Modeling the clinical phenotype of BTK inhibition in the mature murine immune system.

Inhibitors of Bruton's tyrosine kinase (BTK) possess much promise for the treatment of oncologic and autoimmune indications. However, our current knowledge of the role of BTK in immune competence has been gathered in the context of genetic inactivation of btk in both mice and man. Using the novel BTK inhibitor PF-303, we model the clinical phenotype of BTK inhibition by systematically examining the impact of PF-303 on the mature immune system in mice. We implicate BTK in tonic BCR signaling, demonstrate dependence of the T3 B cell subset and IgM surface expression on BTK activity, and find that B1 cells survive and function independently of BTK. Although BTK inhibition does not impact humoral memory survival, Ag-driven clonal expansion of memory B cells and Ab-secreting cell generation are inhibited. These data define the role of BTK in the mature immune system and mechanistically predict the clinical phenotype of chronic BTK inhibition.

Selective inhibition of BTK prevents murine lupus and antibody-mediated glomerulonephritis.

Autoantibody production and immune complex deposition within the kidney promote renal disease in patients with lupus nephritis. Thus, therapeutics that inhibit these pathways may be efficacious in the treatment of systemic lupus erythematosus. Bruton's tyrosine kinase (BTK) is a critical signaling component of both BCR and FcR signaling. We sought to assess the efficacy of inhibiting BTK in the development of lupus-like disease, and in this article describe (R)-5-amino-1-(1-cyanopiperidin-3-yl)-3-(4-[2,4-difluorophenoxy]phenyl)-1H-pyrazole-4-carboxamide (PF-06250112), a novel highly selective and potent BTK inhibitor. We demonstrate in vitro that PF-06250112 inhibits both BCR-mediated signaling and proliferation, as well as FcR-mediated activation. To assess the therapeutic impact of BTK inhibition, we treated aged NZBxW_F1 mice with PF-06250112 and demonstrate that PF-06250112 significantly limits the spontaneous accumulation of splenic germinal center B cells and plasma cells. Correspondingly, anti-dsDNA and autoantibody levels were reduced in a dose-dependent manner. Moreover, administration of PF-06250112 prevented the development of proteinuria and improved glomerular pathology scores in all treatment groups. Strikingly, this therapeutic effect could occur with only a modest reduction observed in anti-dsDNA titers, implying a critical role for BTK signaling in disease pathogenesis beyond inhibition of autoantibody production. We subsequently demonstrate that PF-06250112 prevents proteinuria in an FcR-dependent, Ab-mediated model of glomerulonephritis. Importantly, these results highlight that BTK inhibition potently limits the development of glomerulonephritis by impacting both cell- and effector molecule-mediated pathways. These data provide support for evaluating the efficacy of BTK inhibition in systemic lupus erythematosus patients.

Quantitative analysis of target coverage and germinal center response by a CXCL13 neutralizing antibody in a T-dependent mouse immunization model.

PURPOSE: Study the impact of CXCL13 neutralization on germinal center (GC) response in vivo, and build quantitative relationship between target coverage and pharmacological effects at the target tissue. METHODS: An anti-CXCL13 neutralizing monoclonal antibody was dosed in vivo in a T-dependent mouse immunization (TDI) model. A quantitative site-of-action (SoA) model was developed to integrate antibody PK and total CXCL13 levels in serum and spleen towards estimating target coverage as a function of dose. To aid in the SoA model development, a radio-labeled study using [I(125)] CXCL13 was conducted in mice. Model estimated target coverage was linked to germinal center response using a sigmoidal inhibitory effect model. RESULTS: In vivo studies demonstrated that CXCL13 inhibition led to an architectural change in B-cell follicles, dislocation of GCs and a significant reduction in the GC absolute numbers per square area (GC/mm(2)). The SoA modeling analysis indicated that ~79% coverage in spleen was required to achieve 50% suppression of GC/mm(2). The 3 mg/kg dose with 52% spleen coverage resulted in no PD suppression, whereas 30 mg/kg with 93% coverage achieved close to maximum PD suppression, highlighting the steepness of PD response. CONCLUSIONS: This study showcases an application of SoA modeling towards a quantitative understanding of CXCL13 pharmacology.

