Archway Randomized Phase 3 Trial of the Port Delivery System with Ranibizumab for Neovascular Age-Related Macular Degeneration.

PURPOSE: To evaluate the safety and efficacy of the Port Delivery System with ranibizumab (PDS) for the treatment of neovascular age-related macular degeneration (nAMD). DESIGN: Phase 3, open-label, randomized, visual acuity assessor-masked noninferiority and equivalence trial. PARTICIPANTS: Patients with nAMD diagnosed within 9 months of screening previously treated with and responsive to anti-vascular endothelial growth factor therapy. METHODS: Patients were randomized 3:2 to treatment with the PDS with ranibizumab 100 mg/ml with fixed 24-week (Q24W) refill-exchanges (PDS Q24W) or intravitreal ranibizumab 0.5-mg injections every 4 weeks (monthly ranibizumab). MAIN OUTCOME MEASURES: Primary end point was change in best-corrected visual acuity (BCVA) Early Treatment Diabetic Retinopathy Study letter (letters) score from baseline averaged over weeks 36 and 40 (noninferiority margin,-4.5 letters; equivalence margin, ±4.5 letters). RESULTS: Archway enrolled 418 patients; 251 were randomized to and 248 received treatment with the PDS Q24W, and 167 were randomized to and received treatment with monthly ranibizumab. Baseline BCVA was 74.4 letters (PDS Q24W arm) and 75.5 letters (monthly ranibizumab arm; Snellen equivalent, 20/32). Adjusted mean change in BCVA score from baseline averaged over weeks 36 and 40 was +0.2 letters (standard error [SE], 0.5 letters) in the PDS Q24W arm and +0.5 letters (SE, 0.6 letters) in the monthly ranibizumab arm (difference, -0.3 letters; 95% confidence interval, -1.7 to 1.1 letters). PDS Q24W was both noninferior and equivalent to monthly ranibizumab. Of 246 PDS-treated patients assessed for supplemental ranibizumab treatment, 242 (98.4%) did not receive supplemental ranibizumab treatment before the first refill-exchange procedure, including 4 patients who discontinued treatment before the first refill-exchange procedure. Prespecified ocular adverse events of special interest were reported in 47 patients (19.0%) in the PDS Q24W arm and 10 patients (6.0%) in the monthly ranibizumab arm, which included, in the former arm, 4 (1.6%) endophthalmitis cases, 2 (0.8%) retinal detachments, 13 (5.2%) vitreous hemorrhages, 6 (2.4%) conjunctival erosions, and 5 (2.0%) conjunctival retractions. Most ocular adverse events in the PDS Q24W arm occurred within 1 month of implantation. CONCLUSIONS: Archway met its primary objective and PDS Q24W demonstrated noninferior and equivalent efficacy to monthly ranibizumab, with 98.4% of PDS-treated patients not receiving supplemental treatment in the first 24-week interval.

Ocular Half-Life of Intravitreal Biologics in Humans and Other Species: Meta-Analysis and Model-Based Prediction.

Therapeutic antibodies administered intravitreally are the current standard of care to treat retinal diseases. The ocular half-life (t1/2) is a key determinant of the duration of target suppression. To support the development of novel, longer-acting drugs, a reliable determination of t1/2 is needed together with an improved understanding of the factors that influence it. A model-based meta-analysis was conducted in humans and nonclinical species (rat, rabbit, monkey, and pig) to determine consensus values for the ocular t1/2 of IgG antibodies and Fab fragments. Results from multiple literature and in-house pharmacokinetic studies are presented within a mechanistic framework that assumes diffusion-controlled drug elimination from the vitreous. Our analysis shows, both theoretically and experimentally, that the ocular t1/2 increases in direct proportion to the product of the hydrodynamic radius of the macromolecule (3.0 nm for Fab and 5.0 nm for IgG) and the square of the radius of the vitreous globe, which varies approximately 24-fold from the rat to the human. Interspecies differences in the proportionality factors are observed and discussed in mechanistic terms. In addition, mathematical formulae are presented that allow prediction of the ocular t1/2 for molecules of interest. The utility of these formulae is successfully demonstrated in case studies of aflibercept, brolucizumab, and PEGylated Fabs, where the predicted ocular t1/2 values are found to be in reasonable agreement with the experimental data available for these molecules.

The Port Delivery System with Ranibizumab for Neovascular Age-Related Macular Degeneration: Results from the Randomized Phase 2 Ladder Clinical Trial.

