Engineering platforms for localized long-acting immune modulation

Abstract

Systemic immunotherapeutics have been a clinical staple in the treatment of cancer, infectious diseases, organ and cell transplantation, autoimmunity, and allergies. While their utility remains unquestioned, systemic administration of these drugs is associated with limited efficacy, significant adverse off-target effects, transient activity, and the requirement for frequent repeated dosing. To this end, recent technological advancements have provided novel means for sustained drug delivery to specific tissues and targeted localized approaches for immunotherapeutics. In this article, we present various cutting-edge platform technologies, including implants, multi-reservoir systems, and scaffolds encapsulating immunomodulatory agents for local administration. Examples of their application in cancer, cell transplantation, allergy and infectious diseases, are discussed, highlighting the potential of such systems for innovative immunomodulatory intervention.

Introduction

Immunomodulatory therapies engage immune-relevant targets to manage or treat a variety of diseases. Depending on the disease, such therapies can be conventional small molecule pharmaceutics or biological agents including nucleic acids, proteins, and cells(1). Immunomodulatory agents have demonstrated efficacy in clinical applications such as cancer, infectious diseases, transplantation and autoimmunity, and allergic diseases. However, transient efficacy and unfavorable physiological kinetics upon therapeutic administration poses challenges. Systemic delivery causes treatment dispersion, whereas efficacy hinges on the presence of high therapeutic concentrations and activity within target tissues or at the disease site(2). Due to transient activity or low therapeutic levels, repeated administration is typically required but not always feasible. Further, serious adverse reactions such as on-target side effects due to over-activation or -suppression of the immune system are impediments to successful disease management. Additionally, off-target toxicities attributable to treatment non-specificity further exacerbate safety concerns.

To this end, technological innovations that provide a targeted approach with longer disease management duration are poised to be the new frontier of immunomodulatory therapies. Long-acting approaches could reduce dosage, administration frequency, and toxicities, which in turn improves medication adherence, yielding a long-lasting preventative or curative effect. Of relevance, long-acting disease-targeting agents and platforms are clinically established for chronic conditions such as HIV(3), diabetes, and psychiatric illnesses(4). Considering the success of these approaches, we highlight a few platform technologies developed for long-acting immunomodulation to achieve immune control of cancer, infectious diseases, cell transplantation, and allergy, with a particular focus on localized approaches.

Cancer and Infectious Diseases

Immunotherapy has transformed cancer treatment in an unprecedented manner. However, inherent or acquired immune heterogeneity leads to variable treatment responses. Immune evasion is a leading contributor to treatment failure. Thus, platforms that can educate the immune system to identify foreign, non-self-targets allows for eluding evasion. Pertinent to this, various technologies have been developed in the context of in situ cancer vaccines(5, 6), and their sphere of applicability is broad and extends to vaccines for infectious diseases. These typically rely on biodegradable in situ forming hydrogels, polymers, implants or nanoformulations, capable of sustained delivery of immunomodulators and antigens. In a pre-clinical setting, some of these platforms have been highly efficacious in various tumor models and infectious disease settings and hold much promise for clinical translation(7). However, ultimate clinical success hinges on multiple factors, including the practicality of the approach and patient acceptability amongst others. Of note, some of these systems may require frequent booster administration, which may be unfeasible and limit the clinical relevance of the technology.