IL-21 receptor is required for the systemic accumulation of activated B and T lymphocytes in MRL/MpJ-Fas(lpr/lpr)/J mice.

MRL/MpJ-Fas(lpr/lpr)/J (MRL(lpr)) mice develop lupus-like disease manifestations in an IL-21-dependent manner. IL-21 is a pleiotropic cytokine that can influence the activation, differentiation, and expansion of B and T cell effector subsets. Notably, autoreactive CD4(+) T and B cells spontaneously accumulate in MRL(lpr) mice and mediate disease pathogenesis. We sought to identify the particular lymphocyte effector subsets regulated by IL-21 in the context of systemic autoimmunity and, thus, generated MRL(lpr) mice deficient in IL-21R (MRL(lpr).IL-21R(-/-)). Lymphadenopathy and splenomegaly, which are characteristic traits of the MRL(lpr) model were significantly reduced in the absence of IL-21R, suggesting that immune activation was likewise decreased. Indeed, spontaneous germinal center formation and plasma cell accumulation were absent in IL-21R-deficient MRL(lpr) mice. Correspondingly, we observed a significant reduction in autoantibody titers. Activated CD4(+) CD44(+) CD62L(lo) T cells also failed to accumulate, and CD4(+) Th cell differentiation was impaired, as evidenced by a significant reduction in CD4(+) T cells that produced the pronephritogenic cytokine IFN-γ. T extrafollicular helper cells are a recently described subset of activated CD4(+) T cells that function as the primary inducers of autoantibody production in MRL(lpr) mice. Importantly, we demonstrated that T extrafollicular helper cells are dependent on IL-21R for their generation. Together, our data highlighted the novel observation that IL-21 is a critical regulator of multiple pathogenic B and T cell effector subsets in MRL(lpr) mice.

Tumor Thrombus of Hepatocellular Carcinoma: A Direct Extension From the Liver to the Right Atrium.

Hepatocellular carcinoma (HCC) is a very aggressive type of cancer and can either invade or spread distantly through the portal vein to the inferior vena cava (IVC) and the right atrium (RA). The presentation varies based on the stage of the cancer at the time of diagnosis. Liver transplantation or surgical resection is the ideal management of small lesions without metastases, while systemic therapy can help in extensive cases to decrease the tumor burden to allow surgical resection of the tumor. We present a rare case of HCC with a tumor thrombus (TT) extending to the RA. Unfortunately, the patient did not survive the cancer. We hope that this case report can contribute to saving the lives of future patients with HCC.

Enhanced GITR/GITRL interactions augment IL-27 expression and induce IL-10-producing Tr-1 like cells.

The glucocorticoid-induced TNFR-related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under-stand the role of long-term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five-fold expansion of the Treg-cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T-cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3(-) IL-10-producing type 1 regulatory T (Tr-1)-like cells that suppress naïve T-cell proliferation in an IL-10-dependent fashion. Increased IL-27 production from Tg APCs and activation of c-Maf in the Tr1-like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T-cell responses, which includes promoting the differentiation of Tr-1-like cells, which contribute to the maintenance of peripheral T-cell tolerance.

IL-21 receptor is critical for the development of memory B cell responses.

Development of long-term humoral immunity, characterized by the formation of long-lived plasma cells (PCs) in the bone marrow and memory B cells, is a critical component of protective immunity to pathogens, and as such it is the major goal of vaccination. However, the mechanisms involved in the generation of long-term humoral immunity remain poorly understood. In this study, we used IL-21R-deficient (IL-21R.KO) mice to examine the role of the IL-21 pathway in the development of the B cell memory response. Primary IgG serum Ab responses to the T cell-dependent Ag 4-hydroxy-3-nitrophenylacetyl (NP) hapten conjugated to chicken γ globulin were delayed in IL-21R.KO mice, but reached normal titers within 3 to 4 wk of immunization. IL-21R.KO mice formed germinal centers and generated normal numbers of PCs in their bone marrow. Additionally, memory B cell formation was similar in wild-type and IL-21R.KO mice. However, NP-specific memory B cells and PCs failed to expand following secondary immunization of IL-21R.KO mice, and consequently, secondary IgG Ab responses to NP hapten conjugated to chicken γ globulin were significantly impaired. These results identify the IL-21 pathway as a critical component of the memory B cell response.

