Ocular Inflammation in Cynomolgus Macaques Following Intravenous Administration of a Human Monoclonal Antibody

General Toxicity Studies Conducted in Cynomolgus Macaques With CNTO95

To support clinical dosing of CNTO95 in phase I and phase II clinical trials in oncology patients, we conducted 3 repeated-dose toxicology studies (studies 1–3, Table 1). In these studies, saline or CNTO95 (10 or 50 mg/kg) was administered intravenously once per week to cynomolgus macaques (3–6 per sex per treatment group) for 4 to 26 weeks. These studies have been described in detail elsewhere ¹⁴ and are referred to here only in the context of the ocular evaluations that were conducted as part of the general toxicity assessments. Ophthalmic examinations were conducted prestudy and after animals had received at least 4 weekly doses of CNTO95 (during weeks 4 and 8 for study 1, during week 8 for study 2, and during weeks 12, 25, and 36 for study 3). At the end of the study periods, a comprehensive anatomic pathology examination was conducted. A subset of animals in studies 1 and 3 was allowed a treatment-free period prior to necropsy. At necropsy, eyes were collected from all animals and preserved in Davidson’s fixative for optimum fixation. The eyes were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy.

Laser-Induced CNV Model

The model of laser-induced CNV used in this study has been described previously ¹⁵ and is similar to the model described by Krzystolik et al. ¹⁶ In both models, intravitreal administration of an anti-VEGF antibody Fab fragment was shown to reduce CNV.

For the evaluation of the effects of CNTO95 on laser-induced CNV, CNTO95 was administered either intravenously at a dose of 50 mg/kg or intravitreally at a dose of 500 μg per eye (50 μL; study 4, Table 1). The doses of 50 mg/kg intravenously and 500 μg per eye were selected to produce serum and vitreal concentrations of CNTO95, respectively, that are sufficient to fully saturate α_ν-integrin receptor binding. ¹⁴ Five male cynomolgus macaques weighing 3.2 to 5.8 kg received slow bolus intravenous injections of CNTO95. The animals in the intravenous treatment group were not injected intravitreally. Eight male cynomolgus macaques weighing 3.6 to 5.1 kg received intravitreal injections of CNTO95 into the right eye and an intravitreal injection of an equivalent volume of saline (50 μL) in the left eye. In these animals, the left eye was used as the control. An additional 8 cynomolgus macaques weighing 3.4 to 4.2 kg received a saline injection into the left eye and were used throughout the study as controls only. Animals were administered CNTO95 by intravitreal or intravenous injection or saline by intravitreal injection on days 1, 15, 29, and 43.

For the intravitreal injections, animals were anesthetized with ketamine and xylazine, and the eyes were rinsed with sterile balanced salt solution. A topical anesthetic (0.5% proparacaine) was instilled in each eye before dose administration. The eyes were cleaned with a 25% betadine solution prepared with 0.9% sodium chloride for injection, USP (2.5% providone iodine) and then rinsed with a physiological salt solution. Eyes were injected intravitreally using a 1-cc tuberculin syringe and 30-gauge needle. A topical antibiotic (Tobrex) was instilled in each eye after dose administration. Animals were observed cage side throughout the study period for any clinical signs, with particular attention to the eyes.

On day 8 or 9, the macula of each eye underwent laser treatment using methods similar to those described previously. ^15–17 Color ocular photographs were taken on the day of laser induction (after the laser treatment) and on day 41. The color ocular photographs included the retina and pertinent ocular abnormalities, stereoscopic photographs of the posterior pole, and nonstereoscopic photographs of 2 mid-peripheral fields (temporal and nasal).

Fluorescein angiography was conducted on days 22, 27, and 41. Animals were anesthetized with ketamine and maintained on isoflurane, and the eyes were dilated with a mydriatic agent. Animals were given an intravenous injection of fluorescein.

