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(Investigative Ophthalmology and Visual Science. 1999;40:2225-2234.)
© 1999 by The Association for Research in Vision and Ophthalmology, Inc.

Human Anti-Transforming Growth Factor-ß2 Antibody: A New Glaucoma Anti-Scarring Agent

M. Francesca Cordeiro, Jennifer A. Gay and Peng T. Khaw

From the Wound Healing Research and Glaucoma Units, Moorfields Eye Hospital and Institute of Ophthalmology, London, United Kingdom.

Abstract

PURPOSE. Currently available anti-scarring regimens for glaucoma filtration surgery have potentially blinding complications and thus the need for alternative and safer agents. The effects of a new antibody to transforming growth factor (TGF)-ß2 on in vitro and in vivo conjunctival scarring and after glaucoma filtration surgery were investigated.

METHODS. The activity of a novel recombinant monoclonal neutralizing antibody (mAb) to human TGF-2 (rhAnti-TGF-ß2 mAb) was studied in conjunctival fibroblast-mediated proliferation, migration, and collagen contraction. Its safety in subconjunctival administration was assessed in vivo, and, in a rabbit model of glaucoma filtration surgery, its effects on conjunctival scarring and filtration surgery outcome were investigated.

RESULTS. The rhAnti-TGF-ß2 mAb effectively inhibited TGF-ß2–mediated conjunctival scarring activity in vitro, at 50% inhibitory concentrations (IC50) of less than 1 nM. It significantly improved glaucoma filtration surgery outcome in an animal model of aggressive conjunctival scarring compared with control (P = 0.0291) and was clinically safe, nontoxic, and well tolerated after subconjunctival administration.

CONCLUSIONS. Subconjunctival rhAnti-TGF-ß2 mAb treatment significantly affects surgical outcome and effectively reduces conjunctival scarring both in vitro and in vivo. It appears safe for subconjunctival administration and when compared with mitomycin-C treatment histologically, much less destructive to local tissue. rhAnti-TGF-ß2 mAb may have potential as a new anti-scarring agent for use in glaucoma filtration surgery.

Transforming growth factor (TGF)-ß is a multifunctional growth factor that is a potent stimulator of scarring throughout the body.1 2 3 4 In the eye, of the three human isoforms (TGF-ß1, TGF-ß2, and TGF-ß3), TGF-ß2 is predominant5 6 and has been implicated in several ocular scarring processes including proliferative vitreoretinopathy, cataract formation, and conjunctival wound healing, especially that occurring after glaucoma filtration surgery.7 8 9 10 11

In glaucoma filtration surgery, excessive postoperative scarring at the wound site significantly reduces surgical success (Fig. 1) .12 13 14 15 Although anti-scarring agents such as mitomycin-C (MMC) and 5-fluorouracil help prevent postsurgical scarring and improve glaucoma surgical outcome, they do so by causing widespread fibroblast cell death and apoptosis16 17 and are associated with severe and potentially blinding complications.18 19 20 More specific anti-scarring treatments are therefore required.



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  		Figure 1. Scarring occurring after filtration surgery is a common cause of surgical failure. (A) A typical bleb appearance after successful surgery is seen (arrows). (B) Any scarring at the surgical wound site impedes the flow of aqueous, leading to flattening, vascularization and contraction of the bleb (arrows). Although antimetabolites such as MMC have improved filtration surgery results, their use is associated with severe complications such as thin and cystic blebs (arrows, C) that are at risk of wound leakages and infection.

 
The use of more physiological and alternative agents such as neutralizing TGF-ß antibodies, although not studied in the eye, has been shown to reduce experimental dermal scarring.21 However, previous investigators have used polyclonal antibodies, which are not suitable for human use.21

We investigated a novel, recombinant human anti-TGF-ß2 monoclonal antibody (rhAnti-TGF-ß2 mAb), distinct from other TGF-ß mAbs, particularly because of its human sequence and high affinity and specificity for the active form of the TGF-ß2 isoform. The production of this mAb is unusual because it does not involve the process of immunization. Instead, it is isolated using phage display repertoire technology in Escherichia coli, being finally expressed as a recombinant, fully human, monoclonal IgG4 antibody,22 23 which made it a suitable agent for therapeutic manipulation of conjunctival wound healing. Although studies have shown rhAnti-TGF-ß2 mAb to be safe and nontoxic with systemic and intraocular administration, it has not been clinically assessed for subconjunctival application. We therefore investigated its safety and role in inhibiting conjunctival scarring after glaucoma filtration surgery.

