Kelly Byrnes-Blake

Phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis C virus infection.

Interferon lambda 1 (IFN‐λ1) is a type III IFN that produces intracellular responses similar to those of IFN‐α but in fewer cell types because of differences in the receptor distribution pattern, and this could potentially result in an improved safety profile. This was an open‐label three‐part study of patients with chronic hepatitis C virus (HCV) genotype 1 infection. Part 1 evaluated single‐agent pegylated interferon lambda (PEG‐IFN‐λ) at 1.5 or 3.0 μg/kg administered every 2 weeks or weekly for 4 weeks in patients who had relapsed after previous IFN‐α‐based treatment. Part 2 evaluated weekly doses of PEG‐IFN‐λ ranging from 0.5 to 2.25 μg/kg in combination with ribavirin (RBV) for 4 weeks in treatment‐relapse patients. Part 3 evaluated weekly PEG‐IFN‐λ at 1.5 μg/kg in combination with RBV for 4 weeks in treatment‐naive patients. Fifty‐six patients were enrolled: 24 patients in part 1, 25 patients in part 2, and 7 patients in part 3. Antiviral activity was observed at all PEG‐IFN‐λ dose levels (from 0.5 to 3.0 μg/kg). Two of seven treatment‐naive patients (29%) achieved rapid virological response. Treatment was well tolerated with minimal flu‐like symptoms and no significant hematologic changes other than RBV‐associated decreases in hemoglobin. The most common adverse events were fatigue (29%), nausea (12%), and myalgia (11%). Six patients experienced increases in aminotransferases that met protocol‐defined criteria for dose‐limiting toxicity (DLT) or temporarily holding therapy with PEG‐IFN‐λ. Most DLT occurred in patients with high PEG‐IFN‐λ exposure. Conclusion: Weekly PEG‐IFN‐λ with or without daily RBV for 4 weeks is well tolerated with minimal adverse events and hematologic effects and is associated with clear antiviral activity across a broad range of doses in patients with chronic HCV. (HEPATOLOGY 2010;)

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.

Targeting Immune Complex-Mediated Hypersensitivity with Recombinant Soluble Human FcγRIA (CD64A)

Binding of Ag-Ab immune complexes to cellular FcγR promotes cell activation, release of inflammatory mediators, and tissue destruction characteristic of autoimmune disease. To evaluate whether a soluble FcγR could block the proinflammatory effects of immune complexes, recombinant human (rh) versions of FcγRIA, FcγRIIA, and FcγRIIIA were prepared. Binding of rh-FcγRIA to IgG was of high affinity (KD = 1.7 × 10−10 M), whereas rh-FcγRIIA and rh-FcγRIIIA bound with low affinity (KD = 0.6–1.9 × 10−6 M). All rh-FcγR reduced immune complex precipitation, blocked complement-mediated lysis of Ab-sensitized RBC, and inhibited immune complex-mediated production of IL-6, IL-13, MCP-1, and TNF-α by cultured mast cells. Local or systemic delivery only of rh-FcγRIA, however, reduced edema and neutrophil infiltration in the cutaneous Arthus reaction in mice. 125I-labeled rh-FcγRIA was cleared from mouse blood with a rapid distribution phase followed by a slow elimination phase with a t1/2γ of ∼130 h. The highest percentage of injected radioactivity accumulated in blood ∼ liver ∼ carcass > kidney. s.c. dosing of rh-FcγRIA resulted in lower serum levels of inflammatory cytokines and prevented paw swelling and joint damage in a murine model of collagen Ab-induced arthritis. These data demonstrate that rh-FcγRIA is an effective inhibitor of type III hypersensitivity.

Recombinant Soluble Human FcγR1A (CD64A) Reduces Inflammation in Murine Collagen-Induced Arthritis

Binding of immune complexes to cellular FcγRs can promote cell activation and inflammation. In previous studies, a recombinant human (rh) soluble FcγR, rh-FcγRIA (CD64A), was shown to block inflammation in passive transfer models of immune complex-mediated disease. To assess whether rh-FcγRIA could block inflammation in a T cell- and B cell-dependent model of immune complex-mediated disease, the efficacy of rh-FcγRIA in collagen-induced arthritis was evaluated. Mice with established arthritis were treated with a single s.c. injection of rh-FcγRIA (0.2–2.0 mg/dose) given every other day for 11 days. Relative to mice injected with vehicle alone, mice treated with rh-FcγRIA exhibited lower serum concentrations of IL-6, anti-type II collagen Abs, and total IgG2a. These changes were correlated with lower levels of paw swelling and joint damage in the rh-FcγRIA-treated mice and occurred in the presence of a significant murine Ab response to rh-FcγRIA. Comparison of the serum rh-FcγRIA concentration vs time profiles for rh-FcγRIA administered at two dose levels by i.v. and s.c. injection revealed that the bioavailabilty of s.c. administered rh-FcγRIA was 27–37%. Taken together, these data show that rh-FcγRIA is an effective inhibitor of inflammation in a model of established arthritis in mice.