PURPOSE: To evaluate the safety and efficacy of the Port Delivery System with ranibizumab (PDS) for neovascular age-related macular degeneration (nAMD) treatment. DESIGN: Phase 2, multicenter, randomized, active treatment-controlled clinical trial. PARTICIPANTS: Patients diagnosed with nAMD within 9 months who had received 2 or more prior anti-vascular endothelial growth factor intravitreal injections and were responsive to treatment. METHODS: Patients were randomized 3:3:3:2 to receive the PDS filled with ranibizumab 10 mg/ml, 40 mg/ml, 100 mg/ml, or monthly intravitreal ranibizumab 0.5-mg injections. MAIN OUTCOME MEASURES: Time to first implant refill assessed when the last enrolled patient completed the month 9 visit (primary efficacy end point), improvement in best-corrected visual acuity (BCVA) and central foveal thickness (CFT), and safety. RESULTS: The primary analysis population was 220 patients, with 58, 62, 59, and 41 patients in the PDS 10-mg/ml, PDS 40-mg/ml, PDS 100-mg/ml, and monthly intravitreal ranibizumab 0.5-mg arms, respectively. Median time to first implant refill was 8.7, 13.0, and 15.0 months in the PDS 10-mg/ml, PDS 40-mg/ml, and PDS 100-mg/ml arms, respectively. At month 9, the adjusted mean BCVA change from baseline was ‒3.2 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, ‒0.5 ETDRS letters, +5.0 ETDRS letters, and +3.9 ETDRS letters in the PDS 10-mg/ml, PDS 40-mg/ml, PDS 100-mg/ml, and monthly intravitreal ranibizumab 0.5-mg arms, respectively. At month 9, the adjusted mean CFT change from baseline was similar in the PDS 100-mg/ml and monthly intravitreal ranibizumab 0.5-mg arms. The optimized PDS implant insertion and refill procedures were generally well tolerated. After surgical procedure optimization, postoperative vitreous hemorrhage rate was 4.5% (7/157; 1 event classified as serious). There was no evidence of implant clogging. CONCLUSIONS: In the phase 2 Ladder trial, the PDS was generally well tolerated and demonstrated a dose response across multiple end points in patients with nAMD. The PDS 100-mg/ml arm showed visual and anatomic outcomes comparable with monthly intravitreal ranibizumab 0.5-mg injections but with a reduced total number of ranibizumab treatments. The PDS has the potential to reduce treatment burden in nAMD while maintaining vision.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

Retinal Fluid and Thickness Fluctuations in Archway Trial for Port Delivery System With Ranibizumab vs Monthly Ranibizumab Injections.

PURPOSE: To determine proportion of eyes with neovascular age-related macular degeneration (nAMD) with retinal fluid and/or central subfield thickness (CST) fluctuations and evaluate their impact on best-corrected visual acuity (BCVA) in eyes treated with the Port Delivery System with ranibizumab (PDS) versus monthly intravitreal ranibizumab injections. DESIGN: Post-hoc analyses of phase 3 Archway trial (NCT03677934). PARTICIPANTS: Adults with nAMD responsive to anti-vascular endothelial growth factor therapy. INTERVENTION: 418 patients randomized 3:2 to the PDS (100 mg/mL) with refill-exchanges every 24 weeks (Q24W) or monthly intravitreal ranibizumab (0.5 mg) for 96 weeks. OUTCOMES: Proportion of eyes in each treatment arm with subretinal and/or intraretinal fluid (SRF/IRF) overall and in central 1-mm; BCVA changes from baseline by treatment arm and fluid presence/location; proportion of eyes with CST fluctuations from baseline to week 48, week 48 to 96, and baseline to week 96; effects of CST fluctuations on BCVA. RESULTS: 415 eyes were assessed. In the PDS versus monthly ranibizumab arm, proportion of eyes with SRF/IRF, central SRF, and central IRF were 47.6% versus 50.9%, 29.0% versus 19.2%, and 11.7% versus 12.6% at baseline, and 57.8% versus 56.1%, 21.6% versus 14.8%, and 7.0% versus 8.4% at week 96. BCVA changes from baseline to week 96 were -1.1 letters with the PDS versus -1.4 with monthly ranibizumab in eyes with SRF/IRF, and -1.9 versus -1.8 in eyes with central SRF. In eyes with central IRF, BCVA changes from baseline to week 96 were -2.1 with the PDS versus -6.9 with monthly ranibizumab, respectively (mean BCVA at 96 weeks 68.9 [20/40] versus 64.6 [20/50]). CST fluctuations occurred in 32.1% and 29.7% of PDS versus monthly ranibizumab eyes; corresponding BCVA changes from baseline to week 96 were -2.5 versus -2.6 (mean BCVA at 96 weeks 72.7 [20/35] versus 71.5 [20/38]). CONCLUSIONS: PDS Q24W maintained BCVA to 96 weeks regardless of SRF/IRF, central SRF, central IRF, or CST fluctuations, comparable with monthly ranibizumab, thus supporting the use of the PDS in stabilizing retinal anatomy without the need for monthly treatment in patients with nAMD.

