An anti-TNF-glucocorticoid receptor modulator antibody-drug conjugate is efficacious against immune-mediated inflammatory diseases.

Glucocorticoids (GCs) are efficacious drugs used for treating many inflammatory diseases, but the dose and duration of administration are limited because of severe side effects. We therefore sought to identify an approach to selectively target GCs to inflamed tissue. Previous work identified that anti-tumor necrosis factor (TNF) antibodies that bind to transmembrane TNF undergo internalization; therefore, an anti-TNF antibody-drug conjugate (ADC) would be mechanistically similar, where lysosomal catabolism could release a GC receptor modulator (GRM) payload to dampen immune cell activity. Consequently, we have generated an anti-TNF-GRM ADC with the aim of inhibiting pro-inflammatory cytokine production from stimulated human immune cells. In an acute mouse model of contact hypersensitivity, a murine surrogate anti-TNF-GRM ADC inhibited inflammatory responses with minimal effect on systemic GC biomarkers. In addition, in a mouse model of collagen-induced arthritis, single-dose administration of the ADC, delivered at disease onset, was able to completely inhibit arthritis for greater than 30 days, whereas an anti-TNF monoclonal antibody only partially inhibited disease. ADC treatment at the peak of disease was also able to attenuate the arthritic phenotype. Clinical data for a human anti-TNF-GRM ADC (ABBV-3373) from a single ascending dose phase 1 study in healthy volunteers demonstrated antibody-like pharmacokinetic profiles and a lack of impact on serum cortisol concentrations at predicted therapeutic doses. These data suggest that an anti-TNF-GRM ADC may provide improved efficacy beyond anti-TNF alone in immune mediated diseases while minimizing systemic side effects associated with standard GC treatment.

Self-Immolative Carbamate Linkers for CD19-Budesonide Antibody-Drug Conjugates.

Antibody-drug conjugates consist of potent small-molecule payloads linked to a targeting antibody. Payloads must possess a viable functional group by which a linker for conjugation can be attached. Linker-attachment options remain limited for the connection to payloads via hydroxyl groups. A releasing group based on 2-aminopyridine was developed to enable stable attachment of para-aminobenzyl carbamate (PABC) linkers to the C21-hydroxyl group of budesonide, a glucocorticoid receptor agonist. Payload release involves a cascade of two self-immolative events that are initiated by the protease-mediated cleavage of the dipeptide-PABC bond. Budesonide release rates were determined for a series of payload-linker intermediates in buffered solution at pH 7.4 and 5.4, leading to the identification of 2-aminopyridine as the preferred releasing group. Addition of a poly(ethylene glycol) group improved linker hydrophilicity, thereby providing CD19-budesonide ADCs with suitable properties. ADC23 demonstrated targeted delivery of budesonide to CD19-expressing cells and inhibited B-cell activation in mice.

TLR5-mediated sensing of gut microbiota is necessary for antibody responses to seasonal influenza vaccination.

Systems biological analysis of immunity to the trivalent inactivated influenza vaccine (TIV) in humans revealed a correlation between early expression of TLR5 and the magnitude of the antibody response. Vaccination of Trl5(-/-) mice resulted in reduced antibody titers and lower frequencies of plasma cells, demonstrating a role for TLR5 in immunity to TIV. This was due to a failure to sense host microbiota. Thus, antibody responses in germ-free or antibiotic-treated mice were impaired, but restored by oral reconstitution with a flagellated, but not aflagellated, strain of E. coli. TLR5-mediated sensing of flagellin promoted plasma cell differentiation directly and by stimulating lymph node macrophages to produce plasma cell growth factors. Finally, TLR5-mediated sensing of the microbiota also impacted antibody responses to the inactivated polio vaccine, but not to adjuvanted vaccines or the live-attenuated yellow fever vaccine. These results reveal an unappreciated role for gut microbiota in promoting immunity to vaccination.

Immunity to viruses: learning from successful human vaccines.

For more than a century, immunologists and vaccinologists have existed in parallel universes. Immunologists have for long reveled in using 'model antigens', such as chicken egg ovalbumin or nitrophenyl haptens, to study immune responses in model organisms such as mice. Such studies have yielded many seminal insights about the mechanisms of immune regulation, but their relevance to humans has been questioned. In another universe, vaccinologists have relied on human clinical trials to assess vaccine efficacy, but have done little to take advantage of such trials for studying the nature of immune responses to vaccination. The human model provides a nexus between these two universes, and recent studies have begun to use this model to study the molecular profile of innate and adaptive responses to vaccination. Such 'systems vaccinology' studies are beginning to provide mechanistic insights about innate and adaptive immunity in humans. Here, we present an overview of such studies, with particular examples from studies with the yellow fever and the seasonal influenza vaccines. Vaccination with the yellow fever vaccine causes a systemic acute viral infection and thus provides an attractive model to study innate and adaptive responses to a primary viral challenge. Vaccination with the live attenuated influenza vaccine causes a localized acute viral infection in mucosal tissues and induces a recall response, since most vaccinees have had prior exposure to influenza, and thus provides a unique opportunity to study innate and antigen-specific memory responses in mucosal tissues and in the blood. Vaccination with the inactivated influenza vaccine offers a model to study immune responses to an inactivated immunogen. Studies with these and other vaccines are beginning to reunite the estranged fields of immunology and vaccinology, yielding unexpected insights about mechanisms of viral immunity. Vaccines that have been proven to be of immense benefit in saving lives offer us a new fringe benefit: lessons in viral immunology.

