U.S. patent application number 10/243265 was filed with the patent office on 2003-05-15 for specific binding members for tgfbeta1.
Invention is credited to Braddock, Peta Sally Helena, Conroy, Louise Anne, Du Fou, Sarah Leila, Lennard, Simon Nicholas, McCafferty, John Gerald, Tempest, Philip Ronald, Thompson, Julia Elizabeth, Wilton, Alison Jane.
Application Number | 20030091566 10/243265 |
Document ID | / |
Family ID | 22451886 |
Filed Date | 2003-05-15 |
United States Patent
Application |
20030091566 |
Kind Code |
A1 |
Thompson, Julia Elizabeth ;
et al. |
May 15, 2003 |
Specific binding members for TGFbeta1
Abstract
The invention provides specific binding members, for example in
the form of antibody variable domains, based on the CDR3 sequences
of the antibody VH regions of SL15 (SEQ ID NO:4) and JT182 (SEQ ID
NO:10). The antibodies have strong neutralising activity for
TGF.beta..sub.1 and are useful in treating conditions associated
with excess TGF.beta..sub.1 activity, such as fibrosis, immune
responses and tumor progression
Inventors: |
Thompson, Julia Elizabeth;
(Cambridgeshire, GB) ; Lennard, Simon Nicholas;
(Linton, GB) ; Wilton, Alison Jane; (Cambridge,
GB) ; Braddock, Peta Sally Helena; (Huntingdon,
GB) ; Du Fou, Sarah Leila; (Herfordshire, GB)
; McCafferty, John Gerald; (Cambridgeshire, GB) ;
Conroy, Louise Anne; (Cambridge, GB) ; Tempest,
Philip Ronald; (Cambridgeshire, GB) |
Correspondence
Address: |
Nabeela R. McMillian
MARSHALL, GERSTEIN & BORUN
Sears Tower
233 S. Wacker Drive, Suite 6300
Chicago
IL
60606-6357
US
|
Family ID: |
22451886 |
Appl. No.: |
10/243265 |
Filed: |
September 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10243265 |
Sep 13, 2002 |
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09560198 |
Apr 28, 2000 |
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60131983 |
Apr 30, 1999 |
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Current U.S.
Class: |
424/145.1 ;
530/388.25 |
Current CPC
Class: |
A61P 27/06 20180101;
C07K 2317/21 20130101; A61P 27/02 20180101; A61P 35/02 20180101;
C07K 2317/622 20130101; A61P 37/00 20180101; C07K 16/22 20130101;
A61P 37/02 20180101; A61P 35/04 20180101; A61P 29/00 20180101; A61P
35/00 20180101; A61P 17/02 20180101; A61P 27/12 20180101; A61P
11/06 20180101; A61K 2039/505 20130101; C07K 2317/565 20130101;
A61P 13/12 20180101 |
Class at
Publication: |
424/145.1 ;
530/388.25 |
International
Class: |
A61K 039/395; C07K
016/22 |
Claims
We claim:
1. An isolated specific binding member capable of binding
TGF.beta..sub.1, wherein said specific binding member comprises an
antigen binding domain comprising a VH CDR3 with an amino acid
sequence substantially as set out as the VH CDR3 of SL15 (SEQ ID
NO:13) or the VH CDR3 of JT182 (SEQ ID NO:15).
2 The specific binding member of claim 1 which further comprises a
VH CDR1 or a VH CDR2 with an amino acid sequence substantially as
set out as one or both of the CS37 VH CDR1 (SEQ ID NO:11) and CS37
VH CDR2 (SEQ ID NO:12).
3. The specific binding member of claim 1 which comprises a CDR1
sequence substantially as set out as the CS37 VH CDR1 (SEQ ID NO
11) and CS37 VH CDR2 (SEQ ID NO:12)
4 The specific binding member of claim 3 wherein said CDR1, CDR2
and CDR3 sequences are carried by a human antibody framework.
5 An isolated specific binding member capable of binding
TGF.beta..sub.1, wherein said specific binding member comprises the
SL15 VH domain substantially set out in SEQ ID NO:4
6. An isolated specific binding member capable of binding
TGF.beta..sub.1, wherein said specific binding member comprises the
JT182 VH domain substantially set out in SEQ ID NO:10.
7. An isolated specific binding member capable of binding
TGF.beta..sub.1, wherein said specific binding member comprises the
SL15S VL domain substantially set out in SEQ ID NO:8.
8 An isolated specific binding member capable of binding
TGF.beta..sub.1, wherein said specific binding member comprises:
(i) a VH domain selected from the group of the SL15 VH domain
substantially set out in SEQ ID NO:4 and the JT182 VH domain
substantially set out in SEQ ID NO 10, and (ii) a VL domain
selected from the group of the SL15S VL domain substantially set
out in SEQ ID NO 8 and the SL15A VL domain substantially set out in
SEQ ID NO 6.
9. The isolated specific binding member of claim 8 wherein the VH
domain is the SL15 VH domain substantially set out in SEQ ID NO
4
10. The isolated specific binding member of claim 1 in the form of
a single chain Fv (scFv)
11. The isolated specific binding member of claim 8 in the form of
a single chain Fv (scFv).
12. The isolated specific binding member of claim 1 in the form of
an IgG.
13. The isolated specific binding member of claim 12 wherein said
IgG is an IgG1 or IgG4.
14. The isolated specific binding member of claim 8 in the form of
an IgG
15. The isolated specific binding member of claim 14 wherein said
IgG is an IgG1 or IgG4
16. A pharmaceutical composition comprising the specific binding
member of claim 1 in association with a pharmaceutically acceptable
excipient, carrier, buffer or stabiliser.
17. A pharmaceutical composition comprising the specific binding
member of claim 8 in association with a pharmaceutically acceptable
excipient, carrier, buffer or stabiliser
18. A method of treating a condition associated with extracellular
matrix deposition in a patient in need of treatment, the method
comprising administering to said patient an effective amount of the
specific binding member of claim 1 or claim 8.
19. The method of claim 18 wherein said condition is selected from
glomerulonephritis, keloid and hypertrophic scarring, proliferative
vitreoretinopathy, glaucoma drainage surgery, corneal injury and
cataracts.
20. A method of modulating the immue or inflammatory response in a
patient in need of treatment, the method comprising administering
to said patient an effective amount of the specific binding member
of claim 1 or claim 8.
21 A method of treatment of a tumor, in a patient in need of
treatment, the method comprising administering to said patient an
effective amount of the specific binding member of claim 1 or claim
8
22. The method of claim 21 wherein treatment of said tumor inhibits
anglogenesis within or metastasis of said tumor
23. The method of claim 22 wherein said tumor is a breast,
prostate, ovarian, stomach, colerectal, skin, lung, cervical and
bladder tumor, or a leukemia or sarcoma
24. A method of treatment of asthma in a patient in need of
treatment, the method comprising administering to said patient an
effective amount of the specific binding member of claim 1 or claim
8.
25. A method of determining the amount of TGF.beta..sub.1 in a
sample which comprises bringing the sample into contact with a
specific binding member according to claim 1 or 8, and determining
the amount of binding of the specific binding member to
TGF.beta..sub.1 in the sample.
25. An isolated nucleic acid comprising a sequence which encodes
the specific binding member of claim 1 or claim 8.
26. An method of preparing a specific binding member capable of
binding TGF.beta..sub.1, said method comprising expressing the
nucleic acid of claim 25 in a host cell under conditions to provide
for expression of said nucleic acid, followed by recovery of said
specific binding member
Description
[0001] This application claims priority from U.S. provisional
application No. 60/131,983 filed Apr. 30, 1999, whose contents are
hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to specific binding members,
particularly antibodies and fragments thereof, which bind to
transforming growth factor 1 (TGF.beta..sub.1). More particularly,
the invention is concerned with specific binding members which
include the VH CDR3 of the antibody SL15 (the antibody formerly
known as Kylie), especially the SL15 VH domain, which may be in
combination with the SL15A or SL15S VL domains. Furthermore, the
invention relates to use of such specific binding members in
pharmaceutical preparations, particularly for the treatment of
fibrotic disease, the modulation of wound healing and the treatment
of cancer.
BACKGROUND TO THE INVENTION
[0003] PCT/GB96/02450 published as WO97/13844 discloses the
isolation of human antibodies specific for human TGF.beta..sub.1
and human antibodies specific for human TGF.beta..sub.2. It
describes antibodies with the 31G9 VH domain and variants of the
domain. More specifically the application described the antibody
CS37 that comprises the 31G9 VH domain together with the CS37 VL
and variants of this domain, including antibodies which:
[0004] (i) compete in ELISA with CS37 for binding to
TGF.beta..sub.1,
[0005] (ii) bind TGF.beta..sub.1 preferentially with respect to
TGF.beta..sub.3, and
[0006] (iii) neutralise TGF.beta..sub.1.
DISCLOSURE OF THE INVENTION
[0007] The present invention is based on identification of
antibodies which are related to CS37, but which have unexpectedly
advantageous properties with respect to binding and neutralisation
of TGF.beta..sub.1. They do not bind to, or neutralise,
TGF.beta..sub.2 or TGF.beta..sub.3.
[0008] Antibodies of the present invention strongly neutralise
active TGF.beta..sub.1. The epitope for these antibodies lies in
the C-terminal region of TGF.beta..sub.1 (residues 83-112) and
includes the loop consisting of residues 92-98 of TGF.beta..sub.1,
also known as finger 2, a region which has been identified as
interacting with the receptor for TGF.beta.. The antibodies bind
preferentially to active TGF.beta..sub.1 with respect to latent
TGF.beta.1
[0009] Variants of SL15S that strongly neutralise TGF.beta..sub.3
are also disclosed herein. These vary mainly by amino acid
substitutions in the CDR3 of VH or VL domains. There are, however,
other sites where substitutions may be made, e.g. at residue 25 in
the light chain an alanine may be substituted, generating the IgG4
antibody, SL15A IgG4, CAT-192. Several substitutions may be made
that are compatible with the retention of strong neutralising
activity. The antibodies of this invention will be particularly
useful for treatment of fibrotic diseases, e.g. lung fibrosis,
modulation of the scarring response, e.g. in wound healing and
corneal scarring, and in other contexts discussed further below
such as the treatment of tumors.
[0010] Specific binding proteins such as antibodies which are based
on the complementarity-determining regions (CDRs) of the
advantageous antibody VH and VL domains identified herein,
particularly the CDR 3 regions, will be useful for the purposes
discussed, and represent aspects of the present invention.
[0011] The most preferred embodiments of the present invention in
its various aspects are based on the VH CDR3 of the SL15 VH domain
identified herein, VH domains including the SL15 VH CDR3,
especially the SL15 VH domain itself, and pairings of such VH
domains with VL domains, especially the SL15A or SL15S VL domains,
or other VL domain comprising the SL15 VL CDR3. The antibody
antigen-binding domain SL15 (in whichever format, e.g. scFv or
IgG4) consists of the SL15 VH and, in two variants, either the
SL15A VL (CS37) or SL15S VL (CS37 with A25S). In either variant,
SL15 is the antibody formerly known as Kylie. SL15S scFv is also
known as CAT 191; SL15A IgG4 is also known as CAT-192; and SL15S
IgG4 is also known as CAT-193.
[0012] Further embodiments of the invention in its various aspects
are based on the JT182 VH CDR3, VH domains including the JT182 VH
CDR3, especially the JT182 VH domain, and pairings of such VH
domains with VL domains, especially the CS37 VL domain. JT182 is
not as effective as SL15, but still has unexpectedly improved
properties over CS37.
[0013] In a first aspect the present invention provides an isolated
specific binding member capable of binding TGF.beta..sub.1, wherein
said specific binding member comprises an antigen binding domain
comprising a VH CDR3 with an amino acid sequence substantially as
set out as VH CDR3 of SL15 or JT182 in Table 1 or Table 2.
1TABLE 1 Anti-TGFb1 Clones-CDR3s & Relative Potencies of scFv
Approx IC50 CLONE (RRA) VH CDR3 VL CDR3 CS37 10-15 nM
TGEYSGYDTSGVEL LQDSNYPLT (SEQ ID NO.14) (SEQ ID NO:18) JT182 0.5-1
nM TGEYSGYDTPASPD CS37 (SEQ ID NO:15) SL15 0.1 nM TGEYSGYDTDPQYS
CS37(+L25 A to S) (SEQ ID NO:13)
[0014] Residues that differ between the scFv fragments are
underlined.
[0015] The invention further provides said isolated specific
binding member which further comprises a VH CDR1 or VH CDR2 with
amino acid sequences substantially as set out as one or both of the
VH CDR1 and VH CDR2 of the CS37 VH, preferably both (Table 2).
2TABLE 2 CDR Sequences of CDRs of CS37, SL15 and JT182 Domain CDR1
CDR2 CDR3 CS37 SYGMH VISYDGSIKYYADS TGEYSGYDTSGVEL VH (SEQ ID
NO:11) VKG (SEQ ID NO:14) (SEQ ID NO:12) SL15 SYGMH VISYDGSIKYYADS
TGEYSGYDTDPQYS VH (SEQ ID NO:11) VKG (SEQ ID NO:13) (SEQ ID NO:12)
JT182 SYGMH VISYDGSIKYYADS TGEYSGYDTPASPD VH (SEQ ID NO:11) VKG
(SEQ ID NO:15) (SEQ ID NO:12) CS37 RASQGIGDDLG GTSTLQS LQDSNYPLT VL
(SEQ ID NO:16) (SEQ ID NO:17) (SEQ ID NO:18) SL15S RSSQGIGDDLG
GTSTLQS LQDSNYPLT VL (SEQ ID NO:19) (SEQ ID NO:17) (SEQ ID
NO:18)
[0016] In a preferred embodiment, the binding domains are carried
by a human antibody framework. One preferred example of such an
embodiment is a VH domain with an amino acid sequence substantially
as set out as the JT182 VH domain of which the sequence is set out
in SEQ ID NO:10. A further preferred embodiment is a VH domain with
an amino acid sequence substantially as set out as the SL15 VH
domain, of which the sequence is set out in SEQ ID NO:4.