Exudation in Patients With Neovascular Age-Related Macular Degeneration Treated With the Port Delivery System or Monthly Injections.

PURPOSE: To evaluate for the presence, severity, and type of exudation at each study visit for a subgroup of patients with neovascular age-related macular degeneration from the Archway and Portal trials. DESIGN: Retrospective analysis of prospectively obtained data. METHODS: Spectral-domain optical coherence tomography scans from each study visit of 44 patients from the Port Delivery System (PDS) arm and 32 patients from the monthly injection arm of Archway were evaluated, and composites of horizontal scans through the fovea were created. Each composite was graded for the presence, type, and severity of exudation and impact on best-corrected visual acuity. RESULTS: After PDS implantation, 20 of 44 eyes (45%) never showed any exudation in the fovea, 2 (5%) never showed exudation in the fovea but had several missed visits, whereas 15 (34%), 3 (7%), and 4 (9%) showed mild, moderate, or severe exudation at 1 or more study visits, respectively. When exudation was present, it was most commonly subretinal fluid (50%). Of 32 patients randomized to monthly injections, 15 (47%) had no exudation in the fovea during monthly injections or after PDS implantation. Fluctuation of exudation in the fovea over time was seen in some patients after PDS implantation or during monthly injections with little or no identifiable impact on best-corrected visual acuity. In the 7 eyes with moderate or severe exudation in the fovea after PDS implantation, final vision was good in 5 (20/25 in 3, 20/40 in 1, and 20/50 in 1) and 2 had reduced vision from submacular hemorrhage. CONCLUSIONS: The PDS provides excellent control of exudation in the fovea in patients with neovascular age-related macular degeneration, and when exudation occurs, it often resolves without a negative impact on vision.

Supplemental Intravitreal Ranibizumab Injections in Eyes Treated with the Port Delivery System with Ranibizumab in the Archway Trial.

PURPOSE: To determine the proportion and characteristics of eyes with neovascular age-related macular degeneration (nAMD) treated with the Port Delivery System (PDS) with ranibizumab that receive supplemental intravitreal ranibizumab injections because of changes in best-corrected visual acuity (BCVA) or central subfield thickness (CST), or both, and to investigate the safety and efficacy of supplemental injections in eyes with the PDS. DESIGN: Post hoc analyses of data from the phase III, randomized, multicenter, open-label, active-comparator Archway trial (NCT03677934). PARTICIPANTS: Adults with nAMD diagnosed within 9 months of screening previously responsive to anti-VEGF therapy. INTERVENTION: Four hundred eighteen patients were randomized to the PDS with ranibizumab 100 mg/ml with fixed refill-exchanges every 24 weeks (Q24W) or monthly intravitreal ranibizumab 0.5 mg for 96 weeks. RESULTS: Of the 246 eyes treated with the PDS Q24W and assessed for supplemental treatment criteria, the vast majority (94.6%-98.4%) did not receive supplemental treatment during each retreatment interval, with 87.4% not receiving supplemental treatment at any point during the trial. Of the 31 eyes receiving supplemental treatment, 58.1% received 1 injection and 32.3% received 2. At baseline, eyes receiving supplemental treatment were significantly more likely to have thicker retinas (mean CST, 370.5µm vs. 304.4µm; P = 0.0001), subretinal fluid (54.8% vs. 21.2%; P < 0.0001), and larger pigment epithelial detachment height (215.7 µm vs. 175.9 µm; P = 0.003). These features have previously been associated with difficult-to-treat nAMD. Although BCVA and CST generally remained constant throughout the trial in eyes without supplemental treatment, the small number of eyes receiving supplemental treatment on average lost 1 line of vision from baseline to week 96 (mean, -5.7 ETDRS score letters) and CST continued to increase over time. Absolute BCVA at week 96 was similar irrespective of supplemental treatment status (71.1 and 73.7 letters). Best-corrected visual acuity and CST generally improved within 28 days of supplemental treatment. CONCLUSIONS: Although the PDS Q24W effectively maintains vision and retinal stability in most eyes with nAMD, a small proportion of patients with features of difficult-to-treat nAMD may benefit from supplemental intravitreal anti-VEGF injections and initial close monitoring is recommended. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

