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Cancer Immunology, Immunotherapy : CII logoLink to Cancer Immunology, Immunotherapy : CII
. 1993 Nov;36(6):403–408. doi: 10.1007/BF01742257

The development and purification of a bispecific antibody for lymphokine-activated killer cell targeting against the rat colon carcinoma CC531

Peter J K Kuppen 1,, Alexander M M Eggermont 2, Katinka M Smits 1, Jaap D H van Eendenburg 1, Sylvia P G Lazeroms 1, Cornelis J H van de Velde 3, Gert Jan Fleuren 1
PMCID: PMC11038143  PMID: 8500112

Abstract

In vivo targeting of lymphokine-activated killer (LAK) cells to tumour deposits by bispecific monoclonal antibodies (bimAb) may be a way to improve adoptive immunotherapy. We developed a bimAb against adherent LAK (ALAK) cells and colon tumour CC531 in Wag rats. The bimAb was produced by somatic hybridization of two mouse hybridomas, one producing monoclonal antibodies (mAb) against CD8 (IgG2b, OX8), and the other producing mAb against a CC531-associated antigen (IgG1, CC52). A bimAb-producing clone was selected by an enzyme-linked immunosorbent assay with CC531 tumour cells. BimAb were purified from ascitic fluid by protein A affinity chromatography. Each of five pooled peak fractions was analysed by flow cytometry for the presence of bimAb. Most bimAb were found in a fraction that was eluted at pH 4.5 from protein A. FPLC analysis of this fraction revealed that no parental antibodies were present. The OX8 × CC52 bimAb greatly increased conjugate formation in vitro between ALAK cells and CC531. Results of51Cr-release assays with CC531 as target cells and ALAK cells as effector cells were not significantly different in the presence or in the absence of the bimAb. The methods we used here, a cell enzyme-linked immunosorbent assay and flow cytometry, are simple methods for development and purification of a bimAb when a functional selection method is not a priori available. The OX8 × CC52 bimAb we developed this way may increase in vivo tumour targeting of ALAK cells and thus augment antitumour effect in vivo.

Key words: Bispecific monoclonal antibody, Lymphokine-activated killer cell, Rat, CD8