To this end, various technologies have been developed, capable of addressing the need for long-term drug(8) and antigen delivery. Among them, the NanoLymph was designed as a localized immunomodulatory system for immune cell homing and/or reprogramming against specific targets(9). The NanoLymph is a 3D printable nanofluidic implant designed for subcutaneous deployment with dual refillable reservoirs, each for sustained immunomodulator elution and antigen or cell presentation. The small scale (~8 mm diameter, 2 mm thickness) “D-shaped” agnostic platform offers flexibility for use of a wide spectrum of immune-modulatory agents and antigens. When the reservoirs are each loaded with GM-CSF and resiquimod (a TLR 7/8 agonist), and peptide antigen, dendritic cells (DC) are recruited on site and activated as antigen presenting cells. Thereafter, the activated DC migrate to the lymph nodes to initiate an antigen-specific T cell response. The versatility in immunomodulators, antigens, and/or cells loaded will permit the orchestration of a targeted immune cell-mediated response across a range of chronic diseases, acting as an in situ therapeutic or prophylactic vaccine with clear applicability to cancer and infectious diseases. In the context of infectious diseases, various antigens can be adopted to generate immunity against specific pathogens. In the context of cancer, autologous whole tumor cell lysates are used as antigen source, facilitating the generation of an immune response directed towards the unique antigenic repertoire in the patient’s own tumor(10). Of note, the site of deployment may be crucial in generating a potent immune activation. In this context, peritumoral implantation may be more effective as compared to a distal subcutaneous site, albeit such placement would compromise the accessibility for antigen and adjuvant loading and refilling. The refillability aspect of the technology is advantageous for long-term treatment.

Other platforms including biodegradable polymers, nanoparticle systems, and microencapsulation have been designed to gradually release vaccine components (antigens and adjuvants) against various pathogens, ensuring sustained immunization(11, 12). As compared to traditional vaccines, these systems have shown enhanced immune response with fewer doses, which aspects are particularly relevant in resource-limited areas. However, challenges include limited stability and viability of vaccine components, precise control of release rates, and the need for extensive testing and development.

An alternative form of immunomodulation, adoptive T cell therapy, engineers T cells ex vivo to target tumor antigens in vivo. However, upon reinfusion, only a small fraction of the engineered T cells actually reaches the tumor, where they encounter difficulty penetrating the tumor mass(13). More importantly, the heterogeneity of tumor surface antigens increases as it proliferates, resulting in short-lived responses to adoptive T cell therapy. To address these challenges, an immune engineered biomaterials-based platform, termed synergistic in situ vaccination enhanced T cell depot (SIVET), was developed(14). The SIVET platform advances beyond previous in situ vaccination work centered solely on dendritic cells. Composed of an alginate-collagen hybrid cryogel, the SIVET allows intratumoral controlled release of adoptively transferred T cells and immunostimulants to attract antigen-presenting cells (APC). The T cell depot facilitates tumor debulking, where dying cancer cells serve as the antigen source. Simultaneous local release of GM-CSF or FLT3L and CpG stimulates continual APC recruitment and activation, respectively. This synergistic in situ vaccination approach enables protection against tumor antigen escape and long-term T cell activation, representing an innovative intervention for long-acting immunomodulation.

Cell Transplantation

Transplant patients rely on viable allografts to restore dysfunctional organs or tissues. This is the case for both cell as well as organ transplantation. However, host immune rejection remains a barrier to widespread clinical adoption. Graft rejection results in the destruction of transplanted tissues, triggered by even the slight mismatches in human leukocyte antigen (HLA) alleles. As such, systemic immunosuppressive regimens are commonly used to avoid rejection. Unfortunately, these agents are associated with severe adverse effects and increased risks of infections, neoplasms, and organ damage. To address this challenge, numerous approaches have been developed to achieve local immune modulation, sparing the body from the deleterious effect of life-long immune suppression(15). These include minimizing immunogenicity of transplanted cells via CRISPR-Cas9 genome editing, leveraging RNA therapeutics and cytokine delivery to induce immune tolerance, local co-transplantation with immunomodulatory cells such as T regs, Sertoli cells, mesenchymal stem cells (MSC), as well as local delivery of immunomodulatory agents(15). These approaches share the need for suitable technologies to generate an ideal site for cell transplantation that achieves cell retention while providing sustained modulation of the tissue immune microenvironment.

