Joseph L. Kuijper

Interleukin 21 and its receptor are involved in NK cell expansion and regulation of lymphocyte function.

Cytokines are important in the regulation of haematopoiesis and immune responses, and can influence lymphocyte development. Here we have identified a class I cytokine receptor that is selectively expressed in lymphoid tissues and is capable of signal transduction. The full-length receptor was expressed in BaF3 cells, which created a functional assay for ligand detection and cloning. Conditioned media from activated human CD3+ T cells supported proliferation of the assay cell line. We constructed a complementary DNA expression library from activated human CD3+ T cells, and identified a cytokine with a four-helix-bundle structure using functional cloning. This cytokine is most closely related to IL2 and IL15, and has been designated IL21 with the receptor designated IL21R. In vitro assays suggest that IL21 has a role in the proliferation and maturation of natural killer (NK) cell populations from bone marrow, in the proliferation of mature B-cell populations co-stimulated with anti-CD40, and in the proliferation of T cells co-stimulated with anti-CD3.

Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice

Correction of the obese state induced by genetic leptin deficiency reduces elevated levels of both blood glucose and hypothalamic neuropeptide Y (NPY) mRNA in ob/ob mice. To determine whether these responses are due to a specific action of leptin or to the reversal of the obese state, we investigated the specificity of the effect of systemic leptin administration to ob/ob mice (n = 8) on levels of plasma glucose and insulin and on hypothalamic expression of NPY mRNA. Saline-treated controls were either fed ad libitum (n = 8) or pair-fed to the intake of the leptin-treated group (n = 8) to control for changes of food intake induced by leptin. The specificity of the effect of leptin was further assessed by 1) measuring NPY gene expression in db/db mice (n = 6) that are resistant to leptin, 2) measuring NPY gene expression in brain areas outside the hypothalamus, and 3) measuring the effect of leptin administration on hypothalamic expression of corticotropin-releasing hormone (CRH) mRNA. Five daily intraperitoneal injections of recombinant mouse leptin (150 μg) in ob/ob mice lowered food intake by 56% (P < 0.05), body weight by 4.1% (P < 0.05), and levels of NPY mRNA in the hypothalamic arcuate nucleus by 42.3% (P < 0.05) as compared with saline-treated controls. Pair-feeding of ob/ob mice to the intake of leptin-treated animals produced equivalent weight loss, but did not alter expression of NPY mRNA in the arcuate nucleus. Leptin administration was also without effect on food intake, body weight, or NPY mRNA levels in the arcuate nucleus of db/db mice. In ob/ob mice, leptin did not alter NPY mRNA levels in cerebral cortex or hippocampus or the expression of CRH mRNA in the hypothalamic paraventricular nucleus (PVN). Leptin administration to ob/ob mice also markedly reduced serum glucose (8.3 ± 1.2 vs. 24.5 ± 3.8 mmol/l; P < 0.01) and insulin levels (7,263 ± 1,309 vs. 3,150 ± 780 pmol/l), but was ineffective in db/db mice. Pair-fed mice experienced reductions of glucose and insulin levels that were < 60% of the reduction induced by leptin. The results suggest that in ob/ob mice, systemic administration of leptin inhibits NPY gene overexpression through a specific action in the arcuate nucleus and exerts a hypoglycemic action that is partly independent of its weight-reducing effects. Furthermore, both effects occur before reversal of the obesity syndrome. Defective leptin signaling due to either leptin deficiency (in ob/ob mice) or leptin resistance (in db/db mice) therefore leads directly to hyperglycemia and the overexpression of hypothalamic NPY that is implicated in the pathogenesis of the obesity syndrome.

Interleukin 31, a cytokine produced by activated T cells, induces dermatitis in mice

T cell–derived cytokines are important in the development of an effective immune response, but when dysregulated they can promote disease. Here we identify a four-helix bundle cytokine we have called interleukin 31 (IL-31), which is preferentially produced by T helper type 2 cells. IL-31 signals through a receptor composed of IL-31 receptor A and oncostatin M receptor. Expression of IL-31 receptor A and oncostatin M receptor mRNA was induced in activated monocytes, whereas epithelial cells expressed both mRNAs constitutively. Transgenic mice overexpressing IL-31 developed severe pruritis, alopecia and skin lesions. Furthermore, IL-31 receptor expression was increased in diseased tissues derived from an animal model of airway hypersensitivity. These data indicate that IL-31 may be involved in promoting the dermatitis and epithelial responses that characterize allergic and non-allergic diseases.

