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. 1993 Jan;37(1):54–60. doi: 10.1007/BF01516942

A critical comparison of three internalization assays applied to the evaluation of a given mAb as a toxin-carrier candidate

P Casalini 1, M Caldera 1, S Canevari 1, S Ménard 1, D Mezzanzanica 1, E Tosi 1, M Gadina 1, M I Colnaghi 1,✉
PMCID: PMC11038973  PMID: 8099847

Abstract

In the attempt to define a strategy for screening new monoclonal antibodies (mAb) that could be appropriate for clinical application in oncology, we evaluated the suitability of three methods: a direct internalization assay (DIA), an indirect internalization assay (IIA) and an indirect cytotoxicity assay (ICA), by applying them to already selected mAb. The latter were directed against three antigenic systems [38-kDa glycoprotein (gp38), epidermal growth factor receptor, and theneu oncogene product], which, according to their tumor selectivity, could be considered suitable for mAb-guided therapy. The dose-dependent and time-dependent binding, as well as the low intraassay variability, demonstrated the reliability of the three tests. However, a certain degree of inter-assay variability was observed in each one, the highest value being that found when IIA was applied. Furthermore, the degree of variability, as well as the predictability, seemed to be more related to the mAb/antigen (Ag) combination used rather than to the test applied. From the overall data we suggest a procedure to be applied for screening purposes. As a first approach applied to the raw material, ICA is only suitable for screening in the case of an already selected toxin whereas IIA may be helpful to eliminate the true negative mAb. After purification of the relevant mAb a repeated analysis using DIA could allow the selection of true internalizing mAb. However, this second screening should be followed by a further analysis of the fate of the Ag−Ab complex after internalization.

