Fibroblast growth factor receptor 1/Klothoß agonist BFKB8488A improves lipids and liver health markers in patients with diabetes or NAFLD: A phase 1b randomized trial.

BACKGROUND AND AIMS: BFKB8488A is a bispecific antibody targeting fibroblast growth factor receptor 1c and Klothoß. This phase 1b study assessed safety, tolerability, pharmacokinetics, immunogenicity, and pharmacodynamics of BFKB8488A in patients with type 2 diabetes mellitus (T2DM) or NAFLD. APPROACH AND RESULTS: Patients were randomized to receive multiple doses of BFKB8488A at various dose levels and dosing intervals (weekly, every 2 weeks, or every 4 weeks) or placebo for 12 weeks. The primary outcome was the safety of BFKB8488A. Overall, 153 patients (T2DM: 91; NAFLD: 62) were enrolled and received at least one dose of treatment. Of these, 102 patients (62.7%) reported at least one adverse event (BFKB8488A: 83 [68.6%]; placebo: 19 [59.4%]). BFKB8488A exhibited nonlinear pharmacokinetics, with greater than dose-proportional increases in exposure. The treatment-emergent antidrug antibody incidence was 22.7%. Overall, trends in exposure-dependent increases in high-density lipoprotein (HDL) and decreases in triglyceride levels were observed. Decreases in alanine aminotransferase and aspartate aminotransferase were 0.7% and 9.2% for medium exposure and 7.3% and 11.2% for high-exposure tertiles, compared with increases of 7.5% and 17% in the placebo group, respectively, at Day 85. In patients with NAFLD, the mean decrease from baseline liver fat was 13.0%, 34.5%, and 49.0% in the low-, medium-, and high-exposure tertiles, respectively, compared with 0.1% with placebo at Day 85. CONCLUSIONS: BFKB8488A was adequately tolerated in patients with T2DM or NAFLD, leading to triglyceride reduction, HDL improvements, and trends in improvement in markers of liver health for both populations and marked liver fat reduction in patients with NAFLD. ( ClinicalTrials.gov : NCT03060538).

Antibody-mediated activation of the FGFR1/Klothoß complex corrects metabolic dysfunction and alters food preference in obese humans.

Fibroblast growth factor 21 (FGF21) controls metabolic organ homeostasis and eating/drinking behavior via FGF receptor 1/Klothoß (FGFR1/KLB) complexes expressed in adipocytes, pancreatic acinar cells, and the nervous system in mice. Chronic administration of recombinant FGF21 or engineered variants improves metabolic health in rodents, nonhuman primates, and humans; however, the rapid turnover of these molecules limits therapeutic utility. Here we show that the bispecific anti-FGFR1/KLB agonist antibody BFKB8488A induced marked weight loss in obese cynomolgus monkeys while elevating serum adiponectin and the adipose expression of FGFR1 target genes, demonstrating its action as an FGF21 mimetic. In a randomized, placebo-controlled, single ascending-dose study in overweight/obese human participants, subcutaneous BFKB8488A injection caused transient body weight reduction, sustained improvement in cardiometabolic parameters, and a trend toward reduction in preference for sweet taste and carbohydrate intake. These data suggest that specific activation of the FGFR1/KLB complex in humans can be used as therapy for obesity-related metabolic defects.

Development of a therapeutic anti-HtrA1 antibody and the identification of DKK3 as a pharmacodynamic biomarker in geographic atrophy.