The purposes of this study were to determine the effects of anti-TGF-ß2 mAb on ocular scarring in vitro (in human Tenon’s capsular fibroblasts [HTFs]), to assess the safety and tolerance of rhAnti-TGF-ß2 mAb in vivo (subconjunctival administration in the rabbit), and to investigate the effects of subconjunctival rhAnti-TGF-ß2 mAb using an animal model of aggressive subconjunctival scarring after glaucoma filtration surgery.

Methods

All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. For human cell culture experiments, the tenets of the Declaration of Helsinki were followed, and institutional human experimentation committee approval was granted.

Cell Culture
HTFs were propagated from 0.5-cm3 tissue explants,24 as previously described. Fibroblasts from the same initial population, between the third and sixth passages, were cultured in monolayers at 37°C in 5% humidified CO2 in air and fed every 3 days with renewed medium (Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 µl/ml penicillin, 100 µg/ml streptokinase, 0.25 µg/ml fungizone, and 50 µg/ml gentamicin (all from Gibco Life Technologies, Paisley, UK).

In Vitro Assays
In all assays, treatment groups consisted of TGF-ß2 at 10-12 M (R&D Systems, Oxon, UK) in Dulbecco’s modified Eagle’s medium-1% bovine serum albumin (BSA) preincubated with different concentrations of rhAnti-TGF-ß2 mAb and a null IgG4 antibody at 0, 0.01, 0.1, 1, 10, and 20 µg/ml (Cambridge Antibody Technology, Cambs, UK) in phosphate-buffered saline (PBS).

The assay (WST-1; Boehringer Mannheim, East Sussex, UK), which is a colorimetric assay based on the cleavage of the tetrazolium salt—4-[3-(iodophenyl)-2(4-ntrophenyl)-2H-5-tetrazolio]-1,3-benzene disulphonate—by mitochondrial dehydrogenase in viable cells, was used to assess cell proliferation in this study. Experiments were performed in sextuplicate in 96-well plates at an initial cell density of 3000 cells/well. Antibody treatments were applied to cell monolayers. Cell number for each treatment was determined directly from a WST-1calibration curve (WST-1 absorbance of known cell densities performed for each experimental run).

Ocular fibroblast migration (chemotaxis) was assessed with an assay system with tissue culture inserts (Transwell; Costar, High Wycombe, UK) for 24-well plates, using a method previously described.25 Cells (initial density 10,000 cells/well) were allowed to settle, after which treatments were added to the outer chamber. After 16 hours at 37°C in 5% CO2 in air, the number of cells that had migrated to the undersurface of each membrane was counted.

TGF-ß2 antibody collagen contraction studies were performed in 48-well plates. One milliliter free-floating collagen type-1 (Sigma, Dorset, UK) lattices were prepared, using a method previously described.26 Aliquots (125 µl) of the collagen–cell suspension mixture (250,000 cells/ml) were pipetted into single wells and allowed to polymerize, after which 500 µl of each antibody treatment was administered. Measurement of lattices was performed at 3 days and calculated as the percentage reduction in the original lattice surface area.

All results were calculated as means with 95% confidence intervals (CIs). The data were analyzed using statistical computer software (SPSS for Windows; SPSS, Chicago, IL) at individual time points, using a one-way analysis of variance.