[0017] In a second aspect, the invention provides an isolated
specific binding member capable of binding TGF.beta..sub.1, wherein
said specific binding member comprises an antigen binding domain
comprising a VL domain with an amino acid sequence substantially as
set out as the SL15S VL domain, of which the sequence is set out in
SEQ ID NO:8.
[0018] In a further aspect, the invention provides a specific
binding member capable of binding TGF.beta..sub.1, comprising a VH
domain as set out above with respect to the first aspect, and a VL
domain, preferably wherein the VL domain has an amino acid sequence
substantially as set out as the CS37 VL (SL15A), of which the
sequence is set out in SEQ ID NO:6, or the SL15S VL, of which the
sequence is set out in SEQ ID NO:8.
[0019] In a particularly preferred embodiment, the invention
provides a specific binding member comprising the CS37 VL domain
and a VH domain selected from JT182 VH and SL15 VH, most preferably
SL15 VH. In a further particularly preferred embodiment, the
invention provides a specific binding member comprising SL15 VH and
SL15A VL (CS37 VL) or SL15S VL.
[0020] Preferred embodiments of the present invention provide
specific binding members comprising the JT182 VH or SL15 VH domain
in which 1, 2, 3, 4 or 5 amino acid substitutions have been made in
a CDR, e.g. CDR3, and/or FR, which specific binding members retain
ability to bind TGF.beta..sub.1. Further preferred embodiments
provide specific binding members comprising the SL15A (CS37) VL or
SL15S VL, or SL15A or SL15S VL domain in which 1, 2, 3, 4 or 5
amino acid substitutions have been made in a CDR, e.g. CDR3, and/or
FR which specific binding members retain ability to bind
TGF.beta..sub.1 Such amino acid substitutions are generally
"conservative", for instance substitution of one hydrophobic
residue such as isoleucine, valine, leucine or methionine for
another, or the substitution of one polar residue for another, such
as arginine for lysine, glutamic for aspartic acid, or glutamine
for asparagine. At certain positions non-conservative substitutions
are allowable.
[0021] Such specific binding members are able to bind
TGF.beta..sub.1. Preferred embodiments lack significant
cross-reactivity with TGF.beta..sub.2 and/or TGF.beta..sub.3,
preferably TGF.beta..sub.2 and TGF.beta..sub.3.
[0022] Preferred embodiments strongly neutralise TGF.beta..sub.1,
having a potency of at least 5 times better than does CS37, more
preferably about 10 times, 15 times, 20 times, 50 times, 75 times,
100 times or 150 times better, in a radioreceptor assay (Lucas C et
al (1991) Meth in Enzymology 198, 303-16). Potency is measured with
the antibody under study and CS37 in equivalent molecular formats,
e.g. as monovalent antibodies (scFv or Fab) or as bivalent
antibodies (IgG1 or IgG4) Preferred embodiments bind active
TGF.beta..sub.1 preferentially to latent TGF.beta..sub.1.
[0023] Variants of the VH and VL domains and CDRs of which the
sequences are set out herein and which can be employed in specific
binding members for TGF.beta..sub.1 can be obtained by means of
methods of sequence alteration or mutation and screening. Such
methods are also provided by the present invention.
[0024] In addition to antibody sequences, the specific binding
member may comprise other amino acids, e.g. forming a peptide or
polypeptide, such as a folded domain, or to impart to the molecule
another functional characteristic in addition to ability to bind
antigen. Specific binding members of the invention may carry a
detectable label, or may be conjugated to a toxin or enzyme (e.g.
via a peptidyl bond or linker).
[0025] In further aspects, the invention provides an isolated
nucleic acid which comprises a sequence encoding a specific binding
member as defined above, and methods of preparing specific binding
members of the invention which comprise expressing said nucleic
acids under conditions to bring about expression of said binding
member, and recovering the binding member.
[0026] Specific binding members according to the invention may be
used in a method of treatment or diagnosis of the human or animal
body, such as a method of treatment (which may include prophylactic
treatment) of a disease or disorder in a human patient which
comprises administering to said patient an effective amount of a
specific binding member of the invention. Conditions treatable in
accordance with the present invention are described below.
[0027] These and other aspects of the invention are described in
further detail below.
[0028] All documents mentioned herein are incorporated by
reference. Sequences described herein are shown and referred to in
the conventional 5' to 3' and N to C terminal notation for nucleic
acid and amino acids sequences respectively, unless specifically
indicated otherwise.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIG. 1: Neutralisation of TGF.beta..sub.1 but not
TGF.beta..sub.2 or .beta..sub.3 by SL15S scFv in a proliferation
assay using TF1 cells. The neutralisation by Genzyme mAb (Mab
1D.11.16-squares) or SL15S scFv (diamonds) of TGF.beta..sub.1,
.beta..sub.2 or .beta..sub.3-induced inhibition of proliferation of
TF1 is shown.
[0030] FIG. 2: Inhibition of [.sup.125I]TGF.beta..sub.1 binding to
A549 cells by anti-TGF.beta..sub.1 scFv. ScFv preparations of
SL15S, JT183 and CS37 were titrated 2-fold and tested for their
ability to inhibit [.sup.125I]TGF.beta..sub.1 binding to A549
cells. SL15S and JT182 were used as his-preps, CS37 was
fplc-purified.
[0031] FIG. 3: Inhibition of [.sup.125I]TGF.beta..sub.1 binding to
A549 cells by anti-TGF.beta..sub.1 antibodies. SL15S scFv, SL15S
IgG4 and SL15A IgG4 were compared with Genzyme mAb in their ability
to inhibit [.sup.125I]TGF.beta..sub.1 binding to A549 cells Data
are the average of 3 experiments, using a three-fold dilution
series.
[0032] FIG. 4: Inhibition of [.sup.125I]TGF.beta..sub.1 binding to
A549 cells by scFv in the presence of latent TGF.beta..sub.1. SL15S
scFv and SL15A IgG4 were tested for their ability to inhibit
[.sup.125I]TGF.beta..sub.1 binding to A549 cells. To investigate
whether SL15S recognised latent TGF.beta..sub.1, the standard
experiment was performed in the presence of 0.1 nM latent
TGF.beta..beta..sub.1, active TGF.beta..sub.1 or acid-activated
latent TGF.beta..sub.1. Data are expressed as % max for each set of
conditions.
[0033] FIG. 5: Neutralisation of TGF.beta..sub.1-induced inhibition
of TF1 cell proliferation by scFv and IgG in the presence of latent
TGF.beta..sub.1. The ability of SL15S scFv and SL15A IgG4 to
neutralise the growth inhibition induced by latent TGF.beta..sub.1,
active TGF.beta..sub.1 or acid-activated TGF.beta..sub.1 was
compared in the TF1 assay. In (a) varying concentrations of
TGF.beta..sub.1 formats were compared where as in (b) scFv and IgG
were compared against 20 pM TGF.beta..sub.1 formats. Data expressed
as % control (growth in the absence of TGF.beta..sub.1). The
inhibition induced by latent TGF.beta..sub.1 is due to the small
amount of active TGF.beta..sub.1 present in the latent
preparation.
[0034] FIG. 6: Inhibition of binding of CS37 scFv displayed on
phage to TGF.beta..sub.1 using chimaeric TGF.beta.s. Binding of
CS37 scFv displayed on phage to TGF.beta..sub.1 was assayed by
ELISA in the presence of: the TGF.beta..sub.1 isoform
(TGF.beta..sub.1); the TGF.beta..sub.2 isoform (TGF.beta..sub.2);
TGF.beta..sub.1/.beta..sub.2 (83-112) (1-1-2);
TGF.beta..sub.2/.beta..sub.1 83-112 (2-2-1);
TGF.beta..sub.1-.beta..sub.2 (40-112) (1-2-2); or
TGF.beta..sub.1-.beta..- sub.2(92-98) (92-98)
[0035] FIG. 7: Inhibition of binding of SL15S (Kylie scFv)
displayed on phage to TGF.beta..sub.1 using chimaeric TGF.beta.s.
Binding of SL15S (Kylie) scFv displayed on phage to TGF.beta..sub.1
was assayed by ELISA in the presence of the TGF.beta..sub.1 isoform
(TGF.beta..sub.1); the TGF.beta..sub.2 isoform (TGF.beta..sub.2);
TGF.beta..sub.1/.beta..sub.2 (83-112) (1-1-2);
TGF.beta..sub.2/.beta..sub.1 83-112 (2-2-1);
TGF.beta..sub.1-.beta..sub.2 (40-112) (1-2-2); or
TGF.beta..sub.1-.beta..- sub.2(92-98) (92-98).
[0036] FIG. 8: The effect of CAT192 on corneal re-epithelialisation
was investigated following an excisional trephine wound of bovine
isolated cornea in the air interface organ culture model. Bovine
cornea were treated with 100 l of either serum free Medium 199
(control) or medium containing vehicle (for antibody and
TGF.beta..sub.1). Null isotype-matched antibody (10.mu.g),
TGF.beta..sub.1 (1 ng) or CAT192 (10 .mu.g) were administered
immediately after wounding and at 12 h intervals thereafter. CAT
192 caused a significant increase in rate of re-epithelialisation
of wounded bovine cornea whereas TGF.beta..sub.1 caused a
significant decrease in this variable. Data are expressed as
percentage re-epithelialisation of the corneal wound. Each point
represents the mean value and the vertical bars show s.e.mean of 8
cornea per point. The effect of the different treatments were
compared at each time point using repeated measures ANOVA with
Bonferroni test. *P<0.01 compared to the null antibody treatment
group; t P<0.01 compared to the vehicle treated group.
[0037] FIG. 9: The effect of CAT192 on corneal re-epithelialisation
was investigated following an excisional trephine wound of bovine
isolated cornea in the air interface organ culture model. CAT192
(0.001-10 .mu.g) was administered immediately after wounding and at
12 h intervals thereafter. CAT192 caused a significant dose-related
increase in re-epithelialisation of wounded bovine cornea. The EC50
for CAT192 was between 0.01 and 0.1 .mu.g Data are expressed as the
percentage change in re-epithelialisation of the vehicle treated
control group. The dotted lines show the s.e. mean values of the
vehicle control group. Each point represents the mean value and and
the vertical bars show s.e.mean of 6 cornea per point. The effect
of the different doses of CAT192 were compared to control treatment
using one way ANOVA and Dunnett's test, *P<0.01.
DETAILED DESCRIPTION OF THE INVENTION TERMINOLOGY
[0038] Specific Binding Member
[0039] This describes a member of a pair of molecules which have
binding specificity for one another. The members of a specific
binding pair may be naturally derived or wholly or partially
synthetically produced. One member of the pair of molecules has an
area on its surface, or a cavity, which specifically binds to and
is therefore complementary to a particular spatial and polar
organisation of the other member of the pair of molecules. Thus the
members of the pair have the property of binding specifically to
each other. Examples of types of specific binding pairs are
antigen-antibody, biotin-avidin, hormone-hormone receptor,
receptor-ligand, enzyme-substrate. This application is concerned
with antigen-antibody type reactions.
[0040] Antibody
[0041] This describes an immunoglobulin whether natural or partly
or wholly synthetically produced. The term also covers any
polypeptide or protein having a binding domain which is, or is
substantially homologous to, an antibody binding domain. These can
be derived from natural sources, or they may be partly or wholly
synthetically produced. Examples of antibodies are the
immunoglobulin isotypes and their isotypic subclasses; fragments
which comprise an antigen binding domain such as Fab, scFv, Fv,
dAb, Fd; and diabodies.
[0042] It is possible to take monoclonal and other antibodies and
use techniques of recombinant DNA technology to produce other
antibodies or chimeric molecules which retain the specificity of
the original antibody. Such techniques may involve introducing DNA
encoding the immunoglobulin variable region, or the complementarity
determining regions (CDRs), of an antibody to the constant regions,
or constant regions plus framework regions, of a different
immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or
EP-A-239400. A hybridoma or other cell producing an antibody may be
subject to genetic mutation or other changes, which may or may not
alter the binding specificity of antibodies produced.
[0043] As antibodies can be modified in a number of ways, the term
"antibody" should be construed as covering any specific binding
member or substance having a binding domain with the required
specificity. Thus, this term covers antibody fragments,
derivatives, functional equivalents and homologues of antibodies,
including any polypeptide comprising an immunoglobulin binding
domain, whether natural or wholly or partially synthetic. Chimeric
molecules comprising an immunoglobulin binding domain, or
equivalent, fused to another polypeptide are therefore included.
Cloning and expression of chimeric antibodies are described in
EP-A-0120694 and EP-A-0125023.
[0044] It has been shown that fragments of a whole antibody can
perform the function of binding antigens Examples of binding
fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI
domains; (ii) the Fd fragment consisting of the VH and CHI domains;
(iii) the Fv fragment consisting of the VL and VH domains of a
single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature
341:544-546 (1989)) which consists of a VH domain, (v) isolated CDR
regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two
linked Fab fragments (vii) single chain Fv molecules (scFv),
wherein a VH domain and a VL domain are linked by a peptide linker
which allows the two domains to associate to form an antigen
binding site (Bird et al, Science, 242, 423-426, 1988; Huston et
al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain
Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or
multispecific fragments constructed by gene fusion (WO94/13804; P.