Genetic polymorphisms in the region of the trimeric serine hydrolase high-temperature requirement 1 (HTRA1) are associated with increased risk of age-related macular degeneration (AMD) and disease progression, but the precise biological function of HtrA1 in the eye and its contribution to disease etiologies remain undefined. In this study, we have developed an HtrA1-blocking Fab fragment to test the therapeutic hypothesis that HtrA1 protease activity is involved in the progression of AMD. Next, we generated an activity-based small-molecule probe (ABP) to track target engagement in vivo. In addition, we used N-terminomic proteomic profiling in preclinical models to elucidate the in vivo repertoire of HtrA1-specific substrates, and identified substrates that can serve as robust pharmacodynamic biomarkers of HtrA1 activity. One of these HtrA1 substrates, Dickkopf-related protein 3 (DKK3), was successfully used as a biomarker to demonstrate the inhibition of HtrA1 activity in patients with AMD who were treated with the HtrA1-blocking Fab fragment. This pharmacodynamic biomarker provides important information on HtrA1 activity and pharmacological inhibition within the ocular compartment.

Prognostic Power of Pathogen Cell-Free DNA in Staphylococcus aureus Bacteremia.

BACKGROUND: Staphylococcus aureus is a leading global cause of bacteremia that can cause invasive tissue infections with high morbidity and mortality despite appropriate antibiotic therapy. Clinicians lack sufficient tools to rapidly identify patients with a poor prognosis to guide diagnostic workup and treatment decisions. Host cell-free DNA provides prognostic value across a spectrum of critical illnesses, including S. aureus bacteremia and sepsis. Metrics of high bacterial load are associated with disease severity in S. aureus bacteremia, and the objective of this study was to evaluate whether incorporating quantitation of cell-free bacterial DNA would provide additive prognostic value when combined with biomarkers of the inflammatory response. METHODS: S. aureus cell-free DNA was measured by quantitative polymerase chain reaction (PCR) in baseline serum samples from an observational cohort of 111 patients with complicated S. aureus bacteremia and correlated with host inflammatory markers and clinical outcomes. RESULTS: High levels of S. aureus cell-free DNA at the time of positive index blood culture were prognostic for all-cause and attributable mortality and persistent bacteremia and were associated with infective endocarditis. However, they did not provide additive value to biomarkers of the host response to infection in multivariate analysis. CONCLUSIONS: Measurements of bacterial load by PCR are a clinically feasible candidate biomarker for stratifying patients at higher risk for complications and poor outcomes. Their diagnostic and prognostic value for identifying foci of infection and influencing treatment remain to be evaluated in additional cohorts.

Sustained Circulating Bacterial Deoxyribonucleic Acid Is Associated With Complicated Staphylococcus aureus Bacteremia.

BACKGROUND: Staphylococcus aureus (SA) bacteremia often requires a long treatment duration with antibiotics to prevent relapse due to the ability of SA to establish reservoirs of infection in sites such as heart and bone. These metastatic sites of infection cannot be serially sampled to monitor the clearance of SA infection. This study aimed to establish a link between persistence of circulating SA deoxyribonucleic acid (SA-DNA) and tissue reservoirs in patients with SA bacteremia. METHODS: A highly sensitive quantitative polymerase chain reaction was used to measure whole blood SA-DNA and plasma-derived SA cell-free DNA (SA-cfDNA) in a set of longitudinal samples from 73 patients with confirmed SA bacteremia and correlated with clinical features. RESULTS: Blood SA-DNA was detected for longer than the duration of positive blood cultures. Longer duration of circulating bacterial DNA was observed in complicated SA bacteremia infections, such as endocarditis and osteoarticular infections, compared with uncomplicated bloodstream infections. In contrast, traditional blood cultures demonstrated similar time to clearance regardless of foci of infection. Plasma-derived SA-cfDNA showed concordance with blood SA-DNA levels. Baseline levels of SA-DNA were higher in patients presenting with greater clinical severity and complicated bacteremia. CONCLUSIONS: Prolonged levels of circulating SA-DNA in patients with complicated tissue reservoirs after clearance of blood cultures observed in this single-center study should be validated in additional cohorts to assess the potential utility for monitoring clearance of infection in patients with SA bacteremia.

A Prognostic Model of Persistent Bacteremia and Mortality in Complicated Staphylococcus aureus Bloodstream Infection.

