–656

    Article  PubMed  CAS  Google Scholar 

  34. Reichert JM, Valge-Archer VE (2007) Development trends for monoclonal antibody cancer therapeutics. Nat Rev Drug Discov 6:349–356

    Article  PubMed  CAS  Google Scholar 

  35. Rodrigues ML, Snedecor B, Chen C, Wong WL, Garg S, Blank GS, Maneval D, Carter P (1993) Engineering Fab′ fragments for efficient F(ab)2 formation in Escherichia coli and for improved in vivo stability. J Immunol 151:6954–6961

    PubMed  CAS  Google Scholar 

  36. Saif MW, Zalonis A, Syrigos K (2007) The clinical significance of autoantibodies in gastrointestinal malignancies: an overview. Expert Opin Biol Ther 7:493–507

    Article  PubMed  CAS  Google Scholar 

  37. Salazar MD, Ratnam M (2007) The folate receptor: what does it promise in tissue-targeted therapeutics? Cancer Metastasis Rev 26:141–152

    Article  PubMed  CAS  Google Scholar 

  38. Toffoli G, Cernigoi C, Russo A, Gallo A, Bagnoli M, Boiocchi M (1997) Overexpression of folate binding protein in ovarian cancers. Int J Cancer 74:193–198

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the Italian Ministry of University and Research (MUR). We thank Dr. Angela Coliva for assistance in radiolabeling antibody fragments and Ms. Gloria Bosco for manuscript preparation.

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Authors and Affiliations

  1. Unit of Molecular Therapies, Department of Experimental Oncology and Laboratories, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133, Milan, Italy

    Mariangela Figini, Renata Ferri, Elena Luison, Elena Ripamonti, Alberto Zacchetti, Mimosa Mortarino & Silvana Canevari

  2. Dompé Pharma, L’Aquila, Italy

    Franck Martin, Vito Di Cioccio, Giovanni Maurizi & Marcello Allegretti

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  1. Mariangela Figini
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  2. Franck Martin
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  3. Renata Ferri
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  4. Elena Luison
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  11. Silvana Canevari
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Correspondence to Silvana Canevari.

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Figini, M., Martin, F., Ferri, R. et al. Conversion of murine antibodies to human antibodies and their optimization for ovarian cancer therapy targeted to the folate receptor. Cancer Immunol Immunother 58, 531–546 (2009). https://doi.org/10.1007/s00262-008-0575-5

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  • DOI: https://doi.org/10.1007/s00262-008-0575-5

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