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Clinical Cancer Research Vol. 11, 6959-6965, October 1, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Absence of Adverse Effects in Cynomolgus Macaques Treated with CNTO 95, a Fully Human Anti-{alpha}v Integrin Monoclonal Antibody, Despite Widespread Tissue Binding

Pauline L. Martin1, Qun Jiao1, Joel Cornacoff1, William Hall2, Bradley Saville3, Jeffrey A. Nemeth1, Allen Schantz1, Marielena Mata1, Haishan Jang1, Adedigbo A. Fasanmade1, Lisa Anderson2, Martin A. Graham1, Hugh M. Davis1 and George Treacy1

Authors' Affiliations: 1 Centocor, Radnor, Pennsylvania; 2 Pathology Associates, Frederick, Maryland; and 3 Sierra Biomedical, Sparks, Nevada

Requests for reprints: Pauline L. Martin, Department of Toxicology and Investigational Pharmacology, Centocor, Inc., 145 King of Prussia Road, Radnor, PA 19087. Phone: 610-240-8733; Fax: 610-651-6798; E-mail: Pmarti27{at}cntus.jnj.com.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: CNTO 95 is a fully human anti-{alpha}v integrin monoclonal antibody that inhibits macaque and rodent angiogenesis and inhibits human tumor growth in rodents. The purpose of these studies was to evaluate the preclinical safety of long-term administration of CNTO 95 in cynomolgus macaques.

Experimental Design: The in vitro binding profiles of CNTO 95 to human and macaque tissues and the in vivo binding to macaque tissues was evaluated by immunohistochemistry. The preclinical safety of CNTO 95 (10 and 50 mg/kg, i.v.) was evaluated in macaques treated once per week for up to 6 months. Safety was evaluated by clinical observations, ophthalmic and physical examinations (including heart rate, blood pressure, and electrocardiogram), clinical pathology (including coagulation parameters), and comprehensive anatomic pathology. The effect of CNTO 95 (50 mg/kg, i.v.) on incisional wound healing was evaluated in macaques.

Results: The tissue binding studies showed that CNTO 95 bound with mild to moderate intensity to macaque and human endothelial cells, epithelial cells, and vascular smooth muscle cells in most normal tissues examined. CNTO 95 showed strong to intense staining to the positive control tissue, human placenta. Despite the widespread binding to normal tissues, treatment of cynomolgus macaques with CNTO 95 produced no signs of toxicity and no histopathologic changes in any of the tissues examined (including ovaries and bone growth plates). CNTO 95 did not impair wound healing.

Conclusion: These studies show that CNTO 95 is safe and, unlike some other angiogenesis inhibitors, does not seem to inhibit normal physiologic angiogenesis.

CNTO 95 is a fully human monoclonal antibody that binds to and blocks {alpha}v integrins. Integrins are essential components of angiogenesis and facilitate the adhesion of stimulated endothelial cells to the extracellular matrix (1, 2). The {alpha}vß3 and {alpha}vß5 integrins are both important in angiogenesis. Evidence indicates that {alpha}vß3 and {alpha}vß5 promote angiogenesis via distinct pathways, {alpha}vß3 is involved in the response to basic fibroblast growth factor and tumor necrosis factor {alpha}, and {alpha}vß5 is involved in the response to vascular endothelial growth factor (VEGF) and transforming growth factor {alpha} (TGF{alpha}; ref. 3).

CNTO 95 binds to human {alpha}vß3 and {alpha}vß5 integrins with high affinity and also binds to integrins {alpha}vß1 and {alpha}vß6 (4). By binding and blocking the {alpha}vß3 and {alpha}vß5 integrins, CNTO 95 can inhibit cell adhesion, migration, proliferation, and invasion of both tumor cells and endothelial cells in vitro and has shown antitumor activity against human tumors in rodents. CNTO 95 is currently being evaluated clinically for the treatment of solid tumors (5, 6).