BACKGROUND: Staphylococcus aureus is a leading cause of bacteremia, yet there remains a significant knowledge gap in the identification of relevant biomarkers that predict clinical outcomes. Heterogeneity in the host response to invasive S. aureus infection suggests that specific biomarker signatures could be utilized to differentiate patients prone to severe disease, thereby facilitating earlier implementation of more aggressive therapies. METHODS: To further elucidate the inflammatory correlates of poor clinical outcomes in patients with S. aureus bacteremia, we evaluated the association between a panel of blood proteins at initial presentation of bacteremia and disease severity outcomes using 2 cohorts of patients with S. aureus bacteremia (n = 32 and n = 124). RESULTS: We identified 13 candidate proteins that were correlated with mortality and persistent bacteremia. Prognostic modeling identified interleukin (IL)-8 and CCL2 as the strongest individual predictors of mortality, with the combination of these biomarkers classifying fatal outcome with 89% sensitivity and 77% specificity (P < .0001). Baseline IL-17A levels were elevated in patients with persistent bacteremia (P < .0001), endovascular (P = .026) and metastatic tissue infections (P = .012). CONCLUSIONS: These results demonstrate the potential utility of selected biomarkers to distinguish patients with the highest risk for treatment failure and bacteremia-related complications, providing a valuable tool for clinicians in the management of S. aureus bacteremia. Additionally, these biomarkers could identify patients with the greatest potential to benefit from novel therapies in clinical trials.

Pharmacokinetics and pharmacodynamics of DSTA4637A: A novel THIOMAB™ antibody antibiotic conjugate against Staphylococcus aureus in mice.

DSTA4637A, a novel THIOMAB™ antibody antibiotic conjugate (TAC) against Staphylococcus aureus (S. aureus), is currently being investigated as a potential therapy against S. aureus infections. Structurally, TAC is composed of an anti-S. aureus antibody linked to a potent antibiotic, dmDNA31. The goal of the current study was to characterize the pharmacokinetics (PK) of TAC in mice, assess the effect of S. aureus infection on its PK, and evaluate its pharmacodynamics (PD) by measuring the bacterial load in various organs at different timepoints following TAC treatment. Plasma concentrations of 3 analytes, total antibody (TAb), antibody-conjugated dmDNA31 (ac-dmDNA31), and unconjugated dmDNA31, were measured in these studies. In non-infected mice (target antigen absent), following intravenous (IV) administration of a single dose of TAC, systemic concentration-time profiles of both TAb and ac-dmDNA31 were bi-exponential and characterized by a short distribution phase and a long elimination phase as expected for a monoclonal antibody-based therapeutic. Systemic exposures of both TAb and ac-dmDNA31 were dose proportional over the dose range tested (5 to 50 mg/kg). In a mouse model of systemic S. aureus infection (target antigen present), a single IV dose of TAC demonstrated PK behavior similar to that in the non-infected mice, and substantially reduced bacterial load in the heart, kidney, and bones on 7 and 14 d post dosing. These findings have increased our understanding of the PK and PK/PD of this novel molecule, and have shown that at efficacious dose levels the presence of S. aureus infection had minimal effect on TAC PK.

Sustained Brown Fat Stimulation and Insulin Sensitization by a Humanized Bispecific Antibody Agonist for Fibroblast Growth Factor Receptor 1/ßKlotho Complex.

