Agonist Conjugate Research Articles

Self-Adjuvanting TLR7/8 Agonist and Fentanyl Hapten Co-Conjugate Achieves Enhanced Protection against Fentanyl Challenge.

Currently approved pharmacotherapies for opioid use disorders (OUDs) and overdose reversal agents are insufficient to slow the spread of OUDs due to the proliferation of fentanyl. This is evident in the 31% rise in drug overdose deaths from 2019 to 2022, with rates increasing from 21.6 to 28.3 overdoses per 100,000 deaths. Vaccines are a potential alternative or adjunct therapy for the treatment of several substance use disorders (nicotine, cocaine) but have shown limited clinical success due to suboptimal antibody titers. In this study, we demonstrate that coconjugation of a Toll-like receptor 7/8 (TLR7/8) agonist (UM-3006) alongside a fentanyl-based hapten (F1) on the surface of the carrier protein cross-reactive material 197 (CRM) significantly increased generation of high-affinity fentanyl-specific antibodies. This demonstrated enhanced protection against fentanyl challenges relative to an unconjugated (admix) adjuvant control in mice. Inclusion of aluminum hydroxide (alum) adjuvant further increased titers and enhanced protection, as determined by analysis of fentanyl concentration in serum and brain tissue. Collectively, our findings present a promising approach to enhance the efficacy of antiopioid vaccines, underscoring the need for extensive exploration of TLR7/8 agonist conjugates as a compelling strategy to combat opioid use disorders.

Abstract 1863: Evaluation of BDB101, a first-in-class TLR7/8 dual agonist conjugated to an anti-PD-L1 mAb, in cancer treatment

Abstract Background Responses to antiPDL1 checkpoint inhibitors (CPIs) in many tumors are governed by the PDL1 expression on tumor and immune cells in the tumor microenvironment (TME). Increasing PDL1 expression on cells may enhance the response to CPIs. Tolllike receptor (TLR) agonists have shown to induce PDL1 expression in the TME and may enhance the antitumor effects of CPIs. The effective dose of TLR agonist administered to patients is limited by systemic toxicities. To overcome this limitation, one approach is to conjugate the TLR agonist to an antibody. This allows for delivery and release of higher doses of the TLR agonist in the TME while limiting systemic exposure.BDB001 is an intravenously (IV) delivered TLR7/8 dual agonist that activates both the innate and adaptive immune system. In clinical trials, BDB001 has been well tolerated and shown promising antitumor responses in monotherapy and in combination with antiPDL1 therapy (Patel M. et al, SITC 2020 and SITC 2021). To further enhance the antitumor effect and allow for increased concentration of the TLR7/8 dual agonist in the TME, we developed BDB101, a proprietary firstinclass TLR7/8 dual agonist - antiPDL1 monoclonal antibody (mAb) conjugate using our TollLike receptor Agonist Conjugate (TLAC) platform. Here we report on the preclinical and nonhuman primate (NHP) evaluation of BDB101. Methods BDB101 utilizes a cleavable linker, allowing for the TLR7/8 dual agonist payload to be released in the TME and enabling a bystander effect. BDB101 was assessed for in vitro TLR7/8 dual agonist activity and PDL1 binding, and in vivo antitumor efficacy in immune competent mouse models. The pharmacokinetic and pharmacodynamic effects of BDB101 was evaluated in NHP. Results The conjugated TLR7/8 dual agonist did not change the PDL1 binding specificity of the antibody, and the uncleaved conjugate did not activate TLR7 or TLR8. In the C26 mouse model, a BDB101 mouse surrogate showed a significantly more robust antitumor effect compared to the TLR7/8 dual agonist or the antiPDL1 mAb alone. In NHP, a single IV infusion of BDB101 showed no significant differences in PK parameters between BDB101 and the total antiPDL1 mAb. BDB101 demonstrated remarkable in vitro stability in human plasma for up to 21 days. In NHP, BDB101 was also very stable with minimal release of free TLR7/8 dual agonist payload. Pharmacodynamic studies in NHP were consistent with PK behavior. Conclusions BDB101 exhibits robust preclinical efficacy and prolonged stability in NHP. The low rate of free TLR7/8 dual agonist payload released into the systemic circulation from the conjugated form of BDB101, should minimize the risk of systemic TLR7/8 dual agonist exposure, while maintaining the ability to deliver the payload to the TME. These results indicate that BDB101 will likely have a good in vivo therapeutic window and strongly support the clinical development of BDB101. Citation Format: Jing Yang, Robert H. Andtbacka, Walter Lau, Lixin Li. Evaluation of BDB101, a first-in-class TLR7/8 dual agonist conjugated to an anti-PD-L1 mAb, in cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1863.