One such platform termed NICHE, is a dual reservoir 3D-printed nylon implant, designed for subdermal transplantation of pancreatic islets for the management of type 1 diabetes (T1D). In their native microenvironment, islets are densely vascularized via intraislet capillaries and obtain ~20% of the pancreatic blood supply. Upon implantation, the NICHE relies on the angiogenic properties of MSC to generate a dense blood vessel network capable of supplying oxygen, nutrients and rapid glucose and insulin exchange suitable for the long-term viability and function of pancreatic islets(16). Immune rejection is prevented via local sustained delivery of immunosuppressants such as thymoglobulin, CTLA4Ig, and Anti-CD40-L, which have the function of depleting or impeding activation of the co-stimulatory pathway for T cells, respectively. Notably, the NICHE allows for transcutaneous reloading of the drug reservoir, extending functionality long-term. In this context, the platform allows for the use of different agents (e.g. growth factors, immunoadjuvants, cytokines) alone or in combination, and their simultaneous or sequential delivery, to support transplanted cells during the phases of engraftment and local tissue remodeling. In an allogenic pancreatic islet transplantation model, in immunocompetent diabetic rats, the NICHE achieved T1D reversal with no sign of systemic immunosuppression or adverse effects throughout the 180 days of analysis(17).

Local immune modulation can also be achieved via co-delivery of immune adjuvant-releasing microparticles or gels with transplanted cells(18). As a notable example, FasL-modified microgels conferred long term immune protection to pancreatic islet allografts co-transplanted in non-human primates. Here, the Fas receptor/Fas ligand (FasL) pathway was leveraged to confer immune privilege and tolerance to self-antigens by inducing apoptosis in infiltrating lymphocytes and inflammatory cells(19).

These clinically translatable technologies could be adopted for the transplantation and delivery of cell therapeutics for clinical applications beyond T1D, including neurodegenerative and cardiovascular diseases and pathologies associated with hormone deficiencies. Their adaptability for various agents, flexibility in dosing timing and duration, as well as the ability to localize distribution renders these platforms significant in the development of novel immunomodulatory approaches for cell therapeutics. However, given their multi-component nature, the regulatory approval of these strategies will require complex translational efforts.

Allergy

Allergic disorders affect over one third of the population and remain a therapeutic challenge. Although allergen avoidance is an effective method of prevention, it is not always possible, and symptomatic medications may not be adequate. Over the past century, allergen immunotherapy (AIT) has evolved to be an effective antigen-specific treatment for IgE-mediated hypersensitivity disorders(20). The conventional AIT strategy for many aeroallergens or stinging insect allergens is the repeated subcutaneous injection of allergen extracts, and more recently with recombinant allergens, chemically altered allergens (allergoids), or T cell-targeted peptides. Sublingual immunotherapy (SLIT) is efficacious with some aeroallergens, and oral AIT is approved only for peanut allergy desensitization. Epicutaneous, microneedle patch, mRNA vaccine, or intralymphatic immunotherapy strategies are under investigation, and a growing number of additional innovative strategies to improve AIT remain exploratory. These include combining allergen with immune modulating TLR agonists, monoclonal antibodies against type 2 cytokines or cytokine receptors.

The goal of AIT is to induce antigen-specific tolerance (20). Mechanistically, AIT-induced tolerance is manifested by induction of regulatory dendritic cells (DCreg), B cells (Breg), and T cells (Treg) which produce IL-10 and TGFb and are capable of inhibiting type 2 helper T cells (Th2) and type 2 innate lymphoid cells (ILC2) hence reducing type 2 cytokines and inducing IgG blocking antibodies that inhibit the binding of IgE to allergen. Efforts to enhance the tolerance-inducing efficacy and safety of AIT are exploring novel long-acting immunomodulatory delivery platforms such as nanoparticles, microneedle patches(21), and programmable subcutaneous implants for delivery of allergen and immunomodulatory agents.