Dissipating excess calories as heat through therapeutic stimulation of brown adipose tissues (BAT) has been proposed as a potential treatment for obesity-linked disorders. Here, we describe the generation of a humanized effector-less bispecific antibody that activates fibroblast growth factor receptor (FGFR) 1/ßKlotho complex, a common receptor for FGF21 and FGF19. Using this molecule, we show that antibody-mediated activation of FGFR1/ßKlotho complex in mice induces sustained energy expenditure in BAT, browning of white adipose tissue, weight loss, and improvements in obesity-associated metabolic derangements including insulin resistance, hyperglycemia, dyslipidemia and hepatosteatosis. In mice and cynomolgus monkeys, FGFR1/ßKlotho activation increased serum high-molecular-weight adiponectin, which appears to contribute over time by enhancing the amplitude of the metabolic benefits. At the same time, insulin sensitization by FGFR1/ßKlotho activation occurs even before the onset of weight loss in a manner that is independent of adiponectin. Together, selective activation of FGFR1/ßKlotho complex with a long acting therapeutic antibody represents an attractive approach for the treatment of type 2 diabetes and other obesity-linked disorders through enhanced energy expenditure, insulin sensitization and induction of high-molecular-weight adiponectin.

TMEFF2 is a PDGF-AA binding protein with methylation-associated gene silencing in multiple cancer types including glioma.

BACKGROUND: TMEFF2 is a protein containing a single EGF-like domain and two follistatin-like modules. The biological function of TMEFF2 remains unclear with conflicting reports suggesting both a positive and a negative association between TMEFF2 expression and human cancers. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that the extracellular domain of TMEFF2 interacts with PDGF-AA. This interaction requires the amino terminal region of the extracellular domain containing the follistatin modules and cannot be mediated by the EGF-like domain alone. Furthermore, the extracellular domain of TMEFF2 interferes with PDGF-AA-stimulated fibroblast proliferation in a dose-dependent manner. TMEFF2 expression is downregulated in human brain cancers and is negatively correlated with PDGF-AA expression. Suppressed expression of TMEFF2 is associated with its hypermethylation in several human tumor types, including glioblastoma and cancers of ovarian, rectal, colon and lung origins. Analysis of glioma subtypes indicates that TMEFF2 hypermethylation and decreased expression are associated with a subset of non-Proneural gliomas that do not display CpG island methylator phentoype. CONCLUSIONS/SIGNIFICANCE: These data provide the first evidence that TMEFF2 can function to regulate PDGF signaling and that it is hypermethylated and downregulated in glioma and several other cancers, thereby suggesting an important role for this protein in the etiology of human cancers.

A new ELISA for use in a 3-ELISA system to assess concentrations of VEGF splice variants and VEGF(110) in ovarian cancer tumors.

BACKGROUND: Vascular endothelial growth factor (VEGF), which affects tumor angiogenesis, is expressed as different splice variants, including the major isoforms VEGF(165) and VEGF(121), and can be cleaved by plasmin to generate VEGF(110). The amount of VEGF(121) and VEGF(110) in biological samples has not been well studied. METHODS: We developed an ELISA that detects VEGF(165) and VEGF(121) equally, but does not detect VEGF(110). We used this ELISA together with 2 other ELISAs, one detecting VEGF(165) and the other detecting VEGF(165), VEGF(121), and VEGF(110) equally, to assess the concentrations of VEGF(121) and VEGF(110) in ovarian cancer tumors. RESULTS: The median concentrations in ovarian cancer tumor lysates were 0.61 (range <0.055-74) fmol/mg protein for VEGF(165), 1.4 (range <0.20-500) fmol/mg protein for VEGF(165) plus VEGF(121), and 2.3 (range <0.079-520) fmol/mg protein for total VEGF including VEGF(110) (n = 248). VEGF concentrations measured by the 3 ELISAs were highly correlated (r = 0.91-0.94). Median estimated VEGF(121) and VEGF(110) concentrations were 0.77 and 0.58 fmol/mg protein, respectively. In lysates with measurable VEGF(165) and total VEGF concentrations, mean VEGF(165) was approximately 31% (SD 23%) of the total VEGF (n = 217). In contrast, VEGF(165) constituted approximately half of the total circulating VEGF. CONCLUSION: VEGF(165), VEGF(121), and VEGF(110) may be present at significant amounts in ovarian cancer tumors.