Key words: Internalization assay, Monoclonal antibody, Tumor-associated antigen

References

  • 1.Anderson RGW, Kamen BA, Rothberg KG, Lacey SW. Potocytosis: sequestration and transport of small molecules by caveolac. Science. 1992;255:410. doi: 10.1126/science.1310359. [DOI] [PubMed] [Google Scholar]
  • 2.Basu A, Murthy U, Rodeck U, Herlyn M, Mattes L, Das M. Presence of tumor-associated antigens in epidermal growth factor receptors from different human, carcinomas. Cancer Res. 1987;47:2531. [PubMed] [Google Scholar]
  • 3.Calafat J, Molthoff C, Janssen H, Hilkens J. Endocytosis and intracellular routing of an antibody-ricin A chain conjugate. Cancer Res. 1988;48:3822. [PubMed] [Google Scholar]
  • 4.Casalini P, Mezzanzanica D, Canevari S, Della Torre G, Miotti S, Colnaghi MI, Matzku S. 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. 1991;48:284. doi: 10.1002/ijc.2910480222. [DOI] [PubMed] [Google Scholar]
  • 5.Centis F, Tagliabue E, Uppugunduri S, Pellegrini R, Martignone S, Mastroianni A, Ménard S, Colnaghi MI. p185 HER2/neu epitope mapping with murine monoclonal antibodies. Hybridoma. 1992;11:267. doi: 10.1089/hyb.1992.11.267. [DOI] [PubMed] [Google Scholar]
  • 6.Chignola R, Colombatti M, Dell'Arciprete L, Candiani C, Tridente G. Distribution of endocytosed molecules to intracellular acidic environments correlates with immunotoxin activity. Int J Cancer. 1990;46:1117. doi: 10.1002/ijc.2910460628. [DOI] [PubMed] [Google Scholar]
  • 7.Cogliati T, Brusa P, Canevari S, Caldera M, Mezzanzanica D, Dosio F, Cattel L, Colnaghi MI. Preparation and biological characterization of conjugates consisting, of ricin and a tumor-specific nonintermalizing mAb. Anticancer Res. 1991;11:417. [PubMed] [Google Scholar]
  • 8.Conde FP, Orlandi R, Canevari S, Mezzanzanica D, Ripamonti M, Muñoz SM, Jorge P, Colnaghi MI. TheAspergillus toxin restrictocin is a suitable cytotoxic agent for generation of immunoconjugates with monoclonal antibodies directed against human carcinoma cells. Eur J Biochem. 1989;178:795. doi: 10.1111/j.1432-1033.1989.tb14511.x. [DOI] [PubMed] [Google Scholar]
  • 9.Coney LR, Tomassetti A, Carayannopoulos L, Frasca V, Kamen BA, Colnaghi MI, Zurawski VR. Cloning of a tumor-associated antigen: MOv 18 and MOv 19 antibodies recognize a folate-binding protein. Cancer Res. 1991;51:6125. [PubMed] [Google Scholar]
  • 10.Dasgupta JD, Watkins S, Slayter H, Yunis EJ. Receptor-like nature of class I HLA: endocytosis via coated pits. J Immunol. 1988;141:2577. [PubMed] [Google Scholar]
  • 11.Della Torre G, Canevari S, Orlandi R, Colnaghi MI. Internalization of a monoclonal antitbody against human breast cancer by immunoelectron microscopy. Br J Cancer. 1987;55:357. doi: 10.1038/bjc.1987.72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Derbyshire EJ, Wawrzynczak EJ. Monoclonal antibodies recognising the cluster 2 antigen associated with human small cell lung cancer mediate the toxic effects of ricin A chain in an indirect assay of immunotoxin cytotoxicity. Br J Cancer. 1991;63:74. [PMC free article] [PubMed] [Google Scholar]
  • 13.Ey PL, Prowse SJ, Jenkins CR. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry. 1978;15:429. doi: 10.1016/0161-5890(78)90070-6. [DOI] [PubMed] [Google Scholar]
  • 14.Fendly BM, Winget M, Hudziak RM, Lipari MT, Napier MA, Ullrich A. Characterization of murine monoclonal antibodies reactive to either the human epidermal growth factor receptor or HER2/neu gene product. Cancer Res. 1990;50:1550. [PubMed] [Google Scholar]
  • 15.Fodstad O, Kvalheim G, Pihl A, Godal A, Funderud S. New indirect approach to the therapeutic use of immunotoxins. JNCI. 1988;80:439. doi: 10.1093/jnci/80.6.439. [DOI] [PubMed] [Google Scholar]
  • 16.Foon KA. Biological response modifiers: the new immunotherapy. Cancer Res. 1989;49:1621. [PubMed] [Google Scholar]
  • 17.Garnett MC, Baldwin RW. Endocytosis of a monoclonal antibody recognizing a cell surface glycoprotein antigen visualized using fluorescent conjugates. Eur J Cell Biol. 1986;41:214. [PubMed] [Google Scholar]
  • 18.Goldmacher VS, Scott CF, Lambert JM, McIntyre GD, Blatter WA, Collison AR, Stewart JK, Chong LD, Cook S, Slayter HS, Beaumont E, Watkins S. Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis. J Cell Physiol. 1989;141:222. doi: 10.1002/jcp.1041410129. [DOI] [PubMed] [Google Scholar]
