Immunoengineering approaches for cytokine therapy.

Since the discovery of cytokines, much effort has been put forth to achieve therapeutic translation for treatment of various diseases, including cancer and autoimmune diseases. Despite these efforts, very few cytokines have cleared regulatory approval, and those that were approved are not commonly used due to their challenging toxicity profile and/or limited therapeutic efficacy. The main limitation in translation has been that wild-type cytokines have unfavorable pharmacokinetic and pharmacodynamic profiles, either eliciting unwanted systemic side effects or insufficient residence in secondary lymphoid organs. In this review, we address protein-engineering approaches that have been applied to both proinflammatory and anti-inflammatory cytokines to enhance their therapeutic indices, and we highlight diseases in which administration of engineered cytokines is especially relevant.

Entropic Control of Receptor Recycling Using Engineered Ligands.

Receptor internalization by endocytosis regulates diverse cellular processes, from the rate of nutrient uptake to the timescale of essential signaling events. The established view is that internalization is tightly controlled by specific protein-binding interactions. However, recent work suggests that physical aspects of receptors influence the process in ways that cannot be explained by biochemistry alone. Specifically, work from several groups suggests that increasing the steric bulk of receptors may inhibit their uptake by multiple types of trafficking vesicles. How do biochemical and biophysical factors work together to control internalization? Here, we show that receptor uptake is well described by a thermodynamic trade-off between receptor-vesicle binding energy and the entropic cost of confining receptors within endocytic vesicles. Specifically, using large ligands to acutely increase the size of engineered variants of the transferrin receptor, we demonstrate that an increase in the steric bulk of a receptor dramatically decreases its probability of uptake by clathrin-coated structures. Further, in agreement with a simple thermodynamic analysis, all data collapse onto a single trend relating fractional occupancy of the endocytic structure to fractional occupancy of the surrounding plasma membrane, independent of receptor size. This fundamental scaling law provides a simple tool for predicting the impact of receptor expression level, steric bulk, and the size of endocytic structures on receptor uptake. More broadly, this work suggests that bulky ligands could be used to drive the accumulation of specific receptors at the plasma membrane surface, providing a biophysical tool for targeted modulation of signaling and metabolism from outside the cell.

Cysteine-binding adjuvant enhances survival and promotes immune function in a murine model of acute myeloid leukemia.

ABSTRACT: Therapeutic vaccination has long been a promising avenue for cancer immunotherapy but is often limited by tumor heterogeneity. The genetic and molecular diversity between patients often results in variation in the antigens present on cancer cell surfaces. As a result, recent research has focused on personalized cancer vaccines. Although promising, this strategy suffers from time-consuming production, high cost, inaccessibility, and targeting of a limited number of tumor antigens. Instead, we explore an antigen-agnostic polymeric in situ cancer vaccination platform for treating blood malignancies, in our model here with acute myeloid leukemia (AML). Rather than immunizing against specific antigens or targeting adjuvant to specific cell-surface markers, this platform leverages a characteristic metabolic and enzymatic dysregulation in cancer cells that produces an excess of free cysteine thiols on their surfaces. These thiols increase in abundance after treatment with cytotoxic agents such as cytarabine, the current standard of care in AML. The resulting free thiols can undergo efficient disulfide exchange with pyridyl disulfide (PDS) moieties on our construct and allow for in situ covalent attachment to cancer cell surfaces and debris. PDS-functionalized monomers are incorporated into a statistical copolymer with pendant mannose groups and TLR7 agonists to target covalently linked antigen and adjuvant to antigen-presenting cells in the liver and spleen after IV administration. There, the compound initiates an anticancer immune response, including T-cell activation and antibody generation, ultimately prolonging survival in cancer-bearing mice.

Synthetically mannosylated antigens induce antigen-specific humoral tolerance and reduce anti-drug antibody responses to immunogenic biologics.

Immunogenic biologics trigger an anti-drug antibody (ADA) response in patients that reduces efficacy and increases adverse reactions. Our laboratory has shown that targeting protein antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a strategy to increase delivery of otherwise immunogenic biologics to the liver via conjugation to a synthetic mannose polymer, p(Man). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production, which is maintained throughout subsequent administrations of the native biologic. We find that p(Man)-antigen treatment impairs the ADA response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten immunodominance or control by T regulatory cells. We identify increased T cell receptor signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment via transcriptomic analyses. This modular platform may enhance tolerance to biologics, enabling long-term solutions for an ever-increasing healthcare problem.

A serine-conjugated butyrate prodrug with high oral bioavailability suppresses autoimmune arthritis and neuroinflammation in mice.