Tolerance of Subconjunctival rhAnti-TGF-ß2 mAb in Rabbit
Eleven New Zealand rabbits, aged 12 to 14 weeks and weighing 2 to 2.4 kg, were randomly assigned to treatment consisting of subconjunctival injections of 100 µl of either rhAnti-TGF-ß2 mAb (1.0 mg/ml) , null antibody (1.0 mg/ml), or PBS (carrier). These were administered to animals under topical anesthesia (amethocaine 1% eye drops) using a 25-G needle (Myjector 100u; Terumo, Tokyo, Japan), in the left eye at the same site (5 mm behind the limbus at the nasal margin of the superior rectus muscle). The first dose was administered on day 0 and a second 1.5 hours later. Further injections were administered on days 1, 2, 3, and 7. The rationale for this dose regimen was first to attempt to achieve local availability of the antibody at the critical time for postoperative fibrosis, and second, to mimic the clinical situation when subconjunctival injections may be conveniently administered in preparation for surgery and on subsequent visits up to 7 days after surgery as an outpatient. The contralateral eye acted as control. Animals were killed 30 days after the first dose, using a lethal intravenous injection of pentobarbitone administered with animals under a general anesthetic.

Subconjunctival Administration of rhAnti-TGF-ß2 mAb in Rabbit Filtration Surgery
Seventy-two female New Zealand rabbits, aged 12 to 14 weeks and weighing 2 to 2.4 kg, were randomly assigned to treatment that consisted of 100 µl of either 1.0 or 0.1 mg/ml rhAnti-TGF-ß2 mAb (dissolved in PBS). As the control, 1.0 mg/ml null antibody or PBS carrier was administered. Subconjunctival injections were administered by passing the needle from the same point in each eye, as described, and injecting into the filtration bleb on day 0 before filtration surgery, immediately after surgery, and then on days 1, 2, 3, and 7 after surgery.

Filtration surgery was performed in the left eye of all rabbits, using a technique previously described,27 with insertion of a 22-gauge, 25-mm Venflon 2 (Ohmeda, Sweden) intravenous cannula through a scleral tunnel into the anterior chamber. One drop of atropine sulfate 1% and betnesol-N ointment were instilled at the end of surgery.

Evaluation of Tolerance to rhAnti-TGF-ß2 mAb
Tolerance of rhAnti-TGF-ß2 mAb was investigated by assessment of local toxicity and ocular intolerance in rabbit eyes after repeated subconjunctival injections of rhAnti-TGF-ß2 mAb, using clinical parameters such as anterior chamber activity, measurement of intraocular pressure (IOP), and macroscopic appearance of the injected area and conjunctival vascularity, as well as histologic characteristics.

Examination by a masked observer was made daily for the first 2 weeks and every 3 days thereafter until animals were killed on day 30. Both eyes were examined, with the contralateral untreated eye acting as control. Anterior chamber inflammation was assessed by slit lamp photography and graded as follows: 0, no cells; +1, cells present; +2, fibrin formation; and +4, a hypopyon. A general description was recorded of the injected area in terms of complications such as lid edema, chemosis, hemorrhage, and corneal toxicity. Vascularity was assessed by dividing the conjunctival areas into quadrants—superior, nasal, and temporal—and scoring the appearance (0, avascular; +1, normal vascularity; +2, hyperemic; +3, very hyperemic). Avascularity was also assessed and graded binomially as follows: 1, presence of avascularity in any area of the eye, or 0, no avascularity. Measurement of IOP in both eyes was made using the Mentorô Tonopen (Mentor, Norwall, MA) after topical installation of 0.4% benoxinate HCl local anesthetic. Three recordings per eye were made per time point, and a mean reading was documented.

Conjunctival vascularity per quadrant was analyzed using the repeated measures procedure and the generalized linear model (SPSS). Avascularity was assessed using Pearson’s {chi}2 test to compare treatment groups. Finally, IOP was analyzed using the multivariate analysis of variance, with Bonferroni’s modification to compare differences between treatments and the effects of time and treatment.

Clinical Evaluation of rhAnti-TGF-ß2 mAb Effects in Filtration Surgery
All animals were examined by a masked observer at set times after surgery. Examination of both eyes (contralateral untreated eye used as control) was made daily from day 0 to day 4 and at regular periods until death. Bleb characteristics including bleb width, height, and length were assessed, as in previous studies,27 as were recordings of IOP, anterior chamber activity, and conjunctival vascularity, as described. Anterior chamber depth was assessed subjectively, graded, and recorded as either deep (+2), shallow (+1), or flat (0).

