Antibody-Mediated Endocytosis of Polysialic Acid Enables Intracellular Delivery and Cytotoxicity of a Glycan-Directed Antibody-Drug Conjugate

Abstract

The specific targeting of differentially expressed glycans in malignant cells has emerged as an attractive anticancer strategy. One such target is the oncodevelopmental antigen polysialic acid (polySia), a polymer of α2,8-linked sialic acid residues that is largely absent during postnatal development but is re-expressed during progression of several malignant human tumors, including small-cell and non-small cell lung carcinomas, glioma, neuroblastoma, and pancreatic carcinoma. In these cancers, expression of polySia correlates with tumor progression and poor prognosis and appears to modulate cancer cell adhesion, invasiveness, and metastasis. To evaluate the potential of PolySia as a target for anticancer therapy, we developed a chimeric human polySia-specific mAb that retained low nanomolar (nmol/L) target affinity and exhibited exquisite selectivity for polySia structures. The engineered chimeric mAb recognized several polySia-positive tumor cell lines in vitro and induced rapid endocytosis of polySia antigens. To determine whether this internalization could be exploited for delivery of conjugated cytotoxic drugs, we generated an antibody-drug conjugate (ADC) by covalently linking the chimeric human mAb to the tubulin-binding maytansinoid DM1 using a bioorthogonal chemical reaction scheme. The resulting polySia-directed ADC demonstrated potent target-dependent cytotoxicity against polySia-positive tumor cells in vitro. Collectively, these results establish polySia as a valid cell-surface, cancer-specific target for glycan-directed ADC and contribute to a growing body of evidence that the tumor glycocalyx is a promising target for synthetic immunotherapies. SIGNIFICANCE: These findings describe a glycan-specific antibody-drug conjugate that establishes polySia as a viable cell surface target within the tumor glycocalyx.

Figure 1.. Binding specificity of the ch735 antibody.

(a) Antigen binding activity for recombinant purified ch735 determined by ELISA with either NCAM or endoN-treated NCAM immobilized as antigens. ELISA signals (Abs₃₇₀) were obtained with anti-human IgG-HRP conjugate. (b) Glycoprotein-binding specificity of ch735 was probed using an array of ~50 glycoproteins. Antibodies were assayed at 1 μg/mL and detected with anti-human IgG antibodies. (c) Glycan-binding specificity of ch735 was measured against CFG glycan microarray (version 5.4) that contained ~585 natural and synthetic mammalian glycans (http://www.functionalglycomics.org). Antibodies were assayed at 10 μg/mL and detected with anti-human IgG antibodies. All data are the average of three replicate experiments and error bars are the standard deviation of the mean. (d) Same as in (c) but only showing data for glycan structures containing α2,8-linked sialic acid (see Supplementary Table 1 for a list of the corresponding structures).

Figure 2.. Immunostaining of antibody ch735 to polySia-expressing cancer cells.

(a) External levels of polySia on a panel of cancer cell lines with or without endoN treatment measured by flow cytometry using ch735 and fluorescent anti-human secondary. Data are the geometric mean fluorescence intensity (MFI), with the values reported as the average of three replicates and the error represented as the standard deviation of the mean. (b) Confocal microscopic images of endoN-treated and non-treated polySia expressing cancer cell lines to assess ch735 binding on the cell surface. Cells were stained with ch735 (green), wheat germ agglutinin (WGA, red) to stain the cell membrane, and Hoescht (blue) to stain nuclei. Scale bars, 10 μm. (c) Formalin-fixed, paraffin-embedded (FFPE) human tissue sections of SCLC and (d) adjacent normal tissue stained for polySia with mo735. Scale bars, 200 μm.

Figure 3.. Internalization of ch735 into polySia-positive cancer cells.

(a) Internalization of ch735 in polySia-positive cell lines SH-SY5Y, SW2, H69, and H82, and in polySia-negative MCF7 cells after 1 h. Data are reported as the mean percent internalization and error bars are the standard deviation of the mean (n = 3). (b) Time course of antibody internalization in polySia-positive cell line SH-SY5Y treated with ch735 or isotype control. Data reported as the mean percent internalization and error bars are the standard deviation of the mean (n = 3). (c) Confocal microscopy images of SH-SY5Y cells incubated for 1 h with ch735 labeled with AF488 and transferrin labeled with AF647. Nuclei were stained by Hoescht (blue). Scale bar, 10 μm. Fluorescence intensity was measured across the dotted white line and normalized to the maximum value in each channel. White arrows indicate regions of colocalization. The inset shows only the ch735 (green) and DNA (blue) channels of the boxed region. (d) Confocal microscopy images of SH-SY5Y cells incubated for 1 h with ch735 labeled with AF488 and anti-LAMP-3 labeled with AF647. Nuclei were stained by Hoescht (blue). Scale bar, 10 μm. Fluorescence intensity was measured across the dotted white line and normalized to the maximum value in each channel. White arrows indicate regions of colocalization. The inset show only the ch735 (green) and DNA (blue) channels of the boxed region. (e) Confocal microscopy images of SH-SY5Y cells incubated for 120 min with ch735. Lysosomes were stained with anti-LAMP-1 and A647-labeled anti-rabbit antibody (red), ch735 was stained with AF488-labeled anti-human antibody (green), and nuclei were stained by Hoescht (blue). Scale bar, 10 μm. Fluorescence intensity was measured across the dotted white line and normalized to the maximum value in each channel. White arrows indicate regions of colocalization. The top right inset shows only the ch735 (green) and DNA (blue) channels of the boxed region.

Figure 4.. Target-mediated in vitro cytotoxicity of glycan-directed ADC.

(a) Overview of the two-step ADC synthesis strategy used to generate ch735-Py-DM1. The first step involved conjugation of NHS-PEG4-tetrazine (NHS-Tz) to free lysines and the second step involved the reaction of the trans-cyclooctene (TCO) group on the TCO-maleimide-DM1 drug linker (TCO-mal-DM1) with the Tz on the antibody. (b) Chemical structure of the non-cleavable drug linker with DM1. (c) Viability of SH-SY5Y (polySia+) and MCF7 (polySia−) cells following treatment with different concentrations of ch735-Py-DM1 or isotype-Py-DM1. Percent viability is calculated based on the signal relative to untreated control cells. Representative data depicts mean percent viability and error bars are the standard deviation of the mean (n = 3). (d) Viability of SKOV3 (HER2+) and SH-SY5Y (HER2-) cells following treatment with different concentrations of T-Py-DM1 and isotype-Py-DM1. Representative data depicts mean percent viability and error bars are the standard deviation of the mean (n = 3).