Holliger et al, Proc. Natl Acad. Sci. USA 90 6444-6448, 1993). Fv,
scFv or diabody molecules may be stabilised by the incorporation of
disulphide bridges linking the VH and VL domains (Y. Reiter et al,
Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFv
joined to a CH3 domain may also be made (S. Hu et al, Cancer Res,
56, 3055-3061, 1996).
[0045] Diabodies are multimers of polypeptides, each polypeptide
comprising a first domain comprising a binding region of an
immunoglobulin light chain and a second domain comprising a binding
region of an immunoglobulin heavy chain, the two domains being
linked (e.g by a peptide linker) but unable to associate with each
other to form an antigen binding site: antigen binding sites are
formed by the association of the first domain of one polypeptide
within the multimer with the second domain of another polypeptide
within the multimer (WO94/13804).
[0046] Where bispecific antibodies are to be used, these may be
conventional bispecific antibodies, which can be manufactured in a
variety of ways (Holliger, P. and Winter G. Current Opinion
Biotechnol. 4, 446-449 (1993)), e g. prepared chemically or from
hybrid hybridomas, or may be any of the bispecific antibody
fragments mentioned above. It may be preferable to use scFv dimers
or diabodies rather than whole antibodies. Diabodies and scFv can
be constructed without an Fc region, using only variable domains,
potentially reducing the effects of anti-idiotypic reaction.
[0047] Bispecific diabodies, as opposed to bispecific whole
antibodies, may also be particularly useful because they can be
readily constructed and expressed in E. coli. Diabodies (and many
other polypeptides such as antibody fragments) of appropriate
binding specificities can be readily selected using phage display
(WO94/13804) from libraries. If one arm of the diabody is to be
kept constant, for instance, with a specificity directed against
antigen X, then a library can be made where the other arm is varied
and an antibody of appropriate specificity selected. Bispecific
whole antibodies may be made by knobs-into-holes engineering (J. B.
B. Ridgeway et al, Protein Eng., 9, 616-621, 1996).
[0048] Diabodies may be made with one binding site for
TGF.beta..sub.1 formed by VH and VL domains as disclosed in this
application and the other binding site being for TGF.beta..sub.2
The TGF.beta..sub.2 binding site may be formed for instance from
the VH and VL domains of the antibody 6B1 (WO97/13844).
[0049] Antigen Binding Domain
[0050] This describes the part of an antibody which comprises the
area which specifically binds to and is complementary to part or
all of an antigen. Where an antigen is large, an antibody may only
bind to a particular part of the antigen, which part is termed an
epitope. An antigen binding domain may be provided by one or more
antibody variable domains (e.g. a so-called Fd antibody fragment
consisting of a VH domain). Preferably, an antigen binding domain
comprises an antibody light chain variable region (VL) and an
antibody heavy chain variable region (VH).
[0051] Specific
[0052] This may be used to refer to the situation in which one
member of a specific binding pair will not show any significant
binding to molecules other than its specific binding partner(s).
The term is also applicable where e.g. an antigen binding domain is
specific for a particular epitope which is carried by a number of
antigens, in which case the specific binding member carrying the
antigen binding domain will be able to bind to the various antigens
carrying the epitope.
[0053] Comprise
[0054] This is generally used in the sense of include, that is to
say permitting the presence of one or more features or
components.
[0055] Isolated
[0056] This refers to the state in which specific binding members
of the invention, or nucleic acid encoding such binding members,
will be in accordance with the present invention. Members and
nucleic acid will be free or substantially free of material with
which they are naturally associated such as other polypeptides or
nucleic acids with which they are found in their natural
environment, or the environment in which they are prepared (e.g.
cell culture) when such preparation is by recombinant DNA
technology practised in vitro or in vivo. Members and nucleic acid
may be formulated with diluents or adjuvants and still for
practical purposes be isolated--for example the members will
normally be mixed with gelatin or other carriers if used to coat
microtitre plates for use in immunoassays, or will be mixed with
pharmaceutically acceptable carriers or diluents when used in
diagnosis or therapy. Specific binding members may be glycosylated,
either naturally or by systems of heterologous eukaryotic cells
(e.g. CHO or NSO (ECACC 85110503) cells, or they may be (for
example if produced by expression in a prokaryotic cell)
unglycosylated.
[0057] By "substantially as set out" it is meant that the relevant
CDR or VH or VL domain of the invention will be either identical or
highly similar to the specified regions of which the sequence is
set out herein. By "highly similar" it is contemplated that from 1
to 5, preferably from 1 to 4 such as 1 to 3 or 1 or 2, or 3 or 4,
substitutions may be made in the CDR and/or VH or VL domain.
[0058] The structure for carrying a CDR of the invention will
generally be of an antibody heavy or light chain sequence or
substantial portion thereof in which the CDR is located at a
location corresponding to the CDR of naturally occurring VH and VL
antibody variable domains encoded by rearranged immunoglobulin
genes. The structures and locations of immunoglobulin variable
domains may be determined by reference to (Kabat, E. A. et al,
Sequences of Proteins of Immunological Interest. 4th Edition. US
Department of Health and Human Services. 1987, and updates thereof,
now available on the Internet (http://immuno.bme.nwu.edu)). CDRs
are generally as defined by Kabat. Additionally, in CDR grafting
residues of the loop defined by Chothia adjacent the Kabat VH CDR1
may be grafted. For SL15S this would comprise residues 26 to 30 of
the heavy chain (GFTGS).
[0059] Preferably, an amino acid sequence substantially as set out
in Table 1 is carried as the CDR3 in a human heavy chain variable
domain or a substantial portion thereof.
[0060] Variable domains employed in the invention may be derived
from any germline or rearranged human variable domain, or may be a
synthetic variable domain based on consensus sequences of known
human variable domains. CDR-derived sequences of the invention may
be introduced into a repertoire of variable domains lacking CDR3
regions, using recombinant DNA technology.
[0061] For example, Marks et al (Bio/Technology, 1992, 10:779-783)
describe methods of producting repertoires of antibody variable
domains in which consensus primers directed at or adjacent to the
5' end of the variable domain area are used in conjunction with
consensus primers to the third framework region of human VH genes
to provide a repertoire of VH variable domains lacking a CDR3.
Marks et al further describe how this repertoire may be combined
with a CDR3 of a particular antibody. Using analogous techniques,
the CDR3-derived sequences of the present invention may be shuffled
with repertoires of VH or VL domains lacking a CDR3, and the
shuffled complete VH or VL domains combined with a cognate VL or VH
domain to provide specific binding members of the invention The
repertoire may then be displayed in a suitable host system such as
the phage display system of WO92/01047 so that suitable specific
binding members may be selected. A repertoire may consist of from
anything from 10.sup.4 individual members upwards, for example from
10.sup.6 to 10.sup.8 or 10.sup.10 members.
[0062] Analogous shuffling or combinatorial techniques are also
disclosed by Stemmer (Nature, 1994, 370:389-391), who describes the
technique in relation to a .beta.-lactamase gene but observes that
the approach may be used for the generation of antibodies.
[0063] A further alternative is to generate novel VH or VL regions
carrying a CDR-derived sequences of the invention using random
mutagenesis of, for example, the SL15 or JT182 VH gene or SL15A or
SL15S VL genes to generate mutations within the entire variable
domain. Such a technique is described by Gram et al (1992, Proc.
Natl. Acad. Sci., USA, 89:3576-3580), who used error-prone PCR.
[0064] Another method which may be used is to direct mutagenesis to
CDR regions of VH or VL genes. Such techniques are disclosed by
Barbas et al, (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and
Schier et al (1996, J. Mol. Biol. 263:551-567).
[0065] All the above described techniques are known as such in the
art and in themselves do not form part of the present invention.
The skilled person will be able to use such techniques to provide
specific binding members of the invention using routine methodology
in the art.
[0066] A further aspect of the invention provides a method for
obtaining an antibody antigen binding domain specific for
TGF.beta..sub.1 and preferably one or more of the additional
properties disclosed herein for specific binding members according
to embodiments of the invention, the method comprising providing by
way of addition, deletion, substitution or insertion of one or more
amino acids in the amino acid sequence of a VH domain set out
herein (SL15 or JT182) a VH domain which is an amino acid sequence
variant of the VH domain, combining the VH domain thus provided
with one or more VL domains, and testing the VH/VL combination or
combinations to identify an antibody antigen binding domain
specific for TGF.beta..sub.1 and optionally with one or more of
said preferred properties. Said VL domain may have an amino acid
sequence which is substantially as set out for SL15A VL (CS37) or
may have an amino acid sequence which is substantially as set out
for VL.
[0067] An analogous method may be employed in which one or more
sequence variants of a VL domain disclosed herein are combined with
one or more VH domains.
[0068] A further aspect of the invention provides a method of
preparing a specific binding member specific for TGF.beta..sub.1,
which method comprises:
[0069] a) providing a starting repertoire of nucleic acids encoding
a VH domain which either include a CDR3 to be replaced or lack a
CDR3 encoding region;
[0070] b) combining said repertoire with a donor nucleic acid
encoding an amino acid sequence substantially as set out herein for
SL15 or JT182 VH CDR3 such that said donor nucleic acid is inserted
into the CDR3 region in the repertoire, so as to provide a product
repertoire of nucleic acids encoding a VH domain;
[0071] c) expressing the nucleic acids of said product repertoire;
and
[0072] d) selecting a specific binding member specific for
TGF.beta..sub.1; and
[0073] e) recovering said specific binding member or nucleic acid
encoding
[0074] Again, an analogous method may be employed in which a VL
CDR3 of the invention is combined with a repertoire of nucleic
acids encoding a VL domain which either include a CDR3 to be
replaced or lack a CDR3 encoding region.
[0075] Similarly, one or more, or all three CDRs may be grafted
into a repertoire of VH or VL domains which are then screened for a
specific binding member or specific binding members specific for
TGF.beta..sub.1.
[0076] A substantial portion of an immunoglobulin variable domain
will comprise at least the three CDR regions, together with their
intervening framework regions. Preferably, the portion will also
include at least about 50% of either or both of the first and
fourth framework regions, the 50% being the C-terminal 50% of the
first framework region and the N-terminal 50% of the fourth
framework region. Additional residues at the N-terminal or
C-terminal end of the substantial part of the variable domain may
be those not normally associated with naturally occurring variable
domain regions. For example, construction of specific binding
members of the present invention made by recombinant DNA techniques
may result in the introduction of N- or C-terminal residues encoded
by linkers introduced to facilitate cloning or other manipulation
steps. Other manipulation steps include the introduction of linkers
to join variable domains of the invention to further protein
sequences including immunoglobulin heavy chains, other variable
domains (for example in the production of diabodies) or protein
labels as discussed in more details below.
[0077] Although in a preferred aspect of the invention specific
binding members comprising a pair of VH and VL domains are
preferred, single binding domains based on either VH or VL domain
sequences form further aspects of the invention It is known that
single immunoglobulin domains, especially VH domains, are capable
of binding target antigens in a specific manner.
[0078] In the case of either of the single chain specific binding
domains, these domains may be used to screen for complementary
domains capable of forming a two-domain specific binding member
able to bind TGF.beta..sub.1.
[0079] This may be achieved by phage display screening methods
using the so-called hierarchical dual combinatorial approach as
disclosed in WO 92/01047 in which an individual colony containing
either an H or L chain clone is used to infect a complete library
of clones encoding the other chain (L or H) and the resulting
two-chain specific binding member is selected in accordance with
phage display techniques such as those described in that reference.
This technique is also disclosed in Marks et al, ibid.
[0080] Specific binding members of the present invention may
further comprise antibody constant regions or parts thereof For
example, a VL domain such as SL15A VL or SL15S VL may be attached
at their C-terminal end to antibody light chain constant domains
including human C.kappa. or C.lambda. chains, preferably C.lambda.
chains. Similarly, specific binding members based on SL15 VH may be
attached at their C-terminal end to all or part of an
immunoglobulin heavy chain derived from any antibody isotype, e.g.
IgG, IgA, IgE and IgM and any of the isotype sub-classes,
particularly IgG1 and IgG4. IgG4 is preferred.
[0081] Antibodies of the invention may be labelled with a
detectable or functional label. Detectable labels include
radiolabels such as .sup.131I or .sup.99Tc, which may be attached
to antibodies of the invention using conventional chemistry known
in the art of antibody imaging. Labels also include enzyme labels
such as horseradish peroxidase. Labels further include chemical
moieties such as biotin which may be detected via binding to a
specific cognate detectable moiety, e.g. labelled avidin.
[0082] Antibodies of the present invention are designed to be used
in methods of diagnosis or treatment in human or animal subjects,
preferably human.
[0083] Antibodies specific for human TGF.beta..sub.1 have been
shown to be effective in animal models for the treatment of
fibrotic diseases and other diseases where TGF.beta..sub.1 is
overexpressed such as rheumatoid arthritis or cancer. Antibodies
against TGF.beta..sub.1 have been shown to be effective in the
treatment of glomerulonephritis (W. A. Border et al., Nature,
346:371-374, 1990); neural scarring (A. Logan et al., Eur. J.
Neurosci. 6: 355-363, 1994); dermal scarring (M. Shah et al.,
Lancet 339:213-214, 1992; M. Shah et al., J. Cell. Science
107:1137-1157,1994; M. Shah et al., 108:985-1002, 1995), and
pulmonary fibrosis (Giri et al., Thorax 48-959-966, 1993). Further,
antibodies cross-reactive with the isoforms, TGF.beta.s 1,2 and 3,
have been shown to be effective in models of lung fibrosis,
radiation induced fibrosis (Barcellos-Hoff, U.S. Pat. No. 5,616,561
(1997)), myelofibrosis, burns, Dupuyen's contracture, gastric
ulcers and rheumatoid arthritis (Wahl et al., J. Exp. Medicine
177:225-230, 1993).
