Inhibition of glial scarring in the injured rat brain by a recombinant human monoclonal antibody to transforming growth factor-β2

Inhibition of glial scarring in the injured rat brain by arecombinant human monoclonal antibody to transforminggrowth factor-b2

Ann Logan, Jonathan Green1, Allison Hunter2, Ronald Jackson1 and Martin Berry2

Department of Medicine, University of Birmingham, Birmingham B15 2TT, UK1Cambridge Antibody Technology Limited, The Science Park, Melbourn, Royston, Cambridgeshire SG8 6JJ, UK2Department of Anatomy and Cell Biology, UMDS, Guy's Hospital, London SE1 9RT, UK

Keywords: central nervous system, ®brosis, ®brotic neuropathology, immunoneutralization, transforming growth factor b

Abstract

The transforming growth factor-bs (TGF-bs) are potent ®brogenic factors implicated in numerous central nervous system (CNS)pathologies in which ®brosis and neural dysfunction are causally associated. In this study, we aim to limit the ®brogenic process in amodel of CNS scarring using a recombinant human monoclonal antibody, derived from phage display libraries and speci®c to theactive form of the TGF-b2 isoform. The implicit inference of the work was that, as such antibodies are potential pharmacologicalagents for the treatment of human CNS ®brotic diseases, validation of ef®cacy in a mammalian animal model is a ®rst step towardsthis end. Treatment of cerebral wounds with the anti-TGF-b2 antibody led to a marked attenuation of all aspects of CNS scarring,including matrix deposition, formation of an accessory glial-limiting membrane, in¯ammation and angiogenesis. For example, in thewound, levels of: (i) the connective tissue components ®bronectin, laminin and chondroitin sulphate proteoglycan; and (ii) wound-responsive cells including astrocytes and macrophages/microglia, were markedly reduced. Our ®ndings suggest that such syntheticanti-®brotic TGF-b antibodies are potentially applicable to a number of human CNS ®brotic diseases to arrest the deposition ofexcessive extracellular matrix components, and maintain and/or restore functional integrity.

Introduction

Fibrosis is caused by the excessive deposition of extracellular matrix,

which can compromise the function of the tissue involved. The

transforming growth factor-bs (TGF-bs) are potent ®brogenic factors

which have been implicated in a broad diversity of biological actions,

including enhancement of wound healing, stimulation of extracellular

matrix synthesis, modulation of in¯ammatory cell in®ltration,

immunosuppression and neuroprotection (Cui & Ackhurst, 1996;

Pratt & McPherson, 1997). A role for TGF-bs has been suggested in

numerous central nervous system (CNS) pathologies in which ®brosis

and neural dysfunction are causally associated. For example, in post-

trauma brain and spinal cord scarring (Logan et al., 1992, 1994); post-

surgical arachnoiditis (Logan & Berry, 1994), haemorrhagic stroke

(Krupinski et al., 1996) and sub-arachnoid haemorrhage (Kitazawa &

Tada, 1994); TGF-bs may also promote plaque development in

Alzheimer's disease and Downs syndrome (Wyss-Coray et al., 1997).

In all of these conditions, TGF-b levels are raised in the cerebrospinal

¯uid (CSF) and locally in damaged neural tissue. For example, after

CNS traumatic damage, we have demonstrated an elevation of the

TGF-b1 isoform, initially derived from haematogenous cells and later

supplemented by endogenous local synthesis by neurons and glia in

the damaged neuropil, and by choroid plexus cells of the impaled

lateral ventricle leading to raised cytokine levels in the CSF (Logan

et al., 1992, 1994). As part of a separate study, to be reported

elsewhere, we have also shown that the TGF-b2 mRNA and protein is

strongly upregulated to a peak of expression in wounds at 5±7 days

following penetrating CNS injury (C. Lagord, M. Berry and A.

Logan, unpublished results). In particular, TGF-b2 is found localized

to reactive astrocytes of the damaged neuropil. These observations

imply a potential role for both TGF-b isoforms in the CNS injury

response.

The most direct evidence to date for a ®brogenic role for TGF-bisoforms in the pathophysiology of CNS ®brosis comes from

experiments in the lesioned brain. On the one hand, raised levels of

TGF-b1 are correlated with the deposition of scar material in such

lesions, whilst immunoneutralization with a turkey polyclonal TGF-

b1 antibody markedly inhibits ®brogenic scarring, albeit with an

accompanying enhanced in¯ammatory response (Logan et al., 1994).