In the analysis of rhAnti-TGF-ß2 mAb in rabbit filtration surgery, the primary efficacy end point was taken as bleb survival. Bleb failure was defined as the appearance of a flat, vascularized, scarred bleb in association with a deep anterior chamber. Kaplan–Meier and log rank statistics were used to compare treatment groups in bleb and IOP failure (defined as the return of the IOP in the surgical eye to baseline level). Bleb area and height, anterior chamber depth and activity, and conjunctival vascularity per quadrant were all analyzed using the repeated measures procedure and the generalized linear model. This allowed comparison of treatment groups over the whole study period, using between-subjects tests. Avascularity and IOP were analyzed as has been described.

Analysis of Rabbit Tissues
On days 3, 8, 14, and 30 after surgery, rabbits were killed and both eyes exenterated. The eyes were fixed in 10% buffered formal saline and then embedded in paraffin wax. Sequential sections (5 µm) were then cut and histologic staining performed including: hematoxylin and eosin (for cellularity); Gamori’s trichrome, picrosirius red, and anti-collagen III immunohistochemistry (for collagen and scar formation); aldehyde fuchsin (for elastin and elaunin fibers) and oxidation aldehyde fuchsin (for oxytalan). Grading of the following parameters was performed by a masked observer: total cellularity and fibroblast count gauged with hematoxylin staining,27 scar formation and architecture assessed with picrosirius red staining,21 collagen density and orientation (trichrome staining characteristics),21 collagen III deposition (immunostaining intensity), and oxytalan and elastic-related fiber deposition, judged by aldehyde fuchsin staining.27 The scoring system used was originally described by Shah et al.21 (Scale: -4 to +4; 0, same as control eye; 1, 1%–25% of control; 2, 26%–50% of control; 3, 51%–75% of control; 4, >75% of control: prefix +, more than; prefix -, less than). All operated eyes were compared with nonsurgical contralateral eyes. This method of grading was used because the wound-healing process in skin is believed to be similar to that occurring elsewhere in the body. It therefore seemed appropriate to use a grading system well characterized in skin to assess histologic differences. Mean scores of histologic parameters for each treatment group per time point were calculated, and these semiquantitative data were analyzed using analysis of variance statistics (SPSS).

Results

Antibody Effects In Vitro
The rhAnti-TGF-ß2 mAb was found to inhibit ocular scarring effectively in vitro, when assessed by its effects on HTFs.

Fibroblast Proliferation.
Treatment with rhAnti-TGF-ß2 mAb significantly reduced cell numbers compared with the control antibody treatment at all concentrations studied (Fig. 2 ; P < 0.05). The 50% inhibitory concentration (IC50) was found to occur at 0.13 µg/ml, equivalent to 0.885 nM, where the baseline was taken as the mean number of cells/well in the serum-free–only control group (3948 ± 356 [95% CI]) and maximum activity as the mean number of cells/well in the TGF-ß2–only treatment group (5492 ± 93.5).



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  		Figure 2. The rhAnti-TGF-ß2 mAb effectively inhibited in vitro human ocular fibroblast proliferation, migration, and contraction. Assessment of HTF proliferation was made by determining the cell number for each treatment directly from a WST-I calibration curve. Determination of the neutralization activity of rhAnti-TGF-ß2 mAb was performed at 3000 cells and showed a significant reduction in cell number compared with control antibody at all concentrations studied (0.01, 0.1, 1, 10, and 20 µg/ml). The IC50 neutralization activity of rhAnti-TGF-ß2 mAb was found to occur at 0.13 µg/ml, equivalent to 0.885 nM. Error bars, 95% CI; *activity of rhAnti-TGF-ß2 mAb significantly different from that of null antibody (P < 0.05).

 
Fibroblast Migration.
HTF migration was significantly reduced after treatment with the TGF-ß2 antibody, which inhibited HTF migration at concentrations of 0.1 µg/ml and higher compared with that with the control antibody (P < 0.05). The IC50 was found to occur at 0.092 µg/ml, equivalent to 0.627 nM (baseline control group, 15.75 ± 3.20 cells; maximum in the TGF-ß2–only group, 61.75 ± 6.48 cells).

