Aerosol pulmonary immune engineering

Abstract

Aerosolization of immunotherapies poses incredible potential for manipulating the local mucosal-specific microenvironment, engaging specialized pulmonary cellular defenders, and accessing mucosal associated lymphoid tissue to redirect systemic adaptive and memory responses. In this review, we breakdown key inhalable immunoengineering strategies for chronic, genetic, and infection-based inflammatory pulmonary disorders, encompassing the historic use of immunomodulatory agents, the transition to biological inspired or derived treatments, and novel approaches of complexing these materials into drug delivery vehicles for enhanced release outcomes. Alongside a brief description of key immune targets, fundamentals of aerosol drug delivery, and preclinical pulmonary models for immune response, we survey recent advances of inhaled immunotherapy platforms, ranging from small molecules and biologics to particulates and cell therapies, as well as prophylactic vaccines. In each section, we address the formulation design constraints for aerosol delivery as well as advantages for each platform in driving desirable immune modifications. Finally, prospects of clinical translation and outlook for inhaled immune engineering are discussed.

Keywords: Asthma; Biologics; COPD; COVID-19; Cancer; Cystic fibrosis; Immune engineering; Immunotherapy; Particulates; Pulmonary delivery; Small molecules.

Figure 1:

Engineered delivery to the pulmonary immune microenvironment. The lung is divided into three major zones: Upper, Lower, and Alveolar regions. These regions host distinct cellular populations critical to induce immune responses via activation of lymphoid resident T and B cells. Delivery of therapeutics to these regions is limited primarily to aerosol aerodynamic size ranging from >10 μm to ~1 μm. Mac. = macrophage, DCs = dendritic cells, ILCs = innate lymphoid cells, Ig = Immunoglobulin. Figure created with BioRender.

Figure 2:

Inhalable immunotherapeutic workflow. Current immunotherapy agents can be formulated into an aerosol delivery platform with proper consideration and testing. First, inhalable immunotherapeutic vehicles can be administered as dry powder formulations via dry powder inhalers (DPI) or as liquid formulations using a nebulizer or metered dose inhaler (MDI). Aerosol deposition in the lung through these devices can then be predicted in vitro using equipment such as the cascade impactors and in silico using computational fluid-particle dynamics (CFPD). The biological activity of reagents in the pulmonary environment can then be tested in vitro using advanced models such as realistic air-liquid interface (ALI) cultures or lung on chip microfluidic devices. Metrics of immunity can be characterized via methods such as flow cytometry, immunoassays, tissue histology, and genetic sequencing experiments. Further preclinical trials using murine models (C57BL/6 or BALB/c) through to large animal studies evaluating the in vivo efficacy are required before the formulation can undergo clinical trials. Figure created with BioRender.

Figure 3.

Compilation of relevant examples of Inhaled Biologies. A) Percent of initial IFN doses (5 μg/kg in 1 mL and 0.15 mL of sterile saline for i.v and i.t delivery respectively) in lung tissue homogenate (Top) and BAL (Bottom) at various times. PEGylated IFN-α [PEG12-IFNα2b (31kDa) and PEG40-IFNα2a (60 kDa)] shows a significant increase bioavailability in the pulmonary tissue site when compared to naked IFN [158]. B) H&E stains of mice lungs and quantification of tumor lesion versus total lung area. Tumor suppression in mice models after i.t delivery of IL-10 loaded onto micro-particles [159]. C) Lung distribution of anti-EGFR mAbs, cetuximad-AF 750, after 10 mg/kg dosing through i.v or pulmonary route. Aerosolized mAbs show increased bioavailability in lung tumor site after administration [160]. D) Concentration of anti-VEGF mAbs, G6-31, in serum after single dose (10 mg/kg) administration by either pulmonary route or i.v route. Here, inhaled delivery of anti-VEGF mAbs minimizes systemic exposure [161]. Data reproduced with permission.

Figure 4:

Electron microscopy of selected inhaled immunotherapy formulations. A) Liquid and dry powder formulation of Advax-adjuvanted WIV (from top to bottom): I.) TEM images of Advax adjuvant, II.) SEM images of spray-freeze dried whole inactivated influenza A virus strain NIBRG 23, III.) SEM images of spray-freeze dried Advax-adjuvanted WIV. [239] B) SEM image of alginate-coated CS NP. Scale bar represents 500 nm [240]. C) TEM images of CS nanocomplex with aPD-L1. Scale bar represents 200 nm.[241] D) SEM image of aluminum particles loaded with OVA. Scale bar represents 1 μm[242]. Data reproduced with permission.

Figure 5:

Particulate-based pulmonary immunotherapies examples. A) Cytokine production in spleen increases with PLGA concentration in pulmonary delivery of Hepatitis B vaccine (HBsAg). NP-A(PLA based), NP-B (PLGA 85:15 based), NP-C (PLGA 50:50 based)[228]. B) Mice demonstrate synergistic reduction in lung metastasis following pulmonary delivery of NPs loaded with STING agonist (cGAMP) and fractionated radiation (IR). Mice lung metastasis were allowed to grow for 5 days before treatment and then results represent whole lungs from euthanized mice (n=6/group) on day 18 (top middle). Expression of OVA peptide SIINFEKL-MHC-I molecule Kb on CD103+ DCs in tumor-draining lymph nodes. Measurements on day 9 (top right).[286] C) Immune response after pulmonary delivery of CS nanocomplex with aPD-L1 demonstrated reduced lung metastasis in H&E stains (bottom left) and higher cytokine production compared to other controls (bottom right).[241] Data reproduced with permission.

Figure 6.

Compilation of relevant examples for Inhaled Prophylactic Vaccines. A) pDNA delivered through SAW nebulizer. Atomic force microscopy of plasmid before (a) and after (b) nebulization. Ethidium bromide agarose gel (c) and quantification (d) of any damage caused by SAW nebulization. Lanes 1 and 4 are controls, lanes 2 and 3 are nebulized at 30 Mhz, and lanes 3 and 6 and nebulized at 20 Mhz. Lanes 2 and 5 had a concentration of 85 μg/mL, and lanes 3 and 6 had a concentration of 50 μg/mL[315]. B) Antibody response after vaccination with inulin-stabilized influenza vaccine. Inhaled dry powder (p.i.) vaccination generated a more robust immune response than inhaled nebulized and i.m vaccination [316]. C) Immunization with VLP after 34 months of dry powder storage at room temperature. Mice were immunized using a VLP control (MS2), an experimental VLP (MS2-16L2) stored as dry powder for 34 months at room temperature and reconstituted in phosphate buffer, or fresh liquid MS2-15L2. Despite extensive room temperature storage, dry powder MS2-15L2 induced antibody production [317]. D) Saliva antibody levels of anionic liposome (AL)/CS/DNA vaccines after intranasal administration. Significant and lasting mucosal immunity was established with this complex compared to naked DNA group.[318] Data reproduced with permission.