Expression and Purification
VRS-317 (XTEN1–rhGH–XTEN2) was expressed as a fusion protein of rhGH and hydrophilic, unstructured amino acids, XTEN. The XTEN domains of VRS-317 increased the solubility and stability of the rhGH domain. Unlike rhGH, VRS-317 was soluble in the cytoplasm of E. coli
and no inclusion bodies were observed. Purification was facilitated by the increased acid stability of VRS-317. After cell lysis, treatment with acetic acid caused precipitation of most host cell proteins with minimal VRS-317 loss. The negative charge from the glutamate residues in the XTEN domain resulted in a pI
of approximately 3 for VRS-317, as compared with the pI
of 5.2 for rhGH.19
These properties enabled the use of ion-exchange methods to remove host cell proteins, DNA, and aggregates of VRS-317. As shown in , the final purified material was a single band on a nonreducing SDS-PAGE gel slightly larger than its calculated molecular weight (MW) of 119 kDa and did not contain aggregates as measured by size exclusion chromatography. The large size and unstructured nature of the XTEN domains provide a large hydrodynamic radius as noted by the early elution time in the size exclusion chromatogram,17
as confirmed by size exclusion chromatography with multiangle light scattering (112,290 Da; data not shown). The large hydrodynamic radius introduced by the XTEN sequence and the low pI
of VRS-317 also cause VRS-317 to migrate less than the globular protein markers on the SDS-PAGE gel (e.g., gamma-globulin, 158 kDa, pI
~ 6.9). The XTEN1–rhGH construct was expressed and purified yielding a similar high-purity fusion protein for further characterization.
Figure 1 (a) SDS-PAGE of 2, 5, and 10 μg of VRS-317 compared with a molecular weight marker. (b) Size exclusion HPLC analysis of VRS-317 (solid curve) compared with a set of globular molecular weight standards (dashed curve, Bio-Rad Laboratories, Hercules, (more ...)
An early construct of VRS-317 was designed to maximize the binding to the hGH receptor in vitro. This construct was a fusion protein of a long XTEN sequence at the N-terminus (83.6 kDa) and rhGH at the C-terminus (XTEN1–rhGH; 105.6 kDa). To assess the impact of reduced receptor binding, a short XTEN domain (XTEN2; 13.3 kDa) was added to the C-terminus of rhGH along with the long XTEN (XTEN1) domain at the N-terminus. There was no significant effect of the long N-terminal XTEN (XTEN1–rhGH) on binding affinity when compared with rhGH in an ELISA-based receptor binding assay. However, VRS-317 (XTEN1–rhGH–XTEN2) had an approximately 11-fold reduction in affinity (IC50 = 27 nM) compared with rhGH (IC50 = 2.5 nM) (a). The ability of VRS-317 to bind to two hGH receptors and cause dimerization that results in intracellular signaling was tested using a cell line engineered to proliferate in response to rhGH. In this in vitro potency assay, the EC50 of VRS-317 was 12-fold (6.8 nM) greater than rhGH (0.6 nM) indicating a lower in vitro potency for VRS-317 (b).
Because the XTEN constructs had significantly larger molecular mass than rhGH, the analyses below were performed using both mass (mg/kg) and molar (nmol/kg) dosing to provide an estimate of the rhGH dose administered in each case (rhGH MW: 22 kDa). VRS-317 (119 kDa) had a molecular mass approximately fivefold greater than rhGH.
To assess the PK of VRS-317 and XTEN1–rhGH, studies were performed in rats and monkeys (). Rats were dosed s.c. with approximately equal molar doses of rhGH, XTEN1—rhGH, or VRS-317. In addition, XTEN1–rhGH and VRS-317 were dosed i.v. to measure the t1/2 of each protein. As shown in a, rhGH has a rapid clearance after s.c. administration. XTEN1–rhGH and VRS-317 had a slower absorption phase, greater Cmax, and a longer t1/2 than rhGH. The terminal elimination half-lives of XTEN1–rhGH and VRS-317 determined after i.v administration in rats were 6.8 and 15 h, respectively. The PK of XTEN1–rhGH and VRS-317 was also determined in monkeys after a single s.c. dose. As shown in b, both the XTEN1–rhGH and VRS-317 achieved a similar Cmax within 48 h postdose, but VRS-317 had a significantly longer half-life (XTEN1–rhGH: 48.6 h; VRS-317: 110 h).
Figure 3 (a) Pharmacokinetics in rats after subcutaneous (s.c.) administration of 0.3 mg/kg (13.5 nmol/kg) rhGH (•), 1.5 mg/kg (13.2 nmol/kg) of XTEN1–rhGH (; dashed line), or 1.5 mg/kg (12.6 nmol/kg) of VRS-317 (). (b) The pharmacokinetics (more ...)