Antibody conjugates for targeted delivery of Toll-like receptor 9 agonist to the tumor tissue.

Imiquimod, a Toll-like receptor 7 (TLR7) agonist is routinely used for topical administration in basal cell carcinoma and stage zero melanoma. Similarly, the TLR agonist Bacillus Calmette-Guérin is used for the local treatment of bladder cancer and clinical trials showed treatment efficacy of intratumoral injections with TLR9 agonists. However, when administered systemically, endosomal TLR agonists cause adverse responses due to broad immune activation. Hence, strategies for targeted delivery of TLR agonists to the tumor tissue are needed to enable the widespread use of endosomal TLR agonists in the context of tumor immunotherapy. One strategy for targeted delivery of TLR agonist is their conjugation to tumor antigen-specific therapeutic antibodies. Such antibody-TLR agonist conjugates act synergistically by inducing local TLR-mediated innate immune activation which complements the anti-tumor immune mechanisms induced by the therapeutic antibody. In this study, we explored different conjugation strategies for TLR9 agonists to immunoglobulin G (IgG). We evaluated biochemical conjugation of immunostimulatory CpG oligodesoxyribonucleotides (ODN) to the HER2-specific therapeutic antibody Trastuzumab with different cross-linkers comparing stochastic with site-specific conjugation. The physiochemical make-up and biological activities of the generated Trastuzumab-ODN conjugates were characterized in vitro and demonstrated that site-specific conjugation of CpG ODN is crucial for maintaining the antigen-binding capabilities of Trastuzumab. Furthermore, site-specific conjugate was effective in promoting anti-tumor immune responses in vivo in a pseudo-metastasis mouse model with engineered human HER2-transgenic tumor cells. In this in vivo model, co-delivery of Trastuzumab and CpG ODN in form of site-specific conjugates was superior to co-injection of unconjugated Trastuzumab, CpG ODN or stochastic conjugate in promoting T cell activation and expansion. Thereby, this study highlights that site-specific conjugation of CpG ODN to therapeutic antibodies targeting tumor markers is a feasible and more reliable approach for generation of conjugates which retain and combine the functional properties of the adjuvant and the antibody.

Self-Assembled TLR7/8 Agonist-Mannose Conjugate as An Effective Vaccine Adjuvant for SARS-CoV-2 RBD Trimer.

Small synthetic TLR7/8-agonists can be used as vaccine adjuvants to enhance cell and humoral-mediated immune responses to specific antigens. Despite their potency, after local injection they can be dispersed to undesired body parts causing high reactogenicity, limiting their clinical applications. Here we describe a vaccination strategy that employs the covalent conjugate of a mannose and TLR7/8 agonist as a vaccine adjuvant to take advantage of mannose binding C-type lectins on dendritic cells to enhance the vaccine's immunogenicity. The mannose-TLR7/8 agonist conjugate can self-assemble into nanoparticles with the hydrophilic mannose on the outside and hydrophobic TLR7/8 agonist inside. Although its ability to stimulate HEK-BlueTM hTLR7/8 cells dropped, it can efficiently stimulate mouse bone marrow-derived dendritic cells as indicated by the up-regulation of CD80 and CD86, and higher cytokine expression levels of TNF-α, IL6, and IL-12p70 than the native TLR7/8 agonist. In vivo, vaccination using the SARS-CoV-2 RBD trimer as the antigen and the conjugate as the adjuvant induced a significantly higher amount of IgG2a. These results suggest that the mannose-TLR7/8-agonist conjugate can be used as an effective vaccine adjuvant.