Butyrate-a metabolite produced by commensal bacteria-has been extensively studied for its immunomodulatory effects on immune cells, including regulatory T cells, macrophages and dendritic cells. However, the development of butyrate as a drug has been hindered by butyrate's poor oral bioavailability, owing to its rapid metabolism in the gut, its low potency (hence, necessitating high dosing), and its foul smell and taste. Here we report that the oral bioavailability of butyrate can be increased by esterifying it to serine, an amino acid transporter that aids the escape of the resulting odourless and tasteless prodrug (O-butyryl-L-serine, which we named SerBut) from the gut, enhancing its systemic uptake. In mice with collagen-antibody-induced arthritis (a model of rheumatoid arthritis) and with experimental autoimmune encephalomyelitis (a model of multiple sclerosis), we show that SerBut substantially ameliorated disease severity, modulated key immune cell populations systemically and in disease-associated tissues, and reduced inflammatory responses without compromising the global immune response to vaccination. SerBut may become a promising therapeutic for autoimmune and inflammatory diseases.

Membrane-localized neoantigens predict the efficacy of cancer immunotherapy.

Immune checkpoint immunotherapy (ICI) can re-activate immune reactions against neoantigens, leading to remarkable remission in cancer patients. Nevertheless, only a minority of patients are responsive to ICI, and approaches for prediction of responsiveness are needed to improve the success of cancer treatments. While the tumor mutational burden (TMB) correlates positively with responsiveness and survival of patients undergoing ICI, the influence of the subcellular localizations of the neoantigens remains unclear. Here, we demonstrate in both a mouse melanoma model and human clinical datasets of 1,722 ICI-treated patients that a high proportion of membrane-localized neoantigens, particularly at the plasma membrane, correlate with responsiveness to ICI therapy and improved overall survival across multiple cancer types. We further show that combining membrane localization and TMB analyses can enhance the predictability of cancer patient response to ICI. Our results may have important implications for establishing future clinical guidelines to direct the choice of treatment toward ICI.

Topically-applied collagen-binding serum albumin-fused interleukin-4 modulates wound microenvironment in non-healing wounds.

Non-healing wounds have a negative impact on quality of life and account for many cases of amputation and even early death among patients. Diabetic patients are the predominate population affected by these non-healing wounds. Despite the significant clinical demand, treatment with biologics has not broadly impacted clinical care. Interleukin-4 (IL-4) is a potent modulator of the immune system, capable of skewing macrophages towards a pro-regeneration phenotype (M2) and promoting angiogenesis, but can be toxic after frequent administration and is limited by its short half-life and low bioavailability. Here, we demonstrate the design and characterization of an engineered recombinant interleukin-4 construct. We utilize this collagen-binding, serum albumin-fused IL-4 variant (CBD-SA-IL-4) delivered in a hyaluronic acid (HA)-based gel for localized application of IL-4 to dermal wounds in a type 2 diabetic mouse model known for poor healing as proof-of-concept for improved tissue repair. Our studies indicate that CBD-SA-IL-4 is retained within the wound and can modulate the wound microenvironment through induction of M2 macrophages and angiogenesis. CBD-SA-IL-4 treatment significantly accelerated wound healing compared to native IL-4 and HA vehicle treatment without inducing systemic side effects. This CBD-SA-IL-4 construct can address the underlying immune dysfunction present in the non-healing wound, leading to more effective tissue healing in the clinic.

Synthetically mannosylated antigens induce antigen-specific humoral tolerance and reduce anti-drug antibody responses to immunogenic biologics.

Immunogenic biologics trigger an anti-drug antibody (ADA) response in patients, which reduces efficacy and increases adverse reactions. Our laboratory has previously shown that targeting protein antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a strategy to increase delivery of otherwise immunogenic biologics to the liver via conjugation to a synthetic mannose polymer (p(Man)). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production, which is maintained throughout subsequent administrations of the native biologic. We found that p(Man)-antigen treatment impairs the ADA response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten immunodominance or control by Tregs. We identify increased TCR signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment via transcriptomic analyses. This modular platform may enhance tolerance to biologics, enabling long-term solutions for an ever-increasing healthcare problem.

Engineered IL-7 synergizes with IL-12 immunotherapy to prevent T cell exhaustion and promote memory without exacerbating toxicity.

