Oral spleen tyrosine kinase/Janus Kinase inhibitor gusacitinib for the treatment of chronic hand eczema: Results of a randomized phase 2 study.

BACKGROUND: Gusacitinib is an oral inhibitor of Janus and Spleen tyrosine kinases. METHODS: The efficacy and safety of gusacitinib were evaluated in a double-blind, placebo-controlled, multicenter, phase 2 study in 97 chronic hand eczema patients randomized (1:1:1) to placebo or gusacitinib (40 or 80 mg) for 12 weeks (part A). Then, in part B (through week 32), the patients received gusacitinib. RESULTS: At week 16, patients receiving 80 mg gusacitinib showed a 69.5% (P <.005) decrease in the modified total lesion-symptom score versus 49.0% for 40 mg (P =.132), and 33.5% for placebo. Considerable improvement in Physician's Global Assessment was seen in 31.3% of patients receiving 80 mg versus 6.3% of placebo (P <.05). A 73.3% decrease in the hand eczema severity index versus placebo (21.7%) occurred in patients receiving 80 mg (P <.001). Patients receiving 80 mg experienced a considerable decrease in hand pain (P <.05). As early as week 2, considerable reductions over placebo in modified total lesion-symptom score (P <.005), Physician's Global Assessment (P =.04), and hand eczema severity index (P <.01) were observed (80 mg gusacitinib). Adverse events included upper respiratory infection, headache, nausea, and nasopharyngitis. CONCLUSIONS: Gusacitinib showed rapid improvement in chronic hand eczema patients and was well tolerated, warranting further investigations.

Oral Janus kinase/SYK inhibition (ASN002) suppresses inflammation and improves epidermal barrier markers in patients with atopic dermatitis.

BACKGROUND: Moderate-to-severe atopic dermatitis (AD) has been associated with significant disease burden and systemic abnormalities and often requires systemic treatments. Currently, safe and effective oral systemic treatments for moderate-to-severe AD are not yet available. ASN002 is an oral inhibitor of the Janus kinase/spleen tyrosine kinase signaling pathways, targeting several cytokine axes (TH2/TH22/TH17/TH1) and epidermal differentiation. OBJECTIVE: We sought to evaluate the effect of ASN002 on the cellular and molecular biomarker profile of patients with moderate-to-severe AD and to correlate changes in biomarkers to improvements in clinical severity measures and pruritus. METHODS: Thirty-six patients with moderate-to-severe AD were randomized to groups with dose escalation of ASN002 (20, 40, and 80 mg) and a placebo group. Skin biopsy specimens were performed at baseline, day 15, and day 29. Gene expression studies were conducted by using microarray and quantitative RT-PCR, and cellular infiltrates and protein expression were studied by using immunohistochemistry. RESULTS: ASN002 reversed the lesional skin transcriptome toward a nonlesional phenotype. It also rapidly and significantly suppressed key inflammatory pathways implicated in AD pathogenesis, including TH2 (IL4 receptor [IL4R], IL13, CCL13/monocyte chemoattractant protein 4, CCL17/thymus and activation-regulated chemokine, CCL18/pulmonary and activation-regulated chemokine, CCL22/macrophage-derived chemokine, and CCL26/eotaxin-3), TH17/TH22 (lipocalins, PI3/elafin, CCL20, S100A7/S100A8/S100A9, and IL36G/IL36RN), and TH1 (IFNG, CXCL9/CXCL11, and MX1) axes and barrier-related measures (filaggrin [FLG] and CLDN23). Significant improvements in AD gene signatures were observed predominantly in the 40- and 80-mg groups. Smaller and largely nonsignificant molecular changes were seen in the 20-mg and placebo groups. CONCLUSION: The Janus kinase/spleen tyrosine kinase inhibitor ASN002 significantly suppressed key AD inflammatory pathways, corresponding to clinical response. ASN002 might be an effective novel therapeutic agent for moderate-to-severe AD.

ASN004, A 5T4-targeting scFv-Fc Antibody-Drug Conjugate with High Drug-to-Antibody Ratio, Induces Complete and Durable Tumor Regressions in Preclinical Models.

The 5T4 oncofetal antigen (trophoblast glycoprotein) is expressed in a wide range of malignant tumors but shows very limited expression in normal adult tissues. ASN004 is a 5T4-targeted antibody-drug conjugate (ADC) that incorporates a novel single-chain Fv-Fc antibody and Dolaflexin drug-linker technology, with an Auristatin F hydroxypropylamide payload drug-to-antibody ratio of approximately 10-12. The pharmacology, toxicology, and pharmacokinetic properties of ASN004 and its components were investigated in vitro and in vivo ASN004 showed high affinity for the 5T4 antigen and was selectively bound to and internalized into 5T4-expressing tumor cells, and potent cytotoxicity was demonstrated for a diverse panel of solid tumor cell lines. ASN004 induced complete and durable tumor regression in multiple tumor xenograft models, derived from human lung, breast, cervical, and gastric tumor cell lines having a wide range of 5T4 expression levels. A single dose of ASN004, as low as 1 mg/kg i.v., achieved complete tumor regression leading to tumor-free survivors in the A431 cervical cancer model. In head-to-head studies, superior activity of ASN004 was demonstrated against trastuzumab-DM1, in a low-5T4/high-HER2 expressing gastric tumor model, and 10-fold greater potency was found for ASN004 against the 5T4-targeted ADC PF-06263507 in a lung tumor model. In marmoset monkeys, ASN004 was well tolerated at doses up to 1.5 mg/kg Q3W i.v., and showed dose-dependent exposure, linear pharmacokinetics, and markedly low exposure of free payload drug. Taken together, these findings identify ASN004 as a promising new ADC therapeutic for clinical evaluation in a broad range of solid tumor types.