Nothing is known of the role of the TGF-b2 isoform in the ®brogenic

process in the CNS. It is implicit from previous observations that

attenuation of either excess or inappropriate matrix deposition using

TGF-b-related anti-®brotic agents may limit the progress of the

pathogenic process, with anticipated clinical bene®ts. Although the

principle of inhibition of ®brogenesis by TGF-b immunoneutraliza-

tion is established, the reported exacerbation of in¯ammation

associated with the use of animal-derived polyclonal antibodies in

general, and TGF-b1 polyclonal antibodies in particular, may limit

their therapeutic potential for the treatment of patients with ®brotic

disease.

Selection from phage display libraries of human single-chain Fv

fragments allows the preparation of speci®c neutralizing recombinant

human monoclonal antibodies against human self antigens. Although

their therapeutic effectiveness in vivo remains to be established, such

Correspondence: A. Logan, as above.E-mail: [email protected]

Received 31 July 1998, revised 22 February 1999, accepted 1 March 1999

European Journal of Neuroscience, Vol. 11, pp. 2367±2374, 1999 ã European Neuroscience Association

synthetic antibodies would be preferred therapeutic molecules due to

their increased speci®city and reduced immunogenicity. Here, we

report for the ®rst time in vivo ef®cacy of a recombinant human

monoclonal antibody, isolated from a phage display library and

speci®c to the active form of the TGF-b2 isoform. The antibody

signi®cantly inhibits ®brogenesis, glial scarring and, interestingly,

in¯ammation in penetrating incisional wounds of the rat brain.

Methods

Neutralizing TGF-b2 antibody

6B1 IgG4 (immunoglobulin) is a recombinant antibody with a fully

human sequence, directed against TGF-b2. The VH and VL variable

regions of 6B1 were obtained by selection on active human TGF-b2

from phage display libraries of human single-chain Fv antibody

molecules at Cambridge Antibody Technology (Melbourn, Cambrid-

geshire, UK). A whole antibody molecule of the IgG4 isotype was

then constructed by recombinant techniques and expressed in NS0

myeloma cells and puri®ed. The recombinant antibody, termed 6B1,

has been well characterized (Thompson et al., 1999). For example: (i)

it has a high af®nity for TGF-b2 with a dissociation constant of

0.89 nM, as determined by binding to TGF-b2 using the BIACore

biosensor (Table 1); (ii) it shows approximately 9% cross-reactivity

with TGF-b3 (dissociation constant, 10 nM) compared with TGF-b2;

(iii) it has no detectable binding to TGF-b1; (iv) it is speci®c for the

active form of TGF-b2 and does not signi®cantly bind the latent

form; (v) it strongly neutralizes the antiproliferative effect of TGF-b2

in bioassays using TF1 human erythroleukaemia cells (Randall et al.,

1993) (Table 1) with an IC50 of 1±2 nM; (vi) it has strong inhibition of

binding of TGF-b2 to cell surface receptors in a radioreceptor assay

using A549 cells (Lucas et al., 1991) (Table 1); (vii) it has some

ability to neutralize and inhibit TGF-b3 binding, as would be

expected from the 9% cross-reactivity; (viii) it has no signi®cant

ability to inhibit or neutralize TGF-b1 binding as would be expected

from the undetectable binding by BIACore; (ix) it shows no

detectable cross-reactivity with related or unrelated antigens by

immunocytochemistry and ELISA; and (x) it binds to active TGF-b2

from human, rat, mouse, pig and rabbit.

The control antibody used in these experiments, termed 2G6 IgG4,

is an isotype-matched recombinant human IgG4 antibody containing

the variable heavy (VH) variable region of a humanized antibody,

B1.8, directed against 4-hydroxy-3-iodo-5-nitrophenylacetic acid

(NIP) and the variable light (VL) variable region of a humanized

antibody, D1.3, directed against lysozyme. 2G6 IgG4, expressed in

NS0 cells and puri®ed as for 6B1 IgG4, does not bind to TGF-bisoforms and has been shown by immunocytochemistry not to bind to

a panel of human tissues.

