Glatiramer acetate enhances tumor retention and innate activation of immunostimulants.

Cancer immunotherapy aims to stimulate immune cells to recognize and attack tumor tissue. The immunostimulatory polyanions polyI:C and CpG induce potent pro-inflammatory immune responses as TLR3 and TLR9 agonists, respectively. Clinical trials of TLR agonists, however, have been fraught with immune-related adverse events, even when injecting intratumorally in an effort to minimize systemic exposure. We identified Glatiramer Acetate (GA), a positively-charged polypeptide approved for multiple sclerosis, as a delivery agent capable of complexing with polyI:C or CpG and reducing the mobility of these actives. Small nanoparticles termed polyplexes form when mixing positively-charged GA and negatively-charged immunostimulant (polyI:C or CpG). The ratio of GA to immunostimulant directly affected the potency of TLR activation and the mobility of these actives in simulated tumor tissue. Polyplexes of GA and CpG were injected intratumorally in a tumor model of head and neck cancer (HNC) and significantly mitigated tumor growth as compared to the vehicle controls. Intratumoral injections of CpG showed the slowest tumor growth but exhibited dramatically higher systemic proinflammatory cytokine levels compared to polyplexes of GA with CpG. Sequencing of RNA from resected tumors revealed a similar pattern of upregulated proinflammatory cytokines for CpG and polyplexes, a finding supported by histological tumor staining showing similar infiltration of immune cells induced by these treatments. Intratumoral administration of polyplexes of GA with immunostimulant represents a translational approach to enhance local immune responses while mitigating systemic immune-related adverse events.

Human intratumoral therapy: Linking drug properties and tumor transport of drugs in clinical trials.

Cancer therapies aim to kill tumor cells directly or engage the immune system to fight malignancy. Checkpoint inhibitors, oncolytic viruses, cell-based immunotherapies, cytokines, and adjuvants have been applied to prompt the immune system to recognize and attack cancer cells. However, systemic exposure of cancer therapies can induce unwanted adverse events. Intratumoral administration of potent therapies utilizes small amounts of drugs, in an effort to minimize systemic exposure and off-target toxicities. Here, we discuss the properties of the tumor microenvironment and transport considerations for intratumoral drug delivery. Specifically, we consider various tumor tissue factors and physicochemical factors that can affect tumor retention after intratumoral injection. We also review approved and clinical-stage intratumoral therapies and consider how the molecular and biophysical properties (e.g. size and charge) of these therapies influences intratumoral transport (e.g. tumor retention and cellular uptake). Finally, we offer a critical review and highlight several emerging approaches to promote tumor retention and limit systemic exposure of potent intratumoral therapies.

Immunostimulant Complexed With Polylysine Limits Transport and Maintains Immune Cell Activation.

Activation of the immune system to treat cancer has emerged as a powerful therapy tool, however, treatments must overcome the immunosuppressive microenvironment established by tumors. Toll-like receptor (TLR) agonists like CpG and polyI:C are potent stimulators of non-specific, pro-inflammatory immune responses, targeting TLR9 and TLR3, respectively. While these immunostimulants seem promising, systemic exposure can eventually induce severe side effects. Adverse inflammatory reactions in healthy tissues may be avoided by delivering and retaining immunostimulants in proximity to tumors or to primary sites of tumor metastases. Immunostimulants such as CpG and polyI:C cannot be completely immobilized, however, since the target TLR9 and TLR3 are located intracellularly. Previously, polycations like poly-l-lysine (PLL) have been complexed to the anionic CpG or polyI:C with the purpose of improving intracellular delivery and potency. Here, the relationship between PLL molecular weight and immunostimulant complexation, TLR activation, and transport in a simple, model tumor microenvironment was investigated. The polyplexes could be formulated to dramatically limit immunostimulant transport suggesting the potential for injection site retention and minimized systemic exposure of immunostimulants. The molecular weight of PLL and ratio of PLL to immunostimulant affected the accessibility of the immunostimulant within the polyplex and polyplex interaction strength.

Adjuvants as Delivery Systems in Antigen-Specific Immunotherapies.