International consortium for innovation and quality: An industry perspective on the nonclinical and early clinical development of intravitreal drugs.

The eye, which is under constant exposure to environmental pathogens, has evolved various anatomic and immunological barriers critical to the protection of tissues lacking regenerative capacity, and the maintenance of a clear optic pathway essential to vision. By bypassing the ocular barriers, intravitreal (IVT) injection has become the mainstay for the delivery of drugs to treat conditions that affect the back of the eye. Both small molecules and biotherapeutics have been successfully administered intravitreally, and several drugs have been approved for the treatment of (wet) age-related macular degeneration and diabetic macular edema. However, IVT injection is an invasive procedure, which requires sufficient technical expertise from the healthcare professional administering the drug. Potential side effects include bleeding, retinal tear, cataracts, infection, uveitis, loss of vision, and increased ocular pressure. Pharmaceutical companies often differ in their drug development plan, including drug administration techniques, collection of ocular tissues and fluids, ophthalmology monitoring, and overall conduct of nonclinical and clinical studies. The present effort, under the aegis of the Innovation & Quality Ophthalmic Working Group, aims at understanding these differences, identifying pros and cons of the various approaches, determining the gaps in knowledge, and suggesting feasible good practices for nonclinical and early clinical IVT drug development.

Population Pharmacokinetics of Ranibizumab Delivered via the Port Delivery System Implanted in the Eye in Patients with Neovascular Age-Related Macular Degeneration.

The port delivery system with ranibizumab (PDS) is designed to continuously deliver ranibizumab to maintain therapeutic drug concentrations in the vitreous of the eye for an extended duration. The PDS has been evaluated for the treatment of neovascular age-related macular degeneration in the Ladder (PDS 10, 40, and 100 mg/mL, with refill exchanges as needed, versus monthly intravitreal ranibizumab 0.5 mg), Archway (PDS 100 mg/mL with 24-week refill exchanges, versus monthly intravitreal ranibizumab 0.5 mg), and ongoing Portal (PDS 100 mg/mL with 24-week refill exchanges) clinical trials. Data from Ladder, Archway, and Portal were used to develop a population pharmacokinetics (PK) model to estimate the ranibizumab release rate from the PDS implant, describe ranibizumab PK in serum and aqueous humor, and predict the concentration in vitreous humor. A model was developed to adequately describe the serum and aqueous humor PK data, as suggested by goodness-of-fit plots as well as visual predictive checks. In the final model, the first-order implant release rate was estimated to be 0.00654 (1/day), corresponding to a half-life of 106 days, consistent with the implant release rate determined in vitro. The model-predicted vitreous concentrations achieved with PDS 100 mg/mL given every 24 weeks were below the intravitreal peak concentration and above the intravitreal trough concentration of ranibizumab over the entire 24-week refill interval. The results demonstrate a durable release of ranibizumab from the PDS with a half-life of 106 days, providing vitreous exposure to ranibizumab for at least 24 weeks that is within the range of exposure for monthly intravitreal treatment.

In-vitro characterization of ranibizumab release from the Port Delivery System.