Surgery and experimental procedures

Surgical procedures and animal care were licensed and carried out

according to British Home Of®ce guidelines. Stereotactic lesioning of

the cerebral cortex and intraventricular cannulation were executed

exactly as described by us elsewhere (Logan et al., 1994; Logan &

Berry, 1994). Adult female 200±250 g Wistar rats were assigned to

three treatment groups, each receiving: (i) vehicle (saline) plus 0.1%

autologous rat serum to negate the protein concentration of the test

reagents (10 animals); (ii) vehicle plus 200 ng/day 2G6 (an irrelevant

IgG4) immunoglobulin (six animals); (iii) vehicle plus 200 ng/day

human anti-TGF-b2 (6B1 IgG4) immunoglobulin (nine animals). On

day 0 of the experiment, a stereotactically de®ned unilateral

incisional lesion was placed through the cerebral cortex into the

lateral ventricle at the same time as ipsilateral placement of a

permanent intraventricular cannula. Reagents (5 mL) were perfused

into the lesion by immediate and subsequent daily injection for

10 days under halothane anaesthesia through a cannula into the lateral

ventricle. The body weights of each animal were monitored daily and

no signi®cant differences were noted between the treatment groups at

any time point. After 14 days, animals were killed and their brains

processed for either ¯uorescence or peroxidase immunohistochemical

analysis of the lesion site.

Histology and immunohistochemistry

Brains were processed into polyester wax and 7-mm sections of the

lesion site stained by ¯uorescent immunohistochemistry to detect

glial ®brillary acidic protein (GFAP)-positive astrocytes (using a

polyclonal rabbit anti-GFAP antibody from Dakopatts, Ely, Cambs.,

UK at a dilution of 1 : 250), ED1-positive macrophages and microglia

(using a monoclonal mouse anti-rat ED1 antibody from Serotec,

Oxford, UK at a dilution of 1 : 200), ®bronectin (using a polyclonal

rabbit anti-®bronectin antibody from Dakopatts at a dilution of

1 : 100), laminin (using a polyclonal rabbit anti-laminin antibody

from Dakopatts at a dilution of 1 : 100), and peroxidase immunohis-

tochemistry to detect chondroitin sulphate proteoglycan (CSP) (using

a monoclonal anti-CSP antibody, C-8035, from Sigma, Poole, UK,

which recognizes CS56 at a dilution of 1 : 200). Both methods are

described in detail elsewhere (Logan et al., 1994; Logan & Berry,

1994). Note that in all sections viewed under ¯uorescent light,

in¯ammatory macrophages and microglia showed red auto¯uores-

cence. Also, the laminin antibody detected the basement membrane

component of both the glia limitans and the neuropil microvascu-

lature.

Quantitation of immunohistochemistry

In all cases, the effects of each treatment on the CNS wounding

response were quanti®ed by image analysis of ¯uorescently-labelled

sections taken from a de®ned anatomical plane through the lesion site

(Logan et al., 1994; Logan & Berry, 1994) using a Leitz confocal

microscope linked to a Biorad MRC500 laser scanning system. The

relative intensity of ¯uorescence in terms of the mean integrated pixel

intensity was expressed as the integrated ¯uorescent intensity/mm2 in

de®ned and exactly equivalent areas at a constant magni®cation for

each animal. Similarly, the density of peroxidase staining/mm2 was

measured at a constant magni®cation in sections under bright ®eld

using a Leitz microscope video-linked to a Macintosh computer and

National Institutes of Health Image Analysis software.

Results

Cellular effects of TGF-b2 immunoneutralization

By 14 days following an untreated penetrating lesion of the cerebral

cortex, a mature contracted scar is formed at the wound site, which

has a dense, matrix-rich core which is surrounded by a limiting glial

membrane (Fig. 1). Residual ED1-positive macrophages and

TABLE 1. Binding and biological activity properties of 6B1 IgG4 for TGF-bisoforms

TGF-b1 TGF-b2 TGF-b3

Kd (dissociation constant) (nM) 0.89 10.0IC50, TF1 neutralization assay (nM) > 100 2 11IC50, A549 radioreceptor assay (nM) > 400 0.05 4

2368 A. Logan et al.

Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 2367±2374

FIG. 1. Appearance of a mature untreated®brotic scar in the lesioned cerebralhemisphere. A section through a de®ned planeof the site of an untreated cerebral lesion at14 dpl, stained with antibodies that bind GFAP(a marker of reactive astrocytes), laminin,®bronectin and ED1 (a marker of macrophagesand microglia). Antibody binding is detectedwith ¯uorescein-conjugated secondaryantibodies to show the dense ®brous matrix inthe core of the wound which is surrounded bya glial membrane. The scar tissue extendsdown from the cerebrum surface between thecut surfaces of the neuropil of the cortex andreveals the extent of the de®ned lesion. Bar,100 mm.