Ophthalmic examinations were conducted before the initiation of treatment, 1 day after the first treatment (day 2), 5 days after the first treatment (day 6), and 1 day after the last (fourth biweekly) treatment (day 44). Animals were anesthetized with ketamine, and the eyes were dilated with a mydriatic agent. The adnexa and anterior portion of both eyes was examined using a handheld slit-lamp biomicroscope. The ocular fundus of both eyes was examined using an indirect ophthalmoscope. Aqueous and vitreal cell scores were assigned using the same estimate of cells per single 0.2-mm field of the focused slit-lamp beam (0 = no cells, trace = 1-5 cells, 1+ = 5–25 cells, 2+ = 25–50 cells, 3+ = 50–100 cells, and 4+ = more than 100 cells). Aqueous flare was scored as follows: 0 = no visible protein in anterior chamber; trace = anterior chamber protein is visible only to an experienced observer using a small, bright focal light source and magnification; 1+ = mild; 2+ to 3+ = moderate protein, with 3+ more severe than 2+ ; and 4+ = severe (modified from Hogan et al ¹⁸ ).

On day 44, serum and vitreal samples were collected for analysis of CNTO95 concentration and were analyzed using methods described previously. ¹⁴

Ocular Inflammation Follow-Up Studies

A follow-up study was conducted to identify a noeffect dose level for clinical ophthalmic findings following intravenous administration of CNTO95 (study 5, Table 1). In this study, the effects of intravenously administered CNTO95 were compared with a saline control, vehicle control, and an isotype (human IgG1)–matched mAb (trastuzumab) control.

Male and female cynomolgus macaques weighing 2.1 to 3.9 kg were assigned to 5 groups of 3 animals per group. Animals were dosed by bolus intravenous injection on days 1, 8, 15, and 22. Group 1 animals received saline control (0.9% sodium chloride for injection, USP; sterile saline) on days 1 and 8 and vehicle control (0.01 M sodium phosphate, 10% (w/v) sucrose [pH 6], 0.0001% polysorbate 80) on day 15. On day 22, group 1 animals received CNTO95 (50 mg/kg). Groups 2, 3, and 4 received CNTO95 at 2, 10, or 50 mg/kg, respectively (except group 4 animals on day 22 received CNTO95 vehicle). Group 5 received trastuzumab at 50 mg/kg. Doses were administered at volumes ranging from 0.1 to 2.5 mL/kg.

Ophthalmic examinations were conducted prior to initiation of dosing, on the day following dosing, and 7 days after each dose. Body temperature and hematology evaluations were conducted on the same days as the ophthalmic examinations. Clinical observations and body weight were evaluated throughout the study. Animals were returned to the colony at the end of the observation periods.

An additional 9 male cynomolgus macaques weighing 2.5 to 2.8 kg received a single intravenous bolus injection or 2-hour infusion of CNTO95 (50 mg/kg) on day 1 (study 6, Table 1). Ophthalmic examinations were conducted on days 2, 3, 4, 5, and 8 to evaluate the time course and resolution of the ocular inflammation. Animals were returned to the colony at the end of the observation periods.

Additional Toxicology Studies

In 2 additional studies, ophthalmic examinations were conducted in cynomolgus macaques at 24 hours after the first administration of CNTO95 (studies 7 and 8, Table 1). In study 7, 2 male (3.9–4 kg) and 1 female (2.5 kg) cynomolgus macaques received a slow bolus intravenous injection of CNTO95 (10 mg/kg) on day 1 followed by ophthalmic examinations (by the staff veterinarian) on day 2. In study 8, CNTO95 (20 mg/kg) or saline was administered intravenously to 3 male (2.6–3 kg) and 3 female (3.6–4.5 kg) cynomolgus macaques per treatment group once per week for 4 weeks. This study also included a group of 3 male (2.8–3 kg) and 3 female (2.7–3.5) animals that received bevacizumab (anti-VEGF mAb) 20 mg/kg once per week for 4 weeks. The purpose of the bevacizumab group was to compare the toxicity of CNTO95 to that of a second antiangiogenesis inhibitor. Ophthalmic examinations were conducted 24 hours after the first dose, and histopathological examinations were conducted at the end of the 4-week treatment period.