Pharmacokinetics and pharmacodynamics of pegylated interferon lambda-1 in cynomolgus monkeys.

Type III lambda interferons (IFNs) have activity similar to type I IFNs, but a more restricted receptor distribution. A pegylated human IFN lambda-1 (pegIFNλ) is under development for chronic hepatitis C. Induction of receptor signaling (STAT1 phosphorylation) and expression of interferon-stimulated genes (ISGs) by pegIFNλ were assessed in, respectively, cynomolgus monkey leukocyte subsets and hepatocytes stimulated in vitro. ISG induction by pegIFNλ or IFNα was also assessed in peripheral leukocytes and liver biopsies after single and repeat (x3) dosing of pegIFNλ (0.03, 0.3, 3.0 mg/kg) or unpegylated IFNα-2b (10(7) IU/kg). Single-dose pharmacokinetics of pegIFNλ were evaluated. Strong ISG induction occurred in cultured hepatocytes and liver biopsies with both pegIFNλ and IFNα. However, STAT1 phosphorylation, MHC class 1 upregulation, and ISG induction in leukocytes only occurred with IFNα. Serum neopterin was unaffected by pegIFNλ; however, β-2-microglobulin was elevated at all doses. The terminal half-life of pegIFNλ was 23 h with a 59 mL/kg volume of distribution, consistent with other pegylated IFNs. Serum exposure was dose-proportional across the dosing range. These data demonstrate the suitability of cynomolgus monkeys for the preclinical evaluation of pegIFNλ. Additionally, the absence of pegIFNλ pharmacologic activity in leukocytes is consistent with its low receptor expression in blood.

Preclinical Safety, Pharmacokinetics, and Pharmacodynamics of Recombinant Human Interleukin-21 in Cynomolgus Macaques (Macaca fascicularis)

Interleukin-21 (IL-21), a pleiotropic immunostimulatory type I cytokine, has anticancer effects in animal models. Preclinical studies designed to assess the safety of recombinant human IL-21 (rIL-21) for use in phase I oncology studies are described. The rIL-21 (≤3.0 mg/kg per dose) was given intravenously to cynomolgus monkeys (Macaca fascicularis) once daily for 5 days, followed by 9 nondosing days (1 cycle) for ≤4 cycles. The rIL-21 pharmacokinetics was dose-dependent. Accumulation was not observed after repeated dosing, consistent with the short elimination half-life (t 1/2,λz; 0.4-0.8 hours). Safety findings included cyclical anemia and thrombocytopenia, clinical pathology changes consistent with acute-phase response, leukocyte infiltrates in hepatic sinusoids, and sporadic serum transaminase elevations (typically <3 times upper-limit of normal); all were reversible upon cessation of treatment. Decreased pharmacodynamic responses with time corresponded to the development of anti-rIL-21 antibodies; effects varied among individuals and were dose-dependent. These studies demonstrated rIL-21 to be generally well-tolerated when administered to cynomolgus monkeys, and all adverse effects were reversible upon treatment cessation. However, development of anti-rIL-21 antibodies may limit the use of this species in long-term studies.

Nonclinical Profile of BLZ-100, a Tumor-Targeting Fluorescent Imaging Agent:

BLZ-100 is a single intravenous use, fluorescent imaging agent that labels tumor tissue to enable more complete and precise surgical resection. It is composed of a chlorotoxin peptide covalently bound to the near-infrared fluorophore indocyanine green. BLZ-100 is in clinical development for intraoperative visualization of human tumors. The nonclinical safety and pharmacokinetic (PK) profile of BLZ-100 was evaluated in mice, rats, canines, and nonhuman primates (NHP). Single bolus intravenous administration of BLZ-100 was well tolerated, and no adverse changes were observed in cardiovascular safety pharmacology, PK, and toxicology studies in rats and NHP. The single-dose no-observed-adverse-effect-levels (NOAELs) were 7 mg (28 mg/kg) in rats and 60 mg (20 mg/kg) in NHP, corresponding to peak concentration values of 89 400 and 436 000 ng/mL and area-under-the-curve exposure values of 130 000 and 1 240 000 h·ng/mL, respectively. Based on a human imaging dose of 3 mg, dose safety margins are >100 for rat and monkey. BLZ-100 produced hypersensitivity reactions in canine imaging studies (lethargy, pruritus, swollen muzzle, etc). The severity of the reactions was not dose related. In a follow-up study in dogs, plasma histamine concentrations were increased 5 to 60 minutes after BLZ-100 injection; this coincided with signs of hypersensitivity, supporting the conclusion that the reactions were histamine based. Hypersensitivity reactions were not observed in other species or in BLZ-100 human clinical studies conducted to date. The combined imaging, safety pharmacology, PK, and toxicology studies contributed to an extensive initial nonclinical profile for BLZ-100, supporting first-in-human clinical trials.