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. 2019 Jun 7;11(6):789.
doi: 10.3390/cancers11060789.

An Fc-Optimized CD133 Antibody for Induction of Natural Killer Cell Reactivity against Colorectal Cancer

Bastian J Schmied  1   2 , Fabian Riegg  3   4 , Latifa Zekri  5   6 , Ludger Grosse-Hovest  7 , Hans-Jörg Bühring  8 , Gundram Jung  9 , Helmut R Salih  10   11
Affiliations

An Fc-Optimized CD133 Antibody for Induction of Natural Killer Cell Reactivity against Colorectal Cancer

Bastian J Schmied et al. Cancers (Basel). .

Abstract

The introduction of monoclonal antibodies (mAbs) has largely improved treatment options for cancer patients. The ability of antitumor mAbs to elicit antibody-dependent cellular cytotoxicity (ADCC) contributes to a large extent to their therapeutic efficacy. Many efforts accordingly aim to improve this important function by engineering mAbs with Fc parts that display enhanced affinity to the Fc receptor CD16 expressed, e.g., on natural killer (NK) cells. Here we characterized the CD133 mAb 293C3-SDIE that contains an engineered Fc part modified by the amino acid exchanges S239D/I332E-that reportedly increase the affinity to CD16-with regard to its ability to induce NK reactivity against colorectal cancer (CRC). 293C3-SDIE was found to be a stable protein with favorable binding characteristics achieving saturating binding to CRC cells at concentrations of approximately 1 µg/mL. While not directly affecting CRC cell growth and viability, 293C3-SDIE potently induced NK cell activation, degranulation, secretion of Interferon-γ, as well as ADCC resulting in potent lysis of CRC cell lines. Based on the preclinical characterization presented in this study and the available data indicating that CD133 is broadly expressed in CRC and represents a negative prognostic marker, we conclude that 293C3-SDIE constitutes a promising therapeutic agent for the treatment of CRC and thus warrants clinical evaluation.

Keywords: ADCC; CD133; NK cells; antibody; colorectal cancer; immunotherapy; prominin-1.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Comparative binding analysis of different CD133 monoclonal antibodies (mAbs) to colorectal cancer (CRC) cells. (A) Specific binding of three different commercially available CD133 mAbs (293C3, AC133, and W6B3C1) was determined by flow cytometry using B16F10-CD133 and B16F10-control transfectants. Cells were incubated with 5 µg/mL CD133 mAb followed by a goat anti-mouse phycoerythrin (PE) conjugate. Shaded peaks: CD133 mAbs; open peaks: controls. (B) Relative CD133 mRNA expression in five CRC cell lines—which were used as a model for mAb binding analyses—was determined by quantitative PCR as described in the method section. (C) The CD133 mAb surface binding was comparatively analyzed in flow cytometry experiments by incubating CD133 mRNAhigh Caco-2 cells with increasing mAb concentrations of the different CD133 mAbs or isotype controls followed by a goat anti-mouse PE conjugate. Specific fluorescence intensity (SFI) levels were calculated as described in the method section. (D,E) The CD133 mAb binding to the panel of five CRC cell lines was comparatively analyzed by flow cytometry. Cells were incubated with 1 µg/mL CD133 mAb or isotype controls followed by a goat anti-mouse PE conjugate. Specific fluorescence intensity (SFI) levels (not applicable for SW-620 cells due to bimodal CD133 expression) and histograms are depicted in (D,E), respectively. Representative data of one experiment from a total of at least two with similar results is shown.
Figure 2
Figure 2
Generation and characterization of 293C3-SDIE in colorectal cancer (CRC). (A) Schematic illustration of 293C3-SDIE. (B) Purified 293C3-SDIE was analyzed by size exclusion chromatography (left) and SDS-PAGE (right). mAU: milli absorption unit; R: reduced; NR: non-reduced; M: marker. (C) B16F10-CD133 and B16F10-control transfectants were incubated with 10 µg/mL 293C3-SDIE or Iso-SDIE followed by an anti-human phycoerythrin (PE) conjugate. Shaded peaks: 293C3-SDIE; open peaks: Iso-SDIE. (D) B16F10-CD133 transfectants were incubated with the indicated concentrations of 293C3 or 293C3-SDIE and their respective isotype controls followed by an anti-mouse or anti-human PE conjugate. (E) The colorectal cancer (CRC) cell lines Caco-2 (CD133high: left), HCT-116 (CD133int: middle), and HT-29 (CD133low: right) were incubated with the indicated concentrations of 293C3-SDIE or Iso-SDIE followed by an anti-human PE conjugate. Int: intermediate; SFI: specific fluorescence intensity.
Figure 3
Figure 3
Direct effects of 293C3-SDIE on colorectal cancer (CRC) cell viability. (A) CD133 and epidermal growth factor receptor (EGFR) expression on CRC cell lines were comparatively analyzed by flow cytometry using 293C3-SDIE, cetuximab and panitumumab and their respective isotype controls (all at 1 µg/mL) followed by an anti-human phycoerythrin (PE) conjugate. Shaded peaks: specific mAbs; open peaks: isotype controls. (B) Caco-2 (left), HCT-116 (middle), and HT-29 (right) cells were incubated with 1 µg/mL of the indicated mAbs for three days. ATP levels were then determined by CellTiterGlo assays. Representative data of one experiment from a total of at least two with similar results are shown. C: cetuximab; int: intermediate; ns: not significant; P: panitumumab; *: significant (p-value < 0.05).
Figure 4
Figure 4
Induction of natural killer (NK) cell activity by 293C3-SDIE in the presence of colorectal cancer (CRC) cells. Peripheral blood mononuclear cells (PBMC) of healthy donors were cultured with or without the indicated CRC cell lines at an effector to target ratio of 2.5:1 in the presence or absence of 293C3-SDIE/Iso-SDIE (1 µg/mL). On the left, exemplary results obtained in a single experiment with PBMC of a single donor are shown; right panels depict combined analyses of data obtained with PBMC from five independent donors (bars represent respective means). (A) Activation of NK cells identified as CD14-CD56+CD3- cells within PBMC was determined after 24 h by flow cytometry for CD69. (B) Cells were cultured for 4 h in the presence of anti-human CD107a-PE/GolgiStop/GolgiPlug and NK cells subsequently analyzed by flow cytometry for CD107a as surrogate marker for degranulation. (C) Cells were cultured for 6 h before supernatants were analyzed for Interferon (IFN)-γ by an enzyme-linked immunosorbent assay. Int: intermediate; ns: not significant; *: significant (p-value < 0.05).
Figure 5
Figure 5
Induction of natural killer (NK) cell mediated colorectal cancer (CRC) cell lysis by 293C3-SDIE. Peripheral blood mononuclear cells (PBMC) of healthy donors were cultured with Caco-2 (left), HCT-116 (middle), and HT-29 cells (right) in the presence or absence of 293C3-SDIE/Iso-SDIE (1 µg/mL). Tumor cell lysis was measured after 2 h by Europium based cytotoxicity assays. In the top exemplary data over a broad range of effector to target (E:T) ratios with PBMC of one donor and in the bottom pooled data (bars represent respective means) at an E:T ratio of 80:1 with PBMC of five different donors are shown. Int: intermediate; ns: not significant; *: significant (p-value < 0.05).
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