Bispecific T-Cell Engagers Targeting Membrane-Bound IgE

Abstract

The increased incidence of allergies and asthma has sparked interest in IgE, the central player in the allergic response. Interaction with its high-affinity receptor FcεRI leads to sensitization and allergen presentation, extracellular membrane-proximal domain in membrane IgE can act as an antigen receptor on B cells, and the interaction with low-affinity IgE receptor CD23 additionally influences its homeostatic range. Therapeutic anti-IgE antibodies act by the inhibition of IgE functions by interfering with its receptor binding or by the obliteration of IgE-B cells, causing a reduction of serum IgE levels. Fusion proteins of antibody fragments that can act as bispecific T-cell engagers have proven very potent in eliciting cytotoxic T-lymphocyte-mediated killing. We have tested five anti-IgE Fc antibodies, recognizing different epitopes on the membrane-expressed IgE, for the ability to elicit specific T-cell activation when expressed as single-chain Fv fragments fused with anti-CD3ε single-chain antibody. All candidates could specifically stain the cell line, expressing the membrane-bound IgE-Fc and bind to CD3-positive Jurkat cells, and the specific activation of engineered CD3-overexpressing Jurkat cells and non-stimulated CD8-positive cells was demonstrated for 8D6- and ligelizumab-based bispecific antibodies. Thus, such anti-IgE antibodies have the potential to be developed into agents that reduce the serum IgE concentration by lowering the numbers of IgE-secreting cells.

Keywords: Fcε; T-cell activation; anti-IgE antibodies; bispecific T-cell engagers; cytotoxic T-lymphocyte mediated killing; extracellular membrane-proximal domain.

Figure 1

Organization of cell-expressed IgE-Fc and schematic depiction of the location of epitopes of targeting antibodies (CD19 for blinatumomab). IC: intracellular domain, TM: transmembrane domain, EMPD: extracellular membrane–proximal domain, Cε2–4: Fcε domains 2–4, 3× FLAG: 3 repeats of FLAG-tag.

Figure 2

Purified bispecific BiTE antibodies: (a) HPLC-SEC profiles of purified bispecific BiTE antibodies. MW (molecular weight) standard (Bio-RAD) includes thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa), and vitamin B12 (1.3 kDa); (b) SDS-PAGE analysis (first lane: Mark12 unstained standard (Thermo Fisher Scientific, Waltham, MA, USA) with MW in kDa).

Figure 3

Test of Fcε expression using staining with Xolair^®: Full circles-full line: doxycycline-induced Ramos-Fcε cell line, empty circles-full line: doxycycline-induced Ramos cell line transformed with an empty vector, full circles-dashed line: uninduced Ramos-Fcε cell line, full diamond: cells stained with secondary reagent only, empty diamond: cells only. All measurements were done at least in duplicates. MFI: mean fluorescence units resulting from bound secondary reagent anti-human-IgG-phycoerythrin (PE).

Figure 4

Cell surface staining experiments: (a) Binding of BiTE antibodies to the Ramos-Fcε cell line (black: blinatumomab, red: omalizumab, blue: 8D6, green: ligelizumab, orange: MEDI4212, purple: quilizumab); (b) Binding of quilizumab to Ramos-Fcε cell line (full circles-full line: induced Ramos-Fcε cell line, empty circles-full line: doxycycline-induced Ramos cell line transformed with an empty vector, full circles-dashed line: uninduced Ramos-Fcε cell line, full diamond: cells stained with secondary reagent only, empty diamond: cells only); (c) Binding of blinatumomab to: induced Ramos-Fcε cell line (full circles-full line) and induced Ramos cell line transformed with an empty vector (empty circles-full line). Full diamond: cells stained with secondary reagent only, empty diamond: cells only; (d) Binding of BiTE antibodies to the Jurkat E6-1 cell line (color scheme as in (a)). All measurements were done at least in duplicates. MFI: mean fluorescence units resulting from the signal of bound antibody conjugates (a,c) anti-pentahis-AlexaFluor^® 488, (b) anti-human-IgG-phycoerythrin (PE), (d) anti-pentahis-AlexaFluor^® 647.

Figure 5

Immunofluorescence microscopy to observe the binding to cell-bound targets of bispecific antibodies: Binding of BiTE antibodies to the Ramos-Fcε cell line; the Ramos cell line transformed with the empty vector, CD3-positive Jurkat E6-1 cells, and CD3-negative TIB-153 cells. Cells stained with secondary reagent only (2nd only) were used as a negative control. Scale bar represents 10 µm.

Figure 6

Internalization of Fcε, expressed on Ramos-EHRB cells, upon incubation with BiTE antibodies. Mean fluorescence intensity (MFI) values resulting from binding of the anti-FLAG antibody detected with an anti-mouse-FITC conjugate are shown for the cells treated with BiTEs on ice (blue bars) or 37 °C (red bars), in triplicate measurements (mean ± S.D.).

Figure 7

T-cell activation assays with Ramos-Fcε—targeting BiTE-like fragments: T-cell activation as a response to IgE-targeting BiTE-fragments using graded concentrations of BiTE starting with 400 pM for omalizumab-, MEDI4212-, and quilizumab-based molecules, and 2 nM for 8D6- and ligelizumab-based BiTEs (full bars: Fcε-cell line, empty bars: control cell line) and T-cell activation caused by dilutions of blinatumomab starting at 1 nM, when bound to Ramos-Fcε cells with the response of TIB-153 as a negative cell line. NFAT: nuclear factor of activated T cells, IL-2: interleukin 2, E:T: effector to target cell ratio.

Figure 8

Activation of CD8-positive T cells. Percent CD69-positive and CD25-positive cytotoxic T cells when incubated with (a) 8D6- and ligelizumab-based BiTE or without antibody; (b) blinatumomab. Full bars: Ramos-Fcε cells, empty bars: Ramos cells, transformed with empty vector; duplicate measurements are presented (mean ± S.D.).