An Intelligent Nanovehicle Armed with Multifunctional Navigation for Precise Delivery of Toll-Like Receptor 7/8Agonist and Immunogenic Cell Death Amplifiers to Eliminate Solid Tumors and Trigger Durable Antitumor Immunity.

Cancer immunotherapy is revolutionary in oncology and hematology. However, a low response rate restricts the clinical benefits of this therapy owing to inadequate T lymphocyte infiltration and low delivery efficiency of immunotherapeutic drugs.Herein, an intelligent nanovehicle (folic acid (FA)/1-(4-(aminomethyl) benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (IMDQ)-oxaliplatin (F/IMO)@CuS) armed with multifunctional navigation is designed for the accuratedelivery of cargoes to tumor cells and dendritic cells (DCs), respectively. The nanovehicle is based on a near infrared-responsive inorganic CuS nanoparticles, acting as a photosensitizer and carrier of the chemotherapeutic agent oxaliplatin, and enters tumor cells owing to the presence of folic acid on the surface of CuS upon intratumoral injection. Furthermore, a toll-like receptor (TLR) 7/8 agonist-conjugated polymer, anchored on the surface of CuS, is modified with mannose to bind with DCs in the tumor microenvironment. Upon exposure to laserirradiation, nanovehiclesdisassemble, releasing oxaliplatin, to ablate tumor cells and amplify immunogenic cell death in combination with photothermal therapy. Mannose-modified polymer-TLR7/8 agonist conjugates are subsequently exposed, leading to the activation of DCs and proliferation of T cells. Collectively, these intelligent nanovehicles reducetumor burden, exert a robust antitumor immune response, and generatelong-term immune protectionto prevent tumor recurrence.

Antibody–Pattern Recognition Receptor Agonist Conjugates: A Promising Therapeutic Strategy for Cancer (Adv. Biology 3/2022)

Antibody–Drug Conjugate In article 2101065, Xu, Li, and Du study a promising therapeutic strategy for cancer. The innate immune response is the body's primary defense against pathogenic infections and other stimulations. Pattern recognition receptors (PRRs) are crucial co-stimulatory molecules expressed in innate immune cells and play an important role in boosting immune activation. Therefore, applying antibody-PRR agonist conjugates in cancer-targeted therapy has great potential.

Design, Synthesis, and Targeted Delivery of an Immune Stimulant that Selectively Reactivates Exhausted CAR T Cells.

Although chimeric antigen receptor (CAR) T cells have demonstrated significant promise in suppressing hematopoietic cancers, their applications in treating solid tumors have been limited by onset of CAR T cell exhaustion that accompanies continuous CAR T cell exposure to tumor antigen. To address this limitation, we have exploited the abilities of recently designed universal CARs to bind fluorescein and internalize a fluorescein-TLR7 agonist conjugate by CAR-mediated endocytosis. We demonstrate here that anti-fluorescein CAR-mediated uptake of a fluorescein-TLR7-3 conjugate can reactivate exhausted CAR T cells, leading to dramatic reduction in T cell exhaustion markers (PD-1+ Tim-3+ ) and shrinkage of otherwise resistant tumors without inducing systemic activation of the immune system. We conclude that CAR T cell exhaustion can be reversed by administration of a CAR-targeted TLR7 agonist, thereby enabling the CAR T cells to successfully treat solid tumors without incurring the systemic toxicity that commonly accompanies administration of nontargeted TLR7 agonists.