Fibroblast-Mediated Collagen Contraction.
Treatment with hAnti-TGF-ß2 significantly reduced HTF-mediated collagen contraction at concentrations of 0.1 µg/ml and higher compared with control antibody treatment, (P < 0.05). The IC50 of the test antibody was found to occur at 0.12 µg/ml, equivalent to 0.818 nM (baseline control group, 31.9 ± 3.24%; maximum TGF-ß2–only group, 55.23 ± 2.82%).

Effects of Antibody In Vivo
The rhAnti-TGF-ß2 mAb was found to improve glaucoma filtration surgery outcome significantly in the rabbit in a model of aggressive postsurgical scarring in glaucoma and appeared safe and nontoxic after repeated subconjunctival injections.

Local Tolerance of Subconjunctival rhAnti-TGF-ß2 mAb.
Single and repeated subconjunctival injections of rhAnti-TGF-ß2 mAb (100 µl of 1 mg/ml) were well tolerated in New Zealand rabbits. No intraocular inflammation was seen in any rabbit at any time in the study. Administration of rhAnti-TGF-ß2 mAb, null antibody, and PBS control caused early chemosis in all treatment groups but resolved after the 7-day period of repeated injections. No significant differences in conjunctival vascularity were found between rhAnti-TGF-ß2 mAb and null antibodies. All injections were administered in the superior quadrant, the site of maximum vascularity, with a significant difference only on days 1 and 2 between both rhAnti-TGF-ß2 and null antibody groups compared with the PBS carrier treatment (P < 0.05). No significant differences were found in the extent of nonperfused, avascular areas or mean IOPs between treatment groups (P > 0.05), or at any time point after injection.

Histologically, no evidence of inflammatory cells in the aqueous or vitreous gel was seen in any eye. No statistical differences were found in histologic parameters between rhAnti-TGF-ß2 mAb and null antibody treatments.

Effects on Filtration Surgery.
rhAnti-TGF-ß2 mAb was associated with successful filtration surgery, evidenced by bleb morphology. Because any subconjunctival scarring occurring at the filtration site causes flattening and a decrease in surface area of the bleb, important indicators of effective filtration surgery include the presence of a raised and a well-formed bleb. Figure 3 shows the typical appearances of filtration blebs at day 30 of this study. Treatment with rhAnti-TGF-ß2 mAb was associated with an elevated, diffuse, fleshy-looking bleb (Fig. 3A) compared with the flat, scarred bleb in the PBS control group (Fig. 3B) .



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  		Figure 3. The rhAnti-TGF-ß2 mAb was found to improve glaucoma filtration surgery outcome in the rabbit significantly and appeared safe and nontoxic after repeated subconjunctival injections. Rabbits undergoing filtration surgery with rhAnti-TGF-ß2 mAb treatment characteristically had an elevated, diffuse, fleshy-looking bleb 30 days after treatment (A), compared with the flat, scarred blebs treated with PBS control (B). (Black arrows demarcate edges of the bleb, and white arrowheads indicate cannula).

 
rhAnti-TGF-ß2 mAb significantly prolonged bleb survival after filtration surgery, compared with control. The Kaplan–Meier curve in Figure 4 A, shows the effect of rhAnti-TGF-ß2 mAb treatment on bleb survival compared with control (log rank statistics; P = 0.0291). Table 1 demonstrates the rates of bleb failure in rabbits. At day 30, bleb failure was present in only two rabbits (33%) treated with rhAnti-TGF-ß2 mAb (1.0 and 0.1 mg/ml) but was present in five rabbits (83%) undergoing control treatments. The mean bleb survival within treatment groups was as follows: rhAnti-TGF-ß2 mAb (1 mg/ml), 26.83 days; rhAnti-TGF-ß2 mAb (0.1 mg/ml), 26.50 days; null antibody (1 mg/ml), 24.17 days; and PBS carrier, 21.83 days. This suggests that rhAnti-TGF-ß2 mAb on average increased bleb survival by 5 days. Comparison between the two concentrations of the rhAnti-TGF-ß2 mAb showed no significant difference in bleb survival.