The Port Delivery System with ranibizumab (PDS) consists of an implant that is a permanent, indwelling drug delivery device that can be refilled through a self-sealing septum and is designed to continuously release a customized formulation of ranibizumab into the vitreous by passive diffusion through a porous titanium release control element. Target release rates of ranibizumab via the implant used in studies of the PDS in patients with neovascular age-related macular degeneration were selected based on clinical and pharmacokinetic (PK) data from previously conducted intravitreal ranibizumab injection studies. In-vitro testing was performed to verify release rates with a range of ranibizumab concentrations before the phase II Ladder (NCT02510794) and phase III Archway (NCT03677934) trials of the PDS. Implants were filled with ranibizumab and were regularly transferred to new buffer-containing tubes to represent ocular ranibizumab clearance and release kinetics. Ranibizumab concentrations were measured and release rates calculated. Release rate data were fit to an exponential model and compared with expected release kinetics of diffusion. Release profiles of the implant releasing ranibizumab at concentrations of 10 mg/mL, 40 mg/mL, and 100 mg/mL were determined in the pre-phase II in-vitro studies. At day 3.5, mean (SD) ranibizumab release rates were 1.75 (0.07), 6.42 (0.35), and 16.69 (0.67) µg/d for PDS 10 mg/mL, 40 mg/mL, and 100 mg/mL, respectively. At month 6, mean (SD) release rates were 1.68 (0.05) and 4.16 (0.05) µg/d for PDS 40 mg/mL and 100 mg/mL, respectively. Measured release rates were within 90% of theoretical release rates during the course of drug release. PDS 100 mg/mL released 73% (SD, 1.92) of drug by month 6. In the pre-phase III in-vitro studies, mean (SD) release rates with PDS 100 mg/mL were 17.97 (0.90), 4.44 (0.11), and 2.45 (0.08) µg/d at 3.5 days, 6 months, and 9 months, respectively. Cumulative release (SD) was 73% (1.92) by month 6 and 87% (1.88) by month 9. The sustained, continuous, and reproducible release from the PDS observed in the in-vitro studies was also observed in Ladder and Archway. In conclusion, in-vitro studies were a powerful tool for characterizing and verifying ranibizumab release from the PDS implant and supported clinical evaluation of the PDS. PDS 100 mg/mL, which was associated with the longest therapeutic-level delivery of ranibizumab among the concentrations tested, was selected for evaluation in the pivotal phase III Archway trial.

Pharmacokinetics of the Port Delivery System with Ranibizumab in the Ladder Phase 2 Trial for Neovascular Age-Related Macular Degeneration.

INTRODUCTION: Ladder was a phase 2 trial that evaluated the Port Delivery System with ranibizumab (PDS) for neovascular age-related macular degeneration. Serum and aqueous humor samples were collected to characterize the pharmacokinetics (PK) of ranibizumab delivered through the PDS. METHODS: Ladder was a multicenter, randomized, active treatment-controlled, phase 2 clinical trial. Patients with neovascular age-related macular degeneration (n = 220) were randomized (3:3:3:2) to PDS 10 mg/ml, PDS 40 mg/ml, PDS 100 mg/ml, or monthly intravitreal ranibizumab 0.5 mg. Serum PK samples were collected in all arms and analyzed for ranibizumab concentration using an enzyme-linked immunosorbent assay. The main PK analyses were conducted in the PK-evaluable population (n = 68), which excluded patients who received fellow eye intravitreal treatment, supplemental ranibizumab treatment, or had previous treatment with bevacizumab in either eye within 9 months of randomization. RESULTS: In the PDS 10 mg/ml arm, median serum ranibizumab concentrations were below the serum trough concentration (Ctrough; 130 pg/ml) expected with monthly intravitreal ranibizumab 0.5 mg at all time points. In the PDS 40 mg/ml and 100 mg/ml arms, median serum ranibizumab concentrations were above the Ctrough expected with monthly intravitreal ranibizumab 0.5 mg (130 pg/ml) through month 3 and month 12 after implantation, respectively, and remained above the lower limit of quantification through month 15 and month 16 after implantation, respectively. CONCLUSIONS: These PK data indicate that the implant in the PDS 100 mg/ml arm maintained ranibizumab concentrations within the range of monthly intravitreal ranibizumab 0.5 mg injections (130-2220 pg/ml) through month 12 after implantation. TRIAL REGISTRATION: ClinicalTrials.gov identifier, NCT02510794.

Leveraging patient-centric sampling for clinical drug development and decentralized clinical trials: Promise to reality.

Advances in the technologies to enable patient-centric sampling (PCS) have the potential to improve blood sample collection by enabling clinical trial participants to collect samples via self-collection or with the help of a caregiver in their home. Typically, blood samples to assess pharmacokinetics and pharmacodynamics of a drug during clinical development are collected at a clinical site via venous blood draw. In this position paper by the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ), the potential value PCS can bring to patients, to the clinical datasets generated, and to clinical trial sponsors is discussed, along with considerations for program decision making, bioanalytical feasibility, operations, and regulatory implications. With an understanding of the value of PCS and considerations when implementing during clinical drug development, we can bring the promise of PCS closer to reality and enable decentralized clinical trials.