To determine the rate and extent of absorption of VRS-317 after s.c. or i.m. injection, the PK of VRS-317 was studied in monkeys after i.v. (n = 4), s.c. (n = 3) or i.m. (n = 3) administration of 1.5 mg/kg (12.6 nmol/kg) VRS-317. VRS-317 was rapidly absorbed after either s.c. or i.m. administration (). The Cmax relative to that following i.v. injection was 78.5% and 89% for s.c. and i.m., respectively. The bioavailability based on AUC0–t was 101% and 103% for s.c. and i.m., respectively. These results demonstrated that VRS-317 was rapidly and near completely absorbed after s.c. administration.
The dose proportionality of the PKs of VRS-317 was studied after s.c. administration in cynomolgus monkeys as part of a safety study. The PK profile of VRS-317 after single s.c. administration to monkeys was approximately linear with dose (). These results indicate that the absorption and clearance of VRS-317 was not dependent upon dose level between 1 and 25 mg/kg (8.4–210 nmol/kg).
Pharmacokinetic Parameters (Mean ± SD) Estimated by Noncompartment Methods After Single Subcutaneous Doses of VRS-317 in Cynomolgus Monkeys
Repeat dose PK was evaluated in juvenile cynomolgus monkeys (two per sex per group) administered s.c. doses of 0.4 or 1.4 mg/kg (3.4 or 11.8 nmol/kg) VRS-317 every 28 days or 0.05 mg/kg/day rhGH for 28 days. The PKs of VRS-317 were determined after each dose and the PKs of rhGH were determined after first (Day 1) and last (Day 28) dose as shown in . A consistent and dose proportional Cmax (0.4 mg/kg: 40 ± 17 nM; 1.4 mg/kg: 100 ± 34.5 nM) was noted after each administration. No accumulation was noted on the basis of comparable Cmax and exposure (AUC-0.4 mg/kg: 4440 ± 1209 nM-h; 1.4 mg/kg: 16800 ± 3586 nM-h) after each administration.
Figure 5 (a) Pharmacokinetics of VRS-317 dosed s.c. at 0.4 (□) or 1.4 () mg/kg (3.4 or 11.8 nmol/kg) VRS-317 every 28 days in juvenile cynomolgus monkeys (genders combined). (b) Pharmacokinetics of 0.05 mg/(kg day) rhGH dosed s.c. in juvenile (more ...)
Weight gain in a hypophysectomized-rat-administered daily rhGH for 6 or 7 days was the standard historical method for assessment of rhGH potency. To assess the in vivo potency of VRS-317, different dose regimens of VRS-317 were compared with a fixed dose of daily rhGH in hypophysectomized rats dosed s.c.
In the first experiment, VRS-317 dosed every other day at 0.34 mg/kg (12 nmol/kg total) produced the same total weight gain as daily dosing of 0.10 mg/kg hGH (35 nmol/kg total). There was also no difference in the thickness of the tibial epiphyses between the two treatment groups (). Higher doses of VRS-317 produced an increased weight gain and tibial epiphyses thickness compared with daily rhGH treatment. These results indicated a greater in vivo potency of VRS-317 relative to rhGH on a molar basis. In addition, VRS-317 demonstrated a direct effect on bone growth in this model.
Potency Assessment of Different Doses and Regimens of VRS-317 Compared with rhGH and Placebo in Hypophysectomized Rats
To evaluate the relationship between VRS-317 plasma levels and potency in hypophysectomized rats, an additional dose ranging study was performed comparing lower doses of VRS-317 with daily-administered rhGH. A total dose of 1 mg/kg (9 nmol/kg) of VRS-317 [3 nmol/kg every other day or 1.5 nmol/kg per day] produced a weight gain slightly greater than a total hGH dose of 35 nmol/kg (Experiment 2, ). Similar effects were also noted on the thickness of tibial epiphyses after sacrifice of the rats on Day 7. Using single dose PK data following s.c. administration of VRS-317 or rhGH in rats, the relationship between plasma exposure and efficacy in hypophysectomized rats was modeled to calculate the steady-state plasma concentration (Css) of VRS-317. These results indicate that sustaining a level of VRS-317 above 1 nM provides a growth response comparable to a daily dose of rhGH in hypophysectomized rats.