Design, Synthesis, and Targeted Delivery of an Immune Stimulant that Selectively Reactivates Exhausted CAR T Cells

Although chimeric antigen receptor (CAR) T cells have demonstrated significant promise in suppressing hematopoietic cancers, their applications in treating solid tumors have been limited by onset of CAR T cell exhaustion that accompanies continuous CAR T cell exposure to tumor antigen. To address this limitation, we have exploited the abilities of recently designed universal CARs to bind fluorescein and internalize a fluorescein-TLR7 agonist conjugate by CAR-mediated endocytosis. We demonstrate here that anti-fluorescein CAR-mediated uptake of a fluorescein-TLR7-3 conjugate can reactivate exhausted CAR T cells, leading to dramatic reduction in T cell exhaustion markers (PD-1+Tim-3+) and shrinkage of otherwise resistant tumors without inducing systemic activation of the immune system. We conclude that CAR T cell exhaustion can be reversed by administration of a CAR-targeted TLR7 agonist, thereby enabling the CAR T cells to successfully treat solid tumors without incurring the systemic toxicity that commonly accompanies administration of nontargeted TLR7 agonists.

Antibody-Pattern Recognition Receptor Agonist Conjugates: A Promising Therapeutic Strategy for Cancer.

Antibody-drug conjugates (ADCs) are composed of monoclonal antibodies linked to cytotoxic payload drugs, each of which can be diversely designed in accordance with pharmacological and clinical requirements. The use of ADCs is effective for the treatment of different diseases, including cancers, and is gaining widespread attention. To date, 12 ADCs have been approved by the U.S. Food and Drug Administration for treating cancer and improving the quality of life of patients. To expand the application of ADCs and improve their treatment efficiency, various formats have recently been manufactured, including pattern recognition receptor (PRR) agonist-based ADCs. The antibody has a unique structure that enables the specific delivery of PRR agonists to the tumor area, and this improves the therapeutic efficacy while minimizing systemic toxicity. This review briefly discusses the current landscape and future perspectives of antibody-PRR agonist conjugates for cancer therapy.

Design, Synthesis, Pharmacokinetics, and Biodistribution of a Series of Bone-Targeting EP4 Receptor Agonist Prodrugs for Treatment of Osteoporosis and Other Bone Conditions

A series of bone-targeting EP4 receptor agonist conjugate prodrugs were prepared wherein a potent EP4 receptor agonist was bound to a biologically inactive, bisphosphonate-based bone-targeting moiety. Singly and doubly radiolabeled conjugates were synthesized and were shown to be stable in blood, to be rapidly eliminated from the bloodstream, and to be effectively taken up into bone in vivo after intravenous dosing. From these preliminary studies a preferred conjugate 4 (also known as C3 and Mes-1007) was selected for follow up biodistribution and elimination studies. Doubly radiolabeled conjugate 4 was found to partition largely to the liver and bones, and both labels were eliminated from liver at the same rate indicating the conjugate was eliminated intact. Quantification of the labels in bones indicated that free EP4 agonist (EP4a)(2a) was released from bone-bound 4 with a half-time of about 7 days. When dosed orally, radiolabeled 4 was not absorbed and passed through the gastrointestinal tract essentially unchanged, and only traces of radiolabeled 4 were found in the liver, blood, or bones. 4 was found to bind rapidly and completely to powdered bone mineral or to various forms of calcium phosphate, forming a stable matrix suitable for implant and that could made into powders or solid forms and be sterilized without decomposition or release of 4. Basic hydrolysis released free EP4 agonist 2a quantitatively from the material.