Systems biological approaches to measure and understand vaccine immunity in humans.

Recent studies have demonstrated the utility of using systems approaches to identify molecular signatures that can be used to predict vaccine immunity in humans. Such approaches are now being used extensively in vaccinology, and are beginning to yield novel insights about the molecular networks driving vaccine immunity. In this review, we present a broad review of the methodologies involved in these studies, and discuss the promise and challenges involved in this emerging field of "systems vaccinology."

TLR7 enables cross-presentation by multiple dendritic cell subsets through a type I IFN-dependent pathway.

Conjugation of TLR agonists to protein or peptide antigens has been demonstrated in many studies to be an effective vaccine formula in inducing cellular immunity. However, the molecular and cellular mediators involved in TLR-induced immune responses have not been carefully examined. In this study, we identify Type I IFN and IL-12 as critical mediators of cross-priming induced by a TLR7 agonist-antigen conjugate. We demonstrate that TLR7-driven cross-priming requires both Type I IFN and IL-12. Signaling through the IFN-αßR was required for the timely recruitment and accumulation of activated dendritic cells in the draining lymph nodes. Although IL-12 was indispensable during cross-priming, it did not regulate DC function. Therefore, the codependency for these 2 cytokines during TLR7-induced cross-priming is the result of their divergent effects on different cell-types. Furthermore, although dermal and CD8α(+) DCs were able to cross-prime CD8(+) T cells, Langerhans cells were unexpectedly found to potently cross-present antigen and support CD8(+) T-cell expansion, both in vitro and in vivo. Collectively, the data show that a TLR7 agonist-antigen conjugate elicits CD8(+) T-cell responses by the coordinated recruitment and activation of both tissue-derived and lymphoid organ-resident DC subsets through a Type I IFN and IL-12 codependent mechanism.

The capacity to induce cross-presentation dictates the success of a TLR7 agonist-conjugate vaccine for eliciting cellular immunity.

Covalent conjugation of TLR agonists to protein Ags often facilitates the generation of a CD8(+) T cell response. However, mechanisms underlying the efficacy of the conjugate over its unconjugated counterpart have been largely uninvestigated. In this study, we show that conjugation of a TLR7 agonist enhances CD8(+) T cell responses without affecting Ag persistence and with minimal impact on cellular uptake of the Ag in vivo. Instead, the conjugated form induced a robust accumulation of dendritic cells (DCs) in regional lymph nodes. Perhaps more importantly, cross-presentation in DCs was detected only when the Ag was delivered in the conjugated form with the TLR7 agonist. Collectively, these data represent the first demonstration that a TLR agonist-Ag conjugate elicits CD8(+) T cell responses based not on its capacity to induce DC maturation or Ag persistence and uptake, but on the engagement of DC cross-presentation pathways.

Baculovirus-infected insect cells expressing peptide-MHC complexes elicit protective antitumor immunity.

Evaluation of T cell responses to tumor- and pathogen-derived peptides in preclinical models is necessary to define the characteristics of efficacious peptide vaccines. We show in this study that vaccination with insect cells infected with baculoviruses expressing MHC class I linked to tumor peptide mimotopes results in expansion of functional peptide-specific CD8+ T cells that protect mice from tumor challenge. Specific peptide mimotopes selected from peptide-MHC libraries encoded by baculoviruses can be tested using this vaccine approach. Unlike other vaccine strategies, this vaccine has the following advantages: peptides that are difficult to solublize can be easily characterized, bona fide peptides without synthesis artifacts are presented, and additional adjuvants are not required to generate peptide-specific responses. Priming of antitumor responses occurs within 3 days of vaccination and is optimal 1 wk after a second injection. After vaccination, the Ag-specific T cell response is similar in animals primed with either soluble or membrane-bound Ag, and CD11c+ dendritic cells increase expression of maturation markers and stimulate proliferation of specific T cells ex vivo. Thus, the mechanism of Ag presentation induced by this vaccine is consistent with cross-priming by dendritic cells. This straightforward approach will facilitate future analyses of T cells elicited by peptide mimotopes.

Maturational stage-dependent thymocyte responses to TCR engagement.

Thymocyte positive and negative selection are dependent on avidity-driven TCR-mediated recognition events in the thymus. High-avidity recognition events result in negative selection, while low-avidity recognition events result in positive selection. However, it has not been established how thymocytes maturation stages affect their responses to TCR signals of different avidities. We gained insight into this question when we reduced thymocyte selection to an in vitro system, in which full maturation of developmentally synchronized immature double-positive thymocytes was induced on a cloned line of thymic epithelial cells. Our analysis of the kinetics of thymocyte development supports a multi-phasic model of thymic selection. In it, thymocyte maturation stages as well as interaction avidity control the outcome TCR stimulation. Positive selection is initiated during a primary recognition event that proceeds independently of the TCR avidity. During a secondary recognition event the final fate of thymocyte, full maturation versus negative selection, is determined by TCR avidity.