The effect of sustained VRS-317 exposure compared with daily rhGH administration was also assessed in juvenile cynomolgus monkeys. As shown in , 1.4 mg/kg (11.8 nmol/kg) VRS-317 stimulated an IGF-I and IGFBP-3 response comparable to 0.05 mg/kg/day rhGH in juvenile cynomolgus monkeys over 28 days (Total rhGH dose: 1.4 mg/kg or 64 nmol/kg). The pharmacodynamic response was achieved at approximately a sixfold lower molar dose than daily hGH indicating that the sustained levels of VRS-317 stimulated the pharmacodynamic response continuously over the 28 days. The reduced pharmacodynamic response noted around 14 days after dosing of 0.4 mg/kg (3.4 nmol/kg) VRS-317 correlated with the plasma levels of VRS-317 decreasing below 1 nM (119 ng/mL VRS-317; a).
Figure 6 IGF-I (a) and IGFBP-3 (b) changes from baseline in juvenile cynomolgus monkeys after administration of placebo (×), 0.05 mg/(kg day) rhGH for 28 days (; 1.4 mg/kg rhGH; 64 nmol/kg), 0.40 (□) or 1.4 mg/kg (; 3.4 or 11.8 (more ...)
A similar IGF-I response was noted in adult cynomolgus monkeys (four males per group) administered a single s.c. dose of VRS-317. A dose dependent increase in IGF-I levels was noted in monkeys administered 0.3, 1.5, or 7.5 mg/kg (2.5, 12.6, or 63 nmol/kg, respectively) VRS-317 with mean maximum increases above baseline (predose) of 71%, 204%, and 335%.
Safety Assessment of VRS-317
To date, VRS-317 has been administered to rats, pigs, and monkeys as single doses and to rats and monkeys as repeat doses up to 25 mg/kg (63 nmol/kg). No observable clinical signs or symptoms have been noted in these studies.
In the juvenile cynomolgus monkey study, no test article-related effects among lipid parameters were identified in any group at any interval. All individual animal variations were considered to be within the range of normal biologic variability for this species. Therefore, no adverse effects were noted in monkeys dosed with 0.40 or 1.4 mg/kg VRS-317 every 28 days for 84 days. At Day 85, two monkeys dosed 1.4 mg/kg VRS-317 developed low titer antibodies against VRS-317 that were specific to the rhGH domain. This antibody response was probably caused by the lack of homology between cynomolgus GH and rhGH. No anti-XTEN antibodies were detected. There were no detectable differences in VRS-317 PK or the pharmacodynamics response in these monkeys, suggesting that the low titer antibody responses did not have a significant clinical effect.
In the 14-week GLP monkey toxicology study, monkeys were administered placebo or 1, 5, or 25 mg/kg VRS-317 every other week. There were no VRS-317-related effects on the following parameters: clinical observations, dermal irritation scores, body weights, ophthalmoscopic examinations, respiratory rate, electrocardiographic parameters, coagulation parameters, clinical chemistry analytes, urinalysis parameters, macroscopic observations, or organ weights. There were no adverse histopathology changes attributed to VRS-317. In particular, no significant differences in glucose or lipid metabolism, mammary or prostate tissues, or brain tissues were noted between placebo and VRS-317 treated animals. None of the effects noted during this study were considered adverse. Therefore, the No-Observed-Adverse-Effect Level (NOAEL) for this study was 25.0 mg/kg per dose of VRS-317. The proposed highest dose in the ongoing single administration Phase 1 trial in adult GHD patients is 0.8 mg/kg (based on a 70 kg human), which on a human equivalent dose basis is approximately 10-fold below the NOAEL established in this repeat-dose toxicology study in monkeys.
Because lipoatrophy has been noted for PEGylated rhGH, the potential for lipoatrophy caused by s.c. administration of VRS-317 was extensively studied in pigs and monkeys. Four healthy naïve female Sinclair miniature swine were injected s.c. at three injection-site grids per pig (12 total grids). In each injection-site grid, there was a placebo control, an rhGH control (injected every 8 h), 20 mg methylprednisolone, or 5.4 or 27 mg of VRS-317. Injection sites were harvested by biopsy from 24 to 96 h postdose. Histology and histomorphometry were conducted on each biopsy sample. There were no visible changes except for the rhGH control in which inflammation and tissue damage resulted from repeated injections. There was no significant lipoatrophy observed for any of the treatments at any of the time points by morphometry. These data suggest that VRS-317 does not cause overt lipoatrophy.
No lipoatrophy has been noted following the administration of VRS-317 in the monkey studies conducted to date. Injection-site biopsies from the repeat dose juvenile monkey study were analyzed to further assess the potential for VRS-317 to cause lipoatrophy. The fat content of skin biopsies from the injection sites was quantitatively evaluated. Differences in fat content appeared related to individual and/or sampling variations, and there were no discernible VRS-317 and/or time effect. Overall the qualitative evaluation correlated to the quantitative histomorphometric evaluation. In addition, injection-site analyses from the 14-week GLP monkey toxicology study also did not reveal any notable lipoatrophy.