Antibodies directed against the {alpha}vß3 integrin or against VEGF have been shown to disrupt intratumoral neovessels and cause regression of vascularization (7, 8). Vitaxin (an anti-{alpha}vß3 integrin monoclonal antibody) is currently being evaluated in clinical trials for the treatment of solid tumors (9). Avastin (an anti-VEGF monoclonal antibody), in combination with 5-fluorouracil, is currently approved for the first-line treatment of patients with metastatic carcinoma of the colon or rectum. The safety of long-term treatment with Avastin has been evaluated in patients and in animals. Treatment of patients with Avastin has been associated with an increased incidence of hemorrhage, hypertension, thromboembolic events, proteinuria, congestive heart failure, and impaired wound healing. Animal studies with VEGF inhibitors have shown epiphyseal dysplasia in immature animals and inhibition of follicular angiogenesis and ovulation in sexually mature females (10–15). VEGF is an important angiogenic factor in both physiologic and pathologic angiogenesis and is a vascular permeability factor (8, 16). The effects seen in normal animals are most likely due to inhibition of physiologic angiogenesis.

These studies were designed to evaluate the safety of long-term administration of CNTO 95 in the cynomolgus macaque to support dosing in clinical trials. CNTO 95 has previously been shown to bind to cynomolgus macaque aortic endothelial cells with an affinity similar to that for binding to human cells (KD, 5 nmol/L; EC50, 1 µg/mL; ref. 4). In addition, CNTO 95, when administered i.v. (10 mg/kg) to cynomolgus macaques, was shown to inhibit pathologic angiogenesis into s.c. implanted basic fibroblast growth factor containing matrigel plugs (4). Based on this information, the cynomolgus macaque was considered a biologically relevant species for the evaluation of the safety of this human therapeutic monoclonal antibody.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
All in-life procedures were conducted at Charles River Laboratories in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare. Study protocols were reviewed and approved by the Institutional Animal Care and Use Committees.

Immunolocalization of CNTO 95 binding to human and cynomolgus tissues. The in vitro binding of CNTO 95 to human and cynomolgus tissues and the in vivo binding of CNTO 95 to cynomolgus tissues were evaluated. For the in vitro studies, 37 tissues from three anonymous human donors (Cooperative Human Tissue Network, Charlotte, NC, and National Disease Research Interchange, Philadelphia, PA) and 11 tissues from one to three cynomolgus macaques were evaluated. The human tissues evaluated included all the tissues recommended on the list of normal tissues to be used for immunohistochemical investigations of cross-reactivity in the Food and Drug Administration document "Points to Consider in the Manufacture and Testing of Monoclonal Products for Human Use." Cryosections of human placenta were used as a positive control tissue.

For the in vivo study, CNTO 95 was administered to macaques at a dose of 50 mg/kg, i.v. Three animals were euthanized 4 days after dosing for collection of tissues for immunolocalization of CNTO 95 binding (see Incisional wound healing study).

Fresh unfixed tissue samples were embedded in Tissue-Tek optimal cutting temperature medium, frozen, sectioned at ~5 µm, and fixed immediately in acetone for 10 minutes. Sections were fixed in 10% neutral buffered formalin for 10 seconds just before staining. After washing the slides in PBS, tissue endogenous peroxidase was blocked with a solution containing sodium azide (1 mmol/L), glucose oxidase (1 units/mL), and glucose (10 mmol/L) for 60 minutes at 35°C. Nonspecific binding of antibodies was blocked with sequential changes of avidin and biotin for 15 minutes each. A protein block, containing PBS, 1% bovine serum albumin, 0.5% casein, and 1 mg/mL of heat-aggregated rabbit IgG, was applied to the slides for 20 minutes at 63°C. Slides were incubated with CNTO 95 (1 or 10 µg/mL) for 60 minutes. CNTO 95 binding to tissues was detected by an indirect immunoperoxidase procedure using a mouse anti-idiotypic antibody against CNTO 95 (C508) followed by biotinylated goat anti-mouse IgG and then avidin-biotin complex. Slides were rinsed with PBS and avidin-biotin complex reagent (Vector Laboratories, Burlingame, CA) was applied. Slides were washed with PBS and then reacted with the chromogen diaminobenzidine. Slides were washed, counterstained with hematoxylin, dehydrated, cleared, and mounted in a resinous medium.

Slides were examined by light microscopy. The staining intensity of the slides was graded semiqualitatively using the following scale: 0 (negative), ± (equivocal), 1+ (weak), 2+ (moderate), 3+ (strong), and 4+ (intense).