Immunolocalization of CNTO95 in Macaque and Human Eye Tissues In Vitro

The in vitro binding of CNTO95 to cryosections of macaque and human eye tissues was evaluated. Human placenta was included as a positive control tissue. Eye tissues were obtained at necropsy from untreated cynomolgus macaques or at autopsy from normal human donors. Fresh unfixed tissue samples were placed in molds and frozen on dry ice in OCT embedding medium. Tissues were maintained below −70°C until sectioning. Sections were cut at approximately 5 μm and fixed in room temperature in acetone for 10 minutes. Just prior to staining, the slides were fixed in 10% neutral buffered formalin for 10 seconds. An indirect immunoperoxidase procedure was performed to evaluate CNTO95 localization using methods described previously. ¹⁴ Briefly, cryosections were blocked sequentially with avidin, biotin, and a protein block designed to reduce nonspecific binding. CNTO95 was applied to the tissues in vitro at a concentration of 10 μg/mL. CNTO95 binding was detected using a mouse antiidiotypic antibody toward CNTO95 followed by goat antimouse antibody and then avidin-biotin complex. All slides were counterstained with hematoxylin, dehydrated, and coverslipped for interpretation. Slides were examined by light microscopy and grade using the following scale: 0 = negative, ± = equivocal, 1+ = weak, 2+ = moderate, 3+ = strong, and 4+ = intense.

Serum and Vitreous CNTO95 Concentration Analysis

Serum and vitreous CNTO95 concentrations were measured using a validated sandwich enzyme immunoassay (EIA). The limit of quantification of the assay was 0.05 μg/mL (for a 1/10 dilution) of CNTO95 in the matrix. Two different murine antiidiotypic antibodies against CNTO95 were used for this assay, one for capture and one for detection. Animal serum samples diluted in phosphate-buffered saline (PBS), and 1% bovine serum albumin (BSA) was added to the capture antibody-coated plates in triplicate. Biotin-coupled detection antibody diluted in BSA + PBS buffer was used as the secondary antibody followed by streptavidin-conjugated horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, Pennsylvania). Plates were washed and tetramethylbenzidine (Kirkegaard and Perry Labs, Gaithersburg, Maryland) substrate was added. The colorimetric reaction was stopped with 4N H₂SO₄. The 450- to 650-nm OD values of the wells were determined using a spectrophotometric microplate reader. The concentration of each unknown was calculated from a standard curve.

General Toxicity Studies

The histopathological examination of eyes from macaques treated with CNTO95 or bevacizumab for 4 to 26 weeks (studies 1, 2, 3, and 8) did not reveal any treatment-related ocular toxicities. In these repeated-dose toxicology studies, the ophthalmic examinations that were conducted following the first-dose administration in 1 study (study 8) and following multiple-dose administration in the other 3 studies (studies 1–3) revealed no ophthalmologic findings (refer to Table 1).

Laser-Induced CNV Model

Administration of CNTO95 either intravenously or intravitreally at the described dose levels did not inhibit laser-induced CNV in this model (study 4; results not shown). No evidence of retinal toxicity was apparent by clinical examination or by evaluation of either the color photographs or the fluorescein angiograms.

Ophthalmic examinations indicated that CNTO95, administered intravitreally at a dose of 500 μg or intravenously at a dose of 50 mg/kg, produced an acute (24 hours postdose), intense inflammatory response in the anterior chamber of the eyes (Figure 1). Inflammatory cells in the anterior chamber were graded as 3 to 4+, and aqueous flare was graded as 1 to 4+. The anterior segment inflammation was largely resolved by day 6, at which time inflammatory cells could be visualized in the vitreous. The inflammation observed in CNTO95-treated animals on days 2 and 6 occurred prior to the laser-induced injury, which was induced on day 8 or 9. Additional administration of CNTO95 on days 15, 29, and 43 resulted in a greatly diminished anterior chamber inflammatory response, even after laser-induced injury (day 8 or 9), so that by 24 hours after the fourth injection (day 44, the next ophthalmic examination time point), the inflammatory response was comparable to that of saline control eyes. In the animals that received intravitreal injections of saline on days 1, 15, 29, and 43, 0 to trace inflammatory cells were detected in the anterior chamber on days 2, 6, and 44.

Posterior segment inflammation in the vitreous exhibited a similar pattern to that seen in the anterior segment, although, as expected, inflammatory cells in the vitreous were slower to appear.

Conjuctival hyperemia was seen only in eyes injected intravitreally with CNTO95 or saline. One day after the first injection, the hyperemia was graded as moderate (2+) in the CNTO95-injected eyes and trace in the saline-treated eyes. One day after the fourth injection, hyperemia was graded as mild in all injected eyes.

Two of 8 eyes receiving CNTO95 intravitreally and 2 of 10 eyes receiving CNTO95 intravenously had corneal keratic precipitates 1 day after the first dose. Keratic precipitates were not present on ophthalmic examinations conducted on days 6 and 44.