Phase 1 Study of the Anti-HtrA1 Antibody-binding Fragment FHTR2163 in Geographic Atrophy Secondary to Age-related Macular Degeneration.

PURPOSE: FHTR2163 is a novel antigen-binding fragment (Fab) directed against high-temperature requirement protein A1 (HtrA1). HTRA1 inhibition may preserve retinal integrity and slow disease progression in geographic atrophy (GA) secondary to age-related macular degeneration (AMD). This study examined the safety, pharmacokinetics, immunogenicity, and changes in the HTRA1-specific substrate Dickkop-related protein 3 (DKK3) in patients with GA who received FHTR2163. DESIGN: Phase I, open-label, single ascending dose escalation and multiple-dose expansion study. METHODS: Adults aged ≥ 50 years with GA secondary to AMD with best corrected visual acuity ranging between Snellen 20/125 and 20/400 were enrolled. In the first stage, a single intravitreal injection of FHTR2163 was given in 5 dose-escalation cohorts ranging from 1 to 20 mg (n = 3 patients/cohort; n = 15 total patients). The second stage evaluated the maximum tested dose of 20 mg administered every 4 weeks for 3 doses (n = 13 patients). RESULTS: No dose limiting toxicities or ocular serious AEs were reported. The most frequently reported AEs in the study eye were conjunctival hemorrhage (n = 7), conjunctival hyperemia (n = 4), and eye pain (n = 2). No non-ocular or ocular AEs were assessed as drug related. There were no clinically significant changes in ocular exams. A sustained pharmacodynamic effect of anti-HtrA1 was observed in the aqueous humor, as measured by levels of cleaved DKK3. CONCLUSIONS: FHTR2163, a novel Fab directed against HtrA1, was well tolerated with no DLTs or significant ocular AEs. The molecule when injected intravitreally for 3 doses showed a sustained pharmacodynamic effect at the maximum tested dose of 20 mg.

Protein engineering to increase the potential of a therapeutic antibody Fab for long-acting delivery to the eye.

To date, ocular antibody therapies for the treatment of retinal diseases rely on injection of the drug into the vitreous chamber of the eye. Given the burden for patients undergoing this procedure, less frequent dosing through the use of long-acting delivery (LAD) technologies is highly desirable. These technologies usually require a highly concentrated formulation and the antibody must be stable against extended exposure to physiological conditions. Here we have increased the potential of a therapeutic antibody antigen-binding fragment (Fab) for LAD by using protein engineering to enhance the chemical and physical stability of the molecule. Structure-guided amino acid substitutions in a negatively charged complementarity determining region (CDR-L1) of an anti-factor D (AFD) Fab resulted in increased chemical stability and solubility. A variant of AFD (AFD.v8), which combines light chain substitutions (VL-D28S:D30E:D31S) with a substitution (VH-D61E) to stabilize a heavy chain isomerization site, retained complement factor D binding and inhibition potency and has properties suitable for LAD. This variant was amenable to high protein concentration (>250 mg/mL), low ionic strength formulation suitable for intravitreal injection. AFD.v8 had acceptable pharmacokinetic (PK) properties upon intravitreal injection in rabbits, and improved stability under both formulation and physiological conditions. Simulations of expected human PK behavior indicated greater exposure with a 25-mg dose enabled by the increased solubility of AFD.v8.

Determination of Cellular Processing Rates for a Trastuzumab-Maytansinoid Antibody-Drug Conjugate (ADC) Highlights Key Parameters for ADC Design.

Antibody-drug conjugates (ADCs) are a promising class of cancer therapeutics that combine the specificity of antibodies with the cytotoxic effects of payload drugs. A quantitative understanding of how ADCs are processed intracellularly can illustrate which processing steps most influence payload delivery, thus aiding the design of more effective ADCs. In this work, we develop a kinetic model for ADC cellular processing as well as generalizable methods based on flow cytometry and fluorescence imaging to parameterize this model. A number of key processing steps are included in the model: ADC binding to its target antigen, internalization via receptor-mediated endocytosis, proteolytic degradation of the ADC, efflux of the payload out of the cell, and payload binding to its intracellular target. The model was developed with a trastuzumab-maytansinoid ADC (TM-ADC) similar to trastuzumab-emtansine (T-DM1), which is used in the clinical treatment of HER2+ breast cancer. In three high-HER2-expressing cell lines (BT-474, NCI-N87, and SK-BR-3), we report for TM-ADC half-lives for internalization of 6-14 h, degradation of 18-25 h, and efflux rate of 44-73 h. Sensitivity analysis indicates that the internalization rate and efflux rate are key parameters for determining how much payload is delivered to a cell with TM-ADC. In addition, this model describing the cellular processing of ADCs can be incorporated into larger pharmacokinetics/pharmacodynamics models, as demonstrated in the associated companion paper.

