GIPR gene expression in testis is mouse specific and can impact male mouse fertility.

BACKGROUND: Glucose-dependent insulinotropic polypeptide receptor (Gipr) gene expression has been reported in mouse spermatids and Gipr knockout male mice have previously been reported to have decreased in vitro fertilization, although the role of Gipr signaling in male mouse fertility is not well understood. OBJECTIVES: The purposes of these studies were to determine the role of glucose-dependent insulinotropic polypeptide receptor in male fertility using Gipr knockout mice and anti-glucose-dependent insulinotropic polypeptide receptor antibody-treated wild-type mice and to determine if the expression of Gipr in mouse testes is similar in non-human and human primates. METHODS AND MATERIALS: Adiponectin promoter-driven Gipr knockout male mice (GiprAdipo-/- ) were assessed for in vitro and in vivo fertility, sperm parameters, and testicular histology. CD1 male mice were administered an anti-glucose-dependent insulinotropic polypeptide receptor antibody (muGIPR-Ab) prior to and during mating for assessment of in vivo fertility and sperm parameters. Expression of Gipr/GIPR mRNA in the mouse, cynomolgus monkey, and human testes was assessed by in situ hybridization methods using species-specific probes. RESULTS: GiprAdipo-/- male mice are infertile in vitro and in vivo, despite normal testis morphology, sperm counts, and sperm motility. In contrast, administration of muGIPR-Ab to CD1 male mice did not impact fertility. While Gipr mRNA expression is detectable in the mouse testes, GIPR mRNA expression is not detectable in monkey or human testes. DISCUSSION: The infertility of GiprAdipo-/- male mice correlated with the lack of Gipr expression in the testis and/or adipocyte tissue. However, as administration of muGIPR-Ab did not impact the fertility of adult male mice, it is possible that the observations in genetically deficient male mice are related to Gipr deficiency during development. CONCLUSION: Our data support a role for Gipr expression in the mouse testis during the development of sperm fertilization potential, but based on gene expression data, a similar role for glucose-dependent insulinotropic polypeptide receptor in non-human primate or human male fertility is unlikely.

MRGPRX2 activation as a rapid, high-throughput mechanistic-based approach for detecting peptide-mediated human mast cell degranulation liabilities.

Mast cells play key roles in allergy, anaphylaxis/anaphylactoid reactions, and defense against pathogens/toxins. These cells contain cytoplasmic granules with a wide spectrum of pleotropic mediators that are released upon activation. While mast cell degranulation (MCD) occurs upon clustering of the IgE receptor bound to IgE and antigen, MCD is also triggered through non-IgE-mediated mechanisms, one of which is via Mas-related G protein-coupled receptor X2 (MRGPRX2). MRGPRX2 can be activated by many basic biogenic amines and peptides. Consequently, MRGPRX2-mediated MCD is an important potential safety liability for peptide therapeutics. To facilitate peptide screening for this liability in early preclinical drug development, a rapid, high-throughput engineered CHO-K1 cell-based MRGPRX2 activation assay was evaluated and compared to histamine release in CD34+ stem cell-derived mature human mast cells as a reference assay, using 30 positive control and 29 negative control peptides for MCD. Both G protein-dependent (Ca2+ endpoint) and -independent (ß-arrestin endpoint) pathways were assessed in the MRGPRX2 activation assay. The MRGPRX2 activation assay had a sensitivity of 100% for both Ca2+ and ß-arrestin endpoints and a specificity of 93% (ß-arrestin endpoint) and 83% (Ca2+ endpoint) compared to histamine release in CD34+ stem cell-derived mature human mast cells. These findings suggest that assessing MRGPRX2 activation in an engineered cell model can provide value as a rapid, high-throughput, economical mechanism-based screening tool for early MCD hazard identification during preclinical safety evaluation of peptide-based therapeutics.

Glucose-Dependent Insulinotropic Polypeptide Receptor Therapies for the Treatment of Obesity, Do Agonists = Antagonists?

Glucose-dependent insulinotropic polypeptide receptor (GIPR) is associated with obesity in human genome-wide association studies. Similarly, mouse genetic studies indicate that loss of function alleles and glucose-dependent insulinotropic polypeptide overexpression both protect from high-fat diet-induced weight gain. Together, these data provide compelling evidence to develop therapies targeting GIPR for the treatment of obesity. Further, both antagonists and agonists alone prevent weight gain, but result in remarkable weight loss when codosed or molecularly combined with glucagon-like peptide-1 analogs preclinically. Here, we review the current literature on GIPR, including biology, human and mouse genetics, and pharmacology of both agonists and antagonists, discussing the similarities and differences between the 2 approaches. Despite opposite approaches being investigated preclinically and clinically, there may be viability of both agonists and antagonists for the treatment of obesity, and we expect this area to continue to evolve with new clinical data and molecular and pharmacological analyses of GIPR function.

Current Concepts in Natural Killer Cell Biology and Application to Drug Safety Assessments.