Corrination of a GLP-1 Receptor Agonist for Glycemic Control without Emesis

Glucagon-like peptide-1 receptor (GLP-1R) agonists used to treat type 2 diabetes mellitus often produce nausea, vomiting, and in some patients, undesired anorexia. Notably, these behavioral effects are caused by direct central GLP-1R activation. Herein, we describe the creation of a GLP-1R agonist conjugate with modified brain penetrance that enhances GLP-1R-mediated glycemic control without inducing vomiting. Covalent attachment of the GLP-1R agonist exendin-4 (Ex4) to dicyanocobinamide (Cbi), a corrin ring containing precursor of vitamin B12, produces a "corrinated" Ex4 construct (Cbi-Ex4). Data collected in the musk shrew (Suncus murinus), an emetic mammal, reveal beneficial effects of Cbi-Ex4 relative to Ex4, as evidenced by improvements in glycemic responses in glucose tolerance tests and a profound reduction of emetic events. Our findings highlight the potential for clinical use of Cbi-Ex4 for millions of patients seeking improved glycemic control without common side effects (e.g., emesis) characteristic of current GLP-1 therapeutics.

A Self-Adjuvanting Vaccine Platform: Optimization of Site-Specific Sortase A Mediated Conjugation of Toll-Like Receptor 2 Ligands onto the Carboxyl or Amino terminus of Recombinant Protein Antigens.

Self-adjuvanting vaccines, consisting of recombinant protein antigens and covalently attached Toll-like receptor (TLR) agonists, have the ability to simultaneously and efficiently deliver antigen and TLR adjuvant to antigen presenting cells (APCs). Here, an enzyme-mediated ligation approach was used to overcome difficulties in producing homogeneous, molecularly defined self-adjuvanting vaccine products under native conditions. This process was optimized to allow the incorporation of the lipopeptide TLR2 agonist fibroblast-stimulating lipopeptide (FSL)-1 onto the N- or C-termini of recombinant protein antigens, employing the enzyme Staphylococcus aureus sortase A (SrtAsa) penta mutant. In addition, because SrtAsa-mediated ligations are reversible, a tryptophan zipper derived sequence was introduced into both reactants, which was demonstrated to improve ligation efficiency through the formation of a β-hairpin structure that hinders the reverse reaction. Finally, it was demonstrated that N- or C-terminal conjugation, and the incorporation of the β-hairpin structure, did not affect the TLR2-agonist activities of protein antigen TLR agonist conjugates. Overall, this SrtAsa-mediated ligation platform enabled production of antigen TLR2 agonist conjugates with enhanced ligation efficiency, with the conjugates demonstrating potent TLR2 signaling activation (EC50 <1nM).

Semisynthetic, self-adjuvanting vaccine development: Efficient, site-specific sortase A-mediated conjugation of Toll-like receptor 2 ligand FSL-1 to recombinant protein antigens under native conditions and application to a model group A streptococcal vaccine

Protein antigens are, in general, weakly immunogenic, and therefore require co-delivery with adjuvants to stimulate potent immune responses. The fusion of (poly)peptide antigens to immunostimulatory adjuvants (e.g. Toll-like receptor (TLR) agonists) has been demonstrated to greatly improve vaccine potency compared to mixtures of antigen and adjuvant. Chemical approaches, to enable the rapid, site-specific and high-yielding linkage of TLR2 ligands to recombinant protein antigens, have been previously optimized. These approaches require the use of denaturing conditions to ensure high reaction yields, which limits their application, as maintenance of native protein folding is necessary to elicit antibodies against conformational epitopes. Here, this work aimed to optimize an alternative method, to ensure the efficient bioconjugation of TLR2 ligands onto folded protein antigens. An enzyme-mediated approach, using Staphylococcus aureus sortase A (or a penta mutant with enhanced efficiency), was optimized for reaction yield and time, as well as enzyme type and amount. This approach enabled the site-specific conjugation of the TLR2-agonist fibroblast-stimulating lipopeptide-1 (FSL-1) onto a model group A Streptococcus (GAS) recombinant polytope antigen under conditions that maintain protein folding, yielding a homogeneous, molecularly-defined product, with ligation yields as high as 90%. Following intramuscular (IM) administration of the ligation product to humanized plasminogen AlbPLG1 mice, high-titer, antigen-specific IgG antibodies were observed, which conferred protection against subcutaneous challenge with GAS strain 5448. In comparison, mixtures of the GAS antigen with aluminum hydroxide or FSL-1 failed to provide protection, with the FSL-1 mixture yielding ~1000-fold lower antigen-specific IgG antibody titers, and the mixture with alum yielding a Th2-biased response compared to the more balanced Th1/Th2 responses observed with the FSL-1 conjugate. Overall, a FSL-1 bioconjugation method for the efficient production of antigen-TLR2 agonist conjugates, which maintain protein folding, was produced, with broad utility for the development of self-adjuvanting vaccines against subunit protein antigens.