[0084] There are a number of further conditions associated with
extracellular matrix deposition that may be ameliorated by
administration of an antibody directed to TGF.beta..sub.1 These
include systemic sclerosis, postoperative adhesions, keloid and
hypertrophic scarring, proliferative vitreoretinopathy, glaucoma
drainage surgery, corneal injury, cataract, Peyronie's disease,
diabetic nephropathy, adult respiratory distress syndrome,
cirrhosis of the liver, post myocardial infarction, post
angioplasty restenosis, keloid scars, scarring after subarachnoid
haemorrhage, multiple sclerosis, fibrosis after laminectomy,
fibrosis after tendon and other repairs, tatoo removal, sclerosing
cholangitis, pericarditis, pleurisy, tracheostomy, penetrating CNS
injury, eosinophilic myalgic syndrome, vascular restenosis,
veno-occlusive disease, pancreatitis and psoriatic arthropathy. In
some cases treatment in combination with an antibody directed
against TGF.beta..sub.2, such as 6B1 IgG4 (CAT-152) (see
WO97/13844) may be valuably employed, for instance in the treatment
of dermal scarring. The efficacy of SL15A IgG4 in treatment of
corneal epithelial wound healing is demonstrated in Example 6.
[0085] The success of CAT-192 in promoting corneal wound healing
provides indication of its usefulness in other conditions where
promotion of re-epithelialisation is beneficial. These include
diseases of the skin, such as venous ulcers, ischaemic ulcers
(pressure sores), diabetic ulcers, graft sites, graft donor sites,
abrasions and burns, diseases of the bronchial epithelium, such as
asthma, ARDS, diseases of the intestinal epithelium, such as
mucositis associated with cytotoxic treatment, oesophagual ulcers
(reflex disease), stomach ulcers, small intestinal and large
intestinal lesions (inflammatory bowel disease).
[0086] TGF.beta. also inhibits endothelial proliferation so
anti-TGF.beta. antibodies may be used to stabilise atherosclerotic
plaques and speed healing of vascular anastomoses. TGF.beta. may
stimulate smooth muscle proliferation so anti-TGF.beta. treatment
may be additionally appropriate or arterial disease and for
asthma.
[0087] Asthma is a chronic inflammatory disorder of the airways
manifesting as intermittent airflow obstruction, which over time
may become progressive. Both in the allergic and non-allergic forms
of the disease, there is evidence of an altered local T cell
response in favour of the Th-2 cytokine release resulting on B-cell
isotype switching to IgE, mast cell, eosinophil and basophil
recruitment and activation and release of a wide range of
inflammatory mediators. However, it has become clear that by
itself, inflammation is not able to explain many of the features
characteristic of chronic asthma and that restructuring of airway
wall is also required (Holgate ST et al., J. Allergy Clin. Immunol.
2000 (in press)). This "remodelling" response accounts for the
incomplete therapeutic efficacy of corticosteroids, with persistent
bronchial hyperresponsiveness (BHR) (Lundgren R et al., (1988) Eur.
Respir. J. 1(10):883-889), and the progressive decline in pulmonary
function over time, which occurs in those asthmatics with more
chronic and severe disease (Lange P et al., (1998) N. Engl. J. Med.
339(17): 1194-1200).
[0088] Subepithelial and submucosal fibrosis is implicated in
asthma. When assessed by high resolution CT, patients with severe
asthma have thicker airways when compared to normal subjects or
those with mild disease (Awadh N et al., (1998) Thorax
53(4):248-253). This involves thickening and increased density of
the SBM collagen layer, increases in smooth muscle and
microvascular networks (Carroll N et al., (1993) Am. Rev. Respir.
Dis. 147(2):405-410) On the basis of measurements made in human
airways and in a guinea pig model of chronic antigen exposure, SBM
thickening reflects that of the entire airway wall. SBM-collagen
thickness has been shown to correlate with disease severity,
chronicity and BHR.
[0089] SBM thickening is due to the deposition of interstitial
collagens Types I, III and V and fibronectin in the lamina
reticularis and originates from the myofibroblasts whose numbers
and activity are increased in asthma (Brewster CE et al., (1990)
Am. J. Respir. Cell Mol. Biol. 3(5):507-511) and further enhanced
by allergen exposure (Gizycki M J et al., (1997) Am. J. Respir.
Cell Mol. Biol. 16(6):664-673). Bronchial biopsy and lavage studies
have provided a compelling case for the epithelium as a potential
source of profibrogenic growth factors. In asthma, immunostaining
for TGF.beta. and b-FGF show both epithelial localisation and
extensive staining of matrix, indicative of important interactions
between these growth factors and matrix proteoglycans through
specific glycosaminoglycan (GAG) binding sites (Redington A E et
al., (1998) J. Pathol. 186(4): 410-415). Both TGF.beta..sub.1 and
b-FGF are found in increased concentrations in lavage fluid, with
further increases occurring after allergen exposure (Redington AE
et al., (1997) Am. J. Respir. Crit. Care Med. 156(2, Pt
1):642-647). Tissue analysis of growth factors, cytokines and
chemokines have shown that these are mainly present in complex,
high molecular weight forms Latency associated peptide has been
identified as the TGF.beta. binding molecule and using a bronchial
explant model, it has been shown that allergen exposure of
asthmatic mucosal tissue results in activation of TGF.beta. that is
dependant upon plasmin activity, while b-FGF is released in soluble
form by heparin and heparinase from mast cells and eosinophils
respectively (McConnell W et al., Eur. Respir. J. 152s. 1999, Ref
Type: Abstract).
[0090] Asthma is also involved in epthelial injury and airways
remodelling. A characteristic feature of the remodelled airways in
asthma is extensive epithelial damage caused by inflammatory cell
products (Laitinen LA et al, (1985) Am. Rev. Respir. Dis.
131(4):599-606). Mucosal damage not only allows tissue damaging
molecules to pass unimpeded into the airways wall, but also causes
the epithelium to become "activated" with expression of a variety
of proinflammatory chemokines, autacoid mediators and adhesion
molecules which contribute to chronic inflammation (Holgate ST et
al., J. Allergy Clin. Immunol. 2000 (in press)). Injured and
repairing epithelial cells are also important regulators of airway
remodelling through increased production of fibroproliferative and
profibrogenic growth factors including TGF.beta. isoforms (Zhang S
et al., (1999) Lab. Invest. 79(4):395-405) It has recently been
found that impairment of epidermal growth factor receptor
(EGFR)-mediated epithelial repair causes greatly increased release
of TGF.beta..sub.2 by damaged epithelial cells and a marked
enhancement of collagen III gene expression when conditioned medium
is added to myofibroblast cultures. As TGF.beta. isoforms are
potent inhibitors of epithelial cell proliferation, excessive
production of TGF.beta. in asthma may account for the unexpectedly
low level of expression of the proliferation marker PCNA found in
asthmatic epithelium (Demoly P et al., (1994)
[0091] Am. J. Respir. Crit. Care Med. 150(1):214-217). In this way,
conditions that favour collagen biosynthesis by the sub-epithelial
myofibroblasts, may also contribute to disease chronicity by
retarding epithelial repair. Hence reducing TGF.beta. levels by the
use of specific binding members of the present invention would be
expected to address the need in chronic and severe asthma to
prevent matrix protein biosynthesis by bronchial myofibroblasts, as
well as promoting bronchial epithelial repair in order to restore a
non-activated epithelial phenotype and normal barrier function.
[0092] TGF.beta..sub.1 also modulates immune and inflammatory
responses, for instance in response to malignancy and infection. An
antibody against TGF.beta..sub.1 may be used for improving the
immune response to infections such as hepatitis B, hepatitis C or
tuberculosis or for reducing immunosuppression induced, for
instance, by tumors or AIDS infection or granulomatous diseases. An
antibody against TGF.beta..sub.1 may be useful for treatment of
acute and chronic rejection of organ transplant and malignant
tumors, and may be used in prevention of the spread of cancer cells
induced by treatment with cyclosporine.
[0093] An antibody against TGF.beta..sub.1 may be used as an
adjuvant for immunotherapy.
[0094] An antibody against TGF.beta..sub.1 may be used for
inhibition of angiogenesis, for instance in treatment of tumors The
majority of tumor cells express detectable levels of
TGF.beta..sub.1 (Wojtowicz-Praga 1997, J. Immunother. 20 (3):
165-77). Furthermore, cells that produce higher levels of
TGF.beta..sub.1 have a higher metastatic (Blanckaert et al., 1993,
Cancer. Res. 53 (17): 4075-81) or invasive (Arteaga et al., 1993,
Cell Growth and Differentiation 4(3): 193-201) potential. In many
cancers TGF.beta..sub.1 plasma levels are correlated with disease
progression. Thus the source of TGF.beta..sub.1 can be the tumor
cells as well as surrounding tissue.
[0095] TGF.beta. is a potent suppressor of malignant transformation
in normal healthy epithelial tissue and can inhibit proliferation.
However, many advanced cancers become resistant to the
growth-inhibitory actions of TGF.beta. as a result of abnormalities
in the type II TGF.beta. receptor (Markowitz et al., 1995, Science
268 (5215): 1336-8) or SMAD signal transduction (Hata et al., 1998,
Mol. Med. Today 4(6): 257-62).
[0096] Tumours require a blood supply for growth in excess of 1
mm.sup.3 and for metastasis (Folkman 1995, Breast Cancer Res.
Treat. 36 (2): 109-18). This has led to the rapid development of
anti-angiogenic treatments for solid tumors.
[0097] TGF.beta..sub.1 has been shown to cause angiogenesis
indirectly by up regulating VEGF production in vitro and in vivo.
Breast cancers contain large numbers of infiltrating macrophages.
The role and function of these cells within the tumor remain
unclear, but a number of studies have found an association with
poor prognosis. Both tumor cells and tumor macrophages produce VEGF
in vitro and production is up regulated by TGF.beta..sub.1. Serum
VEGF level is enhanced in patients with breast cancer and these
levels directly correlate to serum TGF.beta.1 levels. Thus,
TGF.beta..sub.1 expression by breast cancer cells and
cancer-associated macrophages may elicit an angiogenic response
through generation of VEGF Donovan et al., 1997, Ann. Surg. Oncol 4
(8):621.7; Harmey et al., 1998, Ann. Surg. Oncol. 5(3):
271-278).
[0098] Ueki et al. (1992, Japanese Journal of Cancer Research
84(6):589-93) demonstrated that TGF.beta..sub.1 enhanced tumor
growth in vivo. This group transected CHO cells with the
TGF.beta..sub.1 gene resulting in overexpression of
TGF.beta..sub.1. TGF.beta..sub.1-secreting CHO cells were shown to
grow more rapidly that non-transected cells when injected
subcutaneously into nude mice. Prominent vascularisation was
observed in tumors derived from TGF.beta..sub.1-transected cells;
vascularisation was diminished in the non-transected cells. In
addition, an anti-TGF.beta..sub.1 neutralising antibody was able to
inhibit both growth and angiogenesis in the tumors derived from
TGF.beta..sub.1-transected cells. Thus, overproduction of
TGF.beta..sub.1 by tumor cells contributed to tumor growth and
neovascularisation.
[0099] It is known that patients who have cancer also have a
defective immune system. Recently TGF.beta..sub.1 has been
suggested to play a key role in tumor-associated immunosuppression.
This topic has been the focus of a recent review (Wojtowicz-Praga,
1997, ibid). Certainly, TGF.beta..sub.1 appears to be a potent
immunosuppressor, and it has been consistently detected from a
variety of tumor cell lines and in plasma of tumor-bearing
hosts.
[0100] Neutralisation of TGF.beta..sub.1 by monoclonal antibodies
or inhibition of production by antisense results in attenuation of
tumor growth and metastatic ability in animal models. Growth of
MCF-7 breast cancer cells transected with TGF.beta..sub.1 in mice
is prevented with 2G7 (repeated i.p. does), an anti-TGF.beta..sub.1
2 3 antibody (Arteaga et al., ibid). Growth of normal MCF-7 cells
is prevented by 2G7 but only when treatment started at the time of
tumor cell inoculation. Further, more convincing evidence for an
action of anti-TGF.beta. antibodies to relive tumor-induced
immunosuppression has been provided (Arteaga et al., J. Clin.
Invest. 92 (6):2569-2576). MDA-23 1, a human breast cancer cells
line caused a decrease in spleen natural killer (NK) cell activity
in nude mice following i.p. inoculation. 2G7 (200 .mu.g every 2
days, i.p.) attenuated intra-abdominal tumors and lung metastasis
as well as markedly increasing the activity of spleen NK cell
activity. Furthermore, conditioned medium from cultures of MDA-231
tumor cells inhibited the NK cell activity of human blood, again
2G7 prevented this. Growth of subcutaneous xenografts of MDA-231
cells were only transient inhibited by 2G7. The action of 2G7 on
tumor growth, metastasis, and NK cell activity were absent in beige
NK cell-deficient nude mice (Arteaga et al., J. Clin. Invest. 92
(6):2569-2576).
[0101] In a further study, the 2G7 anti-TGF.beta. antibody (500
.mu.g i.p. every other day) in combination with IL-2 (10000U i. p.