Pharmacokinetics and immune antibody response. Before the initiation of the multiple-dose safety studies in macaques, the pharmacokinetics and immune antibody responses to CNTO 95 were evaluated. Nine cynomolgus macaques with three animals in each group received a slow bolus i.v. injection (over 90 seconds) of 2, 10, or 50 mg/kg of CNTO 95. Serum samples were obtained before dosing on day 1, 0.5, 2, and 6 hours postdose, and on days 2, 3, 4, 8, 15, 22, 29, and 36. Serum samples were analyzed for CNTO 95 concentrations using an enzyme-linked immunoassay. Samples collected on days 8, 15, 22, 29, and 36 were also analyzed for anti–CNTO 95 antibodies.

Serum CNTO 95 concentration assay. Serum CNTO 95 concentrations were measured using a validated sandwich enzyme-linked immunoassay. The limit of quantification of the assay was 0.05 µg/mL (for a 1:10 dilution) of CNTO 95 in the matrix. The capture antibody was a murine anti-idiotypic antibody to CNTO 95 (C585). Animal serum samples diluted in PBS and 1% bovine serum albumin were added to the plates in triplicate. Biotin-coupled C508 (murine anti-idiotypic antibody against CNTO 95) diluted in bovine serum albumin + PBS buffer was used as the secondary antibody followed by Streptavidin-conjugated horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA). Plates were washed and tetramethylbenzidine (Kirkegaard and Perry Labs, Gaithersburg, MD) substrate was added. The colorimetric reaction was stopped with 4 N H2SO4. The 450 to 650 nm absorbance values of the wells were determined using a spectrophotometric microplate reader. The concentration of each unknown was calculated from a standard curve.

Pharmacokinetic analysis. Noncompartmental analysis was used to calculate the pharmacokinetic parameters of CNTO 95 (WinNonlin, Version 4.0.1, Pharsight Corporation, Mountain View, CA). The maximum serum concentration (Cmax) was obtained from inspection of the individual serum concentration versus time profile. The area under the serum concentration versus time curve (AUC) from time zero to the last quantifiable serum concentration [AUC(0-tz)] was obtained by the trapezoidal rule. The AUC from time 0 to infinity was determined as AUC(0-tz) + Cz / {lambda}z, where Cz is the last measurable serum concentration and {lambda}z is the terminal rate constant, which was determined by least-squared regression analysis of the last three concentration data points. Mean residence time was obtained by dividing the area under the first moment serum concentration-time curve by AUC. The total body clearance (CL) was calculated as dose / AUC. Volume of distribution during the terminal phase (Vz) was calculated by CL / {lambda}z.

Anti–CNTO 95 immune response assay. Because the development of monkey anti–CNTO 95 antibodies could potentially interfere with the pharmacokinetic and safety evaluation of CNTO 95, the ability of animals to mount an immune response against CNTO 95 was determined by evaluation of macaque sera from CNTO 95–treated animals for anti–CNTO 95 antibodies. The immune response assay consisted of a bridging enzyme-linked immunoassay capable of detecting 25 ng/mL of purified cynomolgus anti–CNTO 95 antibody (equivalent to a serum concentration of 250 ng/mL after accounting for sample dilution). Briefly, enzyme-linked immunoassay microtiter wells were coated with CNTO 95 and blocked with bovine serum albumin. Wells were washed with PBS + 0.05% Tween 20 between each step. Controls and test sera diluted 10-fold with PBS + bovine serum albumin were permitted to interact with CNTO 95–coated microtiter wells in triplicate. If the sample contained antibodies to CNTO 95, these would capture CNTO 95 covalently coupled to biotin in the next step. Streptavidin labeled with horseradish peroxidase was used to tag antibody complexes for colorimetric detection via catalysis of tetramethylbenzidine, the product of which absorbs light of 450 nm. The antibody titers are expressed as the reciprocal of the greatest dilution of serum found to generate a positive immune response assay result.

Preclinical safety evaluations. The safety of weekly i.v. administered CNTO 95 was evaluated in cynomolgus macaques. Male and female macaques (3-5/sex/group) were administered CNTO 95 (10 or 50 mg/kg) or physiologic saline (2.5 mL/kg) via bolus i.v. injection once weekly for 4, 9, or 25 weeks (total number of animals per study 30, 24, and 30, respectively). At the onset of each of the studies, all animals were experimentally naive and weighed ~2 to 3 kg for the females and 2 to 5 kg for the males. The first day of dosing was designated day 1. The animals underwent physical examinations, including measurements of blood pressure, heart rate, respiratory rate, body temperature, and electrocardiographic evaluations, and ophthalmic examinations before dosing and following CNTO 95 treatment. Animals were evaluated throughout the study for changes in clinical signs, body weight, qualitative food consumption, and clinical pathology indices (standard clinical chemistry and hematology panels and coagulation parameters – activated partial thromboplastin time and prothrombin time). Urine samples for standard urinalysis were collected from all animals at necropsy. Serum samples were collected at various times throughout the studies for evaluation of serum CNTO 95 concentration and assessment of antibodies to CNTO 95.

