Developing simulation games to advance public health nursing competence in baccalaureate education.

We describe the development of an innovative baccalaureate nursing education strategy for public health nursing. Virtual simulation pedagogy is known to be effective for acute care nursing practice while less known for public health nursing. Three Canadian nursing schools, the Canadian Association of Schools of Nursing (CASN), and the Canadian Alliance of Nurse Educators using Simulation (CAN-Sim) partnered to develop three public health nursing virtual simulation games. Learners work through unfolding population health scenarios, simulating public health nursing practice focused on entry level public health nursing competencies. Each game fosters clinical reasoning and collaborative, community decision-making to respond to population health issues during community assessment, evidence-informed health promotion planning, and evaluation processes. A companion guide was developed to support best practices in implementing virtual simulation and promote optimum student learning using the public health nursing games.

Toward a Combinatorial Approach for the Prediction of IgG Half-Life and Clearance.

The serum half-life and clearance of therapeutic monoclonal antibodies (mAbs) are critical factors that impact their efficacy and optimal dosing regimen. The pH-dependent binding of an mAb to the neonatal Fc receptor (FcRn) has long been recognized as an important determinant of its pharmacokinetics. However, FcRn affinity alone is not a reliable predictor of mAb half-life, suggesting that other biologic or biophysical mechanisms must be accounted for. mAb thermal stability, which reflects its unfolding and aggregation propensities, may also relate to its pharmacokinetic properties. However, no rigorous statistical regression methods have been used to identify combinations of physical parameters that best predict biologic properties. In this work, a panel of eight mAbs with published human pharmacokinetic data were selected for biophysical analyses of FcRn binding and thermal stability. Biolayer interferometry was used to characterize FcRn/mAb binding at acidic and neutral pH, while differential scanning calorimetry was used to determine thermodynamic unfolding parameters. Individual binding or stability parameters were generally weakly correlated with half-life and clearance values. Least absolute shrinkage and selection operator regression was used to identify the combination of two parameters with the best correlation to half-life and clearance as being the FcRn binding response at pH 7.0 and the change in heat capacity. Leave-one-out subsampling yielded a root mean square difference between observed and predicted half-life of just 2.7 days (16%). Thus, the incorporation of multiple biophysical parameters into a cohesive model may facilitate early-stage prediction of in vivo half-life and clearance based on simple in vitro experiments.

Rapid Purification of Human Bispecific Antibodies via Selective Modulation of Protein A Binding.

Methods to rapidly generate high quality bispecific antibodies (BsAb) having normal half-lives are critical for therapeutic programs. Here, we identify 3 mutations (T307P, L309Q, and Q311R or "TLQ") in the Fc region of human IgG1 which disrupt interaction with protein A while enhancing interaction with FcRn. The mutations are shown to incrementally alter the pH at which a mAb elutes from protein A affinity resin. A BsAb comprised of a TLQ mutant and a wild-type IgG1 can be efficiently separated from contaminating parental mAbs by differential protein A elution starting from either a) purified parental mAbs, b) in-supernatant crossed parental mAbs, or c) co-transfected mAbs. We show that the Q311R mutation confers enhanced FcRn interaction in vitro, and Abs harboring either the Q311R or TLQ mutations have serum half-lives as long as wild-type human IgG1. The mutant Abs have normal thermal stability and Fcγ receptor interactions. Together, the results lead to a method for high-throughput generation of BsAbs suitable for in vivo studies.

Modulation of protein A binding allows single-step purification of mouse bispecific antibodies that retain FcRn binding.

The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies.

Functional optimization of agonistic antibodies to OX40 receptor with novel Fc mutations to promote antibody multimerization.

Immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising targets for cancer immunotherapies. To optimize the agonism of therapeutic antibodies to these receptors, Fc engineering of antibodies was applied to facilitate the clustering of cell surface TNFRs to activate downstream signaling pathways. One engineering strategy is to identify Fc mutations that facilitate antibody multimerization on the cell surface directly. From the analyses of the crystal packing of IgG1 structures, we identified a novel set of Fc mutations, T437R and K248E, that facilitated antibody multimerization upon binding to antigens on cell surface. In a NF-κB reporter assay, the engineered T437R/K248E mutations could facilitate enhanced agonism of an anti-OX40 antibody without the dependence on FcγRIIB crosslinking. Nonetheless, the presence of cells expressing FcγRIIB could facilitate a boost of the agonism of the engineered antibody with mutations on IgG1 Fc, but not on the silent IgG2σ Fc. The Fc engineered antibody also showed enhanced effector functions, including antibody-dependent cell-meditated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity, depending on the IgG subtypes. Also, the engineered antibodies showed normal FcRn binding and pharmacokinetic profiles in mice. In summary, this study elucidated a novel Fc engineering approach to promote antibody multimerization on a cell surface, which could enhance agonism and improve effector function for anti-TNFR antibodies as well as other therapeutic antibodies.