2.times. daily) was able to reduce B16 melanoma lung metastasis but
was not as effective i.v. inoculation. 2G7 alone also reduced the
number of lung metastasis but was not as effective as the combined
therapy Plasma TGF.beta..sub.1 levels were significantly reduced in
the antibody treated animals (Wojtowicz-Praga et al., 1996, J.
Immunother. Emphasis Tumor Immunol. 19(3):169-175). Two previous
studies either failed to show an effect or only caused a small
effect using the combination of anti-TGF.beta..sub.1 and 1L-2
(Gridley et al., 1993, Cancer Biother. 8 (2): 159-170; Mao et al,
1994, Cancer Biother. 9(4):317-327, respectively), however, the
doses of anti-TGF.beta..sub.1 antibodies used in these studies were
small (10 ng and 1 .mu.g respectively). Thus, combination of
anti-TGF.beta. therapy with immunostimulation would appear, from
animal model data, to provide proof of concept for this therapeutic
approach to cancer
[0102] Hoefer and Anderer (1995, Cancer Immunol. Immunother. 41
(5): 302-308) demonstrated that the human carcinoma cell line,
SLU-1, and the highly metastatic sub-line SLU-M1, resulted in
metastasis in nude mice following s.c. inoculation. The incidence
of metastasis as well as primary tumor growth was reduced by
treatment with anti-TGF.beta..sub.1 antibodies (treatment from day
3, every 3-4 days with 100 .mu.g s.c. at the tumor site) The
authors suggested that the antibodies reversed
TGF.beta..sub.1-induced immunosuppression leading to inhibition of
tumor growth and metastasis.
[0103] Previous work has also demonstrated that anti-TGF.beta.
antibodies can reduce tumor metastasis in vivo (Arteaga et al.,
1993, J. Clin., Invest. 92(6):2569-2576; Hoefer & Anderer,
1995, ibid; Wojtowicz-Praga et al, 1996, ibid). Initial conclusions
from these studies suggested that TGF.beta..sub.1-induced
immunosuppression permitted metastasis of these tumor xenografts.
However, a recent paper suggests that TGF.beta..sub.1 may enhance
the invasive and metastatic potential of cells directly (Hojo et
al., 1999, Nature 397:530-534). Cyclosporin dose-dependently
induces TGF.beta..sub.1 release from human pulmonary adenocarcinoma
cells in culture, however, the mechanism of TGF.beta. production by
cyclosporin is not known. Treatment of adenocarcinoma cells with
cyclosporin (or TGF.beta..sub.1) results in membrane ruffling,
pseudopodia formation, anchorage-independent (invasive) growth and
motility. An anti-TGF.beta..sub.1 antibody inhibits these cell
morphology and motility changes in vitro. Similar observations were
made for renal cells adenocarcinoma, mammary gland epithelial cells
and mink lung epithelial lung cells. In immunodeficient SCID-beige
mice (deficient in T cells, B cells, NK cells), cyclosporin
increased the number of metastasis following incubation (i v.) Of
murine renal cell adenocarcinoma, Lewis lung carcinoma or human
bladder cancer cells Treatment with the anti-TGF.beta..sub.1 2 3
neutralising antibody, 1D11.16 (200 .mu.g per pay, initial dose 1
day prior to tumor cell inoculation) significantly reduced the
number of pulmonary metastasis in cyclosporin treated mice (to
levels below that of the non-cyclosporin treated control group)
Thus, it appears and in that cyclosporin can, through an action
dependant on TGF.beta..sub.1 production, increase invasion and
metastasis in animal models independent of the host's immune system
(Hojo et al., 1999, ibid).
[0104] Evidence from in vitro and in vivo models suggests that
TGF.beta..sub.1, can enhance tumor formation utilising three main
mechanisms, angiogenesis, immunosuppression and phenotypic changes
of tumor cells to increase invasive and metastatic behaviour. Thus,
inhibition of TGF.beta..sub.1 would be expected to inhibit
malignancy in man and within a single molecule, deliver a combined
anti-cancer therapy.
[0105] Cancers in which TGF.beta..sub.1 have been implicated
include breast, prostate, ovarian, stomach, colerectal, skin, lung,
cervical and bladder cancers, as well as various leukemias and
sarcomas, such as Kaposi's Sarcoma. Accordingly, antibodies of the
invention may be administered for the treatment of cancers in which
TGF.beta..sub.1 is implicated either angiogensis, metastasis or
tumor progression, including cancers of the foregoing conditions.
It will of course be appreciated that in the context of cancer
therapy, "treatment" includes any medical intervention resulting in
the slowing of tumor growth or reduction in tumor metastases, as
well as partial remission of the cancer in order to prolong life
expectancy of a patient
[0106] Antibody therapy for the treatment of cancer is an
established treatment in the art. Three anti-cancer antibodies are
currently licenced for clinical use in the US and/or Europe
(Panorex for the treatment of colorectal cancer, Rituxan for B-cell
lymphoma and Herceptin for breast cancer), in addition to numerous
other anti-cancer antibodies currently in Phase I, II or III
clinical trials. These antibodies are often used in late stage
treatment and are considered effective by the criteria of the
preceding paragraph, as well as, in some cases, providing complete
remission of the tumor.
[0107] Accordingly, further aspects of the invention provide
methods of treatment comprising administration of a specific
binding member as provided, pharmaceutical compositions comprising
such a specific binding member, and use of such a specific binding
member in the manufacture of a medicament for administration, for
example in a method of making a medicament or pharmaceutical
composition comprising formulating the specific binding member with
a pharmaceutically acceptable excipient.
[0108] In accordance with the present invention, compositions
provided may be administered to individuals. Administration is
preferably in a "therapeutically effective amount", this being
sufficient to show benefit to a patient. Such benefit may be at
least amelioration of at least one symptom. The actual amount
administered, and rate and time-course of administration, will
depend on the nature and severity of what is being treated
Prescription of treatment, eg decisions on dosage etc, is within
the responsibility of general practioners and other medical
doctors. Appropriate doses of antibody are well known in the art;
see Ledermann J. A. et al. (1991) Int J. Cancer 47. 659-664;
Bagshawe K. D. et al. (1991) Antibody, Immunoconjugates and
Radiopharmaceuticals 4:915-922.
[0109] A composition may be administered alone or in combination
with other treatments, either simultaneously or sequentially
dependent upon the condition to be treated.
[0110] Antibodies of the present invention may be administered to a
patient in need of treatment via any suitable route, usually by
injection into the bloodstream or directly into the site to be
treated, e.g. cornea, wound, tumor, etc The precise dose will
depend upon a number of factors, including whether the antibody is
for diagnosis or for treatment, the size and location of the area
to be treated (e.g. wound), the precise nature of the antibody
(e.g. whole antibody, fragment or diabody), and the nature of any
detectable label or other molecule attached to the antibody. A
typical antibody dose will be in the range 0.5 mg to 100 g for
systemic applications, such as treatment of fibrosis in
glomerulonephritis or in the treatment of cancers and 10 .mu.g to 1
mg for local applications such as treatment of dermal scarring.
Typically, the antibody will be a whole antibody, preferably the
IgG4 isotype. This is a dose for a single treatment of an adult
patient, which may be proportionally adjusted for children and
infants, and also adjusted for other antibody formats in proportion
to molecular weight. Treatments may be repeated at daily,
twice-weekly, weekly or monthly intervals, at the discretion of the
physician
[0111] It is presently preferred that a whole antibody of the IgG4
isotype is used for systemic and local applications but for local
applications a scFv antibody may be particularly valuable.
[0112] Specific binding members of the present invention will
usually be administered in the form of a pharmaceutical
composition, which may comprise at least one component in addition
to the specific binding member
[0113] Thus pharmaceutical compositions according to the present
invention, and for use in accordance with the present invention,
may comprise, in addition to active ingredient, a pharmaceutically
acceptable excipient, carrier, buffer, stabiliser or other
materials well known to those skilled in the art. Such materials
should be non-toxic and should not interfere with the efficacy of
the active ingredient. The precise nature of the carrier or other
material will depend on the route of administration, which may be
oral, or by injection, e.g. intravenous.
[0114] Pharmaceutical compositions for oral administration may be
in tablet, capsule, powder or liquid form. A tablet may comprise a
solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical
compositions generally comprise a liquid carrier such as water,
petroleum, animal or vegetable oils, mineral oil or synthetic oil.
Physiological saline solution, dextrose or other saccharide
solution or glycols such as ethylene glycol, propylene glycol or
polyethylene glycol may be included. Formulation as eye drops may
be valuable for prevention or treatment of ocular fibrosis or
scarring.
[0115] For intravenous, injection, or injection at the site of
affliction, the active ingredient will be in the form of a
parenterally acceptable aqueous solution which is pyrogen-free and
has suitable pH, isotonicity and stability Those of relevant skill
in the art are well able to prepare suitable solutions using, for
example, isotonic vehicles such as Sodium Chloride Injection,
Ringer's Injection, Lactated Ringer's Injection. Preservatives,
stabilisers, buffers, antioxidants and/or other additives may be
included, as required.
[0116] The antibody may be administered from a sustained delivery
system to prevent fibrosis or may be coated onto prosthetic
devices, such as hip replacements, to prevent development of
fibrosis associated with their insertion.
[0117] A composition may be administered alone or in combination
with other treatments, either simultaneously or sequentially
dependent upon the condition to be treated. Other treatments may
include the administration of suitable doses of pain relief drugs
such as non-steroidal anti-immflamatory drugs (e.g. asprin,
paracetamol, ibuprofen or ketoprofen) or opitates such as morphine,
or anti-emetics.
[0118] The present invention provides a method comprising causing
or allowing binding of a specific binding member as as provided
herein to TGF.beta..sub.1. As noted, such binding may take place in
vivo, e.g. following administration of a specific binding member,
or nucleic acid encoding a specific binding member, or it may take
place in vitro.
[0119] The amount of binding of specific binding member to
TGF.beta..sub.1 may be determined. Quantitation may be related to
the amount of TGF.beta..sub.1 in a test sample, which may be of
diagnostic interest, which may be of diagnostic interest, for
example, measurement of TGF.beta..sub.1 has also been proposed as
an indicator for atherosclerosis, low concentrations being
correlated with advanced atherosclerosis.
[0120] The reactivities of antibodies on a sample may be determined
by any appropriate means. Radioimmunoassay (RIA) is one
possibility. Radioactive labelled TGF.beta..sub.1 is mixed with
unlabelled TGF.beta..sub.1 (the test sample) and allowed to bind to
the antibody. Bound TGF.beta..sub.1 is physically separated from
unbound TGF.beta..sub.1 and the amount of radioactive
TGF.beta..sub.1 bound to the antibody determined. The more
TGF.beta..sub.1 there is in the test sample the less radioactive
TGF.beta..sub.1 will bind to the antibody. A competitive binding
assay may also be used with non-radioactive TGF.beta..sub.1, using
TGF.beta..sub.1 or an analogue of TGF.beta..sub.1 linked to a
reporter molecule. The reporter molecule may be a fluorochrome,
phosphor or laser dye with spectrally isolated absorption or
emission characteristics. Suitable fluorochromes include
fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable
chromogenic dyes include diaminobenzidine.
[0121] Other reporters include macromolecular colloidal particles
or particulate material such as latex beads that are colored,
magnetic or paramagnetic, and biologically or chemically active
agents that can directly or indirectly cause detectable signals to
be visually observed, electronically detected or otherwise
recorded. These molecules may be enzymes which catalyse reactions
that develop or change colors or cause changes in electrical
properties, for example. They may be molecularly excitable, such
that electronic transitions between energy states result in
characteristic spectral absorptions or emissions. They may include
chemical entities used in conjunction with biosensors.
Biotin/avidin or biotin/streptavidin and alkaline phosphatase
detection systems may be employed.
[0122] The signals generated by individual antibody-reporter
conjugates may be used to derive quantifiable absolute or relative
data of the relevant antibody binding in samples (normal and
test).
[0123] The present invention also provides the use of a specific
binding member as above for measuring TGF.beta..sub.1 levels in a
competition assay, that is to say a method of measuring the level
of TGF.beta..sub.1 in a sample by employing a specific binding
member as provided by the present invention in a competition assay.
This may be where the physical separation of bound from unbound
TGF.beta..sub.1 is not required. Linking a reporter molecule to the
specific binding member so that a physical or optical change occurs
on binding is one possibility. The reporter molecule may directly
or indirectly generate detectable, and preferably measurable,
signals. The linkage of reporter molecules may be directly or
indirectly, covalently, e.g. via a peptide bond or non-covalently.
Linkage via a peptide bond may be as a result of recombinant
expression of a gene fusion encoding antibody and reporter
molecule.
[0124] The present invention also provides for measuring levels of
TGF.beta..sub.1 directly, by employing a specific binding member
according to the invention for example in a biosensor system.
[0125] The mode of determining binding is not a feature of the
present invention and those skilled in the art are able to choose a
suitable mode according to their preference and general
knowledge.
[0126] The present invention further extends to a specific binding
member which competes for binding to TGF.beta..sub.1 with any
specific binding member which both binds TGF.beta..sub.1 and
comprises a V domain including a CDR with amino acid substantially
as set out herein or a V domain with amino acid sequence
substantially as set out herein. Competition between binding
members may be assayed easily in vitro, for example by tagging a
specific reporter molecule to one binding member which can be
detected in the presence of other untagged binding member(s), to
enable identification of specific binding members which bind the
same epitope or an overlapping epitope. Competition may be
determined for example using the TGF.beta..sub.1 ELISA as described
in Example 1.