Natural killer (NK) cells are lymphocytes capable of cytotoxicity against virally infected cells and tumor cells. The display of effector function by NK cells is the result of interactions between germline encoded activating/inhibitory NK cell receptors and their ligands (major histocompatibility complex class I, major histocompatibility complex class I-like, viral, and cellular stress-related surface molecules) expressed on target cells. Determination of NK cell number and function is a common element of the immunotoxicology assessment paradigm for the development of certain classes of pharmaceuticals across a range of modalities. This article summarizes the evidence associating NK cell dysfunction with infectious and cancer risks, reviews emerging NK cell biology, including the impact of immunogenetics on NK cell education and function, and provides perspectives about points to consider when assessing NK cell function in different species in the context of safety assessment.

Biological Characterization of a Stable Effector Functionless (SEFL) Monoclonal Antibody Scaffold in Vitro.

The stable effector functionLess (SEFL) antibody was designed as an IgG1 antibody with a constant region that lacks the ability to interact with Fcγ receptors. The engineering and stability and pharmacokinetic assessments of the SEFL scaffold is described in the accompanying article (Jacobsen, F. W., Stevenson, R., Li, C., Salimi-Moosavi, H., Liu, L., Wen, J., Luo, Q., Daris, K., Buck, L., Miller, S., Ho, S-Y., Wang, W., Chen, Q., Walker, K., Wypych, J., Narhi, L., and Gunasekaran, K. (2017) J. Biol. Chem 292). The biological properties of these SEFL antibodies were assessed in a variety of human and cynomolgus monkey in vitro assays. Binding of parent molecules and their SEFL variants to human and cynomolgus monkey FcγRs were evaluated using flow cytometry-based binding assays. The SEFL variants tested showed decreased binding affinity to human and cynomolgus FcγRs compared with the wild-type IgG1 antibody. In addition, SEFL variants demonstrated no antibody-dependent cell-mediated cytotoxicity in vitro against Daudi cells with cynomolgus monkey peripheral blood mononuclear cells, and had minimal complement-dependent cytotoxicity activity similar to that of the negative control IgG2 in a CD20+ human Raji lymphoma cell line. SEFL mutations eliminated off-target antibody-dependent monocyte phagocytosis of cynomolgus monkey platelets, and cynomolgus platelet activation in vitro These experiments demonstrate that the SEFL modifications successfully eliminated Fc-associated effector binding and functions.

Manipulation of regulatory T-cell function by immunomodulators: a boon or a curse?

Regulatory T cells (Tregs) constitute a subset of lymphocytes that have the capability of suppressing immune responses in vivo and in vitro both directly by cell-cell contact and indirectly through the production of anti-inflammatory cytokines, such as interleukin-10 and tumor growth factor-ß. Tregs constitute a small subset of T lymphocytes, yet their presence can prevent and control autoimmune disease and organ transplant rejection and contribute to maternal tolerance of fetal alloantigens, whereas their absence results in uncontrolled inflammation. But Treg function may not always be considered beneficial: There is growing evidence that the immunosuppressive effects of Tregs are also associated with growth of tumor cells. Thus, Tregs are of considerable medical interest as targets for the treatment of both inflammatory diseases and cancer. In this review of published literature, we describe some well-characterized immunomodulatory drugs and environmental toxicants that can either positively or negatively affect the number and/or function of Tregs in animal models and/or human patients. The targeted suppression or enhancement of Treg function needs to be carefully considered in immunotoxicity evaluations as manipulation of this immune cell population could result in undesired consequences, including decreased host resistance, decreased fertility, or increased incidence of inflammatory disease.

A synthetic TLR4 antagonist has anti-inflammatory effects in two murine models of inflammatory bowel disease.

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel disease is due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonistic activity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proinflammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularly secondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-alpha release. Importantly, treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: the dextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between enteric bacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease.

Bacterial flagellin is a dominant antigen in Crohn disease.

Chronic intestinal inflammation, as seen in inflammatory bowel disease (IBD), results from an aberrant and poorly understood mucosal immune response to the microbiota of the gastrointestinal tract in genetically susceptible individuals. Here we used serological expression cloning to identify commensal bacterial proteins that could contribute to the pathogenesis of IBD. The dominant antigens identified were flagellins, molecules known to activate innate immunity via Toll-like receptor 5 (TLR5), and critical targets of the acquired immune system in host defense. Multiple strains of colitic mice had elevated serum anti-flagellin IgG2a responses and Th1 T cell responses to flagellin. In addition, flagellin-specific CD4(+) T cells induced severe colitis when adoptively transferred into naive SCID mice. Serum IgG to these flagellins, but not to the dissimilar Salmonella muenchen flagellin, was elevated in patients with Crohn disease, but not in patients with ulcerative colitis or in controls. These results identify flagellins as a class of immunodominant antigens that stimulate pathogenic intestinal immune reactions in genetically diverse hosts and suggest new avenues for the diagnosis and antigen-directed therapy of patients with IBD.