alpha(1,3)Fucosylated oligosaccharides represent components of leukocyte counterreceptors for E- and P-selectins and of L-selectin ligands expressed by lymph node high endothelial venules (HEV). The identity of the alpha(1,3)fucosyltransferase(s) required for their expression has been uncertain, as has a requirement for alpha(1,3)fucosylation in HEV L-selectin ligand activity. We demonstrate here that mice deficient in alpha(1,3) fucosyltransferase Fuc-TVII exhibit a leukocyte adhesion deficiency characterized by absent leukocyte E- and P-selectin ligand activity and deficient HEV L-selectin ligand activity. Selectin ligand deficiency is distinguished by blood leukocytosis, impaired leukocyte extravasation in inflammation, and faulty lymphocyte homing. These observations demonstrate an essential role for Fuc-TVII in E-, P-, and L-selectin ligand biosynthesis and imply that this locus can control leukocyte trafficking in health and disease.

E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by alpha(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV(-/-)/FucT-VII(-/-) mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII(-/-) mice is revoked in FucT-IV(-/-)/FucT-VII(-/-) mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.

The Gal␣133Gal structure is displayed on the zona pellucida glycoprotein ZP3 on murine oocytes. This trisaccharide has been implicated in sperm-zona pellucida adhesive events thought to be essential to fertilization in the mouse. To determine directly if this molecule is required for fertilization, we have generated mice that are deficient in a gene (␣1,3GT) encoding the UDPGal:␤-D-Gal-␣133Gal-galactosyltransferase enzyme responsible for Gal␣133Gal synthesis and expression. These mice develop normally and exhibit no gross phenotypic abnormalities. The Gal␣133Gal epitope is absent from the vascular endothelium and other tissues in ␣1,3GT (؊/؊) adult mice. By contrast, ␣1,3GT (؊/؊) mice, like humans, develop naturally occurring anti-␣-galactoside antibodies normally absent in wild type mice. Female ␣1,3GT (؊/؊) mice yield oocytes that are devoid of the Gal␣133Gal epitope; however, these mice are fully fertile. These observations indicate that the Gal␣133Gal moiety is not essential to sperm-oocyte interactions leading to fertilization or to essentially normal development. They further suggest that ␣1,3GT (؊/؊) mice will find utility for exploring approaches to diminish anti-Gal-dependent hyperacute xenograft rejection, which presents a major barrier to the use of porcine and other non-primate organs for xenotransplantation in humans.Fertilization in mammals involves an adhesive interaction between sperm and the zona pellucida, a glycoprotein-containing shell that surrounds the oocyte. Sperm receptor activity of the murine oocyte resides in the zona pellucida glycoprotein ZP3 (1). Sperm recognition of murine ZP3 depends upon Olinked oligosaccharides displayed by ZP3 (Ref. 2;. Treatment of purified egg ZP3 and ZP3-derived O-linked oligosaccharides with ␣-galactosidase eliminates sperm receptor activity (6). These observations have been taken to imply that terminal ␣-galactosides on ZP3 glycoconjugates are critical for sperm binding activity (6). This notion is supported by more recent observations demonstrating that structurally defined bi-and tetraantennary blood group I-related oligosaccharides containing terminal Gal␣133Gal moieties inhibit binding of sperm to eggs in a dose-dependent manner (7).In the mouse, at least one UDP-Gal:␤-D-Gal-␣133Gal-galactosyltransferase (␣1,3GT) 1 is responsible for the synthesis of terminal Gal␣133Gal␤134GlcNAc trisaccharides from common lactosamine-terminated glycoconjugates (8, 9). Mice and other placental mammals express the Gal␣133Gal␤134GlcNAc trisaccharide products of ␣1,3GT on a variety of glycoproteins and in a variety of tissues (10, 11). Aside from the postulated role of Gal␣133Gal moiety in murine fertilization, the function(s) of this structure are not known.By contrast, humans, apes, and Old World monkeys lack the ability to synthesize these oligosaccharide moieties, because the genetic homologues of the murine ␣1,3GT locus are pseudogenes incapable of encoding a functional ␣1,3GT (12, 13). Consequently, these latter species are reciprocally replete with immunoglobulins...

We believe these studies are the first to consistently demonstrate prevention of a secondary humoral response after cell or organ transplantation in a pig-to-primate model. The development of sensitization to the murine elements of the anti-CD40L monoclonal antibodies suggests that nonresponsiveness to cell membrane-bound antigen (e.g., alphaGal) is a specific phenomenon and not a general manifestation of immunological unresponsiveness. T cell costimulatory blockade may facilitate induction of mixed hematopoietic chimerism and, consequently, of tolerance to pig organs and tissues.