Abstract IA29: Peptide-TLR-7/8 agonist conjugate vaccines chemically programmed for nanoparticle self-assembly to enhance the magnitude and breadth of anticancer neoantigen CD8 T cell immunity

Abstract T cells recognizing tumor-specific neoantigens can mediate effective anti-tumor immunity and provide the rationale for personalized cancer vaccines (PCVs). Current approaches for PCVs include synthetic long peptides given with adjuvants or RNA vaccines. Such approaches induce de novo CD8 T cell responses in ~10% or less of predicted neoantigens. Here, we focused on how peptide based neoantigen vaccines could be altered to enhance the magnitude and breadth of CD8 T cells. A key finding was that particulate peptide antigens, unlike soluble peptide antigens, are more efficiently taken up by dendritic cells in draining lymphoid tissue for increasing CD8 T cell expansion. However, due to the broad range of neoantigen physicochemical properties, ensuring consistent particle vaccine formulations with neoantigen peptides is a major challenge. Therefore, we developed a new PCV platform based on charge-modified peptide-TLR-7/8 agonist conjugates that are chemically programmed for self-assembling into nanoparticles (“SNP-7/8a”) of a defined size (20–50 nm) irrespective of the underlying neoantigen peptide composition. Bioinformatics analysis of 72 million human genome-derived neoantigens shows that SNP-7/8a ensures consistent nanoparticle formation with neoantigens at extremes of charge and hydropathy. Vaccination of mice with SNP-7/8a using predicted neoantigens (n = 179) from three tumor models induced CD8 T cells in ~50% of those with high predicted MHC-I binding affinity (IEDB consensus score &amp;lt; 0.5). Moreover, therapeutic vaccination with SNP-7/8a enhanced tumor clearance in tumor models using neoantigens, self-antigens or viral antigens. Overall, the SNP-7/8a PCV is a universal approach for co-delivering peptide antigens and immune adjuvants in nanoparticles to enhance anticancer T cell immunity. Citation Format: Geoffrey M. Lynn, Faezzah Baharom, Yaling Zhu, Vincent Coble, Andrei Ramirez-Valdez, Hide Yamane, Kennedy Tobin, Brennan Decker, Andrew Ishizuka, Robert Seder. Peptide-TLR-7/8 agonist conjugate vaccines chemically programmed for nanoparticle self-assembly to enhance the magnitude and breadth of anticancer neoantigen CD8 T cell immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA29.

Lipid conjugation of TLR7 agonist Resiquimod ensures co-delivery with the liposomal Cationic Adjuvant Formulation 01 (CAF01) but does not enhance immunopotentiation compared to non-conjugated Resiquimod+CAF01