Essential role for the lymphostromal plasma membrane Ly-6 superfamily molecule thymic shared antigen 1 in development of the embryonic adrenal gland.

Thymic shared antigen 1 (TSA-1) is a plasma membrane protein of the Ly-6 superfamily expressed on thymocytes, thymic stromal cells, and other cells of the hematopoietic system. TSA-1 is also expressed in other nonhematopoietic tissues, in particular, embryonic and adult adrenal glands. To address the function of TSA-1, we generated mutant mice in which TSA-1 expression was inactivated by gene targeting. Here we show that deletion of both TSA-1 alleles results in abnormal adrenal gland development and midgestational lethality due to cardiac abnormalities. We also report that TSA-1-deficient adrenal glands have significantly reduced levels of the catecholamines noradrenaline and adrenaline. We conclude that TSA-1 is required for normal embryonic development but that deletion of its expression does not obviously impair lymphoid development.

Effects of LPS-mediated bystander activation in the innate immune system.

LPS induces dendritic cell (DC) activation, but the precise in vivo mechanism is unclear since DCs express low levels of TLR4. Here, it is shown that DCs can be activated in response to LPS through a bystander mechanism. This result was obtained using chimeric mice reconstituted with LPS-responsive and nonresponsive bone marrow cells. Thus, after indirect in vivo conditioning by LPS, bystander-activated DCs (LPS nonresponsive) up-regulated CD86. This up-regulation occurred even when LPS-responsive cells were MyD88 deficient. Functional analysis demonstrated that in vivo LPS conditioning endowed both the LPS-responsive and bystander cells with the ability to produce IFN-gamma in response to TLR9 stimulation in vitro. IFN-gamma production was also shown to be important for enhanced T-bet gene expression but not important for up-regulation of CD86. To investigate aspects of the mechanism, we used intracellular cytokine staining and found that NKDCs were responsible for at least some of the IFN-gamma production. Thus, our in vivo results demonstrated that bacterial LPS can bridge activation of various cellular populations of the innate immune system through a bystander mechanism.

Dendritic cell-T cell interactions in the generation and maintenance of CD8 T cell memory.

Dendritic cells (DCs) are the critical antigen-presenting cells involved in initiating CD8 T cell responses to microbial and viral pathogens. Hence the generation of memory T cells from naïve T cells is intricately intertwined with DCs at every level. This review broadly addresses DC-CD8 T cell interactions that result in the generation and maintenance of CD8 memory T cells.

Heat shock protein gp96 is a master chaperone for toll-like receptors and is important in the innate function of macrophages.

gp96 is an endoplasmic reticulum chaperone for cell-surface Toll-like receptors (TLRs). Little is known about its roles in chaperoning other TLRs or in the biology of macrophage in vivo. We generated a macrophage-specific gp96-deficient mouse. Despite normal development and activation by interferon-gamma, tumor necrosis factor-alpha, and interleukin-1beta, the mutant macrophages failed to respond to ligands of both cell-surface and intracellular TLRs including TLR2, TLR4, TLR5, TLR7, and TLR9. Furthermore, we found that TLR4 and TLR9 preferentially interacted with a super-glycosylated gp96 species. The categorical loss of TLRs in gp96-deficient macrophages operationally created a conditional and cell-specific TLR null mouse. These mice were resistant to endotoxin shock but were highly susceptible to Listeria monocytogenes. Our results demonstrate that gp96 is the master chaperone for TLRs and that macrophages, but not other myeloid cells, are the dominant source of proinflammatory cytokines during endotoxemia and Listeria infections.

The lipopolysaccharide adjuvant effect on T cells relies on nonoverlapping contributions from the MyD88 pathway and CD11c+ cells.

Bacterial LPS is a natural adjuvant that induces profound effects on T cell clonal expansion, effector differentiation, and long-term T cell survival. In this study, we delineate the in vivo mechanism of LPS action by pinpointing a role for MyD88 and CD11c(+) cells. LPS induced long-term survival of superantigen-stimulated CD4 and CD8 T cells in a MyD88-dependent manner. By tracing peptide-stimulated CD4 T cells after adoptive transfer, we showed that for LPS to mediate T cell survival, the recipient mice were required to express MyD88. Even when peptide-specific CD4 T cell clonal expansion was dramatically boosted by enforced OX40 costimulation, OX40 only synergized with LPS to induce survival when the recipient mice expressed MyD88. Nevertheless, these activated, but moribund, T cells in the MyD88(-/-) mice acquired effector properties, such as the ability to synthesize IFN-gamma, demonstrating that effector differentiation is not automatically coupled to a survival program. We confirmed this notion in reverse fashion by showing that effector differentiation was not required for the induction of T cell survival. Hence, depletion of CD11c(+) cells did not affect LPS-driven specific T cell survival, but CD11c(+) cells were paramount for optimal effector T cell differentiation as measured by IFN-gamma potential. Thus, LPS adjuvanticity is based on MyD88 promoting T cell survival, while CD11c(+) cells support effector T cell differentiation.