Elevated Free Fatty Acids Induce Uncoupling Protein 3 Expression in Muscle: A Potential Explanation for the Effect of Fasting

The newly described uncoupling protein 3 (UCP3) may make an important contribution to thermogenesis in humans because of its high level of expression in skeletal muscle. Contrary to expectations, fasting, a condition that reduces resting energy expenditure, has been reported to increase UCP3 expression in muscle. We have confirmed that a 10-fold increase in UCP3 mRNA levels occurs in rat quadriceps muscle between 12 and 24 h of food removal. A less consistent twofold increase in muscle UCP2 mRNA levels was observed in animals fasted for up to 72 h. Administration of recombinant leptin to prevent a fall in circulating leptin levels did not eliminate the fasting-induced increase in quadriceps UCP3 expression. Administration of a high dose of glucocorticoid to fed animals to mimic the increase in corticosterone induced by fasting did not reproduce the increase in UCP3 expression observed in fasted animals. In contrast, elevation of circulating free fatty acid levels in fed animals by Intralipid plus heparin infusion caused significant increases in the UCP3/actin mRNA ratio compared with saline-infused fed controls in both extensor digitorum longus (2.01 ± 0.34 vs. 0.68 ± 0.11, P = 0.002) and soleus muscles (0.31 ± 0.07 vs. 0.09 ± 0.02, P = 0.014). We conclude that free fatty acids are a potential mediator of the increase in muscle UCP3 expression that occurs during fasting. This seemingly paradoxical induction of UCP3 may be linked to the use of free fatty acid as a fuel rather than an increased need of the organism to dissipate energy.

Vstm3 is a member of the CD28 family and an important modulator of T-cell function

Members of the CD28 family play important roles in regulating T‐cell functions and share a common gene structure profile. We have identified VSTM3 as a protein whose gene structure matches that of the other CD28 family members. This protein (also known as TIGIT and WUCAM) has been previously shown to affect immune responses and is expressed on NK cells, activated and memory T cells, and Tregs. The nectin‐family proteins CD155 and CD112 serve as counter‐structures for VSTM3, and CD155 and CD112 also bind to the activating receptor CD226 on T cells and NK cells. Hence, this group of interacting proteins forms a network of molecules similar to the well‐characterized CD28–CTLA‐4–CD80–CD86 network. In the same way that soluble CTLA‐4 can be used to block T‐cell responses, we show that soluble Vstm3 attenuates T‐cell responses in vitro and in vivo. Moreover, animals deficient in Vstm3 are more sensitive to autoimmune challenges indicating that this new member of the CD28 family is an important regulator of T‐cell responses.

Engineering of stable bispecific antibodies targeting IL-17A and IL-23

Bispecific antibodies (bsAbs) present an attractive opportunity to combine the additive and potentially synergistic effects exhibited by combinations of monoclonal antibodies (mAbs). Current challenges for engineering bsAbs include retention of the binding affinity of the parent mAb or antibody fragment, the ability to bind both targets simultaneously, and matching valency with biology. Other factors to consider include structural stability and expression of the recombinant molecule, both of which may have significant impact on its development as a therapeutic. Here, we incorporate selection of stable, potent single-chain variable fragments (scFvs) early in the engineering process to assemble bsAbs for therapeutic applications targeting the cytokines IL-17A/A and IL-23. Stable scFvs directed against human cytokines IL-23p19 and IL-17A/A were isolated from a human Fab phage display library via batch conversion of panning output from Fabs to scFvs. This strategy integrated a step for shuffling V regions during the conversion and permitted the rescue of scFv molecules in both the V(H)V(L) and the V(L)V(H) orientations. Stable scFvs were identified and assembled into several bispecific formats as fusions to the Fc domain of human IgG1. The engineered bsAbs are potent neutralizers of the biological activity of both cytokines (IC(50) < 1 nM), demonstrate the ability to bind both target ligands simultaneously and display stability and productivity advantageous for successful manufacture of a therapeutic molecule. Pharmacokinetic analysis of the bsAbs in mice revealed serum half-lives similar to human mAbs. Assembly of bispecific molecules using stable antibody fragments offers an alternative to reformatting mAbs and minimizes subsequent structure-related and manufacturing concerns.

Leptin does not fully account for the satiety activity of adipose tissue-conditioned medium

To determine whether leptin alone accounts for the satiety activity secreted by native adipose tissue, we prepared culture media conditioned by microdissected adipose tissue from overfed Long-Evans rats, fa/ farats, or db/ dbmice ( media A, B, and C, respectively). Medium A significantly suppressed food intake following intracerebroventricular delivery to Long-Evans rats (2-h chow intake = 68 ± 5% of baseline, P< 0.001). Media B and C significantly suppressed food intake following intraperitoneal delivery to ob/ obmice (24-h chow intake = 56 ± 7% of baseline for medium B, P= 0.001; 4-day chow intake = 78 ± 3% of baseline for medium C, P= 0.004). Using a leptin receptor-based bioassay, we determined that the leptin concentration of medium C was 392 ± 18 ng/ml. This concentration was 20-fold lower than the concentration of recombinant murine leptin required to produce a similar degree of feeding suppression following 5 days of administration to ob/ obmice. Neither medium conditioned by adipose tissue from ob/ obmice nor medium conditioned by adipose tissue from fa/ farats and subsequently immunodepleted of leptin had significant satiety activity. We conclude that leptin is necessary but not sufficient to account for the satiety activity of native adipose tissue, perhaps due to the production by adipocytes of a cofactor that augments the ability of leptin to suppress feeding.