Pattern recognition receptors, including the Toll-like receptors (TLRs), are important in the induction and activation of two critical arms of the host defence to pathogens and microorganisms: the rapid innate immune response (as characterised by the production of Th1 promoting cytokines and type 1 interferons) and the adaptive immune response. Through this activation, ligands and agonists of TLRs can enhance immunotherapeutic efficacy. Resiquimod is a small (water-soluble) agonist of the endosome-located Toll-like receptors 7 and 8 (TLR7/8). However due to its molecular attributes it rapidly distributes throughout the body after injection. To circumvent this, these TLR agonists can be incorporated within delivery systems, such as liposomes, to promote the co-delivery of both antigen and agonists to antigen presenting cells. In this present study, resiquimod has been chemically conjugated to a lipid to form a lipid-TLR7/8 agonist conjugate which can be incorporated within immunogenic cationic liposomes composed of dimethyldioctadecylammonium bromide (DDA) and the immunostimulatory glycolipid trehalose 6,6′ – dibehenate (TDB). This DDA:TDB-TLR7/8 formulation offers similar vesicle characteristics to DDA:TDB (size and charge) and offers high retention of both resiquimod and the electrostatically adsorbed TB subunit antigen Ag85B-ESAT6-Rv2660c (H56). Following immunisation through the intramuscular (i.m.) route, these cationic DDA:TDB-TLR7/8 liposomes form a vaccine depot at the injection site. However, immunisation studies have shown that this biodistribution does not translate into notably increased antibody nor Th1 responses at the spleen and draining popliteal lymph node compared to DDA:TDB liposomes. This work demonstrates that the conjugation of TLR7/8 agonists to cationic liposomes can promote co-delivery but the immune responses stimulated do not merit the added complexity considerations of the formulation.

Lymph-Node-Targeted Immune Activation by Engineered Block Copolymer Amphiphiles-TLR7/8 Agonist Conjugates.

Small molecule immuno-modulators such as agonists of Toll-like receptors (TLRs) are attractive compounds to stimulate innate immune cells toward potent antiviral and antitumor responses. However, small molecules rapidly enter the systemic circulation and cause "wasted inflammation". Hence, synthetic strategies to confine their radius of action to lymphoid tissue are of great relevance, to both enhance their efficacy and concomitantly limit toxicity. Here, we demonstrate that covalent conjugation of a small molecule TLR7/8 agonist immunomodulatory to a micelle-forming amphiphilic block copolymer greatly alters the pharmacokinetic profile, resulting in highly efficient lymphatic delivery. Moreover, we designed amphiphilic block copolymers in such a way to form thermodynamically stable micelles through π-π stacking between aromatic moieties, and we engineered the block copolymers to undergo an irreversible amphiphilic to hydrophilic transition in response to the acidic endosomal pH.

Chemogenetic Approach Using Ni(II) Complex-Agonist Conjugates Allows Selective Activation of Class A G-Protein-Coupled Receptors.

Investigating individual G-protein-coupled receptors (GPCRs) involved in various signaling cascades can unlock a myriad of invaluable physiological findings. One of the promising strategies for addressing the activity of each subtype of receptor is to design chemical turn-on switches on the target receptors. However, valid methods to selectively control class A GPCRs, the largest receptor family encoded in the human genome, remain limited. Here, we describe a novel approach to chemogenetically manipulate activity of engineered class A GPCRs carrying a His4 tag, using metal complex–agonist conjugates (MACs). This manipulation is termed coordination tethering. With the assistance of coordination bonds, MACs showed 10–100-fold lower EC50 values in the engineered receptors, compared with wild-type receptors. Such coordination tethering enabled selective activation of β2-adrenoceptors and muscarinic acetylcholine receptors, without loss of natural receptor responses, in living mammalian cells, including primary cultured astrocytes. Our generalized, modular chemogenetic approach should facilitate more precise control and deeper understanding of individual GPCR signaling pathways in living systems.

