Dual Antibody-Conjugated Amyloid Nanorods to Promote Selective Cell-Cell Interactions

Abstract

Grafting biomolecules on nanostructures' surfaces is an increasingly used strategy to target pathogenic cells, with both diagnostic and therapeutic applications. However, nanomaterials monofunctionalized by conjugating a single type of ligand find limited uses in pathologies/therapies that require two or more targets/receptors to be targeted and/or activated with a single molecular entity simultaneously. Therefore, multivalent nanomaterials for dual- or multitargeting are attracting significant interest. This study provides a proof of concept of such nanostructures. We have recently developed a modular methodology that allows obtaining amyloid-based materials decorated with active globular domains. Here, this approach is exploited to generate functional amyloid fibrils displaying antibody capture moieties. A high antibody binding affinity and capacity for the resulting nanofibrils, whose size can be manipulated to obtain homogeneous nanorods with high biocompatibility, are demonstrated. These nanorods are then used for specific antibody-mediated targeting of different cell types. Simultaneous conjugation of these nanorods with different antibodies allows obtaining a mimic of a bispecific antibody that redirects T lymphocytes to tumoral cells, holding high potential for immunotherapy. Overall, the work illustrates a modular and straightforward strategy to obtain preparative quantities of multivalent antibody-functionalized nanomaterials with multitargeting properties without the need for covalent modification.

Keywords: amyloid; antibody; dual- or multitargeting; multivalency; nanomaterials; nanorods.

Figure 1

Biophysical characterization of Sup35-Z fibrils. Sup35-Z and Z-domain proteins were incubated for 5 days and analyzed by measuring (A) Th-T fluorescence emission and (B) CR absorbance. Z-domain and Sup35-Z are shown in blue and red, respectively. Phosphate-buffered saline (PBS) alone was included as a control (black line). Representative TEM images of incubated proteins upon negative staining: (C) Z-domain and (D) Sup35-Z. The scale bar represents 1 μm and 0.5 μm, respectively.

Figure 2

Antibody binding affinity of Sup35-Z fibrils. Representative fluorescence microscopy image of fibrils incubated with single IgG labeled with Alexa 488: (A) Sup35 peptide fibrils and (B) Sup35-Z fibrils. The scale bar represents 50 μm. (C) Fluorescence emission spectra of incubated fibrils at 0.4 μM. The blue line represents the fluorescence spectra of the antibody alone. (D) A linear plot of the fluorescence intensity of incubated fibrils as a function of the fibrils’ concentration.

Figure 3

Double antibody binding of Sup35-Z fibrils. The representative fluorescence image of the Sup35-Z fibrils (upper panel) and Sup35 peptide fibrils (bottom panel) incubated simultaneously with two secondary antibodies: rabbit antimouse antibody labeled with Alexa 488, and goat antirabbit antibody labeled with Alexa 555. The scale bar represents 50 μm.

Figure 4

Representative TEM images of sonicated Sup35-Z amyloid fibrils upon negative staining. The scale bar corresponds to 500 and 200 nm, respectively.

Figure 5

Binding selectivity of functionalized Sup35-Z nanorods to human cells. (A) Representative confocal microscopy images of HeLa cells treated with nanorods conjugated with an anti-CD3 antibody (NRs-anti-CD3, Alexa 488) (upper panel) or an anti-EGFR antibody (NRs-anti-EGFR, Alexa 555) (middle panel) and T lymphocytes incubated with nanorods conjugated with an anti-CD3 antibody (NRs-anti-CD3, Alexa 488) (lower panel). The scale bar corresponds to 50 μm. (B) Quantitative analysis of fluorescein fluorescence on HeLa cells and T lymphocytes by flow cytometry. HeLa cells or T lymphocytes were incubated with anti-EGFR-loaded Sup35-Z nanorods (NRs-anti-EGFR, green line), anti-CD3 antibody-loaded nanorods (NRs-anti-CD3, red line), and free nanorods (NRs, blue line). HeLa cells or T lymphocytes treated with PBS were used as a control. Alexa Fluor 488-labeled antibody was used as a fluorescence probe to calculate the proportion of cells bound to nanorods using a FITC detector.

Figure 6

Double mAbs-conjugated nanorods redirect the CD3 expressing T cells to EGFR expressing HeLa cells. (A) Representative microscopy images of EGFR expressing HeLa cells and CD3 expressing T cells in the presence of anti-EGFR and anti-CD3-bound nanorods (anti-EGFR-Alexa Fluor 555, anti-CD3-Alexa Fluor 488, lower panel) and anti-EGFR and antirabbit-bound nanorods (anti-EGFR-Alexa Fluor 555, antirabbit-Alexa Fluor 488, upper panel), respectively. The white arrows show the presence of T lymphocytes with circular and round shapes. The anti-EGFR antibody and anti-CD3 antibody are labeled with Alexa Fluor 555 (red color) and Alexa Fluor 488 (green color), respectively. (B) Representative microscopy images of HeLa and T cells expressing EGFR and CD3 receptors, respectively, in the presence of unlabeled anti-EGFR-NRs-anti-CD3 (upper panel) and unlabeled anti-EGFR-NRs-antirabbit (lower panel) nanorods. T cells are specifically stained with an anti-CD28 IgG labeled with FITC (green fluorescence), and cell nuclei are stained with Hoechst (blue color). The scale bar represents 50 μm.

Figure 7

Schematic representation of the dual-targeting functionality of the mAbs-nanorod complex. The construct of Sup35-Z fusion consists of a Sup35 SAC (green square) and a Z-domain (green ball) that acts as an antibody capture domain, linked with a flexible linker (black line); Sup35-SAC induces the self-assembly of the fusion protein into antibody-binding nanofibrils. Nanorods bound to two monoclonal antibodies (mAbs) direct the TCR/CD3 complex-positive T lymphocytes to EGFR expressing tumor cells. Activated T lymphocytes would kill tumor cells.