Nanoparticle formulations can be optimized for a variety of physical and chemical factors including size, pH, antigen loading, and optimal cellular targeting and uptake(22). Importantly, nanoparticle platforms offer the potential for topical, oral, or systemic administration. Murine studies with allergen-loaded nanoparticles support the potential for inducing allergen-specific tolerance (23). Microneedle patches can be engineered to potentially improve upon passive epicutaneous allergen delivery to antigen presenting cells in the skin, thereby enhancing the tolerance-inducing efficacy of epicutaneous AIT. Murine studies with peanut allergen support the potential for this platform to induce antigen-specific tolerance (24).

There is also potential for implantable microchambers, such as the programmable 3D-printed NanoLymph platform noted above for potential cancer immunotherapy applications (3), to be exploited for enhanced AIT. Such an approach would entail subcutaneous implantation of the NanoLymph in which one chamber is allergen-loaded and one chamber is cytokine-loaded with GM-CSF to promote migration and activation of dendritic cells and with IL-10 and TGFb to promote differentiation of allergen-specific Treg and allergen-specific tolerance.

Overall, the convergence of biologic and engineering strategies is leading to highly innovative approaches for AIT. Such interdisciplinary collaborations are likely to yield novel technological advances for treating allergic disorders in the near future.

Conclusion

In conclusion, innovations in drug and antigen-delivery technologies have broadened the spectrum of applicability of immunomodulation for the prevention, management and treatment of diseases. In this context, long-acting and tunable immunomodulatory approaches can extend disease management duration while simplifying dosing regimen and improving quality of lives. As evident in the field of cancer immunotherapy, cell transplantation and allergy management, the success of long-acting immunomodulatory platforms have the potential to have a long-lasting, transformative impact in clinical care. Notably, these technologies could enhance cost-effectiveness in medical treatments and contribute to healthcare equity. Enhanced efficacy, reduced drug doses and lower frequency of administration could not only lower direct treatment costs but also decrease associated healthcare expenses, such as those related to administration and patient care.

Figure 1.

Localized long-acting immunomodulatory platforms to control immune responses for long-term disease management and treatment. These platforms include implants, multi-reservoir systems, or scaffolds containing immunomodulatory agents and/or antigens. Examples of therapeutic applications of localized immunomodulatory platforms include A) antitumor immunity, B) cell graft tolerance and C) allergy tolerance. A) For cancer and infectious diseases, the goal is to generate and sustain antitumor immune responses, primarily through the activation of dendritic cells, CD8 cytotoxic T cells and M1 macrophages with the suppression of Tregs and M2 macrophages. B) In cell transplantation, localized immunomodulation creates an immunosuppressive microenvironment inclusive of tolerogenic dendritic cells (TolDC), Tregs, Bregs and M2 macrophages, along with suppression of CD8 T cells and M1 macrophages to protect against graft rejection. C) For allergy treatment, localized immunomodulatory platforms could yield allergen-specific tolerance through the continuous suppression of Type 2 allergic inflammation (via increase in Th1 CD4 T cells, Bregs, and Tregs, B cell isotype switch from IgE to IgG and IgA). Figure created with Biorender.com.

Abbreviations:

AIT

Allergen immunotherapy

APC

Antigen-presenting cells

Bregs

Regulatory B cells

Cas9

CRISPR associated protein 9

CRISPR

Clustered regularly interspaced short palindromic repeats

CTLA4Ig

Cytotoxic T lymphocyte-associated antigen-4-Ig

DC

Dendritic cells

FLT3L

FMS-like tyrosine kinase 3 ligand

GM-CSF

Granulocyte-macrophage colony-stimulating factor

HIV

Human immunodeficiency virus

HLA

Human leukocyte antigen

IgG

Immunoglobulin G

ILC2

Type 2 innate lymphoid cells

mRNA

Messenger RNA

MSC

Mesenchymal stromal cell

RNA

Ribonucleic acid

SIVET

In situ vaccination enhanced T cell depot

SLIT

Sublingual immunotherapy

T1D

Type 1 diabetes

TGFb

Transforming growth factor beta

Th2

Type 2 helper T cells

TLR

Toll-like receptor

TolDC

Tolerogenic dendritic cells

Tregs

Regulatory T cells