In the animals receiving CNTO95 intravenously, there were no outward signs of toxicity; pupillary miosis was not observed, the conjunctiva was not hyperemic, and there were no other outward manifestations of ocular inflammation (eg, blepharospasm, tearing, or other signs of discomfort).

Follow-Up Studies Evaluating Ocular Inflammation Following Intravenous Injection of CNTO95

Follow-up studies were conducted to further evaluate the nature of the ocular inflammation observed following intravenous dosing of CNTO95 (studies 5 and 6).

In study 5, intravenous administration of CNTO95 or an isotype-matched human monoclonal antibody (anti-HER2 mAb, traztuzumab) had no effect on clinical observations, body weight, body temperature, or hematology evaluations. Administration of CNTO95 resulted in intraocular inflammation in all treated animals on the day following dosing (∼24 hours postdose; Figure 2). Overall, this reaction was less in the 2-mg/kg treatment group than in the 10-mg/kg and 50-mg/kg treatment groups, but the small sample size precluded making unequivocal dose–response comparisons, and 1 or more animals in each of the 3 groups exhibited a considerable inflammatory response (2 to 3+ aqueous flare/4+ anterior chamber cell). This inflammatory response rapidly and spontaneously improved. By day 8, all eyes in all 3 CNTO95–treated groups had either no or only trace anterior chamber cell responses. With additional doses of CNTO95, the anterior chamber inflammatory response was markedly diminished, and all 3 CNTO95-treated groups exhibited almost no or only trace anterior chamber cell responses on all examinations after day 2. Vitreal cell scores tended to parallel those in the anterior chamber, although the peak vitreal cell scores were typically not achieved until 1 week after inflammatory cells first appeared in the anterior chamber. Vitreal cells also tended to be slower to resolve than those in the aqueous humor. Many of the vitreal cells after day 8 were brown in color, suggesting that they represented previous inflammation rather than persistent active inflammation. Group 1 animals that received CNTO95 on day 22 only showed ophthalmic observations on day 23 similar to those seen in group 4 animals on day 2 (1 day after the first dose).

Additional findings on day 2 included the deposition of a very small clump of pigment on the corneal endothelium of 1 eye of 1 animal in the 2-mg/kg group and the presence of white, inflammatory keratic precipitates on the corneal endothelium of both eyes of 2 animals in the 10-mg/kg group and 1 animal in the 50-mg/kg group. These deposits were secondary to anterior segment inflammation, and only the minute pigment clump persisted past day 2. In all CNTO95-treated groups, pupillary miosis was not observed, the conjunctiva was not hyperemic, and there were no other outward manifestations of ocular inflammation (eg, blepharospasm, tearing, or other signs of discomfort).

There were no changes from baseline ophthalmic observations for animals given sterile saline, vehicle control article, or the isotype-matched human monoclonal antibody control (traztuzumab).

Studies examining the time course and resolution of the ocular inflammation following a single dose (study 6) showed that the anterior chamber inflammatory response was greatest 1 day after dosing and spontaneously and steadily improved over the 1-week follow-up period (Figure 3). The intensity and the time course of the inflammation were similar following an intravenous bolus injection and following a 2-hour intravenous infusion.

Immunolocalization of CNTO95 Binding to Normal Macaque and Human Eye Tissues

Table 2 summarizes the immunolocalization of CNTO95 to cryosections of normal macaque and human eyes. CNTO95 bound to various elements in both macaque and human eyes, and the staining patterns were similar in both macaques and humans.

In the eye, the staining was described as cytoplasmic staining, whereas in the positive control tissue, human placenta, both cytoplasmic and membrane staining was described.

Concentrations of CNTO95 in Serum and Vitreous

Table 3 summarizes the concentrations of CNTO95 measured in serum and vitreous at 24 hours after the fourth biweekly intravitreal or intravenous administration of CNTO95. Low levels of CNTO95 were detected in the serum following intravitreal injection of CNTO95. The concentrations in the serum following intravitreal administration were approximately 3000-fold lower than in the serum following intravenous administration. Following intravenous administration of CNTO95, 100-fold lower levels of CNTO95 were detected in the vitreous when compared with the intravitreal injection. The concentrations of CNTO95 in the vitreous following intravenous administration of CNTO95 were approximately 1000-lower than the serum concentrations.