Formation of multimeric antibodies for self-delivery of active monomers

Abstract

Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.

Keywords: Protein drug release; antibodies; bioactivity; multimers; self-delivery.

Figure 1.

ThT spectra (100 μM) of native and self-assembled Igs (0.15 mg/ml) preparations. Preparation symbols are detailed in the inset box. (A) IgG and (B) IgY.

Figure 2.

(A) CD spectra of native (gray) and self-assembled (black) IgG (left) and IgY (right) preparations (20 μg/ml). (B) Images of non-reducing gel electrophoresis for native and self-assembled IgG (left panel) and IgY (right panel).

Figure 3.

AFM images of native (A) versus self-assembled (B, 24 h; C, 48 h; D, 72 h preparation times) IgGs (1 mg/ml). Blue lines mark of discrete IgGs, green lines mark native IgG, all IgGs in the field were considered for height averaging. Native IgGs and self-assembled IgGs were diluted ×8000 and ×1000, respectively, prior to imaging. Bar graphs demonstrate mean height (±standard deviation) of measured IgGs in the field. Similarly, blue and green height charts are presented.

Figure 4.

(A) Binding activity of self-assembled IgG (left panel) and IgY (right panel), following 24 h preparation (see text for dilution protocols). (B) Binding activity of lower fraction versus total binding activity) of 1 mg/ml IgG or IgY (left and right panels, respectively), after ultracentrifugation at 300 000g. (C) Competition assay for self-assembled versus native IgG binding to HER2 + expressing SKBR3 cells. FACS spectra of native IgG (black) versus native and self-assembled IgGs (red) binding capacity. The average drop in the native IgG binding activity is given in the bar graph.

Figure 5.

Binding activity after long incubation periods of 1 mg/ml self-assembled IgG (left panel) and IgY (right panel). Binding is compared to the native antibodies binding capacity under similar conditions. Images in the inset panel represent vails containing the native (left vail) and self-assembled (right vail) suspensions after 40 (IgG) or 42 (IgY) days.