At the end of the dosing period, animals were euthanized and a comprehensive anatomic pathology examination was conducted. A standard complete set of tissues and organs were collected and preserved in neutral-buffered 10% formalin (except for the eyes, which were preserved in Davidson's fixative for optimum fixation). All tissues were embedded in paraffin, sectioned, stained with H&E, and examined by light microscopy. Two animals per group in the 1- and 6-month studies were allowed a treatment free period of 4 or 12 weeks, respectively, before necropsy to evaluate reversibility of any treatment-related findings.

Incisional wound healing study. An incisional wound healing study was done to evaluate the effects of CNTO 95 on wound repair and healing in cynomolgus macaques.

Four males and four female macaques received saline and six males and six females received 50 mg/kg CNTO 95 as a single, slow bolus, i.v. injection on day 1 ~30 minutes before the initiation of wound creation. On day 1, all animals received atropine sulfate (0.04 mg/kg, i.m.) before anesthesia with ketamine HCl (10 mg/kg, i.m.). The analgesic Ketorolac (1 mg/kg, i.m.) was administered before surgery. Animals were intubated and maintained on anesthesia with inhaled isoflurane. The back and the sides of each animal were shaved and the shaved area was prepared for aseptic surgery. Six full-thickness incisional wounds, measuring ~1 cm long by 3 mm deep, were created on the back of each animal using a surgical scalpel. Wounds were closed with skin closure strips and covered. Animals were allowed to recover from anesthesia and were observed for clinical signs for up to 2 weeks postsurgery. Blood samples were collected for standard hematology and coagulation (prothrombin time, activated partial thromoplastin time, and fibrinogen) evaluation and C-reactive protein and serum amyloid A analysis (as biomarkers for inflammation) before treatment and before necropsy. Blood samples were also collected for measurement of CNTO 95 serum concentrations. Two animals from the saline control group and three animals from the CNTO 95–treated group were euthanized on days 4, 7, 11, and 15. Following deep anesthesia with sodium pentobarbital, four wound sites for tensile strength evaluation were removed. Wounds were evaluated for tensile strength using a tensile strength machine with a UMC612 digital weight indicator. Following removal of the wound sites for tensile strength testing, each animal was administered 300 units/kg of heparin and was perfused with 0.9% saline. Following perfusion, one wound site from each animal was fixed in 10% neutral buffered formalin and stained with H&E for histopathology and one wound site from three animal euthanized on day 4 was embedded in optimal cutting temperature medium for immunolocalization of CNTO 95 binding.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Immunolocalization of CNTO 95 binding to human and cynomolgus tissues. The in vitro tissue cross-reactivity studies showed that CNTO 95 reacted strongly (3+) to intensely (4+) with syncytiotrophoblasts, cytotrophoblasts, and Hofbauer cells of the positive control human placental tissue (Table 1). The staining was primarily to the cell membrane but was also cytoplasmic. Cytoplasmic staining of some of the placental vascular endothelial cells was also observed.


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  		Table 1. Immunolocalization of CNTO 95 binding to sections of selected human tissues when applied in vitro at concentrations of 1 and 10 µg/mL

 
Specific immunolocalization of CNTO 95 to normal human and macaque tissues was observed as mostly weak to strong cytoplasmic staining in epithelial cells, endothelial cells, and smooth muscle cells from the majority of tissues examined. Results from selected human tissues are shown in Table 1 and from macaque tissues are summarized in Table 2. The epithelial staining was generally along the basement membrane or basal lamina. Neural cell staining consisted of glial cells, Purkinje cells, and cells of the molecular and granular layers of the cerebellum. Additionally, there was staining of the basal lamina surrounding Schwann cells in some of the peripheral nerves and nerve roots. Other cell types with immunostaining included bone marrow and macrophage lineage cells, such as Kupffer cells in the liver.