Modification of the Fc Region of a Human Anti-oncostatin M Monoclonal Antibody for Higher Affinity to FcRn Receptor and Extension of Half-life in Cynomolgus Monkeys.

The purpose of this study was to evaluate the pharmacokinetics (PK) of anti-oncostatin M (OSM) IgG1 monoclonal antibodies, CNTO 1119 and its Fc variant (CNTO 8212), which incorporates the LS(Xtend) mutation to extend terminal half-life (T1/2 ), after a single intravenous (IV) or subcutaneous (SC) administration in cynomolgus monkeys, and to predict human PK. In study 1, single doses of CNTO 1119 and CNTO 8212 were administered IV or SC at 3 mg/kg to cynomolgus monkeys (n = 3 per group). In study 2, single doses of CNTO 8212 were administered IV at 1, 5 or 20 mg/kg, or SC at 5 mg/kg to cynomolgus monkeys (n = 5 per group). Serial blood samples were collected for assessment of serum concentrations of CNTO 1119 and/or CNTO 8212. A two-compartment population PK model with first-order elimination was utilized to simultaneously describe the serum concentrations of CNTO 1119 and CNTO 8212 over time after IV and SC administration in cynomolgus monkeys. The typical population PK parameter estimates for CNTO 1119 in cynomolgus monkeys were clearance (CL) = 2.81 mL/day/kg, volume of distribution of central compartment (V1 ) = 31.3 mL/kg, volume of distribution of peripheral compartment (V2 ) = 23.3 mL/kg, absolute bioavailability (F) = 0.84 and T1/2 = 13.4 days. In comparison, the typical population PK parameter estimates for CNTO 8212 in cynomolgus monkeys were CL = 1.41 mL/day/kg, V1 = 39.8 mL/kg, V2 = 32.6 mL/kg, F = 0.75 and T1/2 = 35.7 days. The mean CL of CNTO 8212 was ~50% lower compared with that for CNTO 1119 in cynomolgus monkeys. The overall volume of distribution (V1 +V2 ) for CNTO 8212 was about 32% larger compared with that for CNTO 1119, but generally similar to the vascular volume in cynomolgus monkeys. The T1/2 of CNTO 8212 was significantly (p < 0.05) longer by about 2.7-fold than that for CNTO 1119 in cynomolgus monkeys. Thus, the modification of the Fc portion of an anti-OSM IgG1 mAb for higher FcRn binding affinity resulted in lower systemic clearance and a longer terminal half-life in cynomolgus monkeys. CNTO 8212 demonstrated linear PK after a single IV dose (1-20 mg/kg) in cynomolgus monkeys. The predicted human PK parameters suggest that CNTO 8212 is likely to exhibit slow clearance and long terminal half-life in human beings and may likely allow less frequent dosing in the clinical setting.

Functional, Biophysical, and Structural Characterization of Human IgG1 and IgG4 Fc Variants with Ablated Immune Functionality.

Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically "silent".

Human and non-human primate intestinal FcRn expression and immunoglobulin G transcytosis.