[0127] Preferred specific binding members for TGF.beta..sub.1
compete for binding to TGF.beta..sub.1 with CAT 191, CAT 192 and/or
CAT 193
[0128] Preferred embodiments strongly neutralise TGF.beta..sub.1,
having a potency of at least 5 times better than does CS37, more
preferably about 10 times, 15 times, 20 times, 50 times, 75 times,
100 times or 150 times better, in a radioreceptor assay (Lucas C et
al (1991) Meth in Enzymology 198:303-316). Potency is measure with
the antibody under study and CS37 in equivalent molecular formats,
e.g. as monovalent antibodies (scFv or Fab) or as bivalent
antibodies (IgG1 or IgG4).
[0129] In one aspect, a specific binding member according to the
present invention binds a peptide including the amino acid sequence
of residues 92-98 of TGF.beta..sub.1 (the same epitope as
CS37).
[0130] In testing for this, a peptide with this sequence plus one
or more amino acids at either end, may be used. Such a peptide may
be said to "consist essentially" of the specified sequence.
Specific binding members according to the present invention may be
such that their binding for TGF.beta..sub.1 is inhibited by a
peptide with or including the sequence given. In testing for this,
a peptide with either sequence plus one or more amino acids may be
used.
[0131] Specific binding members which bind a specific peptide may
be isolated for example from a phage display library by panning
with the peptide(s).
[0132] The present invention further provides an isolated nucleic
acid encoding a specific binding member of the present invention.
Nucleic acid includes DNA and RNA. In a preferred aspect, the
present invention provides a nucleic acid which codes for a CDR or
VH or VL domain of the invention as defined above.
[0133] The present invention also provides constructs in the form
of plasmids, vectors, transcription or expression cassettes which
comprise least one polynucleotide as above.
[0134] The present invention also provides a recombinant host cell
which comprises one or more constructs as above. A nucleic acid
encoding any CDR, VH or VL domain, or specific binding member as
provided itself forms an aspect of the present invention, as does a
method of production of the encoded product, which method comprises
expression from encoding nucleic acid therefor. Expression may
conveniently be achieved by culturing under appropriate conditions
recombinant host cells containing the nucleic acid. Following
production by expression a VH or VL domain, or specific binding
member may be isolated and/or purified using any suitable
technique, then used as appropriate.
[0135] Specific binding members, VH and/or VL domains, and encoding
nucleic acid molecules and vectors according to the present
invention may be provided isolated and/or purified, e.g. from their
natural environment, in substantially pure or homogeneous form, or,
in the case of nucleic acid, free or substantially free of nucleic
acid or genes origin other than the sequence encoding a polypeptide
with the required function. Nucleic acid according to the present
invention may comprise DNA or RNA and may be wholly or partially
synthetic. Reference to a nucleotide sequence as set out herein
encompasses a DNA molecule with the specified sequence, and
encompasses a RNA molecule with the specified sequence in which U
is substituted for T, unless context requires otherwise.
[0136] Systems for cloning and expression of a polypeptide in a
variety of different host cells are well known. Suitable host cells
include bacteria, mammalian cells, yeast and baculovirus systems.
Mammalian cell lines available in the art for expression of a
heterologous polypeptide include Chinese hamster ovary cells, HeLa
cells, baby hamster kidney cells, NSO mouse melanoma cells and many
others A common, preferred bacterial host is E. coli.
[0137] The expression of antibodies and antibody fragments in
prokaryotic cells such as E. coli is well established in the art.
For a review, see for example Pluckthun, A. Bio/Technology
9:545-551 (1991). Expression in eukaryotic cells in culture is also
available to those skilled in the art as an option for production
of a specific binding member, see for recent reviews, for example
Reff, M. E. (1993) Curr. Opinion Biotech. 4: 573-576, Trill J. J.
et al. (1995) Curr. Opinion Biotech 6:553-560.
[0138] Suitable vectors can be chosen or constructed, containing
appropriate regulatory sequences, including promoter sequences,
terminator sequences, polyadenylation sequences, enhancer
sequences, marker genes and other sequences as appropriate. Vectors
may be plasmids, viral e.g. phage, or phagemid, as appropriate. For
further details see, for example, Molecular Cloning: a Laboratory
Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor
Laboratory Press Many known techniques and protocols for
manipulation of nucleic acid, for example in preparation of nucleic
acid constructs, mutagenesis, sequencing, introduction of DNA into
cells and gene expression, and analysis of proteins, are described
in detail in Short Protocols in Molecular Biology, Second Edition,
Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures
of Sambrook et al. and Ausubel et al. are incorporated herein by
reference.
[0139] Thus, a further aspect of the present invention provides a
host cell containing nucleic acid as disclosed herein. A still
further aspect provides a method comprising introducing such
nucleic acid into a host cell. The introduction may employ any
available technique. For eukaryotic cells, suitable techniques may
include calcium phosphate transfection, DEAE-Dextran,
electroporation, liposome-mediated transfection and transduction
using retrovirus or other virus, e.g. vaccinia or, for insect
cells, baculovirus. For bacterial cells, suitable techniques may
include calcium chloride transformation, electroporation and
transfection using bacteriophage.
[0140] The introduction may be followed by causing or allowing
expression from the nucleic acid, e.g. by culturing host cells
under conditions for expression of the gene.
[0141] In one embodiment, the nucleic acid of the invention is
integrated into the genome (e.g. chromosome) of the host cell.
Integration may be promoted by inclusion of sequences which promote
recombination with the genome, in accordance with standard
techniques.
[0142] The present invention also provides a method which comprises
using a construct as stated above in an expression system in order
to express a specific binding member or polypeptide as above.
[0143] The following examples illustrate aspects and embodiments of
the present invention.
[0144] Example 1 Identification of SL15S scFv (CAT-191) and
JT182
[0145] Example 2 Construction of Cell Lines Expressing the Antibody
SL15A IgG4 (CAT-192) and SL15S IgG4 (CAT-193)
[0146] Example 3 Assessment of neutralisation properties of SL15S
scFv (CAT-191) and SL15A IgG4 (CAT-192) and SL15S IgG4
(CAT-193)
[0147] Example 4 Binding of the antibody SL15S scFv (CAT-191) and
SL15A IgG4 (CAT-192) to active and latent TGF.beta..sub.1
[0148] Example 5 Epitope mapping of the antibodies SL15S scFv and
CS37 scFv
EXAMPLE 1
Identification of SL15S SCFV (CAT-191) and JT182
[0149] The present inventors have identified antibody CDRs and VH
and VL domains related to those of the CS37 antibody disclosed in
WO97/13844, but with unexpectedly good properties.
[0150] The CS37 VH (31G9) domain sequence and encoding nucleic acid
therefor are shown in SEQ ID NO:2 and SEQ ID NO:1, respectively
[0151] The SL15 (a.k.a. KYLIE) VH domain sequence of the present
invention and encoding nucleic acid therefor are shown in SEQ ID
NO:4 and SEQ ID NO:3, respectively.
[0152] The respective VH CDR3 sequences are shown in Table 1, also
Table 2 which includes CDR1 and CDR2 sequences for both VH and VL
domains.
[0153] Comparison of CS37 (31 G9) and SL15 (Kylie) VH domains shows
three further differences in framework residues, at residues 1
(glutamine CS37 to glutamate SL15), 6 (glutamine CS37 to glutamate
SL15) and 44 (glycine CS37 to glutamate SL15).
[0154] The SL15 VH domain may be paired with different VL domains,
and two such SL15 variants have been identified. One, known as
SL15A, includes the CS37 VL. The other, known as SL15S, includes a
VL which corresponds to the CS37 VL save for the presence of Serine
at residue 25 in SL15S compared with alanine in SL15A (CS37).
[0155] The SL15A VL (CS37) domain sequence and encoding nucleic
acid therefor are shown in SEQ ID NO:6 and SEQ ID NO:5,
respectively.
[0156] The SL15S VL domain sequence and encoding nucleic acid
therefor are shown in SEQ ID NO:8 and SEQ ID NO:7,
respectively.
[0157] The JT182 VH domain sequence and encoding nucleic acid
therefor are shown in SEQ ID NO:10 and SEQ ID NO:9,
respectively.
[0158] SL15S scFv, CS37 scFv and a related antibody JT182 were
screened as phage supernatants in ELISA assays for the ability to
bind TGF.beta..sub.1. ELISA plates (96 well; Falcon) were uncoated,
or coated with recombinant TGF.beta..sub.1 (0.2 .mu.g/ml). Phage
that bound specifically to the antigen coated plate were detected
using a sheep anti-fd antiserum (Pharmacia), followed by alkaline
phosphatase conjugated anti-sheep (Sigma) and p-nitrophenyl
phosphate (pNPP) substrate (Sigma).
[0159] The scFv fragments were subsequently tested for their
ability to neutralise binding of .sup.125I-TGF.beta..sub.1 to A549
cells in a radioreceptor binding assay (RRA), using the protocol
described in Example 3 (see below). For the RRA, individual clones
were expressed as soluble scFv and subsequently purified from
periplasmic preparations by immobilised metal affinity
chromatography (IMAC) followed by fractionation of monomeric scFv
by gel filtration FPLC on a Superdex 75 column (Pharmacia).
[0160] SL15 scFv (SL15 VH/SL15 VL) has the highest neutralisation
potency in the RRA, having an IC.sub.50 of 100 pM which is at least
100 fold better than CS37. The complete specificity of SL15 scFv
for the TGF.beta..sub.1 isoform has been confirmed in the TF1 assay
where no interaction with TGF.beta..sub.2 or TGF.beta..sub.3 was
detected.
[0161] The scFv antibodies SL15S (SL15 VH/SL15 VL; CAT-191 a k a.
Kylie scFv) and SL15A (SL15 VH/CS37VL) were converted into whole
antibody.
EXAMPLE 2
Construction of Cell Lines Expressing the Antibody S115A IGG4
(CAT-192) and SL15S IGG4 (CAT-193)
[0162] For the construction of cell lines expressing human IgG4,
.kappa. antibodies, SL15 scFV heavy and light chain variable
domains were cloned into mammalian expression vectors containing
human IgG4 and human kappa constant domains respectively Two
versions were prepared, SL15A IgG4 (CAT-192) and SL15S IgG4
(CAT-193) The antibodies are also termed Kylie IgG.
[0163] Heavy Chain Expression vector
[0164] The VH from the SL15S scFv DNA was PCR-amplified with
oligonucleotides P80 (SEQ ID NO:25) and P64 (SEQ ID NO:22) and
joined by overlapping PCR to a 159 bp DNA fragment containing a
signal sequence, splice sites and intron from M13VHPCR1 (Orlandi et
al., 1989, Proc. Natl Acad. Sci. USA 86:3833-3837) using
oligonucleotides P10 (SEQ ID NO:20) and P64 (SEQ ID NO:22). The 558
bp PCR product was cut with HindIII and ApaI and cloned into
HindIII-ApaI cut pGamma4 (obtained from Lonza Biologics). Ligated
DNA was transformed into E. coli TG1 and ampicillin-resistant
colonies screened. A plasmid with the correct insertion was
identified and designated pKylieVH.gamma.4.
[0165] Light Chain Expression Vector
[0166] The V.kappa. from the CS37 scFv DNA or from SL15S scFv was
PCR-amplified with oligonucleotides P65 (SEQ ID NO:23) and P66 (SEQ
ID NO:24) and joined by overlapping PCR to a 168 bp DNA fragment
containing a signal sequence, splice sites and intron from
M13VKPCR1 (Orlandi et al., 1989, Proc. Natl Acad. Sci. USA
86;3833-3837) using oligonucleotides P11 (SEQ ID NO:21) and P66
(SEQ ID NO:24). The 510 bp PCR product was cut with BstBI and BsiWI
and cloned into BstBI-BsiWI cut pMR15.1. Ligated DNA was
transformed into E. coli TG1 and ampicillin-resistant colonies
screened. A plasmid with the correct insertion was identified and
designated pCS37.kappa. or pKylie.kappa..
[0167] Tandem Expression Vector
[0168] A single plasmid containing both heavy and light chain DNAs
and the gs selectable marker was constructed for each of the Kylie
variants The heavy chain vector, pKylieVH.gamma.4, was digested
with BamHI and NotI and the 4497 bp fragment containing H chain DNA
purified. The light chain vector, pCS37V.kappa. or pKylie.kappa.,
was similarly cut with BamHI and NotI and the 9611 bp fragment
containing L chain DNA was isolated. The two purified fragments
were ligated together, transformed into E. coli TG1 cells and
ampicillin-resistant colonies screened. A plasmid with the correct
insertions was identified and the V regions confirmed by
sequencing. The final expression vector was designated
pKylieg4.gamma.s. Two versions were prepared containing SL15S VL or
CS37 VL.
[0169] Expression of SL15S IgG4 and SL15A IgG4
[0170] SL15S IgG4 and SL15A IgG4 were expressed in the mouse
myeloma cell line NSO (ECACC 85110503). Fifty .mu.g of
pKylieg4.gamma.s were linearised by digestion with PvuI, ethanol
precipitated and dissolved in 100 .mu.l water. 10.sup.7 NS0 cells
were washed in PBS, resuspended in 0.9 ml PBS, mixed with the
vector DNA and held in ice for 5 min. The cells were then
electroporated with a single pulse of 250 V at 960 .mu.Fd and
incubated in ice for 10 min. The transfected cells were then added
to 30 ml Dulbecco's modified Eagle's medium (DMEM) containing 2 mM
glutamine and 10% dialysed foetal calf serum (FCS) as described by
Bebbington et al. (1992) Bio/Technology, 10 169-175, and 50 .mu.l
aliquots distributed into 6.times.96-well plates. 24 h later
glutamine-free DMEM/10% FCS (Bebbington et al. 1992) was added to
each well.