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  		Table 2. Immunolocalization of CNTO 95 binding to sections of cynomolgus macaque tissues when applied in vitro at a concentrations of 1 or 10 µg/mL or when administered to macaques in vivo at a dose of 50 mg/kg i.v

 
Immunolocalization of CNTO 95 bound to tissues from macaques treated with CNTO 95 in vivo generally showed a localization pattern similar to that observed when CNTO 95 was applied in vitro to tissues from untreated animals (Table 2). Minor differences included increased distribution in the interstitial fluid and increased localization to stromal cells locally. Neural cell binding in the cerebellum was not observed in cynomolgus macaques when CNTO 95 was administered i.v.

Pharmacokinetics and immune antibody response. The results from the single-dose cynomolgus macaque pharmacokinetic study are shown in Fig. 1 and summarized in Table 3. As shown in Fig. 1, the serum concentration-time profile of CNTO 95 after i.v. dosing exhibited a short, rapid distribution phase of ~1 day and a second elimination phase. The second elimination phase was prolonged at higher doses. A third phase was also observed in most of the macaques where CNTO 95 concentrations declined rapidly from the serum. The onset of the rapid elimination phase seemed to occur when serum concentrations dropped below ~30 µg/mL. The rapid elimination phase was noted to begin by day 3, 14, or 28 following doses of 2, 10, and 50 mg/kg, respectively.



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  		Fig. 1. Serum concentration of CNTO 95 in cynomolgus macaques following a single i.v. dose of CNTO 95 at 2, 10, or 50 mg/kg. Each curve represents data from a single animal.

 

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  		Table 3. Results from the single-dose pharmacokinetic analysis of CNTO 95 in macaque serum

 
The Cmax increased proportionally with the administered dose (Table 3). However, the AUC values increased nonlinearly with the dose. Terminal elimination half-life, calculated over the last three serum concentration time points, increased with dose and the clearance decreased with dose.

Antibodies to CNTO 95 were detected in only one (2 mg/kg) of nine animals receiving a single dose of CNTO 95.

Preclinical safety evaluations. Following weekly i.v. administration of CNTO 95 at doses of 10 and 50 mg/kg for 1 to 6 months, there were no unscheduled deaths and no clinical signs or changes in food consumption indicative of an adverse effect of CNTO 95. Body weights were unaffected by treatment and there were no findings from the physical or ophthalmic examinations that were CNTO 95 related. Similarly, there were no CNTO 95–related changes in physiologic indices, electrocardiograms, or clinical pathology parameters, including coagulation assays and urinalysis. No CNTO 95–related lesions were observed in any of the tissues and organs examined histologically at necropsy, including ovaries and uterus and the epiphyseal plate of the femur in both males and females. CNTO 95 produced no treatment-related changes in organ weights, including ovary weights, compared with control animals.

In all three repeat-dose safety studies, CNTO 95–treated animals received extensive exposure to CNTO 95 throughout the dosing period. Serum concentrations of CNTO 95 in the subset of animals assigned to a treatment-free period before necropsy are illustrated in Figs. 2 and 3. Peak serum concentrations of CNTO 95 increased between the first and the last doses. In the 25-week study, evaluation of trough serum concentrations indicated that steady state was achieved after the sixth dose.



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  		Fig. 2. Serum concentration of CNTO 95 in cynomolgus macaques following four weekly i.v. doses of CNTO 95 at 10 or 50 mg/kg. Arrows, days of CNTO 95 administration. Trough values only were collected between days 7 and 21. Points, mean of data from four animals (two males and two females); bars, SE.

 


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  		Fig. 3. Serum concentration of CNTO 95 in cynomolgus macaques following 25 weekly i.v. doses (days 1-168) of CNTO 95 at 10 or 50 mg/kg. Trough values only were collected between days 7 and 161. Points, mean of data from four animals (two males and two females); bars, SE.

 
Antibodies to CNTO 95 were detected in only 1 (50 mg/kg) of 40 CNTO 95–treated animals in which anti–CNTO 95 antibodies were measured.

Incisional wound healing study. Administration of 50 mg/kg CNTO 95 in the wound healing study was well tolerated. There were no CNTO 95–related effects noted in clinical observations, body weights, clinical pathology, coagulation parameters, C-reactive protein, and serum amyloid A (results not shown).

The tensile strength of wounds from CNTO 95–treated animals and saline-treated animals were similar (Fig. 4). Immunohistochemical staining of the wound sites showed mostly negative to weak binding of CNTO 95 compared with the positive control human placental tissues that showed moderate to strong binding (Table 2).



