Luspatercept in Patients with Lower-Risk Myelodysplastic Syndromes.

BACKGROUND: Patients with anemia and lower-risk myelodysplastic syndromes in whom erythropoiesis-stimulating agent therapy is not effective generally become dependent on red-cell transfusions. Luspatercept, a recombinant fusion protein that binds transforming growth factor ß superfamily ligands to reduce SMAD2 and SMAD3 signaling, showed promising results in a phase 2 study. METHODS: In a double-blind, placebo-controlled, phase 3 trial, we randomly assigned patients with very-low-risk, low-risk, or intermediate-risk myelodysplastic syndromes (defined according to the Revised International Prognostic Scoring System) with ring sideroblasts who had been receiving regular red-cell transfusions to receive either luspatercept (at a dose of 1.0 up to 1.75 mg per kilogram of body weight) or placebo, administered subcutaneously every 3 weeks. The primary end point was transfusion independence for 8 weeks or longer during weeks 1 through 24, and the key secondary end point was transfusion independence for 12 weeks or longer, assessed during both weeks 1 through 24 and weeks 1 through 48. RESULTS: Of the 229 patients enrolled, 153 were randomly assigned to receive luspatercept and 76 to receive placebo; the baseline characteristics of the patients were balanced. Transfusion independence for 8 weeks or longer was observed in 38% of the patients in the luspatercept group, as compared with 13% of those in the placebo group (P<0.001). A higher percentage of patients in the luspatercept group than in the placebo group met the key secondary end point (28% vs. 8% for weeks 1 through 24, and 33% vs. 12% for weeks 1 through 48; P<0.001 for both comparisons). The most common luspatercept-associated adverse events (of any grade) included fatigue, diarrhea, asthenia, nausea, and dizziness. The incidence of adverse events decreased over time. CONCLUSIONS: Luspatercept reduced the severity of anemia in patients with lower-risk myelodysplastic syndromes with ring sideroblasts who had been receiving regular red-cell transfusions and who had disease that was refractory to or unlikely to respond to erythropoiesis-stimulating agents or who had discontinued such agents owing to an adverse event. (Funded by Celgene and Acceleron Pharma; MEDALIST ClinicalTrials.gov number, NCT02631070; EudraCT number, 2015-003454-41.).

Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21.

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.

Unliganded fibroblast growth factor receptor 1 forms density-independent dimers.

Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Förster resonance energy transfer combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full-length FGFR1 at the cell-surface with or without the coreceptor ßKlotho. Using this approach we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR) and was further increased by the addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and ßKlotho. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface.

Venetoclax and Cobimetinib in Relapsed/Refractory AML: A Phase 1b Trial.

BACKGROUND: Therapies for relapsed/refractory acute myeloid leukemia remain limited and outcomes poor, especially amongst patients who are ineligible for cytotoxic chemotherapy or targeted therapies. PATIENTS AND METHODS: This phase 1b trial evaluated venetoclax, a B-cell lymphoma-2 (BCL-2) inhibitor, plus cobimetinib, a MEK1/2 inhibitor, in patients with relapsed/refractory acute myeloid leukemia, ineligible for cytotoxic chemotherapy. Two-dimensional dose-escalation was performed for venetoclax dosed daily, and for cobimetinib dosed on days 1-21 of each 28-day cycle. RESULTS: Thirty patients (median [range] age: 71.5 years [60-84]) received venetoclax-cobimetinib. The most common adverse events (AEs; in ≥40.0% of patients) were diarrhea (80.0%), nausea (60.0%), vomiting (40.0%), febrile neutropenia (40.0%), and fatigue (40.0%). Overall, 66.7% and 23.3% of patients experienced AEs leading to dose modification/interruption or treatment withdrawal, respectively. The composite complete remission (CRc) rate (complete remission [CR] + CR with incomplete blood count recovery + CR with incomplete platelet recovery) was 15.6%; antileukemic response rate (CRc + morphologic leukemia-free state/partial remission) was 18.8%. For the recommended phase 2 dose (venetoclax: 600 mg; cobimetinib: 40 mg), CRc and antileukemic response rates were both 12.5%. Failure to achieve an antileukemic response was associated with elevated baseline phosphorylated ERK and MCL-1 levels, but not BCL-xL. Baseline mutations in ≥1 signaling gene or TP53 were noted in nonresponders and emerged on treatment. Pharmacodynamic biomarkers revealed inconsistent, transient inhibition of the mitogen-activated protein kinase (MAPK) pathway. CONCLUSION: Venetoclax-cobimetinib showed limited preliminary efficacy similar to single-agent venetoclax, but with added toxicity. Our findings will inform future trials of BCL-2/MAPK pathway inhibitor combinations.

Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP).

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

Tethered-variable CL bispecific IgG: an antibody platform for rapid bispecific antibody screening.

Bispecific antibodies offer a clinically validated platform for drug discovery. In generating functionally active bispecific antibodies, it is necessary to identify a unique parental antibody pair to merge into a single molecule. However, technologies that allow high-throughput production of bispecific immunoglobulin Gs (BsIgGs) for screening purposes are limited. Here, we describe a novel bispecific antibody format termed tethered-variable CLBsIgG (tcBsIgG) that allows robust production of intact BsIgG in a single cell line, concurrently ensuring cognate light chain pairing and preserving key antibody structural and functional properties. This technology is broadly applicable in the generation of BsIgG from a variety of antibody isotypes, including human BsIgG1, BsIgG2 and BsIgG4. The practicality of the tcBsIgG platform is demonstrated by screening BsIgGs generated from FGF21-mimetic anti-Klotho-ß agonistic antibodies in a combinatorial manner. This screen identified multiple biepitopic combinations with enhanced agonistic activity relative to the parental monoclonal antibodies, thereby demonstrating that biepitopic antibodies can acquire enhanced functionality compared to monospecific parental antibodies. By design, the tcBsIgG format is amenable to high-throughput production of large panels of bispecific antibodies and thus can facilitate the identification of rare BsIgG combinations to enable the discovery of molecules with improved biological function.

An efficient route to bispecific antibody production using single-reactor mammalian co-culture.

Bispecific antibodies have shown promise in the clinic as medicines with novel mechanisms of action. Lack of efficient production of bispecific IgGs, however, has limited their rapid advancement. Here, we describe a single-reactor process using mammalian cell co-culture production to efficiently produce a bispecific IgG with 4 distinct polypeptide chains without the need for parallel processing of each half-antibody or additional framework mutations. This method resembles a conventional process, and the quality and yield of the monoclonal antibodies are equal to those produced using parallel processing methods. We demonstrate the application of the approach to diverse bispecific antibodies, and its suitability for production of a tissue specific molecule targeting fibroblast growth factor receptor 1 and klotho ß that is being developed for type 2 diabetes and other obesity-linked disorders.

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.