Optimizing design parameters of a peptide targeted liposomal nanoparticle in an in vivo multiple myeloma disease model after initial evaluation in vitro.

Despite ligand-targeted liposomes long garnering interest as drug delivery vehicles for cancer therapeutics, inconsistency in successful outcomes have hindered their translation into the clinic. This is in part due to discrepancies between in vitro design evaluations and final in vivo outcomes. By employing a multifaceted synthetic strategy to prepare peptide-targeted nanoparticles of high purity, reproducibility, and with precisely controlled quantity of functionalities, we systematically evaluated the individual roles that peptide-linker length, peptide hydrophilicity, peptide density, and nanoparticle size play on cancer cell uptake and tumor targeting both in vitro and in vivo, and how the results correlated and contrasted. These parameters were analyzed using a VLA-4-targeted liposome system in a multiple myeloma mouse xenograft model to evaluate in vivo biodistribution and tumor cell uptake. The results showed that using in vitro models to optimize targeted-nanoparticles for maximum cellular uptake was helpful in narrowing down the particle characteristics. However, in vitro optimization fell short of achieving enhanced results in animal models, rather had negative consequences for in vivo targeting. This outcome is not surprising considering that the receptor being targeted is also present on healthy lymphocytes and increasing targeting peptide valency on particle surfaces results in an increase in non-selective, off-target binding to healthy cells. Hence, further optimization using in vivo models was absolutely necessary, through which we were able to increase the uptake of peptide-targeted liposomes by cancerous cells overexpressing VLA-4 to 15-fold over that of non-targeted liposomes in vivo. The results highlighted the importance of creating a comprehensive understanding of the effect of each liposome design parameter on multifactorial biological endpoints including both in vitro and in vivo in determining the therapeutic potential of peptide-targeted liposomes.

Covalent Heterobivalent Inhibitor Design for Inhibition of IgE-Dependent Penicillin Allergy in a Murine Model.

Drug allergies occur when hapten-like drug metabolites conjugated to serum proteins, through their interactions with specific IgE, trigger allergic reactions that can be life threatening. A molecule termed covalent heterobivalent inhibitor (cHBI) was designed to specifically target drug hapten-specific IgE to prevent it from binding drug-haptenated serum proteins. cHBI binds the two independent sites on a drug hapten-specific Ab and covalently conjugates only to the specific IgE, permanently inhibiting it. The cHBI design was evaluated via ELISA to measure cHBI-IgE binding, degranulation assays of rat basophil leukemia cells for in vitro efficacy, and mouse models of ear swelling and systemic anaphylaxis responses for in vivo efficacy. The cHBI design was evaluated using two separate models: one specific to inhibit penicillin G-reactive IgE and another to inhibit IgE specific to a model compound, dansyl. We show that cHBI conjugated specifically to its target Ab and inhibited degranulation in cellular degranulation assays using rat basophil leukemia cells. Furthermore, cHBIs demonstrated in vivo inhibition of allergic responses in both murine models. We establish the cHBI design to be a versatile platform for inhibiting hapten/IgE interactions, which can potentially be applied to inhibit IgE-mediated allergic reactions to any drug/small-molecule allergy.

Nanoallergens: A multivalent platform for studying and evaluating potency of allergen epitopes in cellular degranulation.

Degranulation caused by type I hypersensitivity (allergies) is a complex biophysical process, and available experimental models for studying relevant immunoglobulin E binding epitopes on allergen proteins lack the ability to adequately evaluate, rank, and associate these epitopes individually and with each other. In this study, we propose a new allergy model system for studying potential allergen epitopes using nanoallergens, liposomes modified to effectively display IgE binding epitopes/haptens. By utilizing the covalently conjugated lipid tails on two hapten molecules (dinitrophenol and dansyl), hapten molecules were successfully incorporated into liposomes with high precision to form nanoallergens. Nanoallergens, with precisely controlled high-particle valency, can trigger degranulation with much greater sensitivity than commonly used bovine serum albumin conjugates. In rat basophil leukemia cell experiments, nanoallergens with only 2% hapten loading were able to trigger degranulation in vitro at concentrations as low as 10 pM. Additionally, unlike bovine serum albumin-hapten conjugates, nanoallergens allow exact control over particle size and valency. By varying the nanoallergen parameters such as size, valency, monovalent affinity of hapten, and specific IgE ratios, we exposed the importance of these variables on degranulation intensity while demonstrating nanoallergens' potential for evaluating both high- and low-affinity epitopes. The data presented in this article establish nanoallergen platform as a reliable and versatile allergy model to study and evaluate allergen epitopes in mast cell degranulation.

Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.