References

  • 1.Basse P, Herberman RB, Nannmark U, Johansson BR, Hokland M, Wasserman K, Goldfarb RH. Accumulation of adoptively transferred adherent, lymphokine-activated killer cells in murine metastases. J Exp Med. 1991;174:479. doi: 10.1084/jem.174.2.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Beun GDM, van Eendenburg JDH, Corver WE, Van de Velde CJH, Fleuren GJ. T-cell retargeting using bispecific monoclonal antibodies in a rat colon carcinoma model. I. Significant bispecific lysis of syngeneic colon carcinoma CC531 is critically dependent on prolonged preactivation of effector T-lymphocytes by immobilized anti-T-cell receptor antibody. J Immunother. 1992;11:238. doi: 10.1097/00002371-199205000-00002. [DOI] [PubMed] [Google Scholar]
  • 3.Chambers WH, Vujanovic NL, DeLeo AB, Olszowy MW, Herberman RB, Hiserodt JC. Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells. J Exp Med. 1989;169:1373. doi: 10.1084/jem.169.4.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Eggermont AMM, Marquet RL, de Bruin RWF, Jeekel J. Effects of the interferon-inducer ABPP on colon cancer in rats: importance of tumor load and tumor site. Cancer Immunol Immunother. 1986;22:217. doi: 10.1007/BF00200036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Felgar RE, Hiserodt JC. In vivo migration and tissue localization of highly purified lymphokine-activated killer cells (A-LAK Cells) in tumor-bearing rats. Cell Immunol. 1990;129:288. doi: 10.1016/0008-8749(90)90205-6. [DOI] [PubMed] [Google Scholar]
  • 6.Ferrini S, Prigione I, Miotti S, Ciccone E, Cantoni C, Chen Q, Colnaghi MI, Moretta L. Bispecific monoclonal antibodies directed to CD16 and to a tumor-associated antigen induce target-cell lysis by resting NK cells and by a subset of NK clones. Int J Cancer. 1991;48:227. doi: 10.1002/ijc.2910480213. [DOI] [PubMed] [Google Scholar]
  • 7.Carcía de Palazzo I, Gercel-Taylor C, Kitson J, Weiner LM. Potentiation of tumor lysis by a bispecific antibody that binds to CA19-9 antigen and the Fcgamma receptor expressed by human large granular lymphocytes. Cancer Res. 1990;50:7123. [PubMed] [Google Scholar]
  • 8.Gorter A, Krüse KM, Schrier PI, Fleuren GJ, Van de Griend RJ. Enhancement of the lytic activity of cloned human CD8 tumour-infiltrating lymphocytes by bispecific monoclonal antibodies. Clin Exp Immunol. 1992;87:111. doi: 10.1111/j.1365-2249.1992.tb06422.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Grimm EA, Mazumder A, Zhang HZ, Rosenberg SA. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin-2-activated autologous human peripheral blood lymphocytes. J Exp Med. 1982;155:1823. doi: 10.1084/jem.155.6.1823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Herberman RB, Hiserodt JC, Vujanovic NL, Balch CM, Lotzová E, Bolhuis RLH, Golub SH, Lanier LL, Phillips JH, Riccardi C, Ritz J, Santoni A, Schmidt RE, Uchida A. Lymphokine-activated killer cell activity. Characteristics of effector cells and their progenitors in blood and spleen. Immunol Today. 1987;8:178. doi: 10.1016/0167-5699(87)90035-1. [DOI] [PubMed] [Google Scholar]
  • 11.Kuppen PJK, Eggermont AMM, Lazeroms SPG, Marinelli A, Van de Velde CJH, Fleuren GJ. The effect of antibodies recognizing adherent-lymphokine-activated killer (ALAK) cells on lysis of P815 and YAC-1 (abstract) Proc Am Assoc Cancer Res. 1991;32:1623. [Google Scholar]
  • 12.Kuppen PJK, Eggermont AMM, Marinelli A, de Heer E, Van de Velde CJH, Fleuren GJ. Induction of lymphokine-activated killer activity in rat splenocyte cultures: the importance of 2-mercaptoethanol and indomethacin. Cancer Immunol Immunother. 1991;33:28. doi: 10.1007/BF01742524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Kuppen PJK, Marinelli A, Camps JAJ, Pauwels EKJ, Van de Velde CJH, Fleuren GJ, Eggermont AMM. Biodistribution of lymphokine-activated killer (LAK) cells in Wag rats after hepatic artery or jugular vein infusion. Int J Cancer. 1992;52:266. doi: 10.1002/ijc.2910520219. [DOI] [PubMed] [Google Scholar]
  • 14.Melder RJ, Walker ER, Herberman RB, Whiteside TL. Surface characteristics, morphology, and ultrastructure of human adherent lymphokine-activated killer cells. J Leukocyte Biol. 1990;48:163. doi: 10.1002/jlb.48.2.163. [DOI] [PubMed] [Google Scholar]
  • 15.Rosenberg SA. Adoptive immunotherapy of cancer using lymphokine activated killer cells and recombinant interleukin-2. In: DeVita VT Jr., Hellman S, Rosenberg SA, editors. Important advances in oncology 1986. Philadelphia: Lippincott; 1986. p. 55. [PubMed] [Google Scholar]
  • 16.Rosenberg SA, Lotze MT, Yang JC, Aebersold PM, Linehan WM, Seipp CA, White DE. Experience with the use of high-dose interleukin-2 in the treatment of 652 cancer patients. Ann Surg. 1989;210:474. doi: 10.1097/00000658-198910000-00008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Tandon N, Dinsdale C, Tamatani T, Miyasaka M, Weetman AP. Adhesion molecule expression by the FRTL-5 rat thyroid cell line. J Endocrinol. 1991;130:451. doi: 10.1677/joe.0.1300451. [DOI] [PubMed] [Google Scholar]
  • 18.Titus JA, Perez P, Kaubisch A, Garrido MA, Segal DM. Human K/natural killer cells targeted with hetero-cross-linked antibodies specifically lyse tumor cells in vitro and prevent tumor growth in vivo. J Immunol. 1987;139:3153. [PubMed] [Google Scholar]
  • 19.Van den Brink MRM, Hunt LE, Hiserodt JC. In vivo treatment with monoclonal antibody 3.2.3 selectively eliminates natural killer cells in rats. J Exp Med. 1990;171:197. doi: 10.1084/jem.171.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Van Dijk J, Warnaar SO, van Eendenburg JDH, Thienpont M, Braakman E, Boot JHA, Fleuren GJ, Bolhuis RLH. Induction of tumor-cell lysis by bi-specific monoclonal antibodies recognizing renal-cell carcinoma and CD3 antigen. Int J Cancer. 1989;43:344. doi: 10.1002/ijc.2910430230. [DOI] [PubMed] [Google Scholar]
  • 21.Vujanovic NL, Herberman RB, Maghazachi AA, Hiserodt JC. Lymphokine-activated killer cells in rats. III. A simple method for the purification of large granular lymphocytes and their rapid expansion and conversion into lymphokine-activated killer cells. J Exp Med. 1988;167:15. doi: 10.1084/jem.167.1.15. [DOI] [PMC free article] [PubMed] [Google Scholar]

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