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Natural killer cells are crucial for the efficacy of Icon (factor VII/human IgG1 Fc) immunotherapy in human tongue cancer - PubMed

  • ️Fri Jan 01 2010

Natural killer cells are crucial for the efficacy of Icon (factor VII/human IgG1 Fc) immunotherapy in human tongue cancer

Zhiwei Hu et al. BMC Immunol. 2010.

Abstract

Background: Icon is a novel, dual neovascular- and cancer cell-targeting immunotherapeutic agent and has shown efficacy in the treatment of cancer, wet form macular degeneration and endometriosis. However, its underlying mechanism remains to be investigated. The objective of this study is to elucidate the mechanism of Icon immunotherapy in cancer using a squamous carcinoma human tongue cancer line TCA8113 in vitro and in vivo in severe combined immunodeficiency (SCID) mice.

Results: We showed that Icon, as a chimeric factor VII and human IgG1 Fc immunoconjugate, could separately induce murine natural killer (NK) cells and activate complement to kill TCA8113 cancer cells in vitro via antibody dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, Icon-NK ADCC had a significantly stronger effect than that of Icon-CDC. Moreover, Icon could completely eradicate established human tongue tumour xenografts in vivo in the CB-17 strain of SCID mice that have functional NK cells at a normal level, whereas it was less effective in SCID/Beige mice that do not have functional NK cells.

Conclusions: We conclude that NK cells are crucial for the efficacy of Icon immunotherapy in the treatment of cancer. The results also suggest that impaired NK level/activity could contribute to the resistance to therapeutic antibodies that are currently under investigation in preclinical and clinical studies.

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Figures

Figure 1
Figure 1

Production and binding activity of Icon protein. a. Molecular weight of the Icon protein produced by CHO cells analysed by SDS-PAGE. fVII: mouse factor VII with K341A mutation; H: hinge region of a human IgG1 Fc; CH2 and CH3: the second and third domains of the constant region on the heavy chain of a human IgG1 Fc. b. Binding activity of Icon protein to human tongue cancer TCA8113 cells by flow cytometry. Control: TCA8113 cancer cells were not incubated with Icon but with secondary antibody FITC. Mouse Icon: the cells were incubated with Icon protein then with the secondary antibody to human IgG Fc FITC. c. Immunoprecipitation Western-blotting analysis of Icon protein production by TCA8113 cells one day after infection with AdmIcon (lane 1) or AdBlank (lane 2). The serum free culture medium from uninfected TCA8113 cells was used as uninfected control (lane 3). M: Protein markers (Bio-Rad All blue). Molecular weights (kDa) of the protein markers are indicated. Data are representative of two experiments.

Figure 2
Figure 2

Lysis of TCA8113 cancer cells by Icon-dependent NK cell-mediated ADCC and complement-mediated CDC in vitro. a. Isolation of murine NK cells from a single cell suspension of splenocytes from SCID/CB-17 mice. b. Icon-dependent NK cell-mediated ADCC. c. Icon-dependent complement-mediated cytotoxicity. Note that % specific lysis was derived from the % of cytotoxicity of Icon + NK cell or Icon + complement subtracted by % of cytotoxicity of NK cell alone or complement alone. P values and no significance (ns) were analysed by unpaired t test (b) or one way ANOVA with Tukey's Multiple Comparison Test (c). Data are representative of two experiments.

Figure 3
Figure 3

Icon protein has no effect on cancer cell proliferation in the absence of NK cell and complement. After incubation with mouse Icon protein for 1 hr followed by overnight growth, cancer cell proliferation was determined by CellTiter One Solution reagent, as described in Methods. P values obtained were of no significance as determined by one-way ANOVA with Tukey's Multiple Comparison Test. Data are representative of two experiments.

Figure 4
Figure 4

NK cells are crucial for the efficacy of Icon immunotherapy of human tongue cancer in vivo in SCID mice. Human tongue tumour xenografts were generated separately in SCID/CB-17 (a) and SCID/Beige mice (b) by subcutaneous injection of the TCA8113 tumour cells into the right flank of each mouse. After tumour formed, adenoviral vector encoding mouse Icon (AdmIcon) or control vector (AdBlank) were injected intratumourally with 1 × 1010 viral particles (vp) per injection for immunotherapy on the days as indicated below. a: In CB-17 SCID mice, AdmIcon (◯, n = 4) or AdBlank (Δ, n = 3) was injected on days 0, 3, 6, 9, 12, 15, 18, 21. Additional intratumoural injections of AdmIcon were administered in 2 partially responded mice on days 54, 84, 93, 99 and 135. P < 0.001 on days 6 and 9 and < 0.001 on days 12-48 for AdmIcon vs. AdBlank by two-way ANOVA. b: In Beige SCID mice, AdmIcon (◯, n = 5) or AdBlank (Δ, n = 5) was intratumourally injected on days 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 36 and 42. The control mice were euthanised on day 53 and the Icon-treated mice on day 74. P < 0.01 on day 36 and < 0.001 on days 39-51 by two-way ANOVA for AdmIcon vs. AdBlank.

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