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  		Figure 4. The rhAnti-TGF-ß2 mAb significantly prolonged bleb survival after filtration surgery, compared with control, as shown in the Kaplan–Meier survival curve (A; log rank statistics, P = 0.0291). Anti-TGF-ß2 mAb–treated eyes tended to have larger blebs, and a significant difference in bleb height was found between rhAnti-TGF-ß2 mAb andcontrol treatments (P = 0.029; B), but not in bleb base area (P = 0.059; C).

 

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  		Table 1. Incidence of Bleb Failure in Rabbits Undergoing Filtration Surgery with rhAnti-TGF-ß2 mAb and Control Treatments

 
Bleb size was also affected by rhAnti-TGF-ß2 mAb treatment. Analysis of bleb area and height using the repeated measures procedure by the generalized linear model, revealed a significant difference between rhAnti-TGF-ß2 mAb and control in bleb height (P = 0.029; Fig. 4B ). Anti-TGF-ß2 mAb–treated eyes tended to have larger blebs—that is, consistently higher with greater bleb base area—although bleb area did not reach statistical significance (P = 0.059; Fig. 4C ).

There was a trend for rhAnti-TGF-ß2 mAb treatment groups to have shallower anterior chamber depths than control, reflecting increased drainage of aqueous. However, comparison between treatment groups did not achieve statistical significance (P = 0.109).

Local reaction to rhAnti-TGF-ß2 mAb injections was assessed by the degree of anterior chamber inflammation and conjunctival vascularity. No significant difference was found between treatment groups in anterior chamber activity (P = 0.911), when judged by slit lamp microscopy. Comparison of treatment groups in the vascularity in each quadrant showed no significant difference (superior, P = 0.275; temporal, P = 0.849; nasal, P = 0.488) throughout the study period.

One of the features of existing cytotoxic anti-scarring regimens (e.g., MMC) is their production of nonperfused, avascular areas in the locally treated tissues. These areas of avascularity are associated with thin-walled, cystic blebs and the attendant risks of leakage through the bleb walls and infection. Therefore, all rabbit eyes were studied for the presence of avascularity. Comparison between treatment groups using {chi}2 tests showed a significant difference on day 7 only (P = 0.036), when rhAnti-TGF-ß2 mAb treatment was associated with an increase in the degree of avascularity, but at no other time point.

Finally, analysis of IOP survival and mean IOPs in the surgical eyes showed no significant difference between treatment groups over the whole study period (P > 0.05).

Histologic Effects.
The rhAnti-TGF-ß2 mAb appeared to be associated with reduced scarring activity at a microscopic level. In the control groups, scarring in the subconjunctival areas at the filtration site consisted characteristically of densely packed layers of collagen and fibroblasts (Fig. 5 A, 5B). In comparison, the rhAnti-TGF-ß2 mAb–treated eyes showed much looser architecture and visible evidence of subconjunctival bleb formation (Fig. 5C 5D) . Total scar formation and architecture, judged by staining characteristics with picrosirius red, was significantly reduced in rhAnti-TGF-ß2 mAb treatment compared with control treatment on day 30 (P = 0.001), as was collagen density, assessed by staining with Gamori’s trichrome (P = 0.021). In addition, an increase in elastic fiber deposition was found in control-treated eyes on day 8 (P = 0.028) but at no other time point. However, no significant differences were found between antibody and control treatment groups in total cellularity, fibroblast count, and collagen III and oxytalan deposition.



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  		Figure 5. Histologic features of 30-day rabbit filtration blebs showing rhAnti-TGF-ß2 mAb is associated with reduced scarring activity at a microscopic level. In the control groups (A, B), scarring in the subconjunctival areas at the filtration site consisted characteristically of densely packed layers of collagen and fibroblasts. In comparison, the rhAnti-TGF-ß2 mAb–treated eyes showed much looser architecture and visible evidence of subconjunctival bleb formation (C, D). Sections are stained with picrosirius red (A, C) and hematoxylin and eosin (B, D). Magnification, x20. C, conjunctiva; b, subconjunctival bleb; S, scleras.