The assignment of the human insulin receptor-related receptor gene (INSRR) to chromosome 1q21→q23 by the use of radiation hybrid mapping

The insulin receptor-related receptor (INSRR) was first identified as a genomic sequence encoding a novel member of the insulin receptor family (Shier and Watt, 1989). Unlike other members of the receptor family, the expression of INSRR is relatively restricted. INSRR mRNA and protein are localized in subsets of neuron (Reinhardt et al., 1993), in renal distal tubules (Reinhardt et al., 1993) and in the enterochromaffinlike cells of the fundic stomach (Tsujimoto et al., 1995). Most recently, expression of INSRR was found in the islets of Langerhans (Ozaki, 1998) suggesting INSRR may be involved in the physiological functions of insulin-secreting cells. Previously, the INSRR locus was localized to chromosome 1 by the use of a human hamster somatic cell panel (Shier et al., 1990). As a first step to assess a possible role of INSRR in heritable forms of diabetes or neurodegenerative disease, we carried out a regional mapping of the human INSRR locus. Our results map INSRR to chromosome 1q21→q23, a region that has been shown by two recent studies to be involved in susceptibility to type II diabetes (Hanson et al., 1998; Elbein et al., 1999). Materials and methods

Abstract 4550: CD80 vIgD-Fc proteins combine checkpoint antagonism and costimulatory signaling for potent antitumor immunity

Introduction: PD-1 pathway antagonists have revealed the importance of checkpoint pathways in regulating antitumor immunity, but an existing immune response is generally required for clinical efficacy. Specific T cell costimulation through CD28 is central to this process, but the CD28 ligands CD80 and CD86 are often poorly expressed in the tumor microenvironment, accounting for a second important mechanism of immune evasion by tumors. In contrast, PD-L1 expression has been found extensively in multiple tumor cell types. Therapeutics that combine PD-L1/PD-1 antagonism coupled with PD-L1 dependent CD28 agonism may therefore provide a more potent, yet safe immunotherapeutic approach. Experimental Procedures: The variant Ig Domain (vIgD) TM platform has generated a diversity of human CD80 variants using yeast display affinity maturation and selections against all three CD80 counterstructures CD28, CTLA-4, and PD-L1. CD80 vIgDs were produced in a mammalian expression system as recombinant Fc fusion proteins (CD80 vIgD-Fc proteins) and their binding properties were quantified by flow cytometry. Functional activity was determined in vitro by assessing responses from human primary T cells or an IL-2-luciferase Jurkat T cell reporter line stimulated with PD-L1-expressing artificial antigen presenting cells (aAPCs). In vitro human T cell cytotoxicity assays with a human PD-L1-expressing tumor line were also performed. Antitumor activity was assessed in vivo with mice implanted with human PD-L1 transduced MC38 tumors. Data Summary: The human CD80 IgV fragment was found to be optimal for high affinity PD-L1 and CD28 binding. A large panel of CD80 vIgD-Fc proteins demonstrated a range of binding towards CD28, PD-L1, and/or CTLA-4, and CD80 vIgD-Fc proteins with high affinity for PD-L1 antagonized the PD-L1/PD-1 interaction. Some CD80 vIgD-Fc proteins agonized CD28 in a PD-L1 dependent fashion with increased luciferase activity in the Jurkat reporter assay as well as increased cytokine production by primary human T cells when stimulated with a PD-L1 expressing aAPC in vitro. The same candidates also showed specific killing of human PD-L1 expressing tumor cells in vitro compared to the parental tumor line lacking PD-L1 expression. Importantly, selected CD80 vIgD-Fc proteins caused significant tumor reduction in the MC38 in vivo tumor model. Conclusion: Engineered CD80 vIgD-Fc proteins that deliver a localized CD28 costimulatory signal to T cells while simultaneously antagonizing the inhibitory PD-L1/PD-1 pathway may provide a transformative mechanism of action to drive potent, tolerable antitumor immunity. Preclinical development of therapeutic candidates is under way. Citation Format: Ryan Swanson, Mark F. Maurer, Chris L. Navas, Chelsea J. Gudgeon, Joseph L. Kuijper, Martin Wolfson, Katherine E. Lewis, Stacey R. Dillon, Steve D. Levin, Michael G. Kornacker. CD80 vIgD-Fc proteins combine checkpoint antagonism and costimulatory signaling for potent antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4550.