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  		Fig. 4. Effect of CNTO 95 treatment (50 mg/kg, i.v. on day 1) on tensile strength of incisional wounds in the cynomolgus macaque.

 
Serum concentrations of CNTO 95 during the wound healing study ranged from 657 ± 26 µg/mL 1 day postdose (day 2) to 104 ± 11 µg/mL 14 days postdose (day 15).

Histopathologic examination of the wound sites showed no notable differences in the healing response between CNTO 95–treated animals and saline-treated animals.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
These studies show that CNTO 95 can be safely administered to cynomolgus macaques by weekly i.v. administration without any signs of adverse effects. The suitability of the cynomolgus macaque as the species of choice for preclinical safety evaluation of CNTO 95 was based on similar binding affinity to macaque and human cells (4) and similar binding profiles to human and macaque tissues in vitro.

The immunolocalization studies to human and macaque tissues showed that CNTO 95 bound to vascular endothelial cells, vascular smooth muscle cells, and epithelial cells in most tissues evaluated. The pattern of in vitro tissue binding was similar in human and macaque tissues and the binding of CNTO 95 to cynomolgus tissue sections in vitro was similar to that observed following i.v. administration.

CNTO 95 was more rapidly eliminated from serum following i.v. dosing at 2 mg/kg than at 10 or 50 mg/kg. The nonlinear pharmacokinetics of CNTO 95 in cynomolgus macaques is consistent with the reported nonlinear pharmacokinetics reported for the anti-{alpha}vß3 integrin monoclonal antibody, Vitaxin (9). A rapid elimination phase of CNTO 95 seemed to occur when serum concentration fell below ~30 µg/mL. This rapid disappearance of CNTO 95 from the serum may be related to the binding of CNTO 95 to tissues as described above. An earlier quantitative analysis of CNTO 95 binding showed that CNTO 95 exhibited saturable binding to monkey endothelial cells in vitro with an EC50 value of ~1 µg/mL (4). To maintain consistent exposure of macaques to CNTO 95 over the study periods, it was necessary to maintain the serum concentrations of CNTO 95 above the threshold concentration below which a rapid elimination profile was observed. At the doses of CNTO 95 (10 and 50 mg/kg, i.v.) selected for the macaque safety evaluation studies, serum concentrations were maintained at above 30 µg/mL and are, therefore, expected to produce adequate saturation of tissue binding throughout the dosing period.

Although monkey anti–CNTO 95 antibodies could also result in rapid clearance of CNTO 95 from serum, this is unlikely to be the cause of the rapid terminal elimination. Antibodies to CNTO 95 were detected in only two animals in these studies. Although the presence of CNTO 95 in the serum could potentially interfere with the detection of antibodies to CNTO 95, the treatment-free washout period was sufficient in 16 animals to positively confirm that they were antibody negative. Additional animals were analyzed for antibodies to CNTO 95; however, the results were inconclusive because, in most instances, necropsy was done before washout, leaving substantial serum concentrations of CNTO 95 that could have interfered with the antibody assay. Monkey immune responses to CNTO 95 were evaluated in these studies to support the interpretation of the pharmacokinetic and safety evaluations. The immunogenicity of human immunoglobulin proteins in monkeys is not considered to be predictive of immunogenicity in humans (17, 18).

Although CNTO 95 was shown to bind to epithelium, endothelium, and vascular smooth muscle cells throughout the body, this binding seemed to have no adverse consequence. In most cases, the binding intensity was mild to moderate and was localized to the cytoplasm rather than to the membrane. Intense membrane binding was seen only in the human placenta, where active angiogenesis is ongoing.

Macaques treated with CNTO 95 for up to 6 months at doses up to 50 mg/kg showed no signs of toxicity. There were no adverse effects of treatment throughout the repeat dose studies and microscopic examination of an extensive set of tissues and organs revealed no treatment-related pathologic findings. These results differ from observations seen with angiogenesis inhibitors with a different mechanism of action, such as inhibitors of VEGF. Studies conducted in cynomolgus macaques with Avastin showed epiphyseal dysplasia in the growth plates in immature animals and decreased ovary and uterine weights with an absence of corpora lutea in females (11). These effects were proposed to result from inhibition of normal physiologic angiogenesis in the growth plates of immature animals and to inhibition of angiogenesis in normal ovarian cycling. The cynomolgus macaques used in the Avastin studies were of a similar age and weight range to the animals used in these CNTO 95 studies. Therefore, similar effects would have been expected in the CNTO 95–treated animals if physiologic angiogenesis was being inhibited.