Steady state dendritic cells present parenchymal self-antigen and contribute to, but are not essential for, tolerization of naive and Th1 effector CD4 cells.

Bone marrow-derived APC are critical for both priming effector/memory T cell responses to pathogens and inducing peripheral tolerance in self-reactive T cells. In particular, dendritic cells (DC) can acquire peripheral self-Ags under steady state conditions and are thought to present them to cognate T cells in a default tolerogenic manner, whereas exposure to pathogen-associated inflammatory mediators during the acquisition of pathogen-derived Ags appears to reprogram DCs to prime effector and memory T cell function. Recent studies have confirmed the critical role of DCs in priming CD8 cell effector responses to certain pathogens, although the necessity of steady state DCs in programming T cell tolerance to peripheral self-Ags has not been directly tested. In the current study, the role of steady state DCs in programming self-reactive CD4 cell peripheral tolerance was assessed by combining the CD11c-diphtheria toxin receptor transgenic system, in which DC can be depleted via treatment with diphtheria toxin, with a TCR-transgenic adoptive transfer system in which either naive or Th1 effector CD4 cells are induced to undergo tolerization after exposure to cognate parenchymally derived self-Ag. Although steady state DCs present parenchymal self-Ag and contribute to the tolerization of cognate naive and Th1 effector CD4 cells, they are not essential, indicating the involvement of a non-DC tolerogenic APC population(s). Tolerogenic APCs, however, do not require the cooperation of CD4(+)CD25(+) regulatory T cells. Similarly, DC were required for maximal priming of naive CD4 cells to vaccinia viral-Ag, but priming could still occur in the absence of DC.

Residual antigen presentation after influenza virus infection affects CD8 T cell activation and migration.

Activated virus-specific CD8 T cells remain in the lung airways for several months after influenza virus infection. We show that maintenance of this cell population is dependent upon the route of infection and prolonged presentation of viral antigen in the draining lymph nodes (DLN) of the respiratory tract. The local effects on T cell migration have been examined. We show retention of virus-specific CD8 T cells in the mediastinal lymph node (MLN) and continuing recruitment of blood-borne migrants into the lung airways during antigen presentation. These data show that antigen that is retained after pulmonary influenza virus infection controls the migratory pattern and activation state of virus-specific CD8 T cells near the site of virus amplification.

CD4 T cells inhibit the CD8 T cell response during low-dose virus infection.

CD4 T cells are not thought to play a significant role in generating an effective primary CD8 T cell response to most viral infections. We have challenged this view by demonstrating that antigen-specific CD4 T cells can indeed suppress the proliferation of antigen-specific naive CD8 T cells in response to low doses of vesicular stomatitis virus. This finding is in contrast to the established observations that at high antigen loads CD4 T cells play little role in generating CD8 T cell responses, and that in non-infectious model systems CD4 T cells actually help the CD8 T cell response. Our results suggest that at low infectious doses, CD4 T cells play a much larger role in controlling infections than previously appreciated.

CCR6-mediated dendritic cell activation of pathogen-specific T cells in Peyer's patches.

T cell activation by dendritic cells (DCs) is critical to the initiation of adaptive immune responses and protection against pathogens. Here, we demonstrate that a specialized DC subset in Peyer's patches (PPs) mediates the rapid activation of pathogen specific T cells. This DC subset is characterized by the expression of the chemokine receptor CCR6 and is found only in PPs. CCR6(+) DCs were recruited into the dome regions of PPs upon invasion of the follicle associated epithelium (FAE) by an enteric pathogen and were responsible for the rapid local activation of pathogen-specific T cells. CCR6-deficient DCs were unable to respond to bacterial invasion of PPs and failed to initiate T cell activation, resulting in reduced defense against oral infection. Thus, CCR6-dependent regulation of DCs is responsible for localized T cell dependent defense against entero-invasive pathogens.

Dendritic cells maximize the memory CD8 T cell response to infection.

Costimulatory signals from dendritic cells (DCs) are required for naive T cells to respond to antigenic stimulation. To what extent DCs reactivate memory T cells during recall responses is not known. Here, an in vivo depletion system has been used to analyze the role of DCs in reactivating CD8 memory T cells during recall responses to three different microbial infections. We show a profound decrease in the numbers of responding memory CD8 T cells in both lymphoid and nonlymphoid tissues during the recall responses to infection with vesicular stomatitis virus, Listeria monocytogenes (Lm), or influenza virus. These data show that interaction with DCs is a major mechanism driving T cell reactivation in vivo, even during a tissue-specific infection of the respiratory tract.