 
We also compared histologic and electron microscopic characteristics of a rabbit eye treated with MMC at its clinically used concentration (0.4 mg/ml) with that treated with rhAnti-TGF-ß2 mAb (1 mg/ml). The MMC treatment was associated with destructive changes in normal conjunctival architecture (Fig. 6 A), including abnormal and absent areas of epithelium and markedly reduced cellularity, with minimal numbers of fibroblasts (Fig. 6A , inset), as has been reported.16 27 28 rhAnti-TGF-ß2 mAb, however, preserved conjunctival architecture (Fig. 6B) , with the appearance of normal, although reduced, numbers of fibroblasts (Fig. 6B , inset), compared with control.



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  		Figure 6. Ultrathin sections showing subconjunctival bleb area in an MMC-treated rabbit (clinically used dose of 0.4 mg/ml MMC, A) compared with that treated with rhAnti-TGF-ß2 mAb (1 mg/ml, B), with transmission electron micrographs in insets. MMC treatment was associated with destructive changes in normal conjunctival architecture (A), including abnormal and absent areas of epithelium and markedly reduced cellularity, with minimal numbers of fibroblasts (A, inset). rhAnti-TGF-ß2 mAb, however, preserved conjunctival architecture (B), with the appearance of normal although reduced numbers of fibroblasts (B, inset) compared with control. Magnification, x20 (insets, x500).

 
Discussion

Important drawbacks in currently used anti-scarring strategies in eyes, such as MMC, are their side effects, associated complications, and extensive microscopic and destructive cellular effects.29 30 31 This study demonstrated a new, more physiological agent that effectively inhibited TGF-ß2–mediated conjunctival scarring activity in vitro and appeared clinically safe, nontoxic, and well tolerated, when administered subconjunctivally in vivo. It significantly improved glaucoma filtration surgery outcome in an animal model of aggressive conjunctival scarring.

HTFs perform an essential role in conjunctival scarring, provide a useful cellular in vitro model,32 and are particularly important in the subconjunctival scarring response after glaucoma filtration surgery. The rhAnti-TGF-ß2 mAb was effective in inhibiting three important fibroblast functions: TGF-ß2 (at 10-12 M) stimulated fibroblast-mediated contraction, proliferation, and migration. The IC50 concentrations of rhAnti-TGF-ß2 mAb for all these assays were similar at less than 1.0 nM.

Compared with TGF-ß1, the effects of TGF-ß2 on ocular-mediated contraction are less well established.33 34 However, Pena et al.,35 using rabbit dermal-fibroblast–populated collagen gels as a model of proliferative vitreoretinopathy, found TGF-ß2 to be comparable to TGF-ß1 in stimulating contraction at a concentration of 2 x 10-10 M. In that same study, anti-TGF-ß1 and anti-TGF-ß2 neutralizing antibodies were shown to reduce contraction in a dose-dependent manner with maximal inhibition at 50 µg/ml. Stimulation of ocular fibroblast proliferation has been shown to occur with TGF-ß1,9 TGF-ß2, and TGF-ß3,10 although TGF-ß1-induced proliferation has mainly been studied in human foreskin and human dermal and mouse fibroblast proliferation.36 37 38 39 40 41 Effects of TGF-ß on ocular cell migratory activities include stimulation of HTF with TGF-ß19 ; trabecular meshwork cells with a mixture of TGF-ß1 and -ß242 ; and corneal cell migration,43 44 although TGF-ß1 has also been implicated in inhibiting keratocyte (corneal fibroblast) migration.45 Cell migration induced by TGF-ß has been demonstrated in several other cell types including neutrophils and peripheral monocytes.46 47

Rabbit models of glaucoma filtration surgery, although commonly used experimentally, represent a very aggressive scarring response compared with that in humans.48 49 Therefore, virtually every agent shown to reduce scar tissue formation in the rabbit is effective in humans.48 49 50 51 The particular surgical procedure used in this study characteristically maximizes the scarring response to the level of the subconjunctiva (as opposed to the sclera).27 This is achieved by maintaining a patent channel through the sclera for aqueous to pass from the anterior chamber of the eye to the subconjunctival space. Localization of scar deposition thus occurs in the subconjunctival area overlying the filtration site.

