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Basic Sciences

Murine Tumor Cell Lysis by Antibody-dependent Macrophage-mediated Cytotoxicity Using Syngeneic Monoclonal Antibodies

Ichiro Kawase, Kiyoshi Komuta, Takeshi Ogura, Hiromi Fujiwara, Toshiyuki Hamaoka and Susumu Kishimoto
Ichiro Kawase
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Kiyoshi Komuta
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Takeshi Ogura
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Hiromi Fujiwara
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Toshiyuki Hamaoka
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Susumu Kishimoto
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DOI:  Published April 1985
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Abstract

Four monoclonal antibodies to MH134 murine syngeneic hepatoma cells, 3H1, 7C2, 11G2, and 12A2, were produced by hybridomas constructed by fusing P3-X63-Ag8-U1 murine myeloma cells with spleen cells of a C3H/HeN mouse immunized with the syngeneic tumor cells. Immunodiffusion analysis with rabbit anti-mouse immunoglobulin antisera showed that 3H1, 7C2, 11G2, and 12A2 are IgG2a, IgM, IgG1, and IgG2a, respectively. Enzyme-linked immunosorbent assay using cells of five syngeneic tumor lines, MH134, MM102, MM46, MM48, and X5563, and lymph node cells of C3H/HeN and C57BL/6 mice showed that 3H1 specifically bound to MH134 tumor cells, whereas 7C2, 11G2, and 12A2 reacted not only with MH134 but also with MM102 and MM46 tumor cells. None of these monoclonal antibodies bound either to cells of MM48 or X5563 tumor lines or to normal lymph node cells. These results strongly suggest that MH134 tumor cells display at least two kinds of tumor-associated antigens on their cell surfaces: one is expressed uniquely by MH134 tumor cells, which are recognized by 3H1; the other is commonly shared by MH134, MM102, and MM46 tumor cells, which are determined by the other three antibodies.

3H1, 11G2, and 12A2 but not 7C2 were found to be able to induce antibody-dependent cellular cytotoxicity (ADCC) against MH134 tumor cells. Target specificity of ADCC induced by these monoclonal antibodies was identical with that seen in enzymelinked immunosorbent assay. 3H1, 7C2, and 12A2 but not 11G2 exhibited complement-dependent cytotoxicity, showing the same specificity in target cell lysis as that seen in enzyme-linked immunosorbent assay or ADCC. Pretreatment of MH134 tumor cells with 7C2 inhibited ADCC of both 11G2 and 12A2. Pretreatment of the tumor cells with 11G2 inhibited complement-dependent cytotoxicity of both 7C2 and 12A2. Neither ADCC nor complement-dependent cytotoxicity of 3H1 was inhibited by the pretreatment of the cells with 7C2 or 11G2. These results strongly suggest that tumor-associated antigens recognized by 3H1 are located apart from that recognized by 7C2, 11G2, and 12A2 and that the binding sites of the latter three antibodies are closely associated with, or identical with, each other in the tumor-associated antigen.

The ability of 12A2 to induce ADCC against MH134 tumor cells was significantly stronger than that of 3H1 or 11G2. Effector cell analysis of ADCC induced by 12A2 revealed that thioglycolate-induced peritoneal macrophages exhibited high cytotoxicity, whereas peritoneal resident macrophages of normal mice showed low cytotoxicity. Neither normal spleen cells nor non-adherent cells obtained from thioglycolate-induced peritoneal cells mediated cytotoxicity, indicating that cytotoxicity is mediated by macrophages and that stimulation of macrophages is essential to augment the cytotoxicity. These monoclonal antibodies, derived from the syngeneic tumor immune system, will contribute to investigation of combined cancer therapy with monoclonal antibodies and biological response modifiers which can activate macrophages.

Footnotes

  • ↵1 Supported by a Grant-in-Aid for Cancer Research from Ministry of Education, Science and Welfare.

  • ↵2 To whom requests for reprints should be addressed.

  • Received August 31, 1984.
  • Revision received January 7, 1985.
  • Accepted January 9, 1985.
  • ©1985 American Association for Cancer Research.
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April 1985
Volume 45, Issue 4
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Murine Tumor Cell Lysis by Antibody-dependent Macrophage-mediated Cytotoxicity Using Syngeneic Monoclonal Antibodies
Ichiro Kawase, Kiyoshi Komuta, Takeshi Ogura, Hiromi Fujiwara, Toshiyuki Hamaoka and Susumu Kishimoto
Cancer Res April 1 1985 (45) (4) 1663-1668;

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Murine Tumor Cell Lysis by Antibody-dependent Macrophage-mediated Cytotoxicity Using Syngeneic Monoclonal Antibodies
Ichiro Kawase, Kiyoshi Komuta, Takeshi Ogura, Hiromi Fujiwara, Toshiyuki Hamaoka and Susumu Kishimoto
Cancer Res April 1 1985 (45) (4) 1663-1668;
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