Utomilumab (PF-05082566) is an agonistic mAb that engages the immune costimulatory molecule 4-1BB/CD137. In this first-in-human, phase I, open-label, multicenter, multiple-dose study (NCT01307267) we evaluated safety, tolerability, pharmacokinetics, preliminary clinical activity, and pharmacodynamics of single-agent utomilumab in patients with advanced malignancies. Dose escalation was based on a standard 3+3 design for doses of utomilumab from 0.006 to 0.3 mg/kg every 4 weeks and a time-to-event continual reassessment method for utomilumab 0.6 to 10 mg/kg every 4 weeks. The primary study endpoint was dose-limiting toxicity (DLT) in the first two cycles. Utomilumab demonstrated a well-tolerated safety profile ( = 55). None of the patients experienced a DLT at the dose levels evaluated. The most common treatment-related adverse events were fatigue, pyrexia, decreased appetite, dizziness, and rash (<10% of patients). Only one (1.8%) patient experienced a grade 3-4 treatment-related adverse event (fatigue), and no clinically relevant elevations in transaminases were noted. Utomilumab demonstrated linear pharmacokinetics at doses ranging from 0.006 to 10 mg/kg, with similar safety and pharmacokinetics in anti-drug antibody (ADA)-negative and ADA-positive patients. The overall objective response rate was 3.8% (95% CI, 0.5%-13.0%) in patients with solid tumors and 13.3% in patients with Merkel cell carcinoma, including a complete response and a partial response. Circulating biomarkers support 4-1BB/CD137 engagement by utomilumab and suggest that circulating lymphocyte levels may influence probability of clinical benefit. The favorable safety profile and preliminary antitumor activity demonstrated by utomilumab warrant further evaluation in patients with advanced malignancies. .

Xenotransplantation could overcome the severe shortage of allogeneic organs, a major factor limiting organ transplantation. Unfortunately, transplantation of organs from pigs, the most suitable potential donor species, results in hyperacute rejection in primate recipients, due to the presence of anti–Galα1-3Gal (Gal) natural antibodies (NAbs) in their sera. We evaluated the ability to tolerize anti-Gal NAb–producing B cells in α1,3-galactosyltransferase knockout (GalT KO) mice using bone marrow transplantation (BMT) from GalT+/+ wild-type (WT) mice. Lasting mixed chimerism was achieved in KO mice by cotransplantation of GalT KO and WT marrow after lethal irradiation. The levels of anti-Gal NAb in sera of mixed chimeras were reduced markedly 2 wk after BMT, and became undetectable at later time points. Immunization with Gal+/+ xenogeneic cells failed to stimulate anti-Gal antibody production in mixed chimeras, whereas the production of non–Gal-specific antixenoantigen antibodies was stimulated. An absence of anti-Gal–producing B cells was demonstrated by enzyme-linked immunospot assays in mixed KO+WT→ KO chimeras. Thus, mixed chimerism efficiently induces anti-Gal–specific B cell tolerance in addition to T cell tolerance, providing a single approach to overcoming both the humoral and the cellular immune barriers to discordant xenotransplantation.

Human natural Abs against Galα1-3Galβ1-4GlcNAc (Gal) epitopes are a major barrier to xenotransplantation. Studies in this report, which use combined multiparameter flow cytometric sorting and enzyme-linked immunospot assay, demonstrate that anti-Gal IgM-producing cells are found exclusively in a small B cell subpopulation (i.e., CD21−/low IgMhigh B220low CD5− Mac-1− 493− cells) in the spleens of α1,3-galactosyltransferase-deficient mice. All IgM-producing cells were detected in a similar splenic subpopulation of α1,3-galactosyltransferase-deficient and wild-type mice. A higher frequency of B cells with anti-Gal surface IgM receptors was observed in the peritoneal cavity than in the spleen, but these did not actively secrete Abs, and showed phenotypic properties of B-1b cells (CD21−/low IgMhigh CD5− CD43+ Mac-1+). However, these became Mac-1− and developed anti-Gal Ab-producing activity after in vitro culture with LPS. The splenic B cells with anti-Gal receptors consisted of both Mac-1+ B-1b cells and Mac-1− B-1b-like cells. The latter comprised most anti-Gal IgM-producing cells. Our studies indicate that anti-Gal natural IgM Abs are produced by a B1b-like, Mac-1− splenic B cell population and not by plasma cells or B-1a cells. They are consistent with a model whereby B-1b cells lose Mac-1 expression upon Ag exposure and that these, rather than plasma cells, become the major IgM Ab-producing cell population.

Purpose: This phase Ib study (NCT02179918) evaluated the safety, antitumor activity, pharmacokinetics, and pharmacodynamics of utomilumab, a fully human IgG2 mAb agonist of the T-cell costimulatory receptor 4-1BB/CD137 in combination with the humanized, PD-1-blocking IgG4 mAb pembrolizumab in patients with advanced solid tumors.Experimental Design: Utomilumab (0.45-5.0 mg/kg) and pembrolizumab (2 mg/kg) were administered intravenously every 3 weeks. Utomilumab dose escalation was conducted using the time-to-event continual reassessment method.Results: Twenty-three patients received combination treatment with no dose-limiting toxicities. Treatment-emergent adverse events were mostly grades 1 to 2, without any treatment-related discontinuations. Six patients (26.1%) had confirmed complete or partial responses. Pharmacokinetics and immunogenicity of utomilumab and pembrolizumab were similar when administered alone or in combination. A trend toward higher levels of activated memory/effector peripheral blood CD8þ T cells was observed in responders versus nonresponders.Conclusions: The safety, tolerability, and clinical activity demonstrated by utomilumab in combination with pembrolizumab support further investigation in patients with advanced solid tumors.