[0171] Three to six weeks after transfection colonies were screened
by ELISA for the ability to secrete human IgG. Wells of ELISA
plates (Immulon 4, Dynatech) were coated in 50 mM sodium
bicarbonate/carbonate pH 9.6 with 100 ng per well of goat
anti-human IgG antibodies (Harlan). Supernatant from wells
containing transfected colonies was added to the wells in PBS
containing 0.05% (v/v) Tween 20 (PBST) for 1 h. The plates were
washed 3 times with PBST and captured human IgG was detected with
100 .mu.l 1:2000 dilution horseradish peroxidase (HRP)-conjugated
goat anti-human kappa antibodies in PBST (Harlan). After 30 min at
room temperature the plates were washed 3.times. PBST and 100 .mu.l
OPD substrate added. Reactions were stopped after 5-10 min by the
addition of 50 .mu.l 12.5% (v/v) sulphuric acid and the A 490 nm
measured.
[0172] Transfectants secreting the highest amounts of IgG were
expanded for growth in glutamine-free medium in reduced FCS, in
gammaglobulin-free FCS or no FCS. Cell lines were subsequently
cloned by limiting dilution.
[0173] Purification of IgG
[0174] Human IgG4 antibodies were purified by protein A affinity
chromatography followed by size-exclusion chromatography (SEC).
Supernatant from the growth of transfected NSO cells secreting IgG
was clarified by centrifugation and filtration through a 0.22 .mu.m
membrane. A column of protein A Sepharose Fast Flow matrix
(Pharmacia) was equilibrated with 0.3 M NaCl, 50 mM sodium
phosphate pH 8.0 and the supernatant applied. The column was then
extensively washed with 50 mM sodium phosphate pH 8.0. Human IgG
was eluted with 0.1 M glycine-HCl pH 3.0. Eluted fractions were
neutralised with 1 M Tris HCl pH 9.0 and protein containing
fractions identified by measuring the absorbance at 280 nm.
Purification by SEC was on a Superdex 200 column in PBS. The IgG
was finally concentrated by diafiltration against pyrogen-free PBS
using a YM30 MWCO filter (Amicon).
EXAMPLE 3
Assessment of Neutralisation Properties of SL15S SCFV (CAT-191) and
SL15A IGG4 (CAT-192) and SL15S IGG4 (CAT-193)
[0175] The potency of neutralisation of TGF.beta..sub.1 was
measured for SL15S scFv (Kylie scFv) and its derivatives using a
radioreceptor assay and a cell proliferation assay (TF1).
[0176] Materials
[0177] [.sup.125I]TGF.beta..sub.1 was supplied by Amersham
(specific activity range 800-2200Ci/mmol). Recombinant human
TGF.beta..sub.1, .beta..sub.2,.beta..sub.3, latent TGF.beta..sub.1
GM-CSF and IL-5 were obtained from R&D Systems (Minneapolis
USA). Genzyme murine mAb against TGF.beta..sub.1, .beta..sub.2 and
.beta..sub.3 was obtained from Genzyme (Cambridge, Mass., USA). The
TF1 cell line was supplied by Robin Thorpe (NIBSC, UK) and cultured
as detailed below. The A549 human lung epithelial carcinoma cell
line was obtained from ATCC and grown in DMEM with 10% FCS and 2 mM
glutamine. All other reagents were supplied by Sigma.
[0178] Methods
[0179] Radioreceptor Assay
[0180] A549 cells were seeded into 24-well plates at
2.times.10.sup.5 cells per well for 24 hours in order to achieve
>90% confluency. Immediately before the assay, monolayers were
washed twice with buffer (1.1 DMEM:Hams-F12) and 0.5 ml assay
buffer (1 :1 DMEM:Hams F12+0.1% BSA) was added. Two or three-fold
serial dilutions of antibodies were prepared in assay buffer and
added to an equal volume of 40 pM [.sup.125I]TGF.beta..sub.1 in
assay buffer.
[0181] After 1 hour at room temperature, 0.5 ml of this
antibody/[.sup.125I]TGF.beta..sub.1 mixture was added in duplicate
to the cells (already in 0.5 ml assay buffer) and incubated for 1
hour at 37.degree. C. Final concentration of
[.sup.125I]TGF.beta..sub.1 was 10 pM. Controls were included as
maximum binding (to cells, no antibody) and minimum binding (wells
incubated with buffer but no cells) in at least triplicate.
[0182] Finally plates were washed 4.times. with ice-cold PBS before
0.8 ml solubilisation buffer (25 mM Tris pH 7.5, 10% glycerol, 1%
Triton x100) was added. Plates were left for at least 20 minutes on
a rocking platform before the contents of each well were counted
using a gamma counter.
[0183] Data were expressed after subtraction of minimum binding, as
% of maximum binding.
[0184] In the study using latent TGF.beta..sub.1, % maximum was
calculated for each set of conditions (eg in the presence of latent
TGF.beta..sub.1, acid-activated latent TGF.beta..sub.1 or active
TGF.beta..sub.1).
[0185] (Lucas C et al (1991) Meth in Enzymology 198, 303-16)
[0186] TF1 Assay
[0187] TF1 cells were routinely grown in RPMI1640 containing 5% FCS
and 2 mM glutamine (growth medium) with 2 ng/ml GM-CSF. Immediately
before the experiment, cells were washed twice and resuspended at
4.times.10.sup.5 cells/ml in fresh medium supplemented with 4 ng/ml
IL-5 either with or without TGF.beta..sub.1, .beta..sub.2 or
.beta..sub.3 (each at 50 pM) and 100 .mu.l aliquots transferred to
96-well plates. scFv preparations used in this assay were
FPLC-purified fractions which had had endotoxin removed.
[0188] Antibodies (two fold dilution series) were prepared in
growth medium and 100 .mu.l added to cells in duplicate. Controls
were cells with TGF.beta. only (no antibody, maximum inhibition of
growth) and cells with no TGF.beta. and no antibody (minimum
inhibition). Cells were incubated for 48 hours at 37.degree. C.
[0189] At the end of the assay cell number was assayed using
CellTiter96 (Promega) and data expressed as % neutralisation i.e. 1
%neutralisation = (testvalue - maximuminhibition)
(minimuminhibition - maximuminhibition) .times. 100
[0190] In the study of latent TGF.beta..sub.1 data were expressed
as % of control (growth in the absence of TGF.beta..sub.1) as the
amount of active TGF.beta..sub.1 in each test condition varied.
[0191] (Randall LA et al (1993) J Immunol Meth 164, 61-7)
[0192] Production of Antibodies
[0193] ScFv antibodies and IgG4 antibodies were prepared and
purified as described above.
[0194] Results
[0195] Potency of SL15S scFv (CAT-191) and SL15A IgG4 (CAT-192) in
a Bioassay (TF1 Assay)
[0196] The ability of SL15S scFv (CAT-191) to recognise
TGF.beta..sub.1 but not TGF.beta..sub.2 or .beta.3 in the TF1 assay
was investigated.
[0197] SL15S scFV (also known as Kylie scFV) neutralised the growth
inhibition induced by TGF.beta..sub.1 but not that induced by
TGF.beta..sub.2 or TGF.beta..sub.3 (FIG. 1) As a control a
monoclonal antibody, Genzyme Mab 1.D.11.16, was used (Genzyme,
Dasch, J. R., et al, J. Immunol., 142, 1536-1541, 1989), which
neutralises TGF.beta..sub.1, TGF.beta..sub.2 and TGF.beta..sub.3.
Mab 1.D.11.16 has been shown to be effective in models of lung
fibrosis, radiation induced fibrosis (Barcellos-Hoff, U.S. Pat. No.
5,616,561, 1997) and rheumatoid arthritis (Wahl et al, J. Exp.
Medicine, 177, 225-230, 1993). SL15S scFv shows comparable potency
to the Genzyme Mab 1.D.11.16 control against TGF.beta..sub.1.
[0198] The activity of SL15A IgG4 (CAT-192; also termed Kylie IgG4)
was also shown in the TF1 assay (FIG. 5b) in the study using latent
TGF.beta..sub.3.
[0199] Potency of SL15S scFv (CAT-191). SL15A IgG4 (CAT-192) and
SL15S IgG4 (CAT-193) in the Radioreceptor Assay
[0200] The ability of SL15S scFv to recognise TGF.beta..sub.1 and
neutralise binding of TGF.beta..sub.1 to A549 cells was measured in
the radioreceptor assay.
[0201] In a comparision of his-preps of scFv, SL15S (Kylie) was
compared to the parental antibodies CS37 and JT182 (FIG. 2) and was
found to be 100- to 150-fold and 10-fold more active. SL15 was
reformatted as IgG in two forms, SL15A IgG4 (CAT-192) and SL15S
IgG4 (CAT-193). Purified preparations of SL15S scFv, SL15A IgG4,
SL15S IgG4 and Mab 1D.11.16 (Genzyme mAb) were also analysed (FIG.
3).
[0202] SL15S scFv has comparable potency to the Genzyme Mab
1.D.11.16, which is effective in animal models. In summary, SL15S
scFv is a highly potent, neutralising antibody for TGF.beta..sub.1,
with IC.sub.50 values in the range 0.03 to 0.1 nM.
EXAMPLE 4
Binding of the Antibody SL15S SCFV (CAT-191) and SL15A IGG4
(CAT-192) to Active and Latent TGF.beta..sub.1
[0203] The experiments described in this example demonstrated that
SL15S scFv and SL15A IgG4 bind to and neutralise active, but not
latent, TGF.beta..sub.1.
[0204] Latent TGF.beta..sub.1 is the biologically inactive form in
which TGF.beta..sub.1 is secreted from cells, and is composed of a
latency associated peptide dimer (consisting of two 249 amino acid
monomers) and a mature TGF.beta..sub.1 dimer (consisting of two 112
amino acid monomers) Latent TGF.beta..sub.1 is not recognised by
cell surface TGF.beta. receptors Active TGF.beta..sub.1 is thought
to be released by the action of proteases in vivo which can be
mimicked by acidification in vitro.
[0205] Latent TGF.beta..sub.1, has a reported ED50 of 0.43 nM and 2
pM before and after acidification respectively, as assayed in a
proliferation assay. Active TGF.beta..sub.1 has a reported ED50 in
a proliferation assay of approximately 2 pM Presumably the effect
of latent TGF.beta..sub.1 before acidification is due to the
presence of some active TGF.beta..sub.1. This residual activity
must be considered on interpretation of the results.
[0206] The potency of SL15S scFv and SL15A IgG4 was tested using
the radioreceptor assay and TF1 proliferation assay in the presence
of varying amounts of latent TGF.beta..sub.1, acid-activated
TGF.beta..sub.1 and active TGF.beta..sub.1. If the antibodies
recognise latent TGF.beta..sub.1, their potency should be reduced.
Interpretation of both assays is complicated by the fact that any
active TGF.beta..sub.1 present in the latent preparation will
compete with [.sup.125I]TGF.beta..sub.1 for binding and have
biological activity on the cells.
[0207] Latent TGF.beta..sub.1 was acid-activated by the addition of
2 .mu.l 0.5M HCl to 1 ml latent TGF.beta..sub.1 for 15 min at room
temperature then neutralised with 43 .mu.l 0.05 M NaOH/0 01 M
Hepes.
[0208] Results show that in the radioreceptor assay there is no
significant effect of latent TGF.beta..sub.1 (0.1 nM) on the
potency of SL15S scFv (Kylie scFv) or SL15A IgG4 (Kylie IgG),
whereas acid-activation of the latent TGF.beta..sub.1, or an
equivalent concentration of active TGF.beta..sub.1 reduces the
ability of the antibody to neutralise [.sup.125I]TGF.beta..sub.1
binding (FIG. 4).
[0209] The TF1 proliferation assay was very sensitive to the
activity of the active TGF.beta..sub.1 in the latent
TGF.beta..sub.1 preparation. Nevertheless, the SL15S scFv or SL15A
IgG4 were found to be able to neutralise the small proportion of
active TGF.beta..sub.1 of the latent preparation and when the
latent TGF.beta..sub.1 is acid-activated neutralise this
effectively (FIG. 5).
[0210] Therefore, SL15, in the scFv or IgG format, binds and
neutralises only active but not latent TGF.beta..sub.1 as measured
by both the radioreceptor and the TF1 neutralisation assay.
EXAMPLE 5
Epitope Mapping of the Antibodies SL15S SCFV and CS37 SCFV
[0211] In this example the epitope on TGF.beta..sub.1 to which the
antibodies CS37 and SL15S binds was determined.
[0212] TGF.beta..sub.1 and TGF.beta..sub.2 have similar structures
but differ in their binding affinity for the TGF.beta. type II
receptor and their potency in a number of biological assays (O. G.
Ottmann & L. M. Pelus J Immunol. 140:2661-2665, 1988; J. R
Merwin et al., Am. J. Pathol., 138:37-51, 1991; K. C. Flanders et
al., Development 113:183-191, 1991; L. Suardet et al Cancer Res.
52:3705-3712, 1992). Qian et al (J. Biol. Chem. 271: 30656-30662,
1996) have taken advantage of the differences in affinity to
identify the key residues involved in the high affinity binding of
TGF.beta..sub.1 to the type II receptor. This has been done by
making a series of chimeric molecules between TGF.beta..sub.1 and
TGF.beta..sub.2 and identifying those species which retain high
affinity receptor binding in vitro (Qian et al, supra) and in vivo
(J. K. Burmester et al Growth Factors 15:231-242, 1998) In this
way, the C terminal region encompassed by residues 83-112 of
TGF.beta..sub.1 was identified as sufficient to retain efficient
receptor binding.