Inhibition of glial scarring in the injured brain 2369


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FIG. 3. Quantitative image analysis of the wounding response after cerebral lesion. The effects of saline, control IgG4 (2G6) and anti-TGF-b2 IgG4 (6B1) on thelevels of immunoreactive ®bronectin (i), laminin (ii), CSP (iii), ED1-positive macrophages and microglia (iv), and GFAP-positive astrocytes (v), within cerebralwounds at 14 dpl. Fluorescent- and peroxidase- (CSP only) stained images of sections through a de®ned plane of the lesion site were digitized, and either theintegrated ¯uorescent intensity or the density of peroxidase-related staining quanti®ed in exactly equivalent areas. *Signi®cant reduction compared with salinecontrol, P < 0.05. **Signi®cant reduction compared with control 2G6 IgG4, P < 0.05. ***Signi®cant reduction compared with saline control and control 2G6 IgG4,P < 0.05.



activated microglia are still apparent at this time point. In rats treated

with control 2G6 IgG4, the ®brotic scars were qualitatively

equivalent to or indistinguishable from those in lesioned untreated

or saline-treated rats. When quanti®ed, there was an apparent

reduction in immunoreactive laminin and ®bronectin in the wounds

compared with controls, but this reduction was marginal and never

reached statistical signi®cance. The scar contracted into a typical

dense permanent trilaminar glial/®brotic complex (Fig. 2ai-v), the

core of which was laid down by meningeal ®broblasts, and was rich

in ®bronectin (Fig. 2ai) and CSP (Fig. 2aiii). Residual macrophages

and microglia occupied the core and bordering viable neural tissue

(Fig. 2aiv), both of which were separated by a laminin-rich basal

lamina of the glia limitans externa (Fig. 2aii) and undercoated by end-

feet and processes of reactive astrocytes of the glia limitans (Fig.

2av). These three layers extended throughout the lesion and became

contiguous with the complementary laminae of the glia limitans

externa at the pial surface of the cerebrum. The basement membrane

of neuropil blood vessels was also visualized with the anti-laminin

antibody and revealed the angiogenic response in the tissue

surrounding the wound (Fig. 2aii).

In contrast, in every anti-TGF-b2 (6B1 IgG4)-treated rat, the glia

limitans (comprising abutted astrocyte processes and a basal lamina)

was reconstructed only in the sub-pial layers of the lesioned cortex,

and thus normal wound closure was limited to this site. In the deeper

cortical layers, the numerous activated astroglia neither organized

into the expected limiting membrane (Figs 2bv and 3v), nor laid down

a laminin-rich basal lamina in the wound (Fig. 2bii). Little or no

®bronectin was deposited in the core of the wound (Figs 2bi and 3i),

and there was signi®cantly less CSP deposited in the scar and

surrounding neuropil (Figs 2biii and 3iii). Hence, the cut neuropil

surfaces became apposed without the intervention of a glial/matrix

scar. The angiogenic activity of TGF-bs was also suppressed as the

laminin-rich basal laminae of wound-related blood vessels were

rarely seen in the neuropil surrounding a TGF-b2 neutralized wound,

although the normal small diameter microvasculature was apparent

(Fig. 2bii). Finally, there was a signi®cant reduction in the numbers of

EDI-positive macrophages and microglia in the wound and

juxtaposed neuropil at 14 days post-lesion (dpl, Figs 2biv and 3iv),

indicating immunosuppression.

Statistics

The data of each group were compared using the Wilcoxon

ranking test. Comparison of the anti-TGF-b2 antibody-treated data

with those of the saline control showed a signi®cant reduction in

speci®c ¯uorescence/peroxidase staining (P < 0.05) for ED1,

®bronectin, laminin and CSP. There was a statistically insignif-

icant reduction of ®bronectin, laminin and CSP deposition by the

control irrelevant 2G6 IgG4 when compared with the saline

control. Nonetheless, the immuno¯uorescence/peroxidase staining

observed in the group treated with 6B1 IgG4 compared with that

treated with 2G6 IgG4 was signi®cantly less (P < 0.05) for ED1,

GFAP and ®bronectin. Total scarring was determined by

combining the data for ®bronectin- and laminin-stained sections.