Evolution of Antibody-Drug Conjugate Tumor Disposition Model to Predict Preclinical Tumor Pharmacokinetics of Trastuzumab-Emtansine (T-DM1).

A mathematical model capable of accurately characterizing intracellular disposition of ADCs is essential for a priori predicting unconjugated drug concentrations inside the tumor. Towards this goal, the objectives of this manuscript were to: (1) evolve previously published cellular disposition model of ADC with more intracellular details to characterize the disposition of T-DM1 in different HER2 expressing cell lines, (2) integrate the improved cellular model with the ADC tumor disposition model to a priori predict DM1 concentrations in a preclinical tumor model, and (3) identify prominent pathways and sensitive parameters associated with intracellular activation of ADCs. The cellular disposition model was augmented by incorporating intracellular ADC degradation and passive diffusion of unconjugated drug across tumor cells. Different biomeasures and chemomeasures for T-DM1, quantified in the companion manuscript, were incorporated into the modified model of ADC to characterize in vitro pharmacokinetics of T-DM1 in three HER2+ cell lines. When the cellular model was integrated with the tumor disposition model, the model was able to a priori predict tumor DM1 concentrations in xenograft mice. Pathway analysis suggested different contribution of antigen-mediated and passive diffusion pathways for intracellular unconjugated drug exposure between in vitro and in vivo systems. Global and local sensitivity analyses revealed that non-specific deconjugation and passive diffusion of the drug across tumor cell membrane are key parameters for drug exposure inside a cell. Finally, a systems pharmacokinetic model for intracellular processing of ADCs has been proposed to highlight our current understanding about the determinants of ADC activation inside a cell.

Dynamic swelling behavior of interpenetrating polymer networks in response to temperature and pH.

Temperature responsive hydrogels based on ionic polymers exhibit swelling transitions in aqueous solutions as a function of shifting pH and ionic strength, in addition to temperature. Applying these hydrogels to useful applications, particularly for biomedical purposes such as drug delivery and regenerative medicine, is critically dependent on understanding the hydrogel solution responses as a function of all three parameters together. In this work, interpenetrating polymer network (IPN) hydrogels of polyacrylamide and poly(acrylic acid) were formulated over a broad range of synthesis variables using a fractional factorial design, and were examined for equilibrium temperature responsive swelling in a variety of solution conditions. Due to the acidic nature of these IPN hydrogels, usable upper critical solution temperature (UCST) responses for this system occur in mildly acidic environments. Responses were characterized in terms of maximum equilibrium swelling and temperature-triggered swelling using turbidity and gravimetric measurements. Additionally, synthesis parameters critical to achieving optimal overall swelling, temperature-triggered swelling, and sigmoidal temperature transitions for this IPN system were analyzed based on the fractional factorial design used to formulate these hydrogels.

A Flow Cytometric Clonogenic Assay Reveals the Single-Cell Potency of Doxorubicin.

Standard cell proliferation assays use bulk media drug concentration to ascertain the potency of chemotherapeutic drugs; however, the relevant quantity is clearly the amount of drug actually taken up by the cell. To address this discrepancy, we have developed a flow cytometric clonogenic assay to correlate the amount of drug in a single cell with the cell's ability to proliferate using a cell tracing dye and doxorubicin, a naturally fluorescent chemotherapeutic drug. By varying doxorubicin concentration in the media, length of treatment time, and treatment with verapamil, an efflux pump inhibitor, we introduced 10(5) -10(10) doxorubicin molecules per cell; then used a dye-dilution assay to simultaneously assess the number of cell divisions. We find that a cell's ability to proliferate is a surprisingly conserved function of the number of intracellular doxorubicin molecules, resulting in single-cell IC50 values of 4-12 million intracellular doxorubicin molecules. The developed assay is a straightforward method for understanding a drug's single-cell potency and can be used for any fluorescent or fluorescently labeled drug, including nanoparticles or antibody-drug conjugates.