PURPOSE: To evaluate transcytosis of immunoglobulin G (IgG) by the neonatal Fc receptor (FcRn) in adult primate intestine to determine whether this is a means for oral delivery of monoclonal antibodies (mAbs). METHODS: Relative regional expression of FcRn and localization in human intestinal mucosa by RT-PCR, ELISA & immunohistochemistry. Transcytosis of full-length mAbs (sandwich ELISA-based detection) across human intestinal segments mounted in Ussing-type chambers, human intestinal (caco-2) cell monolayers grown in transwells, and serum levels after regional intestinal delivery in isoflurane-anesthetized cynomolgus monkeys. RESULTS: In human intestine, there was an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression. In cynomolgus, serum mAb levels were greater after ileum-proximal colon infusion than after administration to stomach or proximal small intestine (1-5 mg/kg). Serum levels of wild-type mAb dosed into ileum/proximal colon (2 mg/kg) were 124 ± 104 ng/ml (n = 3) compared to 48 ± 48 ng/ml (n = 2) after a non-FcRn binding variant. In vitro, mAb transcytosis in polarized caco-2 cell monolayers and was not enhanced by increased apical cell surface IgG binding to FcRn. An unexpected finding in primate small intestine, was intense FcRn expression in enteroendocrine cells (chromagranin A, GLP-1 and GLP-2 containing). CONCLUSIONS: In adult primates, FcRn is expressed more highly in distal intestinal epithelial cells. However, mAb delivery to that region results in low serum levels, in part because apical surface FcRn binding does not influence mAb transcytosis. High FcRn expression in enteroendocrine cells could provide a novel means to target mAbs for metabolic diseases after systemic administration.

Correlations between pharmacokinetics of IgG antibodies in primates vs. FcRn-transgenic mice reveal a rodent model with predictive capabilities.

Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.

Factors influencing alginate gel biocompatibility.

Alginate remains the most popular polymer used for cell encapsulation, yet its biocompatibility is inconsistent. Two commercially available alginates were compared, one with 71% guluronate (HiG), and the other with 44% (IntG). Both alginates were purified, and their purities were verified. After 2 days in the peritoneal cavity of C57BL/6J mice, barium (Ba)-gel and calcium (Ca)-gel beads of IntG alginate were clean, while host cells were adhered to beads of HiG alginate. IntG gel beads, however, showed fragmentation in vivo while HiG gel beads stayed firm. The physicochemical properties of the sodium alginates and their gels were thoroughly characterized. The intrinsic viscosity of IntG alginate was 2.5-fold higher than that of HiG alginate, suggesting a greater molecular mass. X-ray photoelectron spectroscopy indicated that both alginates were similar in elemental composition, including low levels of counterions in all gels. The wettabilities of the alginates and gels were also identical, as measured by contact angles of water on dry films. Ba-gel beads of HiG alginate resisted swelling and degradation when immersed in water, much more than the other gel beads. These results suggest that the main factors contributing to the biocompatibility of gels of purified alginate are the mannuronate/guluronate content and/or intrinsic viscosity.

Engineering host cell lines to reduce terminal sialylation of secreted antibodies.

Covalently-linked glycans on proteins have many functional roles, some of which are still not completely understood. Antibodies have a very specific glycan modification in the Fc region that is required for mediating immune effector functions. These Fc glycans are typically highly heterogeneous in structure, and this heterogeneity is influenced by many factors, such as type of cellular host and rate of Ab secretion. Glycan heterogeneity can affect the Fc-dependent activities of antibodies. It has been shown recently that increased Fc sialylation can result in decreased binding to immobilized antigens and some Fcγ receptors, as well as decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activity. In contrast, increased Fc sialylation enhances the anti-inflammatory activity of antibodies. To produce antibodies with increased effector functions, we developed host cell lines that would limit the degree of sialylation of recombinantly-expressed antibodies. Towards this end, the catalytic domain of the Arthrobacter ureafaciens sialidase (sialidase A) was engineered for secreted expression in mammalian cell lines. Expression of this sialidase A gene in mammalian cells resulted in secreted expression of soluble enzyme that was capable of removing sialic acid from antibodies secreted into the medium. Purified antibodies secreted from these cells were found to possess very low levels of sialylation compared with the same antibodies purified from unmodified host cells. The low sialylated antibodies exhibited similar binding affinity to soluble antigens, improved ADCC activity, and they possessed pharmacokinetic properties comparable to their more sialylated counterparts. Further, it was observed that the amount of sialidase A expressed was sufficient to thoroughly remove sialic acid from Abs made in high-producing cell lines. Thus, engineering host cells to express sialidase A enzyme can be used to produce recombinant antibodies with very low levels of sialylation.

Characterization of golimumab, a human monoclonal antibody specific for human tumor necrosis factor α.

We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018).  The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry.  In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration.  In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.

Role of protein contaminants in the immunogenicity of alginates.