Because of the differences in intensity of staining of the two

molecules, the data for the ®bronectin and laminin images were

ranked separately for each animal (from 1 for the lowest to 25

for the highest). A composite score was then derived by adding

the two ranked scores for each rat. The analysis showed a near-

signi®cant reduction in total scarring for the 6B1 IgG4-treated

group compared with the control 2G6 IgG4-treated group

(P < 0.1).

Discussion

The technique of preparation of human antibodies against human

self-antigens (Grif®ths et al., 1993; Marks et al., 1991; Vaughan et al.,

1996) was adapted to select VH and VL variable regions of the

neutralizing recombinant human monoclonal anti-TGF-b2 antibody

from phage display libraries of human single-chain Fv antibody

molecules. We have shown that the antibody prepared binds to active

TGF-b2 from rat, mouse, pig and rabbit, as well as human. Over the

past decade, rodent antibodies against human proteins have been

humanized by transplantation of mouse CDRs into human frame-

works (Adair & Bright, 1995) leading to the application of, e.g. an

antibody against the product of the HER2/neu protooncogene

(Baselga et al., 1996) in clinical trials of metastatic breast cancer.

Fully human antibodies directly isolated from phage display libraries

are therapeutically effective in animal models against foreign

antigens, e.g. respiratory syncytial virus (Crowe et al., 1994), and

have been used to image tumours expressing human CEA in a mouse

xenograft model (Jackson et al., 1998) or ®bronectin ED-B domain

(conserved between human and mouse) in a grafted murine

teratocarcinoma mouse model (Neri et al., 1997). However, few or

no reports have been made of the therapeutic effectiveness of human

antibodies prepared against human self-antigens in vivo.

The technique of cerebral injury in rats, fully documented by us

elsewhere (Logan et al., 1994; Logan & Berry, 1994), provides a

well-characterized experimental model of wound healing and CNS

®brosis in particular, in which the anti-®brotic ef®cacy of the

recombinant monoclonal anti-TGF-b2 antibody can be tested. The

lesion penetrates the lateral ventricle, allowing antibody injected into

the ventricular cerebrospinal ¯uid to perfuse the lesion. Within these

standard lesions, a sequential cellular response occurs characterized

by haemorrhage, in¯ammation, the formation of a trilaminar glial-

matrix scar and an abortive regeneration response by the axons of

compromised neurons, which is essentially complete at 14 dpl in the

rat.

This study demonstrates that acute phase treatment of such wounds

with the recombinant human monoclonal antibody against TGF-b2

leads to a marked attenuation of all aspects of CNS scarring. The

spatially graded response to the antibody from wound depths to

cerebrum surface may re¯ect the gradient of wound perfusion, as

neutralizing IgGs are delivered from the ventricles through the base

of the wound. The suppression of in¯ammation, glial/mesenchymal

scarring and angiogenesis illustrates the widespread activities of

TGF-b2 within CNS wounds. All of the inferred activities of TGF-b2

have precedent in other experimental models. For example, TGF-bs

are known to induce chemokinesis and chemotaxis in vitro and in

vivo (Wahl et al., 1987), upregulate astrocyte production of monocyte

chemoattractant protein-1 (a potent chemoattractant and stimulator of

monocytes) (Hurwitz et al., 1995), and affect cell migration via

modulation of expression of integrins, a major class of cell adhesion

receptors and cell adhesion molecules (Ignotz et al., 1989; Heino &

Massague, 1989). These activities may be of relevance to the

apparent immunosuppression by the anti-TGF-b2 antibody observed

in this model of injury, evidenced by the reduction in both

immunoreactive macrophages and microglia in the wound. In

contrast, we have previously seen that neutralization of the TGF-b1

isoform in the same experimental model resulted in an enhanced

in¯ammatory response, with the microglial population being

particularly responsive to such treatment (Logan et al., 1994). Whilst

TGF-bs are recognized as general immunosuppressors, in the initial

stages of in¯ammation they stimulate monocyte migration (Wahl

et al., 1987), and it may be this activity which the anti-TGF-b2 IgG4



has blocked. It is apparent that the immunosuppression noted with

this isoform-speci®c antibody may be therapeutically advantageous,

as it may reduce the risk of treatment initiating adverse immunolo-

gical reactions.