Caregiver and patient reported outcomes after repair of cleft lip and/or palate in the Philippines.

OBJECTIVES: To establish the feasibility of conducting outcomes research among patients treated during a medical mission and to identify the salient outcomes for patients and caregivers in one region of the Philippines. DESIGN AND SETTING: Prospective structured interview conducted in or near participants' homes on the island of Cebu, Philippines. PARTICIPANTS: Individuals who had surgery at least 6 months earlier within the study region were eligible. Recipients of surgery were located from surgical records and by word of mouth. MAIN OUTCOME MEASURES: (1) Proportion of individuals located. (2) Primary outcomes of oral cleft repair identified for the sample. RESULTS: Of 99 people on a surgical list, 52 (53%) were located, eight were excluded (ineligible, unavailable, or inaccessible), and 44 agreed to participate in the study. Ten participants were identified via word of mouth. Seventy-five interviews were conducted (21 caregiver-patient pairs, one adolescent, and 32 caregivers of a child <7 years). Nearly all participants (99%) would encourage others to pursue surgery. Open-ended questions were coded to identify primary outcomes: improved speech (52%), improved eating (25%), social benefits (14%), and improved appearance (6%). Caregivers (50%) and patients (68%) reported that improved speech was the most important change after surgery. CONCLUSIONS: Patients and caregivers ascribe positive changes related to cleft surgery. Outcomes research requires cooperation with local professionals who can communicate effectively. These data serve to demonstrate feasibility and as a model for future studies of treatment outcomes in follow-up to international medical missions.

Mice expressing a humanized form of VEGF-A may provide insights into the safety and efficacy of anti-VEGF antibodies.

VEGF-A is important in tumor angiogenesis, and a humanized anti-VEGF-A monoclonal antibody (bevacizumab) has been approved by the FDA as a treatment for metastatic colorectal and nonsquamous, non-small-cell lung cancer in combination with chemotherapy. However, contributions of both tumor- and stromal-cell derived VEGF-A to vascularization of human tumors grown in immunodeficient mice hindered direct comparison between the pharmacological effects of anti-VEGF antibodies with different abilities to block host VEGF. Therefore, by gene replacement technology, we engineered mice to express a humanized form of VEGF-A (hum-X VEGF) that is recognized by many anti-VEGF antibodies and has biochemical and biological properties comparable with WT mouse and human VEGF-A. The hum-X VEGF mouse model was then used to compare the activity and safety of a panel of VEGF Mabs with different affinities for VEGF-A. Although in vitro studies clearly showed a correlation between binding affinity and potency at blocking endothelial cell proliferation stimulated by VEGF, in vivo experiments failed to document any consistent correlation between antibody affinity and the ability to inhibit tumor growth and angiogenesis in most animal models. However, higher-affinity antibodies were more likely to result in glomerulosclerosis during long-term treatment.

Cross-species vascular endothelial growth factor (VEGF)-blocking antibodies completely inhibit the growth of human tumor xenografts and measure the contribution of stromal VEGF.

To fully assess the role of VEGF-A in tumor angiogenesis, antibodies that can block all sources of vascular endothelial growth factor (VEGF) are desired. Selectively targeting tumor-derived VEGF overlooks the contribution of host stromal VEGF. Other strategies, such as targeting VEGF receptors directly or using receptor decoys, result in inhibiting not only VEGF-A but also VEGF homologues (e.g. placental growth factor, VEGF-B, and VEGF-C), which may play a role in angiogenesis. Here we report the identification of novel anti-VEGF antibodies, B20 and G6, from synthetic antibody phage libraries, which block both human and murine VEGF action in vitro. Their affinity-improved variants completely inhibit three human tumor xenografts in mice of skeletal muscle, colorectal, and pancreatic origins (A673, HM-7, and HPAC). Avastin, which only inhibits the tumor-derived human VEGF, is approximately 90% effective at inhibiting HM-7 and A673 growth but is <50% effective at inhibiting HPAC growth. Indeed, HPAC tumors contain more host stroma invasion and stroma-derived VEGF than other tumors. Thus, the functional contribution of stromal VEGF varies greatly among tumors, and systemic blockade of both tumor and stroma-derived VEGF is sufficient for inhibiting the growth of tumor xenografts.