Alginate is widely used for cell microencapsulation and transplantation. There is a lack of standardization of alginate purity and composition. In a previous study, we compared different alginate purification methods and concluded that polyphenol and endotoxin contaminants were eliminated efficiently but residual protein contaminants persisted with all of the methods under evaluation. The objective of this study was to test the hypothesis that residual proteins play a role in the immunogenicity of certain alginate preparations. Using preparative size exclusion chromatography (SEC) and a large scale purification protocol that was derived from the findings obtained with SEC, we substantially decreased the protein content of alginate preparations. When implanted into mouse peritoneum, barium alginate beads made of alginates that were purified using SEC or the derived large scale protocol induced significantly less pericapsular cell adhesion than those made with control alginates. In conclusions, these results suggest that removing residual protein contamination may decrease the immunogenicity of certain alginate preparations. The measurement of proteins could be used as a screening method for evaluating alginate preparations.

Tumor-associated and microbial proteases compromise host IgG effector functions by a single cleavage proximal to the hinge.

The successful elimination of pathogenic cells and microorganisms by the humoral immune system relies on effective interactions between host immunoglobulins and Fc gamma receptors on effector cells, in addition to the complement system. Essential Ig motifs that direct those interactions reside within the conserved IgG lower hinge/CH2 interface. We noted that a group of tumor-related and microbial proteases cleaved human IgG1s in that region, and the "nick" of just one of the heavy chains profoundly inhibited IgG1 effector functions. We focused on IgG1 monoclonal antibodies (mAbs) since IgG1 is the most abundant human subclass and demonstrates robust Fc-mediated effector functions. The loss of Fc-mediated cell killing activities was correlated with diminished binding to the Fc gamma family of receptors, but a similar decrease in affinity was not observed toward the FcRn receptor that maintains IgG in circulation. Endogenous human IgG cleavage products of comparable size to mAbs with the single cleavage were detected by Western blot analysis in synovial fluid from patients with rheumatoid arthritis and in breast carcinoma extracts. Their detection is problematic under physiological conditions, since there is no loss of structure, and antigen-binding capability is unaffected. These findings suggest that within the hostile proteolytic microenvironments associated with many diseases, key effector functions of host IgGs, or therapeutic Abs, may be compromised.

Direct effect of alginate purification on the survival of islets immobilized in alginate-based microcapsules.

Alginate purification has been shown to decrease the host immune response to implanted alginate-based microcapsules, but the direct effect of contaminants on islet cell survival remains unknown. Wistar rat islets were immobilized in calcium alginate beads made with crude vs. purified alginate and then incubated in CMRL culture medium. Islet survival was evaluated at 1, 4, 7, 14 and 27 days post-encapsulation. Islet viability was investigated using a dual staining assay (propidium iodide and orange acridine). The islet cell necrosis and the proportion of apoptotic cells were quantified under optical microscopy and with a TUNEL assay, respectively. Islets immobilized in purified alginate were more viable, and had fewer necrotic centers, a smaller area of central necrosis and a lower number of apoptotic cells. At day 14 and 27 post-encapsulation, respectively, 48% and 23% of islets were viable with purified alginate vs. 18% and 8% with crude alginate (p<0.05). At day 14, the surface area of central necrosis and the number of necrotic islets were more important with the impure alginate (65% vs. 45% and 73% vs. 53%, respectively; p<0.05). We conclude that alginate purification improves the survival of islets that are immobilized in alginate-based microcapsules. These findings indicate that caution should be taken in the interpretation of in vivo experiments, as the results could be explained by either a direct effect on islet survival or a modification of the host reaction, or both. Moreover, it suggests that the effect on islet viability should be assessed during the development of biomaterials for cell encapsulation.

Adsorption of human immunoglobulin to implantable alginate-poly-L-lysine microcapsules: effect of microcapsule composition.

Alginate-poly-L-lysine-alginate (APA) microcapsules continue to be the most widely studied device for the immuno-protection of transplanted therapeutic cells. Producing APA microcapsules having a reproducible and high level of biocompatibility requires an understanding of the mechanisms of the immune response towards the implants. Here, we investigate the adsorption of immunoglobulins (IgG, IgM, and IgA) onto the surface of APA microcapsules in vitro after their exposure to human serum and peritoneal fluid. Immunoglobulins (Ig) are considered to be opsonizing proteins, thus they tend to mediate inflammation when adsorbed to foreign surfaces. Ig adsorption was monitored using direct immunofluorescence. The amount of Ig adsorbed to the microcapsule surface was not significantly influenced by the guluronic acid content nor the purity level of the alginate, although microcapsules of intermediate-G purified alginate corresponded with the lowest adsorption levels. Ig adsorption was negligible when the poly-L-lysine membrane was omitted, suggesting that positive charges at the microcapsule surface are responsible for binding Ig.