In contrast to the VEGF inhibitors, which inhibit both normal physiologic and pathologic angiogenesis, CNTO 95 does not seem to inhibit physiologic angiogenesis. There were no effects of treatment with CNTO 95 on the histology of the growth plates and no treatment-related changes in ovary weights or histopathologic differences in the ovaries or uterus compared with saline control-treated animals. Studies conducted in cynomolgus macaques treated with cilengitide, a small molecular mass peptide inhibitor of the {alpha}vß3 and {alpha}vß5 integrins, also did not report any effects on bone growth plates or ovaries (19).

The incisional wound healing study showed that CNTO 95 had no apparent effect on wound healing. The in vitro and in vivo immunolocalization studies showed that the binding of CNTO 95 to the wound sites was generally weak or absent, suggesting that {alpha}v integrins were not up-regulated in the incisional wound sites. The lack of an inhibitory effect on wound healing supports the hypothesis that CNTO 95 does not inhibit the normal physiologic wound healing process of which angiogenesis is a component.

The hypothesis that {alpha}v inhibition can inhibit pathologic angiogenesis but not physiologic angiogenesis is supported by other studies in the literature. Tumastatin, an endogenous inhibitor of the {alpha}vß3 integrin, was shown to inhibit tumor and matrigel-supported angiogenesis but not to inhibit angiogenesis in skin wound healing or regenerating liver (20). The {alpha}vß3 integrin was up-regulated only in pathologic angiogenesis and not physiologic angiogenesis. In mice lacking the gene for the {alpha}v integrin subunit, extensive vasculogenesis and angiogenesis is present in embryos, indicating that {alpha}v integrins are not essential for normal blood vessel development (21). In contrast, the {alpha}5ß1 integrin and VEGF are essential for normal vasculogenesis and angiogenesis (22). Because VEGF is required for normal vessel growth, it can inhibit both physiologic angiogenesis, such as wound healing and reproductive function, as well as pathologic angiogenesis. The {alpha}v integrin inhibitors, on the other hand, do not seem to play an important role in normal physiologic angiogenesis so they could have a more selective effect on tumor angiogenesis and fewer adverse effects than the VEGF inhibitors.

In summary, despite the widespread binding to normal tissues, treatment with CNTO 95 produced no signs of toxicity. These studies show that CNTO 95 is safe in macaques and, unlike some other angiogenesis inhibitors, does not inhibit wound healing and does not seem to inhibit normal physiologic angiogenesis.


   Acknowledgments
 
We thank the following scientists from Charles River Laboratories for their contributions on various aspects of these studies: Mark Nedelman, John Turnier, Kreg Howk, Michelle Horner, Cindy Farman, Michael Tomlinson, Gary Baskin, Glenn Elliott, Doug Kornbrust, David Elmore, Jennifer Rojko, and Uriel Blas-Machado; the following scientists from Centocor, Inc., for their valuable contributions to various aspects of the immunoassays: Li-Pin Kung, Carrie Wagner, Pansy Jordan, Lisa Grimminger, and Christine Gardiner for coordination of studies; Tammy Pawling, Patricia Barr, and Peter Bugelski for scientific discussion and review of the manuscript.