[0213] Comparison of the sequences of TGF.beta..sub.1 and
TGF.beta..sub.2 identified several differences including a loop
consisting of residues 92-98, which includes 4 amino acid
differences between TGF.beta..sub.1 and TGF.beta..sub.2. When these
residues from TGF.beta..sub.2 were introduced into a
TGF.beta..sub.1 backbone, receptor binding was greatly reduced,
identifying these as key residues in the interaction of
TGF.beta..sub.1 with the type II receptor.
[0214] The chimeric molecules were used in a similar way to map the
binding site of CS37 and SL15S scFv (CAT-191) on TGF.beta..sub.1.
TGF.beta..sub.1/.beta..sub.2 (83-112) corresponds to the N-terminal
and central region of TGF.beta..sub.1 from residues 1-82, fused
with the C-terminal region of TGF.beta..sub.2 from residues 83-112.
TGF.beta..sub.2/.beta.183-112 corresponds to the N-terminal and
central region of TGF.beta..sub.2 from residues 1-82, fused with
the C-terminal region of TGF.beta..sub.1 from residues 83-112.
Finally, TGF.beta..sub.1-.beta..sub.2 (40-112) corresponds to the
N-terminal region of TGF.beta..sub.1 from residues 1-39, and the
central and C-terminal region of TGF.beta..sub.2 from residues
40-112. These are referred to hereafter as 1-1-2, 2-2-1 and 1-2-2
respectively, to reflect the relative composition of the
isoforms
[0215] These three molecules along with TGF.beta..sub.1 and
TGF.beta..sub.2 were profiled for inhibition of binding of SL15S
scFv and CS37 scFv to immobilized TGF.beta..sub.1 as follows.
[0216] Phage displaying SL15S scFv or CS37 scFv were prepared from
clones in pCANTAB6, by M13K07 superinfection. Phage were PEG
precipitated and resuspended in PBS containing 2% Marvel. Phage
(2.5 to 5.times.10.sup.10 in 50 .mu.l) was added for 30 minutes to
a preblocked ELISA plate coated with TGF.beta..sub.1 at 0.5
.mu.g/ml.
[0217] For inhibition analysis, the same concentration of phage was
used and a dilution series of TGF.beta. concentrations set up for
each of the TGF.beta. chimeric molecules described above (Gift from
J. Burmester, National Institutes of Health) and the
TGF.beta..sub.1 and 2 isoforms.
[0218] These were incubated overnight at 4.degree. C. before adding
to the ELISA plate. Phage that bound specifically to the antigen
coated plate were detected using a sheep anti-fd antiserum
(Pharmacia), followed by alkaline phosphatase conjugated anti-sheep
immunoglobulin (Sigma) and p-nitrophenyl phosphate (pNPP) substrate
(Sigma).
[0219] The uninhibited value (i.e. 0 nM) for Kylie was 1.67
(Average of 3 readings) and for CS37 was 1.216 (Average of 7
readings).
[0220] The result shown in FIGS. 6 and 7 indicates that the
epitopes recognised both by CS37 scFv and SL15S scFv reside between
residues 83-112 of TGF.beta..sub.1. Furthermore, the mutant
molecule TGF.beta..sub.1-.beta..sub.2(92-98) which comprises
TGF.beta..sub.1 with the TGF.beta..sub.2 sequence from residues
92-98 also inhibits CS37 and SL15S scFv binding to TGF.beta..sub.1
but with reduced effect, especially for SL15S scFv. This
demonstrates that at least part of the epitope recognised by these
antibodies resides within the region 92-98, (which has four
residues, at positions 92, 94, 95 and 98, which differ between
TGF.beta..sub.1 and TGF.beta..sub.2).
EXAMPLE 6
Acceleration of Corneal Epithelial Wound Healing in the Presence of
the Anti-TGF.beta..sub.1 Monoclonal Antibody SL15 A IgG4
(CAT-192)
[0221] TGF.beta.has been implicated in the modulation of wound
healing (both ocular and dermal) and has been shown to inhibit the
rate of corneal re-epithelialisation. Here the effect of topical
application of SL15A IgG4 (CAT-192) on the rate of corneal
epithelial wound healing was assessed using an organ culture model
(DM Foreman et al., Exp. Eye Res. 62:555-564, 1996).
[0222] Excisional trephine wounds (5 mm diameter, 250.mu.m depth)
were created on bovine corneas. Corneal scieral rings were
maintained in a serum-free, air-interface organ culture system for
up to 96 hr. At time of wounding, and twice daily thereafter, 1001
.mu.l of serum-free T8 medium was added dropwise to the epithelial
surface and limbus, containing (i) 10 ng/ml TGF.beta..sub.1; (ii)
1001 .mu.g/ml anti-TGF.beta..sub.1 mAb; (iii) a combination of (i)
and (ii); (iv) 100 .mu.g/ml null antibody (2G6 IgG4), (v) 100
.mu.g/ml antibody diluent, or (vi) no additions (n=9 for each
group). Re-epithelialisation was assessed using an image analysis
system and expressed as percentage of the original wound area
covered by epithelium
[0223] Control corneas that received medium alone, null antibody or
antibody diluent demonstrated complete re-epithelialisation by 72
hr Treatment with 100 g/ml of anti-TGF.beta..sub.1 mAb increased
the rate of re-epithelialisation such that corneas were completely
re-epithelialised by 48 hours (FIG. 8). Corneas treated with 10
ng/ml TGF.beta..sub.1 demonstrated delayed re-epithelialisation by
24 hours and this inhibitory effect was abolished by
anti-TGF.beta..sub.1 mAb (FIG. 9). All differences quoted are
significant (p<0.05) Linear regression was performed on the data
points between 2 and 48 h (see FIG. 8) to determine the rate of
re-epithelialisation. CAT192 caused a significant increase in this
variable (Table 3). Histological analysis supported the image
analysis results and confirmed that addition of
anti-TGF.beta..sub.1 mAb did not adversely affect corneal
architecture
3TABLE 3 The effect of CAT192 or TGF.beta..sub.1 on the rate of
re-epithelialisation of bovine isolated cornea in air interface
organ culture following excisional trephine wound Rate of
Re-epithelialisation Treatment (% h.sup.-1) n Medium 199 (100
.mu.l) 1.56 .+-. 0.07 8 Vehicle (100 .mu.l) 1.57 .+-. 0.08 8 Null
antibodyIgG4 (10 .mu.l) 1.48 .+-. 0.09 8 TGF.beta..sub.1 (1 ng)
1.01 .+-. 0.09.sup..tau. 8 CAT192 (10 .mu.l) 2.11 .+-. 0.09* 8
Table 3 legend: Bovine cornea were treated with 100 .mu.l of either
serum free Medium 199 (control) or medium containing vehicle (for
antibody and TGF.beta..sub.1) null IgG4 isotope-matched antibody,
TGF.beta..sub.1 or CAT192 immediately after wounding and at 12 h
intervals thereafter. Linear regression was performed on the data
points between 2 and 48 h (see FIG. 8) to determine the rate of
re-epithelialisation. CAT 192 caused a significant # increase in
the rate of re-epithelialisation of wounded bovine cornea whereas
TGF.beta..sub.1 caused a significant decrease in this variable
Values shown are the mean value and the s.e. mean. The effect of
the different treatments was compared using one way ANOVA with
Tukey test. *P < 0.01 compared to the null antibody treatment
group; .sup..tau.P0.01 compared to the vehicle treated group
[0224] These results confirm the important role of endogenous
TGF.beta..sub.1 in corneal epithelial repair and provide indication
that the topical application of the anti-TGF.beta..sub.1 mAb SL15A
IgG4 (CAT-192) may be used to promote improved wound healing in
vivo
Sequence CWU 1
1
25 1 369 DNA Homo sapiens 1 caggtgcagc tggtgcagtc tgggggaggc
gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120 ccaggcaagg
ggctggagtg ggtggcagtt atatcatatg atggaagtat taaatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240 ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc
gcgaactggt 300 gaatatagtg gctacgatac gagtggtgtg gagctctggg
ggcaagggac cacggtcacc 360 gtctcctca 369 2 123 PRT Homo sapiens 2
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5
10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Ile Lys Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Gly Glu
Tyr Ser Gly Tyr Asp Thr Ser Gly Val Glu Leu 100 105 110 Trp Gly Gln
Gly Thr Thr Val Thr Val Ser Ser 115 120 3 369 DNA Homo sapiens 3
gaggtccagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
60 tcctgtgcag cctctggatt caccttcagt agctatggca tgcactgggt
ccgccaggct 120 ccaggcaagg agctggagtg ggtggcagtt atatcatatg
atggaagtat taaatactat 180 gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 240 ctgcaaatga acagcctgag
agctgaggac acggctgtgt attactgtgc gcgaactggt 300 gaatatagtg
gctacgatac ggacccccag tactcctggg ggcaagggac cacggtcacc 360
gtctcctca 369 4 123 PRT Homo sapiens 4 Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Glu Leu Glu Trp Val 35 40 45 Ala
Val Ile Ser Tyr Asp Gly Ser Ile Lys Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Thr Gly Glu Tyr Ser Gly Tyr Asp Thr Asp
Pro Gln Tyr Ser 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser 115 120 5 321 DNA Homo sapiens 5 gaaattgtgc tgactcagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgcc
gggcaagtca gggcattgga gatgatttgg gctggtatca gcagaagcca 120
gggaaagccc ctatcctcct gatctatggt acatccactt tacaaagtgg ggtcccgtca
180 aggttcagcg gcagtggatc tggcacagat ttcactctca ccatcaacag
cctgcagcct 240 gaagattttg caacttatta ctgtctacaa gattccaatt
acccgctcac tttcggcgga 300 gggacacgac tggagattaa a 321 6 107 PRT
Homo sapiens 6 Glu Ile Val Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Gly Ile Gly Asp Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Ile Leu Leu Ile 35 40 45 Tyr Gly Thr Ser Thr Leu Gln
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Leu Gln Asp Ser Asn Tyr Pro Leu 85 90 95 Thr
Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 7 321 DNA Homo
sapiens 7 gaaattgtgc tgactcagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60 atcacttgcc ggtcaagtca gggcattgga gatgatttgg
gctggtatca gcagaagcca 120 gggaaagccc ctatcctcct gatctatggt
acatccactt tacaaagtgg ggtcccgtca 180 aggttcagcg gcagtggatc
tggcacagat ttcactctca ccatcaacag cctgcagcct 240 gaagattttg
caacttatta ctgtctacaa gattccaatt acccgctcac tttcggcgga 300
gggacacgac tggagattaa a 321 8 107 PRT Homo sapiens 8 Glu Ile Val
Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Gly Ile Gly Asp Asp 20 25
30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu Ile
35 40 45 Tyr Gly Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln
Asp Ser Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu
Glu Ile Lys 100 105 9 369 DNA Homo sapiens 9 caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag
cctctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120
ccaggcaagg agctggagtg ggtggcagtt atatcatatg atggaagtat taaatactat
180 gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240 ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gcgaactggt 300 gaatatagtg gctacgatac gcccgcctcg
ccggactggg ggcaagggac cacggtcacc 360 gtctcctca 369 10 123 PRT Homo
sapiens 10 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Glu Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser
Ile Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Thr Gly Glu Tyr Ser Gly Tyr Asp Thr Pro Ala Ser Pro Asp 100 105 110
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 11 5 PRT Homo
sapiens 11 Ser Tyr Gly Met His 1 5 12 17 PRT Homo sapiens 12 Val
Ile Ser Tyr Asp Gly Ser Ile Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 13 14 PRT Homo sapiens 13 Thr Gly Glu Tyr Ser Gly Tyr Asp
Thr Asp Pro Gln Tyr Ser 1 5 10 14 14 PRT Homo sapiens 14 Thr Gly
Glu Tyr Ser Gly Tyr Asp Thr Ser Gly Val Glu Leu 1 5 10 15 14 PRT
Homo sapiens 15 Thr Gly Glu Tyr Ser Gly Tyr Asp Thr Pro Ala Ser Pro
Asp 1 5 10 16 11 PRT Homo sapiens 16 Arg Ala Ser Gln Gly Ile Gly
Asp Asp Leu Gly 1 5 10 17 7 PRT Homo sapiens 17 Gly Thr Ser Thr Leu
Gln Ser 1 5 18 9 PRT Homo sapiens 18 Leu Gln Asp Ser Asn Tyr Pro
Leu Thr 1 5 19 11 PRT Homo sapiens 19 Arg Ser Ser Gln Gly Ile Gly
Asp Asp Leu Gly 1 5 10 20 30 DNA Artificial Sequence Description of
Artificial Sequence Oligonucleotide 20 ctaagcttac tgagcacaca
ggacctcacc 30 21 35 DNA Artificial Sequence Description of
Artificial Sequence Oligonucleotide 21 aattttcgaa ctacagttac
tgagcacaca ggacc 35 22 46 DNA Artificial Sequence Description of
Artificial Sequence Oligonucleotide 22 atgggccctt ggtggaagct
gaggagacgg tgaccgtggt cccttg 46 23 53 DNA Artificial Sequence
Description of Artificial Sequence Oligonucleotide 23 ttggatatct
ctccacaggt gtccactccg aaattgtgct gactcagtct cca 53 24 40 DNA
Artificial Sequence Description of Artificial Sequence
Oligonucleotide 24 ctaccgtacg tttaatctcc agtcgtgtcc ctccgccgaa 40
25 52 DNA Artificial Sequence Description of Artificial Sequence
Oligonucleotide 25 ttggatatct ctccacaggt gtccactccg aggtgcagct
ggtggagtct gg 52
* * * * *
References