Localized immune transcriptional response to intratumorally administrated TLR7 agonist conjugates

Induced or circumstantial immunosuppression present opportunities for cancer development, and progression. Recently, multiple therapies have been developed to harness the immune system's ability to selectively eradicate cancer cells. These approaches have primarily targeted features of the adaptive immune response such as checkpoint blockade and vaccination of tumorigenic peptides. Toll‐Like receptors (TLRs) are innate immune receptors that recognize molecules broadly shared by various pathogens. TLR pathways are increasingly targeted in cancer immunotherapy approaches seeking to overcome immune‐evasion by cancer cells. Here, we hypothesize that activation of TLR7, an endosomal receptor which recognizes pathogen associated nucleic acids, can mitigate tumor immunosuppression. TLR7 agonist (TLR7‐A) conjugated to silica nanoparticles will be used as an adjuvant in a mouse colon adenocarcinoma (CT26 cell line derived) in vivo tumor model. Transcriptional targets of adaptive immune cells (CD3, CD4, CD8, Granzyme B and Perforin), antigen presenting cells (MHCII, CD80, CD86 and CD40) and inflammatory cytokine response (TNFα, IL‐6, IP‐10, MCP1, MIP1α and IFNγ) were measured by qPCR in tumor tissue following 21 days of treatment. We found that treatment with TLR7‐A unconjugated and conjugated increased localized transcript levels of inflammatory cytokines and chemokines: IL‐6, IP‐10, MCP1, MIP1α and IFNγ. This project will provide valuable insights on the distinct transcriptional immune interplay in response to TLR7‐A treatment (localized vs systemic), support the decision‐making process of subsequent experimental approaches and evaluate TLR7‐A conjugate's potential for treatment.Support or Funding InformationThis research was supported by the National Cancer Institute of the National Institutes of Health under award numbers:U54CA132384 &amp; U54CA132379This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Bisphosphonate-Linked TrkB Agonist: Cochlea-Targeted Delivery of a Neurotrophic Agent as a Strategy for the Treatment of Hearing Loss.

Hearing loss affects more than two-thirds of the elderly population, and more than 17% of all adults in the U.S. Sensorineural hearing loss related to noise exposure or aging is associated with loss of inner ear sensory hair cells (HCs), cochlear spiral ganglion neurons (SGNs), and ribbon synapses between HCs and SGNs, stimulating intense interest in therapies to regenerate synaptic function. 7,8-Dihydroxyflavone (DHF) is a selective and potent agonist of tropomyosin receptor kinase B (TrkB) and protects the neuron from apoptosis. Despite evidence that TrkB agonists can promote survival of SGNs, local delivery of drugs such as DHF to the inner ear remains a challenge. We previously demonstrated in an animal model that a fluorescently labeled bisphosphonate, 6-FAM-Zol, administered to the round window membrane penetrated the membrane and diffused throughout the cochlea. Given their affinity for bone mineral, including cochlear bone, bisphosphonates offer an intriguing modality for targeted delivery of neurotrophic agents to the SGNs to promote survival, neurite outgrowth, and, potentially, regeneration of synapses between HCs and SGNs. The design and synthesis of a bisphosphonate conjugate of DHF (Ris-DHF) is presented, with a preliminary evaluation of its neurotrophic activity. Ris-DHF increases neurite outgrowth in vitro, maintains this ability after binding to hydroxyapatite, and regenerates synapses in kainic acid-damaged cochlear organ of Corti explants dissected in vitro with attached SGNs. The results suggest that bisphosphonate-TrkB agonist conjugates have promise as a novel approach to targeted delivery of drugs to treat sensorineural hearing loss.

Toll-like Receptor Agonist Conjugation: A Chemical Perspective.

Toll-like receptors (TLRs) are vital elements of the mammalian immune system that function by recognizing pathogen-associated molecular patterns (PAMPs), bridging innate and adaptive immunity. They have become a prominent therapeutic target for the treatment of infectious diseases, cancer, and allergies, with many TLR agonists currently in clinical trials or approved as immunostimulants. Numerous studies have shown that conjugation of TLR agonists to other molecules can beneficially influence their potency, toxicity, pharmacokinetics, or function. The functional properties of TLR agonist conjugates, however, are highly dependent on the ligation strategy employed. Here, we review the chemical structural requirements for effective functional TLR agonist conjugation. In addition, we provide similar analysis for those that have yet to be conjugated. Moreover, we discuss applications of covalent TLR agonist conjugation and their implications for clinical use.