Cancer immunotherapy is moving toward combination regimens with agents of complementary mechanisms of action to achieve more frequent and robust efficacy. However, compared with single-agent therapies, combination immunotherapies are associated with increased overall toxicity because the very same mechanisms also work in concert to enhance systemic inflammation and promote off-tumor toxicity. Therefore, rational design of combination regimens that achieve improved antitumor control without exacerbated toxicity is a main objective in combination immunotherapy. Here, we show that the combination of engineered, tumor matrix-binding interleukin-7 (IL-7) and IL-12 achieves remarkable anticancer effects by activating complementary pathways without inducing any additive immunotoxicity. Mechanistically, engineered IL-12 provided effector properties to T cells, while IL-7 prevented their exhaustion and boosted memory formation as assessed by tumor rechallenge experiments. The dual combination also rendered checkpoint inhibitor (CPI)-resistant genetically engineered melanoma model responsive to CPI. Thus, our approach provides a framework of evaluation of rationally designed combinations in immuno-oncology and yields a promising therapy.

Synthetically glycosylated antigens for the antigen-specific suppression of established immune responses.

Inducing antigen-specific tolerance during an established immune response typically requires non-specific immunosuppressive signalling molecules. Hence, standard treatments for autoimmunity trigger global immunosuppression. Here we show that established antigen-specific responses in effector T cells and memory T cells can be suppressed by a polymer glycosylated with N-acetylgalactosamine (pGal) and conjugated to the antigen via a self-immolative linker that allows for the dissociation of the antigen on endocytosis and its presentation in the immunoregulatory environment. We show that pGal-antigen therapy induces antigen-specific tolerance in a mouse model of experimental autoimmune encephalomyelitis (with programmed cell-death-1 and the co-inhibitory ligand CD276 driving the tolerogenic responses), as well as the suppression of antigen-specific responses to vaccination against a DNA-based simian immunodeficiency virus in non-human primates. Our findings show that pGal-antigen therapy invokes mechanisms of immune tolerance to resolve antigen-specific inflammatory T-cell responses and suggest that the therapy may be applicable across autoimmune diseases.

Masking the immunotoxicity of interleukin-12 by fusing it with a domain of its receptor via a tumour-protease-cleavable linker.

Immune-checkpoint inhibitors have shown modest efficacy against immunologically 'cold' tumours. Interleukin-12 (IL-12)-a cytokine that promotes the recruitment of immune cells into tumours as well as immune cell activation, also in cold tumours-can cause severe immune-related adverse events in patients. Here, by exploiting the preferential overexpression of proteases in tumours, we show that fusing a domain of the IL-12 receptor to IL-12 via a linker cleavable by tumour-associated proteases largely restricts the pro-inflammatory effects of IL-12 to tumour sites. In mouse models of subcutaneous adenocarcinoma and orthotopic melanoma, masked IL-12 delivered intravenously did not cause systemic IL-12 signalling and eliminated systemic immune-related adverse events, led to potent therapeutic effects via the remodelling of the immune-suppressive microenvironment, and rendered cold tumours responsive to immune-checkpoint inhibition. We also show that masked IL-12 is activated in tumour lysates from patients. Protease-sensitive masking of potent yet toxic cytokines may facilitate their clinical translation.

Tumor Cell-Surface Binding of Immune Stimulating Polymeric Glyco-Adjuvant via Cysteine-Reactive Pyridyl Disulfide Promotes Antitumor Immunity.

Immune stimulating agents like Toll-like receptor 7 (TLR7) agonists induce potent antitumor immunity but are limited in their therapeutic window due to off-target immune activation. Here, we developed a polymeric delivery platform that binds excess unpaired cysteines on tumor cell surfaces and debris to adjuvant tumor neoantigens as an in situ vaccine. The metabolic and enzymatic dysregulation in the tumor microenvironment produces these exofacial free thiols, which can undergo efficient disulfide exchange with thiol-reactive pyridyl disulfide moieties upon intratumoral injection. These functional monomers are incorporated into a copolymer with pendant mannose groups and TLR7 agonists to target both antigen and adjuvant to antigen presenting cells. When tethered in the tumor, the polymeric glyco-adjuvant induces a robust antitumor response and prolongs survival of tumor-bearing mice, including in checkpoint-resistant B16F10 melanoma. The construct additionally reduces systemic toxicity associated with clinically relevant small molecule TLR7 agonists.

Suppression of Rheumatoid Arthritis by Enhanced Lymph Node Trafficking of Engineered Interleukin-10 in Murine Models.