Human anti-IgG1 hinge autoantibodies reconstitute the effector functions of proteolytically inactivated IgGs.

A number of proteases of potential importance to human physiology possess the ability to selectively degrade and inactivate Igs. Proteolytic cleavage within and near the hinge domain of human IgG1 yielded products including Fab and F(ab')(2) possessing full Ag binding capability but absent several functions needed for immune destruction of cellular pathogens. In parallel experiments, we showed that the same proteolytically generated Fabs and F(ab')(2)s become self-Ags that were widely recognized by autoantibodies in the human population. Binding analyses using various Fab and F(ab')(2), as well as single-chain peptide analogues, indicated that the autoantibodies targeted the newly exposed sequences where proteases cleave the hinge. The point of cleavage may be less of a determinant for autoantibody binding than the exposure of an otherwise cryptic stretch of hinge sequence. It was noted that the autoantibodies possessed an unusually high proportion of the IgG3 isotype in contrast to Abs induced against foreign immunogens in the same human subjects. In light of the recognized potency of IgG3 effector mechanisms, we adopted a functional approach to determine whether human anti-hinge (HAH) autoantibodies could reconstitute the (missing) Fc region effector functions to Fab and F(ab')(2). Indeed, in in vitro cellular assays, purified HAH autoantibodies restored effector functions to F(ab')(2) in both Ab-dependent cellular cytotoxicity and complement-dependent cytotoxicity assays. The results indicate that HAH autoantibodies selectively bind to proteolytically cleaved IgGs and can thereby provide a surrogate Fc domain to reconstitute cell lytic functions.

Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model.

BACKGROUND: Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model. METHODS: Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 microg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10-25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. RESULTS: Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. CONCLUSIONS: These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.

CNTO 859, a humanized anti-tissue factor monoclonal antibody, is a potent inhibitor of breast cancer metastasis and tumor growth in xenograft models.

Thromboembolic complications are frequently associated with advanced cancer. Interestingly, one of the major initiators of blood coagulation, tissue factor (TF), is reported to be overexpressed in several tumor types and can be found on both tumor cells and tumor vasculature. Although the exact mechanisms have yet to be elucidated, TF expressed on tumor cells can trigger intracellular signaling events through various pathways that can lead to tumor angiogenesis, proliferation, and metastasis. There exists preclinical evidence that disruption of TF dependent signaling can effectively inhibit tumor cell migration, metastasis, and angiogenesis. Here, we report for the first time that an antibody to tissue factor can also prevent tumor growth in vivo. Prophylactic administration of CNTO 859, a humanized anti-human TF antibody, was shown to inhibit experimental lung metastasis of MDA-MB-231 human breast carcinoma cells by over 99% compared to a control antibody. Furthermore, therapeutic doses of CNTO 859 were shown to reduce tumor incidence and growth of orthotopically implanted MDA-MB-231 cells.

Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality.

Although it is now clear that certain Fc glycan structures on immunoglobulin G (IgG) antibodies (Abs) can have a dramatic influence on binding to selected Fcgamma receptors (FcgammaR) and on Fc-mediated immune functions, the effects of all known Fc glycan structures still have not been exhaustively studied. We report that in vitro analyses of pairs of monoclonal human IgG Abs that differ in the amount of sialic acid in their Fc glycans revealed that, for each of the three Ab pairs we examined, higher levels of sialylation were associated with reduced activity in Ab-dependent cellular cytotoxicity (ADCC) assays. This relationship between sialylation and ADCC activity was observed regardless of whether the differences in the extent of sialylation were derived by different Ab production processes, use of a lectin column to separate monoclonal Ab preparations into differentially sialylated fractions, or use of direct in vitro glycoengineering methods to convert a lesser sialylated Ab into a highly sialylated Ab. Subsequent investigations revealed that, depending on the individual Ab and how the differences in sialylation were derived, the lower ADCC potency of the more sialylated variants was apparently due to lower-affinity binding to FcgammaRIIIa on natural killer (NK) cells and/or, more interestingly, lower-affinity binding to cell-surface antigen. Our data provide the first example of an Fc glycan structure impacting antigen binding and suggest that avoiding Fc glycan sialylation can offer another means of optimizing ADCC activity of Abs.