   Footnotes
 
Grant support: Centocor, Inc.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12/20/04; revised 6/ 2/05; accepted 7/11/05.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Eliceiri BP, Cheresh DA. The role of {alpha}v integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J Clin Invest 1999;103:1227–30.[Medline]
  2. Wayner EA, Orlando RA, Cheresh DA. Integrins {alpha}vß3 and {alpha}vß5 contribute to cell attachment to vitronectin but differentially distribute on the cell surface. J Cell Biol 1991;113:919–29.[Abstract/Free Full Text]
  3. Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, Cheresh DA. Definition of two angiogenic pathways by distinct {alpha}v integrins. Science 1995;270:1500–2.[Abstract/Free Full Text]
  4. Trikha M, Zhou Z, Nemeth JA, et al. CNTO 95, a fully human monoclonal antibody that inhibits {alpha}-v integrins, has anti-tumor and anti-angiogenic activity in vivo. Int J Cancer 2004;110:326–35.[CrossRef][Medline]
  5. Davis HM, Prabhakar U, Jang H, et al. A rational approach for a phase I clinical study design to evaluate CNTO 95, a novel, fully human anti-{alpha}v monoclonal antibody (mAb), in patients with solid tumors. Proc Am Soc Clin Oncol 2004;22:A2609.
  6. Jayson GC, Mullamitha S, Ton C, et al. Phase I study of CNTO 95, a fully human monoclonal antibody (mAb) to {alpha}v integrins, in patients with solid tumors. Proc Am Soc Clin Oncol 2004;22:A3119.
  7. Gasparini G, Brooks PC, Biganzoli E, et al. Vascular integrin {alpha}(v)ß3: a new prognostic indicator in breast cancer. Clin Cancer Res 1998;4:2625–34.[Abstract]
  8. Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 2004;25:581–611.[Abstract/Free Full Text]
  9. Gutheil JC, Campbell TN, Pierce PR, et al. Targeted antiangiogenic therapy for cancer using vitaxin: a humanized monoclonal antibody to the integrin {alpha}vß3. Clin Cancer Res 2000;6:3056–61.[Abstract/Free Full Text]
  10. Ryan AM, Eppler DB, Hagler KE, et al. Preclinical safety evaluation of rhuMAbVEGF, an antiangiogenic humanized monoclonal antibody. Toxicol Pathol 1999;27:78–86.[Abstract/Free Full Text]
  11. Fraser HM, Wilson H, Rudge JS, Weigand SJ. Single injections of vascular endothelial growth factor Trap block ovulation in the macaque and produce a prolonged, dose-related suppression of ovarian function. J Clin Endocrinol Metab 2005;90:1114–22.[Abstract/Free Full Text]
  12. Fraser HM, Wulff C. Angiogenesis in the primate ovary. Reprod Fertil Dev 2001;13:557–66.[CrossRef][Medline]
  13. Wulff C, Wilson H, Rudge JS, Wiegand SJ, Lunn SF, Fraser HM. Luteal angiogenesis: prevention and intervention by treatment with vascular endothelial growth factor trapA40. J Clin Endocrinol Metab 2001;86:3377–86.[Abstract/Free Full Text]
  14. Wulff C, Wilson H, Wiegand SJ, Rudge JS, Fraser MH. Prevention of thecal angiogenesis, antral follicular growth, and ovulation in the primate by treatment with vascular endothelial growth factor trap R1R2. Endocrinology 2002;143:2797–807.[Abstract/Free Full Text]
  15. Zimmermann RC, Xiao E, Bohlen P, Ferin M. Administration of antivascular endothelial growth factor receptor 2 antibody in the early follicular phase delays follicular selection and development in the rhesus macaque. Endocrinology 2002;143:2496–502.[Abstract/Free Full Text]
  16. Ferrara N. Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin Oncol 2002;29:10–4.
  17. Bugelski PJ, Treacy G. Predictive power of preclinical studies in animals for the immunogenicity of recombinant therapeutic proteins in humans. Curr Opin Mol Ther 2004;6:10–6.[Medline]
  18. Stephens S, Emtage O, Vetterlein LC, et al. Comprehensive pharmacokinetics of a humanized antibody and analysis of residual anti-idiotypic responses. Immunology 1995;85:668–74.[Medline]
  19. Eskens FALM, Dumez H, Hoekstra R, et al. Phase I and pharmacokinetic study of continuous twice weekly i.v. administration of cilengitide (EMD 121974), a novel inhibitor of the integrins {alpha}vß3 and {alpha}vß5 in patients with advanced solid tumors. Eur J Cancer 2003;39:917–26.
  20. Hamano Y, Zeisberg M, Sugimoto H, et al. Physiological levels of tumastatin, a fragment of collagen IV {alpha}3 chain, are generated by MMP-3 proteolysis and suppress angiogenesis via {alpha}vß3 integrin. Cancer Cell 2003;3:589–601.[CrossRef][Medline]
  21. Bader BL, Rayburn H, Crowley D, Hynes RO. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all {alpha}v integrins. Cell 1998;95:507–19.[CrossRef][Medline]
  22. Yang JT, Rayburn H, Hynes RO. Embryonic mesodermal defects in {alpha}5 integrin-deficient mice. Development 1993;119:1093–105.[Abstract]



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