Prestimulation of monocytes by the cytokines GM-CSF or IL- 2 enhances the antibody dependent cellular cytotoxicity of monoclonal antibody 17-1A[1]

Summary

Previously, we have shown that the cytokines ­IFN-α, IFN-γ and IL- 2 significantly enhance the antibody dependent cellular cytotoxicity (ADCC) exerted by the monoclonal antibody (mAb) 17-1A which recognizes the tumor ­associated antigen EpCAM. ADCC was assessed by a new flow cytometric cytotoxicity assay using the PKH2 labeled colorectal tumor cell line HT29 as target cells and peripheral blood mononuclear cells as effectors. Monocytes are assumed to be one of the major effectors for ADCC. However, isolated monocytes have a rather low ADCC capacity while addition of CD4⁺ lymphocytes optimizes ADCC. Since such an interaction between immune cells may act through cytokines we investigated whether a seven-day-prestimulation of monocytes by the cytokines M-CSF, GM-CSF, IFN-γ, IFN-α and IL- 2 enhances ADCC. Thereafter, we added for three days IL- 2 and IFN-α with or without the mAb 17-1A for terminal activation of monocytes. Interestingly, GM-CSF which was ineffective in terminal activation, significantly enhanced ADCC of monocytes when it was used for prestimulation. Similar results were obtained with IL- 2. IFN-γ and M-CSF were also active but less than GM-CSF. Astonishingly, IFN-γ and IFN-α prestimulation of monocytes suppressed the enhancement of ADCC exerted by GM-CSF and IL- 2, respectively. Our experiments suggest that the timing of cytokine application is critical for the induction of optimal ADCC. Subcutaneous pretreatment with GM-CSF or IL- 2 followed by the combination of IL- 2/IFN-α/17-1A should be evaluated in a phase I clinical trial in patients with colorectal cancer.

Steigerung der antikörperabhängigen Zellzytotoxizität (ADCC) des monoklonalen Antikörpers 17-1A durch Vorstimulation von Monozyten mit GM-CSF oder IL- 2

In Vorversuchen haben wir gezeigt, dass die Zytokine IFN-α, IFN-γ und IL- 2 die antikörpervermittelte Zytotoxizität (ADCC) des monoklonalen Antikörpers 17-1A, der das tumorassoziierte Antigen EpCAM auf der Oberfläche von kolorektalen Tumorzellen erkennt, signifikant steigern. Die ADCC wurde mittels eines neu entwickelten durchflusszytometrischen Zytotoxi­zitätstests bestimmt, wobei als Zielzellen die kolorektale Tumorzelllinie HT29 eingesetzt wurde. Monozyten sind eine der wichtigsten Effektorzellpopulationen, die ADCC ausüben. Diese Zellen zeigen aber nach Magnetseparation eine geringe ADCC, die erst nach Zugabe von CD4⁺ Lymphozyten optimal ist. Da durch Zellinteraktionen eine Produktion von Zytokinen stattfindet, haben wir hier untersucht, ob eine Monozytenvorstimulation mit den Zytokinen M-CSF, GM-CSF, IFN-γ, IFN-α und IL- 2 über 7 Tage zu einer Steigerung der ADCC führt. Da­nach haben wir zur terminalen Aktivierung der Monozyten IFN-α und IL- 2 für 3 weitere Tage gegeben. GM-CSF, welches in der terminalen Aktivierung zu keiner Steigerung der ADCC führte, erwies sich in der Vorstimulation als potenter Aktivator der ADCC. Auch IL- 2 erwies sich als potenter Stimulator der ADCC, während IFN-γ und M-CSF nur eine mäßige Aktivität zeigten. Erstaunlicherweise supprimierten IFN-γ und IFN-α die Steigerung der ADCC durch GM-CSF und­ IL- 2. Unsere Unter­suchungen zeigen, dass die zeitliche Abfolge der Gabe von Zytokinen eine große Rolle für eine optimale ADCC spielt. In der Klinik sollte die Sequenz einer subkutanen Behandlung mit GM-CSF oder IL- 2 und anschließender Gabe von IFN-α/IL- 2 und 17-1A in Phase-I-Studien bei Patienten mit kolorektalem Karzinom überprüft werden.

ADCC - GM-CSF - Interleukin- 2 - Monocyte

Colorectal Carcinoma - Monoclonal Antibodies - Cytokines

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Fußnoten

1 Flieger D et al. Influence of cytokines, monoclonal antibodies and chemotherapeutic drugs on EpCAM and LewisY antigen expression (submitted).

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