OBJECTIVE: Rheumatoid arthritis (RA) is a major autoimmune disease that causes synovitis and joint damage. Although clinical trials have been performed using interleukin-10 (IL-10), an antiinflammatory cytokine, as a potential treatment of RA, the therapeutic effects of IL-10 have been limited, potentially due to insufficient residence in lymphoid organs, where antigen recognition primarily occurs. This study was undertaken to engineer an IL-10-serum albumin (SA) fusion protein and evaluate its effects in 2 murine models of RA. METHODS: SA-fused IL-10 (SA-IL-10) was recombinantly expressed. Mice with collagen antibody-induced arthritis (n = 4-7 per group) or collagen-induced arthritis (n = 9-15 per group) were injected intravenously with wild-type IL-10 or SA-IL-10, and the retention of SA-IL-10 in the lymph nodes (LNs), immune cell composition in the paws, and therapeutic effect of SA-IL-10 on mice with arthritis were assessed. RESULTS: SA fusion to IL-10 led to enhanced accumulation in the mouse LNs compared with unmodified IL-10. Intravenous SA-IL-10 treatment restored immune cell composition in the paws to a normal status, elevated the frequency of suppressive alternatively activated macrophages, reduced IL-17A levels in the paw-draining LN, and protected joint morphology. Intravenous SA-IL-10 treatment showed similar efficacy as treatment with an anti-tumor necrosis factor antibody. SA-IL-10 was equally effective when administered intravenously, locally, or subcutaneously, which is a benefit for clinical translation of this molecule. CONCLUSION: SA fusion to IL-10 is a simple but effective engineering strategy for RA therapy and has potential for clinical translation.

Prolonged residence of an albumin-IL-4 fusion protein in secondary lymphoid organs ameliorates experimental autoimmune encephalomyelitis.

Interleukin-4 (IL-4) suppresses the development of multiple sclerosis in a murine model of experimental autoimmune encephalomyelitis (EAE). Here, we show that, in mice with EAE, the accumulation and persistence in the lymph nodes and spleen of a systemically administered serum albumin (SA)-IL-4 fusion protein leads to higher efficacy in preventing disease development than the administration of wild-type IL-4 or of the clinically approved drug fingolimod. We also show that the SA-IL-4 fusion protein prevents immune-cell infiltration in the spinal cord, decreases integrin expression in antigen-specific CD4+ T cells, increases the number of granulocyte-like myeloid-derived suppressor cells (and their expression of programmed-death-ligand-1) in spinal cord-draining lymph nodes and decreases the number of T helper 17 cells, a pathogenic cell population in EAE. In mice with chronic EAE, SA-IL-4 inhibits immune-cell infiltration into the spinal cord and completely abrogates immune responses to myelin antigen in the spleen. The SA-IL-4 fusion protein may be prophylactically and therapeutically advantageous in the treatment of multiple sclerosis.

Generation of potent cellular and humoral immunity against SARS-CoV-2 antigens via conjugation to a polymeric glyco-adjuvant.

The SARS-CoV-2 virus has caused an unprecedented global crisis, and curtailing its spread requires an effective vaccine which elicits a diverse and robust immune response. We have previously shown that vaccines made of a polymeric glyco-adjuvant conjugated to an antigen were effective in triggering such a response in other disease models and hypothesized that the technology could be adapted to create an effective vaccine against SARS-CoV-2. The core of the vaccine platform is the copolymer p(Man-TLR7), composed of monomers with pendant mannose or a toll-like receptor 7 (TLR7) agonist. Thus, p(Man-TLR7) is designed to target relevant antigen-presenting cells (APCs) via mannose-binding receptors and then activate TLR7 upon endocytosis. The p(Man-TLR7) construct is amenable to conjugation to protein antigens such as the Spike protein of SARS-CoV-2, yielding Spike-p(Man-TLR7). Here, we demonstrate Spike-p(Man-TLR7) vaccination elicits robust antigen-specific cellular and humoral responses in mice. In adult and elderly wild-type mice, vaccination with Spike-p(Man-TLR7) generates high and long-lasting titers of anti-Spike IgGs, with neutralizing titers exceeding levels in convalescent human serum. Interestingly, adsorbing Spike-p(Man-TLR7) to the depot-forming adjuvant alum amplified the broadly neutralizing humoral responses to levels matching those in mice vaccinated with formulations based off of clinically-approved adjuvants. Additionally, we observed an increase in germinal center B cells, antigen-specific antibody secreting cells, activated T follicular helper cells, and polyfunctional Th1-cytokine producing CD4+ and CD8+ T cells. We conclude that Spike-p(Man-TLR7) is an attractive, next-generation subunit vaccine candidate, capable of inducing durable and robust antibody and T cell responses.