Rational design of a SOCS1-edited tumor-infiltrating lymphocyte therapy using CRISPR/Cas9 screens.

Cell therapies such as tumor-infiltrating lymphocyte (TIL) therapy have shown promise in the treatment of patients with refractory solid tumors, with improvement in response rates and durability of responses nevertheless sought. To identify targets capable of enhancing the antitumor activity of T cell therapies, large-scale in vitro and in vivo clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens were performed, with the SOCS1 gene identified as a top T cell-enhancing target. In murine CD8+ T cell-therapy models, SOCS1 served as a critical checkpoint in restraining the accumulation of central memory T cells in lymphoid organs as well as intermediate (Texint) and effector (Texeff) exhausted T cell subsets derived from progenitor exhausted T cells (Texprog) in tumors. A comprehensive CRISPR tiling screen of the SOCS1-coding region identified sgRNAs targeting the SH2 domain of SOCS1 as the most potent, with an sgRNA with minimal off-target cut sites used to manufacture KSQ-001, an engineered TIL therapy with SOCS1 inactivated by CRISPR/Cas9. KSQ-001 possessed increased responsiveness to cytokine signals and enhanced in vivo antitumor function in mouse models. These data demonstrate the use of CRISPR/Cas9 screens in the rational design of T cell therapies.

Imidazo[1,5-a]quinoxalines as irreversible BTK inhibitors for the treatment of rheumatoid arthritis.

Imidazo[1,5-a]quinoxalines were synthesized that function as irreversible Bruton's tyrosine kinase (BTK) inhibitors. The syntheses and SAR of this series of compounds are presented as well as the X-ray crystal structure of the lead compound 36 in complex with a gate-keeper variant of ITK enzyme. The lead compound showed good in vivo efficacy in preclinical RA models.

Identification of natural antibodies to interleukin-18 in the sera of normal humans and three nonhuman primate species.

Natural antibodies to cytokines can be found in the sera of normal healthy individuals in the absence of specific immunostimulation. However, the function, impact, and purpose of natural antibody development have yet to be fully elucidated. Interleukin (IL)-18 is a cytokine that exerts proinflammatory activities and induces natural killer (NK) cell activity. Recombinant human IL-18 (rHuIL-18) is currently in development as a cancer immunotherapy. In this study, the presence of natural antibodies to IL-18 in the sera of normal humans and three nonhuman primate species was evaluated by electrochemiluminescence immunoassay (ECLIA). Of the human sera tested, 6 of 47 samples were positive for natural antibodies to IL-18. Of the nonhuman primate sera tested, 22 of 80 cynomolgus monkey samples, 4 of 31 rhesus monkey samples, and 2 of 20 chimpanzee samples were positive for natural antibodies to IL-18. Natural anti-IL-18 antibodies were neutralizing in 5 of 22 cynomolgus and 2 of 4 rhesus sera. None of the chimpanzee or human sera were able to neutralize IL-18 induction of interferon (IFN)-gamma in vitro. In vivo activity of rHuIL-18 was compared in IL-18 natural antibody-positive and -negative cynomolgus monkeys. The presence of natural antibodies to IL-18 did not alter rHuIL-18 systemic exposure levels, induction of neopterin, or induction of treatment-induced antibodies following intravenous administration of rHuIL-18. In conclusion, our data indicate that, as has been found with other cytokines, natural anti-IL-18 antibodies are relatively common. Moreover, natural anti-IL-18 antibodies do not appear to influence rHuIL-18 activity in vivo and are not predictive of a heightened immune response, suggesting that natural anti-IL-18 antibodies do not impact IL-18 therapy. Finally, our data suggest that the ability to detect natural anti-cytokine antibodies may be a useful measure of the adequacy of an assay for deployment in clinical trials.