Heterodimeric bispecific single-chain variable-fragment antibodies against EpCAM and CD16 induce effective antibody-dependent cellular cytotoxicity against human carcinoma cells

Abstract

A heterodimeric bispecific biological recombinant drug was synthesized by splicing DNA fragments from two fully humanized single-chain variable-fragment (scFV) antibody fragments forming a novel drug simultaneously recognizing the CD16 natural killer (NK) cell marker and the cancer marker epithelial cell adhesion molecule (EpCAM). The drug precipitously enhanced the killing of human carcinomas of the prostate, breast, colon, head, and neck even at very low effector:target ratios. The drug EpCAM16 rendered even nonactivated NK cell-proficient killers and activated them to kill via degranulation and cytokine production. Studies show that bispecific antibodies can be used to induce proficient killing of the carcinoma targets that ordinarily are resistant to NK-mediated killing. Apparently, the innate immune system can be effectively recruited to kill cancer cells using the bispecific antibody platform and EpCAM targeting.

Keywords: ADCC; anti-CD16; bispecific antibody; carcinoma; human NK cells.

FIG. 1.

EpCAM16 was successfully purified from bacterial inclusion bodies. (A) A diagram shows the plasmid p16xEpCAM.pET21 containing the EpCAM16 construct consisting of an Nco1 restriction site, the VH and VL regions of anti-human CD16 (NM3E2), a segment of human muscle aldolase, the VH and VL regions of humanized anti-EpCAM (MOC31), and a XhoI restriction site. (B) The absorbance trace of the refolded protein eluted from the Q-sepharose ion-exchange column using a stepwise NaCl gradient. (C) The gel stained with Coomasie blue showing a single band of about 54 kDa eluted from the second column that we ran (size-exclusion column). Lane 1, molecular-weight standards; Lane 2, nonreduced EpCAM16; Lane 3, nonreduced anti-Ly5.1 monoclonal antibody control. EpCAM, epithelial cell adhesion molecule.

FIG. 2.

The EpCAM scFV of EpCAM16 binds EpCAM-expressing HT-29 colorectal cancer cells. HT-29 cells were incubated with a targeted toxin DTEpCAM consisting of anti-EpCAM scFV spliced to diphtheria toxin. The addition of 100 or 300 nM EpCAM16-bispecific antibody blocked 1 nM DTEpCAM killing, but the addition of 100 or 300 nM of 16×19 bispecific antibody did not. Killing was measured as inhibition of thymidine uptake. scFV, single-chain variable fragment.

FIG. 3.

Enriched NK cells from 2 normal human donors express CD16. Enriched NK cells from donors NK1 and NK2 were treated with 10 μg/mL EpCAM16-FITC or anti-CD16-FITC in a direct immunofluorescence assay using flow cytometry. Cells were highly positive compared to the untreated cell (no stain) or anti-B-cell control DT2219ARL-FITC. In every panel, the histogram from the experimental sample is compared to the untreated blank control. NK, natural killer; FITC, fluorescein isothiocyanate.

FIG. 4.

EpCAM16 engages enriched human NK cells to kill various carcinoma cells in vitro. EpCAM-expressing HT-29 colorectal, MDA-MD-468 breast, PC-3 prostate, and UMSCC-11B head and neck cancer cells were tested in different experiments. Cancer cells were mixed with enriched NK cells from 2 different normal donors (NK donor 1 and NK donor 2) and EpCAM16 in plastic plates. After 4 hours, the cells were pulsed with ⁵¹Cr, and the NK activity was calculated based on the isotope release from lysed target cells. In all instances, EpCAM16 showed vastly enhanced NK-killing activity. Control wells where cells were incubated with anti-CD16 alone, anti-EpCAM alone, control anti-EpCAM×Her2/Neu (EpCAM23), or no drug at all did not enhance NK activity.

FIG. 5.

EpCAM16 does not engage enriched human NK cells to kill EpCAM-negative Daudi B-cell lymphoma cells. Daudi cells were mixed with enriched NK cells from 2 different normal donors (NK donor 1 and NK donor 2) and EpCAM16 in plastic plates. Killing was calculated from ⁵¹Cr release. EpCAM16 did not enhance the NK-killing activity.

FIG. 6.

EpCAM16 markedly enhances the expression of CD107a, considered a marker of NK cell activity and expression of interferon-γ (IFN-γ). SKBR3 human breast cancer cells were incubated with enriched resting NK cells in the presence of 20 μg/mL EpCAM16. (A) Cells were studied by flow cytometry to determine the expression of CD107a, also called lysosomal-associated membrane protein. Levels were elevated, but not in cells treated with control anti-EpCAM, anti-CD16 scFV, or untreated cells. (B) Cells were studied by flow cytometry to determine the expression of IFN-γ. The levels were elevated, but not in cells treated with control anti-EpCAM, anti-CD16 scFV, or untreated cells.

FIG. 7.

EpCAM16 is dose-dependent and active at very low E:T ratios. CaCo₂ human colorectal cancer cells were incubated with enriched resting NK cells in the presence of EpCAM16. (A, B) Decreasing concentrations of drug were added beginning at 5 μg/mL. A drop-off in activity was observed at the 1 μg/mL dose level. (C) Cells treated at 5 μg/mL were tested at very low E:T ratios. Activity was still observed at an E:T of 1:1. (D) Three NK donors were tested simultaneously at the 20 μg/mL dose level. High NK activity occurred with all 3 donors. E:T, effector:target.