Others have shown that TGF-bs affect astrocyte morphology,

proliferation, migration and interaction in vitro (Pratt & McPherson,

1997). TGF-b is a potent astrocytic chemotactic agent, it reduces

cell±cell contacts and increases focal contacts, both precursors for

cell migration (Gagelin et al., 1995), and also stimulates the

development of highly branched cellular processes and multicellular

colonies, an activity reminiscent of the cell association that occurs

during formation of the glial membrane (Flanders et al., 1993;

Labourdette et al., 1990; Toru-Delbauffe et al., 1990). Immunoneu-

tralization of either the TGF-b1 (Logan et al., 1994) or TGF-b2

isoform in this model of CNS injury does not limit the extent of

reactive gliosis, but does prevent the organization of astrocytes into

the limiting glial membrane of the CNS scar. We suggest that in both

cases, the neutralizing antibodies are blocking those TGF-b-mediated

astrocyte activities which relate to astrocytic scar formation.

Many groups have demonstrated the ®brogenic effects of TGF-bs

in various models of peripheral tissue injury (see review by Cui &

Ackhurst, 1996), which supports our observations in the CNS that

inhibition of TGF-b activity causes a marked reduction in the

deposition of every matrix molecule examined, including ®bronectin,

laminin and chondroitin sulphate proteoglycan. The anti-®brotic

actions of the antibody may relate to actions on astrocytes as well as

®broblasts, as both produce a range of matrix molecules in response

to TGF-b in vitro (Flanders et al., 1993; Baghdassarian-Chalaye et al.,

1993; Wrana et al., 1986; Ignotz & Massague, 1989; Varga et al.,

1987). The ®brogenic actions of TGF-bs result from upregulating the

synthesis of multiple extracellular matrix molecules (Baghdassarian-

Chalaye et al., 1993; Wrana et al., 1986; Ignotz & Massague, 1989;

Varga et al., 1987) and protease inhibitors (Laiho et al., 1986; Lund

et al., 1987), downregulating the expression of matrix-degrading

proteases (Kerr et al., 1990), and also modulating integrin expression

and consequent ®broblast traf®cking into the wound (Heino &

Massague, 1989; Gagelin et al., 1995). Finally, the angiogenic

activity of TGF-bs is well established (Pepper, 1997; Roberts et al.,

1986), and probably explains the reduced angiogenic response seen in

the neuropil surrounding a TGF-b2 immunoneutralized CNS wound.

The apposition of the cut neuropil surfaces observed in anti-TGF-

b2 antibody-treated lesions suggests the absence of the physical, and

perhaps biochemical, barrier that the gliotic scar represents to

regenerating axons. In this study, regeneration was not rigorously

studied using axonal tracing methods, however, histologically, no

axons were seen to traverse the scar-inhibited lesion. This observation

presumably re¯ects, at least in part, the limiting supply of appropriate

neurotrophic factors required for regeneration. We suggest that, in

addition to inhibiting cicatrix formation, a strategy to mobilize the

axon growth machinery is required in order to achieve vigorous and

sustained neuron regeneration across a transection site.

In summary, we demonstrate the therapeutic effectiveness of a

recombinant human monoclonal antibody against a human self-

antigen derived from phage display libraries in an animal model. The

ef®cacy of anti-TGF-b2 IgG4 in reducing CNS ®brosis emphasizes

the value of phage display libraries for the rapid isolation of

antibodies which are fully human with the potential for a low

frequency of both immunogenicity and side effects in therapy. Our

®ndings suggest that such TGF-b antibodies could be developed as

therapeutic anti-®brotic agents, broadly applicable to a number of

human CNS diseases in which the deposition of extracellular matrix

components is excessive.

Acknowledgements

The authors are indebted to Andrew Baird who initiated these studies withA.L. Also, the TGF-b antibody engineering and preclinical development teamsat Cambridge Antibody Technology for isolation of the VH and VL variableregions of the antibody 6B1, construction of the mammalian cell lineexpressing 6B1 IgG4, and puri®cation and characterization of the antibody.We are also grateful to Mia Martins for assistance with some of the histologyand Peter Treasure for statistical analysis of the data. This work was funded bythe International Spinal Research Trust and the Wellcome Trust.

Abbreviations

CNS, central nervous system; CSF, cerebrospinal ¯uid; CSP, chondroitinsulphate proteoglycan; dpl, days post-lesion; ED1, a marker of macrophagesand microglia; GFAP, glial ®brillary acidic protein; IgG, immunoglobulin;NIP, 4-hydroxy-3-iodo-5-nitrophenylacetic acid; TGF-b, transforming growthfactor-b; VH, variable heavy; VL, variable light.

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Inhibition of glial scarring in the injured rat brain by a recombinant human monoclonal antibody to transforming growth factor-β2