Redirecting immunity via covalently incorporated immunogenic sialic acid on the tumor cell surface

Abstract

Techniques eliciting anti-tumor immunity are of interest for immunotherapy. We herein report the covalent incorporation of a non-self immunogen into the tumor glycocalyx by metabolic oligosaccharide engineering with 2,4-dinitrophenylated sialic acid (^DNPSia). This enables marked suppression of pulmonary metastasis and subcutaneous tumor growth of B16F10 melanoma cells in mice preimmunized to produce anti-DNP antibodies. Located on the exterior glycocalyx, ^DNPSia is well-positioned to recruit antibodies. Given the high levels of natural anti-DNP antibodies in humans and ubiquitous sialylation across many cancers, ^DNPSia offers a simplified route to redirect immunity against diverse tumors without recourse to preimmunization.

Scheme 1. Schematic for the incorporation of the non-self antigen into glycocalyx. ^DNPSia taken up by the tumors is metabolically transferred to glycoconjugates. The neosialoconjugates sorted to the cell surface enable ^DNPSia to be well-positioned to trigger immunity.

Fig. 1. Incorporation of DNP-Sia diads into cell glycocalyx. Chemical structures of sialosides of ^DNPSia and ^DNP–TzSia displayed on a cell surface (A). B16F10 cells treated with the methyl esters of ^DNP–TzSia or ^DNPSia (0, 1 mM) were stained with biotin-labelled anti-DNP Ab, PE-labelled streptavidin, and DAPI specific for the nucleus, and then analysed by confocal fluorescence microscopy (B) or flow cytometry (C) with the mean channel fluorescence (MF) indicated. Bars: 10 μm. (D) Western blot of the lysate of cells treated with the DNP-Sia diads. Protein loading was verified by Coomassie Blue staining.

Fig. 2. ^DNPSia mediated anti-tumor responses in mice. (A) Effects of ^DNPSia on cell proliferation. B16F10 cells pretreated with ^DNPSia (0–1 mM) were cultured in fresh DMEM for 24 h prior to cell number determination. (B) ELISA of anti-DNP Ab in the serum from C57BL/6 mice untreated or treated with an injection of ^DNPKLH. (C) Suppressed growth of inoculated ^DNPSia⁺ B16F10 cells over ^DNPSia-free cells that are injected in opposite flanks of mice. Tumors were excised 7 days post inoculation. (D) Anti-DNP Ab mediated inhibition of ^DNPSia⁺ B16F10 cells and ^DNPSia^– cells subcutaneously inoculated in mice unimmunized (in dark) or immunized with ^DNPKLH (in red). Assays were performed in triplicate each using 3 mice. Error bars represent ±SD of experimental data on a representative assay.

Fig. 3. Tumor suppression with intravenously injected ^DNPSia in mice. ^DNPKLH-immunized C57BL/6 mice were subcutaneously inoculated with B16F10 cells, and then treated with a tail-vein injection of PBS (100 μL) or ^DNPSia (30 mg kg^–1) on the 3^rd, 6^th, and 9^th day post cell inoculation. The tumor volumes were monitored over time. The inset shows the correlation of early stage tumor volume vs. time. The assays were performed in triplicate, using 3 mice each time. The error bars represent ±SD of the experimental data on a representative assay.

Fig. 4. Glycocalyx-anchored ^DNPSia decreases pulmonary metastasis of B16F10 cells in mice. ^DNPKLH-immunized C57BL/6 mice were untreated or treated with ^DNPSia^–, or ^DNPSia⁺ B16F10 cells by tail-vein injection. The organs were excised 7 days after injection. The arrows denote metastases.

Fig. 5. In vivo distribution of ^DNPSia. C57BL/6 mice bearing subcutaneous B16F10 tumors were injected via the tail-vein with PBS (A), or ^DNPSia (60 mg kg^–1) (B). The tumors (10 mm³) and organs were excised 1 h post-injection, sectioned, and stained with biotin-labelled anti-DNP Ab, PE-labelled streptavidin and DAPI prior to fluorescence analysis.

Fig. 6. Temporal retention of ^DNPSia in tumors. C57BL/6 mice with B16F10 tumors (10 mm³) were injected with ^DNPSia (60 mg kg^–1) in the tail-vein. At 0–24 h after the injection, the tumors were excised, sectioned, and stained with biotin-labelled anti-DNP Ab, PE-labelled streptavidin, and DAPI prior to fluorescence detection.