  • 19.Harris AL. The epidermal growth factor receptor as a target for therapy. Cancer Cells. 1990;2:321. [PubMed] [Google Scholar]
  • 20.Kohler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;246:495. doi: 10.1038/256495a0. [DOI] [PubMed] [Google Scholar]
  • 21.Leoni F, Colnaghi MI, Canevari S, Ménard S, Colzani E, Facheris P, Figini M, Miotti S, Magnani JL. Glycolipids carrying Le are preferentially expressed on small-cell lung cancer cells as detected by the monoclonal antibody MLuC1. Int J Cancer. 1992;51:225. doi: 10.1002/ijc.2910510210. [DOI] [PubMed] [Google Scholar]
  • 22.Marchalonis JJ. An enzymatic method for the trace iodination of immunoglobulin and other proteins. Biochem J. 1969;113:299. doi: 10.1042/bj1130299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Matzku S, Bröcker E-B, Brüggen J, Dippold WG, Tilgen W. Modes of binding and internalization of monoclonal antibodies to human melanoma cell lines. Cancer Res. 1986;46:3848. [PubMed] [Google Scholar]
  • 24.Mezzazanica D, Canevari S, Casalini P, Della Torre G, Colnaghi MI (1989) Monoclonal antibodies in cancer therapy:selection of the most appropriate reagent for different approaches. In: Hum Tumor Antigens Specific Tumor Ther Alan R. Liss. Inc, p. 181
  • 25.Ménard S, Tagliabue E, Canevari S, Fossati G, Colnaghi MI. Generation of monoclonal antibodies reacting with normal and cancer cells of human breast. Cancer Res. 1983;43:1295. [PubMed] [Google Scholar]
  • 26.Miotti S, Canevari S, Ménard S, Mezzanzanica D, Porro G, Pupa SM, Regazzoni M, Tagliabue E, Colnaghi MI. Characterization of human ovarian carcinoma-associated antigens defined by novel monoclonal antibodies with tumor-restricted specificity. Int J Cancer. 1987;39:297. doi: 10.1002/ijc.2910390306. [DOI] [PubMed] [Google Scholar]
  • 27.Muñoz SM, Caldera M, Canevari S, Tosi E, Cogliati T, Colnaghi MI, Conde FP. Fractionation of the ribosome inactivating protein preparations with triazine dyes. Biochem Biophys Res Commun. 1990;173:554. doi: 10.1016/s0006-291x(05)80070-1. [DOI] [PubMed] [Google Scholar]
  • 28.Olsnes S, Sandvig K, Petersen OW, van Deurs B. Immunotoxin entry into cells and mechanisms of action. Immunol Today. 1989;10:291. [PubMed] [Google Scholar]
  • 29.Pastan I, Willingham MC, Fitzgerald DJP. Immunotoxins. Cell. 1986;47:641. doi: 10.1016/0092-8674(86)90506-4. [DOI] [PubMed] [Google Scholar]
  • 30.Tagliabue E, Centis F, Campiglio M, Mastroianni A, Martignone S, Pellegrini R, Casalini P, Lanzi C, Ménard S, Colnaghi MI. Selection of monoclonal antibodies which induce internalization and phosphorylation of p185 and growth inhibition of cells with HER2/neu gene amplification. Int J Cancer. 1991;47:933. doi: 10.1002/ijc.2910470625. [DOI] [PubMed] [Google Scholar]
  • 31.Tausk F, Fey M, Gigli I. Endocytosis and shedding of the decay accelerating factor on human polymorphonuclear cells. J Immunol. 1989;143:3295. [PubMed] [Google Scholar]
  • 32.Till M, May RD, Uhr JW, Thorpe PE, Vitetta ES. An assay that predicts the ability of monoclonal antibodies to form potent ricin A chain-containing immunotoxins. Cancer Res. 1988;48:1119. [PubMed] [Google Scholar]
  • 33.Wargalla UC, Reisfeld RA. Rate of internalization of an immunotoxin correlates with cytotoxic activity against human tumor cells. Proc Natl Acad Sci USA. 1989;86:5146. doi: 10.1073/pnas.86.13.5146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Wawrzynczak EJ. Systemic immunotoxin therapy of cancer: advances and prospects. Br J Cancer. 1991;64:624. doi: 10.1038/bjc.1991.374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Weltman JK, Pedroso P, Johnson SA, Davignon D, Fast LD, Leone LA. Rapid screening with indirect immunotoxin for monoclonal antibodies against human small cell lung cancer. Cancer Res. 1987;47:5552. [PubMed] [Google Scholar]
  • 36.Yokota T, Otsuji E, Noguchi A, Yamaguchi T, Sawai K, Takahashi T. Antigenic modulation and internalization of monoclonal antibody to human colonic carcinoma cells detected by enzyme-linked immunosorbent. Int J Cancer. 1989;44:1095. doi: 10.1002/ijc.2910440626. [DOI] [PubMed] [Google Scholar]
  • 37.Youle RJ. Fatal attraction in vitro and in vivo. JNCI. 1988;80:388. doi: 10.1093/jnci/80.6.388. [DOI] [PubMed] [Google Scholar]

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