Combining autoantigens with immune-modulating drugs has emerged as an attractive approach to selectively reinstate tolerance in autoimmune diseases. The disparate properties of autoantigens and small-molecule immunosuppressants commonly used to treat autoimmune diseases can confound efforts to co-deliver these therapies. However, both components may be co-delivered with adjuvants which have been successful in delivering antigens to immune cells. We evaluated several common adjuvants as vehicles to co-deliver a model antigen and immunosuppressant, ovalbumin (OVA) and dexamethasone (DEX), respectively. Formulations were developed, and the release of DEX from adjuvants was investigated. Next, the effect of adjuvant, DEX, and OVA was tested in vitro using a DC line. A MF59-analog (MF59a) formulation was advanced to more sophisticated co-culture studies using OVA-primed bone marrow-derived dendritic cells and splenocytes or T-cells from OT-II mice. Most of these studies indicated MF59a-based antigen-specific immunotherapies could diminish the markers of inflammation associated with OVA recognition. We rationalized MF59a co-delivery of antigen and drug could reduce the risk of side effects typically associated with these drugs and reinstate immune tolerance, thus prompting continued investigation of emulsion adjuvants as delivery vehicles for antigen-specific immunotherapy of autoimmune diseases.

Antigen-Drug Conjugates as a Novel Therapeutic Class for the Treatment of Antigen-Specific Autoimmune Disorders.

Multiple sclerosis represents the world's most common cause of neurological disability in young people and is attributed to a loss of immune tolerance toward proteins of the myelin sheath. Typical treatment options for MS patients involve immunomodulatory drugs, which act nonspecifically, resulting in global immunosuppression. The study discussed herein aims to demonstrate the efficacy of antigen-specific immunotherapies involving the conjugation of disease causing autoantigen, PLP139-151, and a potent immunosuppressant, dexamethasone. Antigen-drug conjugates (AgDCs) were formed using copper-catalyzed azide-alkyne cycloaddition chemistry with the inclusion of a hydrolyzable linker to maintain the activity of released dexamethasone. Subcutaneous administration of this antigen-drug conjugates to SJL mice induced with experimental autoimmune encephalomyelitis, protected the mice from a symptom onset throughout the 25 day study, demonstrating enhanced efficacy in comparison to dexamethasone treatment. These results highlight the benefits of co-delivery of autoantigens with immunosuppressant drugs as AgDCs for the treatment of autoimmune diseases.

Tocopherol Emulsions as Functional Autoantigen Delivery Vehicles Evoke Therapeutic Efficacy in Experimental Autoimmune Encephalomyelitis.

Contemporary approaches to treating autoimmune diseases like multiple sclerosis broadly modulate the immune system and leave patients susceptible to severe adverse effects. Antigen-specific immunotherapies (ASIT) offer a unique opportunity to selectively suppress autoreactive cell populations but have suffered from marginal efficacy even when employing traditional adjuvants to improve delivery. The development of immunologically active antigen delivery vehicles could potentially increase the clinical success of antigen-specific immunotherapies. An emulsion of the antioxidant tocopherol delivering an epitope of proteolipid protein autoantigen (PLP139-151) yielded significant efficacy in mice with experimental autoimmune encephalomyelitis (EAE). In vitro studies indicated tocopherol emulsions reduced oxidative stress in antigen-presenting cells. Ex vivo analysis revealed that tocopherol emulsions shifted cytokine responses in EAE splenocytes. In addition, IgG responses against PLP139-151 were increased in mice treated with tocopherol emulsions delivering the antigen, suggesting a possible skew in immunity. Overall, tocopherol emulsions provide a functional delivery vehicle for ASIT capable of ameliorating autoimmunity in a murine model.

Practical Considerations, Challenges, and Limitations of Bioconjugation via Azide-Alkyne Cycloaddition.

Interrogating biological systems is often limited by access to biological probes. The emergence of "click chemistry" has revolutionized bioconjugate chemistry by providing facile reaction conditions amenable to both biologic molecules and small molecule probes such as fluorophores, toxins, or therapeutics. One particularly popular version is the copper-catalyzed azide-alkyne cycloaddition (AAC) reaction, which has spawned new alternatives such as the strain-promoted azide-alkyne cycloaddition reaction, among others. This focused review highlights practical approaches to AAC reactions for the synthesis of peptide or protein bioconjugates and contrasts current challenges and limitations in light of recent advances in the field. The conical success of antibody drug conjugates has expanded the toolbox of linkers and payloads to facilitate practical applications of bioconjugation to create novel therapeutics and biologic probes. The AAC reaction in particular is poised to enable a large set of functionalized molecules as a combinatorial approach to high-throughput bioconjugate generation, screening, and honing of lead compounds.