Polymersomes decorated with SARS-CoV-2 spike protein receptor binding domain elicit robust humoral and cellular immunity.

A diverse portfolio of SARS-CoV-2 vaccine candidates is needed to combat the evolving COVID-19 pandemic. Here, we developed a subunit nanovaccine by conjugating SARS-CoV-2 Spike protein receptor binding domain (RBD) to the surface of oxidation-sensitive polymersomes. We evaluated the humoral and cellular responses of mice immunized with these surface-decorated polymersomes (RBDsurf) compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl lipid A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that multivalent surface display of Spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

Polymersomes Decorated with the SARS-CoV-2 Spike Protein Receptor-Binding Domain Elicit Robust Humoral and Cellular Immunity.

The COVID-19 pandemic underscores the need for rapid, safe, and effective vaccines. In contrast to some traditional vaccines, nanoparticle-based subunit vaccines are particularly efficient in trafficking antigens to lymph nodes, where they induce potent immune cell activation. Here, we developed a strategy to decorate the surface of oxidation-sensitive polymersomes with multiple copies of the SARS-CoV-2 spike protein receptor-binding domain (RBD) to mimic the physical form of a virus particle. We evaluated the vaccination efficacy of these surface-decorated polymersomes (RBDsurf) in mice compared to RBD-encapsulated polymersomes (RBDencap) and unformulated RBD (RBDfree), using monophosphoryl-lipid-A-encapsulated polymersomes (MPLA PS) as an adjuvant. While all three groups produced high titers of RBD-specific IgG, only RBDsurf elicited a neutralizing antibody response to SARS-CoV-2 comparable to that of human convalescent plasma. Moreover, RBDsurf was the only group to significantly increase the proportion of RBD-specific germinal center B cells in the vaccination-site draining lymph nodes. Both RBDsurf and RBDencap drove similarly robust CD4+ and CD8+ T cell responses that produced multiple Th1-type cytokines. We conclude that a multivalent surface display of spike RBD on polymersomes promotes a potent neutralizing antibody response to SARS-CoV-2, while both antigen formulations promote robust T cell immunity.

Engineering Targeting Materials for Therapeutic Cancer Vaccines.

Therapeutic cancer vaccines constitute a valuable tool to educate the immune system to fight tumors and prevent cancer relapse. Nevertheless, the number of cancer vaccines in the clinic remains very limited to date, highlighting the need for further technology development. Recently, cancer vaccines have been improved by the use of materials, which can strongly enhance their intrinsic properties and biodistribution profile. Moreover, vaccine efficacy and safety can be substantially modulated through selection of the site at which they are delivered, which fosters the engineering of materials capable of targeting cancer vaccines to specific relevant sites, such as within the tumor or within lymphoid organs, to further optimize their immunotherapeutic effects. In this review, we aim to give the reader an overview of principles and current strategies to engineer therapeutic cancer vaccines, with a particular focus on the use of site-specific targeting materials. We will first recall the goal of therapeutic cancer vaccination and the type of immune responses sought upon vaccination, before detailing key components of cancer vaccines. We will then present how materials can be engineered to enhance the vaccine's pharmacokinetic and pharmacodynamic properties. Finally, we will discuss the rationale for site-specific targeting of cancer vaccines and provide examples of current targeting technologies.

Collagen-binding IL-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours.

Checkpoint-inhibitor (CPI) immunotherapy has achieved remarkable clinical success, yet its efficacy in 'immunologically cold' tumours has been modest. Interleukin-12 (IL-12) is a powerful cytokine that activates the innate and adaptive arms of the immune system; however, the administration of IL-12 has been associated with immune-related adverse events. Here we show that, after intravenous administration of a collagen-binding domain fused to IL-12 (CBD-IL-12) in mice bearing aggressive mouse tumours, CBD-IL-12 accumulates in the tumour stroma due to exposed collagen in the disordered tumour vasculature. In comparison with the administration of unmodified IL-12, CBD-IL-12 induced sustained intratumoural levels of interferon-γ, substantially reduced its systemic levels as well as organ damage and provided superior anticancer efficacy, eliciting complete regression of CPI-unresponsive breast tumours. Furthermore, CBD-IL-12 potently synergized with CPI to eradicate large established melanomas, induced antigen-specific immunological memory and controlled tumour growth in a genetically engineered mouse model of melanoma. CBD-IL-12 may potentiate CPI immunotherapy for immunologically cold tumours.

Publisher Correction: Prolonged residence of an albumin-IL-4 fusion protein in secondary lymphoid organs ameliorates experimental autoimmune encephalomyelitis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.