Association between HER-2/neu and vascular endothelial growth factor expression predicts clinical outcome in primary breast cancer patients.

PURPOSE: Activation or overexpression of HER-2/neu is associated with up-regulation of vascular endothelial growth factor (VEGF) in human breast cancer cells in vitro. Preclinical experiments indicate that increased expression of VEGF may in part mediate the biologically aggressive phenotype of HER-2/neu-overexpressing human breast cancer. It was the purpose of this study to: (a). evaluate the association between HER-2/neu and VEGF expression in a large clinical cohort of primary breast cancer patients; (b). compare the prognostic significance of VEGF isoforms; and (c). analyze the combined effects of HER-2/neu and VEGF on clinical outcome. EXPERIMENTAL DESIGN: HER-2/neu and VEGF were measured by ELISA in primary breast tumor tissue lysates from 611 unselected patients with a median clinical follow-up of 50 months. At least six VEGF isoforms consisting of 121, 145, 165, 183, 189, or 206 amino acids are generated as a result of alternative splicing. The VEGF(121-206) ELISA uses antibodies that bind to VEGF(121) and, therefore, detects all of the VEGF isoforms with 121 and more amino acids. The VEGF(165-206) ELISA uses antibodies that bind to VEGF(165) and, therefore, detects all of the VEGF isoforms with 165 and more amino acids. VEGF(121-206) and VEGF(165-206) were analyzed both as continuous and categorical variables, using detectable expression as a cutoff for positivity. Cell lines with defined HER-2/neu expression levels were used to establish a cutoff point for HER-2/neu overexpression in breast tumor samples. RESULTS: Our findings indicate a significant positive association between HER-2/neu and VEGF expression. VEGF(121-206) and VEGF(165-206) expression was detectable in 88 (77.2%) and 100 (87.7%), respectively, of the 114 patients with HER-2/neu-overexpressing tumors, in contrast to 271 (54.5%) and 353 (71.0%), respectively, of the 497 patients with nonoverexpressing tumors (chi(2) test: P < 0.001 for both VEGF(121-206) and VEGF(165-206)). VEGF(121-206) and VEGF(165-206) demonstrate a comparable prognostic significance for survival in unselected primary breast cancer patients (univariate analysis: VEGF(121-206), P = 0.0068; VEGF(165-206), P = 0.0046; multivariate analysis: VEGF(121-206), P = 0.1475; VEGF(165-206), P = 0.1483). When the analyses were performed separately for node-negative and node-positive patients, VEGF(121-206) and VEGF(165-206) were of prognostic significance for survival only in node-positive patients (univariate analysis: VEGF(121-206), P = 0.0003; VEGF(165-206), P = 0.0038; multivariate analysis: VEGF(121-206), P = 0.0103; VEGF(165-206), P = 0.0150). A biological concentration-effect relationship between VEGF expression and survival (VEGF(121-206), P = 0.0280; VEGF(165-206,) P = 0.0097) suggests that VEGF levels, as determined by ELISA, could be of importance as a predictive marker for therapeutic strategies that target VEGF. Combining HER-2/neu and VEGF(121-206)/VEGF(165-206) results in additional prognostic information for survival (VEGF(121-206), P = 0.0133; VEGF(165-206), P = 0.0092). CONCLUSION: The positive association between HER-2/neu and VEGF expression implicates VEGF in the aggressive phenotype exhibited by HER-2/neu overexpression, and supports the use of combination therapies directed against both HER-2/neu and VEGF for treatment of breast cancers that overexpress HER-2/neu.