The rhAnti-TGF-ß2 mAb was effective in improving bleb survival and surgical outcome in our studies. Two doses of the antibody were used (100 µl of 1 mg/ml and 0.1 mg/ml), both of which showed efficacy throughout the study, although no dose-dependent result was obtained. Bleb failure, rather than the return of IOP to baseline, was chosen as the end point in the study, because this rabbit model of filtration surgery appears to destabilize and persistently lower IOP, probably by causing a breakdown in the blood–aqueous barrier.27 However, evidence of increased aqueous outflow through the filtration site in rhAnti-TGF-ß2 mAb treatment groups compared with control treatment groups, may also be obtained from measurements of anterior chamber depth and bleb morphology. Therefore, shallow anterior chambers and larger blebs indicate improved outflow.

In glaucoma filtration surgery, at least two factors would be expected to alter the amount of TGF-ß secreted and its degree of activation at the filtration wound site: first, the presence of aqueous fluid flowing through the wound site, and second, initiation of the blood clotting cascade and breakdown of the blood–aqueous barrier caused by surgery. The production of TGF-ß2 in aqueous is believed to be derived from local tissues52 53 and is most probably activated by plasmin and thrombospondin released from blood components.54 55 The presence of aqueous at the wound site has long been known to affect the wound-healing response in glaucoma surgery,56 57 and TGF-ß activity in aqueous has been implicated as an important factor.11 This has been supported by the demonstration that compared with other growth factors found in aqueous, TGF-ß has been shown to be the most potent in stimulating HTF activity.9 In addition, aqueous concentrations of TGF-ß2 are increased in glaucomatous eyes.58

Compared with MMC, increasingly used by glaucoma surgeons in the United States59 despite the attendant risks,18 19 30 60 61 the new hAnti-TGF-ß2 mAb investigated in the current project was found to be a potentially more controlled alternative as an anti-scarring agent in glaucoma surgery without any obvious complications. Microscopic characteristics of rhAnti-TGF-ß2 mAb, compared with those of MMC treatment, highlighted these differences. MMC appears to have destructive cellular effects, and it is therefore not surprising that, because of their decreased vascular bed, hypocellularity, and severely impaired scarring response, MMC-treated blebs are prone to having thin and cystic walls.60 These blebs are far more likely to leak, and thus, the increased incidence of infection.20 62 In contrast, rhAnti-TGF-ß2 mAb–treated blebs appeared thicker walled, less cystic, and more vascular, suggesting fewer MMC-type complications.

Although neutralizing agents to TGF-ß have not been used previously in the eye, they have been investigated in dermal scarring. By applying neutralizing antibody injections that were anti-TGF-ß1 and anti-TGF-ß2 (a polyclonal antibody raised in rabbit against porcine platelet TGF-ß2), Shah et al.2 showed that the scarring response could be significantly decreased, to the same degree as the effects produced by exogenous TGF-ß3 in rat dermal excisional wounds. These antibodies, however, have not been studied clinically, because they are unsuitable for human use, and, in particular, the role of TGF-ß2 neutralizing antibodies in postsurgical ocular scarring, specifically after glaucoma filtration surgery, has not been determined. The novel technique used to produce the anti-TGF-ß2 mAb used in this study, made it different from other available TGF-ß mAbs, particularly because of its specificity for human TGF-ß2 and its high affinity with the active rather than the latent form.22 23

Our results show that rhAnti-TGF-ß2 mAb is effective in reducing subconjunctival scarring after glaucoma filtration surgery in the rabbit. This is the first time such a specific agent has been used in filtration surgery and represents an exciting new field for development in inhibiting postsurgical scarring in the eye, without perhaps the severe side effects associated with existing modulating agents.

Footnotes

Supported in part by Wellcome Trust Vision Research Fellowship and the Medical Research Council, London, United Kingdom; and Cambridge Antibody Technology, Melbourn, Cambridgeshire, United Kingdom.

Submitted for publication December 3, 1998; revised April 6, 1999; accepted May 20, 1999.

Proprietary interest category: N.

Corresponding author: M. Francesca Cordeiro, Wound Healing Research and Glaucoma Units, Moorfields Eye Hospital and Institute of Ophthalmology, Bath Street, London EC1V 9EL, UK.

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